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Symposium on Structure, Function and Measurement of Respiratory Cilia

Date: 19660300/P
Length: 191 pages
00097351-00097541
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Author
Bates, D.V.
Kilburn, K.H.
Salzano, J.
Area
MINNEMEYER/R&D PILOT PLANT
Alias
00097351/00097541
Type
PSCI, SCIENTIFIC PUBLICATION
CHAR, CHART/GRAPH
PHOT, PHOTOGRAPH
Copied
Minnemeyer, H.J.
Named Organization
Harvard
Natl Tuberculosis Assn
NCI, Natl Cancer Inst
Trudeau Foundation
Univ of Rochester
American Thoracic Society
Duke Univ
Duke Univ School of Medicine
Date Loaded
05 Jun 1998
Named Person
Aiello
Anderson
Baetjer, A.M.
Ballenger, J.J.
Bang, F.B.
Bang, G.B.
Bates, D.V.
Bates, L.M.
Battista, S.P.
Bereblum
Bernfeld, P.H.
Boren, H.G.
Brinkman, G.L.
Brokaw, C.J.
Carpenter, R.
Carson, S.
Casarett, L.J.
Cember
Corssen, G.
Courington, D.
Dalhamn, T.
Dawson, F.W.
Deruyter, M.G.
Falk, H.
Ferris
Fletcher
Foard, M.A.
George, T.W.
Goldhamer, R.
Goodman
Gosselin, R.E.
Guarneri, J.J.
Hall, F.G.
Harding, H.B.
Heide, A.D.
Hers, Jfp
Hilding, A.C.
Hoorn, B.
Kensler, C.J.
Kilburn, K.H.
Kleinerman
Kotin, P.
Krueger
Laurenzi, G.A.
Leeuwenhoek, A.V.
Miller
Moore, J.A.
Morrow, P.E.
Palmer
Parmele, H.B.
Radford, E.P.
Rhodin, Jag
Ringer
Rivera, J.A.
Rose
Rylander, R.
Salzano, J.
Sleigh, M.A.
Spock, A.
Staub, N.C.
Stead, W.W.
Stuartharris, C.H.
Tyrrell
Voltaire
Watson
Weber
Winternitz
Wolbach
Wright, G.W.
Wynder, E.
Request
R1-004
R1-125
Litigation
Stmn/Produced
Author (Organization)
American Review of Respiratory Dise
American Thoracic Society
Duke Univ Medical Center
Characteristic
ATTE, ATTENDEE LIST
Master ID
00097351/7541

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LL MIMNl1Y1" March 1966 VOLUME 93/NUMBER 3 PART 2 OF TWO PARTS THE AMERICAN REVIEW OF R Respiratory Diseases r i , MEASUREMENT OF RESPIRATORY CILIA SYMPOSIUM ON STRUCTURE, FUNCTION AND Duke University Medical Center Durham, North Carolina February 18-19, 1965 OFFICIAL JOURNAL OF THE AMERICAN THORACIC SOCIETY . Medical Section of the National Tuberculosis Association 0 O ~
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SYMPOSIUM ON STRUCTURE, FUNCTION AND MEASUREMENT OF RESPIRATORY CILIA Duke University Medical Center Durham, North Carolina February 18-19, 1965 Sponsored by Department of Physiology and Pharmacology and Department of Medicine Duke University School of Medicine Durham, North Carolina The publication of this supplenient zcas made possible by a grant from P. Lorillard Company
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FOREWORD Growing public concern about air pollution and cigarette smoking has refocused the at- tention of scientists upon respiratory cilia and their role in the clearance of inhaled materials from the lung, Despite identification of cilia by Anton von Leeuwenhoek in 1676 and fur- ther description by Antonio de Heide a decade later, many gaps exist in our knowledge of these ubiquitous and useful structures which are found in all animal phyla except Nematoda. The usefulness of identifying what is known about respiratory cilia and what methods are useful in their investigation as a guide to future research was recognized by Drs. Tore Dalhamn and Ragnar Rylander of Stockholm who, with Dr. H. B. Parmele of the P. Loril- lard Company, conceived the symposium. The appropriateness of holding the meeting in the United States and within a tobacco-growing state was apparent, and Duke University was most willing to cooperate. Drs. John V. Salzano and Iiaye H. Kilburn became the planning committee's co-chairmen and the first plans were laid for a symposium on cilia. Its final content reflected the progressive education of this original group, together with the contributions of Dr. Edward P. Radford of Harvard, Dr. Hans Falk from the \ ational Cancer Institute, Dr. Hollis G. Boren of the Trudeau Foundation, and Dr. F. G. Hall of Duke as members of the arrangement committee. Examination of two recent monographs (i%7. A. Sleigh, The Biology of Cilia and Flagella, and J. A. Rivera, Cilia, Ciliated Epithelium, and Ciliary Activity) helped phrase questions about structure of the ciliated cell, energetics and motility of cilia, and niethods of meas- uring ciliary activity. Application of information to human disease was reflected in sessions on the effects of physical and chemical irritants on ciliated epithelium and their rela- tionship to bronchitis and cancer and on the effects of bacteria and viruses on cilia and pul- monarv clearance. Three types of participants were invited: those concerned with the forni and function of cilia and related structures; investigators who use ciliary activity as an index of toxicity of various materials in living systems; and physicians who are concerned with ltmg disease in patients which might be related to disturbed ciliary function or deficient pulmonar' v clear- ance, About fifty scientists were invited. Conflicting commitments and recent shifts of the active interests of some investigators away from cilia made the final group a representative one, if not absolutely complete. Sixteen papers were presented in four sections. Discussions after each section were help- ful in exploring concepts and techniques and in settling misunderstandings and deiining differences, especially in methodology. The discussion periods included extemporaneous re- marks as well as prepared discussions from forewarned individuals. A precis of these discus- sions furnishes the reader with some of the spirit of the meeting. Exact quotations have heen avoided, and misstatements of fact or interpretation are the responsibility and regret of the sYmposium editor. References to publications mentioned by discussors have not been repeated if they appeared in the reference lists of papers within the proceedings. Other ref- ,,rences appear at the end of sections of discussion. Despite occasional communication dif- iiculties abetted by specialized language, informal discussions took place. Unfortunately, proceedings of all convivial gatherinas were not recorded. Dr. Anderson C. Hilding reviewed the development of his own interest in the activin• of cilia during the past forty years and welcomed latecomers to the field in an informal eve- ning session. Dr. David Bates wove into his symposium summary the impressions and ideas from di- vergent views of cilia, ciliated epithelium, mucus and pulmonary clearance, and their relationship to human disease, particularly bronchitis and emphysema. His summarY is in- Cluded in the proceedings to indicate how the facts were interpreted and the unsolved prob- lcros defined by a clinical physiologist. Dr. William W. Stead, who served as referee from The Ame>ican Review ~~i Respiratory Diseases, provided sound advice and valuable assistance far beyond his charge of duties. The guidance supplied by the editorial staff of the journal is gratefullY acknowledged. The 1'. Lorillard Company was highly commended by the participants for financially spon~zoring this tYpe of gathering for the exchange of scientific information. The arrangement com- nittee is appreciative of the assistance and encouragement of Dr. H. B. Parmele.' Vice- President and Director of Research of P. Lorillard. llthough the flowers of science are often tilled behind disciplinary fences, glorious blooms ,-ften result from cross-pollination. Deliberate fence-crossing is abetted by symposia in which Deceased. iii
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iv FOREWORD people who use varied methods and skills converge upon a problem. The papers and dis- cussions during this symposium identified many questions and areas of ignorance. However, the participants were optimistic that some hybrid investigations utilizing fresh approaches would emerge from contacts made during the meeting. The proceedings are being published to share the provocation and excitement of new information and questions with a wider audience. IiArE H. IiiLBURN, NI.D., Editor JoH. 1". SALZANO, Ph.D., Co-Editor
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CONTENTS .. ForCWOr . AYE H. IIILBCRN AND JOHN V. SALZANO ....... . .. . ............ . ... . ... iii List of Participants .................................................. . . . ..... vii THE CILIATED CELL E. P. Radford, Jr., .lloderator Ultrastructure and Function of the Human Tracheal Mucosa. JOHANNES A. G. RHODIN ...................................................... I Some Aspects of the Comparative Physiology of Cilia. MICHAEL A. SLEIGH ...... 16 Mechanics and Energetics of Cilia. C. J. BROgAta. . ................. . ........... 32 Physiologic Regulators of Ciliary Motion. ROBERT E. GOSSELIN ................ 41 Discussion. GEOFFREY L. BRINKMAN AND GUENTHER COR9SEN ............. .... .... 60 ME.15URE\fENT OF CILIARY ACTION Tore Dalhamn, Moderator Cultural Methods for Measuring Ciliary Activity. J. J. BALLENGER, H. B. HARDING, F. W. DAR'3ON, M. G. DERUYTER, AND J. A. MOORE... ......... 61 Current Techniques to Measure Alterations in the Ciliary Activity of Intact Respiratory Epithelium. RAGNAR RYLANDER .............. . . . ............. ... 67 Discussion T. W. GEORGE, P. H. BERNFELD, AND A. M. BAETJER .................. 73 RESPONSES OF CILIATED EPITHELIUYI TO IRRITANTS Hans Falk, Jloderator Jlucus Transport in the Respiratory Tract. SrEvEK CARSON, ROBERT CARPENTER, AND RICHARD GOLDHA\IER ............................................ ......... 86 Chemical and Physical Factors Affecting itiIamulalian Ciliary Activity. CHARLES J. KENSLER AND SAJI P. BATTISTA ...... . ............................. 93 Effects of Industrial Dust on Ciliated Epithelinm. GEORGE W. WRIGHT ....,... 103 Effect of Cigarette Smoke on Ciliary Activity. TORE DALHANts . ............ . ... 108 Pathogenesis of Cancer in a Ciliated Mucus-Secreting Epithelium. PAUL KOTIN, DORIS COURINGTON, AND HANS L. FALK. ...................... . . . 115 Discussion. PAUL E. MORROR' ............................................ . . ...... 125 EFFECTS OF BACTERIA AND VIRUSES 0.1; CILIATED EPITHELIUM Hollis Boren, _lloderator A Study of the Mechanisms of Pulmonary Resistance to Infection The Rela- tionship of Bacterial Clearance to Ciliary and Alveolar Macrophage Func- tion. GUSTAVE A. L.aURENZI AND JOSEPH J. GUARNERI ...................... . .. 134 Responses of Upper Respiratory Mucosa to Drugs and Viral Infections. F. B. BANG, B. G. B.,LNG, AND M. A. FOARD.............. ..... ........... 142 Respiratory Viruses, Ciliated Epithelium, and Bronchitis. C. H. STUART-IIARRIS ......................................................... 150 Effects of Some Viruses on Ciliated Cells. B. HOORN AND D. A. J. TYRRELL ... .. . 156 Disturbances of ~the Ciliated Epitllelilun Due to Influenza Virus. J. F. PH. I-IERS .. . . ...... .............................. . ...... . .... ... 162 Discussion. NOR6TAN C. ;T:1UB, E. L. `VYNDER, AND A. SPOCK..................... 172 Perspective and'History of lun-estigation of Cilia in Human Disease. A. C. HILDING ................................................................ 178 ,5r(7TLA2arf of CoRfPrEIiCe. D. \'. 13:1TES ... .. . . . . . . . . . . . . . . . . . . . . . . . . ... _ . 182
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PARTICIPANTS IN THE SYMPOSIUM ON STRUCTURE, FUNCTION, AND MEASUREIIENT OF RESPIRATORY CILIA FEBRUARY 18-19, 1965 AAIELLO, EDWARD ............. Department of Pharmacology, New York Medical College, Fifth Avenue and 106th Street, N ew York, N en- York BALLENGER, JOHN J.......... Northwestern University Medical School, Chicago, Illinois BAETJER, ANNA .............. School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, llaryland BANG, FREDERICx BARRV..... Department of Pathobiology; School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Jiarvland BATES, DAVID V .............. Royal Victoria Hospital, JlcGill University, Montreal, P.Q., Canada BATTISTA, SAn2 P ............. Life Sciences Division, Arthur D. Little, Inc., Cambridge, Massachusetts BERNFELD. P. H .............. Vice President and Director of Research, Bio-Research Institute, Inc., 9 Commercial Avenue, Cambridge, AIassa- chusetts BOREN, IIoLI,IS G.. .......... Director of Research, Trudeau Institute, Saranac Lake, New York BRAIN, J ..................... Department of Physiology, School of Public Health, Har- vard University, Boston, 'Massachusetts BRINKMAN, GEOFFREY L...... Henry Ford Hospital, Detroit, Michigan BROSSW, CHARLES J......... Department of Biology, California Institute of Technology, Pasadena, California BUSSE, E ..................... Research Laboratories, Celanese Corporation of America, Summit, New Jersey CARSON, STEVEN ............. Food and Drug Research Laboratories, Inc., AIaspeth, New York CORSSEN, GLENTxER......... Department of Anesthesiology, University of Michigan, Ann Arbor, Michigan COURINGTON, DoRIS.......... National Cancer Institute, National Institutes of Health, Bethesda. Maryland CRESS, HERTA ............... Departnient of Anatomy, Duke University Medical Center, Durham. North Carolina DALUAVN, ToRE . ............ National Institute of Public Health, Stockholm. Sweden FALK, HaNs .................. National Cancer Institute, U. S. Public IIealth Service, Bethesda, Maryland GEORGE, T. WALLER......... Research Laboratories, Celanese Corporation of America, Summit, N ew Jersey GIEI„ B. G ................... Division of Air Pollution, Department of Health, Educa- tion, and Welfare, IJ. S. Public Health Service, Washington, D.C. GoLnuAMER, R>:cIIARD ....... Food and Drug Research Laboratories, Inc., Maspeth, New Yorl. GoonMAN, D. A ....... ...... Division of Preventive Medicine, Sloan-Iiettering Jnsti- tute for ('uncer Re.,earch, 4-1-1 East 68th Street, New Y ork, New York (;ossE>;IN, ROBERT E.. ... .... Department of Pharmacology, Dartmouth \Iedical ~chool, Il.uiover, New Il;unpshire IIAr.t„ F. G .. ............... Department of 1'hysiology and Pharmacolog}•, Duke I-niversit}- \Iedical Center, Durham, North Carolina IIEn.,, J. F. Ph....... ....... Lahoratory of Itespirator}- 1-irus Research, Department uf Internal Medicine, UniversitY Ilospital of Leiden, Leiden, The Netlierlands vii
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Vlll PARTICIPANTS HII,v1NG, A. C ................ Research Laboratory, St. Luke's Hospital, Duluth, ilinne- sota HostnERCER, F ................ Bio-Research Consultants, Inc., 9 Commercial Avenue, Cambridge, Jlassaehusetts KENsr.ER, C. J ............... Senior Vice-President, Life Sciences Division, Arthur D. Little Company, Inc., Cambridge, AIa."achusetts KIr.BLRN, K. H .. ........... Department of Internal Medicine, Duke C'niversity Jledical Center, Durham, North Carolina Kr.ErrvERSAN, JERrtY......... St. Luke's Hospital, 11311 Shaker Boulevard, Cleveland, Ohio KoTlN, PArr.................. National Cancer Institute, Aational Institutes of Health, U. S. Public Health Nervice. Bethesda, 'Mar}•land LARSON, PAtir,............... Department of Pharmacology, -Medical College of Virginia, Richmond, Virginia LArxESZI, GrsTAVE A........ Director, Division of Respiratory Diseases, New Jerse.- College of Medicine, Jersey City, New Jerse}- Lt-cxslNGER, PETER C........ Veterans Administration Hospital, Washington, D.C. MILLER, C. ELGENE.......... Rheological Mechanics Laboratorl-, Alanhattan College, Bronx, New York Mor;ROw, PArr, E....... ... Department of Radiology, University of Rochester School of 'Medicine and Dentistrv, 260 Crittenden Boulevard, Rochester, New York NIELSON, E .................. 1838 Brantly Street, 1j'inston-Salem, Aorth Carolina PARMEr.E, H. B ............... Director of Research, P. Lorillard Company, New York, New York R4nFOxn, E. P., Jrt........... Institute of Physiology, University of Milan, Via Mangia- galli 32, Milan, Italy Rxonr.r-, JoxaNNES A. G...... Department of Anatomy, New York NIedical College, Fifth Avenue and 106th Streer. New York, New York RYi,:1NnEn„ RAGaAR.......... 'National Institute of Public Health. Stockholm, Sweden SALZANO, JoaN I ... ........... Department of Ph}•siology and Pharmacology, Duke University \Iedical Center, Durham, Aorth Carolina SLEIGH, M. A ................ Department of Zoology, The University of Bristol, Bristol, England SYE.1Ra, A. W ................ P. Lorillard Company, Greensboro, North Carolina SPOCK, Ar,Ex.tNnErt........... Department of Pediatrics, Duke University Medical Center, Durham, North Carolina STALB, AortMAN C............ Cardiovascular Research Institute. Department of Physi- ology, University of California Medical Center, San Fran- cisco, California fiTEAn, WI1.LI.aM S~'........... Jlarquette University School of Medicine, 8700 W. Wis- consin Avenue. Milwaukee, Wisconsin STLT AxT-HARRrs, C. H........ Department of Jiedicine, The University of Sheffield, The Royal Ilospital, Sheffield, England TOL"EY, GEORGE .............. Research Department. Tennessee Eastman Company, Knoxville, Tennessee TYRRELL, D. A. J. Corumon Cold Research Unit. Harvard Hospital, Coombe Road, Salishury, Englcrnd WEBER, .l1AS ..... ......... .. Wissenschaftliehe Forschung>stelle, Ilamburg I, An der Alster 6, Germanv lVxr<<rrr, GEORGE W.......... . -t. Luke's Hospital, 11311 Sli;iker Brnilevard. Cleveland, Ohio 1VYNDEx, E. L ..... . . . . . . . . ... Division of Preventive Medicine. '4loan-Iiettering Institute for ('ancer Research, 444 East 68th Street, New York, New York
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THE AMERICAN REVIEW OF RESPIRATORY DISEASES Clinical and Laboratory Studies of Tuberculosis and Respiratory Diseases VOLUME 93 March 1966 NUMBER 3 THE CILIATED CELL E. P. RADFORD, JR., Moderator ULTRASTRUCTURE AND FUNCTION OF THE HUMAN TRACHEAL MUCOSA' JOHANNES A. G. RHODIN INTRODUCTION The mucous membrane of the respiratory -ract is constantly exposed to the inhaled air _uid its contaminations. Because of this, it is nece,~sary for the mucous membrane to be prepared against any foreign particles, fumes, ,,r smoke. The cells of the respiratory epithelium are well differentiated for this purpose, and rlie way they are associated makes the removal .u particles easy. However, the epithelium also =eerns to have certain weaknesses in this re- -pect. The fine structure of the respiratory epithe- _ium of the rat trachea was discussed in detail by i;hodin and Dalhamn (1) in 1956. A brief re- ;,,-,rt by Rhodin (2) on the human trachea and i,~ fine structure appeared in 1959. However, =ince these reports were communicated, great ;i iprovements have been made in the techniques .nvolved in electron microscopy (3). The pres- :rvation of structures is now much better than (.arlier, and the understanding of various cell ~ructures and their functions has become in- ~,reasingly better. Lately, several reports have -tppeared on the fine structure of cilia and fla- oi1a from a great variety of algae and inverte- ~Tates (4-9). These investigations have greatly ilitated our basic understanding of ciliary -rructure and function. It is therefore justifiable ,, review the ultrastructure and function of i:e ciliated epithelium. However, it seems im- ~:~rtaut, at least to this reviewer, that the human ~~piratory mucosa be kept in the center of ` 1-rom the Department of Anatomy, New York \L lical ColleE~e, \ew York,. \ew York. attention all the time. Whatever investigations have appeared in invertebrates, algae, or lower mammals should all be discussed in the light of increasing the understanding of the ciliated epithelium, particularly the respiratory epithe- lium, in man. The present review is based on the writer's studies of the human tracheal mucosa. MATERIALS AND METHODS With the techniques employed, small bits of the trachea were removed surgically from patients at operations which entailed the removal of the ].arynx. The specimens were immediately fixed in buffered, one per cent solution of osmium tetroxide and subsequently dehydrated in ethyl alcohol and embedded in Epon 812 (10). Sections were cut on an LKB Ultratome® with glass knives. They were stained with lead according to Millonig (11) after mounting on Formvar film. Subsequent to the staining, the sections were covered with carbon. Micrographs were taken with a Siemens Elmiskop I. Tracheal Mucosa The respiratory mucosa is composed of several layers (figure 1). Innermost is the epithelium, which is of the pseudostratified columnar ciliated type. This epithelium rests on a basement menn- brane which in the light microscope is seen as a layer of varying thickness. Analysis of the fine structure of the basement membrane denionstrates it to be composed of one thin, con- tinuous layer, with an average thickness of about 1,000 -1, immediately beneath the base of the epithelial cells. The rest of the light micro- scopic basement membrane is composed mainly of a dense network (feltn•ork) of fine collage- nous and reticular fibrils. Both the thin and the ]
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.r Pic. 1. Longitudinal section of the tracheal epithelium (Ep) with part of the lamina propria (Lp).-Human trachea. Electron micrograph. (Magnification, X650) FIG. 2. The epithelium is composed mainly of four cell types: basal cells (Bc), intermediate cells (Ic), mucous cells (Mc), and ciliated cells (Ci). The length of the cilia averages 6 g.- Human trachea. (Magnification, X1,400) 0009'7359
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ULTRASTRUCTURE AND FUNCTION OF TRACHEAL MUCOSA 3 rhick basement membrane may be penetrated 1?' v occasional nerves and cellular components. lt is presently quite generally accepted to refer only to the thin membrane as the base- ment membrane. The dense network of fibrils is really an integral part of the interstitial con- nective tissue fibrils, although with an unusual, dense arrangement. Beneath the feltwork is the lamina propria of the tracheal mucosa. It con- tains a loose network of collagenous and elastic f.berU, fibroblasts, numerous lymphocytes, mac- rophages, occasional mastcelle, and eosinophils. The capillary network is quite vast and is ac- companied mostly by unmyelinated nerves. It .hould be noted that the capillaries do not come very close to the thin basement membrane; ,:~either do they form extensive venous plexuses ~unilar to those in the nasal mucosa. Glands of the Submucosa The submucosa contains a loose connective 'is~ue in which a varying amount of fat cells :~ located. It is also traversed bv occasional ?hin strands of smooth muscle; but its main ieature is the glands. These are mostly simple ''.Ibulo-alveolar glands with a predominance mucus-producing cells, although a fair num- 'ler of serous cells are mixed in between, often m a demilunar kind of arrangement similar 1o that in the sublingual and submandibular <alivary glands. It is important to keep in mind that the se- oretion of the trachea is derived mostly from -hese glands, although the goblet cells of the ~pithelium obviously also contribute to the tormation of the mucous blanket of the respira- )ry mucosa. It is quite likely that the serous ~-omponent of the mucus comes only from the <itbmucosal _lands, and irritants reaching the ~nucosa are likely to cause these submucosal _lauds to discharge rapidly and often with :-niumes so large that it would be impossible or the goblet cells of the epithelium to produce -;ch a quantity of mucus instantaneously. The Tracheal Epithelium ~ILin' V of the most important functions of the -: irheal mucosa rest with the epithelium. It ::eretore seems appropriate to consider this ~~ini,rane quite thoroughly. There are at least r different cell types in the epithelium ure 2). B.i~al cells form a layer next to the basement membrane. Goblet cells represent the mucus-producing cell of the epithelium. They reach from the basement membrane to the surface of the epithelium. Ciliated cells are pre- dominant; they all face the surface and gen- erally reach the basement membrane. A fourth cell type is frequently noticed in the human tracheal epithelium. It is an intermediate cell located just above the basal cell layer. Basal cells: The basal cells are of a polygo- nal or slightly elongated shape (figure 3). They rest on the thin basement membrane. The lateral surfaces make contacts with neigh- boring basal cells, but also with the basal processes of the goblet cells. These contacts may be in the form of thin cytoplasmic processes making a membrane-to-membrane contact, but more often than not these contacts take the shape of desmosomes. This is a specialized cell contact involving a multilayering of membranes in a button-like arrangement. This kind of cellular contact is common in stratified squa- mous epithelia. The nucleus occupies a large por- tion of the basal cell. The cytoplasm is charac- terized by aa multitude of fine tonofilaments which traverse the cell in many directions and often converge on the desmosomes. lTitochondria are sparse, and those present are small and spherical and scattered about in the cell. Ribo- somes are sparse; the Golgi complex is vestigial if at all present. Some granular structures with high electron density may represent lysosomes. In general, the basal cell represents a poorly differentiated cell similar to the cells found near the basement membrane of the epidermis (ger- minal cells) or the esophagus. In the latter cases, the basal cells give rise to other cells of the epithelium, and there is every reason to be- lieve that this is the case also in the tracheal epithelium. Intermediate cells: Infrequently, there are some cells just above the basal cell layer which resemble the basal cells from an ultrastructural point of view, but which have a more elongated or ~4pindle shape (figures 2 and 3). Mitoses can often be seen within this incomplete cell layer, and it is quite obvious that the cells are derived from the basal cells. .17u.cus-producing cells: The mucus-pro- ducing cells are called goblet cells (figure 4). They are structurally very similar to goblet cells of the intestinal tract. In the respiratory
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JOHANNES A. G. RHODIN
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ULTRASTRUCTURE AND FUNCTION OF TRACHEAL MUCOSA O mucosa of the human trachea, the goblet cells h,lve a more elongated shape than elsewhere, probably because of the rather extreme depth of the epithelium. The base of the goblet cell tapers off considerably, and most of the time it is seen to bend around basal and intermedi- .ti'te cells. The nucleus is located toward the base of the cell. Its shape is round or slightly oval, and it is rarely compressed as it is in the in- testinal goblet cells or in the mucus-producing cells of the tracheal lamina propria. The ! Tolgi complex of the goblet cell is rather prom- inent just above the nucleus. It is presently well established that secretory granules are rormed within the Golgi region (12). Golgi membranes and vacuoles gradually develop into premucous granules, and a subsequent swell- in; transforms them into mature mucous gran- ttles. Gradually, the mucous granules become <lensely packed and fill the upper part of the cell, which becomes expanded, giving rise to the goblet shape. Simultaneously, the limiting membrane of the mucous granules becomes less distinct and fusion occurs between the granules. The discharge occurs by an expulsion of the content of the goblet. The cytoplasm of the goblet cell is rich in ribo~omcs, which gives it a dark and elec- rron-dense appearance compared to the basal and ciliated cells. The ribosomes are frequently -ittached to membranes or sacs, forming the _r;mular endoplasmic reticulum which is en- -aged in the early elaboration of the secretory products (12). Mitochondria are sparse and ~- oval in shape. Lysosomes occur throughout the cytoplasm, and they seem to be more abundant 'oward the end of the secretory cycle. The tapered end of the goblet cell contains re,ular tonofilaments although they are not nearly as abundant as in the basal and inter- me liate cells. C'iliated cells: The ciliated cells are the mo<t common in the human tracheal epithelium ~ li_ure 5). On an average, there are about ciliated cells for each mucus-producing -~,iL Generally, the ciliated cell extends from the basement membrane to the luminal sur- face of the epithelium. The nucleus of the ciliated cell is oval; it is located toward the tapered end of the cell. The cytoplasm is light compared to the goblet cell and contains scattered ribosomes and occasional short pro- files of the granular endoplasmic reticulum (13). The apical part of the cell sometimes displays an abundance of smooth-surfaced or agranular endoplasmic reticulum (13). Within this area, there is always an abundance of mitochondria, which are rather long and even show connec- tions with other mitochondria. Mitochondria also occur in other parts of the ciliated cell but are less abundant than in the apical region. These mitochondria tend to be round or slightly oval. The cytoplasm displays tonofila- ments, but again they are less numerous than in the basal cells. There is a large number of lysosomes in the ciliated cells. These structures are membrane-botmd granules with an internal structure of pronounced pleomorphism. This is most likely explained by the function of the lysosomes, which seems to be associated with processes involving lysis (14). It has been shown that the h-sosome contains a great vari- ety of lytic enzymes. Breakdown products from cellular metabolic processes may therefore be handled by the lysosomes in an autodigestive activity during which all sorts of structural configurations may appear, resulting basically from a reorganization of the proteins and lipids of the cell. The luminal surface of the ciliated cell is provided with an average of 200 cilia (figure 6). In addition, microvilli (figure 5) protrude from the surface in between the cilia at varying lengths. The cilia have an average width of 0.3 u and a length of about 6 µ. Each cilium has a system of longitudinally arranged fibrils emanating from the basal body within the cytoplasm just beneath the surface of the ciliated cell (see figure 10). The cilia of the human trachea display the eame basic pattern as the cilia of the rat trachea (1), although some additional features Fte. 3. Detail of basal cells (Bc) resting on the thin basement membrane (Bm). Beneath is the fine feltwork of reticular fibrils of the lamina propria (Lp). The cytoplasm of both the hasal and intermediate (Ic) cells contains fine tonofilaments and small scattered mitochon- dria (Mi). The cells send fine processes into the narrow intercellular space (I) in which tko terminate free sensorc nerve endings. One such nerve (\e) is shown cross-sectioned, r-nntciining neuro'tubule~. in the insert.-Human trachea. (Jlagnification, X1:3,000. Insert. ~ 52,000 )
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1. The mucous cell (1Ic) reaches the surface of the epithelium where it dischar:res the ~lhnnd:int iuucous granules, b}• breakine the cell membrane (arrow). Ciliated cells (Ci) sur- i ( a,h mucous cell on all sides.-Human trachea. (AIamification, x12,000) (i
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hre. 5. The ciliated cells have numerous mitochondria (RZi) clustered beneath the basal br>dies (Bb) of the cilia (Ci). Several lysosomes (Ly) are usually seen between the mito- rhondria and the Golgi complex (Go). Fine bundles of banded fibrils (asterisks) arise from rhe basal bodies and extend downward in the cell amona the mitochondria. Between the r•ilia (Ci) are seen fine microvilli (Vi) projecting from the cell surface.-Human trachea. (\lagnification, X9,500)
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FIG. 6. Cross section through the cilia displays thcir regular arrangement of appro.imatelly 200 pcr cell. The tips of mucous cells (AIc) and sonic slourhing ciliated ce_1k (Ci) an. ~zccii. -Hituian trachca. (Magnification. 3,400) Fn;. 7. In a section parallel to and near ihe surface of the (,pithelium, tlu, cilia and the f,u~al -)rlws can be cross-sectioned at :ie same time due to thre une~-en ~urface ~~f the ehitht-lium. C r( pre.onts the cross-sectioned free cilium: D is the transitional zr>ne botween fmo ~ cilium ,nd ha:al bod' v, the latter seen at E. At C-D and D-E are scen interincdiatc leN els bencP(•n rhe C, D, and E levels.-Human trachea. (lIa~-nification. 68,000) S
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ULTRASTRUCTURE AND FUNCTION OF TRACHEAL MUCOSA have been revealed owing to better preserva- tion and resolution in the present study. There ;ire nine peripheral filaments with a doublet structure, and two central, single fibrils (figure 7 ). In the basal body, the peripheral doublet fibrils become triplet in nature. Toward the tip of the cilium, the peripheral fibrils become single and seem to fuse (figures 8-10). The two central fibrils of the cilium are surrounded by a matrix, slightly denser than the rest of the ciliary cytoplasm. The fibrils terminate at the level of the cell surface and do not partici- pate in the structures of the basal body, al- thouah the dense matrix around the two central fibrils continues down into the basal body where it fills it entirely. The triplets of the basal cor- puscle continue as rootlets with a banded structure. The doublets of the transitional zone are connected by a thin membrane which con- tinues down between the triplets of the basal bod' v. Also, in the transitional zone, the periph- f,ral crevice of each doublet is provided with a lonaitudinal fender-like structure for about a qiiarter of a micron (figures 8 to 10). Detailed studies of the fine structure of cilia of several algae, mussels, and unicellular or- Yanisms (6-9) have recently provided addi- tional information of structures associated with the fibrils in the stalk of the cilium as well as in the transition zone and in the basal body. These structures may have a direct relation to the understanding of the function of the cilium in these species, particularly with reference to means of conduction and contraction. In the ~hat"t of the cilium, the peripheral fibrils have been demonstrated to have small "arms" ex- tending in a clockwise direction half across from one doublet to another. The two central fibrils oiicu seem to be surrounded bv a thin sheath, ~iud structures have been described which appear fine lines in a spokelike arrangement connect- ine the central fibrils with the peripheral ones. The central as well as the peripheral fibrils <eem to have a beaded appearance at very Lia•h tnarnification; it has been suggested that !nis represents a helical pattern of filaments ~orminr the fibril. On cross section, the fibrils ppear in most instances to be hollow. Ilowever, 1'ea<e (15 ) and Thiery (16), using the negative - aining technique applied to fragmented sperm iik, were able to demonstrate that each fibril -:r ~Pt of fibrils contains 10 to 15 filaments, 9 with an average diameter of about 10 A. It is therefore likely that the beaded appearance actually reflects some of the changed interrela- tionship of these fine filaments in each fibril during contraction. Recently, Manton (8), in a paper on the fine structure of the transition zone of the cilia in green algae, demonstrated clearly that within this zone the doublets are interconnected in a most intriguing way. In cross sections it ap- pears as though two starlike structures were superimposed, connecting the doublets to each other. Manton interprets this as resulting from a helical structure which starts at, say, fibril 1, and then connects with 3, 5, 7, 9. From there it connects with fibril 2, 4, 6, 8, and then back to fibril 1, 3, and so on. After the com- pletion of two turns, all peripheral fibrils have been connected. Gibbons and Grimstone (6) and Gibbons (7) demonstrated fine filamentous connections between the peripheral fibrils and the plasma membrane in the transition zone of the cilia of Anodonta. They also have dis- cussed thin filaments connecting triplet fibrils as well as some with a cart-wheel kind of ar- rangement in the basal body of some cilia. They suggested that these mac serve simply as stiff- eners in this unusually long basal body (figure 11). Obviously, many of these structures could be involved in conduction and coordination ac- tivities related to the ciliary stroke. These structures may be of greater importance in algae and invertebrates, in which it might be expected that the cilia have more autonomy, than in vertebrates and mammals such as the rat and man. Since only some of these structures which presumably have conduction or coordina- tion functions have been reported in man, it may well be that they do not exist. On the other hand, refined techniques may in the future re- veal them to be present also in the cilia of the respiratory epithelium of man. Verves in the Tracheal 1'liticosa '\,Zost of the nerves in the lamina propria are of the nonmyelinated type. Some myelinated nerves also occur. The unmyelinated nerves ac- company arterioles and venules, buf some travel singly through the lamina propria and are seen to approach and penetrate the dense feltwork of reticular and collatrenous fibrils of
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O ~
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ULTRASTRUCTURE AND FUNCTION OF TRACHEAL MUCOSA the thick basement membrane. None of these nerves has been observed to pierce the thin I_,asement membrane, but nerves have been re- corded around the basal ends of the ciliated and mucus-producing cells of the epithelium (fi,ure 3). As in the taste bud of the rat's tongue, as described by Farbman (17), these nerves do not make synaptic contact with the epithelial cells. The observed nerves could be classified as belonging to the free sensory nerve endings of most epithelia. Life Span of the Tracheal Epithelium X1ost epithelia are replaced through a process of sloughing and growth. The actual life span of the individual cells has not yet been deter- mined, although this can easily be done now that autoradiographic techniques have been de- veloped for electron microscopy. .1lucus-produ.cing cells have been demon- ztrated to go through a cycle of secretion, re- covery, replenishment, and secretion; however, many early investigators of goblet-cell cytology were of the opinion that a goblet cell would clisintegrate after the completion of a secretory cvcle. It is not known presently how many ~ecretory cycles each goblet cell can go through, but one might assume that each cell is capable of completing two cycles. Ultimately, the goblet cell will slough or become digested by macro- phages, which have been observed to invade the epithelium from time to time. Ciliated cells are sloughed more frequently Than goblet cells, as judged by the number of disinte~rating cells. The sloughing of ciliated cells is preceded by a blebbing and swelling of the apical cytoplasm by which the cilia are iucorporated into the cytoplasm. Subsequently, mitochondria swell and the cytoplasm of the whole cell is thinned out. As a final step, the hase of the ciliated cell is retracted and the cc1l is pu~hed out (figure 12). 11 Regeneration The regeneration of lost, sloughed ciliated and mucus-producing cells occurs through growth and differentiation of basal and inter- mediate cells. This is the process in all epithelia with basal cells, since the basal cells are poorly differentiated and have an obvious potentiality of developing into other cell types. Mitoses are frequent among the basal and intermediate cells. However, the cytoplasm of the intermediate cells contains fewer tonofilaments and more cell organelles such as mitochondria and lyso- somes than does that of the basal cell. The intermediate cells, in turn, are less specialized than the ciliated or goblet cells, and a further differentiation of the intermediate cell type will make it into either a ciliated cell or a mucus- producing cell. There is no evidence from the present inves- tigation of the human trachea that ciliated cells can turn into mucus-producing cells, as was described by many early investigators and also more recently by Osada (18) in an elec- tron microscopic investigation. It seems obvious that the disintegration of ciliated cells has been taken as evidence for such a transition. In the rat trachea, Rhodin and Dalhamn (1) observed cells which had no, or few, cilia but instead a well-developed system of microvilli. On rare occasions, this cell type has been en- countered in the present human material. It is not known by what means cilia are regenerated or differentiated in the human trachea. Wherever studied, as for instance in the neural tube of the chick embryo (19), or in the visual cells of the tadpole retina (20), the formation of a cilium is always preceded by an outgrowth of cytoplasm in the shape of a microvillus. Subsequently, one of the centrioles moves into juxtaposition to the cell membrane and the microvillus, and induces, by growth or I'm. S. At the tip of the cilium, there is a slight indication of a fusion of some of the periphoral single filaments seen in figure A, here reduced to six in number. alightl' y away irom the tip of the cilium (B) the number of the peripheral filaments is nine, of which five are ~in::lets and four doublets. The two central filaments prevail in both figure A and figure B. Frc. 9. In the middle of the cilium are seen both peripheral and central filaments, and their arran_ement is more clearly seen in cross section in figure C. 1-in. 10. The transitional zone (asterisks) between the cilium and the basal body is marked ~~Y a=iight narrowing of the diameter of the peripheral filaments, as seen also in figure -D. In addition, each doublet is ,loiued bv a membrane, and from the common wall of each ~fouM~let emernes a short membrane. The central filaments have terminated hf-fore this level ~f the cilium is reached. In the basal body, the doublets are transformed into triplets (fture E ), At the same time, the filaments are rotated sli0htly. Figures 8-10 and A-D are all from uuman trachea. (Ma,nifications: figures 8-10, X71,000; figures A-D, X1d0.000)
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12 JOIIA\ \E5 A. G. RIIODIV A s C D © I'ta. 11. This drawing summarizes the fine strtir•ttu•e of a hutuan trachenl ritiiim :utd its 1,:1s:t1 bor1.-. It represents this ;utthor's interpretution of tlie thre-dituensional oricntution of ntainl}- the heripheral tilaments. in the tip of the tilium (:1 and B). in the ntnin stalk (C t, in tlie tr: nsitional zone he'tween the cilium and tlie I,,tsal boda (D), snd in the Ixtsil I,od, , in- t-ludiug the rootlets (E).
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ULTRASTRUCTURE AND FUNCTION OF TRACHEAL MUCOSA Fic. 12. Schematic representation of the pseudostratified ciliated epithelium ot the liu- man trachea. The drawing is baeed on a slightly simplilicd tracing of an electron micraunraph. ~4ume cellular features such as desmosonnes, cell membranes, and intercellular spaces are ,uhanced or enlarged. 13 ~a lier~c ise, the formation of peripheral ciliarY could represent the early forulation of cilia, as rll, ril>. This has not been ob~er~~c~~l to halipen u~a~ mngested by Rhodin ('_2). It ~cas believed i 1I:e tracheal epithelium, but it is believed earli(:r (21) that the centrosome pertitioned, r~);ir tbe observed accumulation of micro%illi uid that tlie re~nltinr basa1 bodies migrated
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i t JOHANNES A. G. RHODIN n the distal cell surface where cilia are -prouted. This reviewer is of the opinion that cilia ~~:innot be regenerated by the same cell from ,.viiich they previously were lost. Therefore, an '1.1upairment of ciliary function may ultimately ",:id to loss of cilia and sloughing of the ciliated cetl prematurely. There is presently no ob- vious reason why, under certain conditions, the epithelium of the trachea is transformed from a pseudostratified columnar ciliated form to a ~rratified squamous, mostly noncornified form. Itt is natural that the basal cells, with their strongly developed system of tonofilaments, inay retain this structure upon reaching the epithelial surface if the stimulus for ciliary regeneration is blocked by agents which im- pair ciliary activity. A vicious circle therefore results and leads ultimately to permanent iuetaplasia of the epithelium. .1leans and Routes of Resorption The viscosity of the mucous blanket can be :dtered, chiefly by evaporation or by adding ~orous secretion. To some extent, a slow process ot cellular uptake of fluid by means of micro- ninocytosis may take place. However, it should I)e remembered that water and gases dissolved in the water may easily diffuse through the plasma membrane of both cilia and microvilli. It was not considered earlier in this paper, but the excitation and conductivity and coor- ciination of ciliary beat may take place through a change in the membrane potential. It may very well be that a diffusion of water-soluble ' otiic agents may upset this membrane conduc- .ivity or membrane potential so as to impair or completely stop ciliary activity. SUMMARY The structure of the mucosa of the human a rachca has been anah-zed by means of elce- iron microscopy. The analysis includes a de- ~ailed discussion of recent works on the fine =t ructure of cilia of invertebrates and algae. In -_-neral, the cells of the pseudostratified ciliated nlnnuiar epithelium of the human trachea are i.,<Oribed and classified as ciliated cclls, miicu~;- Touucing goblet cells, basal cells, and inter- ~~wdiate cells (figure 12). The basal cells rep- --ent the layer from which the other cells ufierentiate, the intermediate cells bt•inz tran-,- formed into either ciliated cells or goblet cells. In no instance could an indication be found of a goblet cell being transformed into a ciliated cell or vice versa. The goblet cells go through secre- tory cycles. The ciliated cells are seen to slough more frequently than the mucous cells. Al- though the fine structure of the cilium and the basal body is not as complex as in inverte- brates, sufficient evidence has been obtained from this study that also in man, in the respiratory epithelium, the cilia and their sub- microscopic fibrils are provided with numerous devices for regulating the conduction and syn- chronization of the ciliary beat. Acknowledgments The writer thanks Dr. John F. Daly, Professor and Chairman, Department of Otolaryngology, New York University School of Medicine, New York City, for making the specimens of the human trachea available for this study; Miss Gunilla Runefeldt for technical assistance in the prepara- tion and the electron microscopy of the specimens, and in preparing the photographic prints; and Mrs. Gunvor Rhodin for making the schematic drawings. REFERENCES (1) Rhodin, J., and Dalhamn, T.: Electron mi- croscop,y of the tracheal ciliated mucosa in rat, Z Zellforsch, 1956, 44, 415. (2) Rhodin, J.: Ultrastructure of the ciliated mucosa in rat and man, Ann Otol Rhinol Laryngol, 1959, GS, 964. (3) Rhodin, J. A. G.: An A'las of Ultrastructure, W. B. Saunders Company, Philadelphia, 1963. (4) Afzelius, B. A.: The fine structure of the cilia from ctenophore swimming plates, J Biophys Biochem C,vtol, 1961, 9. 353. (5) hawcett, D. W.: Cilia and flagella. In The C,ell, vol. II, J. Brachet and A. E. Mirskv, editors, Academic Press, New York, 1961, p. 217. (6) Gibbons, I. R., and Grimstone, A. V.: On flagellar structure in certain flagellates, J Biophys Biochem C' vtol. 1960, 7, 697. (7) Uibbons, I. R.: The relationship between the fine atructure and dircetion of beat in eill rilia of a lamellibranc_li mollusc, J Biophys Biochcm Cytol. 1961, 11, 179. (8) Manton, I.: The possible significance of ,ome details of 11aRe11sr bases in plants, J Roy Micr Soc. 1963. S2, 279. (9) Roth, L. E., and Shigenaka, Y.: The -struc- 1 nre and formation of cilia and filaments in nimen protozoa, J Cell Biol, 1964, 20. 219. (10) Luft, J. H.: Improvements in epoxy resin I ) Opp9'73'71
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ULTRASTRUCTURE AND FUNCTION OF TRACHEAL MUCOSA 15 embedding methods, J Biophys Biochem Cytol, 1961, 9, 409. (11) Millonig, G.: A modified procedure for lead staining of thin sections, J Biophys Bio- chem Cytol, 1961, 11, 736. 12) Caro, L. G., and Palade, G. E.: Protein syn- thesis, storage, and discharge in the pan- creatic exocrine cell: An autoradiographic study, J Cell Biol, 1964, 20, 473. (13) Porter, K. R.: The ground substance : Ob- servations from electron microscopy. In The Cell, vol. II, J. Brachet and A. E. Mirsky, editors, Academic Press, New York, 1961, p. 621. (14) Novikoff, A. B.: Lysosomes and related particles. In The Cell, vol. II, J. Brachet and A. E. Mirsky, editors, Academic Press, New York, 1961, p. 723. (15) Pease, D. C.: Histological Technique for Electron Microscopy, ed. 2, Academic Press, New York, 1964. (16) Thiery, J. P.: Application de la technique de ]a "coloration negative" a 1'etude de quelques structures biologiques, C R Soc Biol (Paris), 1961, 155, 1804. (17) Farbman, A. I.: Electron microscope study of the developing taste bud in rat fungi- form papilla, Develop Biol, 1965, 11, 110. (18) Osada, M.: Electron microscopical observa- tions on the human tracheal epithelium with reference to the ciliated cells, Arch Histol Jap, 1963, 24, 91. (19) Sotelo, J. R., and Trujillo-Cenoz, 0.: Elec- tron microscope study on the development of ciliary components of the neural epi- thelium of the chick embryo, Z Zellforsch, 1958, 49, 1. (20) Nilsson, S.-E.S.: Receptor cell outer segment development and ultrastructure of the disk membranes in the retina of the tadpole (Rana pipiens), J Ultrastruct Res, 1964, 11, 581. (21) Walter, L.: Ciliogenesis in a cystically dilated hydatid of Morgagni, Anat Rec, 1929, 42, 177.
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SOME ASPECTS OF THE COMPARATIVE PHYSIOLOGY OF CILIA' MICHAEL A. SLEIGH INTRODUCTION Cilia, flagella, and sperm tails are three \-ariants of a cellular organelle of characteristic internal structure which has the basic function of moving fluids by lashing movements. When the body bearing the organelle is a small one, it may be propelled through the fluid by a ~in;1e flagellum; larger bodies mav be moved the combined action of many organelles. 1j-hcn the body is attached or is too large to ue moved, the action of the organelles results in a movement of fluid over the ciliated stir- lace. These organelles have been found in :uiimals of all major groups except the Nema- toda, and they have functions connected with locomotion, feeding, digestion, respiration, ex- cretion, reproduction, sensory reception, and die cleansing of surfaces. Cilia performing the i; ~-t of these functions will be the primary con- ~~rn at this conference, but the physiolop~y of ,,iiiary organelles of all types will be relevant ~ o our understanding of these structures. The common structural pattern of ciliary orU~anelles described in the previous paper by Dr. I',hodin is found in both cilia and flagella, aithou~h flagella and sperm tails sometimes i:ave additional structures. The often-quoted textbook distinctions between cilia and flagel]a do not always apply, for flagella are not neces- <nril' v lon;er than cilia and flagella, as Nrell as rilia, may be found in large groups. The pri- mary distinction is in the bending pattern, and this lives a functional difference in the way the Or,anelle moves water. In figure 1 it is seen that the flagellum can have more than one com- hletc wa~-e of undulation within its length, and ,;1,it it moves water along the flagellar axis in a tnanner analogous to the movement of water 1,, v the screw of a ship, whereas the cilium has a _,cndular movement and moves water at right to its length in a manner analovous to ~ L, ~ rowin; oars of a boat. '1-he beating patterns of these organelles, parti- 1ihr1Y of cilia, vary conziderabl}-, hnt the ' v :nay be reduced to a common plan. ArormullY I L'rom the DPPartment of Zoolo,_,c-, The 11ni- r~it}-. Bristol S, England. the movement takes place in a single plane, although some examples of both cilia and flarella are known in which the beat is more or less spiral. Three common features of the movement are important: (1) the beating of the organelle is triggered by some form of ex- citation; (2 ) the functional effect of water movement is achieved bv bending of the or- ganelle, presumably resulting from internal contractions; and (3) the bending is propagated from one end of the organelle to the other. It is likely that variations in the rate and pattern of excitation and in the rate of propagation of the bending wave can be responsible for varia- tions in the beating pattern. These characteris- tics of the functioning of single organelles will he the concern of Dr. Brokaw in the next paper, although the idea of excitation of ciliary con- tractions is essential to much of the present contribution. The purpose of this paper is to discuss the functioning of groups of ciliary organelles and to compare the activity of cilia from different sources to reveal similarities and differences. The orzanelles of a cili.arv tract do not move randomly, but are coordinated; they are also usually under some control by the organism. Some excitatory stimulus triggers the ciliary beat, and the sequence of excitation of the cilia in a tract together with the modification of the rate of excitation-as exemplified by dilTerentpatterns of ciliary coordination and control-are important features of groups of cilia which vary widely from one ciliary tract to :inother. To understand the differences between ;roups of cilia with different physiologic fea- titres it i~; neces-:trv first to cliscuss ciliarv meta- Chronism and control before going on to look at some examples. 14ithin anv ciliary tract all the oraanelles tend to have much the '~ame rate and pattern of beat, and thev tend to beat in the ~ame direc- tion. Cilia do not beat ~Ynchronottslv over the Mhnle tr:ict: thev beatone after aonther, ineta- clronall' v. lt, i.~, perhaps, instructive to look :it the bcatin; of Rroup~ of fla-_,ella to under- sinnrl an origin of this t' ype of nuetachroni4m. L'rnh Gray (1) and Ta,vlor (2) have pointNi 16 0009'73'73
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COMPARATIVE PHYSIOLOGY OF CILIA out that flagellar organelles which lie side by Side tend to beat synchronously (figure 2a) be- cause this pattern of beating gives least mutual interference. If these same flagella are turned to one side to lie almost parallel to the cell surface and still beat with least interference between the undulating flagella, the movement of ad- jacent flagella will be synchronized in the plane at right angles to the flagellar axis, but the group of flagella will show metachronism because the excitations responsible for a partic- ular wave of beat pass in succession from one ~ide of the group to the other (figure 2b). An area of cilia could show very similar metachronism (figure 2c) in which the cilia beat one after another in metachronism along the line of progression of the metachronal waves but beat in phase (synchronously) along lines parallel to the metachronal wave crests at right angles to the line of progression of the waves. Metachronism of cilia based on the same principle of least interference between the moving organelles is easy to understand only in this (symplectic) metachronal pattern where the metachronal waves travel in the same direc- tion as the effective stroke of the ciliarv beat. More complex metachronal patterns are, in fact, more usual: These are the antiplectic pat- tern, in which the metachronal waves move in the opposite direction to the effective stroke of the beat and therefore opposite to the move- a C b 17 FIG. 1. The characteristic bending movement of (a) a flagellum and (b) a cilium. The arrows in- dicate the direction of movement of water. ment of fluid; and the diaplectic pattern, in which the cilia beat at right angles to the line of wave progression, beating either to the left (laeoplectic) or to the right (desioplectic) as one looks along the line in the direction of propagation of the metachronal waves (figure 3). These metachronal patterns were named by Knight-Jones (3), and a study of them is of interest here because different patterns tend to be more effective for particular purposes; systems involving all four patterns will be described later. It was a frequent observation of earlier work- ers that cilia are autonomously active on iso- lated cells, although the same cilia in the whole animal might have been subject to periods of inactivity, to more rapid movement, or even Fta. 2. Mechanical coordination of flagella and cilia: (a) a group of flagella beating syn- rhronouslv; (b) flagella showing rnetachronism across the group; (c) metachroni~m of cilia (Opalina).
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UIC11AEL A. SL1s1C]II a b (+ SYMPLECTIC AYTIP~ECTIC rll ~ ~ r ` C L~ cP~ECTIC J Fic. 3. Patterns of metachronism of cilia. In all three c;ises the inetachronal waves are moving toward the right, and the small arrows indicate the direction of the efiective stroke of the ciliary beat. The diaplectic pattern (c) may be dexioplectic if the beat is toward the reader or laeoplectic if the beat is away from the reader and into the page. to periods of reversed beating. These modifica- tions of beating activity are manifestations of the control that may be exercised by the or- ,?nism over the activity of its cilia. In metazoan ~-nimals the mechanism of this control may be :.ervous or hormonal, and it is normally a .orm of coordination of the ciliary activity that i_ completely independent of the ciliarv metach- ronism, as it may act more or less simultane- ouslv over the whole ciliary tract. The prob- lem in protozoa is often more difficult, but control of ciliary activity is certainly present, Pven in simple flagellates where movements oward light or away from noxious stimuli show that the organism can regulate its loco- motor activity. A variety of examples will show ,ome of the features of ciliary control. EYASZPLES TO ILLUSTRATE THE FUNCTIONING OF GROUPS OF CILIA The Cilia of Opalina Opalina, a multinucleate protozoon from the rectum of the frog, has been classified by many _cuthors with the ciliate protozoa, but it shows more similarities with the flagellates and is w~«• regarded as belonging to a separate group uore clotiely related to flagellates than to iii;ites (4). Individual Opalina vary consider- :J,1v in size-an average individual might he ;50 ,, lona and 200 µ wide-but are always -cerv thin, the total thickness not exceeding about 20 j,t. The body is covered with cilia which are normally about 10 to 15 µ long, but are longer near the posterior end of the body. The cilia are arranged in rows which run roughly along the anteroposterior axis of the body (figure 4a) ; these rows are about 3 µ apart, and the cilia are spaced at intervals of ?3 ,u along the rows. Pitelka (5) and \oirot- Timothce (6) have found fine fibrils connect- ing the cilia within the rows but no fibrous con- nections between the rows. When the animal is moving straight forward, it is seen to be covered with lines which lie transversely across the bodY (figure 4c) and which move steadily backward; these are the metachronal waves which show up very clearly under dark-ground illumination. Between one and four metachronal waves pass a given point each second, and they move at about equal in- tervals and at an even speed of 100 to 200 µ per second over the ciliated surface, although there may be some tendency for waves to dis- appear posteriorly by coalescence with others. The waves move backward in the same direc- tion as the beat, so that the metachronism is s' ymplcctic. The form of beat shown bv these cilia is illustrated in figure 2c, and the close contact between the cilia is very suggestive of mechanical metachronal coordination by least mutual interference. If nletachronal coordina- tion is of this type, it is likely that changes in t lie rate of beat will be reflected closelv in 00097375 ;
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CO\4PARATIVE PHYSIOLOGY OF CILIA + II C d Fic. 4. Opalina: (a) general body shape showing the pattern of ciliary rows; (b) detail showing the rows of cilia with intervening pellicular ridges; (c) metachronal waves on an organism swimming forward; and (d) metachronal waves on an organism turning to the right. (Arrows indicate the movement of the metachronal waves in (c) and (d).) changes in the rate of movement of inetachronal waves. This is clearly shown by the effect of increased viscosity (figure 5), for slowing of the beat with increased viscous resistance is accompanied by a parallel reduction in the rate of movement of the metachronal waves. In all conditions, beating and metachronism ap- pear to go together in this organism. Perhaps the most remarkable feature of the locomotion of Opalina is its ability to change the direction of movement by changing the orientation of beat of the cilia. This is seen in the change in the pattern of metachronal waves which turn through an angle that corresponds to the required change in the direction of move- ment (see, for example, figure 4d). This feature of the movement of Opalina has been the subject of extensive studies by Japanese workers. A major contribution was made by Okajima (7), who found that the amount of change in direction of the metachronal waves was re- !nted to the degree of excitation; increase in xcitation by touching the body surface with a d:rect current electrode led to an increased inclination of the movement of the metachronal ,•,aves away from the normal. It is of interest i hat the application of current to a limited area ~~f the surface of the body led to a local response in the form of a change in direction of the metachronal waves over a restricted area. Chanl-e in direction of the waves is affected a , ii la', %~ / ~O1 / I I1+!lo'I'll ;oill ~i I 1 .~ +I,'o~ ~ +t 19 by ethyl-alcohol poisoning at a lower concen- tration than that which affects beating and metachronism, which disappear together (8). It is clear that the mechanism of change in beat direction has properties different from those of the metachronal coordination. Further studies have shown the close relation- ship between ions and excitability to change in the direction of beat. A close relation to mem- brane potential changes was suggested by the fact that application of isotonic potassium chloride would give a prolonged change in beat direction (9), and that there was a linear rela- tionship between membrane potential and the logarithm of the external 1i* concentration (10). A linkage with Ca'* was suggested by the find- ing that excitability was increased in calcium- rich media and was decreased in calcium-free media (11), whereas the injection of Ca2* pre- cipitants gave prolonged changes in beat direc- tion (12). This could mean that the excitation causing the change in beat direction resulted from a decrease in the internal Ca * concen- tration, and that the membrane potential change with direct current or potassium chlo- ride was only an indirect cause of excitation. Further evidence for the lack of direct connec- tion between change in beat direction and mem- brane potential was 'given by Naitoh (10), who found that at the start of flow of an inward electric current the normal stroke of the cilia
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0 1•0 ~ O T U 0 d > d > 20 0 ~ v C 0 X T ~ 40 ~ ~ d L ~ C w 60 V) a w ~ U W 0 w 80 Q r- z w U rY W (L 100 \4ICIIAEL A. SLEIGH VISCOSITY 2•0 3•0 (c e n t i p o i s e s) 4•0 5•0 6•0 OPALINA Fta. 5. The effect of increased viscositN of the inedium on tfie frequency and metachronal wave velocity of cilia of Opali.na. %% 1~ aut_mented, whereas with outward current iiio beat direction changed; if the membrane ,poieutial «-ere reduced to zero by a change in he concentration of potassium chloride, an o~itWard current stimulus of normal size could -! ill (!ause a change in beat direction. Treat- ~_ ni .~ which affect the integrity of the cell ini)r:1uc by titretching also cause a change in iiD'(rirni i 1:3, 14), and spontaneous reg- ~_'~~rpoLu•izntions from the restin; mem- k nrial level are accompanied by ciirection (15). In every case c:In 1>e reconciled with the in i,(,;,t rlirection depend on ntcrnal Ca=' rnncentration, u,,rm:rllv resiilts from an in- ;ni.r:Ine polential. ~l,l,war= that ,it :r particular membrane potential, all of the cilia beat in ap- proximately the same direction and at a similar frequency; they quickly form into metachronal waves by conforming to an average frequency and beat direction so that there is least mutual interference between the adjacent cilia. Change in membrane potential in response to some environmental influence (or spontaneous change) can spread over the whole surface al- most instantaneously and can cause change in beat direction (perhaps through a change in Ca" concentration acting directly on the cilia?), n•liich i~- very soon seen as a change in the meta- chronal wave pattern. Cilia of the Pha.ry7zx and Palate of the 1•'rog The epithelium of the pharynx and palate in the frog is largely composed of ciliated celk -1 00097377
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COMPARATIVE PHYSIOLOGY OF CILIA 'uid gland cells. The cilia are scattered rather irregularly over the surface of the ciliated cells at intervals of about i/2 µ and are inter- persed with numerous microvilli; striated ciliary rootlets run down into the cytoplasm Of the ciliated cells, but no connections have been found between the cilia (16). The cilia beat with their effective stroke toward the entrance to the esophagus, and mucus from the gland cells, together with particles trapped in it, moves toward the esophagus under the action of the cilia. As the cilia are known to beat in this direction, and the metachronal waves are seen to move in the opposite direc- tion, the metachronism is antiplectic. Beating of the cilia on an excised frog's palate is rather irregular, but they always beat in the same direction and at a frequency of a few beats per second. In the intact frog it is believed that these cilia are quite quiescent when unstimulated (17). When the cilia beat att about 6 to 8 beats per second, the meta- chronal waves move at about 100 to 200 p per second. In a preliminary experiment there was a little evidence that an increase in viscosity of the fluid overlying the cilia had little effect on wave frequency, but caused an increase in wave velocity-this certainly needs confirmation. The excised palate is not very satisfactorv for this sort of study because cilia at different regions of the palate may beat at different frequencies and may be meta- chronalh• coordinated independently of other areas quite close by. The metachronal coordination of these cilia r, ould appear to be by mechanical means of least mutual interference. It is easy to under- ~tand how this could function in a system .showing symplectic metachronism, as in Opa- lina; however, it is less easy to explain in L system showing antiplectic metachronism. It has not been possible so far to see clearly how the beating of the rather short and close- -et cilia of the frog's palate takes place, and -n ~inalo_y must be made with the beating of `iin of some IarQer Paramecium, which show a -uPerficially similar antiplectic metachronism (lyure 6). The successive shapes taken by a ~atuie• cilium may be seen by lookinz aluu_~ 7Lo row shown in figure 6 from ri~ht to left; hittiective stroke is toward the left, and the ?nrtachrrntal waves rnove toward the right. The 21 M. W. Frc. 6. The cilia composing a metachronal wave in Paramecium. The small arrow indicates the effective stroke of the beat, and the metachronal wave is moving toward the right. cilia are widely spaced during the effective stroke, but close together during the recovery stroke when the wave of bending is passing up the cilium and permitting mechanical contact between neighboring cilia. Rhythmical contacts in successive beats can keep the cilia beating at the same frequency; they can also serve to propagate metachronal waves in the direction opposite to the effective stroke be- cause the next cilium to be affected by the bending wave which travels up the cilia is the one that has just completed its effective stroke. This frog palate material is unsatisfactory for many experimental purposes because the ciliary rate is very variable; it is also very easily modified by mechanical stimulation as well as by other factors. If the same area of ciliated epithelium is watched for a few min- utes, the activity of the cilia may be found to fluctuate quite considerably. In a study of the ciliary activity of the frog's palate, Seo (18) found that, in addition to mechanical stimuli, a number of other treatments can cause acceleration of the ciliary beating. In particular, he found that chemical, mechani- cal, thermal, and electrical stimulation of the froh's tongue can produce an accelerated beat of the cilia on the palate. This obviously raises the possibility of nervous control of the ciliary beating-in this case, involving a reflex ac- celeration with the palatine nei-ve acting as the route of motor impulses. Seo was able to make a nerve-cilia preparation with the palate of the frog and the palatine nerve: stimulation of the palatine nerve gave increased rate and amplitude of beat of the cilia and increased rate of movement of inetachronal waves. Seo measured the ciliary activity by noting the speed of movement of red blood corpuscles in ihe cilinrY ~ztream. This movement increased
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MICHAEL A. SLEIGH -('me 7 to 20 times following the stimulation of the palatine nerve. The acceleration of "iiiary beating started after a latent period of l)out a second and continued for ten seconds t)r more. It was previously reported (19) that both ~~ctivation and inhibition were used to control the beating of frog pharynx cilia, but later workers have found only activation. In fact, Lucas (17, 20) found that the cilia in vivo are normally quiescent, but are activated by stim- ~..ii of parasympathetic origin transmitted by ,] e~eventh cranial nerve. This could agree with -lte Iinding (21-23) that low concentrations of :~cetVIcholine would increase the rate of particle 1ransport by these cilia. It is possible that the tlerve endings liberate acetylcholine, which dif- iu<e- to the ciliated cells to give the rather slow ~pon.~e (suggestive of a "local hormone" ac- 'inu :') found by Seo with his nerve-cilia prep- ; rltion. T he evidence here suggests that these cilia ro normalh• inactive unless stimulated by ;1rVe impulses acting cholinergically on the .'r•:u d cells; they may at other times be nuuiated by direct mechanical means. When ,lia beat, they appear to be coordinated llil,ulicallv to produce antiplectic meta- r„nai v,avew. Thr llcntbozat (ii ~,~ oi ~'. Stentor is a complex ciliati•d i,r-.r0>i a roughly trurnpet-~,hahcd \N irr. The body surface is covered ~%itii of -im- ple cilia, and around the hro:td ~,t i !i,i of tho body is a row of lar,e (:;il 11, ;1,i;e I .'n1pound cilia, the membranelles, who>r -tri;- :,ire :uid interconnections (24) are pictunod in :: ~ure 7ti. This row of inembranelles ~tart~ iii ~;, "~suilet" region, where the membrancii. .~ :in=maiier and closer together, and cxti-n(i; wit -iito tlte body surface to form a rin, i large cilia which function in m:iiiitcwniuc w.2tcr currents to bring food to the mo>uii .trt a. Th_ese cilia beat outward at ridht au-,Jl~ to t Le row oi cilia, with an effective stroke conunf•ucin,-, at au approximately vertical po~itiun an(i uiovinc through an angle of about 11U', :nici :, recm - ery stroke which brings the c_iiict uwi, to ti~:e oril-inal position (fi_ures 7c and - , . AIer_i- chronal waves originate in the nuillet n--,ion an,i pass along the row of inetn},riuell- - i,, the (i1~- tal end; the metachroniSm i: thereiun dioxiopl,c- tic. At a temperature of abont 1lie men:- branellev beat on averaee about '7 ~iuie; second, and the metachronal \\-a\c•= ino~-e _,_ 650 u per second (25). The membranelles of Stentor moNc• in rou=i i.• a C d '!:' iir ~-i m,,mbrancllcs and the form and direction of trans- ~- r:til (-Ii tho ~irncture and root connections of the mem- ~• ~!Iki (d) tlio reroverp stroke of a membranelle beat. iThe N"t4 r ("urr-nr, and ihe broken arrow indicate- thr-
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COMPARATIVE PHYSIOLOGY OF CILIA 23 ,:.rallel planes. The possibility that mechani- ,:d interaction between adjacent cilia is respon- <irle for metachronal coordination seems less ikely than in Opalina or frog cilia. In fact, ex- =;eriments on the metachronal organization of : he e membranelles all suggest that metachro :ism here is mediated in a different way. Thus the change in frequency with temperature is quantitatively different from the change in metachronal wave velocity: When the viscosity of the medium is increased to two or three times -iie normal level, the beat frequency is reduced, ~:•hereas the metachronal wave velocity re- mains unchanged ; an increase in 1Ig2+ or Al' concentration in the medium increases the beat frequency although it does not change the meta- chronal wave velocity; and low concentrations of digitoxin increase the metachronal wave velocity but hardly affect the beat frequency (26). The effect of viscosity on the activity of Stentor membranelles (figure 8) is of parti- cular interest, for, although an increase in viscosity to about three and a half times the normal does not affect the metachronal wave velocity significantly and reduces the frequency +1201- X +100 } H U 0 J L.I > +80 U ~ > ~ z +60 Q • } v z ZU +40 O w a_ LL z_ W 20 cD z Q V -20 1•0 3•0 5.0 7•0 9-0 VISCOSITY IN CENTIPOISES Fic. S. The effect of increased viscosity of the medium on the frequency and metachronal n-ave velocitv of the membranelles of Stentor.
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MICHAEL A. SLEIGH : some 20 per cent, a further increase in vis- •,o.~ity produces a dramatic change (27). Both mquency and wave velocity curves turn sitd- aenl.- upward at the same viscosity, probably oecause the increased viscosity gives sufficient lrag between adjacent cilia for coordination by mechanical means to occur. The cilia beat more quickly because they help each other more, and the mechanical coordination be- comes quicker and quicker as the viscosity is r:i i;ed and viscous drag becomes more effective. The increases will stop when the medium is iuo .-i~cous for the cilia to beat fast enough or --ith ~utficient amplitude to affect neighboring oilia. These observations indicate that under ditierent conditions both mechanical and another tyhe of coordination can be involved in the nietachronism of Stentor membranelles; both rypes of coordination give a rather similar wave ippearance, but one is affected by mechanical iactors and the other is not. For many years there has been controversy iwtween workers favoring the two alternatives of nechanical and "neuroid" (internal, but with only some nervelike properties) types of inetach- ,oni'<ni, and it is now evident that both exist- the mechanical metachronism of Opalina cilia Aud the neuroid metachronism of Stentor mem- ranelles at normal viscosity are clear elamples. t-nI'ortunately, we know all too little about the mechanism of neuroid metachronism. One uil~ortant point is that in a row of inem- !)ranelles in which the interciliary spacing varies i rilicti are much closer together at the begin- uin,_- of the row), the frequency of beat and . he uamber of cilia stimulated per second by t _<ingle metachronal wave remain constant Throu,hout the row, although the linear dis- ancc travelled by a inetachronal wave per <ocond :ind the metachronal wave length are much lc~s when the cilia are closer together (28). It nhpears that the metachronal excitation travels from cilium to cilium along the row and that the beating of all the cilia cau lie con- trolled hy a pacemaker cilium at the beginning ,,r tlie row, communic;uing its rhythm of beat zseh-bv-step, cilium-bV-cilium, rigtit nlau- the °_1%,, . If ;i cilium ceases to beat, or it' a cut is ciakle acro;s the row of cilia to interrupt the ~r:1n~mi~sion ot merachronal excitation, the aiiun diz4tIl to the di_Seontinnity l,ecomes a .-rI inaker f(,r rhe di_t:tl part of the row - and may originate a new rhythm with a frequency ot beat different from that iu the protimal region. Although the frequency in the distal region may change, the wave velocity remains the same. An idea of the way in which this type or neuroid metachronism may work is shown ir figure 9. The triggering of each cilium is pre- ceded by the conduction of an impulse from the previous cilium and a build-up of excitation to a threshold level within the cilium. In the absence of a conducted impulse the cilium is spontane- ously excited and becomes a pacemaker itself. Immediate and simultaneous stoppage of al the membranelles may occur on stimulation o- the animal-at the same time the body usuall.\ contracts and the body cilia may reverse Inhibition of membranelles and bodv contrac lion both may result from the rapid spread o a change in membrane potential over the bod7 surface. Work on Stentor using ions, drugs, ant electrical techniques is as yet at an early staPe but it offers interesting prospects. However, th ctenophore material to be described next is per haps easier to study on account of ; ize. Stentor membranelles seem to depend on pacemaker for control of their rate of beat, an, the beating of the pacemaker is influenced b~ mechanical factors such as viscosity, and b; ions. The dexioplectic metachronism is of tht neuroid type arld appears to be a stepwise proc ess involving interciliary and intraciliary com ponents. It has been found to be influenced b the drug digitoxin, but not by a number c other treatments. The Comb Plates of Ctcnophores Ctenophores are well provided «-ith cili which perform niany functions: they posse: the largest known ciliary organelles in the fon of the loeomotorN comb plates. Planktoni ctenophores possess eight lon; rows of com plates (figure Illa), which extend as meridiai around the body from the aboral pole to ne: the oral pole. These rows occur in pairs in eac quadrant of the body. 1~~ rom the aboral end each comb plate row, a narrow tract of cii runs toward the aboral pole, joins with i neighbor of the eame quadrant, and te minates at the apicLl sense organ. Each com plate is a highly compotutd -tructitre built hundreds of thousands of cilia. _lfzelitts iound that a comb plate of Mrnemiol>>is has 00091381
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COMPARATIVE PHYSIOLOGY OF CILIA Cilium Cilium base f t t t t t t t ~ Pacemaker t t Con- trattion -x- Excitation build-up C 0 \G I i t ~ t t t i Con- traction -x- T Excitation build-up 25 Spon- taneous excitation I C Con- traction -x- I Spon- taneous excitation Fic. 9. A theory to account for the features of neuroid metachronal coordination in Stentor discussed in the text. to 100 rows, each containing several thousand cilia. All of the cilia are normal cilia complete with membranes, but they are held together in the rows along the longer transverse axis of the comb plate by lamellar connections between the peripheral fibrils of the cilia, so that the whole comb plate normally functions as a single unit. A comb plate might be 1 mm. by 20 µ at the base and about 2 mm. long; adjacent comb plates might be 1/2 mm. or so apart. A swimming ctenophore normally moves mouth forward, so that the effective stroke of a muuuaui^ N the ciliary beat is toward the aboral pole. Meta- chronal waves commence at the aboral end of the comb plate row and progress orally, and the metachronism is antiplectic. It appears that the metachronal excitation has its origin at the apical sense organ and is transmitted along the ciliary tracts to the comb plate rows, for it is usual to find that the time of appearance of metachronal waves in the two comb rows of a quadrant is closely synchronized. Beating of the comb rows may not be completely regular, and it is often intermittent; but, whenever the APICAL SENSE ORGAN M i V C ~ Fl! ~ ~Fic. 10. Pleurobrachia: (a) general body shape showing the relationship of the comb plate ruws to the apical sense organ; (b) three comb plates; (c) the comb plates composing a metachronal wave.
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'?tj MICHAEL A. SLEIGH rutnh plates at the aboral end of the row begin to 1,o:,t, a rnetachronal wave pa:~ses to the other tnh1 of the row. The comb plates appear to be inactive unless stimulated by a metachronal lm} ulse. There are reasons for believing that 1ik metachronal impulse has its origin at the :tpical sen~e organ, and that the normal pacemaker lies there-a cut across the row can lead to a new pacemaker at the beginning of the distal region. Stimuli controlling the beat nl:tY act directly on the pacemaker at that l,oint; the process does not appear to involve ~nrh :t regular rhythmic activity as in Stentor, !uod normal modifications of speed and direc- rion of movement of ctenophores are doubtless controlled by pacemaker activities in the area oi the apical sense organ. In order to change ,h(, direc.tion of movement, the pacemakers of >uuie rows must initiate more frequent waves ilutn the pacemakers of some other rows. The t},ic;tl organ would seem to be equipped to per- 'orni this function, but further investigations C Q • 6000 V N 0 r V 3000 are necessar}- to define the mechanism ade- qnately. Iu order for this system to work, the meta- chroni.sm of these compound cilia must be of some neuroid type, and further evidence that this is so can be obtained from the effects of vi~cosity on the speed of movement of the metaehronal wave and on the beating activity (figure 11). In the figure the angular velocity of heat is quoted because the beat was inter- mittent and the alnplitude was not seen to change sianificantlv. The metachronal wave velocity is tuichnnged viscosities which slow t1le beating of comb plates, and coordination is therefore not likel}- to be mechanical. Ao good evidence concerninl- the mechanism of this neu- roid coordination is Yet available. The rate of stimulation of cilia during the passage of a metachronal wave in both Pleurobrachia and Beroe is about ISO to 200 per second (30) (at 19-'?0°C.) ; this is about the same rate as that in Stentor, although the linear velocities of the BEROE x 40 E E 35 C T 0 b > 1•0 2•0 3•0 4-0 Viscosity in centipoises I,1c. 11. 'I'hw ct'tccr of inrrc,r~;ed ~ isco~itv of the nudittm on the :ui~:ul:u• vclocit.- of tlm ~if-crm, ~troke of he:it and on the inetachronal wave N-clocit}• of conil) F1larcs of the ctcno- phor" Beroi~.
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COIIPARATIVE PHYSIOLOGY OF CILIA 27 metachronal wave in the individuals measured were 0.65 mm. per second in Stentor, 40.6 mm. per second in Beroe, and 72.9 mm. per second in Pleurobrachia. The similarity of rates of <timnlation suggests that the mechanism of all three may be similar, perhaps like that sug- ar~4ted for Stentor, but that the interciliary conduction phase may be very rapid and the intraciliary excitation phase slow. Mechanical stimulation of ctenophores, parti- cularly in the oral region, leads to a temporary complete stoppage of beat of all the comb pl.rtes. The response appears to be spread over the whole body of the ctenophore from the site of stimulation, and the speed of the spread of inhibition suggests nervous transmission. Hor- ridge and Mackay (31) have obtained inhibi- tion of comb plate movements in Beroe and Pleurobrachia by electrical stimulation and have found nerve endings containing synaptic vesicles among the bases of the ciliated cells of the comb plates. They believe that these nerve endings uelong to the ectodermal nerve net, and that when the animal is stimulated impul'<es in hese nerves are responsible for the prevention --f normal metachronal excitation of the comb l,late<. There is no evidence for nervous activation 14 these cilia, for they appear to beat normally Umder pacemaker control unless some stimulus (wcurs to trigger the inhibitory response com- 1ntuiicated by nerves. Inhibition of a similar :ype is believed to occur in the veliger larva of .lie niollusks, in which the large velar cilia 1"•,rt regularly with a tivell-marked metachronal rhythm. Occasional intermissions of ciliary beat are bclieved to re ult from inhibitory nc rvous stimuli. Treatment with narcoties prevents the occurrence of intermissions more quickly and at lower concentrations than it affects the beating or metachronal activity of the cilia (32). In the early stages of these larvae, the locomotor' v cilia beat continuously before the nervous system becomes functional; once the nervous s , vstem has developed, inhibitory inter- missions of the cilia begin to occur. The comb plates of ctenophores seem to beat at a rate that is controlled by a pacemaker mechanism that is almost certainly more com- plex than that in atentor because it appears to be capable of more precise control. The anti- plectic metachronism is likely to be neuroid and ma, N- be of the same stepwise type as that suggested for Stentor. The comb plates are under direct inhibitory control by nerves which are believed to be those which ramify among the bases of the ciliated cells. Cilia of the Gill Filanzents of 3lytilics Rather brief mention will be made of these cilia, as they are expected to feature prominentlN - in Professor Gosselin's paper. The gill filaments of Mytilus carry three distinct tracts of cilia -the lateral, the frontal, and the laterofrontal cilia (figure 12)-which show rather different features. There is a reasonable amount of in- formation only on the first two of these tracts, aa metachronisin of the compound laterofrontal cilia (if it exi.qs_) has not been adequately de~cribed. LATEROFRONTAL : i ! Via. 12. The arranmement of the thrce main ciliai.v truts on the =il1 Lilzuuents of a Lunelli- hranch cnollu.~k like AIvtihus.
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?8 NfICHAEL A. SLEIGH The lateral cilia form a long band of sim- p1o cilia along either side of the gill filament, ,xirh the cilia arranged in short transverse, lnit slightly oblique, rows of 20 or more cilia. The function of these cilia is to maintain a (,lirrent of water through the spaces between i1ie gill filaments, and the effective stroke of the ciliary beat is roughly along t1le transverse rows of cilia and away from the frontal face of the gill. `3'hen one looks at the frontal surface 4 the gill, metachronal waves can be seen .novinQ up the lateral ciliary band on the left hand side and down on the right of the gill lilament-the metachronism is laeoplectic. Aiello I33) found that, although the beat direction Aas _lightly oblique, the metachronal wave- frontC, were approximately transverse to the lhand of lateral cilia. These cilia on the lateral cells are the best on the gills for most types of study, but they :re much less regular in their beat than, say, ihe membranelles of Stentor. In fact, they give the impression of being less well coordinated ,ilia, using a mechanical means of metachronal ~ ran~tnission, and there is some evidence to _iipport this. Gosselin (34) found that increase in viscosity of the medium around the gill tilaments had little effect on ciliary frequency, aithougll the amplitude was consistently re- duced. The metachronal wave velocity increased markedly, as one might expect if there was increased viscous drag between cilia that were coordinated mechanically. Using some- what lower viscosities, Aiello (33) found a reduction in frequency (with a tendency to reduction in amplitude) and a slight increase in a~ e velocity. T1Ie results of these two workers :ire not entirely contradictory since the main- tenance of a normal frequency may depend on the help given by neighboring cKia to each other during their movement. The same authors (~_33, 35) have reported that 5-hydroxytrypta- niine (5-HT, serotonin) consistently increases ' Le rate of beat of these lateral cilia at low con- ""utrations, and that the inetachronal wave velocit}- increases by a parallel amount. It has %!,<o been reported (,33) that the effect of -: cratrine is to increase both the metachronal ",; ~«-e velocity- and the frequency by simil:ir (nionuts. These findings suggest that the meta- 1ronal wave velocity is cloweh dependent on ; he i reouencly, and that metachronism is prob- ably mechanical. Thus, the primary effect of all of these treatments is to modify the ciliary beat, with changes in metachronal wave veloc- ity following as a consequence of changes in ciliary frequency or changes in the amount of mechanical interaction between cilia. Frontal cilia are borne on a band of cells along the midfrontal face of the gill filament. They beat along the axis of the filament and function to transport mucus, together with entrapped particles, to the end of that section of the gill filament for onward transmission to the mouth. The beating of these cilia is more continuous than that of the lateral cilia. The metachronal waves travel in the opposite direction to the effective stroke of the beat, showing antiplectic metachronism. These cilia are also accelerated by 5-HT (36). It is some- what perplexing that, although wide changes in the concentration of DZg" have little effect on the activity of the frontal cilia, similar changes of \Ig'' concentration affect the activity of the lateral cilia of the same fila- ments dramatically; the lateral cilia beat fastest in the absence of magnesium and cease to beat if the concentration of magnesium is increased. Cilia of the same organism situated on adjacent cells behave differently, both in metachronism and in response to experimental treatments. At various times it llas been suggested that the gill cilia of these mollusks are under nervous control. However, after extensive study, Lucas (37, 38) was unable to find any evidence to confirm this. JZore recently, Aiello (33) has found that if the branchial nerve supplying the gill of AIvtilus is transected on one side, then the lateral cilia of the gill on that side show significantly less activity than the lateral cilia on the intact side at 10 minutes and 60 min- utes after transection. It is conceivable that stimulated nerves liberate, or cause the liber- ation of, 5-HT, which may have the function of a cilio-eticitatory hormone in the gill fila- ments. The molluscan gill filament carries a number of types of cilia which differ in function and metachronal pattern: they appear also to differ in their response to physiologic conditions. As a general hypothesis it may be suggested that, in _llytilus at least, the cilia may be controlled by nervous means, with the rather inactive 00097385
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COMPARATIVE PHYSIOLOGY OF CILIA cilia activated to varying degrees according to the amount of 5-HT liberated in response to nervous stimulation. CONCLtiDING C0ti41IENTS This brief survey of some of the better-known ciliary systems illustrates that there is wide variety in both metachronism and control. Metachronism may take any one of four forms if one considers the relation between the move- ment of the cilia and the movement of the meta- chronal waves. It seems generally true to say that diaplectic metachronism is more effective in the creation of water currents and that the movement of mucus is most usually performed by cilia showing antiplectic metachronism. Sym- plectic metachronism is rare, and the creation of water currents by cilia showing antiplectic metachronism is not infrequent, especially in ciliated protozoa and in forms where cilia are highly specialized-as in ctenophores. The mechanism of metachronism is believed to he sometimes mechanical and sometimes neu- roid. The mechanical type depends on the fact that cilia tend to beat with the least mu- tual interference between adjacent moving cilia, and it results in a frequency of beat dependent on the average activity of all the cilia in a par- ticular area; it is probably not dependent on the ictivity of a pacemaker. The neuroid type, on the other hand, requires a definite pacemaker from which impulses are conducted to excite other cilia i o participate in the formation of metachronal waves; all the cilia will maintain precisely the -ame rhythm. The conduction of the metachronal wave probably involves an internal mechanism --•hich has no connection with the mechanical ac- rivirv of the cilia. Cilia are almost invariably under the con- trol of the organism in some way or other. In ~impler forms the cilia normally beat continu- usly unless inhibited, with the additional pos- -ibility (especially in the Protozoa) of a •iiange in direction of beat in response to ~~hpropriate stimulation. In Metazoa the in- ~il)ition normally involves nervous activity -ince this can give immediate stoppage over lic whole ciliated area. AIthough swimming cilia re often required to be continuoush- active, I' iippears that other cilia are only required '' heat when there is work to be done ; these ili:~ may also be under nervous control, but 29 here the nerves activate rather than inhibit. Thus the frog palate cilia are activated in response to mechanical stimulation, food in the rnouth, et cetera, and the lateral cilia of Mytilus gill filaments may be activated when the shell is open, but not when it is closed; activation in the frog i~ believed to involve acetylcholine, and in mollusks, 5-hydroxytrypt- amine. There can be little doubt that the mechanism of beating is the same in all or,anisms, but the variations in metachronal patterns and mech- anisms and of types of control suggest consider- able evolutionary exploitation. In view of the diversity of mechanisms involved in the coor- dinated activity of ciliary systems, it is evident that the application of findings in one organism to a ciliary system in an unrelated organism is unreliable. Because of reasonably close phylo- genetic relationships, it seems reasonable to suggest that the cilia of the mammalian respira- torv tract function in a rather similar wav to the cilia of the frog's pharynx. It is known that cilia of the mammalian respiratory tract have antiplectic metachronism (39), and it seems likely that the metachronisin is mechani- cal. Some indications of activation of mammalian cilia by acetylcholine (23, 40) do not entirely agree with the finding that the cilia of the trachea of the turtle and duck beat continu- ously and did not appear to be under nervous control (41). It was suggested that nerve stim- ulation and some drugs could affect the se- cretion of mucus but not the ciliary beat in these animals. It does not seem essential for a mammal to be able to modifv the rate of beat of cilia in the respiratory tract if they nor- mally beat continuously and at a fairly high rate. Acknowledgments The writer thanks the organizers of this sym- posium for their invitation to present tnis paper, and Miss M. Nicholas for assistance with the fi<,:ures. REFERENCES (1) Gray, J.: Ciliary Movement, Cambridge Llniversity Press, Cambridge, England, 1928. (2) Taylor, G. L: Analysis of the swimming of microscopic organismc. Proc Roy Soc (A), 1951, ?09, 447 (3) l:night-.Tones, E. W.: Relations between metachronism and the direction of ciliary
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yI ICHAEL A. SLEIGH heat in metazoa, Quart J--\iicr Sci, 1954, 90, 503. -41 Honigberg, B. M., and Committee :A revised classification of the phylum Protozoa, J I'rotozool, 1964. 11, 7. 5) Pitelka, D. 11.: An electron microscope study of cortical structures of Opalina obtti- ,qoicoidea, J Biophys Biochem Cytol, 1956, 2, 423. ( 6) -Noirot-Timothee, C.: Recherches sur Pultra- structure (I'Opalina ranarum, Ann Sci \at Zool, 1959 (Ser 12), 1, 265. Okajima, A.: Studies on the metachronal wave in Opalina. I. Electrical stimulation with the microelectrode, Jap J Zool, 1953, 11, 87. S) Olcajima, A.: Studies on the metachronal wave in Opalina. II. The regulating mech- autism of ciliary metachronism and ciliary r(wereal, Annot Zool Jap, 1954, ',-, 40. W) Kinosita, H.: Electrical potentials and ciliary response in Opalina, J Fac Sci U Tokyo (SecIL),1954,-,,1. 10) Aaitoh, Y.: Direct current stimulation of Opalirta with intracellular microelectrode, ;lnnot Zool Jap, 1958, 31, 59. 11) Okajima, A.: Studies on the inetachronal wave in Opalina. III. Time-change of ef- fectiveness of chemical and electrical stim- iili during adaptation in various media, :lnnot Zool Jap, 1954, !7, 46. 12 ) i"e da, K.: Intracellular calcium and ciliarc reversal in Opalina, Jap J Zool, 1956, 12, 1. l:; )\ aitoh, I.: Relation between the deforma- tion of the cell membrane and the change in beating direction of cilia in Opalina, J Fac Sci U Tokyo (Sec IV), 1959, S, 357. Angriffspunktes vegetativer Gifte. Versuche am Embryonalhertsen und am Flimmer- epithel, Pflueger Arch Ges Physiol., 1931, 228. 281. (22) Ishikawa, S., and Ohzono, M.: Einfluss von verschiedener Pharmaka auf die Flimmer- bewegung, Acta Derm (Kyoto), 1931, 17, 478. (23) Kordik. P., Bulbrinr;, E., and Burn. J. H.: Ciliary movement and acetyl choline, Brit J Pharmacol. 1952, 7, 67. (24) Randall, J. T.. and Jackson, S. F.: Fine structure and function in Stentor poly- nzorphu.s, J Biophys Biochem Cytol, 1958, 4,807. (25) Sleigh, M. A.: The Biology of Cilia and Flagella, Pergamon Press, Osforcl, England, 1962. (26) Sleigh, M. A.: Metachronism and frequency of beat in the peristomial cilia of Stentor, J Exp Biol, 1956, 33, 15. (27) Sleigh. M. A.: An example of mechanical coordination of cilia, -Nature (London), 1961.101, 931. (28) Sleigh, M. A.: Further observations on co- ordination and the determination of fre- cluency in the peris'tomial cilia of Stetttor, J Exp Biol, 1957. 3,~, 106. (29) Afzelius. B. _\.: The fine structure of the cilia from ctenophore swimming-plates, J Biophys Biochem Cytol, 1961, 9, 383. (30) Sleinh, -M. A.: Movements and coordination of thee ciliary comb plates of the etenoph- ores Beroe and Plearobracliia, \atttre (London), 1963. 1!)J. 620. (31) Horridge, G. A., and Mackay, B.: \ euro- ciliat.v synapses in Pleitrobrochia (Ct(,n- 11 t\aitoh, Y.: Effects of external ions on the opltora), Quart J Micr Sci, 1964. 105, 163. ,~iliary responses of 0palina induced by (32) Carter. G. S.: On the nervous control of the .I ang:c in o.motic pressttre, J Fac Sci U velar cilia of the nudibranch veli;;er, J Exp Tol:yo ( Scc IV), 1963,10, 1. Biol. 1926, /j. 1. ~' l:uii;ii-nuchi, T., and Okumura, H.: Ciliary ticit} and electrical properties of Opa- 1,11. J P,tc sci Hokkaido U (Ser VI Zool), 1S. So. i). 1C.. and Porter, K. R.: A study i tiue xtructtre of ciliated cpithelia, \l- ri,li. 1954, 221. ~. .1. M.: Comparison of ciliary activity -- -und in tic~o conditions, Proc ~:V- 13ioil \Ied. 1933, 30, 501 „n the ne_rvous re-ulation mm-ement, Jap J _\Ied Sci, Itti,l. ?.-17. ! . I.1-i~tu'c, (`. E., and Lcnue- \nr:~L ;cn(l chemic:d control 1'n>c Soc 1?sp Biol :jctwation of J Ph}'siol, 1035, '/,in• Pruge dc•s (33) Aiello. E. L.: Factors affectina ciliary ac- ti1-it' v on the gill of the mussel .llYlihcs edulis, Physiol Zool, 1960, 33, 120. (34) Gosselin, R. E.: Influence of viscositv on metachronal rltythm of cilia, Fed Proc, 1958. 17, 372. (35) Gosselin, R. E.: The cilioexcitatory actiN-it-y of serotonin, J Cell Comp Physiol, 1961, oS. 17. (36) Gosselin, R. E., and O'Hara, G.: An ttn- suspected source of error in studies of particle transport by lamellibranch gill cili;i, J Cell C'omp Physiol, 1961, oS, 1. (37) Lucas. .\. -M.: .\n int-esti,,,ation of the nerv- ous system as a pos.sible factor in the reg- ulation of ciliarv activitv of the lamel- libranclt gill. J Morph Physiol. 1931, .51, 147. (38) Lucas. A. AI.: The distributimi of the bran- chial nertr in Jf11tilus edalis and its re- 0009'738'7
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COMPARATIVE PHYSIOLOGY OF CILIA :~1 lation to the problem of nervous control of ciliary activity, J Morph Phlysiol, 1931, 51, 195. (39) Proetz, A. W.: Studies of nasal cilia in the living mammal, Ann Otol, 1933, lj2, 778. ({0) Corssen, G., and Allen, C. R.: Acetylcholine: Its significance in controlling ciliar}ac- tivitv of human respiratorY epithelium i a vih•o, JAppl Physiol, 1959. li, 901. (41) Lucas. A. M., and Douglas, L. C.: Principles underlying ciliary activit}• in the respiratory tract. III. Independence of tracheal cilia in vivo of drug and neurogenous stimnli. Arch Otolaryng (Chicago), 1935, 21, 285.
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MECHANICS AND ENERGETICS OF CILIA'•' C. J. BROKAW INTRODUCTION This review will consider, somewhat specula- tively, the mechanisms involved in the genera- tion and control of bending in cilia. Particular attention will be paid to those properties of the bending mechanisms which appear to be common to cilia and to flagella, since most of the author's own work has been with flagella, and since, in many respects, flagella are more accessible to study than are cilia. Sleigh (1) has already indicated that the differences between the nlorphology of movement of cilia and flagella are small, and extensive electron mi- croscope studies have indicated that cilia and flaaella share the same basic structural pat- tern (1). One of the most significant features of cilia is their occurrence in arrays in which their movements are organized by metachronal waves. Although possibly related interactions have been observed with flagella (2, 3) and may be instructive in understanding the mec1l- anisms which control bending, this review will focus on the behavior of an individual cilium. CILI:1 AS INDEPENDENT ORGANELLES A first consideration is the extent to which a cilium is an autonomous motile organelle. Even in the last few years, statements have been published to the effect that contractions of the striated rootlet fibers of cilia are responsible for ciliary movement (4, 5). However, much evidence has accumulated to indicate that cilia (*an perform bending movements even when de- tached from their basal bodies or associated ~tructures such as the kinetodesmal fibers. In discussing this point in his book on cilia in 192S. Gray summarized nineteenth century oh- ~ervations of movements of detached cilia and ;Icieella (2). These movements were tvpically of l)rief duration, presumably because of the lack of a continuing supply of energy. In the la~t ticeade, procedures based on the work of Hotimann-Berling (6) have been developed =From the Division of Biolo,v, California ln- "itut(l of Technology, Pasadena. Cslifornia. -Suqqiorted iii part I) , v grant \n. HC-(i9(in, Aa- :-,nal In~tittite ot G~n~ral Jli,dical scirncr•a_. t-S_. 1'nldic Healtlt Service. which allow the observation of continuing movement of isolated cilia and flagella. Glyc- erol or detergents are used to destroy the mem- brane permeability barrier which surrounds the cilium, so that externally supplied adenosine triphosphate (ATP) can reach the active ele- ments of the cilium and supply energy for movement. This method has been successfully applied to a variety of materials, including cilia of the frog palate (7), flagella of the phytoflagellates Chlamydomonas (8) and Polytoma (9, 10), and cilia of the protozoa Spirostomum (11) and Tetrahymena (12). In these cases, cilia are removed from the cells and these isolated cilia are motile when put into ATP solutions, often swimming vigorously through the medium. In Polytoma and Tetra- hymena there is some limited evidence that the basal bodies remain in the cell body when the flagella or cilia are detached, but this is a difficult point to determine with certainty. Photographs of the movement of ATP-reac- tivated, isolated flagella of Polytoma (figure 1) show conclusively that the isolated flagella re- tain the ability not only to bend rhythmically, but also to propagate bending waves-which explains their ability to swim (10). The move- ment of isolated flagella appears to differ on1v quantitatively from the normal movements of these flagella. Isolated frog palate cilia, on ATP reactivation, show asymmetric, two-dimen- sional movements basically similar to their nor- mal movements. On the other hand, isolated Tetrahymena cilia have a three-dimensional beat whieh is difficult to analyze unless the cilia become attached to a surface by their bases. The cilia then beat with circular, swinging movements which trace out cones having their apices at the points of attachment to the :urface. This type of movement has been deScribed for protozoan cilia under abnor- mal conditions, and according to Parduez's de- ,~cription of ciliary beating (13), represents on1Y the recovery portion of the stroke cycle. These observations show that at least some of the specific properties which differentiate the movements of a particular type of cilium are a part of the cilium itself and also characterize : 2 0009'7389
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MECHANICS AND ENERGETICS OF CILIA its movements after isolation and ATP-reacti- vation. On the other hand, some of the move- ments of cilia are clearly controlled by external factors. The variable direction of the effective stroke of the cilia of ciliated protozoa appears to depend on some factor which is lacking in the isolated, reactivated cilia. This factor may be merely the mechanical interaction between adjacent cilia, or it may involve more complex intracellular mechanisms. In a few favorable cases, mechanical interactions may persist and coordinate ciliary activity in reactivated cilia preparations (11, 14). On the other hand, no indications of behavior such as the "shock reactions" of Polytoma have yet been observed in isolated flagella. These reactions constitute a primitive behavioral mechanism, in which the normal swimming movements are occasionally interrupted by brief periods of movement with the flagella directed anteriorly, causing the cell to dart backward (10). Little is known about the mechanism of this type of flagellar behavior, but it may be very simple. For in- stance, the planar bending waves of sperma- tozoa of a tunicate, Ciona, in viscous solutions can be converted to helical bending waves merely by adding thiourea to the medium (15). THE MORPHOLOGY OF MOVEMENT Photographs with much better resolution than was previously available have recently been obtained of flagellar bending waves, mostly with invertebrate spermatozoa (16, 17). The bending waves are composed of nearly cir- cular arcs, separated by short, straight regions, as illustrated in figure 2a. They are not, as tinggested formerly (18), sine waves. As these waves pass along the flagellum, active bending (and unbending) occurs sequentially along the flagellum, as illustrated in figure 2b. This figure also represents the time course of the cycle of bending at each locus along the length of the Fia(rellum. The time required for the transition between bent and unbent states cannot be meas- 1ired exactly, btrt it is probably not more than ' m~ec. Each element does approximately the <:uile amount of work against the vi3cous nie- diuin, as it cycles, and must have an internal c~nern• supply for bending and unbending (1S) . 7'he most important feature of this pattern is ,lie fact that it ean be controlled in a simple _)n-or-oi[fashion. 33 FIG. 1. Multiple flash photomicrograph (magni- fication, X2,000) of an isolated flagellum of Poh-- toma reactivated by ATP (10). The flash fre- quency is approximately twice the beat frequency of the flagellum, and the first flash can be identified by its higher intensity. The above conclusions were made possible by the fact that spermatozoa are very cooperative about swimming with their wave planes paral- lel to a surface (18, 19), so that the entire wave can be focused easih• for photography. For several reasons, cilia are more difficult to photograph, and photographs are not yet avail- able which give equally precise and detailed de- scriptions of bending patterns, so speculation is required. A possible pattern of ciliary bending, derived by extrapolation from what occurs with flagella, is shown in figure 2c. An essen- tially similar pattern was used as a model by Harris (20). The best photographs of ciliary bending, siich as those of Gra}- (21), show pat- terns which are superficially similar to this, but do not provide sufficient information to deter- mine whether bending is controlled in a similar manner. The pattern shown in figure 2c is only one of many which can be ;enerated using the sante principle. Its important features are propagation of a primarily unilateral bending, activated in an on-or-off manner, so that each element along the length alternates between two states. The constancy of radius of curvature and propa,ation velocity illustrated in this example are not e,.<-ential features. Witlt sperm fl: vella, situations have been found in which the radius of curvature of the bending waves is modified. The wave patterns of spermatozoa which become attached to a surface by their heads show graduallv increas- inEr radius of curvature and propa,ation veloc-
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34 C. J. BROI:AW a , 2 b ! d I Fic. 2. (a) Idealized flagellar bending wave constructed of circular arcs and straight lines. (b) Sequence of bending corresponding to the wave pattern in (a). (c) Idealized ciliary bending pattern containing bent regions of constant curvature. (d) Sequence of bend- ing corresponding to the bending pattern in (c). In (b) and (d),the ordinate represents the anlount of bending and the abscissa represents the position along the length of the flagellum or cilium. itv as the waves move along the flagellum (17). The spermatozoa of an annelid worm, Chaetopterus, generate flagellar bending waves of greath• reduced radius when placed in viScous solutions (15, 17). These effects are currently being examined in detail in the au- thor's laboratory, as they ma.y provide valuable information about the control of bending and unbending. However, since no analogous ob- servations are available for cilia, these observa- tious will not he discussed anv further here. There is a clear need for the development of photographic techniques to enable =tud' y of the morphologY of movement of individnal cilia. TxE llrcxANra1i oF Bt:N-ntNc, Even before the accumulation of extensive cIcctron micro, scope evidence for the existence uf periplieral, lonritttdinal filaments in cilia and fiaE~ella, it was speculated that their bending niovrnicutc were caused by the contraction 6 7 of mu~cle-like, longitttdinally oriented struc- tures on the sides of t1le shaft (3). Electron microscopy has strengthened and added to these speculations (22, '?i) , but there is still no direct evidence for contractility of the fila- ments. The direct approach of separating the filaments, adding ATP, and_ looking to see if they shorten has been suggested (2d), but the amount of shortening (up to 5 per cent) which would occur diiring maximal normal bending could probably not be resolved microscopically with such small structures. In anY event, the individual filaments appear to be firmh• con- nected (23, 25), and attempt~z to isolate them have been un~uccessfui. tiome evidence for noncontractility of the peripheral filanients hae appeared recently. Satir has developed techniques for fixation of molhtscan gill cilia in active and inactive states for electron microscopy ('?6). These methods ~hoW that in the tip re,ion of a bent cilium, 0009'7391
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MECHANICS AND ENERGETICS OF CILIA i lie filaments extending from the inside of the hend terminate beyond those extending from the outside of the bend (27). This result would he necessary if the filaments were anchored firmh• at the basal end of the cilium and did not change their lengths during bending. Less direct, but more easily reproducible, evidence is provided by an observation of Brokaw (17) on thiourea-inhibited sea urchin spermatozoa. Wave patterns are obtained in which the distal end of the flagellum appears stiff and non- niotile, whereas the proximal half beats ac- tiVel}-. In these cases, the head of the sperma- tozoon maintains a constant orientation with respect to the inhibited distal end of the flagel- lum, in dramatically clear distinction to the behavior during normal movement. This be- havior could be explained by the presence in the flaaellum of a bundle of inextensible fila- ments, which are tied together in the basal re- -ion nnd in the inhibited distal region, but Ire free to slide relative to one another as bending occurs in the active region. On the basis of an electron microscope studY of the tuiique compound cilia which compose the "teeth" of the ctenophore Beroe, Horridge 12S1 li:is also concluded that the peripheral filament~ of cilia do not change their lengths and must therefore slide relative to one another when the cilia bend. These indications of inextensible filaments have not yet provided an alternative mechanism for the generation of bending. As has been sug- ;rsterl in the case of mu: cle (29), the macro- ~copic results (contraction, bending) ma' v not he siuiilar in morphologY to the event~z which occur at the molecular or ultrastructural lev- cl;. ()ne approach to the mechanisnl of bending in cilia ha~z been the search for plt' ysiologic and biochenical similarities bet«•een cilia and mus- cle. Howe.-er, since 1>oth of these are s}'stems which ccun use chemical energy from ATP to producc movement, much biochemical similar- itY is expected which will not neces,'arilr in- dicate -imil,irit' v in the morphology of move- ment :cr t1le molecular level. ILi addition, there nia' v lw >n:in.y difference74 between the enen,y- 1ran.~ducin; romponents of the two svstenls, -implv 1>cc:uise tlte' v are orvanized into ~truc- iiirall.%- dift'erent pattern~;. The different ~olnbil- ~nY l,ropertw~ of m.yosin and thc _aTPa,~e 35 enzyme of flagella (23, 30) may have such a basis. The most productive approach Nirould appear to be a search for similarities to those proper- ties of the actomvosin s vstem «'hich are be- lieved to be most closelY associated ivith con- traction; but isolation of the relevant proteins has only begun to be studied (23, 30-32). The work of Rub' v (33) on an actin-l.ike protein of flagella seems to suggest a particularly prom- ising approach, but further study is needed. An interesting physiologic similarity between cilia and muscle was discovered by Yoneda (34), who measured the effects of viscosity on the movement of individual compound ab- frontal gill cilia of 11Tytilus. When the move- ment Nvas slowed don•n by increased viscositv, the bending moment and energy expenditure in- creased, ,just as the force and energy expendi- ture of muscle can be increased when the short- ening velocity is decreased. Similar effects have been obtained with 4ome tYpes of spermatozoan flagella (3b). However, this simi- larity also may merel' v indicate that the bio- chemistrc of the use of energ}• from ATP is similar in cilia and in muscle, and may not re- veal much about the morphology of movement at the molecular level. EVERGE'1'IC, Since glycerinated cilia and flagella can be reactivated bY ATP and adenosine diphosphate, but not bv a number of other phosphorYlated compoimds, ATP is probabl' v utilized for transport of energy from within the cell to the sites of energy utilization in cilia and flanella. Is the amount of ATP required consistent with this conclusion? The most accurate es- timates of the work done by a,imple cilium or flagelhun n-ainst the viscous resistance of the ~urrounding medium are probably those for sea trchin spermatozoa, «'hich give values of about 3 X 10-' ergs per second per sperm at 16- 1S°C. (17, 36). Since, on tlte aver:i;e, the flagellum of these ~permatozoa contaiiis about one :und a half complcte wavcs, there ;ire three point~z nt which bendimr is occurrin~, :tnd the :tvera~_re rate of werkin; at a hendinv point i-~ then about 10-` ergs per second. if' unbenditlv~ i~ ~is: trmed to he pa!~sive. l or a~iullile cilium. '4lei *h lists values of I to 1.4 X 10-' c,r,s prr secorncl for the rute of workin, durinR the e1Tec-
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36 C. J. BROKAW tive stroke of a ciliary cycle, which contains a single bending point (1). The ATPase activity of preparations of cilia which have been treated to remove mem- brane permeability barriers is not necessarily equivalent to the activity under normal con- ditions, but it still compares reasonably well with estimates of the work performed. Meas- urements on Tetrahymena cilia (23, 25), clam cilia (25), Polytoma flagella (37), and sea urchin sperm flagella (38) all give values close to 10-s mols of ATP dephosphorylated per second per milligram of flagellum protein; but Brokaw, using different conditions, has con- sistently obtained values about four times greater for flagella of Polytoma and Chlamy- domonas (8, 9). Unfortunately, only Brokaw's results have also been expressed in terms of ATPase activity per cilium, by making counts of the number of intact isolated cilia. His determi- nations gave values of about 10-'g mols per sec- ond per flagellum at 18° C. for Polytoma flagella (9). Sea urchin spermatozoa, under similar con- ditions, showed an ATPase activity of about 2.5 X 10-'a mols per second per flagellum, con- sistent with their greater length (35). If sea urchin spermatozoa use ATP at the same rate during normal activity, comparison with the values estimated for the rate of working indi- cates that the utilization of 3 kcal per mol of ATP will supply sufficient energy for move- ment. Studies on glycerinated skeletal muscle have demonstrated a utilization of 1.4 kcal per mol of ATP (39) ; and it has been estimated that during normal muscular function, in which creatine phosphate is the measurable energy donor, the work done requires a utilization of 5.9 kcal per mol (40). Sea urchin sperm flagella, and probably the other flagella and cilia on which ATPase activities have been measured, appear to possess the enzymatic capability which is required to utilize ATP as a source of energy for movement. There is reasonable evidence from electron microscopy that the ATPase activity of cilia is distributed along the length of the cilium (23, 41). If ATP is utilized to yield 3 kcal per mol, the required rate of dephosphorylation will be about 0.8 X 10-'B mols per second at each bending point. Since bending points travel along sea urchin sperm flagella at about 900 µ per second under the conditions used to esti- mate the energy expenditure (17), the passage of a single bending region along the flagellum will require the use of only one molecule of ATP for every 20 A of length along the flagel- lum. Each active site involved in bending prob- ably uses no more than one, or possibly a few, ATP molecules each time a bend passes along the flagellum. A study of the effects of factors such as viscosity, which can mechanically alter the rate of propagation of bending, on the amount of ATP used and the amount of work performed might lead to further understanding of the molecular processes involved in bending. Also of interest in this connection is Rothschild's observation (24) that the heat production of bull spermatozoa is drastically reduced in media of high viscosity. If ATP is needed as an energy substrate all along a cilium, it can get there most simply by diffusion. The requirements for an adequate supply of ATP by diffusion can be examined by the following calculation. The basic equation (42) is: (dc/dt = D (S2c/Sx2) - (QA/L), where c is the concentration of ATP at any point along the cilium, measured by the coor- dinate, x; D is the diffusion coefficient for ATP, approximately 4 X 10-' cm? per second (43) ; Q is the total rate of utilization of ATP by the cilium; A is the cross-sectional area of the cilium which is accessible to ATP, some- what less than 0.03 µ2; and L is the total length of the cilium. Under steady-state con- ditions, (dc/dt) = 0, and integration and evaluation of constants leads to the following result: Cl - Co = (QL/2DA). Cl and Co are the concentrations of ATP at the base and at the tip of the cilium, respec- tively. For a sea urchin spermatozoon, with a rather long flagellum of approximately 42 ,u (1, 17), ATP utilization at the rate of 2.5 X 10-'s mols per second requires that CI - Co be about 0.004M, or somewhat more if some of the flagellar cross section is inaccessible to ATP. This concentration difference is attainable with reasonable cellular concentrations of ATP. Ex- periments with glycerinated flagella suggest 0009'7393 I
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MECHANICS AND ENERGETICS OF CILIA that concentrations of ATP greater than 0.01M may be superoptimal (44), so that these flagella may be operating close to the upper limit at which ATP can be supplied by dif- fusion. This calculation implies that there may be significantly lower ATP concentrations near the far end of the flagellum, even though the bending properties of the flagellum normally remain constant all the way to the end (17, 18). The lack of effect of ATP concentration on bending properties at this level might be test- able with glycerinated preparations. This calculation also suggests that the rate of dephosphorylation of ATP during normal activity is probably not much greater than that which has been measured under experi- mental conditions, and that diffusion consider- ations may limit the useful length of flagella. For simple cilia, which are normally much .~horter, and which use most energy near their bases, the supply of ATP by diffusion will be more than adequate. Compound cilia, which are often fairly long, especially in ctenophores, are another matter; but in these cases the energy requirements may be greatly reduced by me- chanical interactions between adjacent cilia. The wave parameters of the posterior flagel- lum of the dinoflagellate Ceratium (15) in- dicate that its energy expenditure will be about W times that of a sea urchin sperm flagellum. Observations by light microscopy suggest that the diameter of this flagellum is significantly greater than that of most simple flagella, but electron micrographs are needed before the energetics of this flagellum can be discussed further. These considerations of the energetics of cilia and flagella raise no conflict with the con- clusion that ATP normally supplies the chemi- cal energy required for ciliary movement. THE STIFFNESS OF A CILIUli During the effective stroke of a ciliary cycle (position 2 in figure 2c), the viscous resistance to movement through the medium tends to heud the straight, distal portion of the cilium. ,~ince only slight bending is observed in this part of the cilium during the effective stroke, the cilium must have sufficient stiffness to re- ~iq the bending moments imposed on it by the viscous resistance of the medium. If the total hcnd of the straight region is measured by the 37 angle B between the directions of the ciliary axis at each end of the straight inactive region, the stiffness is given by the following approxi- mate formula: a = ll0/4 B= 1.1 r1wL`/4 0 where Mo is the active bending moment pro- duced by the cilium at the base of the in- active portion; L is the length of the inactive portion ; q is the viscosity of the medium ; and o, is the angular velocity of the cilium (34). Data for the simple cilia of Paramecium (1) and the compound cilia of Alytilus (34) lead to estimates of about 10-9 dyne cm. for Mo at normal viscosities, although it may be up to ten times as great at higher viscosities (34). Yoneda's photographs of Mytilus cilia (34) permit a rough estimate of B, and lead to an estimate of about 5 X 10-'2 dyne cm - for the stiffness of the individual cilia in these com- pound cilia. A flagellum must have almost as high a stiffness in the bent regions which maintain constant curvature between bending and unbending, as welll as in the shorter straight regions. This stiffness corresponds to an average elastic modulus of 6 X 108 dynes per cm:. As pointed out by Harris (20), this stiffness cannot be provided by the two central filaments of the ciliary cross sections, as an unreasonably high elastic modulus would be required for these filaments. It cannot be provided by membrane turgidity, as suggested by Harris, since glyc- erinated flagella behave with similar properties. It is probably associated with most of the fila- mentous ultrastructure of the cilium, and even then requires that the components of the cilium possess elastic moduli at the upper end of the range for biologic materials. Active bending of a structure having this stiffness to a radius of 5 µ would require a normal bending moment of at least 10-a dyne em. and an energy expenditure of at least 10' ergs per second at each bending point. This energy would have to be very efficiently re- cl.aiuled during tuibending in order to keep the totnl energy expenditure comparable with the ATI':ise activity and the amount of ATP which can be supplied by diffusion. It teems more probable that the high stiff- ness of the inactive regions of a cilium or flagellum must be greatly- reduced durinQ bend-
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38 C. J. BROKAw ing and unbending. This would suggest that the stiffness is a property of the same structural elements which are responsible for active bend- ing and unbending. An approximately tenfold reduction in stiffness would be required, which is about the maximum which could be achieved by a change which occurs only on one side of the cilium. Better measurements might there- fore be able to test the conventional assumption that bending in one direction results from the activity of structures on one side of the cilium or flagellum and bending in the other direction involves an independent set of structures. THE CONTROL OF BENDING The events which occur locally at active bending points on a cilium or flagellum have been considered in order to define the proper- ties of the mechanism which generates bending. The other important mechanism involved in ciliary activity is that which controls the time of bending. This control system contains oscil- la#ory properties which determine the over-all frequency of the beat cycles, and, in addition, coordinates the activity of the distributed ac- tive bending elements in such a manner that bend,, are propagated along the cilium in a functionally appropriate manner. The nature of this control system has received very little at- tention in recent work, except for two impor- tant papers by 'Machin (3, 45) describing a possible mechanism for the control of wave propagation along flagella. The requirements for a control system can be minimized if the active bending elements dis- tributed along a flagellum are assumed to be independent oscillators which go through cycles of bending and unbending with approxi- mately equal frequencies. Coordination between the elements requires only the establishment of proper phase relationships between the elements in order to generate propagated waves, in a manner very similar to the coordina- tion of autonomously beating cilia into meta- chronal waves. Since the oscillators already would be mechanically coupled by the structure of the flagellum, phase relationships Nti•ould be imposed by mechanical factors, leading to propa- gated bending waves. Although four wave modes are po~zsible in a linear ~}•stem of this nature, onl- v one can exist in a nonlinear systenl «-hich produces waves composed of circular arcs (3, 16), and the proper mode could perhaps be es- tablished bv a dominant oscillator near the base of the flagellum (3). There is little evidence that the more distal active bending elements of a flagellum are able to oscillate independently. Gray mentions an observation of the inhibition of bending in re- gions distal to a mechanically constrained point in the flagellum (18). Unpublished ex- periments carried out by the author with broken glycerinated flagella of sea urchin spermatozoa and Polytoma indicate that only portions which contain the basal end of the flagellum retain normal oscillatory properties. Breaking the flagella is a rather crude pro- cedure, and investigations of this point by more sophisticated methods are under way. If the active bending elements have appro- priate sensitivity to bending induced by passively propagated waves, mechanical coor- dination is possible even if the elements are not able to oscillate independently (45). Au- tonomous oscillation could be limited to a special region near the base of the flagellum. The passive bending waves established by this autonomous oscillator can be locally amplified by nonlinear active bending ele- ments, to produce propagation without dec- rement. The propagation velocity of bending waves in this type of system will be determined by the mechanical properties of the system and by the condition that for passively propagated waves the elastic and viscous bending moments have equal maximum amplitudes. The elastic stiffness required to give the observed propaga- tion velocities can be calculated, and it turns ont to be at least an order of magnitude less than the value for the inactive regions of a cilium or flagellum estimated in the previous section. This discrepancy, which has already been pointed out by Harris (20), makes it un- likeh• that active bending can be coordinated by passive propagation of mechanical waves in the stiff, inactive portions of a cilium or flageilum. An alternative type of control system, whiell is possibly more relevant to the generation of the asymmetric bending patterns •typical of cilia, might assume that active bending at any level occurs only in response to a signal re- ceived from adjacent elements on its basal side. 00097395
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MECHASICS AND ENERGETICS OF CILIA 39 Since the velocit3, of bend propagation can be influenced by external mechanical factors such as the viscositv of the medium, the signal is hrobablY either a direct mechanical result of the bending of adjacent elements (46) or can only he transmitted after the completion of a certain degree of bending of the adjacent elements. Thi:s model also restricts autonomous oscillation to a region near the base. Several alternatives are available for the control of unbendine. The identification of the sianal postulated hY this model may be facilitated by further ~tudy of the relationships between propagation velocit5> and ATP concentration, viscosity, and the amount of bending. The properties of the oscillator also invite further study. It is sensitive to local factors ~ttch as ATP concentration and also to the be- havior of the system as a whole. For instance, esperimetrt: have been carried out on broken -permatozoa of the tunicate Ciona. Short lengths of flagella attached to the head move normally for extended periods (17), but the frequency of heat increases nonlinearly to almost double the normal frequencY as the length of flagellum decreases (:30). Such studies on the nature of the control svs- tem re-emphasize the need for better methods for pltotographing the behavior of individuall cilia, so that cilia, as well as flagella, can be utilized in experimental approaches. REFERE\ CEa ( 1) Sleigh, M. A.: The Biology of Cilia and Flagella, Pergamon Press, Oxford, England, 1962. ~2) Gray, J.: Ciliary Movement, Cambridge University Press, Cambridge, England, 1928. (3) \Iachin, K. E.: Thw control and synchroniza- tion of flagellar movement, Proc Roy Soc [Bioll, 1963, 10S, 88. (8) Brokaw. C. J.: Decrea~ed adenosine tri.- phosphatase activity of flagella from a p.ralyzed mutant of Chlana?1do»aonas moe- rrusii, Exp Cell Res, 1960. 19, 430. (9) Brokaw, C. J.: Movement and nucleoside pol}-phosphatase activitc of isolated fla- gella from Polytoma ut•ella, Exp Cell Res, 1961. ??. 151. (10) Brol:aw, C. J.: AIovement of the flagella of Pollytoraa uz-el(a. J Exp Biol, 1963, ~0, 149. (11) Se_ra~-in, L. A.: The role of ATP in the rhythmic mo~-ement of cilia of infusoria, Biokhimiia, 1961, 26, 160. (12) Gihbon~, I. R.: Reactivation of glycerinated (•ilia from Tch•akymeiia pyriformis, J Cell 13io1. 1965, 'o. 400. (13) Parducz, B.: Reizphy~iologische Untersu- chungen an Ziliaten. II. -Neuere Beitrage zunl Bewegungs- und Iioordinationsmecha- nismu.s der Ziliatur, Acta Biol Acad Sci Hung, 1954, 5. 169. (14) Child, F. M., and Tamm, S.: AIetachronal ciliary coordination in aTP-rcactivated models of .llocliolus gills, Biol Bull, 1963, 1?0. 373. (15) Brokaw, C. J.: Thiourea-induced alterations of flagellar waveforms. J Cell Biol, 1964, .?.3, 15.1. (16) Brokaw, C. J., and Wright, L.: Bending waves of the posterior flagellum of Ceratimn, .Science, 1963, 1.i?, 1169. (17) Brokaw, C. J.: Aon-sinusoidal bending waves of sperni flagella. J Fstl Biol, 1965, _11+3, 155. (18) Gra.-, J.: The movement of sea-urchin ,,permatozoa, J Exp Biol, 1955. 332. 775. (19) Rothschild. Lord: Aon-random distribution of bull spermatozoa in a drop of sperm suspension, \ature (London), 1963, 19S, 1221. (20) Harris, J. E.: The mechanics of ciliarv moN-e- (21) ment. In The Cell and the OrQaaisna, Cam- hridge I-niversitY Press, Cambridge, Eng- lamd. 1961, pp. 22-36. Gray, J.: The mechanism of ciliarp mo\-e- ment. VI. Photographic and stroboscopic anal.-sis of ciliary mmement. Proc Roy Soc [Bioll. 1930. 10-,.313. (22) (.iibbons. I. R., and Grinistone, A. V.: On flagellar struc•ture in certain flagellates, J 4) tieunt,icns, H., and Braams. W. G.: An elc•c- tron-microscopic stud}, of the ciliature of the trochophore larva, \ature (London), Biophys Biochem Cytol, 1960. 7, 697. (23) (;ihbons. I. R.: .4tudies on the protein com- ponents of rilia from Tetrah,wmer2a 1938, i.i?. 611. fur~nis, Proc Nat Acnd Sri, 1963. 50, 1002. !:i) Iiuyper. C.: The Organization of Cellular (21) I3othschild. Lord: Sperm energetics. An ac- :1cti6ty, 185, Elsecier, a m~terdam, 1062, p. cottnr oi' work in progr(~,,. In The Ccll awl the Orgailism. Cambri,ive tniN-ci:sity Pn~-, ti) Hofi'mann-Berlin,. H.: (;(•i~.<elmodelle ttnd Cambridge, I:neland. 1961. pp. 9-21. ldeno~intriphosphatc, Biochim Bioph.•s 1cta, 1950, 1(;_. 116. 7) _llexandrov, C. V., and _lrronet, A. L: AIo- tioncattsed bc adeno,ino triphosphate of cilia in ciliated opithelium killcd hy uI.vc- '•rul I'Xir:u•tion (a "rollular mudrl"), llokl :11cad Nauk 1956, 110, 457. (2,i) Child, F. JI.: Sonte ,tspects of the chemi.~n•y of cili;i ;ind fiaeella. Exp Cell Re?, 1961. '-~nppleincnt S. -I7. (26) Satir, P.: Studies on cilia. The tixation of tlie rnetawiironal J C-11 Biol. 1963. l~', ;45. (27) Satir. P.: Studws on rilia: II. Ex:uuination oi
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40 C. J. BROKAW the distal region of the ciliary shaft and the role of the filaments in motility, J Cell Biol, 1965, 26, 805. (28) Horridge, G. A.: Macrocilia with numerous shafts from the lips of ctenophore Beroe, Proc Roy Soc [Biol], 1965, 162, 351. (29) Huxley, H. E., and Hanson, J.: Changes in the cross-striations of muscle during con- traction and stretch and their structural interpretation, Nature (London), 1954, 173, 973. (30) Brokaw, C. J.: Studies on the flagella of Polytoma uvella, J Gen Physiol, 1962, 46, 358A. (31) Burnasheva, S. A.: Properties of spermosin, the contractile protein of spermatozoa, Biokhimiia, 1958, 23, 558. (32) Pautard, F. G. E.: Biomolecular aspects of spermatozoan motility. In Spermatozoan Motility, American Association for the Ad- vancement of Science, 1962, pp. 189-232. (33) Ruby, A. D.: Unpublished work described at the American Society for Cell Biology Meetings, November, 1962. (34) Yoneda, M.: Force exerted by a single cilium of Mytilus edulis. II. Free motion, J Exp Biol, 1962, 39, 307. (35) Brokaw, C. J.: Unpublished findings. (36) Carlson, F. D.: The motile power of a swimming spermatozoon, Proceedings of the First National Biophysics Conference, Yale University Press, New Haven, 1959, 443. (37) Tibbs, J.: The nature of algal and related flagella, Biochim Biophys Acta, 1957, 23, 275. (38) Mohri, H.: Adenosinetriphosphatases of sea urchin spermatozoa, J Fac Sci Tokyo U, 1958, 8,307. (39) Bowen, W. J., and Martin, H. L.: The ability of glycerol-treated muscle fibers to do work during ATP-induced contractions and the free energy of ATP, Arch Biochem Bio- phys, 1962, 98, 364. (40) Carlson, F. D., Hardy, D. J., and Wilkie, D. R.: Total energy production and phos- phocreatine hydrolysis in the isotonic twitch, J Gen Physiol, 1963, 46, 851. (41) Lansing, A. I., and Lamy, F.: Localization of ATPase in rotifer cilia, J Biochem Bio- phys Cytol, 1961, 11, 498. (42) Crank, J.: The Mathematics of Diffusion, Oxford University Press, Oxford, England, 1956. (43) Bowen, W. J., and Martin, H. L.: The diffu- sion of adenosine triphosphate through aqueous solutions, Arch Biochem Biophys, 1964,107, 30. (44) Bishop, D. W.: Reactivation of extracted sperm cell models in relation to the mecha- nism of motility. In Spermatozoan Motil- ity, American Association for the Advance- ment of Science, 1962, pp. 251-268. (45) Machin, K. E.: Wave propagation along flagella, J Exp Biol, 1958, 35, 796. (46) Brokaw, C. J.: Bend propagation along flagella, Nature, 1966, 209, 161. I
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PHYSIOLOGIC REGULATORS OF CILIARY 1VIOTION'• 2 ROBERT E. GOSSELIN Like other forms of cellular activity, ciliary motion can be modified by a large variety of chemical and physical stimuli. Many environ- mental influences of pathogenic importance are discussed by other participants in this sympo- sium. This report will be concerned only with chemical and neural factors that may have physiologic significance, including internal mechanisms by which an organism may regu- late the activity of its own cilia. Information on this subject is fragmentary, and the iden- tification of a physiologic role for any single cilioactive stimulus is only inferential at the present time. Almost nothing is known about the intrinsic mechanisms that control cilia in higher vertebrates. Based on the responses of primitive organisms, however, it is clear that not all aspects of ciliary movement are equally sensitive to all effective stimuli. In this discus- sion attempts will be made to describe how various factors influence six distinct parameters of ciliary motion, namely, beat frequency, ampli- tude, force or work (velocity of particle trans- port), direction of beat ("reversal"), and meta- chronal wave length and wave velocity. 1. Innervation of Ciliated Cells A nervous system is one of the principal devices by which the activities of multicellular organisms are regulated and coordinated. Some ciliated cells are unquestionably innervated, and the cilia of these cells are responsive to nerve stimulation (e.g., reference 1). Estab- lished examples, however, are extraordinarily rare everywhere in the animal kingdom. Gray (2) and Sleigh (3) have summarized perti- nent observations of several investigators. Both inhibitory and excitatory reactions to nerve ;timulation havee been described. Nelson (4, 5) and Galtsoff (6) have noted abrupt and localized cessation of movement of lateral 'From the Department of Pharmacofo,y and Toxicology, Dartmouth Medical School, Ilanoc•er, \ew Hamp,~i ire. ' The preparation of this paper was supported i)x- research grants from the National Science Fonndation and the II.S. Public Health Service (\ational Institute of General \Iedical Scionces). i_, were many of the experiments described herein. cilia on the oyster gill, as long as the gill tis- sue was not sectioned. Because no other ex- planation could be offered, it was assumed that this effect was mediated by inhibitory nerves. In contrast, Aiello's observations (7) imply that activity in the branchial nerve is required to sustain motion of the lateral cilia on the gill of the edible mussel Ilytilus edulis. Indeed, stimu- lation of the visceral ganglion and of the bran- chial nerve arising from it increases the beat fre- quency of these lateral cilia (8). It is not altogether clear, however, that these and other examples represent direct re- sponses to nerve impulses. For example, it is possible that cilia are influenced by chemical or mechanical changes in their environment re- sulting from stimulation or inhibition of smooth muscle cells; the latter are present in gill, particularly at the base of the filaments (9). In a careful morphologic study, Lucas (10) was unable to find branches of the branchial nerve within the gill itself and concluded that these epithelial cells receive no innervation. Aiello and Guideri (8) have recently referred to unpublished observations in which methyl- ene blue and gold chloride were used success- fully to trace nerve fibers into Mytilus gill fila- ments. Although this demonstration may settle the issue in Mytilus, it is highly improbable that nerves are truly responsible for all alleged instances of neurogenic reactions of cilia. In- vestigators are agreed that under appropriate circumstances most, if not all, cilia are sensi- tive to mechanical stimuli, such as touch or motion of the ambient fluid. As it is difficult to avoid such stimuli during most experimental manipulations, appropriate controls are essen- tial for correctly interpreting the results of such studies. The nature of this tactile sensitivity is unknown, but the phenomenon has long been recognized in protozoa and other oreanisms lacking "nerves." Complex patterns of stimula- tion and inhibition at sites well removed from tlie primary stimulus imply an elaborate communication apparatus. In primitive meta- zoa tliia transmission process has been called '`neuroid" (11). Although relevant studies are few and most 41
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42 ROBERT E. GOsSELI\ SHOCK DURATION (msec.) Fic. 1. Activation of cilia on the excised palate of the frog (Rana pipiens) by single square-wave pulses with a polarity indicated in the diagram. Observations of ciliary motion were restricted to the area designated by ®. The rheobase was determined with shocks longer than 1,000 msec. published data are sketchy, cilia of the respir- atory tract of vertebrates, with the notable exception of the frog, appear to be generally unresponsive to nerve stimulation. As early as 1908, Lommel (12) reported that dog tracheal cilia do not react to bilateral vagal section or vagal stimulation. Perhaps the most complete study of this type, however, was performed on the turtle (13) ; its cilia proved to be insensi- tive to vagal nerve stimulation and to faradic and condensor shocks delivered across the tracheal wall. Tracheal mucous membranes, however, are richly innervated (14), and some of the unmyelinated fibers in the cat trachea appear to end as knobs on ciliated cells (15). Furthermore, Laschkow (15) refers to a body of Russian literature (e.g., reference 16) which describes cilioacceleration in response to vagal stimulation and inhibition in response to sympathetic nerve stimulation (stellate gan- glion). The original reports are not available to this reviewer, but some of the experiments are said to have involved mammals or at least "Warmbliitern." With respect to the frog there is no serious conflict in the evidence. In all species tested, the ciliated cells of the palate appear to be in- nervated. Both direct and reflex stimulation of the efferent palatine nerve~ initiate or inten,~,ify ciliary activity. Lucas (17) reported that the accelerator fibers arise in the brain and travel with the seventh cranial nerve; he regards them as part of the bulbosacral or parasympathetic portion of the autonomic nervous system. In accord with these anatomic conclusions, incom- plete pharmacologic studies imply that these nerves are cholinergic (18 ?0). Presumably because of these submucosal nerve fibers, cilia of the excised frog palate are responsive to electric shocks. In figure 1 are summarized the results of a simple experiment' in which the palate was mounted in frog Ringer solution under a glass coverslip and over flattened silver electrodes. linder these circum- stances the cilia gradually become quiescent, reflecting what is presumed to be their normal resting state (17). Whenever fluid under the coverslip was made to flow, cihary motion was promptly initiated. Activity could also be in- duced abruptly by a single D.C. square-Nvave pulse of appropriate voltage and duration. Threshold shocks are described by the strength- duration curve of figure 1. Although D.C. voltages can cause electro-osmotic flow of fluid, it is unlikelv that these brief shocks were ef- fective by mechanical means. The long chron- axie (2.5 to 8 m.ec. in most preparations) is consistent with small unmyelinated fibers which a re usuallv found in the autonomic nervous s}stem. After these single threshold' shocks, ciliary motion continued for 30 to 60 seconds, mnd then gradually ceased. Activity could be ' Cosselin. R. E.: Original data. 00097399
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PHYSIOLOGIC REGULATORS OF CILIARY MOTION -}3 sustained indefinitely by regularh- spaced repetitive di:.charges, but no simple relation- - ship could be recognized between the frequenc-, of the stimulus and the beat frequency of the cilia. This unusual neuroeffector system de- -er.-es more attention and studv. 2. Xezrrotransmitters and Pacemaker Substances Hormones are another major mechanism by which organisms regulate their various cellular functions. Although estrogens are said to vtim- nlate frog palatal cilia (21), most endocrine 'tttdies of ciliary motion have been concerned on11- with neurohormones and neurotransmit- ters. One can predict with confidence that ciliated cells can be stimulated by drugs that are related chemicallv to whatever neurotrans- mitter substance is found in the nerves which nortnallv innervate them. Many such neuro- transmitters and their chemical congeners have been reported to stimulate the cilia of noninnervated cells also. This observation is not really anomalous because one can cite manly reactions to acet' vlcholine, for example, in tissue: lacking cholinergic innervation most mamtnalian blood vessels). Of all drugs n-hicll modify ciliarr activity reversibly, the lo-called '`autonomic" drugs are gencrallY most eifective and are active in the lowest con- centrations. Furthermore, neurotransmitter sub- -t,tnces related to these drugs are present in detectable amounts «ithin supposedly nonin- nervateci ciliated epithelia. Thus serotonin h:ts bcen found in the lamellibranch gill (7, 22), and acetvleholine and/or various enzymes concerned in its tnetabolism are known to exist in fla~el- lated and ciliated protozoa, molluscan gill, and .-ertehrate tracheal mucosa (summarized in ref- erence i). These observations led Burn ('?:3) to propose tllat the autorhythmicitv of such contractile tissues as ciliated epithelium and heart i., in- diicerl and maintained b' v the presence of one or more pacemaker substances. These so-called ' tocA hortnoues° are prestuned to be utilized \vithin ilie same tissues that elaborate and de- 'tt•oY tltem. Accordina to this II' ypothe-is, the ti-ie concentration of free or active hormone ( I0irrtuines :tt anV tinie the intenAty ot ",pun- ian(1ou~" :utivitv. Whether intrin.~ie nwrve~: or unii-n~tiral vlenu•nt~s ure the ;ite of hormone synthesis is no longer regarded as a crucial feature of the theory. 3. Acetylcholine To illustrate and ?upport this interesting hypothesis, Burn (23) has summarized evidence concerning the rhythmicit' v of mammalian car- diac atria and various ciliated epithelia. At least n-ith respect to cilia, his conclusion thatacetyIcholine functions as a local hormone or pacemaker substance is not well supported by available data. Admittedlv there is no scarcitv of reports describing_ effects of acetylcholine on the activity of cilia (as summarized in reference :3). From protozoa to man, various actions have been reported, including increases and decreases of beat frequency, speeding and slowing of par- ticle transport, and prolongation of ciliary re- versal. However, most of these effects are small and require peculiarly high concentrations of the amine. Aside from studies on the frog palate, per- haps the most dramatic effects in vertebrates were described bv Kordik and co-workers (24). On the mucous menibrane of excised rabbit trachea, particle transport R-as acceler- ated b, v low concentrations of acety1clloline and of physostigmine (10-s and 10' m. per milliliter, re.pectivel' v) and inhibited by high concentrations (10- and 2 X 10' b n. per milliliter). Both atropine :tnd d-tubocurarine depressed or arrested ciliary motion in these preparations. At least with respect to the ac- tions of d-tubocurarine, these observations have been challenged (25) and defended (26, 27). J'Iost attempts to demonstrate responses of trachcal cilia to acet}-lcholine and atropine have been less successful (?3-3? : but see also 33 and 3-1). Wherea.s acetvIcholine and other para- sYmpathomimetic drugs stimulate mucus flow :md protect against the cilioinhibitory effects of irritant v:tpors (:35), it is not established ihat these observations reflect actions on cilia (13). An adeqttate explanation can apparetul}- l,e found in the re~zponses of the <eeretor.v appcira- tus. Thus these agents are reported to incre:i~e the atnount and decrease the ti-iscositv of the ntncits blanket (:i51. That acetylcholine cnn- centrations ,reater than 5 X 10-° AI rfre re- qttired to ::ftect the activr rot:ttion of curled pieces of Inunan tracheal muco<a in ti~sue rul- tun•c (3ti) eon;titutels con"-incinn evidence that
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44 U X 20~- a IS ~ ROBERT E. GOSSELIN SW ACH ( I0-') ACH (10-6 ) OU- 0 L 0.5 S W. MODIOLUS LATERAL CILIA S.W ACH ( l0-b) .., S.W. ACH (10-`) S W. I II f I I II II 1.0 1.5 2.0 2.5 3.0 TIME (HOURS) l ACH (10-') 1 3.5 FIC. 2. Responses of Modiolus lateral cilia to graded amounts of acetylcholine. A small portion of excised gill was mounted in a special optical cell (volume less than 0.5 ml.) ; it was exposed to sea water and drug solutions by continuous perfusion (0.5 ml. per minute). Concentrations of acetylcholine chloride are expressed in grams per milliliter (in sea water). S.W. indicates normal sea water (MBL formula). Ciliary beat frequencies at one arbitrary site are expressed in cycles per second. this amine does not play a physiologic role in the control of these cilia. In spite of assertions to the contrary (8), acetylcholine appears both to excite and to inhibit the cilia of Mytilus gill. Although a moderately intense response of frontal cilia has been reported (37), changes in the velocity of particle transport on the lamellibranch gill can be very difficult to interpret (38). The re- ported reactions of lateral cilia, although much less distinct, are of more interest because these observations were based on stroboscopic de- terminations of beat frequency (37). Both frontal and lateral cilia appeared to be excited by low concentrations of acetylcholine bromide (10-° to 10' gm. per milliliter) and inhibited by higher concentrations. Physostigmine had simi- lar but more intense effects, at least on the frontal cilia. Atropine alone (10-` to 10' gm. per milliliter) paradoxically accelerated parti- cle transport (frontal cilia), whereas d-tubo- curarine (10-b to 10' gm. per milliliter) in- hibited both types of cilia. Most of these effects on Mytilus gill, however, proved to be very small, and their significance is therefore ques- tionable. The lateral cilia of Modiolus gill react to acetylcholine more vigorously than do '-\Iytilus cilia, as illustrated in figure 2` Low concen- trations (10' to 10-4 gm. per milliliter) regu- larly stimulated the beat frequency. Although the reaction to 10' gm. per milliliter was ambiguous here, reversible inhibition was ob- served in another preparation at this con- centration. In the absence of exogenous ace- tylcholine, atropine sulfate (10-° gm. per milliliter) was without effect. However, a new and disturbing phenomenon appeared in these trials. Stimulation by acetylcholine proved to be transient, and the excitatory phase was followed by a return to control levels or by frank inhibition, even while the preparation was continuously flushed with drug solution. Nevertheless, the perfusion fluid remained ac- tive, because, after a brief rest period in nor- mal sea water, the tissue regained its respon- siveness to the drug (figure 2). This loss in tissue reactivity probably cannot be ascribed to any accumulation of hydrolysis products. At least in preliminary trials, the effect oc- curred in sea water with and without glycine buffer (pH 7.8) and in response to carbaminoyl- ' Gosselin, R.. E.: Original data. 00U97401
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PHYSIOLOGIC REGULATORS OF CILIARY MOTION choline (carbachol), an ester relatively resistant to spontaneous and enzymatic hydrolysis. A phenomenon which in other systems has been called tachyphylaxis is illustrated in figure 2. Although several possible explanations can be offered on theoretic grounds, no mechanism has yet been established. Whatever the true ex- planation, it will prove almost certainly to be incompatible with the suggestion that acetyl- choline plays a physiologic role as the pacemaker substance in this tissue. 4. Catecholamines Epinephrine and perhaps norepinephrine are also said to stimulate ciliary motion, but, as in the case of acetylcholine, the evidence is con- flicting. Rather few studies are available for comparison, and most published data are not especially convincing. Particle transport on excised rabbit trachea is said to be accelerated by epinephrine (24, 34) but not by norepin- ephrine (34). Burn (23) has suggested that epinephrine may be effective in this prepara- tion by potentiating the activity of endogenous acetylcholine. This suggestion has particular relevance to the frog palate, in which the cilioacceleratory action of epinephrine can be blocked by atropine (18, 20). On the other hand, many investigators have been unable to demonstrate an excitatory effect of epinephrine on vertebrate tracheal cilia. There were no detectable ciliary responses to parenteral doses in dogs and cats (32) or in turtles (13). On excised horse trachea, epinephrine was inactive at an unspecified concentration (28). Lierle and :Uoore (29, 30) and Proetz (39) report that strong solutions of epinephrine hydro- chloride (1:1,000) inhibit or arrest ciliary movement on the nasal mucous membrane of various mammals, but neither the pH nor the buffer concentration of these solutions was re- ported. In lower phyla, cilioexcitatory effects of epi- nephrine have been observed on the protozoan Stentor (3) and on the tentacles of 2~Ietridium (-10). 1'orepinephrine also stimulated the cilia of \Ietridium, whereas acetylcholine chloride in concentrations of 10-` and 10' (gm. per milliliter ?) was inhibitory. Cilia on the excised ovster ;ill caused it to crawl distinctly more .<lowly in epinephrine solutions (2 x 10~6) than in regular sea water (1937 data of Nomura, as TABLE 1 EFFECTS OF CATECHOLAllINES ON LATERAL CILIA OF MYTILUS GILL 45 Average Beat Frequencies (c.p.s.) t Standard Errors Sequence of i, B= Norepi- Treatmer,ts I A = Epinephri ne* ~ nephrine' ~ 1. Sea water i control 11.3 f 0.8 (7)t~'10.3 t 0.6 (12) 2. Same as 1 1 11.2 f 0.5 (11)~ 9.2 f 0.6 (11) 3. Drug A or ~ 9.1 t 0.5 (9) 110.6 f 0.6 (11) B(6X10-5~ AIl 1 ' 4. Drug A or 10. 6 zL 0.5 (14) 10.5 zl= 0.2 (15) B(fiX10-'~ AI) 5. 5ea water t l 9.9 t 0.5 (13)i 9.7 f 0.5 (11) con ro 6. 5-FIT$ (2.5 X'23.8 f 0.3 (16)I22.4 f 0.2 (17) 10-6 iDI) i * Two tissue preparations (A and B) were exposed to epinephrine and norepinephrine, respectively, according to the treatment sequence specified on the left (see also text). Just before use, bitartrate salts of these two drugs were dis- solved in sea water containing as a preservative sodium bisulfite in approximatelv a 1:1 molar ratio with the catecholamine. All solutions were adiusted to pH 7.7-7.8. t Numerals in parentheses indicate number of readings at each period. These measurements were made in stroboscopic light at randomly selected sites (excluding only the occasional areas where cilia ii-ere not moving). $ Serotonin replotted by Tomita, reference 41) ; but findings in this type of test system should be interpreted with much caution (38). Epineph- rine proved to be even more active than acetylcholine in accelerating particle transport by the frontal cilia of excised Mytilus gill (37). A modest increase (average 14 per cent) in the beat frequency of lateral gill cilia was also re- ported (37) after epinephrine concentrations of 10-' and 10-° gm. per milliliter (salt not specified). The last observation is at variance with re- sults obtained at Dartmouth, as summarized in table 1.' Mytilus gill plates were excised and submerged in sea water within flat-bottomed dishes (22). Beat frequencies of the lateral cilia were measured in stroboscopic light at nine or more sites randomly selected at each period of observation. Separate preparations s Gosselin, R. E.: Original data.
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46 ROBERT E. GOSSELIN were exposed in sequence to various drugs dissolved in sea water (pH adjusted to 7.7-7.8). Each exposure period lasted at least 30 min- utes. The tabulated values are means and standard errors of both early and late meas- urements made within each exposure period. Catecliolamines are unstable in alkaline solu- tions at room temperatures, and solutions were therefore prepared just before use. Al- though small amounts of sodium bisulfite were added here as a preservative, similar results were obtained in trials without bisulfite. Neither epinephrine nor norepinephrine stim- ulated or inhibited these cilia; however, the cilia responded normally to the excitatory ac- tion of serotonin (5-HT in table 1). Other methods of exposure, e.g., intermittent super- fusion at four-minute intervals (according to reference 42) gave similar negative results. In- cidental observations over several years have confirmed these findings. Recently the lateral cilia of Modiolus gill were found also to be in- different to epinephrine concentrations as high as 6 X 10-' M, as long as the solution pH was adjusted to 7.8 to match that of the sea water control. Occasionally concentrat.ions of 10-' and 10-' M appear to inhibit the beat frequency and metachronal wave pattern, but these ef- fects are only transient. Significant stimula- tion has not been observed. Although the data are less extensive, trials with dopamine have been similarly uninteresting. No explanation can be offered for the dis- crepancies between the results at Oxford and at Dartmouth. Even though inactive against cilia :is tested in this laboratory, epinephrine and norepinephrine have significant metabolic ef- fects on isolated gill (43), as described in sec- tion 8 below. Moreover, recent studies (see sec- tion 6 below) demonstrate weak interactions with the tissue receptors that are responsive to ~erotonin, even though the receptors are not activated by these catecholamines. In any case, chinephrine cannot serve as a pacemaker sub- >tance in the lamellibranch gill because cate- cholamines are not found in this tissue (-1:3). 5. Serotonin Of all the potential candidates for a pace- maker substance in ciliated epithelium, none has such consistent and intense effects as ~erotonin (5-h.ydroxytrlyptamine, 5-HT) on the cilia of the lamellibranch gill (7, 42). Increases in beat frequency induced by this substance aree prompt, sustained, reversible, and graded over a wide range of drug concentrations (e.g., 10-8 to 10-5 'M ). At least three of the four different kinds of cilia on the lamellibranch gill are excited by 5-HT (38, 42). Both fresh-water and salt-water mussels are responsive, but in preparations in which lateral cilia have an unusually high autorhythmicity they respond with smaller increments in beat frequency, so that supramaximal rates cannot be obtained. The velar cilia of nudibranch embryos are sen- sitive to even lower 5-HT concentrations than the cilia of lamellibranches (44). Probably all molluscan cilia can be affected by 5-HT, al- though some species show distinct seasonal fluctuations in sensitivity (42). Except in mollusca, 5-HT does not appear to be active against cilia. At least the effects de- scribed in other phyla are unimpressive. The peristomal cilia of Stentor are not affected by it e" Particle transport on the frog palate was uninfluenced by concentrations as large as 5 X 10-° M(42) . The propulsive force generated by cilia in the isolated lien trachea could not be modified by local applications of 5-HT (45). According to the method of Scudi and asso- ciates (31), the in vitro survival of rat tra- cheal cilia was not influenced by a ten-minute exposure to a one per cent solution of 5-HT (46). However, particle transport by the iso- lated and submerged rabbit trachea was ac- celerated modettl' v in the presence of 10-8 M 5-HT, and reserpinized preparations were sen- sitive to even lower concentrations (34). In contrast, 5-HT injected intravenously into anesthetized rabbits and guinea pigs induced tracheal iechemia and a reduction in ciliarv beat frequency as measured in stroboscopic light (47). Although intriguing, the results of this last stud' v cannot be accepted without res- ervation hecause the stroboscope is generall, v regarded as an unsatisfactory instrument for reliable measurements of tracheal ciliary activ- ity. Perhaps because the beat frequencY tends to be slightlY irregular in the mammalian trachea, this reviewer, C. J. Kensler,' and T Dalhamn (48), all working independently, "Sleigh, M. A.: Personal communication. ' Cosselin, R. E.: Unpublished data. " Personal c•ommmnication. 0009'7403 I
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PHYSIOLOGIC REGtiLATORS OF CILIARY \IOTIO\ 47 tl~ive been unable to obtain satisfactory data b.- ! is method. The cilioexcitatorv activity of 5-HT on the ;, tmellibrancll gill is of particular interest be- (-~inse serotonin is a normal constituent of this sue. Its presence has been demonstrated by l)hotofiuorometry, bioassay, and paper chroma- tography (22, 49). It has also been detected in the tracheae of various laboratory rodents (50). Concentrations of serotonin in fresh molluscan gill are small. Analyses of 20 speci- ulcus of 11lytilus edulis revealed a mean level of 1.45 -!- 0.7 / per gram wet weight of gill ; even less was found in Modiolus demissus (22). Pleserpinization and most other attempts to niamipulate the tissue level of endogenous 5-HT were unsuccessful; but 5-hydrosytryptophan (5-HTP) added to the surrounding sea water nas decarbosylated enzymaticallv within the ~ill. During this process the tissue gradually accnmulated and sequestered 5-HT; levels as hi~,h as 5 y per gram were attained in JIytilus -ill. The result was a slowly developing but 1>er~istent cilioacceleration. In figure 3 responses *o :i-HT and 5-HTP are compared; the dif- ierence in latency was much more marked when equimolar concentrations were tested (22). '\o information is yett available to <ugge~t 25 ~ a ~ 2 0 ,- > U z v ¢ U_ 15L I 0 ` e°O ~ b. e.ed 00 0 5 1 2.5 3.0 TIME IN which cells of the lamellibranch gill contain the extractable 5-HT or the decarbosv]ase enzyme by which 5-HT is generated from 5-HTP. -Nerve ganglia from bivalve mollusks are partic- ularly rich in 5-HT (51), and it has long been assumed that the molluscan heart is regu- lated by serotonergic nerves. Even if nerve fibers are the source of 5-HT in the lamelli- branch gilt, it is unlikely that thi~ amine func- tions as a neurotransmitter <ub:~tance in this tissue. Aiello (S), in any case, reports that in Mytilus ciliary responses to branchial nerve stimulation are blocked bv variou.~ drugs which do not affect the excitatory actions of exogenou5 5-HT. The distinction is even clearer in nudi- branch embryos, in which nerve stimulation and 5-HT produce opposite effects on the ac- tivity of velar cilia (1, 44). Information is also less than complete about the biochemical mechanisms by which the lamellibranch gill de- grades endogenous 5-HT. Although 'Mvtilus gill pos.~esces an active 5-h' ydrox - vindole oxidase distinct from monoamine oxidn~e (52), neither enzvme seems to occur in Modiolus gill (22). At least it is certain that some mechanisms are present in the l.amellibranch ~ill both for syn- thesizin; and for degradinr thi~z amine. The possibility that 5-HT ftmctiorr as a physiologic pacemaker subz~tance has thus heen est.iblislled. MYTILUS GILL - LATERAL CILIA N 'g P;°y i aa - ~e ~ a 5~ J m S.W. r 6. S W 5 ~ x I P si S`N. 1- 3.5 NOURS I 'I . >e 4.0 22.0 s= PIG. 3. 13rat frctliuenry of lateral i ilia in ;imas of excised M vtilu~4 eill inruhatrd in scn water wit}i ;iud Witliout 5-HT (2.5 X 10 ' :11) aatci 5-HTP (2.3 v 10-' \I). " The bc~a frequenry is stated in i•' vrlr,; per second The tissue was sul?crfuserl h' y tlir filler paper tecImique (reterence 42). Uieprinted uith pei-mi..,iom Jrom t1ic Juro-wil of (;rnei'al Pkysiology. See roference 22).
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0 4s ROBERT E. GOSSELIN To explore further this hypothesis, the effects i 5-HT blocking drugs have been examined. It !t:zs heen shown (53) that such well known :i-HT antagonists as lysergic acid diethylamide lLSD), brom-LSD, and dihydroergotamine are .!I,le to suppress stimulation of gill cilia by added 5-HT; however, these same substances have proved to be potent cilioaccelerators «-hen Qiven alone. With LSD and brom-LSD, ;timulation of -Modiolus lateral cilia occurred at lower concentrations than those required to block the cilioexcitatory actions of exogenous 5-HT. LSD was more potent than brom-LSD i;oth as a stimulating and as a blocking agent. That receptor blocking drugs may excite the receptor mechanism and so mimic the action of the agonist is a widely recognized pharma- cologic principle. Unfortunately this phe- nomenon neither supports nor contradicts the hlypothesis that 5-HT is a cilioregulatory ltormone in the lamellibranch gill. More recently a newer lysergic acid deriva- ~ire has been found to exhibit, in low con- •outrations, only an inhibitory effect on the i)oat frequency of lateral gill cilia. 1Vlethysergide ;nsleate (UAZL-491) depresses reversibly both he spontaneous activity and the stimulation »d nced by added 5-HTs The latter, however, is :!iiiibited to a significantly greater extent than ?'~.c former, whether the observed changes in r.",t irequency are expressed in absolute or in n,latk-e terms. This discovery still does not _rirntav contradict the hypothesis, because examples can be cited in which block- _~ irui.,s i re found to be more effective against ~ 1n;ccted a,onist than against the same sub- t',-lt:lsed endogenously. 'More discon- ~.- i< rhe observation that methysergide is ~tive in suppressing 5-HTP-indueed ~~ n: ciliary motion than in suppress- :1r'1nerniS activitv of the unstimu- =':r~~ 4 illu~trates reversible and I-,i In-er,ide in an excised has heen stimulated ir-xvtrvptophan (~- ITl> aimo;t cer- ~-~iu~n re- ~, -ncr-Zted within ,'t t!;~r ni<th~~:~r~Tirlo "I , rl, i,,. olh- would inhibit 5-HTP-stimulated and unstimu- lated cilia to a comparable extent. Even the un- expected difference cannot be regarded as com- pletely incompatible with the hypothesis that 5-HT is responsible for the autorhythmicity of these cilia, at least not until it is established that identical 5-HT storage sites are involved in stimulated and control preparations. A more useful tool would be a drug which produced permanent or "irreversible" blockade. Recent studies suggest that phenoxybenzamine (DibenzylineD1) is such a drug'° Exposure of Mytilus and Modiolus gill to solutions of this substance depressed or arrested the spontaneous activity of lateral cilia and prevented stimula- tion by subsequent doses of 5-HT. With proper adjustments in phenoxybenzamine concentra- tion and duration of incubation, spontaneous activity could be restored to normal by washing with sea water, but the preparation remained essentially unresponsive to exogenous 5-HT. Ob- viously it is important to learn whether or not the effects of phenoxybenzamine are mediated through the same tissue receptors that are ex- cited by 5-HT. In mammalian pharmacology, phenoxybenzamine is generally recognized as an antagonist of epinephrine and other drugs that act upon adrenergic receptors, but it has been shown also to attack other kinds of drug re- ceptors in isolated mammalian tissues (e.g., reference 54). At least some of the actions of phenoxy- benzamine on the lamellibranch gill must in- volve the same receptors that combine with exogenous 5-HT. This assertion is based upon experiments such as that illustrated in figure 5, in which the response of lateral cilia to 5-HT has been tested before and after an exposure to phenoxybenzamine. 'Moderately complete blockade is illustrated by the solid points. In the presence of a relatively high concentration of 5-HT, however, phenoxybenzamine was much less effective as a blocking drug, as judged by the post-exposure response of the cilia to the test dose of 5-HT (open circles). When 5-IIT and phenoxybenzamine were present to,riher. the former presumablv occupicd ihe rv(-wor to the exclusion of the latter and in 0u- protected the tissue against thw mon, ~tr reversible effects of the b1orlcin, "c;c,ssehn. R. and li,lwd. 00097•10:i
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PHYSIOLOGIC REGULATORS OF CILIARY MOTION 25 N 20 a 0 S. W. 20 LATERAL CILIA - MYTILUS GILL (a) UML H 40 S. W. S. W. (c) UML S. W. I I I I I I I 80 100 120 140 TIME (minutes) FiG. 4. Beat frequency of lateral cilia at a single target site on each of two preparations derived from an excised Mytilus gill preincubated for 90 minutes in sea water (S.W.) con- taining 5-HTP (10' M). The concentrations of methysergide (UML) in periods a, b, and c were 10-", 10-s, and 10-` M, respectively. The preparations were superfused by the filter paper technique at four-minute intervals (as also in figures 5, 6, and 7). 25 ~ U z W ~ O W a: u_ LATERAL (b) UML 1 60 CILIA - MODIOLUS GILL S.W. I 5-HT I PBZ I 5-HT I S.W. 01 11 1 i 0 20 40 60 80 100 TIME (minutes) Fic. 5. Beat frequency of lateral cilia at a single target site on each of two preparations derived from the same excised AIodiolus gill. The graph shows responses to 5-HT (10-tl M) before and after incubation in a solution of phenoxybenzamine (PBZ, o-,, per milliliter). In the preparation depicted by the open circles, 5-HT (10' A1) was present throuchout the exposure to phenoxybenzamine. S.W. indicates normal sea water. 20 15 10 5 49
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•)0 ROBERT E. GOtitit•:Lt\ 50 microns 21 30 29 21 23 25 C Ptc. 6. A large abfrontal gill cilium of 3lytiltcs edulis, as seen ou high-speed movie film during three successive phases (A, B, C) of a single cycle. The numerals represent elapsed titne in hundredths of a second. The recovery or preparatory stroke at the base is limited to ),hasc B. r,wvl tor protection" experiments provide a ~ ~i:i:ihle nuidcl for probing the mode of action A:\ dIrnl-, th::t exeite< or inhibits cilian• mo- I -!i ii: tla- ti-ui• I-t- li bclotrl. ~~•cni,:,,_ thn• ctfrct, uf 5-I1T nn ;;iil cilia, ,. i. 1- i- ~,~:: pIac-1 ,tl(in I,::rrivlc` trans- .r::l cii:l ; -. ''_'. a'_'t. Uther c:x- : ,n !-u i tnt,rv-t. Like t,, ut;•r,•::-r i)r ,~~,,. :~-iI1' I 1~~~. n( ,t :il- <t:v, it ~,i !: tvr::l ~ie•\ ~::tt()ur iii hr:tt : , -r\,d v.!,i: tn:t-t rrlieet r t:;.~i n- = tn th~• ~~ l:rin• ~~f tn~~tachron:tl T1"• i,,rnt (4 the rilian• b•at al.=o dc.~en-es .• t,•tu ion, t -ptvictll}• t he angular velocity of the -L:tit :1n(1 rh0 :nuplitude of itS excursion. Arei- rlu-r q,t:uuit.y c:in l,e ntensured reliabh- on -1w (r:>ntal or lateral cilia of lamellibr;tnch _ili~. llo«-ever, with high-speed motion pic- t:tr:"• it i~ po~zsible to analyze in considerable ~lcJ;til the rnotion of gnant compound cilia ivirri t on t he abfrontal ,ttrface of gill fila- tncnt~. adtnittedlv these cilia exhibit move- tuwnt.~ that are grossly atypical, as illustrated itl fi:;nre fi, in which is reconstructed a single cYcle of a-1'Iytiltts abfrontal ciliutn beating in :•% iA (r, ntc~l rtli:: Ia.l ::nd tho brat normal sea water. The configuration of this cilium has been traced by projecting single frames of a movie film taken at 400 frames per :~econd. \'umerals in the figure indicate elapsed time in hundredths of seconds. The be- ginning of the cycle (t = 0) was chosen ar- bitruril.• to coincide with the moment the tip of the ciliutn began its excursion to the right. I3cc•rttt~e the >haft w;ts approximately straight t Itrc, tehont ti_ure ttA, this phase of the cycle ,•:ut hr• rce.u•dcd a.~ the "effective stroke." In limure tM thc ba~c of the cilium began moving qnivklY to the left. This phase has been called the rerovrre stroke, but the term "preparatory ; trnke" i-s tnore appropriate (55). In figure 6C the base rever,~ed its direction while the tip oontinucd to the left to complete the cycle in a maneuver suggesting the crack of a whip. This peculiar motion has been described be- fore (e.g., references 55 and 56) ; however, it has onlv recently been noted that these ab- frontal cilia, like the more conventional cilia on the frontal and lateral surfaces, respond to 5-HT by increases in beat frequency u Not all phases of the ciliary cycle, however, are speeded equally. Because the analysis is very, tedious and time-consuminr, only a few cilia have been studied in detail; therefore the following con- " Gosselin, R. E.: Unpublished data. 00097407
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YHI"SIOLOGIC REGULATORS OF CILIARl" 3IOTIOS ,-Lu,~ions are only provisional. At the end of ,-very effective stroke, most if not all Modiolus birontal cilia become motionle,s for several lwndred milliseconds before the beginning of the prepal~atorY stroke. Increases in beat fre- qucncy induced by 5-HT seem to be accom- pli.~lud solcly by shortening the duration of this quiescent interval (called here the inter- kinetic period). No changes in amplitude or angular velocitv have been observed in \Iodiolus. Mvtilus abfrontal cilia also tend to rc,<t at the end of the effective stroke, but onh- :ifter periods of sustained activity; during n•ain~4 of normal cycles there are no inter- kinetic periods. Under the influence of 5-HT the c' Vclc length in Mytilus becomes shorter because nf a reduction in amplitude. The entire reduc- tion ~4eems to be accomplished by interrupting the terminal phase of the effective stroke. Thus ;hc action of 5-HT on both _lIvtilu~: and 11odiolus abfrontal cilia can be described as a prem,tture initiation of the preparatorY stroke. 6. Other Bzogenic A?>ai7zes \[any other naturallv occurring amines untst be etanrined as potential candidates for the role of pacemaker substance in ciliated ehithelia. For example, it has lonn been rt,coanized that histamine can be extracted f ront mammalian mucous membrane ref- (1rence 24). No effect on ciliary motion has heen clemonstrated with this sub~4tance (24, :31, 57), but confirmatorv studie,, would be worth- while. Though less ubiquitous in vertebrates, «;nnm:t aminobutyric acid (GABA) h;r at- ,r,tcted attention because of its inhibitory ef- fect., on various neural meehanisms in both v(•rtebrates ;tud invertebrates. However, when udded to sea water in concentr:rtions as 11401 as 2 X 10' M, it is devoid of inhibitory or e.s- citatorly ,tctions on the lateral gill cilia of marine iuusselti." W the two recognized precur:~zors of 5-]ty- drowtr.yptaminc (5-HT), both 5-IITP ,md I- rrYptoph:ur hrive been detected in p;iper chro- +!i:ttotranis of AI, vtilu.~ ;ill estracti (49). ;Ithou'-i the enzvmatic clecarbo.vlation of 5- iL"CP to yield 5-HT h,is been (,~t:ibli.~hed ('?3), 1licre i~ no evidence th,u the l:urrellibr<<nch gill ,,rin convert tryptophan to 5-IIT (22, -i9). Fur- 1 lurnnore, tr.-ptophan appears to have no cilio- =i ]i. 1;.: t"ut'iil>Ii'lwi l dat,i. 51 accelerator' v activity (?3). Tryptamine, how- ever, is not only capable of stimulating lateral gill cilia but is nearlY as potent as 5-HT,'a at least on Jlodiolus gill. Because tryptamine has not been detected in lamellibranch gills (49), it is highly improbable that it is the pacemaker substance of this tissue. _l po~,ible precursor of epinepltrine, namely, tyramine, is al' so a cilioactive substance. Al- though its presence has not been reported in mollusc,ul gills or in other ciliated epithelia, it is capable of exciting the lateral cilia of both DTytilus and llodiolus gills." In a concentration (10' i1I) at which 5-HT regularh• induces an al- most maximal response, tyramine was inactive; but at 10' 11I it produced intense stimulation, as ilhtstrated in figure 7. Generally reactions to tyramine had somewhat longer latencies than responses to 5-HT. Perhaps more time was also required to dissipate stimulation after tyra- mine, but it is impo~sible to be certain that this difference was real. Of more significance is the question of a po~- sible relationship between the cilioexcitatory action.s of tYramine and of 5-HT. Both were reduced or abolished by phenosybenzamine; but even in the presence of this blockin- drug (5 y per milliliter), lIodioltts gill cilia were excited bt- 5-HT (l0" to 10- \I, a~ in figure 5) and not b}- tvramine (10-' -lll. This and other distinctions, however, appear to be quantitative and not qualitative. As seen in figure S, 10-" -1I of tYralnine protected the 5-HT receptors of \iodiolus gill against the irrever:~ible blocking action ot' phenosybenzalnine This protection, however, proved to be incomplete with 10-- M tyrainine (figure S) and negligible with 10' 'M. These observations are interpreted to mean that 5-HT and tYramine excite the same re- ceptor Inech;inism in the I,3mellibranch gill butthat tllese receptors have a much greater aHin- ity for 5-HT (cf. figures 5 and ,). ILr comparable exheriment' < to be reported elsewhere, epinephrinc, at 10 '\I afforded ex- cellent protection to the 5-HT receptor~ of Modiohts gill cilia. apparenrlY epiuephrine oc- culiic.- thc~r rcc(qtor'~ more ti«htl' V ih:in t' Vra- mine, even thouLIh it cloes not excite them as t}•rautinc does (see ~ection 4 ;ibovr). 1j"h:it- ever tlle iuechau,isln of the tran.4nt <tilnttlu- ~~ H. 1?.: t-npublished ~Iut;i. It. E'.: Oririual (tani.
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.52 ROBERT E. UOaSI?LI\ LATERAL CILIA - MODIOLUS GILL 25 20 y \ 1 .\ . ` S. W. 5-HT S. W. TYR. TYR. S. W. 5•H S.W. l0'6 10-6 IO'3 10-6 0 50 " 120 150 200 250 300 TIME (minutes) FIC. 7. A comparison of 5-HT and tyramine (Tyr) as cilioexcitatory substances on the lateral cilia of 111odiolus gill. As in previous figures, all drugs are dissolved in sea water (S.W.) at a final pH of 7.7 to 7.8. Concentrations are expressed in mols per liter. LATERAL CILIA - MODIOLUS GILL 25 20 ~ d U r 5 OOOOOQ z w ~ .! I 0 w cc u- 10 f- a w m 5 0 S. W. TYR I No TYR 5-HT 5-HT PBZ S.W. 5•H 5-HT S.W. 10-6 I0-5 1O'6 10'S 0 50 100 150 200 250 TIM E (minutes) I+re. 8. Beat frequencies of lateral cilia at a single target site on each of two preparations -lerived from the same excised lTodiolus ;ill. The graph shows responses to 5-HT (molar concentrations) before and after incubation in a solution of phenoxybenzamine (PBZ, 5 ry per milliliter). In the preparation depicted by the open circles, tyramine (10-° DS) was present ihroug hout the exposure to phenoxybenzamine. 00097409
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PHYSIOLOGIC REGULATORS OF CILIARY MOTION tuti bv acetylcholine (figure 2), serotonin re- ,eptora do not appear to be involved because 'Ven 10' NI acetylcholine was completely un- mle to protect them against phenoxybenzamine. I i. Potassium and Calcium _lmong the many physiologic substances that can be extracted from ciliated cells, none has been shown to have more profound or more diverse effects on ciliary motion than the sim- ;)le inorganic cations. It is not established that ~han;es in electrolyte concentrations either in- -ide or outside of ciliated cells are involved in -he physiologic regulation of ciliary activity, ~ut the presumption is a strong one. On the ,)tlier hand, there is no reason to suppose that iuoraanic salts serve directly as pacemaker aibstances in the sense proposed by Burn (23). It is more likely that autorhythmicity is regu- lated by the elaboration of specific organic sub- .~rances. As noted below, the characteristic 1)iologic response to such a pacemaker sub- <tance may be conditioned by or mediated [lirough changes in cellular ionic fluxes or con- •entrations. Accordingly, it is not surprising to find that artificial modifications in the extra- ,ellular electrolyte environment are able to mimic the ciliary effects of alleged pacemaker -ubstances and of drugs related to them. \ o simple statement is adequate to sum- marize the many effects on ciliary activity that have been ascribed to electrolytes. A few gen- ,, ralizations, however, appear to be valid and lielpful. Inorganic cations differ in their ef- fectiveness, but all the common anions are in- active and equally well tolerated (2). Ciliated ~~e11s are even indifferent to many anions which ,re not present normally as major components of the biologic environment, e.g., sulfate, iodide, hromide, nitrate, acetate. Divalent cations are more potent than monovalent ones, and trivalent elements such as Al*** are active at ver' v low concentrations (5S). In comparing alkali metals and alkaline earth elements in terms of their effects on ciliary motion, quali- r'1ri.-e as well as quantitative distinctions exist: indeed it can be shown that the monovalent and divalent cations are capable of antagonizing one another. Finally, important differences in bio- l.n?ic activity exist among the various mono- '- 'Jent cations. In trying to formulate these ,onera lizations, many factors complicate the 53 interpretation of experimental data. For ex- ample, as noted by Gray (2), it is difficult to define the influence on ciliary motion of any electrolyte derangement which compromises se- riously the structural integrity of ciliated epi- thelittm. In the physiologic literature on cilia, the following three cations have received most of the attention: H*, K*, and Ca". The effects of pH have been studied repeatedly and are well summarized by Sleigh (3). Thus, ciliary mo- tion is almost always inhibited by acids, es- pecially weak ones such as carbonic acid, which are presumed to penetrate cells and so reduce the intracellular pH. Ciliary responses to al- kalinitv are nowhere near so dramatic or so widespread. In contrast to the hydrogen ion effect, it is impossible to generalize about the influences of K* and Ca'* on ciliary motion, except to note that some effect can almost al- ways be demonstrated. That these same two cations are implicated in many contractile and secretory mechanisms is undoubtedly more than a coincidence. Potassium effects on cilia are especially ubiquitous. For example, GraN • (2) notes that K' makes the frontal cilia of Mi-tilus gill beat faster, even though this cation is not required since activity persists in K*-free sea water. The lateral cilia of excised \Tytilus, however, cease to beat in normal sea water. Activity can be restored by transferring the preparation to sea water with a high potassium concentration (0.07M instead of 0.011I) or with a low mag- nesium concentration (2). A few experi- ments in this reviewer's laboratory indicate that incubations of one or more hours are necessary to demonstrate unequivocal stimulation by K* and inhibition by Mg**. This long latency suggests that changes in intracellular concen- trations are required. In contrast, the lateral cilia of the fresh water mussel Elliptio com- planatus are activated by only 0.02111 potassium chloride after a lag period of less than five minutes (59). From this observation a sur- face effect might be inferred, perhaps an effect on the intracellular transmembrane potential of the ciliated cell. When lateral cilia are stimu- lated by K* (2, 59), the response is weu sus- tained if the pota.sium level is maintained. On the other hand, the abfrontal cilium of 'Mytilus gill almost immediately increases its beat
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ROBERT E. GOSSELI\ frequency when placed in isotonic potassium chloride, but as the mechanism adapts or accommodates to the high K' concentration, the frequency and amplitude fall rapidly. In a preparation at 27° C., movement ceased within 13 minutes (60). In contrast to _)Iytilus and Elliptio gill cilia, the spontaneous activity of Alodiolus lateral cilia is remarkably insensitive to variations in the K' level of sea water, as demonstrated by unpublished results. These experiments were conducted by W. O. Berndt of this laboratory and are summarized in table 2, which is a rec- ord of average beat frequencies measured on excised portions of Modiolus gill preincubated for one hour in artificial sea water with an excess or deficit of K`. Although none of the spontaneous rates is significantly different from the control, a preliminary study has revealed that those preparations conditioned in the low- K' sea water were unusually responsive to the excitatory action of exogenous 5-HT. It is un- likel.y that there exists any single mechanism by which potassium can induce such a diversity of responses. The actions of ionic calcium have also at- tracted attention. Mytilus gill cilia are said to be rather insensitive to the effects of excess Ca", but activity gradually ceases in Ca"-free .solutions (2). In other lamellibranchiates no ciliary actions appear to have been ascribed to calcium, but there is no evidence that the appropriate solutions have been tested. Cer- tainly ciliated protozoa are sensitive to the extracellular concentration of Ca". For exam- ple, in calcium-free solutions Paramecium (61) and Opalina (62) rapidly lose the ability to reverse the direction of ciliary beat in response EFFECTS to an otherwise adequate stimulus. Such a stimulus is any sudden decrease in the intra- cellular transmembrane potential, whether it occurs spontaneously (63), by the application of depolarizing electrical currents (64), or by immersion in a K'-rich medium (62, 63). The duration of ciliary- reversal induced by K' de- pends upon the calcium concentration both in the test solution and in the solution used to precondition the organisms (61, 62). For each conditioning solution a response of maximal duration requires that the ratio (K')/(Ca")'" has a characteristic value in the test solution, whatever the absolute concentrations (65). These and related observations have led to the hypothesis that Ca" and K' ions compete for binding sites in or on these ciliated cells and that ciliary reversal requires the displacement of bound calcium (65, 66). This displacement and the associated ciliary reaction can also be accomplished by the microinjection of calcium chelating agents, such as potassium oxalate or citrate (67). Injections of ionized calcium salts are poorly tolerated (67), but elevations in the extracellular concentration of Ca" induce hy- perpolarization (6S) after a small and transient decrease in the membrane potential (69). No comparable phenomena have been described or are likely to be described in other phyla because metazoan cilia do not generally retain the ability to change the direction of the ciliary stroke, at least not beyond larval and em- bryonic life (e.g., reference 70). S. Oxygen and Cell Metabolism Ciliar}• motion requires energy. As all sur- viving cilia are submerged in aqueous solutions, the minimal energy requirement for motion is TABLE 2 OF POTASSIliM ON THE LATErtdL CILIA OF E%CISEn MODIOLUS GILL* 'Wean Beat Frequenc}° (c.p.s.) f Standard Deviation Experiment No. Normal IC+ (9 ml4f.) High K• (9o mM.) Low K• (0.9 mM.) 1 13.9 f 2.0 13.1 t 2.1 - 2 9.9 t 1.6 (26)t 9.2 f 1.4 (30) 9.5 -:E 1.5 (30) 3 9.7 f 1.4 (30) 9.5 f 1.3 (29) 9.4 t 1.3 (27) * Each experiment involved a different specimen. The gill was divided into three parts, and one por- t ion was preincubated for one hour in each of three different solutions. The solutions consisted-of normal sea water (\IBL formula No. 4) and sea water in which K+ was substituted for Na- and vice versa to niaintain the osmotic pressure and the pH (= 7.8). After one hour beat frequencies were measured at randomlv selected areas over the gill surface (excluding onlY the occasional areas where cilia were not nioving). t\ umerals in parentheses indicate number of observations. 00097411
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PHYSIOLOGIC REGULATORS OF CILIARY MOTION ,3;j ,quivalent to the work performed against the vi~4cosity of the surrounding water. Of course rhi~, energy must be derived from cell metab- nli~4m. As cited by Brokaw in this symposium, there is much evidence which implicates aden- ,-ine triphosphate (ATP) as the proximal -ource of energy for ciliary movement. To maintain the cellular supply of ATP, it is pre- ?umably necessary for the ciliated cell to metabolize energy-Vielding substrates at a brisk rate. .,ince the original demonstration on Anodonta ,ili in 1866 (i1), molecular oxygen has been re- _arded as necessary for ciliarv motion. For cclls with only a]imited ability to derive energy from anaerobic reactions, this oxygen requirement would appear to reflect merely the necessity of replenishing the ATP supply. This explanation is not so satisfying for ciliated ,pitlielia like lamellibranch gills, which are rapable of vigorous anaerobic glycoh•sis (43). Mvtilus frontal cilia, however, require oxygen ior sn' v activity that is sustained for more than aboitt half an hour (2). The lateral cilia are (,~-en more sensitive to anoxia; in the absence of O., their motion ceases within aa few minutes 17), if not immediately." Although some oiliated parasitic protozoa appear to thrive under essentially anaerobic conditions, the cilia of free-living ciliates cease to beat when osygen is removed (72). It is not established that thc maintenance of a rapid energy metabolism is the only essential role which molecular oxygen l~lays in the support of ciliary motion. Although little or nothing i., known about the metabolic apparatus of mammalian ciliated cells, several investigators have studied the I iochemistry of the heavily ciliated gills in bi- valved mollusks. In this tissue glycoh;sis ap- pears to occur along an Embden-_llcyerhof lp,tthwav, and a Krebs cycle is believed to be -en-ed bz- a conventional cvtochrome system. l'Iie principal evidence for these assertion, is i he ability of various metabolic poisons to inhibit both gill respiration and eiliary activit' v. '1'hu~, cyanide, azide, and monoiodoacetate are (,I. fective a,ain:t JIytilus 611 (2, 7 ), ,ind, in ad- lition, malonate and fluoride have proved to artive when incubated with excised gills of : he J:tpane~e oyster Ostrea gigas Thunberg ~;:,-; til. Similarly, cilia of the fresh water " (~nsselin, R. I:.: 17 npublished data. mussel Dreissenia are inhibited bv sodium fluoride and hy the amide of monoiodoacetic acid (77). In contrast to these inhibitors, 2,4- dinitrophenol suppresses ciliary motion while stimulating oxygen consumption, presumably by uncoupling oxidative phosphorylation (7, 75, 76, 7`3). As might be expected, many glt•colytic intermediates, as well as citric acid, are pres- ent in appreciable concentrations within the oyster gill (79). Ciliary motion of molluscan gill is clearlN - dependent upon metabolic activity; however, the source of this energy has not been fully establislled. O.r•ygen consumption and ciliarv motion persist in excised lamellibranch gills for many hours in the absence of exogenous substrates (22, 80). Although gills remove glucose from the incubation medium, the avail- ability of glucose and other potential substrates, e.g. glutamate, succinate, and a-ketoglutarate, does not enhance physiologic or metabolic ac- tivity (76, S0) . It is apparent that the gill is not continuously dependent upon exogenous substrates, but rather that it utilizes endogenous energy stores. GraY (2) proposed that a glyco- protein might serve this function, because lie was unable to detect glycogen or fat in the gill of llytilus edulis. However, other investi- gators have found significant concentrations of gh-cogen in the gills of -Mytilus (SO) and of other mollu.ks (77, SO). The quantity of glyco- gen in excised gills decreases slowly during in- cubation in oxygenated sea water (50) ; an- aerobiasis accelerates the rate of breakdown several times (77, Sl) . These phenomena are illustrated by the data in table :3 (original ob- servations of K. E. \Ioore and R. E. Gosselin). Gl}•cogc,n, however, is not the only endogenous substrate for respiration because the respiratorv quotient of unstimulated gill, is about O.S5 (2, 77) ;und because glycogen disappearance can account for onl}- 30 to 50 per cent of the ox y- gelt consumed (SO). The other substrates which help to maintain the oxidative metab- olism of gill epithcliitm have not been identi- fied. E'.rn less information is available about the role of energy metabolism in regttlating the physiologic activity of cilia. It is evident thlt the presumptive pacemaker substance of lamel- librinch gill cilia (.~ee section 5) has profound ruetubolic effects on t1lis ti~~ue. At leastt exo,-
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56 ROBERT E. GOSSELI\ TABLL 3 C.ARBOHYnRATE METABOLISM OF 11ODIOLCS (-iILL* Oxygen Consumed Lactate in Glycogen Supernatant in Gill µl. mg. nig. Preincubation valuest - 0 5.90 Oxygen Sea water 1,170 0.014 5.47 5-HT$ (10-s M) 2,170 0.012 5.09 Nitrogen Sea water 0 0.097 4.66 5-HT$ (10-5 _lIl 0 0.324 2.12 * The gills of a single specimen of dtodiolus denaisszts were divided into four parts and incu- bated for three hours at 25° C. in sea water with and without 5-HT and with and without oxygen. All values have been adjusted to the equivalent of one gram of gill (wet weight). t A mean value based on six other specimens; the standard error of this mean was 0.2 mg. per gram. $ Serotonin. enous serotonin (5-HT) accelerates glycogen breakdown moderately and stimulates oxygen consumption markedly (table 3 and reference 80). The precursor substance 5-HTP has simi- lar effects, but only after aa considerable latency, during which the drug is presumably con- verted to 5-HT within the gill (80). Gill respiration is also enhanced by other cilioac- celeratory substances, such as K` (2), veratrum (2, 7), and lysergic acid diethylamide (LSD) (80). On the other hand, acetylcholine with or without eserine is inactive or slightly inhibitory (80). Epinephrine, norepinephrine, and isopro- terenol produce only equivocal changes in the oxygen consumption of Modiolus gills (SO). Thus, with respect to both stimulants and de- pressants, a reasonably good correlation exists between drug effects on ciliary activity and on tissue respiration. To understand the role of any pacemaker substance in the physiologic regulation of ciliary activity, it is important to learn whether control is exerted through the contractile ap- paratus of the cilium, the triggering mechanism which activates it, or the metabolic machinery which provides it «•ith essential energy. With respect to 5-HT and lamellibranch gill cilia, no evidence can be offered in support of the first possibility. The second is suggested by the ob- servation that intact cells are required to demonstrate both the cilioacceleratorv and the metabolic stimulating actions of 5-HT. At least no convincing metabolic effects have been produced by adding 5-HT to gill homogenates incubated aerobically or anaerobically, with or without enrichment (43, 80). On the other hand, a primary action on the metabolic ap- paratus is implicit in the demonstration that 5-HT profoundly stimulates glycolysis when the excised but otherwise intact gill is incubated in the absence of oxygen (43). Because ciliary motion is presumably arrested under these circumstances, metabolic stimulation would not be expected to occur if the only site of ac- tion of 5-HT were the trigger or autorhythmic generator. As measured in terms of glycogen disappear- ance and lactic acid production in 11lodiolus gill, low concentrations of 5-HT (e.g., 10-e M) markedly stimulate anaerobic gh•colysis (table 3). Epinephrine, norepinephrine, and 5-HTP are also active, but only at higher concentra- tions (10-s, 10-s, and 10-° M, respectively). With or without eserine, 10-' M acetylcholine significantly depresses lactic acid production. Brom-LSD (BOL) blocks, but LSD mimics, the metabolic stimulation by 5-HT. Sodium fluoride (10-z M) does not affect control rates of anaerobic lactate production, but it does prevent stimulation by 5-HT. At the same con- centration iodoacetate inhibits intensely, whether 5-HT is present or not. Whatever may prove to be the correct interpretation of these metabolic actions of 5-HT, they cannot be ascribed to the activation of gill phosphorylase. At least no ef- fect on this enzyme was demonstrated when 5-HT was added to homogenates of Mytilus and Modiolus gill or when the gill was preincubated with the amine before homogenization (43). SL'rIDIARY Attention has been directed toward various mechanisms by which an organism may regu- late the activity of its cilia. Examples are cited in which cilia appear to be under nervous control; both inhibitory and excitatory inner- vation have been described. Except in the frog palate, however, vertebrate cilia are not known to be innervated. The hypothesis that the autorhy-thmicity of cilia is controlled by local pacemaker substances is worthy of attention. Various physiologic amines have been consid- ered as potential pacemaker ~ubstances in ciliated epithelia, notably acetylcholine, epi- I c z 00097413
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At jeen Ites , or Jier lp- .hat ien -ed <<ry ~~se uld :!c- nic )n. cs, im °s )es n- er ro !ic ie f- ?n id ?d as i- !d .1s r- i? PHYSIOLOGIC REGULATORS OF CILIARY MOTION 57 nephrine, norepinephrine, serotonin (5-HT), tyramine, tryptamine, and histamine. The evidence for and against a cilioregulatory func- tion for each of these substances has been ex- amined. Presently available evidence does not implicate convincingly any of these amines ex- cept serotonin, which probably functions as a ciliary pacemaker substance in mollusks but not elsewhere. Electrolytes also have profound effects on cilia, and some drugs may be cilioac- tive because they modify cellular ionic fluxes or concentrations. Aside from H', the cation that has attracted the most attention is potas- sium; but calcium also appears to play a key role, at least in protozoan cilia. The intensity of ciliary activity is determined ultimately by the metabolic apparatus that provides the nec- essary energy. Apparently oxygen is always re- quired for sustained ciliary motion, even in tis- sues which can survive complete anaerobiasis because of vigorous glycolysis. Thus the main- tenance of a rapid energy metabolism may not be the only essential function of molecular oxygen in the support of ciliary motion. Tis- sue oxygen consumption and carbohydrate catabolism tend to change in parallel with drug-induced alterations in ciliary activity. Whether the metabolic effects are primary or secondary has not been adequately studied. Essentially nothing is known about the physi- ologic regulation of mammalian cilia. Attempts to study the complex actions of serotonin on lamellibranch gill cilia serve to emphasize that ciliary beat frequency and the velocity of par- ticle transport are not the only important parameters. In general, more sophisticated physiologic and pharmacologic studies of ciliary motion are needed. Acknowledgments For experimental data not reported before, the author gratefully acknowledges the collaborative efforts of several persons, especially Mrs. Jean Day, Mr. Robert Woods, Dr. Kenneth Moore, and Dr. William Berndt. R.EFEtI,E\' CES (1) Carter, G. S.: On the nervous control of the velar c•ilia of the nudibranch veliger, Brit J Exp Biol, 1926, y, 1. Also On the struc- ture of the cells bearing the volar cilia of the nudibranch veliger, Brit J Exp Biol, 1928, 6, 97. (2) Gray, J.: Ciliary Movement, Cambridge University Press, Cambridge, England, 1928. (3) Sleigh, M. A.: The Biology of Cilia and Flagella, Pergamon Press, Oxford, Eng- land, 1962. (4) Nelson, T. C.: Nervous control of lateral cilia in the gill of young spat of the oyster Ostrea virginica, Anat Rec, 1951, 111, 160 (abstract). (5) Nelson, T. C.: The feeding mechanism of the oyster. II. On the gills and palps of Ostrea edulis, Crassostrea sirginica, and C. angu- lata, J Morph, 1960, 107, 163. (6) Galtsoff, P. S.: Coordination of ciliary mo- tion and muscular contraction in the gill of Crassostrea i•irginica, Biol Bull, 1958, 115, 320 (abstract). (7) Aiello, E. L.: Factors affecting ciliary activity on the gill of the mussel 111 ytilus edulis, Physiol Zoo], 1960, 33, 120. (8) Aiello, E. L., and Guideri, G.: Nervous con- trol of ciliary activity, Science, 1964, 146, 1692. (9) Lucas, A. M.: An investigation of the nervous system as a possible factor in the regula- tion of ciliary activity of the lamellibranch gill, J Morph Physiol, 1931, 51, 147. (10) Lucas, A. M.: The distribution of the bran- chial nerve in 37ytilics edulis and its re- lation to the problem of nervous control of ciliary activity, J Morph Physiol, 1931, 51, 195. (11) Parker, G. H.: The Elementary Nervous Sys- tem, J. B. Lippincott Co., Philadelphia, 1919. (12) Lommel, F.: Zur Physiologie und Pathologie des Flimmerepithels der Atmungsorgane, Deutsch Arch Iilin Med, 1908, 9i, 365. (13) Lucas, A. M., and Douglas, L. C.: Principles underlying ciliary activity in the respira- tory tract. III. Independence of tracheal cilia in vivo of drug and neurogenous stimuli, Arch Otolaryng (Chicago), 1935, 21,285. (14) Messerklinger, W.: Die Schleimhaut der oberen Luftwege im Blickfeld neuerer For- schung, Arch Ohr Nas ICehlkopfheilk, 1958, 173, 1. (15) Laschkow, W. F.: Zur Jlorpholo_ie der Inner- vation der Trachealschleimhaut, Z Mikr Anat Forsch, 1955, 61, 229. (16) Schmagina, A. P.: Ciliary Motion (N. A. Rozhanovsky and S. I. Schatov, editors), Medical Publishing House, Moscow, U.S.S.R., 1948. In Russian. (Not examined by this reviewer.) (17) Lucas, A. M.: Neurogenous activation of ciliated epithelium, Amer J Physiol, 1935, 112.468. (18) Plattner, F., and Hou, Ch. L.: Zur Frage des Angriffspunktes vegetativer Gifte. Versuche am Embrvonalherzen und am Flimmer- epithel. Pflueger Arch Ges Physiol, 1931, 228. 281.
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58 ROBERT E. GOSSELI\ (19) Ishikawa, S., and Ohzono, M.: Einfluss von verschiedener Pharmaka auf die Flimmer- bewegung, Acta Derm (Kyoto), 1931, 17, 478. (20) Blaich, W., and Klar, G.: Der Einfluss des Penicillins auf die Flimmerbewegung der Rascherschleimhaut des Frosches, Arch Exp Path Pharm, 1950, 211, 67. (21) Boyd, E. M., Clark, J. W., and Perry, W. F.: Estrogens and their effect on ciliated mu- cosa, Arch Otolaryng (Chicago), 1941, 33, 909. (22) Gosselin, R. E., Moore, K. E., and Milton, A. S.: Physiological control of molluscan gill cilia by 5-hydroxytryptamine, J Gen Physiol, 1962, .1j6, 277. (23) Burn, J. H.: Functions of Autonomic Trans- mitters, The Williams and Wilkins Co., Baltimore, 1956. (24) Kordik, P., Biilbring, E., and Burn, J. H.: Ciliary movement and acetylcholine, Brit J Pharmacol, 1952, 7, 67. (25) Hill, J. R.: The influence of drugs on ciliary activity, J Physiol, 1957, 139, 157. (26) Burn, J. H., and Day, M.: The action of tubocurarine and acetylcholine on ciliary movement, J Physiol, 1958, 141, 520. (27) Milton, A. S.: The action of tubocurarine on ciliary movement, Brit J Pharmacol, 1959, 14,323. (28) Hill, L.: The ciliary movement of the trachea studied in vitro, Lancet, 1928, °, 802. (29) Lierle, D. M., and Moore, P. M.: Effects of drugs on ciliary activity of mucosa of upper respiratory tract, Arch Otolaryng (Chi- cago), 1934, 19, 55. (30) Lierle, D. M., and Moore, P. M.: Further study of the effects of drugs on ciliary activity: A new method of observation in the living animal, Ann Otol, 1935, 44, 671. (31) Scudi, J. V., Kimura, E. T., and Reinhard, J. F.: Study on drug action on mammalian ciliated epithelium, J Pharmacol Exp Ther, 1951, 102, 132. (32) Antweiler, H.: Uber die Funktion des Flim- merepithels der Luftwege, insbesondere unter Staubbelastung, Grundfr Silikose- forsvh, 1957, 2, 509. (33) Umeda, T.: A study of the ciliary movement of ox trachea, Acta Derm (Kyoto), 1929, 1,/1, 629 (English abstract). (34) Tsucltiya, M., and Kensler, C. J.: The effects of autonomic drugs and 2-amino-2-methyl propanol, a choline antagonist, on ciliary movement, Fed Proc, 1959, 18, 453 (ab- stract). (35) Falk, H. L., Kotin, P., and Tremer, H. M.: Protective effect of parasympatheticomi- metic agents on ciliated mucus-secreting epithelium, J Nat Cancer Inst, 1961, 27, 1379. (36) Corssen, G., and Allen, C. R.: Acetylcholine: Its significance in controlling ciliary ac- tivity of human respiratory epithelium in vitro, J Appl l'hy.<ioL 1959, 1_$, 901. (37) Biilbring, E., Burn, J. II., ;ind Shelly, H. J.: Acetylcholine and ciliarv movement in the gill plates of .llytilus edulis, Proc Roy Soo [Biol], 1953. 1 ;1. 11;i. (38) Gosselin, R. I:., and O'Hara, G.: An unsus- pected source of error in studies of particle transport bY L•uuellibranch gill cilia, J Cell Comp Physiol, 1961..iS, 1. (39) Proetz, A. W.: Aasal ciliated epithelium, with reference to infection and treat- special ment, J Laryne, 1931, .',D, 557. (40) Ten Cate, J., Cooinau~, lI. E., and Walop, J. N.: L'influciu(, do quelques substances pharmacologiques sur les mouvements des cils vibratilcs des tentaculcs de Metridium sensile (L.),:1rch Neer Zool, 1955,11, 14. (41) Tomita, G.: On the \omura and Tomita method for measuring the mechanical ac- tivity of cilia, Bull 1larine Biol Station at Asamu~4hi, Tuhoku C, 1955, 7, 159. (42) Gosselin, R. E.: The cilioexcitatory activity of serotonin, J Cell Comp Physiol, 1961, 58, 17. (43) Moore, K. E., and Go' sselin, R. E.: Effects of 5-hydroxytryptamine on the anaerobic metabolism and phosphorylase activity of lamellibranch gill, J Pharmacol Exp Ther, 1962, 1,3s, 115. (44) Koshtoyants. Kh., BuznikoN•, G. A., and Manukhin, B. N.: The possible role of 5- hvdroxy-tryptaminf, in the motor activity of embryos of some marine gastropods, Comp Biochem Physiol, 1961, 3, 20. (45) Gosselin, R. E., and Eytel, C. S.: Propulsive forces generated by tracheal cilia, Fed Proc, 1961, 20, 324 (abstract). (46) Kimura, E. T.: Effects of chemical agents on ciliated tracheal cpithelium, Arch Oto- laryng (Chicago), 1959, 69, 674. (47) Krueger, A. P., and Smith, R. F.: The biolog- ical mechanisms of air ion action. I. 5- Hydroxytryptamine as the endogenous mediator of positive air ion effects on the mammalian trachea, J Gen Physiol, 1960, 43,533. (48) Dalhamn, T.: Mucous flow and ciliary activ- ity in the trachea of healthv rats and rats exposed to respiratory irritant gases. Acta Physiol Scand, 1956, 36 (Supplement 123, p. 1). (49) Aiello, E. L.: Identification of the cilioe.r-cita- tory substance present in the gill of the mussel .1I?itihcs erlulis, J Cell Comp Physiol, 1962, 80, 17. (50) Krueger, A. P., and Smith, R. F.: The bio- logical mechanisms of air ion action. II. Negative air ion effects on the concentra- tion and mctabolism of 5-hydroxytrypta- mine in the mammalian respiratory tract, J Gen Physiol, 1960. -f.,, 269. (51) Welsh, J. H., and \Ioorhead, M.: The quan- titative distribution of 5-hvdro..ytrypta- 0009'7415
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PHYSIOLOGIC REGULATORS OF CILIARY MOTION mine in the invertebrates, especially in their nervous system, J Neurochem, 1960, 6, 146. (52) Blaschko, H., and Milton, A. S.: Oxidation of 5-hydroxytryptamine and related com- pounds by Mytilus gill plates, Brit J Phar- macol, 1960, 15, 42. (53) Gosselin, R. E., and Ernst, M. M.: Action of serotonin on the gill cilia of lamellibranchi- ates, Abstracts of Fall Meeting of American Society for Pharmacology and Experimen- tal Therapeutics, 1958. (54) Innes, I. K.: An action of 5-hydroxytrypta- mine on adrenaline receptors, Brit J Phar- macol, 1962, 19, 427. (55) Kinosita, H., and Kamada, T.: Movement of abfrontal cilia of Mytilus, Jap J Zool, 1939, 8,291. (56) Gray, J.: The mechanism of ciliary move- ment. VI. Photographic and stroboscopic analysis of ciliary motion, Proc Roy Soc [Biol], 1930, 107, 313. (57) Ballenger, J. J.: A study of ciliary activity in the respiratory tract of animals, Ann Otol, 1949, 58, 351. (58) Sleigh, M.: Metachronism and frequency of beat in the peristonal cilia of Stentor, J Exp Biol, 1956, 43, 15. (59) Satir, P.: Studies on cilia. The fixation of the metachronal wave, J Cell Biol, 1963, 18, 345. (60) Kinosita, H.: Response of a single cilium. I. Stimulating effect of isotonic KC1 solution on a large abfrontal cilium of Mytilus, Annot Zool Jap, 1952, 25, 8. (61) Kamada, T., and Kinosita, H.: Calcium- potassium factor in ciliary reversal of Paramecium, Proc Imper Acad Tokyo, 1940, 16,125. (62) Okajima, A.: Studies on the metachronal wave in Opalina. III. Time-change of effec- tiveness of chemical and electrical stimuli during adaptation in various media, Annot Zool Jap, 1954, 27, 46. (63) Kinosita, H.: Electrical potentials and ciliary response in Opalina, J Fac Sci U Tokyo (Sec IV ), 1954, 7, 1. (64) Naitoh, Y.: Direct current stimulation of Opalina with intracellular microelectrode, Annot Zool Jap, 1958, 31, 59. (65) Jahn, T. L.: The mechanism of ciliary move- ment. II. Ion antagonism and ciliary re- versal, J Cell Comp Physiol, 1962, 60, 217. (66) Kamada, T.: Ciliary reversal of Paramecium, Proc Imper Acad Tokyo, 1940,16,241. 59 (67) Ueda, K.: Intracellular calcium and ciliary reversal in Opalina, Jap J Zool, 1956, 12, 1. (68) Ueda, K.: Electrical properties of Opalina. I. Factors affecting the membrane potential, Annot Zool Japon, 1961, 34, 99. (69) Naitoh, Y.: Effect of change in external Ca- concentration on the membrane potential of Opalina, Zool Mag (Dobutsugaku Zas- shi), 1964, 73, 233 (English summary). (70) Twitty, V. C.: Experimental studies on the ciliary action of amphibian embryos, J Exp Zool, 1928, 50,319. (71) Kiihne, W.: Uber den Einfluss der Gase auf die Flimmerbewegung, Arch Mikrosk Anat, 1866, 2, 372. (72) Kitching, J. A.: On the activity of protozoa at low oxygen tensions, J Cell Comp Physiol, 1939,1,11, 227. (73) Usuki, I.: Effects of some inhibitors of ana- erobic glycolysis on the ciliary activity of the oyster gill, Sci Rep Tohoku U [Biol], 1955, 22, 49. (74) Usuki, I.: A comparison of the effects of cyanide and azide on the ciliary activity of the oyster gill, Sci Rep Tohoku U [Biol], 1955, 22, 137. (75) Usuki, I.: Effects of malonate and 2,4- dinitrophenol on the ciliary activity of the oyster gill, Sci Rep Tohoku U [Biol], 1955, 22,143. (76) Okamura, N.: Effects of some metabolic in- hibitors on the oxygen consumption of the oyster gill, Sci Rep Tohoku U[Biol], 1959, 25, 91. (77) Wernstedt, C.: Metabolism of gill epithelium of a freshwater mussel, Nature (London), 1944,15-/,, 463. (78) Weller, H., and Ronkin, R. R.: Effects of 2,4-dinitrophenol upon oxygen consump- tion and ciliary activity in the ctenidia of Alytilus, Proc Soc Exp Biol Med, 1952, 81, 65. (79) Usuki, I., and Okamura, N.: Glycolytic in- termediates in the oyster gill, Sci Rep Tohoku U [Biol], 1955, 22, 225. (80) Moore, K. E., Milton, A. S., and Gosselin, R. E.: Effect of 5-hydroxytryptamine on the respiration of excised lamellibranch gill, Brit J Pharmacol, 1961, 17, 278. (81) Moore, K. E., and Gosselin. R. E.: Effect of 5-hydroxytryptamine (5-HT) on glycolysis in lamellibranch gill, Pharmacologist, 1961, 3, 77 (abstract).
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DISCUSSION Dr. Brinkman: Dr. Watson and I have observed that basal bodies of a ciliated cell from the bronchus of the dog have a characteristic appear- ance with rootlets, so there is a tail even on the cilia. In human bronchi the ciliated cells have basal bodies and striated rootlets, as described by Dr. Rhodin. These rootlets can be traced down to mitochondria. Two uses have been suggested for the rootlets: One is that they anchor the cilia. I didn't think it was necessary to have anchors going way down into the body of the mitochondria. The second use, which was touched on by Dr. Rhodin, is that the rootlets may serve a conducting mecha- nism. We would like to suggest a third function for them-that they may be the route by which energy is transmitted from mitochondria to cilia. Very frequently we have seen them down the center of the mitochondria or running into mitochondria. We have some evidence to support this contention. In normal human bronchi mitochondria are well de- veloped, stain well, and have well-developed cristae inside them. However, in patients with chronic bronchitis, mitochondria stain poorly and the cristae inside them are less well developed. It may be, therefore, that in chronic bronchitis there is less energy available for the cilia to beat; or the reverse, that because the cilia beat less, the mito- chondria do not need to be so well developed. There are certain well-developed structures which we have seen only on ciliated cells. Dr. Rhodin re- ferred to them as microvilli, which I usually think of as resembling a row of pegs. But these are better developed than pegs. They have a single stalk and then branch out more like seaweed. These structures, which for the want of a better name we have called "pellicular structures," are very profuse, extend out about a quarter of the distance of the cilia, and are covered by the sur- face membrane of the cell. They must increase the surface area of each cell immensely. The interesting thing is that in persons with bronchitis this pellic- ulous material is not seen and the cell surface is quite nude. However, there are still the same num- ber of cilia, and for those of you who don't think in Angstrom units, as I don't, by count there are between about 1,500 and 2,000 million cilia per square centimeter on the human bronchus. Dr. Corssen: Clusters of human respiratory epithelium which rotate provided a model in which to measure ciliary activity which Dr. Palmer and I employed to examine the effects of acetvlcholine. The high dosages we used were outside the physi- ologic range Dr. Gosselin mentioned today. How- ever, the stimulation and acceleration of ciliary activity caused by acetvlcholine were completely blocked by atropine, which meant that there was some antagonism between these two compounds in ciliated cells. When rotating ciliated clusters were first immersed in physostigmine, the effects of ,lcetv]choline were augmented. The concentrations were unphysiologic, and this may be a sound ob- jection to our conclusion that acetylcholine ex- ceeded all agents in initiating and maintaining ciliar motion. Dr. Gosselin has described other compounds which may be more important in the initiation and maintenance of ciliary activity than is acetyl- choline. I was very happy to hear the presentation of Dr. Rhodin and to see his beautiful electron microscopic pictures showing neuroidal structures in the intermediate areas between two ciliated cells, indicating that there may be neural control of ciliary motion and synchronization in the human respiratory tract. Certain local anesthetics, lido- caine and procaine, disrupted the synchronism of metachronal wave activity of human respiratory epithelium, whereas others, such as tetracaine and dibucaine, never did at any concentration used. They accelerated and subsequently decelerated and stopped activity, but they never disrupted or dis- turbed the synchronic activity of cilia. In contrast, after lidocaine, areas of increased activity of the ciliated epithelium beat againsteach other and did not permit sYnchronic activity; ciliated clusters stopped rotating, and only after washing out the lidocaine was activity rcinitiated. Dr. Tyrrell: How do you get pure and active cilia, particularly from mammalian cells? Dr. Brokaw: This has not been done in mam- malian cells so far as I know, but fairly pure frog cilia have been prepared. Also, isolation and puri- fication of cilia from Tetrahymena and the flagella from Polytoma have been accomplished. Tricks are required with Tetrahymena cilia to keep the cilia clean and free of materials from broken-up cells. Dr. Kotin: Has particle clearance been studied in patients with carcinoid? How do you demon- strate 5-hydroxytryptamine (5-HT)? Dr. Gosselin: There have been no studies in patients with carcinoid. 5-HT is identified by photofluorometry, by paper chromatography, and by bioassay. Dr. Aiello: In what preparations, other than the gills of Mytilus, have 5-HT and tryptophan been demonstrated? Dr. Gosselin: Dr. Krueger has demonstrated 5- HT in the mucous membrane of the rodent trachea by photofluorometry. Dr. Laurenzi: Some years back we undertook the technically difficult task of reversing a segment of the bronchial tree on one side of a dog. The mortality was high, but in the three survivors early after operation the cilia beat as they had before, backward only in the reversed segment. How- ever, after six weeks all cilia on the operated side heat caudad or distalh-, opposite to the normal cephalad beating cilia of the intact side. This sug- gested that a ciliary pacemaker was located in the mainstem bronchus of the dog. 60 I ~n 11c11 I ir L. 00097417
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lcholine ex- maintaining compounds .e initiation in is acetyl- i-ntation of il electron ' structures iliated cells, control of the human ietics, lido- :'ironism of respiratory {acaine and ~tion used. ;erated and sted or dis- ln. contrast, F ity of the ner and did i d clusters rig out the and active t 1~ in mam- f pure frog i and puri.- Ilie flagella i.~d. Tricks F keep the ibroken-up en studied bu demon- studies in ~ntified by ~aphy, and s than the phan been istrated 5- mt trachea undertook a segment dog. The ivors earlv ad before, nt How- ,rated side ,e normal This sug- .ted in the MEASUREMENT OF CILIARY ACTION TORE DALHAMN, Moderator CULTURAL METHODS FOR 1VfEASURING CILIARY ACTIVITY'• ' J. J. BALLENGER, H. B. HARDING, F. W. DAWSON', M. G. DERUYTER, AND J. A. MOORE ~econd session of this symposium is de- i t o the measurement of respiratory ciliary and my particular task is to describe ;uethods of tissue culture that may be ap- ,i i o this problem. The significance of re- ~=depends upon the method and accuracy of >_ircments. These are the two fundamental ,T~ related to significance of results ob- :,r"d under any experimental condition. The irai methods to be described during this have their particular advantages, it i; hoped that as we discuss them to- i,r the, ~:e advantages will become clear. i:e actual growth in culture of ciliated cells has been accomplished many '1'l is fact is only indirectly related to at least as far as the mammalian i:iut is concerned. If the vitamin A con- i, il~ not carefully controlled, nonciliated, de- ,-renriated cells are produced. The growth we all know, can be recorded by photography, and some idea of the ~,, of cessation of ciliary activity can be ob- n(rl. This is only a qualitative measure. Drs. ~'rn nnd Tyrrell, later, will describe a I'.ud of "organ culture" in which human : ,rei epithelium is grown for weeks without -dif'rerentiating. I will be most interested in i iuy how these workers measure ciliary ri f}' per ,?e. TIIE ROTATING CILIATED EXPLANT I«•oul.d like to devote the major portion of '1V rime to a description of the rotating cili- .i-1 explant method (REM) and its use in !r laboratory (1, 2) to measure ciliary activ- ' Prom the Divisions of Otolaryngology and ~liornhiology, Evanston Hospital Association, 1.%,,lnston, Illinois; and the Departments of 1~tnl;lr.-n~mlogy and Microbiology, Northwestern t'nic-er ity Medical School, Chicago, Illinois. -'Zupported by grants from the Illinois Division, \,norwan Cancer Society, and from the Division ,If Air Pollution, Bureau of States Services, U. S. 1'ilhlir Health S-rvi.ce (Grant No. AP 00321). ity in vitro, as well as the use of the stroboscope for both in vivo and in vitro specimens. The earliest observations of the rotating ciliated explants in a plasma clot were made by Drs. Pomerat, Rose, Corssen, and Allen (3). Any respiratory surface possessing ciliated epi- thelial cells may be the source of the specimen -the trachea, the bronchi, nasal mucosa, or ex- cised adenoids. It has been the preponderant practice of our group to obtain our specimens from children at the time of tonsillectomy. The patients, under general anesthesia, are mo- mentarily paralyzed with succinylcholine chlo- ride prior to the insertion of a laryngeal airway. After exposing the larynx by direct laryngos- copy, a cup forceps is inserted, under direct vision, between the vocal cords. This apparatus then is gently scraped against the tracheal wall from below upward for 5 to 6 cm., and the contents are washed off by immersing the blade in a vial of Gey's or Earl's balanced salt solu- tion (BSS). This procedure is repeated five or six times. Each of the scrapings is done lightly, so only the ciliated epithelial cells are removed and not the underlying mucosal cells. If blood is drawn, the scraping has been too deep. A similar specimen can be obtained from the monkey. With small animals such as rab- bits or guinea pigs it is more feasible to sacri- fice the animal with an overdose of intravenous sodium pentothal and then scrape the ciliated surface through the longitudinally split trachea. In any case, once the specimen has been placed in BSS, the cell suspension may be stored for 12 to 2-1 hours without harm. Our practice has been to prepare explants on a Maximow chamber within an hour or so. Briefly, the explants are prepared as fol- lows: Small pieces of the ciliated tissue are removed from the BSS with a Pasteur pipette and placed onto a coverslip, 43 by 50 mm., containing a drop of chicken plasma.' A clot is ' Difco Laboratories, Detroit, Michigan. 61
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62 BALLENGER, HARDING, DAWSON, DE RUYTER, AND MOORE Frc. 1. Rose chamber. formed by the addition of a drop of 50 per cent chicken embryo estract.` A drop of BSS placed in the «•e1l of the slide ensures a hu- mid environment. The coverslip is then in- verted onto a Maximow slide so that the clot hangs into the well of the slide. After sealing the edges of the coverslip with petrolatum, beeswax, and paraflin (3:1:1), ciliary beatinE~ may con- tinue for as long as a week. The explants are examined periodically at 100 times magnifica- tion for the presence of "turners." These are cell aggregates which assume a spherical or near spherical shape. A localized liquefaction of the clot occurs so that the beating cilia propel the aggregate of cells around a constant axis. 1Can- dering of the esplant does not occur if the clot is firm, although bits of debris occasionally in- terfere with rotation. If undisturbed, rotation ma, N- continue for four to five days (oceasionally longer), thus allowing in vitro experiments dtu'ing this length of time. Only explants of less than 0.2 mm. in ; reatest diameter are used. If the explants arc larger than this, the effects of mass and friction add complicatin_ factors. Once rotation has been noted, the coverflip ' Flow Laboratories, Rockville. Mar.-land. and its clot are transferred to a sterile Rose chamber. This is shown in figure 1. As some of you may not be familiar with it, I would like brieflv to describe the Rose chamber. You can see from the figure that it consists of aluminum top and bottom frames, with open centers, held together by four screws. On the inner surface of each frame lies a coverslip. The coverslips are separated by a rubber dia- phra,m. The Rose chamber provides an air- and water-tight space, with a volume of ap- proximately 2.5 ml., through Which photography or microscopic observations are readily per- formed. It avoids the problem of maintain- ing constant humidity, and the external en- vironmental temperature is easily controlled. The greatest advantage of the Rose chamber is the rubber diaphragm. Through this dia- phrarm, inlet and outlet Vacutainer needles ma, v be inserted to allow perfusion with gaseous or liquid media, as indicated in figure ''. The sterile `?1-ganige inlet and outlet needles are in- serted just within the chamber so that if a line were drawn between the points it would bisect the clot. The inlet needle is connected to the perfusate reservoir by means of a 0009"7419
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~.:ose ,)me wdd L r ou I of pen the ,lip. dla- lair- ap- ~phy 'er- k en- tiled. ~r is idia- : lles [otts Che in- i a ''uld ted a i CULTURAL METHODS Fc. 2. Perfusion apparatus. I sterile polyethYlene intravenous catheter. The chamber is then ready for perfusion, using either liquid or gaseous media. In all e.peri- ments using a liquid perfusate, the lower level of the perfusate is placed 4 cm. above die Rose chamber so as to provide for a more constant rate of flow of perfusate. The per- fusion 1 ime for 4 ml. of liquid is approsi- mately ten minutes. Our observations have been made at 100 or -t:30 times magnification, usin, either phase- contrast or bright-field illumination. When liq- 1iirl perfusates were used, the chamber was first 63 filled witli BSS to estnblish a base line. This hase line was obtained by ascertaining thc time require l, using a stop watch, for the explant to complete ten rotations. Four milliliters of the knuwn test solution were then perfused, and a cnuipari<on to the base line n-as made over a iour- to thirty-hour peried, by stop-watch tim- in ; at three- to five-minute intervals. If the per- fusate was a gaseous me 1ium, the base line was obtained from the rotation within the clot alone. Humidity was maintained by a drop of BSS also placed in the chamber. Speeds of perfusion and quantity of perfusate were standardized as
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64 BALLENGER, HARDING, DAWSON, DE RUYTER, AND \fOORE when using liquid perfusates. In each case the explant served as its own control. Statistically, the P value has been found to be less than 0.175 for repetitive experiments utilizing the rotating explant method of meas- uring ciliary activity under base-line conditions. So far as we know, with the exception of Dalhamn's studies (mentioned below), the other methods have not been evaluated, and a direct comparison is therefore not possible. Apparently the P value does not give the com- plete picture of the reliability of the method. Repeatedly, the rotation of control explants has been followed after perfusing with the vehicle solution (BSS alone), and no significant departure from the base line has been seen over a five-hour period. The actual rotation represents the coordi- nated metachronal activity of the cilia. De- creased rotation may be caused by failure of the frequency of the beat, a decreased ex- cursion, a decreased efficiency of each ciliary cycle, or a loss of the metachronal rhythm. With high-speed photography, it is hoped that some of these problems will be elucidated. The use of the stroboscope may be of even more help here. Apart from the statistical validity and re- liability of the rotating ciliated explant method (REM), we believe this method has several unique advantages: (1) Temperature and hu- midity are easily controlled in contrast to methods employing an open trachea or an ex- cised membrane in air. (2) There is complete avoidance of an indicator which might inter- act with the cilia, either by reason of weight or its chemical nature as in the particle transport method. (3) It can be considered, if you will, a "separate epithelial organ" which is perform- ing work, namely, the rotation of cellular ag- gregates. It is an "organ" which is devoid of muscles, a vascular system, and a nervous sys- tem. (4) The rotational activity may be said to reflect the function of an integral part (an extension of the cell membrane) of ivolated epithelial cells. (5) Ability of the cilia to per- form this work may be measured quantita- tively. This technique has been employed in our laboratory to determine the deleterious effects of oxidized and reduced nicotine on ciliarv ac- tivity (4). Present investigations involve, first, perfusing the rotating explant with the GL strain of influenza B virus, and, later, perfusing oxidized nicotine after 12, 18, and 24 hours of incubation at 37°C. In doing this, we have been able to observe the viral potentia- tion of the deleterious effects of nicotine. We anticipate employing other m~~~oviruses to see whether this is a common property they pos- sess. We are also sensitizing monkeys to agamma globulinemic horse serum (AGHS) and now plan to prepare rotating explants for challenge with AGHS in a search for an immediate or delayed (24- to 48-hour) allergic reaction on the cilia. Similar experiments could be per- formed by challenging a tuberculin-sensitive guinea pig with Old Tuberculin. The result should prove to be interesting. These experi- ments are mentioned to illustrate the relativelv long-term studies which are possible because of the rotating explant method. It should be made clear that, following the initial exposure, the same explant, at times, may be exposed a sec- ond time 12 to 24 hours later. Laurenzi (5) has reported the decreased clearance of staphylo- cocci from the lungs of mice which had been sub- jected to prior exposure to smoke. It is possible that observations of rotating ciliated explants could be utilized to perform a study similar to that of Dr. Laurenzi. Another field in which the REM might be employed is biochemistry. The addition of the coenzyme nicotinamide-adenine dinucleotide (NAD), formerly known as DPN, increased ciliary activity in our laboratory approxi- mately 30 per cent. It is believed that this is an original observation. Apparently the NAD provided an ideal oxidation-reduction poten- tial which produced additional energy, thereby increasing ciliary activity. Gosselin and associ- ates (6) reported that 5-hydroxytryptophan (5-HTP) increased ciliary activity in the gill plates of Mytilus and Modiolus in vivo, but we have been unable to reproduce their re- sults using our in vitro svstem. This may be due to the blocking or lack of an enzyme sys- tem on which the conversion oi 5-HTP is de- pendent. This hypothesis was supported by the fact that in our laboratory, 5-h%•dro.vtrypta- mine, the precursor of 5-HTP, also failed to increase ciliary activity in vitro. The principal disadvantages o,i the RE3I are: (1) It is an in vitro rather than in vivo method, as mentioned earlier. (:.') The mam- I ,_: , 00097421
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CULTURAL METHODS 65 later, d 24 s, we ~ntia- t . We ~ o see pos- ;tmma i now I llenge ,te or K,n on per- sitive : esult xperi- tively tivelv hise of made r•, the ,i sec- o ) has 1 'hylo- ti sub- , -sible nlants ar to tht be r,f the eotide eased proxi- his is NAD oten- ereby ssoci- phan e gill but ir re- ~ay be " sys- s de- I:--the ypta- led to ! are: rivo mam- malian blanket of mucus is absent, so that extrapolation to the mucus transport cleans- ing mechanism found in the intact animal must be made. (3) Unfortunately, a most disturbing species specificity exists. Only man and mon- key, in our experience, provide rotating cili- ated explants. The yield from other animals is too scant to be practical. It is obvious that if an animal source could be found to provide ro- tating explants, the usefulness of the method would be greatly enlarged. THE STROBOSCOPE The use of the stroboscope, especially when synchronized with the motion picture camera, provides many advantages in the measuring of ciliary activity. It is possible by this means to examine not only the frequency and the indi- vidual stroke of the cilium, but also to detect a change in the excursion as differentiated from a change in frequency. For example, I have de- scribed how the rotating explant has ceased to rotate because of the failure of the propulsive force provided by the beating cilia. By means of the stroboscope it should be possible to deter- mine «-hich of the various parameters of ciliary motion is affected: force, amplitude, frequency, or the metachronal wave length and velocity. A-Iuch more valuable information is undoubt- edly forthcoming from this method of meas- uring ciliary activity. The stroboscope is most readily and accu- rately used when the layer of cilia is thin, e.g., in the mussel or clam. Here the individual cilia can be well defined and their cycle ex- amined either in vivo or in vitro under vari- ous experimental conditions. With the mam- malian explant, however, the layer of cilia is thick and the use of the stroboscope is much less successful. So many cilia in a vertical (line of vision) direction are visible that the resulting confusion makes accurate counting difficult.. Repeatedly, we have stroboscopically examined human explants in which a sli;ht fold brings the cilia into "profile," but we could not accuratelv count individual cilia. The stroboscope is most easily used if the flash rate is greater than 10 to 15 per second. Below this range there is a verv hothersome psychologic "after image," although this can be alleviated to some extent bv reducinr the intensity of the illumination. In the human ex- plant, the frequencies observed fall into this difficult 10 to 15 per second range. A factor that detracts from the usefulness of the stroboscope is the time difference between the active and recovery strokes of the cilium. In the mammal, the former is to the latter as 1:3. Thus, if you can succeed in "stopping" the active stroke by synchronizing the flash fre- quency, you have not done so for the more time-consuming recovery stroke. This is of little importance if only frequency is being re- corded, but of more importance if you wish to note the configuration at various points in the cycle. Dalhamn (7) has used the stroboscope in conjunction with the cinecamera to examine the mammalian trachea through a window, and he reports an accuracy of 42 beats per minute when the frequency is in the range of 1,250 beats per minute. Rather than measuring the frequency of individual cilia themselves, he observed the motion of adjacent refractile par- ticles in the overlying blanket of mucus as viewed from above or perhaps reflection from groups of cilia. Dalhamn concluded that the stroboscope was insufficiently accurate to meas- ure the frequency of the ciliary beat in the rat. He considered that the accuracy was en- hanced if he also took a motion picture and calculated the frequency from the film strip. Kreuger and Smith (S, 9) also used the stroboscope to examine fields of ciliary activity from above downward. They report an ac- curacy of ±50 beats per minute when the ini- tial speed is 900 per minute. The description of their technique is not sufficiently clear to us to comment on the justification of this degree of accuracy. Again it is doubtful that they meas- ured the ciliary beat directh•, but rather the correlated motion of adjacent fluid. The clam and mussel do present the stroboscopically desirable thin layers of cilia. Even here, however, certain difficulties are present that interfere with accuracy and in- terpretation. The most readily usable are laterofrontal gill cilia, which are relatively miich longer than mammalian cilia and in reality are agglutinated into groups. This per- mits increased ease of visualization, but one wonders if the results can be translated di- rectlv to the shorter, individual mammalian cilia. In practice, the lateral gill cilia are used for anal}-sis by stroboscopy.
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66 BALLENGER, HARDING, DAWSON, DE RUYTER, AND MOORE Another feature of the gill cilia is their abil- ity under certain circumstances to decrease the frequency and change other features of the ciliary beat. This never occurs, so far as we know, in mammalian respiratory cilia, under normal conditions. Translation of data from an animal in which the nervous connections permit such changes to the mammal in which this does not occur is difficult. The stroboscope does not provide a measure of work done, but rather of one or more param- eters of ciliary activity. The REM does measure work performed. It is disturbing to note, when using the stroboscope to measure mammalian ciliary activity, that adjacent groups of cilia may be beating at significantly different fre- quencies. Thus the observer does not know which to accept as the base line under the exist- ing conditions. Similarly, in the particle trans- port method, adjacent indicator particles may travel at different speeds. The chief advantages of the stroboscope are: (1) Immediate readings can be obtained if a suitable field can be found. (2) There is no problem of species specificity as there is with the REIM; accordingly, mussels, clams, et cetera, can be used for study, and in many cases they can be observed in their natural en- vironment. (3) If proper precautions are taken to ensure humidity, body and ambient heat control, observation can be made via small apertures into the tracheae of intact mam- mals. (4) It might be said that the stroboscope is usable wherever the particle transport method is useful. When using the stroboscope, we keep in mind particularly the following: (1) The stroboscope does not measure the ability of the cilia to perform work, but rather it measures some of the parameters of ciliary movement that enable the cilia to perform this work. (2) The stroboscope usually utilize-, as the object of visualization an adjacent refractile point, at least in mammalian cilia, made to move by the ciliary movement but not the motion of the cilium itself. (3) We have yet to be con- vinced that the stroboscope alone is sufficiently reliable for use with mammalian cilia. REFERENCES (1) Ballenger, J. J.: Experimental effect of ciga- rette smoke on human respiratory cilia, New Eng J Med, 1960, 203, 832. (2) Ballenger, J. J., and Orr, M. F.: Quantitative measurement of human ciliary activity, Ann Otol,1963,72,31. (3) Corssen, H. L., and Allen, C. R.: A comparison of the toxic effects of various local anes- thetic drugs on human ciliated epithelium in vitro, Texas Rep Biol Med, 1958, 16, 194. (4) Ballenger, J. J., Dawson, F. W., DeRuyter, M. G., and Harding, H. B.: Effects of nico- tinc on ciliary activity in zitro, Ann Otol, 1965, ; ;. 303. (5) Laurcnzi, G. A., Guarneri, J. J., Endriga, R. B., and CareY, J. P.: Clearance of bacteria from the lower respiratory tract, Science, 1963, 1,1°, 1572. (6) Goss(,lin, R. E., Moore, K. E., and Milton, A. S.: Physiological control of molluscan gill cilia by 5-hydroxytryptamine, J Gen PII}•siol, 1962, .aG, 277. (7) Dalhamn, T.: Mucous flow and ciliary ac- tivity in the trachea of healthy rats and rats exposed to respiratory irritant gases (SO_ , HCHO), Acta Physiol Scand, 1956, 30 (Supplement 123, p. 1). (8) Kreuger, A. P., and Smith, R. F.: Effect of air ions on isolated rabbit tracheas, Proc Soc Exp Biol Med, 1957, 96, 807. (9) Kreuger, A. P., and Smith, R. F.: The effects of air ions on living mammalian trachea, J Gen Physiol, 1958, .li2, 69. tht ti? ba an ~I: mE eff po tll, to co1 cit th, m feI re; sei ul: ve; mt cil mc tio TI th, fer b:: foI ea- te EcI be i'
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~ovement ork. (2) e object )oint, at Piove bv lotion of be con- liciently ~ of ciga- j Iia, -New I ititative ty, Ann Inparison ~ al anes- i.thelium 18, 194. ~ Ruyter, of nico- in Otol, ; , R. B., bacteria Science, 1lilton, i Aluscan J Gen ,arv ac- ats and ~t gases Scand, ffect of s, Proc e effects ichea, J CURRENT TECHNIQUES TO MEASURE ALTERATIONS IN THE CILIARY ACTIVITY OF INTACT RESPIRATORY EPITHELIUDI' RAGNAR RYLANDER It is aeneraily postulated that ciliostasis in the upper respiratory, tract is of great etiologic vignificance in the development of pulmo- nary disease. Therefore, considerable research has been directed to determine the influence, if any, of various substances on ciliary activity. Many different methods in a variety of experi- mental models have been used to measure the effect of ; ' :ubstances such as tobacco smoke, air pollutant.~_, and pharmacologic compounds. Al- thouPh many- of the basic findings with respect to the effects of such agents agree, considerable controversy remains concerning both the spe- cific results and the conclusions drawn. Fur- thermore, direct comparison between different reports has often been difficult owing to dif- ferences in experimental design. For the same reason the ~igni6cance of many reported ob- servations has often been questionable, partic- ularly with reference to human disease. In light of this, this paper will present a sur- vey of the general principles of the various methods available for the measurement of ciliary- activity. In addition, the most com- monly employed types of epithelial prepara- tion and exposure methods will be outlined. The advantages and disadvantages of each of the above will be discussed with respect to dif- ferent experimental objectives. Finally, on the basis of this analysis, recommendations will be formulated as to the preferred techniques for each experimental aim. As the tissue-culture technique and the clam technique have been de- scribed in a previous paper, these methods will be dealt with only- in the discussion. (iF,tir;R.1L CONSIDERaTIONS At the outset it must be clearly appreciated that current methods for the studv of the activity of the cilia, reported in the literature, involve the observation of two distinctly differ- ent but related physioloaic phenomena. These are ciliarv motilitY and mucus transport. Cili- 'From -he Institute of Hygiene, Karolinska Institutet. the National Instituto of Public Healtli, Stockholm. Sweden; and the Institute of Hagienc, Umea University, Umeai, Sweden. ar}• activity may therefore be estimated di- rectlv through observation of the ciliary beat frequency or indirectly through determination of the mucus transport rate. Very often the essential difference between the two phe- nomena has been ignored. Wrong conclusions have been drawn and the growth of a confusing terminology encouraged. Terminology in this paper will be based upon the intercorrelations between "ciliary motility" and "mucus trans- port," as shown in figure 1. OBSERVATION ]METHODS %llucus Transport Rate The flow or transport of mucus overlying the ciliated epithelium can be studied by mi- croscopic observation of the epithelium ttnder diffuse vertical illumination which falls on the epithelial surface at different angles. The trans- port rate is usually recorded at low magnifica- tion. The time required for a particle to move a certain distance is noted and the rate calcu- lated (e.g., 1, 2, 3). This rate is determined under normal and exposure conditions to dis- cover any variation caused by the test agent. The particles used can be artificial particles such as soot, seeds, or small pieces of metal applied on the epithelial surface. Naturally oc- currinl- particles such as shed cells or small air bubbles can also be used to calculate the transport rate. In certain experiments, barium sulfate or other radiopaque substances have been insufflated in living animals and the transport rate has been followed bv x-rav photonraphy- (4, 5). Inhaled radioactive parti- cles have been followed by tracer methods (6), and insuf$ated dye particles have been directly observed (7, S). Measuring mucus transport by using parti- cles or dye is relatively simple, and the repro- ducibility within a certain experimental design is good. Certain shortcomin.,!s exist. The size and weight of the particle used might influence the transport rate (9). Also, the rate on differ- ent areas of the epithelium examined varies widely (10), and firm criteria for using a spe- G7
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68 RAGNAR RYLANDER CILIARY MOTILITY PHYSICAL PROPERTIES ! I OF MUCUS PHAGOCYTOSIS MUCUS TRANSPORT PULMONARY CLEARANCE Fic. 1. The connection between ciliary motility and mucus transport in pulmonary clearance. cial stream of mucus or observing certain par- ticles while discarding others must be set. If particles are insufflated, only very moder- ate amounts should be used, as the normal cleansing mechanism can be blocked by amounts exceeding 1.5 mg. per lung (11). Ciliary Motility In contrast to the diffuse light source used for the microscopic observation of the mucus flow rate, the estimation of ciliary motility re- quires a parallel light beam. When this is placed so as to fall on the epithelial surface almost along the visual axis, a flickering light reflex can be seen through a microscope. This light reflex is caused by the varying reflection of the incident light from the cilia or from the mucus immediately adjacent to the cilia. At a certain angle during their stroke cycle the cilia reflect the light into the microscope and the area of that group of cilia appears light. A few moments later the cilia have changed their angle-the light falls outside the microscope and the area appears dark. Utilizing these phe- nomena, the frequency of the ciliary beat can be recorded by direct observation, by strobos- copy, photographically, or photoelectrically. In mammalian epithelium the beat frequency is so high (12) that direct observation allows only the registration of great changes in rate of the ciliary beat frequency (13, 14). Therefore, except for observing total arrest of ciliary motility, the direct observation method has severe limitations with regard to quantitative accuracy. The stroboscopic method allows the estima- tion of the beat frequency with greater accu- racy (15). The principle of the method is to ad- just the flickering frequency of a stroboscopic lamp to the flickering of the light reflexes on the ciliated epithelium when observed through a microscope. The beat frequency can be ob- tained immediately, and several measurements can be performed in a short time. Nonetheless, a severe shortcoming of this method, which has been pointed out by Ballenger (16) and Dal- hamn (12), is that the upper limit where the flicker of the stroboscope is estimated to cor- respond to the flicker of the epithelium may vary, and the frequency determination will thus involve a fairly large experimental error. The variation is due to the great number of flickering areas on the epithelium and the vari- ation in flicker frequency between adjacent areas. The photographic method involves the registration of the flickering light reflex on mo- tion picture film, using a high-speed camera. The film is later developed and projected onto a screen at a reduced speed, and the beat frequency can then easily be visualized and counted. The method has been used by several investigators (17, 18), and it has been explored in detail by Dalhamn (12, 19), who used a re- cording rate of 220 frames per second. To an- alyze the various phases of the ciliary stroke, the effect stroke, and the recovery stroke, re- cording rates of 700 frames per second were used. The method allows the estimation of the beat frequency with great accuracy (experi- mental error less than 10 per cent). Practical disadvantages are that the method is rather tedious and necessitates extensive training of technicians. Illumination conditions in the trachea must be very good-even moderate amounts of mucus tend to make registration difficult. A photoelectric method has recently been described (20). Here the flickering light reflex from the epithelium causes voltage variations in a photoelectric cell which is mounted in the picture plane of a microscope camera. The frequency of the voltage variations, which cor- responds to the ciliary beat frequency, is re- corded on an oscilloscope or is counted electron- ically in a rate meter. The method is easy to apply and results are obtained immediately. Its principal drawbacks ari--;e from the ex- treme sensitivity of the apparatus to all kinds of vibrations. Difficulties can thus arise when in vivo preparations are used and the epi- thelium moves as a result of respiratory move- ments. 00097425
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~ ements theless, ich has G I Dal- i re the o cor- :,.i may : n w-ill I error. '.ber of vari- ('jacent ~ the Gn mo- I: mera. Id onto 1, beat Id and r everal tplored ": a re- I'o an- ~troke, ke, re- l were sof the ' xperi- : ctical . ather i ng of ~ the erate ation been reflex tions d in . The cor- is re- tron- sy to i ~=ely. ex- ; inds hen ('pi- ove- MEASUREMENT OF TYPES OF EPITHELIAL PREPARATIONS USED A commonly used ciliated epithelial prep- aration is the frog palate and esophagus in vitro. Experimental models always provide for the high humidity needed, but the epithelium itself may be observed either when exposed to air or when submerged in various solutions (21, 22). The mucus layer may be left undis- turbed or it may be wiped off with different solutions (23). The pretreatment of the epi- thelium with adenosine triphosphate (ATP) has been described by Bernfeld (24). Another in vitro preparation used sub- merged or exposed in air is mammalian trachea (2, 25, 26). The replacement of the natural mucus with an artificial mucus has been de- scribed for this type of epithelium (27). Epi- thelium from adenoids, the nasal sinuses, and the nasal cavity, either extirpated or in situ, has also been used for in vitro investigations (23,28). A special preparation which allows only the registration of the ciliary beat frequency is ob- tained by transverse sectioning of trachea and placing of the tracheal rings in a perfusion chamber (29, 30). The cilia can then readily be observed and the beat frequency calculated. For in vivo preparations, the epithelium is observed in situ on anesthetized animals; ade- quate humidity is obtained by replacing natural enclosures with transparent membranes (14) or by enclosing the animal in a humid box (12). Observations can also be made through in- tact trachea if transillumination is used (31). Inspection through the nose and mouth of in- tact animals and human subjects has also been carried out (8, 32). ExPOSURE METHODS When in vitro preparations are used, the epithelium to be studied can either be sub- merged or kept in the air. If the epithelium is submerged, test substances are dissolved or sus- penderl in the bathing solution (33). Exposure of epithelium in air to gaseous test substances has been carried out by introducing the test substance into the humid chamber (3). Test gaSes may also be passed along the surface at speeds equivalent to those present CILIARY ACTIVITY 69 in the trachea or impinged, sprayed, or ap- plied in solution onto the epithelial surface (2, 28, 34-37). Solid test substances may be placed on the epithelial surface unchanged or dissolved, or they may be suspended in some neutral solvent such as Locke's or Ringer's solution. In vivo exposures have been performed by allowing the animal to inhale the test sub- stance either as a gas (38) or in an aerosol form (30, 39). Test solutions may also be applied on the epithelial surface in vivo (14), or they may be introduced into the body by oral administra- lion or injection (40). DISCUSSION It is apparent from the above survey that a great variety of methods exists to measure the activity of cilia. The correlation between cili- ary motility, properties of mucus, and mucus transport rate (figure 1) must always be borne in mind when one is choosing which observa- tion method and which type of epithelial prep- aration to use in a particular experimental situation. As variations in temperature and humidity alter the frequency of the ciliary beat and the mucus transport rate (12), these conditions must be carefully controlled. It is well known that cilia may beat at an unaltered rate although the mucus transport has come to a complete stop (1, 12). This phenomenon has been noticed both with ab- normally viscous and abnormally watery mu- cus. Whether arrest of the ciliary beat always causes the mucus flow to stop remains contro- versial even though observations on trachea in which certain areas are devoid of cilia appear to support this theory. The cilia themselves are extremely resistant structures, and, except for the effect of drying and temperature changes, they are found to beat at an unaltered rate even at fairly high exposure levels. Mucus se- cretion, on the other hand, is a very sensitive phenomenon. Even a faint touch on the epi- thelitmi or on the outside of the trachea can cause a massive flow of mucus on the epi- thelium. Changes in viscosity occur rapidly and might probably occur even in mucus already secreted. Experimentation with ciliated epi- thelium thus means the utilization of a very sen-~itive and complex tissue, and observed ef- 4
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70 RAGNAR RYLANDER fects can be the result of one or of several primary reactions which themselves may go un- detected. If the effect of a substance on the ciliarv motility per se is to be studied, itt is desirable to avoid trsing techniques in which the mucus layer mightt interfere. Therefore, methods using cilia in tiSsue culture or the clam technique are most suitable for this kind of investigation. An inhibitorv effect on the cilia in a tissue culture can probably be looked upon as an in- dex of cy'tototicity, and this property of vari- ous substances can be tested and compared us- ing the tissue culture method. This method is also suitable for investigating the biochemical mechanism behind the ciliary beat. When substances are to be tested for their effect on the activity of the cilia in the respira- tory system and the clinical significance of the effect is important, intact epithelium with a normally functioning mucus layer should be used. The removal of mucus from the epi- thelium increases the accuracy of the method for estimating effects on the ciliary motility, but it probably decreases the pulmonary sig- nificance of a recorded effect. The same could be true if artificial mucus were added or if the epithelium were pretreated with pharmaceutic preparations. In addition, it has been shown by Lierle (-11), among others, that even if the type of effect is the same in preparations with and without mucus, the dose required for ef- fect is very different. The removal of the mucus layer from the preparations also increases the particle transport rate considerably (25). As the clinical significance of an effect on the ciliary frequency is difficult to evaluate without a simultaneous measurement of the mucus transport rate of a normally function- ing mucus layer, preparations in vivo should be preferred. To limit the experimental error, it might be necessary to control the hydration status of the animal. Under certain circumstances, e.g., when a large number of substances are to be tested, the method using in vivo preparations may be considered too expensive and too "tissue-con- suming." Ln such cases screening can be per- formed on in vitro preparations. That the type of response to several substances is the same, whether animal epithelium in vivo or in vitro is used. has been shown, for example, by Lierle (42). On the other hand, it is important to realize that owing to alterations in characteristics of the mucus or changes in ciliary metabolism, epithelium in vitro is suitable only for screen- ing. Changes in the amount of mucus secreted might occur after death (43), and the protec- tive properties of mucus in vitro probably differ from those present under in vivo conditions. Furthermore, differences in dosages required to produce a certain effect have been observed between in vitro and in vivo preparations (14), and Cralley (35) has shown that cilia become increasingly sensitive with time following the death of the laboratory animal. It has been reported (34, 37) that the reac- tions of frog epithelium and mammalian epi- thelium in vitro are very similar. Frog epi- thelium would appear to be preferable for screening because it can be kept at room tem- perature whereas mammalian tissue should be kept at body temperature. Another reason for preferring the frog epithelium is that the ex- cretion of mucus on mammalian epithelium undergoes changes after the death of the animal, and the transport rate may come to a full stop even when no exposure is carried out (12). To obtain the full significance of an effect on ciliary activity in the respiratory epithelium, it is necessary that the exposure conditions be as realistic as possible. Effects should be tested through inhalation in the animal. Breathing through the mouth should be preferred, espe- cially for gaseous substances, of which the ab- sorption capacity of the nose, at least in rab- bits, is very great (38). It must be emphasized that the application of a test substance either by itself or as a solu- tion directly on the epithelium is an exposure procedure which can cause profound changes in the preparation studied (44). Moreover, it is a type of exposure that is seldom present under realistic conditions. Nasal sprays and similar ex- posures produce an "epithelial load" of a con- siderablv smaller magnitude. If, however, the method ,)pplying test substances in a solution is used when mucus transport is studied, the epi- thelium might be covered with some neutral fluid to prevent the effects of countercurrents (45). Letting aerosols into a chamber for vary- ing lenl-ths of time might not provide adequate air turbulence for the particles to impinge on the epithelial ~urface, and, if ;aseous sub- ,tances are pre~ent simultaneously, difficulties may occur when the results are evaluated. 00097427
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lo realize ristics of tabolism, F screen- secreted protec- ,ly differ nditions. juired to Observed ins (14), become i ing the _Ie reac- lian epi- Irog epi- able for ;,m tem- iould be :son for the ex- i thelium animal, .1111 stop (12). ,ffect on ,lium, it ,s be as ^ tested i eathing a, espe- j the ab- in rab- ~lication j a solu- ~ tposure nges in it is a t under Iilar ex- a con- rer, the irtion is ae epi- neutral !urrents , r vary- 1equate inge on is sttb- iiculties :~d. MEASUREMENT OF CILIARY ACTIVITY 71 TABLE 1 PROPOSED TESTS FOR MEASURING THE EFFECT OF VARIOUS KINDS OF Si:BSTAICES ON CILI aRY ACTIVITY Screening Confi rma tion \ otes Physiologic Tissue culture substances Photoelectric or photographic - method in vivo with simultane- - Lung-damaging Frog ous recording of transport rate Mouth inhalation substances Cellular effects Frog, clam Tissue culture A summary of suitable methods for studying effects on ciliary activity is given in table 1, which is based upon the discussion above. Un- der certain circumstances, one of the techni- cally easier methods which has not been rec- ommended in this table might be suitable for a special kind of experiment. However, the use of such "short cuts" should always be based upon a thorough analysis of the significance of the ef- fects in view of the interrelationship between ciliary motility and mucus transport as has been discussed here. In conclusion, it is suggested that future studies in this field should be undertaken only with full cognizance of the points which have been raised in this review. For effective prog- ress, it must be appreciated that, although the older and more primitive techniques may still yield valuable information, in most instances the use of the more refined and sophisticated methods will be required. REFERENCES (1) Lucas, A. M., and Douglas, L. C.: Principles underlying ciliary activity in the respira- tory tract. II. A comparison of nasal clear- ance in man, monkey, and other mammals, Arch Otolarvng (Chicago), 1934, 20, 518. (2) Mendenhall, W. I., and Shreeve, K. E.: The effect of cigarette smoke on the tracheal cilia, J Pharmacol Exp Ther, 1937, 60, 111. (3) Kensler, C. J., and Battista, S. P.: Com- ponents of cigarette smoke with ciliarv-de- pressant activity, New Eng J Med, 1963, 269, 1155. (4) Ernst, A. AL: Einftuss einiger Narkotika auf den Effekt der Bewegungen des Flimmer- epithels in Trachea und Bronchi, Arch Int Pharmacodyn, 1938, oS, 208. (5) van Dongen, K., and Leusing, H.: The action of opium-alkaloids and expectorants on the viliar' y movements in the air passages, Arch Int Pharmacod' vn, 1953, 93, 261. (6) Holma, B.: The intra- and interindividual variations of hmg-clearance of a monodis- Pulmonary significance not proved perse N-octadecanol aerosol in rabbits, Proceedings of the 14th International Con- gress on Occupational Health, Madrid, Spain, 1963. (7) Ornston, D. G.: Office study of cilia, Arch Otolaryng (Chicago), 1956, 44, 19. (8) van Ree, J. H. L., and van Dishoeck, H. A. E.: Some investigations on nasal ciliarv ac- tivity, Prac Otorhinolaryng (Basel), 1962, 2.;, 383. (9) Stewart, W. C.: Weight-carrying capacity and excitability of excised ciliated epithelium, Amer J Physiol, 1948, 152, 1. (10) Viktorow, K.: Uber die Wirkung einiger Arzmittel auf das Flimmerepithel, Acta Otolaryng (Chicago), 1934, 21, 61. (11) La Belle, C. «"., and Brieger, H.: The fate of inhaled particles in the early postexposure period. II. The role of pulmonary phago- cytosis, Arch Environ Health (Chicago), 1960, 1, 423. (12) Dalhamn, T.: ilZucus flow and ciliary ac- tivity in the trachea of healthy rats and rats exposed to respiratory irritant gases (SO2, H:3N, HCHO), Acta Phvsiol Scand, 1956. 36, (Supplement 123, p. 1). (13) Lucas, A. M., and Douglas, L. C.: Principles underlying ciliary activitv in the respira- tory tract. III. Independence of tracheal cilia in vivo of drug and neurogenous stim- uli, Arch Otolaryng (Chicago), 1935, 21, 255. (14) Lierle, D. M., and Moore, P. M.: Further study of the effects of drugs on ciliary ac- ti~•it~~, Ann Otolarvng. 1935, .;{, 671. (15) Kreuger, A. P., and Smith, R. F.: Effects of air ions on isolated rabbit trachea, Proc Soc Exp Biol .lled, 1957. 98. S07. (16) Ballenger, J. J., and Orr, M. F.: Quantitative measurement of human ciliary activity, Ann Otolaryng, 1963, ',2. 31. (17) Lucas, A. '_1I.: Principles underlying ciliary activity in the respiratory tract. I. A method for direct observation of cilia in silis and its application, Arch Otolaryng (Chica,zo), 1933.15, 516. (1S) Frenckner, P.. and Richtner, N. G.: A method for the study and filming of ciliary activity among animals and human beings, Acta Otolar}-ng (Stockholm), 1939, 668.
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72 RAG\AR RYLANDER (19) Dalhamn, T.: The determination in vivo of the rate of ciliary beat in the trachea, Acta Physiol Scand, 1960, 49,242. (20) Rylander, R.: A photo-electric method for estimating the frequency of ciliary beat, Proceedings of the 14th International Con- gress on Occupational Health, Madrid, Spain, 1963, Excerpta Med Int Congress (Ser 62), p. 1426. (21) Umeda, T.: A study on the ciliary move- ment of ox trachea, Acta Derm (Kyoto), 1929, 1,/, 629. (22) Vorhaus, E. F., and Deyrup, I. J.: The effect of adenosine triphosphate on the cilia of the pharyngeal mucosa of the frog, Science, 1953, 118, 553. (23) Lierle, D. M., and Moore, P. M.: Effects of drugs on ciliary activity of mucosa of up- per respiratory tract, Arch Otolaryng (Chi- cago), 1934, 19, 55. (24) Bernfeld, P., Nixon, C. W., and Homburger, F.: Studies on the effect of irritant vapors on ciliary mucus transport, Toxic Appl Pharmacol, 1964, 6,103. (25) Hill, L.: The ciliary movement of the trachea studied in vitro, Lancet, 1928, 215, 802. (26) Ballenger, J. J.: A study of ciliary activity in the respiratory tract of animals, Ann Oto- laryng, 1949, 58: 351. (27) Battista, S. P., DiNunzio, J., and Kensler, C. J.: Versatile apparatus for studying ef- fects of gases, aerosols and drugs on ciliary activitv in nonimmersed mammalian trachea, Fed Proc, 1962 21, 453. (28) Greenwood, G., Pittenger, R. E., Constant, G. A., and Ivy, A. C.: Effect of zephiran chloride, tyrothricin, penicillin and strepto- mycin on ciliary action, Arch Otolaryng (Chicago), 1946, 43, 623. (29) Scudi, J. V., Kimura, E. T., and Reinhard, J. F.: Study of drug action on mammalian ciliated epithelium, J Pharmacol Exp Ther, 1951, 102, 132. (30) Guillerm, R., Badre, R., and Vignon, B.: Effets inhibiteurs de la fumee de tabac sur 1'activite ciliare de 1'epithelium respiratoire et nature des composants responsables, Bull Acad Nat Med, 1961, 20, 416. (31) Goldhamer, R. E., Barnett, B., and Carson, S.: A method for studying mucus flow in the intact nnimal. Food and Drul, _ Research Lab Publ, \ew I'ork, 1964. (32) Ewert, G.: On the mucus flow rate in the human nose, Acta Otolarl•ng, 1965 (Supple- ment 200). (33) Rakieten, N., Rakieten, M. L., Feldman, D., and Boykin, M. J.: Mammalian ciliated respiratory epithelium. Studies with par- ticular reference to effects of menthol, nico- tine and smoke of mentholated and non- mentholated cigarettes, Arch Otolaryng (Chicago), 1952, 56, 494. (34) Tremer, H. M., Falk, H. L., and Kotin, P.: Effect of air pollutants on ciliated mucus- secreting epithelium, J\rat Cancer Inst, 1959, 23, 979. (35) Cralley, L. V.: The effect of irritant gases upon the rate of ciliary activity. J Indust Hyg Toxicol, 1942, ,2i, 193. (36) Boyd, E. M., Clark, J. W., and Perry, W. F.: Estrogens and their effect on ciliated mu- cosa, Arch Otolaryng (Chicago), 1941, 33, 909. (37) Kordik, P., Bulbring, E., and Burn, J. H.: Ciliary movement and acetylcholine, Brit J Pharmacol, 1952, 7, 67. (38) Dalhamn, T., and Strandberg, L.: Acute ef- fects of sulfur dioxide on the rate of ciliary beat in the trachea of rabbit, Int J Air Water Pollut, 1961, .;, 154. (39) Dalhamn, T., and Rylander, R.: Ciliastatic action of smoke from filter-tipped and non- tipped cigarettes, Nature (London), 1964, 201, 401. (40) Krueger, A. P., and Smith, R. F.: The bio- logical mechanisms of air ion action, Gen Physiol, 1960, 43, 533. (41) Lierle, D. M., and Moore, P. M.: Effects of drugs on ciliary activity of mucosa of up- per respiratory tract, Arch Otolaryng (Chi- cago), 1934, 19, 55. (42) Lierle, D. M., and Evers, L. B.: Effects of certain drugs upon ciliary activity of the mucous membrane of the upper respiratory tract, Laryngoscope, 1944. 5-E, 176. (43) Hilding, A. C.: Phagocytosis, mucous flow and ciliary action, Arch Environ Health (Chicago), 1963, 6, 61. (44) Maxwell, S. S.: Effect of salt solutions on ciliary activity, Amer J Physiol, 1905, 13, 154. (45) Hill, ,J. R.: The influence of drugs on ciliary activity, J Physiol, 1957,1,39, 157. }
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1e e- ed '~- i i- g in 3, .`. DISCUSSION Dr. George: Stroboscopic techniques are useful to determine metachronal wave frequency of lat- eral frontal shimmering of a section of clam gill. Usually a general flow of liquid can be seen along the surface. Unfiltered cigarette smoke stops the ciliary activity of the fresh water clam. Smoke from filter cigarettes has temporary effects-the channel in the gill does not swell and recovery occurs. At times the lateral frontal cilia do not move in even a pseudocoordinated fashion, but by what I de- scribe as flick motion. By focusing into the channel and using green light, refraction changes can be seen which are wavelike on opposite sides of the channel and rotate debris. Fluid flow has a sheer field superimposed upon it. The flick state and metachronal frequency help define another state of ciliary activity which can be observed in the clam gill. 5-Hydroxytryptamine (serotonin) is useful to "normalize" the initial frequency of the metachro- nal wave deep in the channel of clam gills. Tissue is mounted in a Rose chamber similar to that de- scribed by Dr. Ballenger. Frequencies of metachro- nal waves are about 1,000 per minute as estimated from the index of refraction under green light. It takes about ten seconds to adjust the dial on the stroboscope, catch the wave, and stop it. Patience is required to get the fundamental frequency rather than a higher harmonic, but the technique is easily learned. Metachronal wave frequency shows tre- mendous scatter in clams obtained weekly and given this pretreatment except during periods of low water. Serotonin in 100 ml. of very pure water dilutes somewhat the physiologic solution in which gill tissue is floated. After a sequence of five control observations, substandard puffs of about 20 ml. of unfiltered cig- arette smoke are applied to a chamber of about 40 ml. A puff remains in the chamber for ten seconds, then is exhausted and replaced by moist air. Meas- urements are made at constant temperature and humidity with the chamber completely closed. The frequency decays rapidly, and below the 200 per minute level the slowing cannot be accurately fol- lowed. Various substances attack the head of the gill filament, which swells, obscuring the wave mo- tion. The serotonin not only regularizes the fre- quency but also opens the channel so waves are readily observable. Approximately 30 lambda of 0.055 molar of pure phenol solution applied to the gill tissue destroys coordination completely. The lateral cilia and the lateral frontal cilia which were initially beating with a pseudocoordinated motion change to random motion. They all beat during ten seconds of ob- servation but without metachronal waves. The ad- dition of more phenol decreases the number of beating cilia. Many substances produce a sig- moidal curve of cessation of ciliary beating. A method of pretreatment of clam gills is needed to standardize the flick state analogous to the seroto- nin effect on metachronal waves. This would per- mit quantitative description of the decay of un- coordinated motion as toxic substances are applied. There are probably many other states of motion in ciliated tissue, and I think it would be most in- teresting if one could identify and describe them quantitatively. One of Dr. Ballenger's nonrotating clusters appeared to have a flick motion. Uncoordi- nated motion would not be expected to produce rotation. The flick motion of cilia is present in tis- sue from near the human adenoid tonsil. It may be possible to obtain clues as to the biochemical re- quirements for motion and provide a basis for translation of effects from one species to another if states of motion are carefully defined. Dr. Dalhamn: I must strongly stress that we are discussing measurements of ciliary action and not the effect of foreign substances on the ciliary ac- tivity. The methods of measurement of ciliary ae- tivity, and perhaps of mucus transport, are to be discussed. 73
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74 DISCUSSION Ciliary Activity in Mucus-Free and Mucus-Covered Tissues' Dr. Bernfeld: It is a well known fact that the ciliated epithe- lium in most animals is covered by a mucus layer, and that measurement of ciliary activity in such tissues is markedly influenced by the thickness and the viscosity of this mucus coat. In order to define more precisely the interference of the mucus layer with measurements of ciliary activity, it appears interesting to compare results obtained in a mucus- free system with those of a mucus-covered system. Such a comparison can best be carried out when three experimental conditions are fulfilled, namely: 1. An analogous method of measurement of cili- ary activity must be used in both systems. 2. The measurements in both systems must be carried out under identical conditions of exposure. 3. The study should include the effects of both ciliostatic and cilia-promoting agents. Experiments performed in our laboratory permit such a comparison. Frog esophageal tissues (Rana pipiens) served as the mucus-covered ciliated epi- thelium, and the gill of the fresh-water clam (Unio) as the mucus-free ciliated tissue. A simple, accu- rate, and reproducible technique of measuring cili- ary activity was applied to both kinds of tissues, namely, that recently described for frog esophageal preparations' The simple apparatus used to expose the ciliated tissue to cigarette smoke or to gases is seen in fig- ure 1. The tissue preparations (usually two at a time) are pinned to a paraffin layer inside a plastic dish which is placed in a bell-shaped glass chamber of 300 ml. capacity. Smoke is introduced into the exposure chamber from a cigarette by means of negative pressure generated with a water buret, and gases are injected through a syringe. The rhythm of exposure follows a schedule of smoke or gas in- halation, retention, exhalation, and aeration, closely imitating that of human smoking. Ciliary activity is visualized by observing under a dissecting micro- scope the rate of progress of a marker, namely, carbon particles deposited as a suspension on the surface of the tissue preparation, under a depth of 2 to 3 mm. of aqueous liquid (frog Ringer's solu- tion, or, in the case of clam gills, spring water). In the frog preparations, the carbon particles mi- grate with the mucus layer down the esophagus. In the case of the clam tissue, the whole gill is pinned to the paraffin layer, and the carbon particles mi- grate in a downward direction on the inner gills (figure 2) and in an upward direction on the some- what smaller outer gills. This seems surprising since the direction of water flow in the intact clam is 'Prepared by P. Bernfeld, T. F. Kelley, and F. Homburger, Bio-Research Consultants, Inc., Cam- hridge, 3lassachusetts. Supported b.• P. Lorillard Co.. New York. \ew York. z Bernfeld, P., ct al.: Toxic Appl Pharmacol, 1964, 6, 103. known to be at a 90° angle to the flow observed on the excised gills. That the transport of carbon par- ticles is actually due to a water flow down the width of the gill can be demonstrated when a neu- tral food dye is layered over the gill as shown in figure 3. The lower edge of the gill is visible in the center of the slide. The dye was applied at the other end of the gill, and pictures of the progress of dye front were taken on the same photographic plate every ten seconds. In the case of the frog esophageal tissue, we thus observe a mucus flow (table 1), and in the clam gill, a water flow. The effects measured in both in- stances are flow rates visualized by the progress of the carbon particles and expressed in millimeters per second. The presence of ciliostatic substances reduces the flow rates, the effects of which are ex- pressed as the ratio of flow rate after exposure (V,) to flow rate before exposure (Vo). All following data will be expressed in terms of Vl/Vo. To com- pare the ciliostatic potencies of different sub- stances with one another, the terms of the recipro- cal of the Vi/Vo ratio minus one are calculated (Va/V1 - 1). It should be noted that the clam gill lends itself also to exposure of ciliostatic substances in aque- ous solution and, in contrast to the frog tissue, can thus be employed either in a liquid or in a gas phase. When a comparison was made between different gills of the same clam (table 2), it was found that, with smoke from any type of cigarette as the cilio- static agent, the inner gill always gave a slightly but significantly smaller Vi/Vo ratio than the outer gill of the same side, i.e., that the inner gill is slightly more sensitive to cigarette smoke than is Fw. 1. Apparatus used to expose ciliated tissue to cigarette smoke or gases. (Reprinted from Toxi- cology and Applied Pharmacology, 196h, 6, 165) 00097431
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visceral ` i~ mantle ~ ganglion ~ V q~m, intesfina mantb f °°t shell cuixF go~ad branchW A ~~ ...~...wr Pic. 2 L,ngitudinal and transversal diagrammatic cross sections of the fresh-water clam, cxliihitinL r_he t,ositions of the gills. (Reprinted from Storer, I. I, and Usinger, R. L.: General Zoo(ogi,,. ; p. 355, McGraw-Hill, 1'ew York, 1957.) e Fia. 3. A neutral food dye layered over the gill demonstrates that the transfer of carbon particles is due to a water flow down the width of the gill. 75
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76 DISCUSSION TABLE 1 CILIARY a4CTIVITIES I-I Two SYSTEMS Ciliated System Frog Esophagus Clam Gill Operative phase Gas Gas or liquid Phenomenon observed Mucus flow Water flow Effect measured Flow rate:* A' = d/t Flow rate:* V = d/t Effect of added substances Ratioa V,/Vo Ratio:t Y1 No Ciliostatic potency Vo/Vt - 1 1'o/vt - 1 * Distance (d) in millimeters; time (t) in seconds. j Flow rate after exposure (V1) to flow rate before exposure (Vo). TABLE 2 COMPARISON BETWEEN DIFFERENT GILLS OF THE SAME CLAM V r/Vo for First Gill Vi/Vo for Second Gill Inner Gill* Inner Gill Outer Gil1• Inner Gillt Outer Gill Outer Gillt Number of pairs studied 23 10 8 Number of pairs in which Vt/Vo of inner gill is smaller than Vt/ti o of outer gill 23 Mean value 0.873 0.926 0.922 Lowest value$ 0.678 0.825 0.765 Highest value§ 0.985 0.993 1.000 * From same side of clam. t Left to right side of clam; large value was placed in denominator. $ Largest discrepancy between two gills of the same clam. § Smallest discrepancy between two gills of the same clam. 0 w tn \ E E z , o $ s o p 1 ........... ~ ~ ~ I 0 ~ ; 0.00039 0.00156 0.00625 0.025 0.1 0.00019 0.00078 0.003125 0.0125 0.05 3~ 2 ATP CONCENTRATION (%) FIG. 4. Relationship between mucus flow rate on frog esophageal tissue (ordinate) and ATP concentration of the medium (abscissa). Each circle represents the measurement of one individual frog tissue preparation. The horizontal bars are the mean values. (Reprinted from Toxicology and Applied Pharmacology, 1964, 6, 106) 00097433
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DISCUSSION TABLE 3 THE EFFECTS OF SBfOKE FRO-m FILTER AND NONFILTER CIGARETTES O\ ATUCUS-COVERED AND MUCUS-FREE CILIATED EPITIIELIU3i - 77 Vi/Vo Mean Va]ues' Air Control Nonfilter Cigarette Brand At Filter Cigarette Brand B± t-Valuej Frog esophagus 0.668 0.171 (20) 0.285 (20) 4.44 Clam gill 0.961 0.455 (6) 0.565 (18) 3.67 * Number of individual observations in parentheses. t Over-all length, 85 mm. $ Statistical significance of the difference between the two brands of cigarettes. TABLE 4 EFFECT OF PHENOL VAPORS ON CILIARY ACTIVITY, IN THE ABSENCE OR PRESENCE OF CIGARETTE SMOKE Vi/Vo Mean Values' Air Phenolt Smokej Smoke: and Phenolt t-Value Frog esophagus 0.694 (16) 0.638 (12) 2.06 Frog esophagus 0.383 (30) 0.283 (30) 4.19 Clam gill 0.962 (8) 0.913 (2) Clam gill 0.627 (8) 0.571 (12) 2.29 *'_Vumber of individual observations in parentheses. t 115 y of phenol per frog tissue; 102 y of phenol per clam gill. $ From a filter cigarette. TABLE 5 CILIARY MUCUS TRANSPORT IN FROG 1LSOPHAGIIS PREPARATIONS: EFFECTS OF HIGH PHENOL CONCENTRATIONS AND OF PIIENOL TOGETHER WITH I NFILTERED SMOKE Vr/Vu Mean Values* Smoke with Smoke 115 y phenolt 230 y phenolt t-Value Filter cigarette 0.365 (16) 0.272 (16) 3.35 F ilter cigarette 0.272 (16) 0.279 (16) 0.275 Nonfilter cigarette 0.223 (16) 0.194 (16) 1.10 *'-Vumber of individual observations in parentheses, t Per frog tissue. the outer one. There is also a much smaller dis- crepancy between the V,/N'o ratios of the two inner gills of the same clam than between inner and outer gills of the same side. The two outer gills are equally much less different from one another than each one would be from the inner gills. It thus becomes apparent that the accuracy of the clam gill technique can be considerably increased when only one kind of the two gills of each clam is used for the measurements, especially the more sensitive inner gills. All further data reported here were obtained in this way. The comparison of the two systems of ciliated tissue was started with the effects of a cilia-promot- ing substance, adenosine triphosphate (ATP). It had been observed earlier that the mucus flow on irog esophageal tissue preparations was slow, fre- quently completely arrested, and unreproducible. Addition of ATP resulted in a drastic improvement of this situation (figure 4). A definite cilia-promot- ing activity was seen at a concentration of 0.00039 per cent ATP, and an optimal effect was reached at a concentration of about 0.00625 per cent. Once the mucus flow was stimulated by ATP, mucus flow rates were found to remain constant at the high level for periods up to 24 hours. In contrast to the effect of ATP on frog esophageal mucus flow, this ,substance was found to exert no influence Nehatso- ever on the ciliary activity of tlle clam gill; it neither stimulates ciliary activity in freshly excised
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78 DISCUSSION gills, nor restores ciliary activity after partial or complete inhibition of gills by cigarette smoke. While the number of cilia-promoting substances is limited, there are a great many ciliostatic agents. As an example, the effects of smoke from filter and nonfilter cigarettes on mucus-covered and mucus-free ciliated epithelium have been com- pared (table 3). In both systems there is marked ciliostasis by both types of smoke, and in both systems the effect of smoke from the nonfilter cigarette is significantly more toxic than that of smoke from the filter cigarette. When the differ- ences of the effects of smoke from filter and non- filter cigarettes are calculated in terms of cilio- static potencies, it can be seen that both systems react even quantitatively in a very similar fashion. This example represents only one of many which demonstrate the similarity of the response of frog and clam tissues to various types of cigarette smoke. Phenol is another ciliostatic substance. Phenol vapors cause only a small decrease of the mucus flow rate in frog esophageal tissue (table 4), and this decrease is not statistically significant. Phenol vapors have an even smaller effect on the ciliary activity of the clam gill. However, when frog or clam tissue was exposed to phenol vapors in the presence of cigarette smoke from a filter cigarette, the effect of phenol in both systems was amplified by that of the smoke, probably through inter- action of phenol with a component of the smoke. It should be noted that the amplification by smoke of the effects of phenol on ciliary activity pertain only to smoke from filter cigarettes and not to smoke from nonfilter cigarettes (table 5). This may be explained by the finding that, when the amount of phenol is increased, no additional ciliostatic effect is noted. It is probable, therefore. that the relatively large quantities of phenol known to occur in the smoke of nonfilter cigarettes invalidate the effect of added phenol. In interpreting the differences between the two systems of ciliated tissues, we can, of course, not claim that the response of the mucus-covered ciliated tissue to ATP is due to the presence of the mucus layer; on the other hand, we cannot reject such a hypothesis on the basis of our data. It is obvious that a striking similarity exists be- tween the response of mucus-covered and mucus- free tissues toward various ciliostatic agents as well as between their response toward the com- bined action of several agents, as illustrated by the effects of phenol without and with cigarette smoke. It appears likely, therefore, that these effects are actually due to inhibition of the ciliary activity, in the mucus-covered as well as in the mucus-free tissue, rather than to changes in amount or viscosity of the mucus layer. i t t
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DISCUSSION L rSearenacnt of Ciliary Activity in the I~itact Trachea'•' iinitiuc•s for studying the effects of atmos- ,. ilollutants. cigarette smoke, and other en- :mu0ntal fhctors on ciliarv activity have utilized V ri — ues from lower organisms, mamma- ;< <~lls. or tissue in vitro, or exposed ciliated ,,hranes in vivo. Although these procedures many advantages because of their simplicity, lo not duplicate the conditions which exist ;i% inv in'tact animals and man, where the mu- _landa and the blood supply must play an ~~~rtaut role in the cilia-mucus clearance mecha- ~n. In order to study ciliary activity under AIR ~ % RH \\,I, ,~/ 4 L ~ 30 0 \\%tII// The method in general is as follows: Radio- active material is injected into the lower trachea through a small incision in the skin. As the injected material moves up the trachea, it passes in se- quence under two detectors encased in a single collimator which is placed over the neck anterior to the site of injection. The signal from each de- tector is fed through a count rate meter to a re- corder (figure 5), and the rate of movement is calculated from the record. The collimator-detector system described in this paper was designed by Lloyd M. Bates, co-author of this paper. It is applicable in chicks and animals with tracheas about 6 cm. in length between the point of injection and the glottis. Larger detectors could not be used because of the shortness of FIG. 5. Measurement of cilia mucus clearance rate in trachea of chicks. more normal physiologic conditions, a radioiso- tope method was developed recently in this lab- oratory. By this method, the rate of movement of droplets and particles by the ciliated mucous membrane can be measured in the trachea of in- tact chicks and animals and in the upper respira- tory tract of large animals. In addition, this method is being used currently by another in- vestigator in this laboratory for similar studies in the upper respiratury tract in httrrlus. 'Preparcd by Anna M. Ba(•tjer and Llovd M. Bates. From the Departments of Environmental -Medicine and Radiological Science. School of Hy- giene and Public Health, The Johns Hopkins Uni- versitv, Baltimore, Maryland. ' This investigation was supported partly by Pub- lic Health Service Research Grant AI-03997-02 VR and partly by Army Medical Research and Devel- opment Contract DA 49-193-MD-2055, under spon- sorship of the Commission on Environmental Hy- giene of the Armed Forces Epidemiology Board. the trachea. Details of the collimator are shown in figures 6, 7, and 8. Figure 6 shows a top view of the collimator. Figures 7 and 8 show cross- section views along the axis A-A and B-B as indi- cated in figure 6. The collimator was designed on the assumption that the radioactivity in the tra- chea would be 6 mm. below the bottom of the collimator when the latter was placed in contact with the exterior surface of the neck. Calculations based on the detectors described below indicated that the sensitivity and resolution of the system would be adequate if the collimator could "see" a 2-mm. length of trachea at this depth. Since high resolution in a direction transverse to the trachea was not required, and since very accurate ~ alignment of the trachea in this direction was ~ difficult, the openings in the bottom of the col- ~ limator were made in the form of slits, as shown ~ in the figures. The collimator weighs about 15 ~~ pounds and is supported on a metal frame with ~ adjustable legs to allow for different size subjects. To maintain the trachea in a fixed position rela- G~5
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9.8 B ~ 0.6 j I ~ 2.0 -~4-L 2.6 I I I 2 ' i - - 3 l . r . 6.6 ----t- a Frc. 6. Top view of collimator. Dimensions given in centimeters. 6.6 - 2.6-31 2. 0->}f- 2. 6 9. 0.6 6.0 i t; V: ~.. L e a , a 28 48 Fic. 7. Collimator section through A-A (figure 6). Dimensions given in centimeters. K--~- 4.2---30I 1.2 FIG. 8. Collimator section through B-B (figiire 6). Dimensions given in centimeters. "0 1
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DISCUSSION III ~~1I I II~~ ~1 if~ C ~ ~ i~j I F- Fic. 9. Typical records obtained from count rate meters (1 and 3) and integrators (2 and 4). tive to the detectors, the neck is held in place :ieainst the collimator with a sponge-rubber holder consisting of a 6-cm. cube with a channel 1 cm. wide by 2 cm. deep for the neck. Two scintillation detectors are located in the oylindrical recesses in the top section of the col- limator. Each consists of a NaI(TI) crystal one- half inch in diameter and three quarters of an inch thick coupled to a photomultiplier tube three quarters of an inch in diameter. This assem- blv has an outside diameter of one inch and an over-all length of ten inches. The absolute effi- ciency of the detector is quoted as 25 per cent for the gamma radiation from CS13'. and the roent- gen sensitivi'ty as 24,000 c.p.m. per mr. per hour. The signal from each detector is fed to a count rate meter with time constants ranging from 0.5 to 40 seconds and sensitivities ranging from 300 to one million c.p.m. for full scale deflection. A recorder connection on the rate meters provides a sienal of 10 mv. or 1 ma. for full scale deflection of the meter. Each rate meter is connected to one channel of a multichannel recorder through a variable gain D.C. amplifier. Th, gain of the amplifier is a(ljuated to provide maximal recorder deflection (5 cm.) lor full srale deflection of the rate meters. In :iddition, two other channels are used to plot the inteeral of the count rate from each detector, i.e., 81 the total activity observed by each detector after any time interval. The recorder is operated at 1 mm. per second. A typical record showing the curves produced by the count rate nieters and the integrators is given in figure 9. The radioactive material is I13` as \ aI in a saline solution. I'a' has a convenient half-life (8.1 days) and emits gamma rays which are detected by small crystals with good efficiency. The ex- posure dose rate from P01 is 0292 mr. per hour per mc. at one meter. It is available in relatively high specific activities-up to 50 mc. per milliliter. The sensitivity of the collimator-detector system is such that 1 µL. of the solution with a specific activity of 5 µc. per kL. is easily detected even when only a portion of the droplet is being "seen" at one time. The droplets move up the trachea in various forms and at different rzues along the trachea. Hence, the most convenicnt measurement is the rate of movement of the forivard edge of the drop- ~ let. In order to standardize the procedure, this ~ edge is assumed to have reached the first detector ~_ i\hen the count rate is double the background level, and to have reached the serond detector at ~ :ti distance of 3.2 cm. when its count rate is double ,:. its background. The time interval is obtained from (.i the record for calculation of the rate. This equipment is currently used to measure
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F 82 DItiCli SSIO\' the rate of clearance in the trachea of chicks anesthetized with pentobarbitaL Three measure- ments are made on the same chick; but, since complete clearance of the I13' does not occur within a reasonable time, measurements repeated within this time are adjusted for the difference in the background. Furthermore, to obtain consistent rate measurements throughout an experiment, the body temperature is maintained constant. For this purpose, the chick is placed in a clear plastic box, so arranged that the air temperature and humidity and the body temperature can be controlled auto- matically. The deep rectal temperature and the air temperature are monitored and recorded by thermistor-telethermometer systems, and the hu- midity of the inspired air is monitored by an electric hygrometer. Other parameters such as re- spiratory rate and volume are recorded as desired. s a s I no= tra~ ani w e: na- hoi car, ere arr thc thc poi atn tb ( sp, a coi de' cal Tl sei ra! co So:
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DISCUSSION S3 Dr. Tyrrell: I don't like radioisotopes up my nose, and fluorescent dyes are carcinogenic, but a tracer of B. micoides spores is suitable for clear- ance studies. These spores can be assayed on a weak agar plate which will not allow growth of nasopharyngeal organisms or spores derived from house dust. The rate of disappearance of spores can be plotted from the number of spores recov- ered from one standard bacteriologic swab. The arrival of snores in the throat is easily noted, so the time between placing spores in the nose and their arrival in the throat measures rate of trans- port down nasal epithelium. Thus, unanesthetized ambulatory human subjects breathing through their noses can be studied. The rates of decline of spores in the nose and throat can also be used as a measure of clearance. However, the model of complete mixing and dilution as it is applied to determine circulation time is not applicable to calculate the volume the spores were diluted in. This method is less subject to errors than ob- servations of dyes and shows that the clearance rate through the nose is drastically reduced during colds. You may find no spores in the throat after somebody has had a cold for a couple of days. Dr. Hilding: I just want to voice a couple of warnings concerning the methods for testing flow rates in the nose referred_to by Dr. Baetjer and Dr. Tyrrell. The line between the ciliated and the nonciliated epithelium is not a definite boundary. It varies among individual persons and between the sides of an individual's nose. One side of the nose is not a mirror image of the other. The place where the ciliated epithelium begins depends upon the age of the individual, and on the contour of the septum and the turbinates, and perhaps on what type of air the person has been breathing, how active the person is, and a great many other factors. In some persons India ink placed in the nose disappeared completely in two to three min- utes; in other persons it was still there after two hours. In some, half of this dot of ink would dis- appcar and the other half remain. In some it would disappear entirely from one side and remain in the other. This is a very tricky business because we cannot tell what type of epithelium is under- lying the ink. Dr. Bates: I ask the privilege of some questions because it is my job to summarize the conference and we have left a tremendous number of loose ends. Dr. Ballenger, what is the effect of altered vis- cosity of the medium on the speed of rotation of the implanted things that spin round and round? Dr. Bnllenger: Tapping of the Rose chamber or microscope produces a jell-like shimmering of the whole preparation except surrounding the rotating ciliated explant. There the viscosity is much re- duced as rotation occurs, but liquefaction does not extend beyond this vicinity. For three or iour days, or even for a week, the liquefaction is only in the immediate vicinity of the rotating body and does not extend to the jell as a whole. Dr. Bates: Dr. Brokaw, did you distinguish be- tween the energetics of the bending of th~, cilia and the energetics of the forward stroke'? Are they different? Is the forward stroke a different mechanism, or is the whole ciliary movement ex- plicable by one mechanism? Dr. Brokazo : My guess is strictly an extrapola- tion from what appears to occur in flagella. I think there are two possibilities. The one which I favor suggests that during the effective stroke there is a bent region and a region which is not bent. At any instant, only a very short region is actively bending, and this is the only point on the cilium that is bending. The actively bending region is progressively moving along the cilium at a con- stant rate, starting at the base. On the other hand, the picture that Dr. Gosselin showed of the large flagella on Mytilus suggested that during the ef- fective stroke the bending is located very close to the base. The rate at which the bending zone progresses is very slow when the force that it has to overcome is high. This is consistent with what occurs to flagella in viscous media where the re- sistive forces are greater. The alternative is that a large region starts out being straight, and gradu- ally increases its curvature until it is partly bent. This does not occur in flagella and appears there- fore unlikely in cilia. Some good pictures would settle this issue. I think this is relevant to the matter of whether the flexion that occurs during the recovery stroke is a continuation of active bending or whether it is simply passive uncurling of elastic energy which was stored during bending. It has been noted that there is an inconsistenoy between the suggestion that the fibrils of the cilium are able to slide relative to one another so that their relative positions at the tip of the flagellum or cilium can change, and the pictures which Dr. Rhodin presented suggesting that the filaments extended and possibly were continuous at the tip of the cilium. Although this is obvi- ously an inconsistency, these are in very different materials. Dr. Staicb: Dr. Brokaw's observations of cilia filaments slipping sideways past each other in the bending wave are directly contradictory to what Dr. Rhodin said about filaments being continuous over the tip of cilia. Can this conflict be resolved? Fluorescent antibody studies of actomyosin might show whether they exist in filaments or not. If we are going to destroy a model, we must have a new model. I would suggest that all energy is utilized at the base and not propagated out along the cilium, because movement, especiallv in the effec- tive wave, appears to be almost entirely at the base. Perhaps flagella are different from cilia. Other possibilities include a hydraulic model, pumping of fluid in and out of a little tubule, or electrical propagation along membranes within the tubule. These things ought to be studied and the transmembrane potentials of epithelial ciliated cells measured. Measurements can be made in much smaller cells than epithelial eells, so this should not be difficult. The other possibility in energetics, in view of the similarity between this
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84 DISCUSSION and muscle contraction, is the relaxing factor which exists in muscle and is known to uncouple and bind calcium. Dr. Brokaw: What is the similarity of cilia to muscle, particularly to actomyosin? That periph- eral filaments do not contract does not exclude the possibility of a muscle-like property in the system. It suggests onl' only that if there is a contrac- tual system it may be located somewhere other than in the peripheral filament. There may be sliding interaction between filaments such as has been suggested as the basis of the contraction of acto- myosin systems. But, as I said before, the bio- chemical work on similarities between known actomyosin systems and the biochemistry of flagellar proteins has just begun and has been limited by the difficulties of getting sufficient cilia separated free of cell material. Dr. Bates: Dr. Rhodin, in the electron micro- scopic pictures of the surface of ciliated epithe- Iium, presumably the water normally present had been removed. D4y question is, what are the cilia in the normal human trachea actually beating in? Is it water? Dr. Staub: Dr. Rhodin, because electron micro- scopic pictures are very beautiful, we fail to criti- cize them. Fixation for electron microscopy is done in water which is not good for fixing tissue. Perhaps anhydrous fixation would show differences we have not yet seen. Second, if cilia are being replaced on ciliated epithelial cells. then at some time there must be some baby cilia. Dr. Rhodin showed some microvilli and Dr. Brinkman showed some sea weed, but no one showed cilia growing. Dr. Rhodin: Clearly there is mucus in between the ciliary shafts. Most of the mucus and also the serous secretion comes from the glands, not from the goblet cells. These glands may discharge sud- denly and fluid will pour over the surface of the epithelium; in contrast, the mucus goblet cells of the epithelium discharge from time to time. Evaporation occurs at the surface of the mucus where it becomes concentrated. Thus, viscosity may be greater at the tip of cilia and less at the base, perhaps because serous fluid predominates there. Second, reabsorption is taking place back into the ciliated cells, via pinocytotic vesicles. In reference to the formation of the cilia, I think that, as Dr. Sleigh pointed out, they form very quickly. In fetal human trachea some of the centrioles move toward the surface and in this zone multiply very rapidly and induce the cell membrane to evaginate. In the spermatid of the mouse testis there are two centriol,,s: one gives rise to the sperm tail; the other, or sperm cen- triole, forms the base and body, and filaments or fibrils grow from this and push the cell membrane in front of them. We cannot determine whether the cell membrane or the fibril goes first, but flagella are formed quickly. In reference to fixa- tion for electron microscopy, I do not think we have to apologize now 15 years after we started using this techniquP. Although it has been im- proved over the cears, a fixative is needed which does not dissolve the water-soluble parts of the cell. In reference to the thickness of the mucus hlanket-Dr. Dalhamn froze the tracheal epithe- lium and, after drying, found that he could meas- ure the thickness of the mucus blanket. Dr. Luft in Seattle has devised a technique to fix the sur- face layer. He showed that the surfaces of endo- thelial cells in the vascular system are provided with a surface substance which can be made visi- ble and which corresponds to a so-called sticky substance where the clotting mechanism of the blood vessel may be initiated. By similar tech- niques this has been demonstrated on the micro- villi of intestinal rells, that there is a certain surface-covering structure, not necessarily asso- viated directly with the membrane similar to that of the alveolar lining of the lung. Dr. Bates: Dr. Sleigh, if inetachronal wave ac- tivity is mechanically determined, why should Dr. Ballenger's turners continue to beat but fail to rotate after tobacco smoke? Dr. Sleigh: I don't have the original data on the frequency of beat of the cilia or the proportion of cilia that remains beating after the activity of the cigarette smoke on the rotating spheres. How- ever, I would suggest that the metachronal wave activity ceases because some of the cilia involved stopped beating before others and broke up the formation of metachronal waves. Dr. Staub: Dr. Sleigh, what is the normal en- vironment of the cilia-both the mucus layer and the watery layer underneath? You were differ- entiating in some cilia between ciliary beat fre- quency and metachronal wave velocity. If the vis- cositv of the media were changed one would change one but not the other necessarily. But the ques- tion is whether viscosity was being changed be- tween the cilia or just on top. '_1laybe cilia do not let things get down between them. Dr. Sleigh: In many cases viscous fluid does get in between cilia because the frequency of beating slows down fairly quickly after the media around the cilia are changed and is then constant over a long period. I would expect it to change gradually if it were getting in more slowly. There have been a number of studies on the development of cilia suggesting that they develop from centrioles very quickly. It is possible to cut off the flagellum of a flagellate and it will regenerate within three or four hours. Dr. Staub: The little diagram presented by Dr. Rylander is very interesting, especially with re- spect to the difference between ciliary beat fre- quency and mucus transport; but a third aspect should be considered, and that is the effectiveness of the ciliary beat. Even if the mucus is not al- tered and the beat frequency may be the same, how about discoordination? Dr. Alorrozo: I believe Dr. Rylander stated that there was evidence that the size and weight of particles would influence the measurement of mu- cus transport, using particles as an index. What is this evidence? Most investigators have concluded that mucus transport of particles is independent 00097441
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Jt r. t i t 1 DISCUSSION ~ L~ceirht of material, presuming that the rial are inert. ;• L'qlander: Observations on the speed of ~~n~portation of particles on mucus, not trans- rr;ition on cilia free of mucus, indicate that rn tion in density of various particles causes a 1 rence in drag in the fluid and also a difference 1 he depth to which the particle will sink in the ~,;i,-u,. Currents have various velocities and dif- r,nt directions on the epithelium, so that par- Ic placement is important. The other question ~ts on discoordination of the ciliary beat. When ,asuring ciliary motility, one can measure (1) frequency and (:,2) the coordination. With ammalian epithelium or rabbit trachea, dis- ~~~rdination can be visualized fairly easily. It is .n entirely different type of flicker that is ob- -rved through a microscope with incident light. Of course, both of these questions are a bit open -o discussion until we get a reliable method to measure such parameters as mucus viscosity and -hickness of the mucus layer. Dr. Bang: I should like to ask Dr. Ballenger ;~ow he differentiates between destruction of cells which would slow the rate of motion of his rotators znd direct effects on cilia? Dr. Ballenger: Destruction of the cells and a :iirect effect on the cilia both slow the rotating bodies. Exposure of the rotating bodies to cigarette zmoke stops rotation, and about eiglit or ten hours later the cluster disintegrates into individual cells. The initial effect is something that interferes with tlie metachronal rhythm of the beating cilia so that they are beating without the ability to rotate the bodv. Dr. Miller: Being a mechanic and not a biolo- gist or biochemist, I would like to know what con- cept of viscosity is held by the discussors. Appar- ently viscosity does effect motion of mucus and cilia. A Newtonian liquid is a one-constant fluid which can be described on a macroscopic scale by this one constant. However, the mucus which is highly non-Newtonian has yield points, a visco- elastic nature, and is highly thixotropic. This fluid will take so many constants or functions of the material, temperature, hydration, and so forth, to 85 describe it, that in my estimation it has no vis- cositv. Dr. Batlista: We have heard at least a half dozen reasons why it is difficult to get repro- ducible measurements of ciliary activity, and I would add one more-that is, the depth of fluid on the epithelium which bears cilia. If particles of graphite with a density of 1.8 are dusted onto the horizontal trachea along with a very light soot, the heavy particles which sink down will move along and the light particles will stick right on top without any motion. As the fluid level drops, the heavy particles stop and the light particles start moving and cilia are apparently beating at the same rate. The depth of fluid and position of the trachea are important. Dr. Kensler: Dr. Baetjer mentioned that the trachea of her chicken seemed to be rather floppy, which presented a problem for her. In our lab- oratory, Dr. Battista has taken advantage of this to pull the trachea out through the mouth of the chicken and to open up the glottis. Movement of particles can be observed, and the rates are similar to those observed by Dr. Baetjer. Transport is inhibited by cigarette smoke. I got the impression that Dr. George's phenol concentrations were ex- tremel,v high, 5 X 10-' or 10-'. In contrast, Dr. Bernfeld needed 20 or 30 frogs to demonstrate a 10 per cent difference in response. In the rabbit trachea with an isolated preparation, phenol is not very active, although at extremely high concentra- tions it stops particle transport. The maximal amount of saturated phenol in a moist chamber preparation produces inhibition even after a filter cigarette has been added. Dr. Bernfeld: I don't think that our results are in conflict with Dr. George's. He uses an aqueous solution of phenol instead of the gas phase ; there- fore, he had higher concentrations of phenol. There are two possible explanations for Dr. Kens- ler's failure to show an effect of phenol in the presence of filtered cigarette smoke in the rabbit. One is that the rabbit, in contrast to the clam, is unreactive to phenol, an l the other is that the smoke from the cigarette was rich in phenol.
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RESPONSES OF CILIATED EPITHELIUM TO IRRITANTS HANS FALK, Moderator = MUCUS TRANSPORT IN THE RESPIRATORY TRACT" STEVEN CARSON, RICHARD GOLDHAMER, AND ROBERT CARPENTER' INTRODUCTION Exposure of an air-breathing animal to its gaseous environment affects the various cells of the respiratory tract more than it does any organ system. The ciliated epithelium of the respiratory tract and its mucous layer, which acts as an air purifier and protective layer, are in continuous contact with the atmosphere. Atmospheric contaminants may alter the prop- erties of the mucus and/or affect the ciliated cells underlying the mucous blanket by chanaing ciliary frequency and gland and goblet-cell function. The effects of atmospheric pollutants on ciliated epithelium have been studied in intact animals and tissue preparations by measuring the frequency of metachronal waves or other characteristics of ciliary beat (1, 2). As these methods may fail to detect early changes due to surface effects, a simple method was devised to measure the velocity of particles on the mu- cous blanket of living animals. The effects of various substances on mucous velocity studied by this method (3) will be de- scribed. The results help to clarify the muco- ciliary defense mechanisms of the lung and trachea. METHOD Young cats of either sex, weighing 750 to 1,500 gm., were anesthetized with 35 mg. of pentobarhi- tal sodium per kilogram. The trachea was exposed by a 3-cm. incision through the skin and blunt dissection of muscle and fa.~cia to avoid injury to nerves and blood vessels. The animal was placed feet down on a platform over a water bath which maintained body temperature. Exposed tissues were kept moist with saline-soaked cotton pledgcrs. `From the Food and Drug Research Labora- tories, Inc.. Jlaspeth, New York. "These studies were supported by the Philip hZorris ResF•arcli C(mter, Richtnonil, Virginia. and the Aational Cancer Institute of the National In- stitutes of Health, U. S. Puhlic Health Service. ' From the Philip Morris Research Center, Rich- mond, Virginia. SB An inverted binocular dissecting microscope with a calibrated ocular (Wild) was focused on the tra- chea. The tracheal translucence and reflectance were increased by oblique cold light. Lycopodium spores triturated with lamp black provided uni- form particles which were introduced through a 22-gauge needle into the tracheal lumen between the carina and the observation point. The move- ments of this marker material floating on the mucous surface were clearlY visible. AIarker mate- rial was reinserted whenever necessary. Gases, smokes, or aerosols were introduced into a mixing chamber or a 200-ml. face mask with a rubber dam headport assembly which fit cephalad to the exposed trachea. Liquids were aerosolized by a`'aponefrinGk` nebulizer. The mask was removed after the desired dosage exposure. Cigarette smoke was administered from a ciga- rette which had been smoked with two-second, 35-nil. puffs at one-minute intervals. After about 30 mm. of the cigarette length had been burned, the fifth puff was introduced into the face mask, and the animal was allowed three breaths of this smoke mixture. The dose in each cat was equiva- lent to three puffs of a cigarette b}- a man weigh- ing 70 kilograms. RESULTS One hundred control animals, observed for four hours each, had an average particle velocity of 0.25 --t 0.08 mm. per minute. In- dividual animals had a maximal variation of 0.03 mm. per minute during this time. Cats with control velocities outside this range were excluded. The velocity of particle transport after ex- posure to a given agent was expressed as a per cent of initial velocity. The effects of smoke from nonfilter, cellulose acetate filter, and carbon cellulose acetate filter ciE~arettes are shown in figure 1. The smoke from the carbon cellulose acetate filter ciearettes had the least effect on particle velocity, and the velocity was most affected by the nonfiltered ciLmrette smoke. A dose-response curve for smoke from non- filtered cigarettes diltited «'ith air is presented in figure 2. Less than 30 y of ci;arette smoke reduced velocity 50 per cent in the trachea, de- spite nasal and pharyngeal removal. 00o97443 I
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10 8 ii a (ra- ice Im ni- a en "e- he Ir,e- ato ~, a ad ed ed for icle In- of ats 'ere ex- ;a of ter, are )on ast S'as :te. on- ted f.ke tie- 40 20 \~n-ti [er e u e Acetafe (.ar'- Cr M~- Filter F ilter Fio. 1. Effect of smoke from commercial cigarettes on mucus flow in the cat (at 0.5 minutes after exposure). 0 i ml q.s. 5 0 ml concentration, -cR/ml Fia. 2. Effect of air dilution on mucus flow in the cat (nonfilter cigarettes). 87
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88 CARSON, GOLDHAMER, AND CARPENTER Frc. 3. Effects of five puffs of smoke from commercial cigarettes on mucus flow (at 10-mm. intervals of cigarette length). A steady decrease in particle velocity with subsequent recovery to pretreatment values was observed in cats exposed to a total of five puffs taken at 10-mm. intervals from a single cigarette (figure 3). The cigarettes used repre- sent three types of commercially available brands. That velocity slowed and recovered rapidly suggested that tiie effects were due to surface changes. Dose-response curves (figure 4) for several pure compounds from cigarette smoke indi- cated that, for a given dosage, formic acid has the least effect on particle-transit time and acrolein has the greatest effect, although both have markedly irritating properties. The effect of nicotine and two of its salts at different pH levels indicated that the salts had a significantly oreater mucostatic activity than the base alone (figure 5). A study of the time course of various com- pounds on particle velocity revealed three definitive patterns. A characteristic biphasic pat- tern (figure 6a) was obtained for those com- pounds shown in figure 4. Slowing, characterized by a delayed onset and a prolonged duration of effect, was found for phenol (figure 6b) ; however, at the lowest concentrations of phenol an initial acceleration of two to three minutes' duration was observed. The pattern for ciga- rette smoke (figure 6c) was different from the patterns described in figures 6a and 6b and sug- gested a combination of these two. Results obtained with three materials com- monly used for treatment of respiratory dis- orders are presented in table 1. No effects were observed with isoproterenol. At the highest concentration studied, A'-acetvlcysteine re- sulted in a marked slowing, accompanied by a 0ppg'7445
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NIL"CL•> TRANSPORT I\ TIIE RESPIRATORY TRACT Fte. 4. Effect of hure materials on mucus, flow rates in the cat. )m- ree at- )m- zed ion D); mol tes' i~a- he :, lv- ,rrl- dis- ere :est re- :a dJS , . 1 +I "in, in -,") n'.1 ilask Pic. 5. Effccr of nicotine ac varcin; PH's on nnicus flow in the cat. 89
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oo CAR,3OS, GOLDHAMER, AND CARPENTER -2 Cigarette Smoke, Carbon-Cellulose Acetate Filter, 35 rii oulf 5 10 time, minutes 1 5 2 0 25 30 35 60 180 Fra. 6. In Viuo tnucus studY: Pure compounds and cigarette nnoke-a. (Top) Acrolein 0.76 y per milliliter; b. (Center) Phenol 3.2 y per millilitcr; c. (13ottom) Cigarette emoke, carbon-cellulose acetate filter, 35-m1. puff. watery appearance of the mucus. In the-se cats, the marker material moved cephalad during expiration and caudad during inspiration. The effects of lobeline sulfate were similar to those of nicotine salts. A series of salts used as mucolytics produced moderate acceleration, except for vnmonium chloride which caused slowing (table 2). Discossros The mer,surement of mucous surface velocity in the intact living animal is a means of as- sessing the capabilities of the mucociliary ap- paratus. 1lucous surface velocity is, in the final analysis, dependent upon viscosity of the mu- cous la' ver, frequency of ciliary beat, and secre- torv function of the goblet cells. Any analysis of the response of the system to an environ- mental agent must consider the independent and interdependent properties of each of the components. The first contac.tof an inhaled irritant is with the protective mucus of respiratory pas- sages from the nose to the tracheobronchial tree. This barrier must be pa~sed before a sub- stance can reach ciliated or goblet cell~. AIa- 0009'7447
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final mu- ecre- Ilysis iron- _lent the MUCUS TRANSPORT I\ THE RESPIRATORY TRACT terials may react with the mucous layer and alter its rheologic properties directly. Such an effect is postulated for substances which produce transient changes in particle transport rate such as was found for formic acid and acrolein. These substances also slow transport of mucus on the excised trachea (4, 5). The effects may be mediated by cross-linking reactions be- tween the test substance and mucopolysac- charides in mucus. The pattern of response to these materials was characterized by fluctua- tions of the surface velocity, which suggested shearing of the elastic film followed by contrac- tion due to the elastoviscous nature of the material. The period of increased velocity oc- curring during the contraction would be fol- lowed by the front of another layer of film so that the velocity would again be reduced. The minute exposure used in this work caused a rapid damping of this effect. It is probable that longer exposure would produce more prolonged effects, just as higher concentrations apparently produce a greater change in the surface veloc- itG. The effects of ionic substances are some- what different from the effects of substances capable of producing cross-linkage. Sputum- liquefying properties have been attributed to iodides and other halides for some time. Per- haps they increase secretion, lower viscosity, and improve mucus flow. The administration of X-acetylcysteine, in large dosages, produced a watery mucus in which cilia probably beat with little or no effect. The fact that the effects of nicotine and ammonia are increased by salts of these ions emphasizes the importance of chemical structure on the mucociliary trans- port system. In addition to altering mucus, an inhaled irritant may damage the ciliated cell or pro- duce alterations of the ciliarv beat. I7z vitro studies with phenol (4, 6, 7) have demonstrated such an effect. The delayed onset of slowing of particle Velocity after the inhalation of sub- :;tances by intact cats suggested that the ef- fects were not ou the mucus but ou the ciliated cells. Acute exposure to noxious agents slows par- ticle transport and appears to make mucus waterv or viscid. Unfortunately, the secretory re-ponses to these materials, possibly of equal importance, have not been measured by the T aBLr 1 C0.\7PARI5O5 OF RESULTS WITH ACTIVE Co31P01"xD5 Jlucits Velocity--In I-iro in the Cat Compound Stl Observation Time Dose Minutes Delivered ' Control 2 5 10 y norz. per minwe ~,r basal vedoctilv Isoproterenol 35 13.3 110'1011 S04 70 12.5 103 ' 102 ' _A"-AcetyIcys- 35 13.9 98 i 94 91 teinc 70 125 13.0 13.0 106 ; 101 87 ~ 38*i Lobeline S'O, 35 13.0 61 ~ 76 li 91 * Puddling. TABLE 2 IN Plvo .1IrCUS CH:1NcEs Test Material in Distilled Base Line Mucus Water Dose l'elocity rng. inlaa2ed per cent Ammonium chloride 3.25 25 Potassium iodide 3.i-k 147 Potassiutn iodide 0. 75 131 Potassium chloride 3. 74 136 Potassium cliloride 3.24 132 method herein described. Aor have the effects of continued insults on chronic exposures been identifiect. In any attempt to evaluate the effect of en- vironmental contaminants upon mucous surface velocity, it must be realized that the gaseous and particulate components present may alter any one or all three of the mechanisms dis- cussed. 1 et, in spite of the complexity of the clearance system and its environmental inter- actionU, the possibilities of preventing or alle- viatinu, ncLver4e effects upon tnucus transport ,ct'm to exist. REFERENCES (1) Dalhantn, T.: Mucous flow and ciliary ac- ~ ti~~it~~ in the trachea of healthv rats and rats ~ exposed to respiratory irritant -lases (SOz, 0 IH:A, HCHO). Acta Physiol Scand, 1956, 36 ~ (Supplement 123. p. 1). '~ (2) Dalhamn, T., and Rylander, R.: Ciliastatic .:+ action of smoke from filter-tipped and non- 7 tipped cigarettes, \ature (London), 1964, Qt ?01, 401. 7
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92 CARSON, GOLDHAMER, AND CARPENTER (3) Goldhamer, R., Barnett, B., and Carson, S.: A new technique for the study of mucus flow in the intact animal, Fed Proc, 1964, 23,406. (4) Kensler, C. J., and Battista, S. P.: Components of cigarette smoke with ciliary depressant activity, A ew Eng J Med, 1963, °69, 1161. (5) Wynder, E. L., and Hoffmann. D.: Studies with gaseous and particulate phase of tobacco smoke, Proc Amer Ass Cancer Res, 1962, 3, 373. (6) Hoffmann, D., and 11"ynder, E. L.: Filtration of phenols from cigarette smoke, J Nat Cancer Inst, 1963, 30, 67. (7) Bernfeld, P., Nixon, C. W., and Homburger, F.: Studies on the effect of irritant vapors on ciliary mucus transport, Toxic Appl Pharmacol, 1964, 6, 103.
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es, 1962, 3, iltration of rat Cancer f )mburger, nt vapors !xic App] • e S CHEMICAL AND PHYSICAL FACTORS AFFECTING MAMMALIAN CILIARY ACTIVITY' CHARLES J. KENSLER AND SAM P. B:ITTISTA The transport of mucus has long been rec- ognized as an important defense mechanism of the lung for removal of dissolved and par- ticulate material of exogenous origin. Proetz (1), Hilding (2), Dalhamn (3), and others have pointed out that normal ciliary trans- port activity is essential for maintaining proper pulmonary function and that substances which compromise these functions are capable of inducing disease. The reports of Hilding (2), and more recently of Dalhamn (4), Falk and associates (5), Guillerm and associ- ates (6), Kensler and Battista (7), and Gold- hamer and co-workers (8), that cigarette smoke is capable of inhibiting either ciliary transport activity or ciliary beat has stimu- lated increased interest in this problem and has led to suggestions by Hilding (2) and Falk and Kotin (9) that failure of cilia to constantly move the stream of mucus is significant in the pathogenesis of lung cancer, based on the hypothesis that ciliostasis enables environmen- tal carcinogens to reach epithelial cells which line the respiratory tree. It is obvious that the movement of the mucus blanket in the respiratory tract of the intact animal depends not only on ciliary motility, but also on the composition and physical properties of the mucus itself. Consequently, the effects of various noxious agents on transport rates in vivo might represent changes either in ciliary function or in mucus volume and composition, or in some combination of the three. 'Most. workers interested in ciliary motility per se have therefore investigated the effects of vari- ous agents on isolated (in vitro) preparations which permit direct visual observation of particle movement or ciliary beat. In addi- tion, effects on mucus secretion and viscosity can be minimized or eliminated in the isolated tracheal preparation, as shown by Iiensler and Battista (7). The bulk of the agents studied can be con- venieutlt• divided into three gener:rl cate- ' Life Sciences Division, Arthur D. I.ittle, Inc., Cambridge, Massachusetts. 93 gories: (n) enrlogenouS re_ulating (humorall agent~; (b) agents of interest because the.- are neneral air pollutants or local pollutants, e.g., hazardous materials associated with some chemical process or uses, combustion products as in smoke from fires and tobacco smoke: and (c) agents containing special physical properties such as negativelY or positively charged ions or particles. The first category- has been covered in this conference by Dr. Gowselin. In our laboratory, Tsuchiy-a and Kensler (101 have found that such diverse possible local regulators as epi- neplrrine, levarterenol, acetylcholine, choline, and serotonin all produced small (10-15 per cent) but reproducible increases in transport rate at low concentrations and decreases nt high concentrations when added to a bath in which rabbit tracheal preparations were im- mersed. Transport rates in these studies were measured by observing the movement of poppy- seeds. All but serotonin were stimulatory at concent rations of the order of 10' to 10-s '-%I. Serotonin in these experiments produced stim- ulation in concentrations between 10-'° and 10-' '_1I. In our more recent experiments, ef- fects at these low concentrations were seldom seen. When the data from these experiments, which were apparently identical in technique, «•eree re-examined, it «-as noted that the control rates in the latter experiments were almost double those in the earlier ones. Whether the differences in response to these agents were attributable to damaged or "fatigued" cilia or to heavier poppy seeds employed in the early experiments has not been e-tablished, but it mi;ht be recalled that ouabain produces a dramatic increase in the force of contraction of the foiigued papillary muscle and not of the normal one. In any case, we agree with Dr. Gosselin that there is no clear-cut evidence to support the concept that mammalian ciliary motilitY is under the control of local hormones 2~uch ns those found to mediate effects in the s.ympatbetic :urd parasympathetic nervous sys- tem. In the secondary categon-, i.e., those agents
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94 KENSLER AND B:ITTISTA which might 1,e air pollutants (components of tobacco smoke, et cetera), a considerable amount of work has been done using both in vitro and in vivo techniques with ciliated epithelia from clam gills (11), frog esopha- gus (5, 12), and mammalian tracheae (3, 6, i). Many of these agents have been found to be capable of inhibiting either ciliary transport activity and/or ciliary beat frequency. Studies on mollusk and frog ciliary activity have been discussed by others at this sYmposium. Our work has been limited to studies on mammalian and avian tracheal preparations (a) immersed in Tyrode's solution, (b) sus- pended in a moist (air) chamber, or (c), in a few instances, on tracheae which were ex- posed and observed in vivo. In experiments with rabbit tracheae im- mersed in T' vrodc's solution, using popp}- seeds as indicators of transport activity, it was found that transport rate was maximal between 36° and 40°C. and between a pH of 6.5 and 5.5. When these limits were exceeded for an appreciable time period (five to ten min- utes), marked reduction in transport rate was observed. Consequently, the temperature of the bath was carefully maintained at 3S°C., and the pH of the bath was measured after each agent was tested. Formic acid (10-' M), nitro- gen dioxide (3 x 10' M), and ammonia (\H,OH-10-"M) produced 50 per cent inhibi- tions which were associated with, and pre~iuii- ably due to, a change in bath pH. Sodium cyanide was a potent inhibitor-the ED:,, con- centration being 10` M. Nicotine, phenol, and catechol all produced 50 per cent inhibition in concentrations of approximately 10- li. In- hibitions of 50 per cent were observed when the total smoke condensate of three cigarettes was added to the 250-mi. bath and with gas phase (material going through a Cambridge filter) alone when four cigarettes were added. This observation indicated that most, but not all, of the ciliotoxic materials of cigarette ~moke were in the gas phase of the smoke. A coinp,iri- ~on of aa series of ci,_narettes in which the nicotine content of the tob:1cco wati varied in five ateps from 2.98 per cent to 0.53 per cent did not ' vield smoke condensates with tii,_,nificantlv different inhibiting activity. This evidence, tozether with the relativelY lmr cilio.~tatic potency nb~;erved previously, apparently excludes nicotine from consideration as a significant contribiitor to the ciliostatic activity of cigarette smoke. The amount of phenol (10 -\I) required to produce a 50 per cent inhibition would re- quire smoke from 2,000 cirarettes rather than the three cigarettes when total smoke is used. Phenol can also be excluded as a signifi- cant inhibitory component of cigarette smoke under these condition>. Cyanide, on the other hand, is present in the smoke in amounts suffi cient to account 1or almost half of the effect observed with the total cigarette smoke. Bub- blin~4 ozone into a bath containing an immersed trachea did not produce any effects on particle transport. As a major portion of the ciliostatic activity of cigarette smoke was apparently associated with gas-phase components, and as the actual exposure of ciliated cells in the smoke was very short, we searched for a method which would permit long-term (eight hours) survival of untreated preparations (control) in a moist chamber in which the ciliated cells could be expo.ed to ii variety of gases, aerosols, and smokes for both short and long periods of time. Such a system suitable for single ex- posures of short duration was reported (13) in which the tracheae were mounted on an in- cline (9° to 10°) so that the ability of the ciliated cells to move particles uphill, and thus do work, could be measured. In this system, ozone, which previously had been inactive in the immersed preparations, was now a po- tent ciliarv inhibitor. When the exposure time was set for IS seconds, 50 per cent inhibition was caused by 25 p.p.m. Under the same ex- posure conditions. i.e., IS seconds, the 50 per cent inhibitory concentrations for various gases and vapors were as follows: hydrogen cyanide-1S0 p.p.m.; sulfur dioxide-165 p.p.m.: nitro,en dioxide--120 p.p.m.; and hy- drogen sulfide-1,500 p.p.m. Carbon monoxide, carbon dioxide, and nitrous -oxide in high con- centrations (:-)7 per cent) were without effect, as were similar short periods (1S seconds) of anaerobioAs (nitrogen). Although the expla- nation for the lack of effect of ozone on the immersed preparation and its high activity in the moist chamber prepar,ltion is not known, it is of some comfort to note that the effect of ozone on stretched rubber shows a similar diver- gence when the rubber is immersed in water or exposed in air. As would be expected, the concentrations of ihese mases required to pro- 00097451
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. smoke. juired to 7ould re- rather -moke is a signifi- e smoke lie other 'Its suffi- ie effect ce. Bub- ; nmersed i particle ; activity -ociated ie actual )ke was i I which survival in a -Cl cells ~terosols, riods of 't;le ex- (13) in i an in- of the ~nd thus svstem, inactive wapo- are time nhibition ;ame ex- 50 per various iydrogen ide-1G5 and hv- .onoxide, igh con- ,ffect, as nds) of ~ expla- on the : ivity in iown, it ttect of i r diver- n water :cci, the ro pro- EFFECTS OF CHEMICAL AND PHl'tiICAL FACTORS 93 Fic. 1. Photograph of the exposure chamber with trachea in place. duce 50 per cent inhibition of mammalian ciliary transport activity are lower as the exposure period is increased. The product of concentration and time (CT), as, for exam- ple, for sulfur dioxide and ammonia, are con- sistent with the CT reported by Dalhanut (3) in his in viz,o rat trachea experiments. The interest in and support for studies on the ciliostatic effects of cigarette smoke under more realistic experimental conditions led to the developmentt of the moist chamber tech- nique, in which mammalian tracheal prepara- tions could be exposed to multiple puffs of either fresh cigarette smoke or individual gas com- ponents under conditions in which the exposure time could be controlled and varied from '2 to 58 seconds. The method has been described in some detail (7), and a photograph of the chamber in which the preparations are mounted is shown in fi-ure 1. Although most of our work has been con- ducted using rabbit tracheal preparations, we have examined the ciliary transport activity of other species, as shown in table 1. The tracer particles employed were the same for all species; their preparation has been described (7) because the nature, size, weight, et cetera, of the tracer particles has a sinnificant effect on the transport rate. All six species exhibited mean transport rates of greater than 20 tmn. per minute; and the tracheae from the doo, c°;it, and chicken }-iel(led somewhat hi_her rates than those from the rabbit, monkey, and rat. The effects of cigarette smoke have also been examined in these six species, and all were found to be sensitive to inhibitorv com- ponents in cigarette smoke. Since our earlier report (7), which included data on the dor, rabbit, and monkey preparations, we changed the puff delivery system from a pulse pattern in which air flow was constant to one emploving a pressurized bulb which, when released, pro- duced a-10-nt1. puff of smoke from the cigarette, 90 per cent of the volutne being delivered in two seconds. The bulb was then repressurized between puffs to the pressure required to produce the -10-m1. smoke puff. The flow pattern obtained by this method of puffing more nearh- approaches that observed in smokers. The ED:,, (50 per cent inhibition), i.e., the number of puffs, thus relates to the response under the conditions employed and has no nbsolute significance. It mav be seen from table 2 that cigarette smoke was approximately equally inhibitory in all species, although about half of the chicken tracheae appeared to resist complete ciliary inhibition. It can be concluded that the bulk of the inhibitory activity resides in the gas phase of the smoke when either puffin_ procedure is employed. Consistent with t1ii~z observation, the use of appropriate, activated charcoal filters greatly reduces the tions inhibitins activitv in both sittta- . The question of rever~ibility of inhibition C O of ciliarv transport activitv has also been inve.~ti;ated L-~ase, ( for tobacco -moke and various the inhibi- As iv shown in fiettre 2 GC ~ ' , ~ .. tor.y eti'ect of cinarette smoke appears to Eie C.^ N
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96 KE\SLER ASD BATTISTA TABLE 1 jA' I'ITRO CILIARY TRANSPORT RATE (IF DIFFERENT ANIMALS Transport Rate (Control) f,Ylm.jMinute) Animal Mean Range N Cat ...... ........ 3G.1 ~ 30.6 to 45.4 Dog. ..... 36.1 ; 33.6 to :37.5 Chicken . ...... . . ~ 33.1 ~ 27.5 to 42.8 Rabbit- _........ ' 24.(i ~, 21.9 to 31.2 tIonkeN, -. .. ....~ 21.1 17.3 to 28.3 Rat ............. I 20.9 i 20.0 to 21.7 10 3 10 10 4 10 from the exposure chamber. The effects of some agents, e.g., acrolein, when administered in concentrations which produce initial inhibi- tion of approximately 50 per cent or more, exhibit little reversibility between exposures spaced one minute apart. Other agents, such as ammonia and hydrogen cyanide, show inter- mediate activity, with only slight reversibility between equally spaced exposures (i.e., one every minute), but with slow recover' y after the last exposure. Formaldehyde, acetaldehyde, formic acid, and acetic acid all show a high degree of reversibility. We have examined ap- proximately 45 different gases at inhibitor}- concentrations and have found a spectrum of responses which var' v from tho~e that are rap- idlv reversible to those that are apparently irreversible. The degree of reversibilit}• of the respon~;e is independent of the relative inhibi- tor' v potcncy of the gases. For example, hexanal TABLE 2 EFFECTS OF CIG_IRETTE* SMOKE OV THE CILIARY TRANSPORT ACTIVITY OF TIIE ISOLATED TRACHEA OF DIFFERENT AN11t.11.a Mean Responset to Cigarette Smoke I EDso I EDiua Animal (Puffs) I (PuBs) i - Cat ... ........ ........ ~ 3.9 (i.3 ~~ Dog . ............... 1.5+ + 0.01 Chicken . ............. ~ :3.1 5.2 ~~ > 8 . 0 Rabbit. 3.7 5.3 2.0$ ~ 4.0t 'Monkev .............~ 2.1$ 5.0$ Rat ............... ..i 3.5 4.7 N - 5 and iso-butti-raldehyde had less dredtll the inhibitory activity of but were more rapidly reversible lein, with approximatel}• one-ten than form , whe th th one-hun- aldehvde, reas acro- e activity 3 of formaldeh3-de, was ven- slowl y reve rsible. 4 Krueger and his collaborators (15, 16) have 5 reported on several occasions that positively li charged air ions inhibit rabbit tracheal ciliary 2 beat frequency as well as mu cus transport 5 . They also found that negativel' v cha rged ions increased ciliary beat frequency and increased transport activity in their preparation. The-,- further reported that the administration of neratively char,ed air ions prevented or re- versed the ciliostatic activity of cigarette smoke. Consequently, iu collaboration with Dr. Bernard Vonnegut, a colleague well versed in matters of the production and meas- urement of charged air ions and particles, we undertook aa study of thB problem. The chamber shown in figure 1 was modified so that a radioactive probe was mounted above the trachea and served as the source of unipolar air ions. Current flow between the probes and a second electrode placed underneath the trachea was used to measure ion den~itv. The circnitrv emplo' yed is illustr:ued in tisure 9. Radioactivitv fronl polonium 210 and fronm tritium was employed in ion generation. Uni- polar ion densitv as great as or greater than tLlo~e reported b' v Kruezer was emploYerl in the stud>-. Four such experiment.~. with polonium as the source of radioactivity, are illu;trated * Regular size, Iwnfiltered cigarettes. j' One -10-m1. puff per miuute-12-secund ex- posure. $ Earlier experiments in which a ditferent puffing procedure was emplo}'ed. irreversible, except when the exposures are limited to less than a 50 per cent inhibition. The data in figure 3 show that smoke from little ciaars (cigarette size) is similar to ciga- rette smoke. The reversibility of the inhibitory activity of a number of gases which are air pollutants or cor~'iponents of tobacco smoke has also been investigated. Examples of the recovery ui rab- bit tracheal preparations following expo<ure to one or more 1'?-~;econd puffs of different con- centrations of i.~o-butyraldehyde, acrolein, form- aldelivde, and ammoni:t .Ire presente l in fig- ures 4-S. The effects of some of these a,~ent~:, such as iso-bntyraldell' yde, have been found to be readily reversible-th:it, is, within a matter of seconds after purging the ~ apor 0009'74S3
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some ied in nhibi- more, i)~ures ~uch inter- one atter i •hYde, high 1 ap- Jitory I',nl of K rap- rentlv i,f the inhibi- cxanal -httn- hyde, acro- I tivitv -le. ) have tivelv -iliar}• sport. l ions ;reased Thev ion of or re- rarette ~ with well meas- irticles, I. The so that ve the nipolar es and ii the -,-. The ire 9. from t. Uni- r than in the ~'onium - ~rated 100 90 60 ; 70 g 60 50 40 30 2 I 0 +10 EFFECTS OF CHEMICAL AND PHYSICAL FACTORS V M afrr-, Ji 0 4 0 10 12 14 16 18 20 22 24 26 28 30 32 34 36 OBSERVATION TIME (In Minutes) Fic. 2. Effect of regular cigarette smoke on ciliary activity-12-second exposure. 100 NO.OF PUFFS •3 .4 • 3 10 12 14 16 18 20 22 24 26 28 30 32 34 36 OBSERVATION TIME (In Minutes) Fic. 3. Effect of smoke from lit tle c•igars on ciliar}• activity-12-second exposure. in fi,ure 10. -No sirnificvit effect was observed on ciliar}• triusport activity when the tis- sueti were exho<ed to either positivel}• or nc;a- tiveh- cliarsed ions impinmed on the tizsue. ,-4imilarly, no Cffect w.is observed when tllc tr•it- ium ionizing element was employed. The in- ltibitor}• action of ciuarette smoke, which, al- XP~ E i-RECOVERY - ~---CONTROLE } 9/ thou_rh electrically neutral, has been found to carry positi.-e or nerative char,es on approxi- matelY 60 per cent of the particles, is also included. This inhibitoi' v effect of cigarette ~moke is not influence l either by positive or by ncrative ion flow. Thus, our experiments failed to confirm the work of Iirueger and t I + 1
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FIG. 4. Effect of iso-butyraldehyde vapor on ciliary activity--12-second exposure. 100~ 90 80r CONTROI~---+--EXPOSURES---~ RECOVERY 1 I 11 1 { { { FIG. 5. Effect of acrolein vapor on ciliary activity-12-second exposure. sr-0 NO.OF PUFFS ~- .7 .8 •8 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 08SERVATION TIME (In Minutes ) co-workers, who reported that nerativelY and positively char,ed air ions ntarkedhy inHuencel ciliary activin- ot the rabbit trachea. Although most of the experiments on ciliary transport activity referred to ;ibove were done on in z,itro mammalian tracheal prep- arations, a number of .~tudies done under in vivo conditions have vielded results which su_gest that the results obtained in the isolated preparations may have applicability to mam- mali:ut ciliar' v tranSport activity in the intact animal. For example, Dalhamn (3, 4) and his associates have shown that Such air pollutants as sulfur dioxide and ammonia inhibit mucus transport activity in ratz and rabbits and that cil,arette smoke also inhibits ciliary trans- 0009'7455
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EFFECTS OF CHENSICAL AND PHYSICAL FACTORS w ~ ~ 0 a x C0NTR0L-> ~f - RECOVERY 10 i oo-o-o~o-o-o-o 0 •10 8 I+tc. 6. Effect of acrolein vapor on ciliary activity-12-second exposure. 100 90 80 d 70 ~ d 60 = ~ 50 ~ = 40 Y 30~ ~olated mam- intact ~nd his :utants n1uCtIS and rans- 10 0 +10 1 2 Numner ~ pF Puffs 10 12 14 16 18 20 22 24 26 28 30 32 34 36 OBSERVATION TIME (In Minutes) 11 ~ ~ 7 1 NO OF PUFFS t . 1 i ~ 4 99 10 12 14 16 18 20 22 24 26 28 30 32 34 36 OBSERVATION TIME (In Minutes) Fla. 7. Effect of formaldehyde vapor on ciliary activity-12-second exposure. port. Goldhamer snd associates (S) have re- ported that cigarette smoke and such smoke components as hydrogen cyanide, formalde- hyde, and acrolein show about the same rela- tive inhibitorv activity in the intact cat as these compounds show in the isolated rabbit CONTROL----* 4--EXPOSURES -4RECOVERY• 1 i i+ i i i i trachea. In our laboratory we have found ciga- rette ~moke to be inhibitor' v to ciliary transport actiVit}• in viro in the chicken. Our in vitro ob- servation that activated charcoal filters signifi- eantl}• reduce the inhibitorY activity of ciga- rette smoke has been confirmed in in viUo
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100 KENSLER A\D BATTISTA 4 -CONTROL-=.-EXPOSURES-+ - RECOVERY-i # # t t 0 0 # # P: m x 100 90 80 70 60 F5 50 40 0 2 4 NO OF PUFFS 8 •8 .8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 OBSERVATION TIME (In Minules) Frc. 8. Effect of ammonia vapor on ciliary activity-12-second exposure. A KEITHLEY ELECTROMETER (M600) B POWER SUPPLY 0 TO 10,000 V.D.C. C RADIOACTIVE PROBE i POLONIUM 210-500µC NUCLEAR PRODUCTS INC. 2 TRITRIUM FOIL 0.2C V VOLTMETER Frc. 9. Diagrammatic sketch of the equipment used in measuring the effect of unipolar air ions. experiments by Goldhamer and ourselves. Weiss (17) has shown similar results in studies on cilia of paramecium. None of the experiments that have been done to date can be more than suggestive that cigarette smoke and air pollutants could, under certain conditions (concentration, exposure time, frequency of exposure, reversibility of effect) re- sult in impaired ciliary transport activity in man during ordinary smokin, or expo.~ure to environmental air pollutants. The fact that activated charcoal filters can significantly re- move amounts of ciliotoxic materials as well as cytotoxic components from cigarette smoke (18) euggests that their use may be beneficial to smokers. Su.>;aiaxr In studies on immersed tracheal preparations, optimal ciliary transport actiVitV was observed when the temperature was maintained between :36° and -t0°C. and the p1I between 6.5 nnd O0p9'7451
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EFFECTS OF CHEMICAL AND PHYSICAL FACTORS = E E E ` w a ~ Cr 0 ~ N 2 ~ 36 r 32 Cr a 28 " 24 20 16 12 8 4 -0 2 4 6 8 10 12 14 16 18 20 22 24 26 TIME (In Minutes) Regular Cigarette I Puff/Minute 40m1/Puff 28 30 9.4 100 10.7 11.5 12.5 13.6 15.0 I8. I . 21. 25.0 300 Ftc. 10. Effects of positive and negative ions from polonium 210 on ciliary transport activity-8- and 10-minute exposure. 8.5. Conditions outside these limits resulted in significant decreases in transport rate. A variety of neurohormones (cholinergic, ad- renergic, serotonin, et cetera) have been found to produce slight increases (10-15 per cent) in transport rate at low concentrations and decreases at high concentrations (10-' i1i). No clear-cut evidence for neurohormonal control of mammalian ciliary transport activity has been obtained under experimental conditions in which possible effects on volume, composi- tion, and physical properties of mucus have been minimized. A number of agents such as formic and acetic acids appeared to inhibit ciliary trans- port activity only at concentrations which altered the pH, so that alteration in pfI would appear to be the primary factor involved in inhibition. Ciliarv transport activity has been studied ~ Control Exposure No.l Exposure No.2 hange Polarity o I ~ ~ Ion Density (for-) om sxl0-9 p o. 9.7 b. 8.9 c. 8.8 d. 9.3 - + i _ f _T 9.4 10.0 10.7 11.5 12-5 15 ~B 214 25.0 3075 ~ -~-- - ~- --T-~ ~ i C ~ - 77 -- ~- , J _ ~ i -Control - ExposureN o.l Exposure No.2-- Chonge Polarity e Negative Ions ~Pos~hve Ions , 7 Smoke From tA , ~ 101 on tracheal preparations from rabbit, dog, cat, monkey, rat, and chicken. With the moist (air) chamber and tracer particles employed, all species exhibited transport rates of greater than 20 mm. per minute and were similar in their response to the inhibitory effects of ciaarette smoke. Gas-phase components of tobacco smoke, such as hydrogen c, •anide, formaldehyde, and acrolein, but not phenol, appeared to be responsible for the bulk of the inhibitory activity of the total smoke. The removal of the gas-phase com- ponents by activated charcoal filters markedly reduced the ciliostatic activity of the total smoke. An examination of the reversibility of the inhibitory activity of a number of gas-phase coniponents of cigarette smoke and air pollut- ants has shown that inhibition is rapidly reversed on termination of the exposure to
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102 KE\SLER AND BATTISTA some of these components, whereas recovery from others occurs less readilv or not at all. The impingement of positively or negatively charged air ions produced by a tritium or polo- nium 210 radioactive source was without effect on ciliary transport activit~. R EFERENCES (1) Proetz, A.: Some preliminary experiments in the study of cigarette smoke and its ef- fects upon the respiratory tract, Ann Otol, 1939, 4s, 176. (2) Hilding, A. C.: On cigarette smoking, bron- chial carcinoma and ciliara action. II. Ex- perimental study on filtering action of cow's lungs, deposition of tar in bronchial tree and removal by ciliary action, New Eng J Med, 1956, 25,1f, 1155. (3) Dalhamn, T.: '-blucous flow and ciliary ac- tivity in the trachea of healthy rats and rats exposed to respiratory irritant gases (SO>, Ha'-N-, HCHO), Acta Physiol Scand, 1956, 36 (Supplement 123, p. 1). (4) Dalhamn, T.: Studies on tracheal ciliary ac- tivity. Amer Rev Resp Dis, 1964, S9, 870. (5) Falk, H. L., Tremer, H. M., and Kotin, P.: Effect of cigarette smoke and its constitu- ents on ciliated mucus-secreting epithelium, J>`i at Cancer Inst, 1959, 23, 999. (6) Guillerin, R., Badre, R., and Vignon, B.: Ef- fets inhibiteurs de la fumee de tabac sur 1'activite ciliaire de 1'epithelium respiratoire et nature des composants responsables, Bull Acad Nat Med (Paris), 1961, 146, 416. (7) Kensler, C. J., and Battista, S. P.: Compo- nents of cigarette smoke with ciliary-de- pressant activity. Their selective removal bv filters containing activated charcoal granules, New Eng J Med, 1963, 'w69, 1161. (8) Goldhamer, R., Barnett, B., and Carson, S.: A new technique for the study of mucus flow in the intact animal, Fed Proc, 1964, 23,406. (9) Falk, H. L., and Kotin, P.: Symposium on chemical carcinogenesis. III. Chemistry, host entry, and metabolic fate of carcino- gens, Clin Pharmacol Ther, 1963, /, 88. (10) Tsuchiya, M., and Kensler, C. J.: Effects of autonomic drugs and 2-amino-methyl pro- panol, choline antagonist, on ciliary move- ment, Fed Proc. 1959, 1S. 453. (11) NVvnder, E. L., Kaiser, H. E., Goodman, D. A., and Hoffmann, D.: A method for determining ciliastatic components in ciga- rette smoke, Cancer, 1963, 16, 1222. (12) Bernfeld, P., Nixon. C. W., and Homburger, F.: Studies on the effect of irritant vapors on ciliar' v mucus transport, Toxic Appl Pharmacol, 1964, 6, 103. (13) Battista, S. P., DiNunzio, J., and Kensler, C. J.: Versatile apparatus for studying ef- fects of gases, aerosols and drugs on ciliary activitv in non-immersed mammalian tra- chea, Fed Proc, 1962, 21, 453. (14) Kensler, C. J., and Battista, S. P.: Inhibition of mammalian ciliary transport activity by gases and aerosols, with special reference to duration of action, Fed Proc, 1964. 23, 105. (15) Krueger, A. P., and Smith, R. F.: Effects of air ions on isolated rabbit trachea, Proc Soc Exp Biol Med, 1957, 96, 807. (16) Krueger, A. P., and Smith, R. F.: Effects of gaseous ions on tracheal ciliary rate, Proc Soc Exp Biol Med, 1958, 9S, 412. (17) Weiss, W.: The toxicity of tobacco smoke solutions for paramecium, Arch Environ Health, 1965, 10, 904. (18) Thaver, P. S., and Kensler, C. J.: Cigarette smoke : Charcoal filters reduce components that inhibit growth of cultured human cells, Science, 1964, 1If6, 642.
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1:FFECTS OF INDUSTRIAL DUST ON CILIATED EPITHELIUM' GEORGE W. WRIGHT ation by to 105. of 1'roc ; -, of 1'roc ioke iron ette ents nan i,icluding myself, might wonder why be a separate consideration of rt< ot iwdtistrial dusts upon the muco- :(pparatus at a symposium such as i':inse of you interested primarily in the uV Nnd physiology of cilia and mucus ,,)n miLht consider this subject to be of thou,h not y our chief, interest be- *lie lnirpose of the mucociliary appara- ,- been thought to be that of removing, --idly and effectively as possible, those ~. both industrial and otherwise, deposited ~he entire surface of the respiratory sys- Ir is proper to inquire into how effective ;i;>paratus is and what impairs it. Some of .•e more directly concerned with the health .ho,~e persons in whose respiratory apparatus ; has been deposited. We are apt to be most ~rested in the effectiveness of the dust-remov- mechanism. There is an obvious overlap of r interests but .ome differences of aim and 'thocls. \oxious agents usually have a time-con- ~-ntration relationship. This relationship must il.VaVs be considered in any studv of the etiology ~,t "agent-caused" diseases as it is the kevstone n[ their prevention. The general problem of "lung cleansing" applies directly to the time- concentration factor. Removal of dust deposited below the larynx involves not only the mucociliar}- system, but also macrophages that engulf those particles deposited distal to the cilia-bearing ducts. It has been postulated that the mucociliarv sheet actually extends into the alveoli, but proof of this remains to be developed. Some hmg diz~eases, for example silicosis, are caused by particles deposited distal to the mucociliary apparatus and, in general, can be thought of as developing even though the mucociliary system operates normally. These diseases are related more to the effectiveness of the macrophap~e system and to other parts of the reticuloendothelial system than to the mucocilinry apparatus. On the other hand, an impaired mucociliary system would be likely 'From the Department of Medical Research, Saint Luke's Hospital. Ck,veland, Ohio. to augment and favor development of macro- phage-oriented diseases. In contrast, diseases such as acute and chronic bronchitis, or bron- chiolitis and bronchial neoplasia, would appear to be more readily related to altered time-con- centration factors attendant upon impaired transit of dust along the bronchial system. A pertinent question, therefore, is: Does dust deposited upon the mucociliary surface impair the rate and effectiveness of that system to perform its role in lung cleansing? There is little reason to believe that dust composed of biologically inert materials should directl}- influence the activity, or alter the anatomy, of respiratory-tract cilia. Such parti- cles fall upon a layer of mucus interposed between the particle and the cilia. It is equally apparent, however, that inert dust may, by physical or physical-chemical means, alter the character of the film of mucus and thus have a very potent effect upon the actions of the cilia and the effectiveness of their function. It is also apparent that dust composed of less inert or of .oluble material, or composed of inert particles upon which soluble materials may be adsorbed, to be subsequently leeched off, might bring , n active substance capable of penetrat- ing the nnicus film and thus directly affect the cilia-bearin~ cells. Exceedingly fine particles of otherwi.>e rather inert materials, as for example colloidal silica, in some in:~tances have very po- tent pharmacologic actions and conceivably might l,enetrate the film of mucus and thus in- fluence the cells. It is not beyond belief that a dust composed of materials having pharmacologic properties might occur in sufficiently fine suspen- sion to penetrate the alveoli and be deposited there, and that the active a,ent might then pass into the vascular system and subsequently influence the ciliated cell, not by direct attack at the cilia-bearing end. but indirectly via the circulation. There have been rclativelv few direct at- tempts to explore the effect of different dusts upon the mucociliary apparatus. A number of inve,ti;ators have utilized the particles spread upon the mucus surface of eilia-bearin_ regions as a means of ineasurin_ the rate of movement 103
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104 GEORGE W. WRIGIiT of the mucus sheet. Variations in the rate of this movement are thought to reflect changes in ciliary activity. Antweiler (1) has made observations on the rate of movement of several dusts applied in this manner, but the observations are pri- marily qualitative in nature. An attempt to quantitate the concentration of the particular dust used, per unit area and thickness of mucus blanket, was not done, probably be- cause of the difficulties one would encounter in such measurement. Nonhydrophilic dusts, such as coal and carbon black, did not seem to alter movement of the mucus blanket in any way. Antweiler noted a rather important difference, in that, with the hydrophilic dusts such as quartz and submicroscopic-sized amorphous silica, there was a considerable delay between the time of applying the dust and the time at which movement of the mucus blanket, as monitored by the dust travel, began. He attributed this to a rapid change in the char- acter of the mucus blanket induced by the dry- ing effect of the hydrophilic particles. He and others have shown that the dryinr of the mu- cus blanket interferes with its movement. Dalhamn (2), who has made similar ob- servations by exposing the mucus blanket to certain gases, showed that although the motion of the mucus blanket was impeded, the ciliary activity was essentially unaffected. This, of course, points up the possibility for error when one studies ciliary action in terms of rate of mucus blanket motion. Dalhamn pointed out that the mucus blanket has depth and that the luminal surface may be different in char- acter from that immediately in contact with the cilia, and hence that the luminal surface may move at a rate slower than appropriate for the ciliary activity. Antweiler also recorded the observation that tiny glass spheres, when placed on the mucus blanket, remain station- ary for a brief period of time before beginning to move; he attributed this to the smoothness of the glass surface. It Nvou1d have been of considerable interest to observe whether or not a mixture of rJazzs spheres and nonh}-dro- philic dust would be carried at different ini- tial rates for the spheres and the other dust particles. The lack of large variation in the rate of movement of the various dusts placed on the mucociliary escalator probably accounts for the relative lack of interest and searcitv of observations along this line. When considering problems of a biologic nature, one sometimes loses sight of the fact that acute effects and subacute effects of an agent reacting with a system may be quite different from the effects of chronic or long- term periods of contact. Biology is replete with examples of adaptation on the one hand, and of accumulative effects on the other, that might be more important than the initial results of contact between a potential influenc- ing agent and the system to which it is applied. Direct observation of the effect of chronic or persistent and repetitive deposition of dust upon the mucociliary apparatus does not appear to have been reported. I hope infor- mation of this sort will be given at this sympo- sium. In this connection, recently I have had the privilege of reviewing, in preliminarv manuscript form, a work by Drs. LaBelle and Brieger (3) bearing upon this point. Their study indicates that exposure of rabbits to cigarette smoke for ten continuous hours, or for five hours a day, five days a week for three weeks, failed to produce a difference be- tween exposed and control animals using their technique for measuring lung clearance. My citing of this work is not meant to indi- cate my acceptance of their results without some question. Rather it is to indicate that, althou~h study of the physiology of cilia and mucus ~ecretion in acute preparations is of great importance, work with chronically altered preparations is absolutely necessary to any comprehensive assessment of the poten- tial of various agents for causing serious harm to humans. Any comments at this time with regard to the influence of chronic dust exposure upon ciliary activity must be based on very indirect observations. The simple fact that men workinc, in unbelievably dustY conditions are able to maintain patent nasal and bronchial passages indicates that the mucociliar' v equipment func- tions relatively well under conditions that might be thought of as stres,ing it _-~evereh-. How much of this tour de force diould be attributed to ciliary action and how much to other inodes of propellin.g mucus remains to be 0009'74ti1
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nts !ity to on 'ct na° to EFFECTS OF INDtiSTRIAL Dti,T seen. The nose and nasal pharynx capture the preponderance of those dusts that we think of as being visible. Relatively few par- ticles larger than 8 to 10 µ in diameter pass beyond the larynx and into the more distal ramifications of the tracheobronchial apparatus and deeper structures of the lung. The actual mass of the total number of particles existing in a cubic meter of dusty air is, of course, not great. Fifty milligrams per cubic meter would be considered excessive. Nevertheless, anyone who has worked for an eight-hour period in a highly dusty atmosphere is well aware of the large amounts of mucus, heavily stained with dust, that will be removed from the nose during, and for a brief period after, the work- ing episode. A1ore dust undoubtedly passes into the nasopharynx and is swallowed. In terms of mass, relatively little dust ac- tually penetrates and is retained more than briefly in the tracheobronchial system and more distal structures. Policard (4 ), for exam- ple, has estimated that a coal miner working in average dusty conditions may take into the tracheobronchial and more distal structures a mass approximating 6,000 gm. over a 30-year period-less than a gram a day. This is made up of particles which are less than 10 µ in diameter and therefore capable of penetrating this region. The effectiveness of the clearance mechanism is so great that, as has been calcu- lated by Policard and others, one per cent or less of the total burden will be found remainina in the lun4 structures at the end of this lifetime of work. This means, of course, that the mucociliary apparatus assisted by the ah-eolar-macrophare system is busily at work each hour of the dav re- moving 99 per cent of the total burden pre- sented to it. One may quibble about the figures just given, and I am sure no one would maintain that these are more than rough approximations of the amount taken in. The accuracy of the amount found at the end of this lifetime of 0xperience is, of course, of a much higher order of reliability. I cite these fiaures on1v to show that even tinder conditions of very heavy load- ing, the cleansing apparatus seems to per- iorm quite well. I realize that the term "we1P" i< completely relative. There is no doubt that the hmos are cleansed sufficiently to maintain 105 open airways, but I would be the first to admit that quantities of material sufficient to cause harm are, in many circumstances, left behind. In this sense, the lung does not be- have well enough. Can anything be learned from data now at hand with regard to the possible long-term ef- fects of dust upon the mucociliarv apparatus? I have given this question considerable at- tention and must confess that I have found nothing that seems to me to be sufficiently trustworthy and at the same time relevant to the question just posed. For example, we have no data indicating whether or not clearance rates change over a long period of repetitive exposure. It might be possible to study this question if one had the specific dust content of a large number of lungs from a "dusty" trade where the exposure had been reasonably constant over a period of 30 or 40 years. If one were fortunate enough to have a sufficient spread of total years of exposure in this series of lungs, one might be able to discover whether or not the rate of dust accumulation was pro- gressivelv accelerated in a manner parallel to the increasinr years of exposure. I was unable to find any such information. If one cannot learn about the effects of long-term exposure to dust from studies of the residue after years of exposure, are there other methods that might be used? In view of modern abilities to stud' v the efficiency of lung cleansinn at any point in time, I would think that such information could be obtained. For rxample, one could measure the rapid clearance rate using one of the traceable materials in- haled in a known time-concentration dose. A study of workers exposed in the "dustv" trades for various periods of time, carried but in this manner, would be verv interesting and might give us a first approximation to the question of chan_,es, if anv, of the clearance capacity of the mncociliary apparatus in pers_ons who have had var}-in,,, dust experiences. An attempt might al.o be made, perhaps retro~pectively, to give tuore detailed attention to the liistoloaic studY of the bronchial epi- thelium in persons who have been elpo>ed to vvious quantities and types of dust. Detailed histolo,ic cleecriptions of the pneumoconioses do not indicate any unusual appearance of .~ .,. O O -1 N
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106 GEORGE W. wRIGHT the bronchial mucosa attributable to long- term inhalation of dust. I am as well aware as the next person of the fact that one usually finds only what one looks for, and it may be that in the past attention has been focused on the parenchymal lesions of the pneumoconioses and no systematic study has been made of the tracheobronchial tree. I am inclined to believe this might be the case in view of the fact that for years no systematic detailed study was made of the tracheobronchial branches in such dis- eases as emphysema, or even in chronic bron- chitis. Of course, even if such detailed studies were made, one would still need to deal with the problem of other causes of any changes observed in the bronchial mucosa-for example, those that might be caused by infection, or by exposure to gas or to other agents that mi;ht co-exist with dust. Numerous long-term studies of dust exposure have been carried out using laboratory animals, and, again, no one has di- rected our attention to an abnormal appear- ance of the ciliated epithelium in such animals. In this connection, however, Schiller (5) re- ported on dogs housed underground in a coal mine and presumed to be exposed to dust in that environment. Dogs examined after two or three years were reported to show hypertrophy of the bronchial epithelium including the gob- let cells. At six years, however, there was marked atrophy of the epithelium and loss of cilia. It is difficult to accept this data as being the result of dust exposure only. Poten- tially irritating gases exist in coal mines. The question of lack of vitamin A must also be considered, and this report does suggest the need for more study in this direction. That the mucociliary apparatus can be im- paired suf$cieiitly to interfere with lung cleans- ing was shown by Robson and co-workers (6), who produced silicosis in rabbits by com- bined exposure to free crystalline silica and either nitrogen dioxide or sulfur dioxide at levels of silica-dust concentration which failed to cause disease in animals exposed to the silica alone. They describe almost total destruc- tion of cilia and flattening of the epithelial cells in the gassed animals. Schlipkoter and Brockhaus (i) have confirmed this findin_Q~ in rats, in terms of quartz-dust retention by ,tnimals allowed to inhale ;ulfur dioxide. IIon-- ev-er, they failed to describe the degree of ciliated cell injury. Before the studies by Robson and his co-workers are given too much weight, it should be pointed out that his studies were not directly controlled, and, in addition, that the concentration of gases must have been extraordinarily high because the mortality rate among his animals interfered completely with any orderly sacrifice schedules. Never- theless, more work in this direction would be of interest. There are some industrial dusts known to possess the biologic property of causing acute inflammation of the bronchial mucosa. Vanadium pentoxide is one (8). Also, there appear to be persons peculiarly sensitive to dusts which, when inhaled, act as a severe irritant to the bronchial mucosa. Presumably such dust reactions would impair the cleansing mech- anism and could increase the transit time. Some investigators believe that a mild stage of such irritation would speed the cleansing action and reduce the transit time. Reliable and rele- vant evidence of this is lackinT. In conclusion, one can say that, although the question regarding the effect of industrial dust upon the mucociliary apparatus is a per- tinent one, there is insufficient evidence at this time upon which to formulate an answer. Acknolcledgment The assistance of Dr. Premvsl Pelnar in search- ing the literature, especially the foreign litera- ture, for information pertinent to the subject of this paper is gratefully acknowledged. REFERENCES (1) Antweiler, H.: Lber die Funktion des Flim- merepithels der Luftwege, insbesondere un- ter Staubbelastung, Beitr Silikoseforsch (Sonderband), 1958, 3, 509. (2) Dalhamn, T.: Mucous flow and ciliary activ- it}- in the trachea of healthy rats and rats exposed to respiratory irritant gases (SO_, HRN, HCHO), Acta Physiol Scand, 1956, 36 (Supplement 123, p. 1). (3) LaBclle, C. jC., and Brieger, H.: Unpublished d:ita. (-1) Policard, A. (Quoted by Gordonofi, T.) : C R J P'ranc Path Minere. Charbonnages de Prance, Paris, 1960 (Oct(lber). 165. (5) Schiller, E.: Inhalation, Retention and Elimi- nation of Dusts from Dots' and Rats' Lungs. n-iili Special Reference to the Alveolar 0009'7463
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EFFECTS OF INDUSTRIAL DUST 107 Phagocytes and Bronchial Epithelium, In- haled Particles and Vapours, Pergamon Press, New I ork, 1961, pp. 342-344. (6) Robson, W. D., Irwin, D. A., and King, E. J.: Experimental silicosis; Quartz, sericite and irritating gases, Canad Med Ass J, 1934, ~331, 237. ';7) Schlipkoter, H. W., and Brockhaus, A.: Ver- suche uber den Einfluss gasformiger Luft- verunreinigungen auf die Deposition and Elimination inhalierter Staube, Zbl Bakt [Orig], 1963,191,339. (8) Sjoberg, S. G.: Vanadium pentoxide (lust: A clinical and experimental investigation on its effect after inhalation, Acta Med Scand, 19b0,13S (Supplement 238). i i- ar
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EFFECT OF CIGARETTE SMOKE ON CILIARY ACTIVITY' TORE DALHAMN We are all aware of the importance at- tached to tobacco smoke in the current debate on causes of respiratory disorders. Along with general atmospheric pollution, cigarette smoke is a major topic of discussion. It is not surprising, therefore, that much research has been devoted to histopathologic study of the respiratory tract after exposure to cigarette smoke, and to methods of regis- tering physiologic changes in the respiratory mucosa. I do not propose to discuss histopatho- logic questions today. The physiologic func- tions which have attracted most attention are transportation of mucus and ciliary activity, and I shall now try to give a brief account of the observations hitherto published. I shall not attempt to describe the methods in detail, however, as these have already been discussed, and also because many of their authors are present here and are more competent than I to deal with such matters. Tobacco smoke is a composite of many sub- stances. Research has sometimes been con- centrated on the total smoke, and has some- times been concerned with attempts to distin- quish between the effects of a particulate phase and a gas phase, or to study the action of single, chemically well defined substances. Mendenhall and Shreeve described experi- ments with tobacco smoke as long ago as 1937 (1) and 1940 (2). They immersed calf tracheae in Tyrode's solution and observed the move- ment of carmine particles along the mucosa. The smoke was brought into contact with the tracliea mainly by dissolving it in Tyrode's solution, which was then substituted for the original immersing fluid. Alternatively, the smoke was blown along the trachea, which was thereafter placed in Tyrode's solution. In all experiments the transit times were reduced by 17 to 40 per cent of the normal. A similar technique for observing the cilio- static action of cigarette smoke n as presented by Rakieten and his co-workers in 1952 (3). 'From the Institute of Hygienp, I:arolinska. In- stitutet, and the Department of General Hv;icnc, National Institute of I ublic Healdh. Stockholm. Sweden. Respiratory epithelitun from human subjects, rabbits, and rats was placed in Locke-Ringer's solution. Vlenthol, nicotine, and mixtures of these two substances were studied, as well as smoke from mentholated and nonmentholated cigarettes. In each case the test substance was dissolved in Locke-Ringer's solution, which was then applied to the mucosa. Cigarette smoke was produced by a smoking machine and was passed through a series of four ab- sorption flasks containing Locke-Ringer's so- lution. Ciliary activity was noted, but not velocit}• of mucus flow. Menthol in Locke-Ringer's solution (0.04 per cent) had no demonstrable toxic effect on the mammalian epithelium. Nor was any synergis- tic action of menthol and nicotine found when the mucosa was exposed for one hour to this menthol concentration together with nico- tine (5 mg. per kilogram). Nicotine alone, in the same concentration, had no visible effect on ciliarv activity. The effect of cigarette smoke on ciliary activity was the same with mentholated as with nonmentholated ciizarettes. A smoke-dosage machine was described as early as 1939 by Proetz (4). He used it chiefly to study the effect of tar deposit on the respiratory mucosa. Although Proetz re- ported no observations on ciliary function, his ingenious apparatus is well worthy of mention. Hilding (5) devised a technique which sim- ttlated fairly closely the conditions in human cigarette smokers. His apparatus permitted variations in mode of smoking and in distribu- tion of smoke. He studied trachea and hmgs from freshly killed cows. The lower third of both lungs was amputated so that the smoke could hass throii:;h ihe trachea and out through the cut bronchi. The amount of smoke was calculated in different ways, such as number of puffs, length of cigarettes in millimeters, and number of completely amoked cigarettes. By openin; up the trachea and bronchi, the deposition of tar and the transportation of mucus could be observed. Hildina found that niuciis flow ceesed in ~ziY of ten cases and was greatl.v reduced in the other four cases. 108 r, t~ c, l; t! n 0 c F 0009'7465
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EFFECT OF CIGARETTE SMOKE 100 Hilding reported many other interesting ob- servations with respect to deposition of tar in smoking, but these were less relevant to ciliary activity. Highly relevant, on the other hand, were his experiments with scrapings of live epithelium, which were placed in a hanging drop of Ringer's solution and studied under a microscope. In this way it was possible to ob- serve ciliarv movement directly. When the Ringer's solution had first been exposed to ciga- rette smoke, the epithelium reacted with cilio- stasis. In another report from 1956, Hilding (6) took up the problem of what he called "de- ciliated islands." He stated: "There are no large non-ciliated areas below the vocal cords comparable to those in the upper tract, but there are islands of squamous epithelium or metaplasia devoid of cilia." Hilding thought that in cigarette smoking a particularly high concentration of substances in solution might occur over these deciliated islands. Mucus flow there, being already poorer than elsewhere, could cease after exposure to cigarette smoke. Experiments performed by Hilding in vitro suggested that this might be the case. The effect of total cigarette smoke on the cili- ated epithelium of the respiratory tract has been studied also at the Institute of Hygiene, Karolinska Institute (7). These experiments comprised three parts. In the first part, ten live rats were used. The trachea was opened lengthwise and the rats were placed in a chamber with warmed and moistened air. The ciliary movement in the trachea was then observed and was registered by means of a high-speed cinecamera. Contact between smoke and trachea was made in the simplest possible way : An assistant smoked a cigarette, collected the smoke in her mouth and then slowly blew it through a short plastic tube over the trachea. Control tests in which moist- ened and warmed iir was blown over the trachea had previouslY been performed and had shown no statistically demonUtr,ible effect on ciliar.- beating: The mean rate of activity was 1,099 beats per minute before and 1,067 beats per minute after five mintrtes of exposure to air without smoke. Cigarette smoke, by contrast, had a clear eftect on tracheal cilittry :,ctivity. In seven of the ten rats, ciliary beatin, ceased after contact with the smoke of a single ciga- rette. The second part of the experiments com- prised observations on the extirpated tracheae of ten rabbits. The tracheae were placed in the warmed and moistened chamber and were otherwise treated as in the experiments on live rats. The results were in agreement with those from the previous experiments. Ciliary activ- ity ceased in six of the ten extirpated tracheae. Finally, the same technique was used to study seven preparations of extirpated human mucosa from maxillary sinus, ethmoid sinus, and bronchus. In six of the specimens, ciliary beating was arrested by the smoke from one cigarette. All the experiments I have mentioned were concerned with the effect of total tobacco smoke on ciliary function. In general, the results showed that the smoke had a ciliary- depressant effect. At that time, no particular efforts were made to separate the various frac- tions of the smoke and in this way possibly to fix the observed effects to single or multiple constituents. Experiments with these aims in view were carried out in more recent years. In this con- nection, it may be appropriate to single out a group of studies in which the particulate phase of cigarette smoke was compared with the gas phase, various types of filters were investigated, and experiments were made with chemically distinct components of the smoke. Before I review the results of these experi- ments, it is necessary to have a clear concep- tion of what is meant by the particulate and the gas phase of tobacco smoke. The writers who have described practical investigations seem in the main to be in agreement on def- initions. Falk, Tremer, and Kotin (8) stated in 1959 that the gas phase is what remains when the smoke has been filtered as effectively as possible. A similar definition was indicated in 1963 by Iiensler and Battista (9): "The gas phase of cigarette smoke wa:~ studied by inser- tion of aa holder containing a Cambridge ab- solute filter pad between the cigarette and ex- posure chamber." At Iiarolinska Institute we haVe ti~:ed analogous techniques, including the Cntnbriclae filter. In their 1959 report, Falk, Tremer, and Iiotin (S) de~zcribed extensive observations ! I :~
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110 TORE DALHAMti on the effects of whole cigarette smoke, smoke from filter cigarettes, individual components in cigarette smoke, and allied compounds on the physiologic activity of the ciliated mucus- secreting epithelium. The experiments were made in vitro on "ciliated epithelium of the frog and trachea of the rat and rabbit." Two methods of exposure were used. In the first method, the smoke was passed through water, and microdrop quantities of the solution were placed at the downstream end of the tissue for periods of 2 or 30 seconds. In the second method, the smoke was blown onto a small portion of the tissue for periods of 2 seconds (direct impingement). The effect was expressed as rate of mucus flow in millimeters per min- ute. Both the aqueous solution of smoke and the direct impingement of smoke on the mu- cosa produced rapid and distinct slowing of mucus flow. Remarkably, this reaction per- sisted for at least 6 hours when the impinge- ment method was used. Falk and his co-workers investigated the gas phase of cigarette smoke (after passage through millipore and glass wool filters). In these experiments, too, an aqueous solution and direct impingement were used and the exposure times were 2 and 30 seconds. They stated: "In neither instance was any response obtained, as neither stimulation nor inhibition could be observed." The same writers reported ob- servations on repeated exposure of mucosa for different times-30 seconds and 2 seconds. Inhibition and recovery of mucus flow showed only small variations in accordance with the duration of exposure to the gas phase. When the tar which collected in the millipore filter was applied in solution to specimens of mucosa, mucus flow was rapidly inhibited. Although time, unfortunately, does not per- mit a detailed discussion of these extremely interesting experiments, it is important that most of the substances which were tested in- dividually, such as nicotine, ammonium hy- droxide, acetonitrile, and phenol, were found to retard mucus flow. Iicnsler and Battista (9) published obser- vations that were to some extent contradictory to those of Falk and his co-workers. They measured particle transit time on excised tracheal mucosa of rabbits and other animals before and after exposure to total and filtered cigarette =nujke and to individual components of the smoke. A heated exposure chamber was used and to it were connected a smoking machine and a smoke chamber. In order to standardize experimental conditions as far as possible, the trachea was coated with Tyrode's solution in egg white. Exposure to smoke was made by an automatic device and was stand- ardized by means of the smoke-dosage machine. This apparatus delivered the equivalent of two puffs per second into the exposure chamber. As in the studies reported by Falk and co- workers, the filters used by Iieneler and Battista included the Cambridge absolute filter. Seven brands of commercially available cigarettes, five with filter and two without filter, were studied with regard to effect of the smoke on particle transit time. The filters were of the conventional cellulose acetate type, but in one brand there was also a filter sec- tion of paper impregnated with carbon. The tracheae were exposed to the smoke for ten seconds after each two-second (40-ml.) puff. The particle transit time was measured after each twelve-second period. It was re- duced b~, all the tested cigarettes and there was no major difference between the brand.s in this respect. These observations were checked in experi- ments comparing the effects of smoke from re~~iilar length cigarettes with and without a Cambridge filter. No significant difference «as found with regard to the average ED, (50 per cent inhibition) values for the gas phase and the total smoke. Kensler and Bat- tista wrote: "Thus, these experiments indicate, as did earlier work with the immersed prehara- tion, that the major portion of the short-term ciliary depressant action of cigarette smoke is due to components of the gas phase." By means of activated charcoal filters, it was possible to remove at least part of the gas phase. It was shown that this charcoal filter, when used together \ti-ith a cellulose ace- tate filter, ;reatly diminished the ciliary-depres- sant action of cigarette smoke. The same writers studied the inhibitory ac- tion of individual components of the gas phase. Among the substances which seemed to be significantly ciliary-depressant in terms of lheir concentration in cigarette snoke were h}•drogen cvanide, formaldehyde, acrolcin, and :lnlmonla. Interesting aspects of rlie effect of various s ~. I 0009'7467
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EFFECT OF CIGARETTE SMOKE 111 er was noking der to ar as ~ rode's ; e was -tand- ~ chine. ,f two mber. id co- ' ttista ::ilable :thout uf the filters type, ~ see- Le for t)-ml.) ^.~ured is re- I w•as d:4 in ~ <peri- from 1,ut a :rence ED,~ e gas Bat- licate, !para- -term )ke is rs, it ,f the arcoal ace- ~pres- ~- ac- ~hase. o be of were and t rious ~ubstances on ciliary epithelium were presented by Bernfeld, Nixon, and Homburger (10) in 1964. They studied the reaction of ciliary transportation of mucus to irritant vapors. The observations were made on excised frog esophagus. The specimens were immersed in frog Ringer's solution with added adenosine triphosphate (ATP) and stored in a special chamber. When the tests were made, the cover- ing solution was poured off and the tissue preparation was exposed to the various test substances. After the exposure, ATP solution was again added. Particle transit time was measured before and after exposure. In exposure to cigarette smoke, 35 ml. of smoke were introduced into the exposure chamber and allowed to remain there for 15 seconds. After an interval of 60 seconds, another cycle was begun. The cigarettes gen- erally were smoked to a predetermined butt length. These writers found that smoke from filter cigarettes retarded mucus transportation con- siderably less than did unfiltered cigarette smoke. The tissue preparations were exposed also to phenol vapor alone and in combination with cigarette smoke. Whereas the effect on mucus flow was little or none when phenol was used alone, phenol in combination with ciga- rette smoke produced a much stronger effect than had been expected. This was attributed to potentiation, for which the particles in the smoke were responsible. The ciliotoxic action of individual sub- stances in cigarette smoke was investigated by Wynder and his co-workers in experiments which I can only briefly survey here. W}-nder, Iiaiter, Goodman, and Hoffmann (11) applied substances in aqueous solution to the ciliated gill epithelium of the fresh-water mussel Lamellibratichiata 2uaio. The actively beating gill cilia were observed directly using trans mitted light. In 1962, 1C' vnder aml IIoffm;inn (1_'1 ~howcd that the ciliostatic action of cigarette smoke could be considerably reduced by u~ing filters which selectively removed phenol. They fur- ther stated that inhibition of ciliarv move- ment is m,tinly c<amecL b~• free radicals and acidic components and onlV to a niinor extent by volatile aldehydes and ketones. In a later paper, Wynder, Goodman, and Hoffmann (13) specially dealt with the cilio- static effect of carboxylic acids and aldehydes. These substances were chosen because the acidic and the weakly acidic portions of the cigarette smoke had been found to be the most potent depressants of ciliary activity. Application of such derivatives in one per cent aqueous solution to ciliated epithelium of mussels produced inhibition of ciliary activ- ity. In discussing the ciliotoxic action of the gas phase and the particulate phase of cigarette smoke, Wynder and his co-workers ex- pressed the following opinion: The actual distribution of compounds in a puff of cigarette smoke, conveniently thought of as an aerosol consisting of a gas phase and a disperse phase, remains largely a matter of conjecture. Since about 27 per cent of the particulate matter is soluble in water and the principal volatile ciho- toxic components appear to be water-soluble, it is probable that the particulate matter contains volatile ciliotoxic components. Adsorption of gase- ous material by the particles provides a second, though relatively unimportant method of gas transport in the smoke stream. This research group assessed the ciliotoxicity of nuany different substances, but I shall men- tion only that, like Kensler and Battista (14) and the French writers Guillerm, Badre, and Vignone (15), they found that acrolein was highly ciliotoxic. Before I summarize these remarks on the reports by other research workers, I shall briefly describe some further observations made at the Institute of Hygiene in Stockholm. The experiments were based essentially on two techniques. The first involved registra- tion of ciliary activity in the trachea of live rats, guinea pigs, rabbits, or cats with the aid of a high-speed cinecamera (16). When rabbits or cats were used, they could be al- lowed to breathe through the natural respira- tory passages, thus obviating the necessity of heating and moistening the inhaled air. Secondly, a ~moke-dosage machine was used (1 i). Conibination of these two techniques permitted the following experimental condi- tions. 1. Irse of live mamuials. 2. Inhalation of cigarette smoke through the mouth or directly in the trachea.
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112 TORE DALFIAVSN 3. Placing of the cigarettes close to the ani- mals, so as to ensure fresh smoke. 4. Regular interruption of smoke inhalation by intake of fresh air. 5. Cigarette combustion standardization with respect to suction time and suction force. 6. Approximation of the amount of inhaled smoke to that in smoking by humans. 7. Smoking of a representative part of the cigarette. The following were among the experiments made under these conditions (18-20) : The ciliostatic effect of a single type of ciga- rette without and with a cellulose acetate filter was studied with regard to the significance of the interval between each cigarette smoked. The animals "smoked" 25 puffs of each cigarette, and the number of puffs preceding total cessation of ciliary beating was calculated. The intervals between cigarettes were 0, 2, 5, 10, and 15 minutes, and each group of experi- ments comprised five cats. Irrespective of the interval between ciga- rettes, the filter cigarettes were less ciliostatic than were those without filters. The longer the interval, however, the greater was the number of puffs required to produce ciliostasis. This ap- plied both to filter and to nonfilter cigarettes. A control group of cats, which were placed in the experimental apparatus for three hours but were not exposed to cigarette smoke, showed no diminution of ciliary activity. In this way a dose-response curve was obtained for the filter and for the nonfilter cigarettes. There was no statistical difference between the slope of the two curves. Another series of experiments (21) has just been completed at the Institute. This was planned to investigate the ciliostatic effect of ~moke from cigarettes fitted with various types and combinations of filters. The type of tobacco was identical in all the cigarettes and the conditions of the experiments otherwise were the same throurhout. The effect of un- filtcred cigarette smoke wa~ conipared with that of ci,arettes with a cornmercial ccllulose acet,rte filter, a C.ImbridE~e absoliitc filter, a filter c•on- taining only activated charcoal, or commercial filters composed of cellulose acetate plus charcoal; two types of these compound fil- ters were stttdied. In order to te~t the effcc- tiveness of the filters, the smoke which had passed through the various filters was anah•zed by gas chromatography. The mean number of puffs required to produce ciliostasis was 91 when no filter was used and 600 when the cigarette smoke was passed through a Cambridge filter. Cigarettes fitted with a compound charcoal and cellulose filter caused ciliostasis after an average of 512 puffs. The corresponding figures for the ciga- rettes with commercial cellulose acetate or char- coal filters were 194 and 170 puffs. The ciliostatic effect in the various experi- ments was compared with the gas chromato- grams of the smoke. In this connection I shall mention only two substances tar and isoprene. In unfiltered cigarette smoke, the content of tar was 20.3 mg. per kilogram, and the content of isoprene was 56 y X 10-' per ciga- rette. As expected, application of a Cambridge filter resulted in a great reduction of the tar content-to 0.5 mg. per kilogram, but the iso- prene content fell only to 40 y X 10-' per cigarette. Filters of charcoal alone removed very little tar (19.4 mg. per kilogram), but greatly di- minished the content of isoprene (9 y X 10-' per cigarette). Cellulose acetate alone reduced the tar content to 12 mg. per kilogram, but scarcely affected the concentration of isoprene. Cellulose acetate plus charcoal trapped both substances: The content of tar fell from the initial 20.3 to 14.6 mg. per kilogram and that of isoprene from 56 to 8 y X 10-1 per cigarette. These experiments thus ~hon-ed that the ciliotoxic properties of cigarette smoke can be greath reduced by using various types of filters. Tlie Cambrid(ze filter was particularly effective, but it rendered the cigarette flavor- less. A combined cellulo~:e acetate and charcoal filter, however, appreciablly diminished the ciliostatic action of the sinoke. Less striking but clearly significant inhibition of ciliostasis occurred when either charcoal or cellulose ruetate was ired alone. Gas clirmnatoPraphy confirmc l the rcduc- tion of ihe ~:moke components (,-,as phase and particulate phase) that was expected when the various filter type., were u'sed. These remarks on the effect of c i;arette _moke m;iv be summarized bY ~zalying that c ci t i 0009'7469
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EFFECT OF CIGARETTE SMOKE 113 iad zed to +.ras isas ites lose .i12 ,,a- rr- tri- ~ I nd nt he ,:a- ::Pe 'ar -o- ; )er r tle li- 10-' ced 'ut ~e. oth the hat the ,an of rh- or- oal _he .11 (._T, lEis 1 -se the results of some experiments were in agree- ment, whereas others were to some extent con- tradictory. All the experiments on exposure of ciliated epithelium to tobacco smoke showed that the smoke contains substances which can slow trans- portation of mucus by depressing ciliary activ- ity. This is scarcely remarkable when one considers that many gases, vapors, and particles have a similar action. But the special interest of tobacco smoke is bound up with the question of its pathogenic effect in the respira- tory tract. That cigarette smoking is a con- tributory cause of bronchogenic cancer seems to be clear, but whether or not mechanisms other than ciliotoxicity are primarily more important is still a matter of doubt. Many workers, how- ever, believe that the ciliary-depressant action of cigarette smoke is extremely important. A wide field therefore remains for investigations of the detailed mechanism of this effect. Although there is general agreement that cigarette smoke contains ciliotoxic substances, difficulties immediately arise when one at- tempts to assess particulars of published ob- servations. The ultimate aim of all research on smoking, however, is to elucidate the con- ditions in human smokers. A point to be noted is that the transit time of particles along the mucosa is commonly used as an indicator of ciliotoxic effect. Although this may be warrantable, we must bear in mind that the functions of mucus transpor- tation and ciliary beating may be in some degree mutually independent, and that mucus transportation possibly could be reduced al- though ciliary activity is unimpaired. That the two functions may be equally important in clinical physiology is another matter. In addition, the animal species ranged from mussels and oysters to small and large mam- mals. The observations were made on excised tissue preparations in some experiments and on living animals in others. Perhaps the most important \ariations were those in the numerous iuetlrods of exposure to the smoke. These methods ranged from simple blowing of smoke alone the trachea to complicated stnoke-dosa„e machines. Dosage of the inhaled smoke is clearly cui essential fac- tor, but smoke is difficult to dispense accurately :1s compared «-ith individual cheuir.rl sub- stances. Several investigations have been based on a standard volume of about 35 ml. per puff in human smoking, sucked through the cigarette in the space of a second or two. Other workers dissolved tobacco smoke in maximal concentrations in water, et cetera, and applied the solutions to the mucosa. In recent years the efficacy of filters has been intensively studied. In most reports it is suggested that reduction of the particulate phase, for instance by a Cambridge-type filter, appreciably reduced the ciliotoxic action of the smoke. Some writers maintain, however, that this action resides chiefly in the gas phase. My personal opinion is that much could be gained if experimental methods of exposure to total cigarette smoke could be standardized, even if only in general principles. In studies on individual components of the smoke, such as phenols, acrolein, et cetera, standardization is less important, since concentrations can be stated with relative ease. I believe, too, that in presenting results one should clearly state whether the effects of smoke were observed on mucus flow or on ciliary beating. How- ever, it is not necessary to calculate the rate of beat. The ciliotoxic action of the cigarette smoke probably occurs too rapidly to permit observation of ciliary slowing preceding total cessation of activity. REFERENCES (1) Mendenhall, W. L., and Shreeve, K.: The effect of cigarette smoke on the tracheal cilia, J Pharmacol Exp Ther, 1937, 60, 111. (2) Mendenhall, W. L., and Shreeve, K.: The effect of tobacco smoke on ciliary action, J Pliarmacol Exp Ther. 1940, 69, 295. (3) ltakieten, N., Rakieten, M. L., Feldman, D., and Boykin, JI. J.: Zlammalian ciliated respiratory epitlrelitun: Sttuiies with par- ticular reference to effects of menthol, nico- tine and smoke of inentlrolated and non- mentholated cigarettes. Arch Otolaryng (Chicago), 1952, 36, 495. (4) Proetz, A.: tiome preliminarr experiments in the study of ci:,•arette <tuoke an(l its ef- feets npon the r(~,piratorv tract, _lnn Otol, 1939..;S, 176. (5) FIilding, A. C.: On cigaretto smoking, bron- chi.d (;lrcinoma and ciliarv action: II. Ex- perimental stud' v on the filtering action of cow :s hmga, thE• depo.~ition of tar in the bronchial ~tree and removal b' v ciliary ac- tion, New Eng J\led, 1956, 1155. (6) Hilding, A. C.: 011 rigaretto stnoking, bron- , hial carcinrrmu and ciliuly ,-timn: Ill. Ac-
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] 1-1 TORE DALHASIN E cumulation of cigarette tar upon artificialh- produced deciliated islands in the respira- tory epithelium, Ann Otol, 1956, 65, 116. (7) Dalhamn, T.: Tlie effect of cigarette smoke on ciliary activity in the upper respiratory tract, Arch Otolaryng (Chicago), 1959, 70, 166. (8) Falk, H. L., Tremer, H. M., and Kotin, P.: Effect of cigarette smoke and its constitu- ents on ciliated mucus-secreting epithelium, J N at Cancer Inst, 1959, 23, 999. (9) Kensler, C. J., and Battista, S. P.: Compo- nents of cigarette smoke with ciliary-de- pressant activity: Their selective removal by filters containing activated charcoal granules, New Eng J D4ed, 1963, 269, 1161. (10) Bernfeld, P., \ixon, C. W., and Homburger, F.: Studies on the effect of irritant vapors on ciliary mucus transport: I. Phenol and cigarette smoke. Toxic Appl Pharmacol, 1964, 6, 103. (11) Wynder, E. L., Kaiser, H. E., Goodman, D. A., and Hoffmann, D.: A method for determining ciliastatic components in ciga- rette smoke, Cancer, 1963, 16, 1222. (12) Wynder, E. L., and Hoffmann, D.: Studies with the gaseous and particulate phase of tobacco smoke, Proc Amer Ass Cancer Res, 1962, 3, 373. (13) Wynder, E. L., Goodman, D. A., and Hoff- mann, D.: Ciliatoxic components in ciga- rette smoke : II. Carboxvlic acids and aldehydes. (From the Division of Environ- mental Cancerogenesis, Sloan-Kettering Institute, New York.) (14) Kensler, C. .J., and Battista, S. P.: Inhibition of mammalian ciliary transport activity by gases and aerosols with special reference to duration of action, Fed Proc, 1964, °3 (abstract, p. 105). (15) Guillerm, R., Badre, R., and Vignone, B.: Effets inhibiteurs de la fumee de tabac sur 1'activite ciliare do 1'cpithelium respiratoire et nature des composants responsables, Bull Acad Nat Med (Paris), 1961, 20, 416. (16) Dalhamn, T.: The determination in vivo of the rate of ciliary beat in the trachea, Acta Pliysiol Scand, 1960, 1,9, 242. (17) Dalhamn. T., von Essen, E., Kajland, A., and Rylander, R.: A machine for intro- ducing a regulated amount of tobacco smoke into an animal, Int J Air Water Pollut, 1963, 7, 511. (18) Dalhamn, T., and Rylander, R.: Ciliastatic action of smoke from filtertipped and non- tipped cigarettes, Nature (London), 1963, 201, 401. (19) Dalhamn, T.: Studies on tracheal ciliary ac- tivity. Special reference to the effect of cigarette smoke in living animals, Amer Rev Resp Dis, 1964, 80, 870. (20) Dalhamn, T., and Rylander, R.: Ciliastatic action of cigarette smoke at varying cx- posure levels. Accepted for publication in _1rcli Otol. 1965. SI. 379. (21) Dalhamn, T., and Rylander, R.: Cigarette smoke and ciliastasis. Varying composi- tion of smoke. lAccepted for publication in Acta Ph}-?iol Scand.)
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PATHOGENESIS OF CANCER IN A CILIATED MUCUS-SECRETING EPITHELIUM` PAUL KOTIN, DORIS COURINGTON, AND HANS L. FALK The anatomy, ultrastructure, and physiology of ciliated cells and their role in the defense mechanisms of the respiratory tract have been elegantly described in the several presenta- tions to this meeting. I should like to discuss the effects of irritants on the activity of ciliated cells as manifested by (1) alterations in the rate of mucus flow, (2) abnormal deposition and retention, and (3) changes in the histo- anatomy of the respiratory tract, particularly as they may be related to the pathogenesis of pulmonary cancer. The increasing incidence of lung cancer has directed attention to the presence of carcinogenic agents in the respira- tory environment. One of the functions of the mucus blanket covering the respiratory epi- thelium is the protection of the underlying cells against the adverse effect of these and other inhaled materials. This is accomplished by the mechanical barrier provided and by the continuous cephalad flow of the mucus stream transporting deposited particulates as well as dissolved liquid and gaseous substances to the oropharynx. The physiologic rate of flow is sufficient to render unlikely the abnormal ac- cumulation and prolonged residence of partic- ulates at any given site. However, under certain adverse conditions, the inhalation of chemical or biologic agents present in the respiratory environment can alter the normal pattern of both mucus secretion and ciliary action. Ex- perimental data indicate that attenuation of these two components of the physiologic de- fense is uniquely compatible with facilitating the biologic action of inhaled environmental carcinogenic agents and associated materials which enhance their effect. It is generally accepted that, independent of site, neoplasms originate from cells which have retained their ability to divide. In cer- tain tissues, as for example the ~kin and lung, these cells are referred to as ba~al cells both because of their anatomic position and be- 'From the Department of Health. Education, and Welfare. U.S. 1'ublic Health ~crviue, Aational Institutcs of Hcalth, National Conc(~r Institute, 13ethesda, MarYl:md 20014. cause of their ability to divide and differenti- ate. In the respiratory epithelium, the basal cells are mechanically separated from the external environment by an overlying layer or layers of cells and a mucus blanket. In order to ascribe a carcinogenic effect to chemical agents in the respiratory environment, it is necessary to identify a progression of events which will permit their entry into the basal cells. Since carcinogens occur in the environ- ment, either adsorbed on soot particles or as aerosol droplets, the initial requirement for any effect is particle deposition and reten- tion. This is necessary to permit elution of carcinogens from particulates in which they may be adsorbed and to allow sufficient time for cellular entry. Irritants, by virtue of a nonspecific effect on the respiratory epithe- lium, can alter ciliary action and modify the physical and chemical properties of mucus with a resultant reduction in the rate of mucus flow. This, in turn, permits abnormal reten- tion and local concentration of carcinogenic ;lgents due to accumulation of particles. Specific alterations in the physiology of ciliary action and mucus transport have already been described by ourselves and others (1-3). The relevance oi these altera- tions in the carcinogenesis sequence is shown in figures 1-3, which schematically describe the response of the respiratory epithelium to the repetitive action of irritants. Representative histologic sections demonstratinv the se- quence in vivo have been previously reported (4). It was noted that the initial effect of aerosols is limited to the superficial cells, with changes characterized by marked hypersecre- tion followed by desquamation down to, but not inclndinr, the basal cells. Recognizing that cells of the basal cell layer are the re- quired targets for c:u•cinoQenic action, the ~equcnce of desquamation and reRencr:ttion in I fie presence of a carcinoeenic ~timulu~ creates an optimal environment for malignant trans- formation. I'ir~t, appo~ition hetween carcino- gen and basal cell can uccttr: and secend, the lmolifcrative efiect provided hY thc ~timulus 115
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oa 1 !I/ ~ {°1~~ .T L,°1't°]¢VTLI 4. 1 0j 01 0~ . ; C. f3 ~ ~ ~ e e. °y Jn ° I L~ ,~ is 9 f 0 0 M1W -77 Fics. 1-3. Schematic representation of sequential, superficial, epithelial decquamation and regeneration following exposure to respiratory tract irritants. The proKression of changes through basal-cell hyperplasia and ultimate squamowz-cell metaplasiu reprrment rc:~ponse 1o irritant. L`Itirnate development of neoplasia apparently depends on presence of carcinogenic agent in respiratorY epit.helium. 1 t 116
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CANCER PATHOGENESIS IN CILIATED EPITHELIi"JI for tissue repair is conducive to carcinogenic response. Repetition of this sequence can, by ~tages, lead to abnormal repair, with the pro- ;ression of lesions from hyperplasia to meta- ; lasia, to metaplasia (figures 4, 5) with atypical ohanvs, and ultimately to cancer. In the -ame study, the deposition and retention of >oot was verified, and the histologic observa- tion of deposition was confirmed by quantita- tive studies on soot retention in the lungs of rabbits exposed to synthetic smog. The distribution and extent of sites demon- -4rating effects of irritant exposure are depend- ent on several factors. First, the degree of environmental exposure; second, the anatomic structure of the tracheobronchial tree; and third, antecedent or concomitant pulmonary disease. Experimental and clinical studies have established that the bifurcations of the trache- obronchial tree, e.g., miniature corynas, respond in an exaggerated manner as determined by the prominence of early signs of goblet-cell stimulation and ultimately by anatomic changes of metaplasia and in situ neoplasia (figure 6). This observation is compatible with the experimental observation that direct impingement of aerosols on a ciliated mucus- secreting epithelium results in a more pro- nounced effect on the rate of mucus flow than does passive settling of aerosol particles. Pulmonary infection, particularly that char- acterized by a proliferative response, also helps to localize the site of tissue change. The sites of particle impingement and dep- osition in the arborizations of the respiratory tract are, of course, a function of physical laws governing particle movement and settling. It is of interest that under experimental condi- tions, particle deposition occurs more readily in the more distal segments of the tracheo- bronchial tree. At first this observation ap- peared to be incompatible with the assunip- tion that primary bronchial carcinomas had their origin largely in the mainstem bronchi. This belief, however, was based primarily on studies using necropsy material and under- standably included a majority of cases in which tumor size was great and point of origin was difficult to ascertain. However, recent reports seriously question the validity of the concept that most primary lung cancers are hilar or central in origin. Rigler (5), in a roentgen atudY Ili of the evaluation of lung cancer, noted that certain bronchial carcinomas grow eccentri- cally, and that what may present as a hilar tumor to either the roelt,enolog_ist or bronchos- copist is, in fact, a neoplasm that may well have arisen in the segmental bronchi or beyond. In reviewing 153 surgically resected speci- mens, Leibow (6) commented: The definition of "peripheral" lung carcinoma that we have used is "a carcinoma of the htng with its center of mass beyond a segmental bronchus." Obviously, in many of the specimens at the time of resection, the proximal margin of the tumor involved a segmental or even more proximal bronchus, yet it is not difficult to establish with reasonable certainty simply by gross inspection that the origin must have been much farther out in the periphery. Additional evidence is provided in some instances by serial films in our series, as in that of Rigler. In the present material all but five (83 percent) of the 32 tumors with honeycombing were of pe- ripheral origin, while in the other 121, 57 percent were considered peripheral by the same criteria. More recently, Garland and associates (7), in studYing the apparent sites of the origin of primary carcinomas of the lung, observed that of 463 cases of microscopically verified primary lung cancers, the sites could be deter- mined with reasonable certainty in 150. He concluded that the data for his entire series corresponded well with the findings of Leibow and Rigler in similar cases. We thus see that as an increasing number of lung cancers are observed either roentgenographically in an early state or in the surgical pathology labora- tory, ihe central tumor mass appears to be moving peripherally. ;AIacklin (S) and more recenth' Hilding (9) have suggested that the cephalad streaming of mucus results in a relative hyperconcen- tration of deposited carcinogenic materials in the hilar or central areas as the area of the tracheobronchial tree decreases. This central region of the tracheobronchial tree was sn;- gested by llacklin as being "preferred bY bronchial carcinoma because it is here that the concentration of carcino,_zens from inhaled cigarette smoke, etc., is densest in the out- drifting mucus." He further explained the pre- dilection of arborization site~ for maximal ef- feet because '`the' se forkine~ were the scene o>: extra accumulation aud also a lingering of the
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118 KOTIN, COURINGTON, AND FALK , . y 3• FIG. 4. Focal area of metaplasia presumably at site of impingement. Note ciliated epithe- lium on either side of metaplastic area. (Magnification, X125) Frc. 5. Higher po«-er magnification of areaa of squamous metaplasia adjacent to ciliated bronchial epithelium. (:1launification, ,•250) carcino. areat lium." zzignific: in the - effect, tion :i and c( what « The ing d. partict; in thi~ and tl the na tively. one nl 0009'74'75
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! CANCER PATHOGENESIS IN CILIATED EPITHELIli\I Fic. 6. Schematic representation of sites of early and initial elective sites of sequential epithelial changes described. Rough approximation of distribution of particle deposition on basis of size is given. ,_,arcinogen which then have leisure to act with _great strength upon the underlying epithe- iium." The effect of streaming is probably less ~ignificant than originally assumed, in that in the presence of prolonged pulmonary irritant o tfect, particles tend to accumulate at deposi- ;ion sites. To a degree, of course, streaming and concentration are not incompatible with What we observe in human lung specimens. The role of the ciliated epithelium in provid- iii, defense against the irritant action of particulate matter involves, as has been shown in this conference, both the force of the cilia and the chemical and physical properties of t he mucus, both qualitatively and quantita- ~ ivel' v. Despite the title of this symposium, Ine mi,ht havr Nri.~hed that nreater emphasis 119 had been placed on the goblet cells, as mucus flow is really the fundamentall defense barrier; at the same time, of course, it is recognized that on an all-or-none basis, cilia are indis- pensable for mucus flow. The physical and chemical characteristics of the mucus are also critical. We have all observed, at one extreme, wildly beating cilia incapable of propelling a highly viscid, tenacious. thick mucus, and, at tiie other extreme, mildly beating cilia pro- pellin; awaterv fluid mucus at a great rate. The action of chemical irritants is essentially mirrored by that of certain bacterial and viral factor;, and, of tlte two, tlie latter appear to be more significant in the production of changes potentiallv related to carcinogenevis. It is of intere.=t, however, that metaplasia, as seen
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120 KOTIN, COI;RI\GTO\, :1SD FALK classicaliv in bronchiectatic cavities, and hy- perplasia, as seen clinically and experimentally following influenza virus infection, do not progress to lung cancer. To further identify the mechanisms involved in the action of environmental carcinogenic agents in initiating pulmonary cancer, ultra- structural studies were undertaken in an attempt to complement the morphogenetic pat- tern seen in tissue. While it was evident from light microscopy that there is an increase in the ratio of goblet cells to ciliated cells, electron microscopy studies failed to show any consist- ent variations in the structure of the mucus- secreting cells. It is generally recognized that the structure of the epithelium varies in different :egments of the tracheobronchial tree. In the bronchiolar area, in the mouse at least, secretory cells with the classic structure of mucus-secreting cells are not readil5 seen. Instead, the secretory cells, though obviously active, are structurally different, and it has been suggested that these cells are the sources of the surfactant material which lines the alveoli. One can postulate relative ineffectiveness of ciliated cells in this area of the bronchus, roughly in those areas one millimeter in diameter and smaller, by noting the cul-de-sacs in which the ciliated cells are nested so that a satisfactory site for particle accumulation and aggregation is in existence. The contribution made bv this Ftc. 7. Ele tron micrograph of a bronchus from a uwuse ehronicallc ex),~)s(,d t,) c:u-bon dust and "smog," showing gohlet-cell h, yperplasirt. Infrr<ptcnt ciliated cclls (c) lwh their elertron-luscent cytoplasm vontrast with the goblet c-11s (m) whose c.-toplastn is ntore electron-dense and contains satterel muws kranules. A L,t<al cell (h) is also pri >~nt. (O~- mium tetroxide fiscuion; ma>;nification. ~3,675) Ct rr 0009'74'77
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i CANCER PATHOGENESIS IN CILIATED EPITHELIti\i anatomic feature with its possible carcinogenic sequence is in need of investigation. As noted in the opening portion of this pres- entation, apposition of the carcinogenic action with the basal cells was postulated as a neces- sity for initiating the carcinogenic process. Under light microscopy, it was apparent that desquamation of the epithelium offered the most likely explanation. In an attempt to identify ultrastructural changes characteristic of, or specific for, the epithelial response described under the light microscope, electron microscopy studies were undertaken. All tissues were fixed bv intra- 121 tracheal injection of cold one per cent buffered osmium tetroxide (10) and imbedded in a mixture of methyl and butyl methacrylate. Sections were cut with glass knives on either the Porter-Blum or LKB ultramicrotomes. Photographs were taken with RCA Electron Microscopes EMU 1-B and EIIU 3-F. Sec- tions in figures 8 and 9 were additionally treated with lead staining described by Watson (11, 12). As shown in figure 7, the increase in the num- ber of cells is associated with increased secre- tory activity. In figure 8, a higher magnifica- tion of the goblet cells seen in figure 7, an Fre. 8. HiL~her magnification of goblet cells from same bronchus as in figure 7, AhoAvinla an apparent increase in intercellular spaces (i) with detachment of two cells (d) apparentl' v related to a concomitant decrease in cellular adhesiveness. A-mall number of mucns ~,ran- tiles (g) are located in the apical portion of the cell and in the supranuclear region. Proto- plasmic proicetions (p) along the apical surface suggest actice release of secretorv ::rnnules. Parallel rows of rough cndopla=mic reticulum (e) and mitochondria (m) t%irli %-,crcJn~Z pro- files, are present. (O~miunt tetroxide fixation: magnilication. ;~ 8.3a0)
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122 KOTI\, COi'RI\GTO-N, AND FALK FrG. 9. Bronchiolar region of mouse lung, demonstrating the proximity of the dust particle- Ia,den macrophage (M) located in the lamina propria, to the nucleus of the ciliated cell (c). Intervening structures consist of a thin true basement membrane, extracelhilar ground siib- ~tances containing reticulin and collagen fibers, and portions of inesench' N-mal cells. (O~mium tetroxide fixation and lead hydroxide staining; magnification, X8,400) apparent increase in the size of in'tracellul:Ir spaces is seen with detachment of t«-o cells, sue:ne~;tin; a concomit.lnt loss in cellular ad- he.siveness. The proximity of a- dust pttrticle- laden macrophage to the overl}•in, brnnchiol:ir epithelitmi may be timn in fi,irre 9. 'The nificance. of this route for the potential entrY oi' carcirnorcnic arent~ into tlte ha~~ll epitlte- liiim is unknown. The characteri~tic httl~iD; of ct itoucili:tied ccll can he .~een in fiaiu•e 111. Thc rel:ttive depression in wltich tlte ciliated ecll.s nre ~4ituatcd rrlative inetiectivene" in prmiclinz prolntlAon for the overh-in;_ nmciis I,L•mlcet. chan_ob~er~. more tions tailed pnbli- 0009'7479
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CANCER PATHOGENESIS IN CILIATED EPITIIELItiJI 123 I•,IG. 10. Bronchiolar region illustrating the characteristic bulgin;; of the non-cilisted coll (B) of the epithelium which mechanicallk, separates the two adjac(nt ciliated ce1L (c). Alodified mitochondria with few to absent cristae are protuincnt thruue;liout thv, c}-t~) id asm of this cell. (Osmium tetroxide fixation and lead hYdroxi(lr ~;tain; niaunification, x5.-]00) _A --'enertl r!sde-nrcnt of the ultramicroscopic chan;;es revcal; nor ouh- tllat the alterntions observed hy linht microscoPy are capable of Inorc tinite definition, but alvo that tions as to mechanism are identifial,lc. _A tailed report of elcctron nticro~colny i~ to he Puhli~hed elsewhere. The cm-ciuc.-'ct!ir i!ul!licauot!, oi lu,l?ttt;tnts in thc rc=1!ir:ctor\ ~ ('nviromtnent rr-icic in at ]c:t~t twn irli;pen~:ci ic iac•tor~ nr':!tivs to tlte ]t:!ihr, r,ne i ut lt!nvz c:!rcrr. 1,hc tir-t i~' of crn!r.~r, thc rnn-irontncut:!I pr(-ceci• ui, ard the lt!,q eutn- ilI', a,cnt~ 1!ro\~('(1 cXh!'ritnr'nr.tllv to he t!nnur!L'c'tuc :!n(l opiclcvtiolot~ic~cllv to he
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KOTIN, COliRINGTON,_yND FALK associated with an increased liability to the development of the disease. The second factor is the atmospheric existence of host-modifying factors that, by virtue of their effect on the ciliated epithelium of the tracheobronchial tree, make possible the abnormal deposition and retention of particulate matter in the lungs. In the instance of carcinogenic aro- matic polycyclic hydrocarbons, the elution of benzo(a)pyrene from soot by intracellular pro- teins is thereby facilitated. A significant local concentration of desorbed aromatic polycyclic hydrocarbons, including 3,4-benzo(a)pyrene, re- sults. Atmospheric irritants may, in addition, periodically and intermittently cause denuda- tion of the superficial epithelium, so that the basal cell layer is directly exposed to the car- cinogenic stimulus. This periodic epithelial desquamation and regeneration in the presence of an abnormal growth stimulus is regarded as providing a favorable environment for subse- quent abnormal growth patterns. REFERENCES (1) Hilding, A.: The physiology of drainage of nasal mucus: II. Cilia and mucin in the mechanical defense of the nasal mucosa: A motion picture demonstration, Ann Otol, 1932, 41, 52. (2) Dalhamn, T.: Mucous flow and ciliary ac- tivity in the trachea of healthy rats and rats exposed to respiratory irritant gases (SO_ , H,N, HCHO), Acta Physiol Scand, 1956, 86 (Supplement 123, p. 1). (3) Falk, H. L., Kotin, P., and Rowlette, W.: The response of mucus-secreting epithelium and mucus to irritants, Ann NY Acad Sci, 1963, 106, 583. (4) Kotin, P., and Falk, H. L.: The role and ac- tion of environmental agents in the patho- genesis of lung cancer: I. Air pollutants, Cancer, 1959, 12, 147. (5) Rigler, L. G.: A roentgen study of the evo- lution of carcinoma of the lung, J Thorac Surg, 1957, S.f, 283. (6) Leibow, A. A.: Personal communication (March 3, 1961). (7) Garland, L. H., Beier, R. L., Coulson, W., Heald, J. H., and Stein, R. L.: The ap- parent sites of origin of carcinomas of the lung, Radiology, 1962, 78, 1. (8) Macklin, C. C.: Induction of bronchial can- cer by local massing of carcinogen concen- trate in outdrifting mucus, J Thorac Surg, 1956, 31, 238. (9) Hilding, A. C.: On cigarette smoking, bron- chial carcinoma and ciliarv action: II. Ex- perimental study on the filtering action of cow's lungs, the deposition of tar in the bronchial tree and removal by ciliary ac- tion, N ew Eng J Med, 1956, 254, 1115. (10) Palade, G. E.: A study of fixation for elec- tron microscopy, J Exp lled, 1952, 95, 285. (11) Watson, M. L.: Staining of tissue sections by electron microscopy with heavy metals, J Biochem Biophys Cytol. 1958, '1, 475. (12) Watson, M. L.: Staining of tissue sections by electron microscopy with heavy metals: II. Application of solutions containing lead and barium, J Biochem Biophys Cytol, 1958, 4, 727.
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DISCUSSION ia v J Dr. Paul E. Morrow: I would like to begin by rommenting on the papers of Dr. Kofin and Dr. Wright because these two presentations are closest to my comprehension of ciliary activity and mucus transport. Our studies at Rochester give a more or less gross assessment of these processes and de- ;;end upon the measurement of the clearance of radioactive dust particles by in vivo counting. This approach permits the study of clearance in various parts of the respiratory tract of the intact animal over any convenient time period. Regarding the interesting questions raised on the vulnerability of the ciliated epithelium, two points can be made. As Hilding' has indicated, bronchiolar branch points serve to interrupt the flow of ma- terial cleared by the mucus escalator. Figure 1A is an illustration of the rather consistent auto- radiographic observation of accumulation of par- ticles at branches. Less obvious is the condition illustrated in figure 113. In this figure may be seen the autoradiographic manifestations of radioactive particles in the peri- bronchiolar lymphatics. These alpha tracks from plutonium 239 have a range in tissue approximately twice that evident in these photographs. It is readily apparent that almost any radioactive dust residing in the peribronchiolar structures consti- tutes a radiation source to the epithelium, and per- haps more important, to the basal cell layer.' (The autoradiograms were kindly supplied by Dr. L. J. Casarett, University of Rochester.) The presentation of Dr. Wright covered matters of great personal interest, and the questions he raised are particularly compelling. For example, hee asked whether the dust particle affects its own clearance. Before I present some information on this question, permit me to digress in order to acquaint you with our general approach. In figure 2, a person is shown seated in front of a collimated radiation detector which is "focused" on the region of the bifurcation. The output of this detector goes to a pulse height analyzer such as that seen in the background. In figure 3 a display of the radio- active measurement obtained by this analyzer may be seen. The spectrum in this case is due to man- ganese 54; it can be measured temporally in the lungs or tracheobronchial tree or nasopharynx, sub- .iect to our ability to collimate and position the detector. In figure 4 the final product is shown. This curve depicts the clearance pattern of a relatively in- ~;oluble dust, mercuric oxide (Hg"). Two events ~ire represented: a rapid reduction of Hg=O' activity with an apparent half-time of less than 24 hours, which is termed Phase I: and a slower clearance 1 Hilding, A. C.: Acta Otolaryng, 1957, 4S, 26. - Morrow, P. E.. and Casarett, L. J.: Inhaled Particles and Vapours, Pergamon Press, Oxford, I;u,Iand, 1961, pp. 167-170. phase which appears to have a biologic half-time of approximately 28 days, Phase II. The Phase I shown in this figure does not exclu- sively reflect the most rapid ciliary-mucus trans- port occurring. This is because the exposure time (dust) and the duration of the radioactive meas- urement were comparativeh• long (-20 minutes). Of course this need not be the case, but the ex- perimental design must take these factors into ac- count. Phase II clearance is associated with the dust deposited in the lung parenchyma. This clearance process appears to he rate-limited in the parenchy- matous areas and it continues to rely, in part, upon the ciliary mechanism. During either clearance phase, most of the mercuric oxide is transported to the gastrointestinal tract via the ciliated epithe- lium, despite the dissimilarity in kinetics. The rate at which dust in the parenchyma is "coupled" to the mucus escalator, and the rate at which the material enters the lymph and blood are all ap- parently tied together by the physicochemical properties of the dust 3 In the measurement of Phase II, we are measuring the sum of these proc- esses. If we considered other dusts of low solubility, we would find, with plutonium dioxide for example, a similar Phase I clearance rate but a Phase II half- time exceeding one year'; with barium sulfate it would be about 18 hours and 9 days.' respectively. We conclude, therefore, that a particle affects its depo.sition site (by its size, density, et cetera) and thereby indirectly affects its clearance. Possibly more significant, it directly governs the half-time of Phase II but has only marginal effects on the rate of Phase I. In figure 5 another demonstration of these points may be seen. In this clearance study of iron oxide (Fe09), the fecal elimination of FeaD was used to calculate tlre respiratory tract clearance rates. (The practicability of this method depends on ancillary studies (if distribution and excretion.) In any case, the direct measurements and the excretion method both indicated a Phase I half-time of 12 to 18 hours and a Phase II half-time (effective) of 16 and 17 days, respec'tivehY. A final matter of considerable importance should be mentioned. Earlier we were shown a relationship of particle size versus respiratory tract deposition. We noticed, and most of us were probably already aware, that the submicronic particle sizes have a relatively low deposition probability in the naso- pharynx and tracheobronchial regions: the major deposition is pulmonary ( al veolar) °With our ex- ° Morrow, P. E., ct al.: Health Phys, 1964, 10, 513. ' Briir, 1V. .i.. ct al.: Ho,dth Phvs. 1963, 8, 639. 'Cemher, H., et al.: _lrch Indust Health (Chi- (,ago). 11156. 1.;, 170. ° Jlorrow. P. E.: Heath Phys, 1960, 2, 366. 125
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Ut~vF.x,l/\/\/ ...: :.::~~~.~-... Fr(;. lA (Left). This airfnradiograph of an alpha-emit.ting dust was made within minutes after an inhalation exposure. 7'he p;irlivIv~- re.vponsible for the :ill)h;i "sun bur5ts°" ~Norc dopnsited distal to this area and are in the process uf IWing cleared. B (Hit;ht). 1'6is autoradiol;raui was tnade several days after tLe inhalation exposure and shows a delayed clear- ance prou•"s, ly-niphatic removal, whicii serves the lung parenchyma.
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DISCUSSION FIG. 2. A woman is shown scated in front of the shielded and collimated gamma ray detector. This unit can be used for clearance measurements without the need for a steel room and trithout exceeding acceptable rudiationvaluesn pressed interest in smokes, we must bc particularl}• alort to the behavior and contribution of particles below optical resolution. On the other hand, it•e r•an be misled bv such relationships because xre are normal] * v clr•alin,- Ncith a (listsibutimz of particle sizes. Aerosol distributions (lo~4 normal), which have ihe :ame frequencv median diameter (C\ID), tan have entirel}- dilferent dr.position patterns, bot hqttalitativelY and <itiantit<ttiveh-. For example, an aorosol distribntion with a Ci1ID of 0.1 µ with different geornetric standard deviations can pro- 127 duce <0.1 Iter cent nasophar' ynreal ~lcposition on a rna~4.s b:tsi;, or >70 per cciii. In this illustration Ihc Leomcirir standard dociations arv 1.5 and 3.0, re,pooticclv. We have found that on(, parametric ltmction, tlte_ ma~;s mcdian di:ime'ter. provirles a far lucttcr drposition rclationArip. 1nd now n few contment~ anr] ~jttc~;tions about othrr papers: Dr. 1)alhamn di~ctr~sf-d an itnpinge- n , nt tnetlial for tohacco rntokr• r erlir•r. Ilow can Morrow, P. E.: Unpublished data.
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128 nISCi; sSIO\ SUBJECT F. L. IMMEDIATELY AFTER Mn54 02 I N HALATION E U 104 103 PEAK i I 1021 1 1 1 1 1 I I& 10 20 30 40 50 60 70 CHANNEL NUMBER FIG. 3. The monoenergetic gamma radiations of Mn" are represented by the activity peak in channels 38 to 52. Each channel is equivalent to 20 I`;ev, in this figure. TIME IN DAYS FIG. 4. The clearance of Hg°0RO from the lower respiratory tract vms accomplished by a serial measurement of the lateral lung fields and the tracheobronchial region. All measure- ments are expressed as a percentage of the activity immediately following exposure. ~mok than hY e; ;lre - Il1s101 rnou Uoe- ,har2 ?he Dr 1ntt(11 in ti «-hic arV 1 Won~ ilot ' I etr-~ I~at~-
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DISCUSSION ~ z w U Ir w O_ n 10 7 3 0 0 \ CUMULATIVE FECAL ELIMINATION IRON59 OXIDE SINGLE INHALATION - RATS EFFECTIVE HALF-LIFE - 17 DAYS 5 10 15 20 25 30 TIME (DAYS) Ftc. 5. After a Fes680a exposure, the rats were placed in metabolism cages for excreta collections. A few rats were sacrificed at daily to weekly intervals and their lungs were analyzed directly. All values are expressed in terms of the total Fe~' activity at zero time. smoke particles be "impinged" when particles less than 0.5 ,u in diameter are essentially unaffected by either centrifugal separators or gravity? They are separated from the air according to their dif- fusion coefficients. I wonder, therefore, if we know enough about the physical states of tobacco smoke. Does it aggregate? If so, how fast? Is it electrically charged'? Is it hygroscopic? In other words, are these factors which modify the characteristics of tobacco smoke so that it is no longer a smoke in the classic sense? Dr. Carson implied that the changes in ciliar' v- mucus transport he recorded were due to changes in the mucus. In view of Dr. Iiensler's paper in which he showed rapidlY reversible effects on cili- ary function with some of these same agents, I Wonder if he can be sure that it is the mucus and not the cilium which is affected. Dr. Carson also incntioned membrane potentials. It would be in- torestin, to learn if these «-ere transmembrane potentials and how they were measured and to hac-e a clcu•ification of what was shown in the 129 pictures of the potentials. Finally, I may have mis- understood Dr. Carson, but I believe he stated that the sodium cyanide was operating through a pH effect. Is this correct? Several papers have dealt with measurements of particle travel in the trachea. I have often en- countered the statement that tracheal-mucus flow is helical, not coaxia] e Since many of these prep- arations involve longitudinal sections, I wonder if this leads to technical complications? Dr. Bates: Dr. Iiensler reported a mucus-trans- port rate of 36 mm. per minute in isolated cat trachea, opposed to 1#.3 mm. per minute for the intact cat bv Dr. Carson. Would these speakers like to speculate as to Nti h7• there is a threefold diff er ence in transport ratos' hr. I:ensler: I assume that it has something to do with the viscositv of mucus. For example, a ' Rarelav. :1. E., and Franklin. K. J.: J Physiol, 1937. 9G, -tS2.
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130 DISCCSSIO\ drop of Tyrode's solution dilutes mucus and in- creases the transport rate in the chicken trachea preparation. Isolated cat trachea and chicken trachea in vivo are low viscosity systems. Dr. Battista: Dr. Carson, smoking experiments are difficult; the animals' breath-hold differs so that the quantity of smoke varies widely. Secondly, phenol has a high vapor presaure, so that about 70 y per 40 ntl. is the highest level attainable, but you show over 100 y. I wonder how you gen- erate this phenol concentration' Dr. Carson: Breath-holding is influenced greatly by the level of anesthesia. We are not troubled by breath-holding, and have found excellent repro- ducibility. We used a Vaponepherin nebulizer to produce a mixed aerosol-vapor of phenol. Dr. Kensler: Dr. Dalhamn, when one puts a Cambridge filter into a smoking machine and in- creases its volume, dilution of the gases occurs; this could account for considerable changes in con- centration of smoke. Did the unfiltered smoke go through in the same volume as with the Cambridgc filter, or did adding the filter add volume to the circuit? Did the smoke from the charcoal-filtered cigarette go through this volumc too? And do you know what the .•oltune was? Dr. Dalltainn: These anah•ses of concentrations in smoke were done after the filter so that the concentrations measured were those of smoke which had gone through the filters and therefore those to which the trachea was exposed. Dr. Battista: Why did you pick isoprene to measure as a gas component? Dr. Dal1ia»in: We measured many other sub- stances too-the choice of an example was arbi- trary. Dr. Aiello: I'm just wondering about dase- response relationships. An animal the size of an 0.5-kg. rat smokes one cigarette and its effects may be compared with those in a man weighing 70 or 80 kg., who obviorusly has a much bigger lung and who also smokes one cigarette, sometitnes over a rather long period of time. The demonstration of pronounced inhi.bitory effects in the animal but no definite evidence of similar inhibitorY effects in human subjects could be parth- explained by dilu- tion effects in lungs of different size. Dr. Dalhantn: I must stress that we havc not drawn any conclusions about human aniokers from observations on c,cts. Dr. Ca>son: The trachca iu the cat and Dr. IiensLcr's in ritro systetn witlt artificial mucoid material on the trachea are not very similar. Mucus transport appears to bo dependent npon other factors a~: mell as ciliarY beating; the frequency of cilinry beating retuains ahnost con.stant :ts de- termined b~, stroboscope and stroboloom record- ings, but the tissue debris moc•ing on tracheal mucus stops whwn tlie preparation is espos-d to various agents. I do not bclieve t he Fdffert of cc-anide on the tran=membranw potentiati is dttc to hYdrogen ion. but rather thctt it i., 6uo to the concentration nf cYanide anions. Th,vansmwm- brane potetitiaLs tcrrre rccordcd nith ila,~~z rapi]Luy electrodes similar to those used by Dr. Brian Hoff- man. Dr. Falk: May I make one comment on the question that you raised, Dr. Morrow? That is, whether the rapid effects of tobacco smoke are on mucus or cilia. We found that if acetylcholine, which we belie~e acts particularly on mucus-secre- ting cells, was applied to ciliated epithelium it would prevent any major effect of the application of tobacco smoke for hours. If acetylcholine was removed, the protective effect was lost. Any agent N%hiclt inhibited cholinestera.se protected the ciliary epithelium of the frog and the tracheobronchial tree of the rat for hours. This action appeared to be on mucus sec•retion; therefore, mucus-secreting cells must be important in tobacco smoke inactiva- tion of mucus transport. Dr. La,irenzi: In reference to Dr. Kotin's difi'er- entiation of tumors by their location, clinicalh•, we usually divide peripheral tumors from central tumors and classify as central those arising from segmental or larger bronchi. The two types present contrasting clinical and roentgenologic signs, and have dissimilar cell types and different survival times of patients. I would not regard the seg- mental area as a peripheral part of the lung and thus consider that 70 per cent of tumors are in segmental or larger bronchi. Dr. Kotin: «'hat Dr. Laurenzi said is true. We have shared the difficulty the literature describes in getting deposition of particles high in the tracheobronchial tree, and this may have dis- turbed the relevance of our experimental data. It has been very comforting to see that the impres- sions of the site of origin for central tumor masses place them slightlc peripherally, and you will admit that the concept of segmental bronchial lo- cation of most primatY tumors is 5 or at the most 20 .-ears old. Before that, it was always assumed that ]a'te bronchogenic neoplasms which invaded mainstem bronchi approaching the carina began there. Our interest is in relation to particle deposi- tion as a reflection of the site of the action of carcinogen. The electron micrograph showing the macrophages emphasizes that there are two Ni-avs to get at a basal cell-knock off the surface pro- tection or sneak in from the back; and we cer- tainly recognize the possibility of sneaking in frout the back. Dr. Bates: A question for Dr. Morrow. If I under- stood him rightly, in regard to his iron oxide, PL", clearance. he made the point that the lung clear- .u1cr was attributable to the ciliarv clearance mccltanism, yet the period shown extended over da.vs, wecks, and months, and surely is dependont on macrophages. Dr. _1Io,•rc,ic: Particles are going toward the mouth to be ~zwallowed and enter the gastroin- testinal tract Yarticulate material is seen much more fretturntly on rhe epithelial surface than in twriLronchiolar ar(-as. Onl}- a fern hours after ex- posurr dttst is in tl,e lumen. Later, it appears in tlo• f,,,rihron(,hi-)lnr .m;t. Lnt it nw~r,r cf~a~,: to :ippi,ar on 010 c'l~itliPlial -ttrflce. The persistenc(- 0009'748'7 #
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DISCUSSION off- the Un ne, I it ion V aJ' ltnt iarY iial i to I ing iVa- 1N-e ~bes the 1is- t. It res- tcill 1 10- ztost rned aded egan Josi- tl of 1 thc lcays Ipro- ; cer- ~ in i ~der- lear- f ance .,' ut of the material in the peribronchiolar area is the point I was trying to make. There is a continuous redistribution for days, months, and years in- volving the ciliary epithelium, and probably in- t olving almost exclusively material within cells. Dr. Bate.~:_><nd the particles are not going up through the iymphatics? Dr. Jloroar,: Oh, yes. They are doing that also. Dr. lir,ie.; : Do you know l-hich is more impor- tant:' Dr. 31o;rotc: TNo, I do not, but I think our attempt to determine this would indicate that the ciliary mechanism for removal of macrophages or cellular debris, which have been phagocytized and then lost, is a major clearance process, irrespective of time. Dr. Kadjord: I have two questions. First, the point on kinetics raised by Dr. Morrow: The kinetics of the response in Dr. Kensler's prepara- tion cannot_ b(, ascribed to an effect on mucus- secreting cells inasmuch as h(, has provided artifi- cial mucus. It seems to me that immediate re- sponses and ces~ations imph- an effect on ciliary function. I would ask him to conlment. My s~cond point is on this matter of the releN~ant importance of variou~ routes of excretion of material that is inhaled and deposited. A rccent study by- Cember has shown that great differenee~ in hold-tup time, clearance time, and all the rest of the faetor-. cie- pend on the chemistry of material. For e.ample, if polonium oxide (Po 210) has certain clearance rharacteris'tic.~, polonium chloride might have quite different ones, and polonium sulfate might have a hird set. Thus the common factor, polonium, does not detcrmine the clearance characteristics. Dr. kensier: It is my opinion that the gas-phase ,•omponents act rapidly on cilia. To think of the rct.pid ,*as-phase effects in ~ ii•o which Carson and I)alhamn described this morning, one would antici- 1,;tte front tile data of Kotin and DIorrow that the 1uu•ticles ctould not hit the trachea at all, and that :iny oarly tracheal effect would he a gas-pllase ronn•ibution. A later action might be due to de- ttpstrcam of particles containing some of tlr, igonts fronn the gas pha~;e which landed 1'atl hcr ~ kncn. Ut•. I')irrell: It is pos'-ihle to meastre th^ ~inaunt~ of muctts which pati:'nts secrete. Aasal -cretion and ~pututn can h~~ measured in grams. t)n- of the troublc, with sonte of the arguments we un• ha\in_ i< rhat no measurements have been ~nade or tho atuotmts of nntcus secreted. _l rneas- nr~oi tot:tl .nliitn~~ and of atnount of nmroprotein rVoMin t. _1>=;n- methods are ;t~-ai'ab!e to mea<ure ilw riuantit,v mueoprotein ita ri(rn. Thr,e wotdd uot cch''thcr thc nlucus conlinQ out of the r, i;aitic•ly teater,v or sticlc.Y. This tnight a~ ;i r, :~j,nn~'• to irritstion. Clinical mtarurr_- :~~ttr~ ditliwult. We tried to m(,asure huntan ;tl > r, I ioti and i0ermin, ,hr ;tmount tli;lt, 'nnW cnn ~tn• iront cnd. witliout knocrin;' how nnch '-•ni dn%cn the I,ark c.ml. 13nt information ~i iLi: hf" ~ntnllin('d witl, othf•r 'lata, malv hc•Ip utnuu~~ r•. )nlI ~lsiti'~soitili.~ <ituation. Dr. Carson: Mucus is the factor that is first affected by smoke and by phenol aerosols. It is the initial point of impingement and responds itnmediatel' v. Studies in progress of mucus flow, tnucus tran,,it time, and ciliary beat frequency measured sitnultaneottslv may -,how what each rontribute; to responses of the respiratory tract. Studies in patients with subacute and chronic dis- ease are needed in addition to tho=_e in patients with acute di<ease. Dr. Hers: I have a duestion for Dr. Kotin. From clinical esperience in chronic bronc•hitis, we have the impres,,ion in the \etherlands that the inci- dence of carcinoma is increased in these patients. Bronchial tissue from such patients without car- cinoma shows an increase of goblet crlls, an in- crc ase of mucus production in glands, and an in- crc ase of the cosinophilir cr 1L in the submucosa und often scattered RttsAl hodies. Aow, we know frcnt clinical nledic-inw th<tt eo-inophilic cells and Ru.,e:Il bodies may, perhaps, inilirate that the antigc,n-antibod}- r!;lction ui th~, hu,-t is changing. On the other hand, we knot-, in our eaperience with htunan material that th,, augmented number of goblet c:'ll,' docs not prociuce normal nttcopoly- ~:accharide. By mcans of histochcmiral nlethods, t:'inr tlte contLin. d.llrian hlue-pwriodlir• acid-Srhiff method, nv, can c.,timate bv color the c•ontent of polYsacchau•idea and pro'tein. We iound that the protein c•ontent tnrasured by color n-a~; incrcased ill pct:sons with chronic bronchitis. Ao«, my yues- tion to Dr. Kotin i~;, in his Nvork with rats in whic•h Ite showed ,m increa.~e of goblet cells, tcere there changes in the cellular mucus measurable bv histocheniral methocis; and, seconcl. was there an increase of eosinophilic cells or Russell bodies in the lamina propria? The second point relates to experiments showing hyperplasia of augmented cells hearint; cilia in the periphery of lungs of mice infected with adapted strains of different influenza viruses. The normal anatomy of bronchial branch- ing in nlice is certainly not eompar:ible to that in Ituntan beings. Thc next point is that Dr. Kotin's aninlals n-ere kitl[ ~d after 20 cveek.~ and 32 weeks. \ow, irn our prolonged st.udic-; (if f_ rr-t~4 and guinea pig.a, aftcr exposure to inliuenza \ irtt., %ce found tltat the ciliated or angmcnmd (,(•11s disappcared. After 60 weeks, we roulcln't find thrtn. So I think that it is c•pithr-lium Nchirh has grotkn down from tite bronchioli alon; the :th-rolnr tcall.~ cchiclt sub- ~,r~uentl~disappears. at lra~t ill frrrrt~ and pigs. 'Ph , lung; of five huntans n1jo ciiecl of othtr cattses ,~ix month: ufter a.;ovrro infinenza tirus pneu- rtonia Ahonn~d no uttrrations of ci'ittd epithc•littm. 1)r. Kulio : In an<~x r to }our iott.: in onler. Fir<t illr~ r-,Lctionrhi) of Lronrhiti~ to cancer: therr'. ;t hooker in tltis, of cotu-e, tu that th:• ttco nt:tior r nriroulnwnt:ti nlrdia. (•i'_'Zrotn,, ~ntokiti, and ~,lhtt ,l t!rhan air. a<--nci:ttrcI Ncith increar (1 risks for Inne i-.utrrr, a, ^ :1Iso a=<ociater] tcith incmased ri~ks for hronc hiti<. Thu~. I~lo not know th;tt vuu can impute a sequi•nce to Nchat tna,- I- a Lu,ha~zil. -•apabilitv nf the chomicals frnm tli11 c1nVironmm11t. I c•annot voncr•ivc ihat tluo brou( huolarization of
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132 DISCUSSION 11 the alveoli, as common as it is at the autopsy table, especially in patients with chronic diseases, has escaped you. As a corollary, I believe that the bronchiolarization in mice has a human counter- part. Downgrowth of bronchiolar cells along the latticework of the alveolar septa is seen in chronic respiratory disease. While the two lesions maniiest themselves differently, I think they are histogenic counterparts. I'm still unconvinced that there is any meaningful consistent quantitative relation- ship between histochemical analyses of mucus and the histochemical methods of staining. You can only make gross observations of alterations. Now, as far as the persistence of cells, in response to the infection with myxoviruses, I fully agree with you. The 6-, 12-, and 24-week sacrifices you saw were just our acute toxicity studies. Some animals vvere sacrificed later, and in the areas in which ciliated epithelium first underwent involution alveolar septa became two or three cell layers thick. This produced what pathologists through the years have erroneously called squamous metaplasia; but there are no epithelial bridges or keratohyaline granules, and it is only when carcinogenic insults are added that pathologic metaplasia appears. The ciliated cells have a short half-life, and the interesting thing about them is that the ciliated cells which are at the initial site of hyperplasia no longer show localization of influenzal protein by immunoflu- orescence. Thus, neoplasms arise from cells which have never seen the influenza virus, yet the in- fluenza virus is indispensable for development of malignant tumors in our system. Dr. Hers: I should like to come back on your comments and your criticism of the periodic acid- Schiff reaction; I quite agree with you. But, you can do the periodic acid-Schiff reaction several ways. If the _1lcian-blue fixation is done first. not only is the mucopolYsaccharide part of the molecule stained, but the protein part as well. So the periodic acid-Schiff reaction in the rat bronchi in which the protein portion is very big is blue or violet to provide some chemical or histochemical aues to its composition. I agree with you that fine interpretation of this sort of metachromasia is highlY disputable. However, it is certainly seen in our cases of chronic bronchitis and it indicates that the mucus is not normal. Dr. Gossclin: Dr. Tyrrell raised the question not only of the quality of mucus but also of its quantity. I made an attempt 15 years ago to meas- ure the amount of mucus secreted in the trachea of an animal with intact innervation and blood supply. A cat was tracheotomized high in the neck, and a little catheter, with a small balloon near its end, was put, doivn to the carina. Thee halloon was blown up to i.,olate the trachca above it while the animal could still breathe with some extra resist- anee through the inner tube. A Ladancei salt <olu- tion was placed in the isolated space and mixed .vith I,ubbled oxygen. Tliis fluid was sampled from time to time and analyzed for protF•in. IncidFn- tally. these must he insoluble seeretiontz. so-called "insoluble mucin." Protein did appear and atimu- lation by systemic pilocarpine produced no differ- ence. Protein accumulated, but the tracheal mucus membrane is terribly leaky to protein and this al- most certainly was plasma protein, not actively secreted material. Dr. Staab: Yesterday I learned two facts: That the respirator.- tree of man has cilia and that the cilia are depressed by cigarette smoke. And today I learned two more facts. That the respiratory tract of an animal secretes mucus and that mucus flow is depressed by cigarette smoke. I do not be- lieve that further analysis of cigarette smoke is going to add a great deal to these facts. Certainly there is no future in it for our understanding of the mucociliarv mechanism of the respiratory tract. I would like just to propose, though, one or two experiments that could be done that might lend some understanding of the function of this impor- tant clearance mechanism. One is something that was brought up by Dr. Brokaw yesterday: Why not test for the action of purified agents on ciliary stiffness? They might be introduced by micro- pipettes close to cilia or flagella under special cir- cumstances. What is the effect of purified agents on the physiochemical properties of isolated mu- cus? What is their penetration rate through known layers of mucus; their diffusion rates: and do the penetration rates of gaseous or particulate sub- stances differ? These are things that could be studied and would contribute to our understanding of this clearance mechanism. Dr. Ban.g: What is the effect of repeated smoke exposure on mucociliary function? Can one set up any system that will test this question over a period of weeks? Dr. Weber: Dr. Kensler said that the vapor phase is responsible for stopping the integration of ciliary activity. What part of this gaseous phase reaches the lung? There are certain substances which hurt the cilia. Do they get to the cilia or not because of selective trapping in the mouth cavity? We know about selective filtration, so we want to know what we are to take out of smoke. Dr. Rylander: I think that the point Dr. Iiens- ler raised about the particulate phase not being able to impinge on the tracheal surface is opposed by observations of a pipe or cigarette holder. The stream of smoke goes through a channel which is roughly similar to the trachea. If the pipe or holder is opened after smoking, it is filled with filthy solid particles, the tar. So, I believe that solid particles impinge on the trachea inn vivo. We have some experiments which suggest that the higher the boiling point of the tar, the more ciliostatic it is. D,•. Kcii.~lrr: I will discuss Dr. Weber's and Dr. 1ty-lander's comments. The particle of cigarette smoke has a mean diameter of 0.25 µ or less. In undiluted smoke, this diameter will double in 10 or 20 seconds. Diluted smoke is much more stable. Dr. Kotin's and Dr. 'Morrow's data on particulate distribution in the lung suggest that most of these particles go right on by but that some reach the target. Uur work is dedicated to finding out what 0009'7489 rlr rc
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DISCUSSION are the most active components, and what one might do about them. Some components will certainly be absorbed along the oral cavity so that the concentration in the lung will be reduced. But the odds favor some of the gas-phase components reaching the trachea, the major bronchi, and be- yond. Dr. Kotin: I have one very brief comment. Cer- tainly there is an effect on the trachea. I think that the particle resonance time is critical. Dr. 133 Wynder, with his studies in St. Louis, showed that metaplasia did occur in the trachea. Why does this not go on to neoplasia as we assume metaplasia does in some other areas. Certainly there is an effect on the tracheal epithelium, as manifested by the appearance of metaplastic changes; but it is equally certain that primary bronchogenic car- cinoma of the trachea occurs rarely, so I think there is nothing mutually exclusive about what Dr. Ry- lander and Dr. Kensler found.
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EFFECTS OF BACTERIA AND VIRUSES ON CILIATED EPITHELIUAI HOLLIS BOREN, 1L-loderator A STUDY OF THE MECHANISMS OF PULMONARY RESISTANCE TO INFECTION: THE RELATIONSHIP OF BACTERIAL CLEARANCE TO CILIARY AND ALVEOLAR MACROPHAGE FUNCTION', " GUSTAVE A. LAURENZI AND JOSEPH J. GUARNERI N atural resistance to infection i~ an ad- justed composite of manY functions, some whiclt are definite and others which remain obscure because of difficulty in identification or measuranent. The capacity of the respira- tory tract to protect itself presents an inter- estinn and fertile area for study because of the unique character of its defense-the highlv specialized mucociliary apparatus and the alveolar macrophage-and becau~e newer, refined techniques make it possible to study these modalities separately. The individuml effectivenes~ of these processes has been demon.~trated many times, and current infor- mation indicates that together they constitute a hi,hly efficient combination. Evidence for this is the fact that the bronchial secretions of the normal respiratory tract are sterile (1) de- spite recurrent bacterial contamination bv inhalation and the aspiration of upper respir- atory tract secretions. In contrast, a state of chronic bacterial colonization exists in the bronchial ~ecretions of patients with chronic bronchitis during periods of well-being (1) ; and an increa:~e in their numbers has been demonstrated concomitant with an acute exacerbation (2). The difference mav be ex- plained by the belief that previously dam- a,ed tuuco.sa is more susceptible to infection. However, these findings may be related to alter,itions in the action of the mucoeiliar}- ap- parant.< and'or alveolar macropha(,e;. ~rtnclard mcthotL for the sturlY of host rel~i~tnnce to infection etupl.o' v avariety of ex- perimental conditions for alterin~ host stts- ` Fruni the Division oI Re<piratory Dise;11eS. \l~w ,Lr-c•y College of Jlcdicine. J:,rsey Cif}', NewJo~~,•V. 'Tlii; %ccn•k was supported 1,~(;rant \u. 00005-Ol from the Divi<inn oi _1ir Polhition, lict- roau of State Servicc,sz, I-. S. Pnhlic IIrtltIi s;,tirc•, ceptibility; however, an even more funda- mental need is a s, vstem for quantitating the host response to nu infectious challenge. This report describes a method for measuring the clearance of bacteria from the lun-s of mice after implantation by the air-borne route. In addition, the protocol includes the measure- ment of the effects of various experimental conditions on bacterial clearance, mucociliarv function, and alveol:tr maerohhaLe activity. METHODS AND MATERIAL :lerosols of a coagtdase-positire strain of Staphy- lococcccs acareus (FDA 209P) Nrc,re generated from a buffered suspension ( _pH 7.3) of the bacteria con- tained in eight DeVilbiss §-10' glass neubulizers. The coarse spray from the nebulizers was carried thron;th mixing cylinders into a large exposure chamber by a second:u•.v air flow of 100 cubic feet t er minute. The particle size distribution of the aerosol in the exposure chamber air was: 3.3 µ to 5.5 µ, 14 per cent : 2.0 µ to 3.3 µ, 26 per cent ; 1.0 µ to 2.0 s. 53 per cent : 1.0 µ or less, 7 per cent. White Swiss 11'ebster m:de mice were exposed to staplt.vlorocci for a pcriod of 30 minutes. This ,train of Staphyiococc:rs anrcics has relativelc low virulenee for the mous,. After exposure, one group of mice Nvas l:illed immediatelv, and tlce other mice were grouped and killed at hourlc (1, 2 3. 4, 6. 5, 12, 2-1) intervals thereafter. Rapid killing was accomplished by clampine across the trachca. as this method prevents agonal aspiration of e_on- taminated oropliaryn,eal sec-retions. The lun_s front the mice in earh aroup were removed asepti- calh- ut the Icvel of thr ma,ior hronchi, homose- niz ed in gluss lioutoarniz:•r, . and diluted in nu- tri( nt hroth. _l~sar t)ucn• p1at, s nere made of the hmg holnogenates; and. :u'ter incicba'tion for 4S hours :ct 37' C., the ntunbers of culturable har_te- ria in the hin;: of raoli mouse were dcrived i'rom tho prc luct c4 iho niimber of Platp colonies and th:, dilntion factor. Previou:~ to and concomitant witli ~,awh stucv it 1va.s e~tablished that the lun_sz of mic(, from this specific colon_v yielded no cul- tur;thle bac•teri;c. 1)eCilhi ss ('ompanv. Somer~c•t. PennsYlvania. 0009'7491 I
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MECHANISMS OF PtiL\iONARY RESISTANCE TO INFECTION 135 nda- ; tlte This r the mice . In ~ure- tttal i iary ; rom ov- i ~e rs. ~ ried ~~ure :1 of 3.3 !ent; cent. d to This low roup 1ther 2, 3, lling -hea, con- nngs ,pti- oge- nu- ;he 1g •te- rnm ind I ant nes nl- C Deposition refers to the numbers of culturable ~i ~_anisms retained in the lungs at the end of the :posure time. Air samples were collected in 1 osphate buffer in all-glass impinger samplers, nd the deposition numbers were correlated with he bacterial concentrations in the nebulizers and sposure chamber air, and with the lung and body cei6ts of the mice. The accuracy and interrela- ionships of these measurements made it possible lo predict the numbers of culturable staphylococci ~ hat would be retained in the mice's lungs. The mean numbers of culturable bacteria retained at tne designated hourly intervals were also deter- mined; and, by subtracting these %-alues from the uean numbers originally deposited, the mean numbers of staphylococci cleared were derived. In addition, the numbers retained were expressed as a percentage of the deposition, and the per- centage cleared was obtained by subtracting the percentage retained from 100 per cent. In these studies, "clearance" refers to the numbers of bacteria whose presence in a culttrable state can no longer be demonstrated. From these data it was established that there is a rapid decrease in the numbers of culturable staphylococci in the lungs of mice (figure 1) (3). The pttlmonarv clearance of staphylococci was then determined by this method in mice sub- jected to various conditions which are often con- sidered important in respiratory infection: hy- poxia, cigarette smoke, alcohol, barbiturates, cortisone, and carbon black. The percentaQe of deposited organisms which were cleared in four hours by the normal (control) mice was com- pared with corresponding results obtained in mice subjected to each experimental condition. How- ever, the individual and comparative effects of the different variables were made more apparent, quantitatively, by relating the experimental and control results in each circumstance in terms of the ratio of the numbers of bacteria retained: experimental/control. This calculation is desig- nated the relative retention ratio (4). Immediatel,y after bacterial aerosol exposure, the mice were divided at random into control and experimental groups, and the four-hour retention of staphy-lococci in their lungs was determined. Animals were made hypoxic for the four-hour period by placing them in an atmosphere of 10 p(,r cent oxygen and 90 per cent nitrogen. Other groups of mice were placed in a plc~xiglass cham- her through which smoke generated from brand rin:u•ettes was drawn. Barbiturates and ethanol «erc adrninistered intraperitoneall' v, and the doses Nvrre adjusted to the animals' weights. The ethanol concentration ranged from 5 per cent to 21 per cent, and c•orrespondine blood alcohol concentra- tions were determined by a rapid. colorimetric micro-assay.' In the corti~;one studies, mice were iu.iectcd snbcntaneoush- with 15 mg. and 30 mg. Dctermntubc C-.11c., Worthington Biochemical C'nrporation. Fr(,chold. Aew Jersey. of hydrocortisone 21-phosphates in three equally divided doses at 24, 16, and 2 hours prior to aerosol exposure. A constant ratio of 0.60 and 1.22 mg. of cortisone per gram of body weight, respectively, was maintained among the experi- mental animals at the two cortisone levels. Carbon black°, in doses of 2.6 mg. in two equally divided amounts, was injected into the tail veins of mice at five hours and immediately prior to exposure. In general, experiments were done in which 100,000 staphylococci were deposited. No less than 15 mice were used for each observation (immediate, four-hour control, and experimental); and a mini- mum of two complete studies was carried out for each variable. Ciliary function was quantitated by measuring the time required for particles to move a distance of 5 millimeters (cephalad) in the intact tracheae of anesthetized kittens (5). Three-month-old kittens that varied in weight from 0.70 to 1.14 kg. were anesthetized with pentobarbital sodium (55 ing. per kilogram ) and kept in a state of un- consciousness corresponding by external signs to plane 3, stage 3 anesthesia. The observations were made through a dissecting microscope with a calibrated eyepiece, and the objective was directed at the illuminated, exposed anterior wall of the trachea. Heterogeneous carbon particles (finely powdered soot with lycopedium spores) were in- jected distal to thc site of observation. AIultiple measurements were made, and they were averaged and expressed in terms of the number of seconds for the particles to move one millimeter (second per millimeter). Each kitten served as its own control in studies in which the effects of airflow and mask resistance were investigated. The effect of alcohol was determined in separate groups of animals after the intraperitoneal administration of ethanol in doses (6.6 gm. per kilogram) which put the animals in a state of unconsciousness com- parablc to that of the barbiturate kittens. Blood alcohol concentrations were also measured. Harve~its of rabbit and mouse alveolar macro- phages were obtained by a modification of iA1yr- vik's method (6) in which the extirpated lungs were lavaged with a balanced salt solution. Total and differential cell counts were done on the n-ash, and macrophage viability was determined by the capacity of the cells to reject eosin Y stain (6). The respira'tion of aliquots of alveolar macro- pliages was measured in the Warburg microrespi- c•ometer, and the oxygen uptake was expressed as ,aL. O_i 10' cells per hour. The numbers, viability, and respiration of cells from alcoholized (coma- tose) rabbits were also determined. and the effect of alcohol in Vitro was measured. In another set of experiments, these measurements were repeated on macrophages obtained after the intratracheal instillation of carbon particles and Staphylococcus aureu.s (FDA 209P), ceparately. The iia t•itro changrs brought about by these materials were `H}-drorortisone; Merc•k, Sharp, and Dohme, West Point, Pennsvlvania. '; Pclikan, German}•.
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136 LAURENZI AND GUARNERI also measured. To date, studies have been com- pleted which measure the effect of alcohol on the macrophage uptake of carbon particles and bacte- rial cells. This was quantitated in vitro by estimat- ing microscopically the intracellular amounts of these materials. nlacrophages were also obtained from the lungs of mice before and after staphylo- coccal depositions. In addition, the numbers re- covered from exposed mice at zero, one, and two hours after treatment with alcohol were deter- mined. A volume of 0.8 ml. of balanced salt solution was used for each lavage and pooled specimens of 5 ml. were examined. RESULTS As shown in figure 1, the lungs of mice killed at hourly intervals during the post-deposition period showed a rapid decrease in the num- bers of culturable staphylococci. At the end of one hour, 45 per cent of the bacteria de- posited had cleared; after two hours, 70 per cent; after three hours, 80 per cent; after four hours, S8 per cent; and after six hours, HOURS AFTER DEPOSITION Fia. 1. The clearance of staphylococci from the lungs of mioe. Each point on the curve represents the mean percentage of three or more studies with an average of 50 mice in each study. L:xarnple: deposition 100.000 ` 13,000; retained at end of one hour, 55,000 - 7,000 ; two hours, 30,000 - 5,000 ; three hour.=, 20,000 -!- 3.500: four hours, 12,000 -_ 2,500; six hours, 5,000 -!' 1,000; and eight hours, -1.000 - 1,000. (Pepro,l,rrr~1 irithhernzission of pohlish.ers. S. Karqcr _lG. hr~.~~rl; Xeic York, of 3l(diciiia !•l,ora- cali.9. 1.`h;a, °2, ;,) 95 per cent of the staphylococci had disappeared. The clearance curve was remarkably constant for bacterial depositions from 1.0 to 2.5 X 105. The results listed in table 1 show that alcohol and cigarette smoke caused the most marked reduction in bacterial clearance; cortisone and hypoxia showed fewer, but significant ef- fects; barbiturates showed even fewer; and carbon black showed no interference with clearance. Histologic sections of the lungs of mice under control and experimental condi- tions failed to show evidence of infection or inflammation. Clearance was shown to be an active process and not the result of the "natu- ral" death of bacteria exposed to aerosoliza- tion and implantation, by the demonstration of bacterial colonization in lungs incubated immediately after aerosol exposure. The remainder of this report is concerned primarily with the effects of alcohol on bac- terial clearance, mucociliary function, and alveolar Inacrophage activity. Graded inter- ference «•ith bacterial disposal was correlated with blood alcohol concentrations and the state of unconsciousness and muscular incoordination (figure 2). The most marked reduction in clear- ance was observed in mice rendered comatose for three- to four-hour periods. The maximal blood alcohol concentrations and the rapid de- terioration of consciousness were achieved within the first five minutes after ethanol administra- tion. As ~hown in finure 3, maximal interference with bacterial clearance is early-occurring with- in the fir,~t hour. Aspiration was ruled out as a contributory factor, as staphylococci im- planted in the pharynges of alcoholized (comatose) mice did not appear in siQnificant numbers in their lungs. The administration of 100 per cent oxj-gen and 3 per cent carbon dioxide in the inspired air of alcoholized ani- mals did not alter alcohol reduction in clear- .nlce. Carbon particles moved at a verv fast rate (:3.1° = I).16 ~econdk per Inillimeter) in the tracheae (if control (barbiturate) kittens. The rate was markedly decreased in the ethanol- treated animals: the maximum was 10.6 ± 0.52 .-econ4ls per millimeter (fiaure =1). _%s in the rnice, peak blood alcohol concentrations, onset nf coma, and the mo,t marke(l slowine of par- ticle transport occurred within the first few 1;+ m ae al II ot rr ce Pl ta tl '1aos7,19,n
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red. tant 105. I-1101 °:icd and cf- ind %ith of udi- or an ttt- :on :ted i MECHANISMS OF PULMONARY RESISTANCE TO INFECTION 137 TABLE I L IIE 1'.FFECT OF VARIOI:S ESPERI\fE\TAL CuSnITIOAS ON TnE I' C1Un-xoCR CLEARANCE OF ST_1PIIYLOCOCCI FRO]t TNE LUNGS OF MICE Control Group Experimental Group' Number Retained ' Number Retained ~ i Relative ~ Condition Mean f S.E.+ I Cleared ( o) ~ Mean t S.E. Cleared i Retention Ratio IHvposia 12,175 f 1,4-48 ~ 89 29,765 t 4,700 I 70 2.4 i.uminal, 4 ing. 5,500 :i= 488 1 93 10 035 t 1 416 Su 1.8 Secon,t.l, 1 ing. ~ , , I Ethanol. 5 per cent 11,265 f 1,852 1 88 17,895 t-I,241 ~ S2 f 1.6 l:thanot, 12 per cent 13,605 f 2.178 84 25,225 zi~ 3,352 1 70 1.9 Ethanol, 15 per cent 10,665 =E: 2,097 1 88 31,960 zE: 6,063 62 3.0 Ethanol, 19 per cent 11,160 f 1,701 ~ 89 =12,-14o f 3,457 ~ 5S 3.7 Ethtutol, 21 per cent 16,300 ~L- 3,541 ' 83 80,400 t 15,446 I 16 4.9 H, vdrocurtisone, 15 ing. 17,430 f 3,193 88 18,500 f 1,:i1',0 i 8d 1.1 II, N-drocortisone, 30 ing. 13,155 =E- 1,990 1 88 26,395 f 3,978 73 2.0 Carbort black, 2.6 mg. G,430 f 800 92 5,140 ~ 1107 92 ! 0.8 * Itefers to the mice exposed to the condition indicated in colurnn 1. t Standard error. iReprodncecl zcithh perrnission of publishers, S. Karger A. G., Basel/1 ew York, of 1l(,licina Thoracalis, 1965, 22, 48). minutes following injection. Recovery of ciliary activity corresponded with a decrease in blood alcohol and the restoration of consciousness. In addition, mask breathing and higher rates of airflo«- decreased particle transport rates. In agreement with \Iyrvik's observations (6), the total yield of macrophages from each rabbit's lungs ranged from SO to 160 million cells, and the cell harvests were almost com- pleteh• homogeneous. The suspensions con- tained an average polvmorphonuclear leukocyte concentration of 1.56 per cent, and the eosin Y procedure showed that 95 per cent of the cells were viable. The metabolic rate of the alveolar Inacroph,i,_,eti avcr:i;ed ''5.S AL./10` cells per hour, and the administration of alcohol to cell suspensions in vivo did not alter respira- tion sMnificnntly. In addition, the numbers of alveolar macrophages harvested from al- coholized rabbits showed no change from control animals. Separate studies with the intratracheal challenges of carbon black and ,~taphirlococcus oureus did not increase the macrophage y-ield, and a~zain alcohol did not c1lan,e the numbers. Alveolar macrophages ;tVidly ph:+,oc.ytosed carbon black and staphvlo- cocci, ~cparately. in vitro. This wa~ meavlu•ed nt one-half-, one-, and two-hour intervals. In the r~arlv observation<, the maioritv of cells 4loWed ailY traces of these material" hut at two hours, the majority of cells were packed. The addition of alcohol to the cell and par- ticle mixtures did not interfere with phagocy- tosis. A mean number of 1.25 X 10' alveolar macrophages was harvested from the lungs of control mice, and this number increased to 4.0 X 10' one hour after staphylococcal deposi- tion. This increase was blocked by the ad- ministration of alcohol immediately after bac- terial challenge (7). • . I • • • , AWAKE J. a T 0 S E Pm. 2. The i,tzect of ethanol on the clearance of staph.vIococci from the lungs of mice. The relative retention ratio is directlc- correla"ted with biooa alcohol concentrations and the state of uncon- sciottsness of the animals. (Reprorluced with permis.siora of 7t2tb1isher.s, S. I:arger _d(,, 73a.sel-A'etc For1,, of lledicina Thora- calix. 1IN;:i. 22, -iS)
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138 LAURENZI AND GUARNERI DISCUSSION The method described herein offers a pre- dictable, quantitative technique for studying pulmonary resistance to air-borne infection. The method includes: (a) the production of bacterial aerosols of controlled concentrations and particle size from which predictable num- bers of organisms can be implanted in the lungs of laboratory animals; (b) the demon- stration of the rapid clearance of the bacteria; and (c) the measurement (assay) of si;nifi- cant reduction in clearance produced by cer- tain conditions which are often implicated as contributory factors in pulmonary damage and infection. The use of an organism of low virulence which clears rapidly provides a con- venient system in relation to time, avoids complicating infection, and lends greater sig- nificance to conditions which reduce clear- o_~ `J MEAN '_ 5 E CONTROL MICE ~r ~dEAN ' S.E ALCOHOL 'REo~TED MICE 2 3 4 HOURS AFTER :iEFOSITION ~-600 I E I I ~ E I a 10; a 4-~ O CONTROL • ALCOHOL TREATED • 0 200 400 600 800 I,000 1,200 BLOOD ALCOHOL LEVEL (mg%) Fic. 4. The effect of ethanol on the transport of heterogeneous carbon particles by the mucociliary strcam of the cat's trachea. Marked slowing of ciliarv function is demonstrated, and the effect is a graded one which corresponds to the blood alcohol concentration. ance. It is emphasized that clearance refers to the loss of viability and not necessarily to the E removal of the bacterial cells from the respira- tory tract. Under the circumstances of this method, smoke inhalation and alcohol caused the most marked reduction in bacterial clearance. In this report the writers have focused their attention on the effects of alcohol as a test variable by correlating the effect on clearance with its ef- fects on mucociliary function and alveolar macrophage activity. There is a large back- around of clinical information which relates alcohol to decreased resistance to infection. In this regard, many clinical reports have empha- sized the adverse effect that alcoholism haw on the prognosis of pneumonia (S. 9). In 1924, ~ > ~ 450 ~ ~ ~ Z 0 ~ 0 M ~-300 L'IG. 3. The effect of ethanol (19 per cent) on the clearance of staphylococci from the lungs of mice. This curve, dc•picting the nnmbers of staphvlococci remaining in the lunt;s of mice oN-cr the foin•-honr period immediateh- following bacterial deposition, shows that the maximal interfcrenc,• with clc;ur:ince occurs durin,e the first hour; this efi(•ct corresponds to the initial peak blood alcohol concentration. ~iillman first demonstrated that inhaled pneu- mococci persisted in the lungs of alcoholized mice and that 40 per cent of the animals died of septicemia (10). Pickrell folind that rabbit.< were more susceptible to pneumococcal infec- tions after the administration of alcohol (11). He demonstrated that in alcohol-treated ani- mals, the migration of leukocytes into ~ local areas of infection was markedly impaired. ; 0Q091495 f
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-o to rhe ra- aod, lost this tion b-,r ef- olar ick- at es . In Aha- : ou 924, i r_tt- ized 4;ied i) its ifec- 11). mi- wal irod. I MECHANISMS OF PULMONARY RESISTANCE TO INFECTION Laurenzi, Guarneri, Endriga, and Carey (4) and Green aud Kass (12) have previously reported on the quantitation of impaired clear- ance of inhaled staphylococci from the lwrrs of alcoholized mice. In the present report, this effect is carefully correlated with the peak blood alcohol concentrations and the state of con:ciousness; and the maximal interference with clearance is shown to occur surprisingly early after aicoholl administration. As with any form of sedation, aspiration pneumonia is a common occurrence in stuporous alcoholics. However, the pharyngeal implantation ex- periments discredit aspiration as significantly affecting the retention of bacteria in the lungs of mice. The same level of unconsciousness produced b.v barbiturates had much less ef- fect than alcohol on bacterial clearance. Simi- larh-, the persistence of the effect of alcohol dtu•ing the administration of 100 per cent oxy- gen and during the maintenance of a normal minnte ventilation by inspiring 3 per cent carbon dioxide, excludes hypoxia and hypo- ventilation, respectively, as contributory fac- tors. Indeed, the rapidity of onset suggests a to.ic alcohol effect rather than a metabolic one. However, marked interference with mucus trans- port i~ found only with estremely hirh blood al- cohol levels. The findings of Green and Kass (13) con- cerning the disproportionate decrease in radioactivity and staphylococci from the lungs of mice exposed to P-32 tagged staph- ylococci focuses on the importance of the alveolar macrophage. They found that after four hours, the culturable bacteria decreased, while the radioactive count remained at imme- dlate, post-deposition levels. These results show that the rapid decline in culturable bacteria is not due to mechanical removal. Indeed, cul- urrabilitc cannot be equated with the per- ~i.~tence or clelrance of radioactivity; since ]re blcterial cell, alive or dead, must be cleared t> a particle. Brieger and La13el1e (1-4) have dotnnrn~tr-lte'i tlilt this process is adow one: rlro hulf-liic of 2 ;a particles is over 100 hour„ :ur(l the d,ita would fit well «-itlr the particle -izc arnerste,l h}- a DeVilbis~ §-IO nebulizer. llowever, tiro potsibility that radioactivity is u:rn>icrrr~t irnnt dead bacteriA cells to pu1- numar. *„<~,rt' h;is not been clarified. Such rt ,Phenrnnrnon could accotmt for the persi<tence 139 of labeled material. Existing evidence that the alveolar macrophage is bacteriostatic, rather than bactericidal (15), must also be considered in the interpretation of any -studies on bac- terial. clearance. Since we are concerned with viability, the matter is whether or not the alveolar macrophage does kill bacteria; and if so, is it the major cause of bacterial killing, or do bacteria which implant on the bronchial tree die because of local conditions offered by the mucus or its components. A compromise proposal depicts the alveolar macrophage as a transport system-engulfing particles and carrying them to the mucociliary stream for disposal. It has been demonstrated that the alveolar macrophage is a specialized cell with metabolic ;tnd enzymatic characteristics distinct from most other white blood cells with phagocytic properties (16, 17). Its basal oly~en con- sumption is four times that of pol.ymorpho- nuclear lcnkocytes, yet it requires only a 20 per cent increase during active phagocytosis. Peri- toneal macrophages require a 250 per cent increment for similar activities. In addition, the alveolar macrophage depends upon oxida- tive energy metabolism for its phagoc,ytic function, and it has three to four times the 1%-soz' vme, acid phosphatase, and beta-glucu- ronidase activity of peritoneal maerophages (17). This study supports the effective method of NTyrvik and his associates ('6) for harvesting l;tr_e numbers of alveolar macrophaees from rabbits; and the writers also demonstrate that this can be applied to mice. In the rabbit ex- periments, alcohol had no effect on the mo- bilization, metabolic activity, and pha,ocytic capacity of the alveolar macrophage. Indeed, no changc in rabbit m:icropha,e numbers could be demonstrated after intratracheal administra- tion of inert particles and hacteria. However, increased numbers of macrophages were ob- fained i'rorn the luna:; of tnice aiter an aeroaol chalienge with bacteria, and this mobilization was I,locked bv alcohol. Tlte l.rtter findings are in atgeement with those of Lal3elle and asso- ciateS (IS), who showed that increased numbers of plra,_,ocytic cells could he washed from the lunas ot rats after the tracheal instillation and inh;rl.uiom ot cc.irbon pmrticlr-. Pickrell 111i found that the migration of leukocytes into
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140 LAL"RESZI AND GL'ARNERI local areas of infection is markedly impaired after the administration of alcohol. Louria (19) has carefully demonstrated that, in the main, alcohol impairs the mobilization of peritoneal macrophages, although it ako de- creases phagocytic activity and intracellular killing. The present studies with alcohol fail to show that it interferes with macrophage me- tabolism or phagocytosis. In an atmosphere such as ours, which con- tains air-borne particles of many sizes, it seems reasonable to assume that substances which implant on the bronchial tree are cleared by the mucociliary apparatus, and that those which deposit in alveoli are phagocytosed. Therefore, the mode of clearance is dependent upon the site of deposition, which is in turn a function of the particle size. It is difficult to determine the relative roles of these processes in the experimental system described herein, since the range in particle size may not have effected major deposition in one or the other of the critical areas. The lack of specific data relating to particle size and site of deposition in broncho-alveolar models which simulate the dimensions of the lungs of mice puts addi- tional limitations on any predictions. The rela- tive contributions of these factors can best be ascertained by determining pulmonary clear- ance rates after exposure to monodispersed aerosols. However, the use of matched studies of this type in which the effect of the same ex- perimental conditions on bacterial clearance, ciliarv function, and alveolar macronha,e ac- tivitv are correlated may further elucidate the relative roles of these mechanisms in pulmonary resistance to bacteria. SUMMARY This study describes a precise aerosol system for exposin, laboratory animals to air-borne organisms and quantitating the rapid clearance of the inhaled bacteria from the animal's lungs. The high de--ree of predictability offered by this technique makes it especially adaptable as a reference challenge-response system in the studv of pulmonary resistance to infection. In this investigation, smoke inhalation and alcohol administration caused the most marked inter- ference with bacterial clearance. Particle trans- port studies demonstrated a profotmd depres- sion of mucociliary function by alcohol. In addition, alcohol interfered with the mobiliza- tion of alveolar macrophages from mice ex- posed to bacterial aerosols. No effect on macro- phage metabolism or phagocytosis by alcohol could be demonstrated. This experimental scheme, in which the effects of a test variable on ciliarv function and alveolar macrophage activity are related specifically to its effect on bacterial clearance, offers an attractive method of investigation. Ackno zcledgments We are especially indebted to Drs. R. Endriga. S. J. Hricko, and B. J. Collins, and to Mrs. C. B. Shichman for a great deal of this work; and to Mr. S. Yin and Mrs. W. Hikel for their technical assistance. REFEREN CES (1) Laurenzi, G. A., Potter, R. T., and Kass, E. H.: Bacteriologic flora of the lower respiratory tract, New Eng J Dled, 1961. 160, 1273. (2) Gocke, T. M., and Laurenzi, G. A.: Ampicil- lin therapy in acute exacerbations of chronic obstructi.-e lung disease, Proceed- ings of the Fourth Interscience Conference on Antimicrobial Agents and Cheinother- apy, American Society for -Microbiology, 1965,686. (3) Laurenzi, G. A., First, M., Berman, L., and Kass, E. H.: A quantitative study of the deposition and clearance of bacteria in the murine lung, J Clin Invest, 1964, i:i, 759. (4) Laurenzi, G. A., Guarneri, J. J., Endriga, R. B., and Carey. J. P.: Clearance of bac- teria by the lower respiratory tract. Science, 1963, 142, 1572. (5) Goldhamer, R., Barnett, B., and Carson, S.: A new technique for the study of mucus flow in the intact animal. Fed Proc, 1964. 2.3.406. (6) -llyrvik, Q. N., Leake, E. S., and Fariss, B.: Studies on pulmonary alveolar macro- pha,-cs from the normal rabbit: .1 tech- nique to procure them in a high state of pu•ity, J Immun.19o0. 88. 128. (7) Guarneri, J. J., Hricko, S. J.. Collins. B. J., Shichman. C. B., Yin, S., and Laurenzi. G. A.: In preparation. (3) tih:ittnck, F. C., and Lawrence, C. H.: _lcute lobar pneumonia, Boston M anrl S.i, 1918. I; S, 2-15. (9) Van Metro, 'L E.. Jr.: Pncuntococ~al pneu- monia treated with antibiotic~:. N,w F:nr; J 'Med. 195-1. ?51. lOaS. (10) titillman, E. G.: P( c-i~tence of inspirc-d bac- trria in the lun,s of alcoholized mice, J Eap Jled. 1921. .:ri, 353. 11) 1'ickrell. h. I:.: Thw ( fiect 4 alcuhOl intoxi- -;ition and r•ther anesthe<ia on re~i~umce to 0009'749'7
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MECHANISMS OF PtiLMONARY RESISTANCE TO INFECTION za- eti- ro- iol :al ble lge on :od i ,a, B. to ( 'al pneumococcal infection, Bull Hopkins Hosp, 1938, 63, 238. (12) Green, G. M., and Kass, E. H.: Factors in- fluencing the clearance of bacteria by the lung, J Clin Invest, 1964, 43, 769. (13) Green, G. M., and Kass, E. H.: The role of the alveolar macrophage in the clearance of bacteria from the lung, J Exp Med, 1964, 119, 167. (14) Brieger, H., and LaBelle, C. `V.: The fate of inhaled particulates in the early postex- posure period, Arch Ind Health, 1959, 19, 510. (15) Pavillard, E. R. J., and RoR-lec, D.: A com- parison of the phagocytic and bactericidal ability of guinea pig alveolar and mouse peritoneal macrophages, Aust J Exp Biol Med Sci, 1962, 40, 207. 141 (16) Oren, R., Farnham, A. E., Saito, K.. Milofsky, E., and Karnovsky, M. L.: Metabolic pat- terns in three types of phagocytizing celle, J Cell Biol, 1963, 17, 487. (17) Leake, E. S., Gonzalez-Ojeda, D., and Myr- vik, Q. N.: Enzymatic differences between normal alveolar macrophages and oil-in- duced peritoneal macrophages obtained from rabbits, Exp Cell Res, 1964, 33, 553. (18) LaBelle, C. W., and Brieger, H.: Patterns and mechanisms in the elimination of dust from the lung. In Inhaled Particles and Vapours, Pergamon Press, Oxford, Eng- land, 1961. (19) Louria, D. B. Susceptibility to infection dur- ing experimental alcohol intoxication, Trans Ass Amer Physicians, 1963, 76, 102. 01, 11- of 1- ace er- ud he he iga, )ac- -ace, S.: icus 96-1, B.: cro- 'ch- ~ of . J., nzi, •:ite ti18, ^. .e J ..+ ul3
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RESPONSES OF UPPER RESPIRATORY '.VIUCOSA TO DRUGS AND VIRAL INFECTIONS'. z F. B. BANG, B. G. BANG, n.xn M. A. FOARD3 Transportation of foreign particles, includ- ing pathogens, from their point of impact on the nasal mucosa to disposal in the pharynx is a combined function of the cilia, the mu- cus blanket, and the cells of the mucosa. As the rate of motion of the mucus blanket de- pends on the quality and quantity of mucus as well as on the rate of ciliary beat, it is important to know which of these factors is directly affected in a given experiment. For instance, we have found that after severe in- ternal dehydration, mucus may be anchored to the secretory cells by fine strands and that motion of the blanket may thus be re- tarded or stopped. If the stalled blanket is removed, the cilia will transport particles (1). This is germane to the evaluation of records that alcohol affects ciliary action. For example, an old but excellent article by Lommel (2) is often quoted in support of this thesis. He followed the rate of motion of dust particles in the tracheae of anesthetized dogs and found, first, that inhalation of irritating gases produced inflammation and slowed the rate of motion of the blanket. However, if the mucus was removed without disturbing the cilia, par- ticles were rapidly transported; the retarded rate had thus apparently been caused by an increase in the mucus mass or load. Secondly, lie gave two dogs enough alcohol in their drinking water to produce acute intoxica- tion and found the rate of particle transport reduced to ?'-"; or i/ o of normal. As alcohol is known to produce dehydration and to increase the viscosity of mucus, ciliary motion per se mav not have been affected. Although M vitro studies of tlie eltects of drugs on ciliary motion have great valuc, their relevance to intact respiratory mucosa is open to question, since blanket motion may itself remove certain drugv before the.y have ~ From the Department of Pathobiologv, School of H' vviene and Public Health, The Johns Hopkins L'niver~it~-, Baltimore, _Maryland. ' Supported bY a grant-in-aid from the Council for Tobacco Rczcarch-t~.S.<1., and GB 393 from the ',~ational Science Foundation. ° With the technical a~~;ietance of Raoul Spicker. affected the mechanism. Various detergents, for instance, have striking effects on cilia in vitro but none whatever on the blanket mo- tion in chickens, possibly because they fail to reach the cilia. For these reasons we will limit most of this discussion to experiments on one system: the nasal mucosa of normal, unanesthetized chicks. Since a virus must have access to a suscep- tible cell in order to produce infection, we have studied the effects of a few drugs and other agents on muco_~al function to determine whether they might alter susceptibility to a virulent strain of Newcastle Disease virus. To check on which component of the epithelial cell system was most directly affected, we have usually followed four parallel procedures: (No. 1) direct examination of mucosa immediately post mortem to test the rate of ink transport, and then observation of ciliary motion when the mucus is removed; (No. 2) examination of periodic acid-Schitt-Utained whole mounts to observe the effect on the total mucous svs- tem (3); (iVo. 3) examination of mucus for cellular debris; and (No. 4) examination of histologic sections for specific effects on muco- sal and submucosal cells. In table 1 are shown the diiterential effects of sotne pharmacologic and other agents on the components of the mucocili- ary system. Although cocaine is listed as having its primary effect on cilia, this appeared to be true with 10 per cent concentrations while 20 per cent also caused depletion of mucus 30 min- utes aftcr application. Among the early studies of clearance rates and of the directional flow of mucus in the nose of man and other animals were those of Lucas and Douglas (4) and of Yroetz (o). Ratea of motion gener.llly vary from about 0.5 cm. to 1 cm. per minute. As we work with young chicks in which tlie rate is 1 rnt. l,er nlinute, aund ilIe nu~cl~ arc too ~tn;ill to per- iuit the 1'ollo«-in'-, of clearance b~t-ray tnethods, we have therciore u~ed .~ever31 combinations of iii vivo <<nd 1>osunortotn methoda to time the olearancc tate6 of lndi;i ink. Theve are ~;um- nn,irized in table 2. 142 0009'7499
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> IP I 1 RESPO\SES OF MUCOSA TO DRUGS AND VIRAL INFECTIONS Most of our evaluations now depend on method No. 3, so that the actual drug effects are determined in the living animal, R•hic1l is killed at a time when the normal mucosa has TABLE 1 1)II'FEREVl'7-1L 1'.FFECTS OF S011E PII_11tNfAC(/LOCiIC AGEST.; (1N TnE Co1IPOSESr.s OF 'rnE MGC'OC'ILSARY ~YSrE\I Effect Primarily on Mucus SecretionCiliary Function I -- i Cellular Integrity llehYdration Cocaine ~ C}-c1:Iiue Pilocarpine l.N-locaiue Severe vitanlin A deficienc}- Vitvnine A Lowered tem- Viruses deficiencY per,tture Influenza ill fer- rets Ltu'yugotrachei- tis in chicks TABLE 2 ME'1'HOnS OF ;TLUYIVG CLEARASC'E IA t:l'1'Elt PESYIRATURY TRAC'I' 1. Kill, admiuister drug, test clearance with itJk 2. Administer drug, kill, test clearance 3. Administer drug, place ink in nose, kill and examine at 15 ntiuutes 4. Administer drug and iiik iutranas,tllv sinlttl- taneously; kill at 15 nlitlutes 5. Administer ink illtranasallv, followed imrnedi- atelv by druE; kill at 15 minutes 143 been cleared of ink (about 15 minutes). We have used, primarily, chicks from S to 12 days old; newly hatched chicks may have less stable rates. Figures 1 and 2~llow diagrammatically the method of studying the normal flon• of mucus immediately post mortem by meastu•ing the rate of ink clearance from the olfactory fossa. In table 3 the results are shown of testing two different drugs by several of the above method.s. It is to be emphasized that the bars end 30 minutes after the chicks are killed, as we believed that after this time the obser- vations began to lose their value: but the arrows extending from the bars indicate that the drug effect lasted beyond 30 minutes. Cocaine at 10 and 20 per cent concentrations slowed the clearancc rate markedly. but tests performed one and a half hour- after instilla- tion of the drug ~howed a return to normal rates. At the peak effect of 20 per cent concen- tration, whole mounts demonstrated exhaustion of the mucous glands and direct examination of the ciliated surfnce showed that cilia were im- mobilized except in ,~cattered random patches. We concluded that there was direct paraly-sis of the cilia. Although ihis conclusion is by no rneans; ori,_inal, it has direct bearinv on the virus ~tudies. In table 4 we have compared the effect of intranasal instillation of ~everal different drugs, I'IC. 1. The directional flow pattern of the mucus blanket as it is carried bv the aligned rows of cilia on the lateral rnucosae of the chick. The olfactotv conclla lacks cilia, but_. as the fine arrows indicate, secretions, or ink, are pulled away by ciliarv traction at its base. In- sets show the linear flow inside the maxillary turhinate (I(Ift), and the scroll of tllis turbinate as it appears in vertical section (right).
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144 BANG, BANG, A\D FOARD 0 ~lood O~ I~rniautes 2o minu~'PS FIG. 2. Diagram representing the clearance of a pool of diluted India ink placed at the root of the maxillary concha in the olfactory chamber: (1) placement of ink; (2) forward line of ink-laden blanket moving over turbinate; (3) main body of ink-blanket inside scroll or moving into nasopharynx; (4) ink-laden pool of mucus as cut edge of nasopharynx, mucosae essentially clear. Times vary somewhat per age/individual. These chicks were ten days old. TABLE 3 R.ETESTIOS 15 TO 20 MINUTES AFTER 7)RUG Minutes Cocaine 10°jo Method \ o. 1 2 3 4 5 5 10 15 20 25 30 Number of Chicks ~ 5 ~~ 5 10 4 8 Cocaine 20;(' Method No. 3 ~ J Cyclaine 5`;(' Method I~o. 2 2 3 13 5 12 * 4 * Intranasal drug 5 niinutes; saline wash 10; ink. all tested by subsequent intranasal instilla- local anesthetics, had no effect on the rate, but tion of ink in the li1-in, chick. The chicks were that hexylcaine (Cyclaine) had a remarkable killed after ten minutes, and the clearance time inhibitory effect. The acute eiiect la'~ted about was observed. It maY be seen that Pontoc_aine, 24 hours and produced quantities of intra- 1y-locaine, and Carbocaine,' all of which are nasal mucus. Direct examination of such `Pontocaine-tetracaine hydrochloride, Win- Carbocaine-mepivacaine hydrochloride, Win- throp. Yylocaine-lidocaine hydrochlorido. _-lstra. throp. Cutni Salil Ink : Drt1~ Coc: Cocn Cy-ci \'ita: Vita• muc ber~ mou cells cont (figt cell quai T: acin rate y'on( was: tion initi of ti « a vi hatc reta squ; phy basi tior Prl` mel rea~ tur lo'a nnl bu! 9 pei str. (j) ~ 00097501
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RESPONSES OF MUCOSA TO DRUGS AND VIRAL INFECTIONS 145 TABLE 4 CLEARANCE RaTES-blr;Tnon \ o. 3 Minutes 0 5 10 15 20 25 30 Number of Chicks 24 Controls Saline alone 4 Ink alone 6 1)rugs P, 1, Cb* 20 Cocaine 10`je 10 Cocaine 201 ' io ~ 5 Cyclaine 5`icf -~ 13 Vitamin A deprived-3 weeks ~ 10 y'itamin A deprived ~ 5 days u 2 * Yontocaine, 0.51 "; Sylocaine, 41,'~; Carhocaine, 21~ce t Hexvlcaine hydrochloride. mucus showed that it contained large num- bers of ciliated cells and mucous cells. Whole mounts demonstrated widespread loss of mucous cells, including acini; and histologic sections confirmed severe destruction of the epithelium (figures 3 and 4). In this case, then, mucosal cell sloughing was the direct cause of inade- quate clearance. The immediate effect of pilocarpine on acinar cells is hypersecretion so rapid that the rate of blanket motion is accelerated far be- yond the ciliary movement rate by an "over- wash" effect. The normal rate of blanket mo- tion is resumed within half an hour, but the initial effect is tantamount to vigorous flushing of the entire mucosal surface. We have found that if chicks are kept on a vitamin A-deficient diet for three weeks after hatching, the rate of clearance is somewhat retarded (again an expected finding since squamous metaplasia following mucosal atro- phy was shown by Wolbach (6) to be the basic lesion of this deficiency). Ciliary func- tion in the deprived birds was, however, sur- prisingly efficient, although the rate of move- ment was si:nificantlv retarded when it reached the imler-scroll surface of the middle turbinate. (See text fi,ure at ri,ht.) The histo- iny;ic et.unination showed evident :hrinkin(z of the nnico<a and pnrtial dvsfunction of mucous cells, l,ut ncark- all cilia were intact (fi,ures 5;lml 61. The effects of viruses on such mucosa de- pend primarily on the amount of mucosal de- ,truction. Although Stuart-Harris and Francis (7) sllowed the destructive effect of influenza TEXT FIGURE: Diagram of retarded clearance of mucus from inner scroll of turbinate of vitamin A-deprived chick. Dotted line shows thin terminal line of ink-stained blanket which, though visibl.moving, is about twice as slow in clearing this area as the hlanket in control chicks. (See figures 5 and 6.) virus on ferret turbinates, and Hers (8), the types of lesions produced in human and ani- mal mucosa, and although we have studied the influenza-ferret cell interaction with the electron microscope, still too little attention has been paid to the direct effects of viruses on mucosa. Two studies of the effects of recently isolated respiratory viru6es on mucosa come to mind: that of Coates and Chanock on respiratory syncytial virus on ferret mucosa (9), and of Buthala and Soret on parainfju- cnza 3 in these are presented. hamsters (11)l: yet in neither of sequential studies of pathogenesis We have searched for an aaent which would caul~e extensive destruction of chick respira- Il
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146 BANG, BANG, AND FOARD ph: rel: of (1:_ ino 1,0, dot bla chr. s1w av: 10, €~_ FrG. 3. Section through maxillary turbinate of normal ten-da' v chick, showing normal ztate, and pattern, of staining of mucous acini. (HematoKVlin and periodic acid-Schiff stain.) FIG. 4. Section through turbinate of ten-day chick one hour after intranasal instillation of cyclaine 5 per cent (4 drops/nostril). Note sloughing, mucosal shrinkage, acinar gaping. (Hematox}-lin and periodic acid-Schiff stain.) FrG. 5. Histologic section through maxillary turbinate of three-week old chick, showing normal appearance of mucous glands. (Hemato.' vlin and eosin stain.) Frc. 6. Section through turbinate of chick which had been deprived of vitamin A for three weeks since hatching. blucosa of inner scroll surface most Severehy aifected, yett most cilia fionctionall' v intact. Black wisps are ink residue; ink was instilled into nose ten minutes _ hefore decapitation and fixation. (Hematon•lin and eosin stain.) tory nutcosa but would not kill the host. The virus of Izrl-ngotracheitis will, under certain conditions. dcstrol- 1~trge areas of mucosa so that functionnl clcarance is almost abolished and thick mucus accnmulates in the nasal fossa (11). Adhesions in the turbinate mucosa during early reaction often cause cyst-like pocket~ whole mounls (,f hte convalescent tnuco.5a ~,lioW that, a(tcr acute slou!~hing, the normal linear functional pattern may be permanently disorganized (figures 7 and S). We have as yet done no electron microscopic studies of these effects; our clectron micro- photographs of postinfiuenzal ferret mucosa showed that rerenerating ciliated cells in the earh- stages hmd relativellv few cilia l1''). As virus must enter the mucosal cells tliroii,h the blanket, there miil~t be three tra cel: sin tlu thc bla re; thc br- « •1-. it~ W1. C~\ in _ in: rc- m, tl; 00097503
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RESPO\SES OF \1tiCO5A TO DRUGS AND VIRAL INFECTIONS 7 8 Fic. 7. Periodic acid-Schiff-stained whole mount of septum of control chick, showing nor- mal lineation of mucous acini. The clear lines between rows of acini are ciliary pathways. F>;c. 8. Whole mount of septum of chick 13 days old following nasal instillation of laryngo- tracheitis virus, showing ahnormal pattern of regeneration of some areas of mucosa. phases in the sequence of the virus/cell relationship. The first would be penetration of the blanket. We have shown elsewhere (13) that of 10° Newcastle Disease virus cells inoculated into the nose of a chick, onlY about 1,000 reach susceptible cells. If this tremen- dously efficient protection is due to the mucus blanket, alteration of the mechanism by changing the nature of the blanket or by slowing its rate of movement should affect the availability of the susceptible cells (figures 9, 10, and 11). The second demonstrable stage in cell pene- tration is adherence of virus to susceptible cells, a temporary phase, detectable by tryp- sinization, which tends to decrease markedly three hours after infection (figure 1'?). Administration of pilocarpine first increases the susceptibility of cells by disturbing the blanket; then, as new mucus is secreted to replace the old, adherent virus is swept out of the tissues, and the amount of infection is brought back to normal (figure 9). Cocaine, which paralyzes the cilia, increases susceptibil- ity of the mucosa 10 to 50 times, a change which persists for several hours (figure 10). Cyclaine, which causes overwhelming slough- ing of cells, results in an initi,llly low ratio of infected cells in turbinate mucosa ; but, ,t,~ the resistant virus sits on rhe esUcntialli- inimobile muco~al surface, it is ~_,~raduallv absorbed and the tntmber of iutected colls eq~ial~ that of thc control (figure 11). 10,000 1,000 ~ ~ U ~ ~ V ~ loo 0 ~ ~ E z' lo x ® ® virus 30 min. after Pilocarpine • control .. 147 ® 1 3 5 Time in Hours Ftc. 9. The effect of 0.6 per cent pilocarpine on the numbcr of infected cells from the middle turbinates of three chicks inoculated with 10' PFU (plaque-forming units) of -Newcastle Disease virus. 1)fsC[-:?IOS _A-ND COrM4RY Although a variety of a,ents will change the rtficiencv of the ciliated na_~al tuuco-za, viruses seem to act bv destroying the cells of the mucosa, leavin; bchind an epithelium which,
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148 BANG, BANG, :1\I) FOARD ~ ) ~ - I ~ I x I ® virus 5-10 min. affer Cocaine I x virus 30 min. affer Cocaine • control I Time in Hours Frn. 10. The effect of 10 per cent cocaine on the number of infected cells from the middle turbinates of three chicks inoculated with 10' PFU of Newcastle Disease virus. i I ® virus I hr. X virus 30 • control after Cyclaine ~ min. after Cyclainel i Time in Hours FIG. 11. The effect of 5 per cent cyclaine on the number of infected cells from the middle turbinates of three chicks inoculated with 10' PFU of Newcastle DiscaSe virus. durine regeneration, has at first few or no Regeneration of the two component parts of cilia. This destruction, which involves both the epithelial sA•stem does not always proceed ciliated and mucous cells, exposes the denuded at the same rate. As the motion of the muco- mucosa to absorption of other toxic substances. ciliarv blanket depends on the concerted activ- c T U009'7505
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RESPONSES OF MUCOSA TO DRUGS AND VIRAL INFECTIONS 20 10 • 2 4 149 5 Time in Hours Fic. 12. Change in the ratio of infected cells/adherent virus in middle turbinates at in- tervals after intranasal inoculation. This change is understood to reflect cellular incorporation of virus. ity of mucous cells and cilia, lack of movement of the blanket does not necessarily mean that ciliarv action itself has been inhibited. Actual ciliary paralysis induced by cocaine did in- crease the number of infected cells by 10 to 50 times over the control values. Finally, it is _~uggested that repeated respiratorv tract infections which in man destroy ciliated cells may make the mucosa more available to carcinogens, in which case the virus ma' v act as a co-carcinogen rather than as the primary disturber of cellular metabolism. REFERENCES (1) Bang, B. G., and Bang, F. B.: Effect of water deprivation on nasal mucous flow, Proc Soc Exp Biol Med, 1961. 106, 516. (2) Lommel, F.: Zur Physiologie und Pathologie des Flimmerepithels der Atmungsorgane, Deutsc•lc Arch lilin Mud. 1905, 9~, 365. 3) Bang, B. G.: The surface pattern of the nasal mucosa and its relation to mucous iiow, J Morph, 1961, 109, 57. (4) Lucas, A. M., and Dou,las, L. C.: Principles underlyin_ ciliar' v activity in the respira- tor' v tract: II, Arch Otolaryn (Chica(_,o), Ll;l1, 30. 51ti. (5) Proetz, A. IC.: EssaYs on the Applied Phx-s- iology of the 'ose, Annals Publishing Company, St. Louis, Missouri, 1953. (6) Wolbach, S. B., and Bessey, 0. A.: Tissue chang_ es in vitamin deficiencies, Physiol Rev, 1942,?2, 233. (7) Francis, T., Jr., and Stuart-Harris, C. H.: Studies on the nasal histology of epidemic influenza virus infection in the ferret: I, II, III, J Exp Med. 1938, 63, 789. (8) Hers, J. F. Ph.: The Histopathology of the Respiratory Tract in Human Influenza, H. 1:. Stenfert Kroe~e N. V., Leyden, The Netherlands, 1955. (9) Coates, H. V., and Chanock, R. M.: Experi- mental infection with respiratory syncytial virus in several species of animals. Amer J Hy-g, 1962, 76, 302. (10) Buthala, D. A., and Soret, M. G.: Parain- fluenza type 3 virus infection in hamsters: Virologic, serologic, and pathologic studies, J Infect Dis, 1964,114, 227. (11) Bang. B. G., and Bano, F. B.: Responses of upper respiratory mucosae to dehydration and infection, Ann \ Y Acad Sci, 1963. 106, 625. (12) Hotz, G., and Bang, F. B.: Electron micro- scope studies of ferret respiratory cells in- fected with influenza, Bull Hopkins Hosp, 1957, 101, 175. (13) Banc, F. B., and Foard, M.: Interaction of respiratorv epithelium of the chick and \ewrastl(~ disea~,_, 6rus, Amer J II}-g, 1964, 7J, 260.
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RESPIRATORY VIRUSES, CILIATED EPITHELIUM, AND BRO\CHITIS' C. H. STUART-HARRIS All of us present at this symposium are aware by personal experience of the impact of viral infections on the respiratory epithelium. We are reconciled to the recurrent minor miseries afflicting our upper respiratory tract since nowadays we are much less likely than formerly to perish from bacterial infection of the lower respiratory tract. In this sympo- sium we have already listened to papers deal- ing with the effect of noxious environmental agents on the ciliated epithelium, and Dr. Bang has introduced the subject of viral in- fection. It is my task to deal briefly with the state of knowledge of the respiratory viruses, their relationship to human disease, and their action at a cellular level. THE RESPIRATORY VIRUSES OF D1.9\ The whole of our knowledge of the human respiratory viruses has been assembled in the past 30 years, and all of that dealing with viruses other than influenza belongs to the past 1'2 years. In table I the six known groups of ~ iruses are listed in the order of their recovery, their ty pe of nucleic acid, where this is known, and their serologic groups. The application of tissue-culture methods based on the work of Enders and co-workers (1) permitted the discovery of most respiratory viruses other than those of the influenza group. Cultivation in the chick embryo, although still the ac- cepted method of primary isolation of the influenza viruses, is not of assistance with any of the other viruses. Moreover, the majority of these viruses are strict human pathogens, and, with the exception of the Coxsackie viruses, certain adenoviruses, and some parainfluenza strains, they produce no pathologic effects in animals. It is only possible here to deal with certain features of the respiratory viruses and of t he infections which they produce. First of all, these viruses are, for the most part, transient inhabitants of the humrul re- spiratory tr.ict, producine brief self-lunited in- fcction either of the ciliated epithelium itself or of adjacent tissue such as lymphatic tissue or i* P'rom the Department of 1ledicine, The Uni- c-ersity of Sheffield, Sheffield, England. the alveolar epithelium. Only the adenoviruses and the enteroviruses appear to be able to attack epithelium other than that of the re- spiratory tract, yet their degree of involvement of the alimentary tract or of general systemic spread is extremely variable. Moreover, each virus group possesses the potentiality of attack- ing the respiratory tract throughout its whole length, from conjunctiva to alveolus, while maintaining a particular predilection for cer- tain areas. This is well seen in the case of the rhinoviruses, whose effect in previously healthy persons is apparently limited to the nose and throat, although in children the lower respiratory tract is sometimes involved. There is as yet no clue with regard to the reason for this organ predilection, but this is a well known phenomenon of all viral di~eases. The second feature shared by nearlv all the viruses is that they are cultivable from secre- tions of the nose and throat or sputum only during or just before the phase of clinical in- fection. Certain serotypes of the adenoviruses are also cultivable from organs such as tonsils or adenoids removed from children in whom they appear to cause latent infections probabh- persisting from an earlier illness. All the other viruses disappear after producina acute infec- tions and are found relatively uncommonly in the respiratory tract of healthy persons. Perhaps because of the transient character of their infections, the re~zpiraton- viruses pro- duce onlv a temporary immtmit' v. After each infection neutr;tlizinz cintihodies develop in the serum, and the-,- can be detected in the respiratory secretions during convalescence from iutcction. ImmunitV to reinfection is temporarily good and apparenth• dependent on specific .uitibodics, for infection i,y heterotypic ~-erotypes is possible in those who have re- covered from infection by viru-es such as the rliinoviruses (2, 3) which produce a hi;hl.y specific seroloaic response iit the host. Reinfec- tion is possible bly homotYpic strains even in thc presence of neutralizing antibodies in the serum with some viruses such as the respira- tory syncytial and parainfluenza groups (-1, 5). It seems likely that the state of the celk of the 150 00097507
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V7RUSES, CILIATED EPITHELIUM, AND BRONCHITIS 151 TABLE 1 TIIE RESPIRATORY LIRtivER* ttSes ~ to re- inent emic "a ch iack- [ hole -ilile cer- Ot )(Islv the the 1 ved. the i.z a the cre- )niv in- •lses sils ;iom bhiher lfec- v in ,r of pro- -ach in the ,nce is Oll }-pic re- rhe _hly ec- i in ih(' ra - hc N irus Group Date of Discovery Class Serotypest Iuiluenza A 1933 \iYsovirus (xN.a) _1 , .1. :1t least t hree f.Iml Ies of re- lated strains each B 1940 B C 1949 C One type only known Adenovirus 1953 DNA 31 human and tnant• animal t"-pes Parainfluenza 1953 1hYxovirus (ItIN A) Four RespiratorY `~ nc. tial (R5) Picorltavirus Ilespiratur}- enteroviruses 11inovirus 1956 ? Jlvxuvirus (1tN' A) From 1948 It\ A oonwards 1960 RNA At least two Coxsackie A, e.g., .a °_1; sackie B ECeo, e.g., types 11, 20, 28 More than 50 tYpes Cox- * Position of reovirus (Ecuo) uncertain. f Provisional. respiratory epithelium, including their capacity to resist virus attack by secreting substances such as interferon, plays an additional and important role in determining the host's ability to resist respiratory viral infection. The dual acquisition of antibodies and of nonspecific factors of resistance is responsible for a change in the frequency in infection and of its clini- cal character with age. Yet adults seem rela- tively resistant to infection by most adeno- viruses, the parainfluenza viruses, and the respiratory syncytial (RS) viruses, whereas rhinoviruses and influenza viruses subject them to repeated attack throughout life. Subclinical infection in which there is viral multiplica- tion and release without any clinical change occurs in each of the groups, but varies in frequency according to the degree of immunity of the host, the size of the infecting dose, and rlte biolo,ic character or virulence of the partic- til:ir viruses. Before discussing the clinical wn- dromes which are related to the various viruses, it is necessarv to consider their pathologic ''Pi=ects on cells and tissues. CELLULAR EFFECT3 OF '1'III: RESPIRATORY VIRliSES JIcutY inn t•(tro systems, such as ti.-_stte cul- mre; ct)mpol~ed 4 ~tisceprible cells, support the growth of the respiratory viruses and develop pathologic changes. Yet there is no equivalence between viral multiplication and cytopathic changes, and there are many instances of cell systems which remain histo- logically normal but which produce continu- ously large amounts of virus. In fact, the reason for the destructive changes occurring iII cells parasitized by viruses is not known, al- though release of lysosomal enzymes (6) is one possible mechanism. It is known, however, that with a particular cell system and viral species, the physiologic state of the cells, includ- ing their temperature, pH, and oxygenation, may determine both the quantity of viral multiplication and cytopathic effects. In addi- tion, some viruses will grow in a wide range of different cell-culture systems, whereas others are estremely fastidious and will only Inultiply in a particular t' ype of cell culture. Viruses, indeed, are known to exist in human secre- tions because of their biolo,ic effects which have ~:o far resisted all attempts at cultivation in zn ritro svstems. With the respiratory viruses which can be cultivated in tissue cultures, the c}•topathic t•tfects vary from necro~is and cell destruction «-inc cnteroviruses tlirou,h ]es~er degrees of clfange includinr syncytial (,_riant-cell) forma-
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152 C. H. STUART-HARRIS i tion with RS and parainfluenza virus, to the appearances of cells producing hemaggiutinins at their surface without other abnormality in influenza viral infection. The cytopathic effects of adenoviruses seem to be obvious, but the cells which roll off the glass continue to metabolize and produce large amounts of acids for some time. The rhinoviruses produce good cytopathic effects, but only in the right sort of cells at the right temperature and pH. When the pathologic effects of the respira- tory viruses in whole tissues are considered, it is obvious that there is much ignorance. Histo- logic studies in man are confined to biopsies, nasal or pharyngeal cytologic microscopy, and the rare autopsy. It is claimed that the first two methods show epithelial cell necrosis and abnormal detachment of ciliated cells with influenza, adenovirus, and probably other infec- tions (7, S). But the observations are scanty, and it requires the application of fluorescent- staining methods to prove that the desquamated cells are actually the site of viral multiplication (9). Autopsies after viral diseases, in man, apart from those in influenzal and adenovirus pneu- monia, are infrequent. One turns therefore to studies in animals for evidence concerning the. pathologic effects of viral multiplication. Here the model is still that of influenza viral infec- tion in the ferret, which is a disease with a remarkable analogy to human influenza. The serial epithelial changes of influenza in the ferret are well known and were described by Francis and Stuart-Harris in 1938 (10). Forty-eight hours after intranasal inoculation with influenza virus A strains, a rapid necrosis sets in with destruction of the ciliated columnar cells covering the nasal turbinates, diapedesis by leukocytes, and vascular engorgement (fig- ure 1). The basal epithelial cells survive and multiply so that within a week a stratified columnar epithelium several cells deep is re- formed. For a time, the surface cells ma~• be squamous, but soon columnar ciliated cells reappear although goblet cells are present in increased numbers (figure 2). Attention is drawn to the similarity of this change to the cltan,es which occur after ionization intra- nasally with zinc, or even after irrigation with zinc sulfate solution. In the~e, destruction of the superficial nasal epithelial cells is fol- lowed by regeneration (ti,ures 3 and 4). Stunrt-IIarris and Frmlck (11) also found that Fro. 1. A asal turbinates of ferret 48 hours after intranasal infection with PR 8 strain of influenza virus A, showing necrosis of ciliated epithelium. ~ ~ ~ Frc. 2. lt(generated epithrlium of Icrrot's turhi- n;ttc 11 dLivs after in'tranasal infection with 1'1{. 8 virii~. \ote prescnco of cili.urd cell_ ;ind ahro ~•oblct c~~ll format ion. ~ the stratified na.~al epitheliiun pre-ent ~even days after influenza viral infection was resis- tant to zinc ioniz,ltion. The converse did not appl' y, for influenza virn.~ multiplied well in tlte regeneratin; cpithelimn foiind after zinc treatment. 0_ ho inr 00097509
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VIRt".SEB, CILI:ITED EPITHELIUM, :1\D IiROSCHITIS a after i.rnza urbi- et ,C Precisely similar ehithelial chan(zes occur durin~: _ inflttenza in the ferrets' hronchioles~ after infection by a lunn-adapted ~4train of influenza virus under anesthesia. Again, re- cover}• by regeneration follow< the initial epithelial destrtution (figures 5, 6, and i) and similar chanoes are found in the mouse and in nian after influenza viral infection. FIere the story ends, for few viruses other than influenza produce pathologic effects in ani- mals. The parainfluenza virus strains «-hich will ~row in the mouse lun~ are believed to be Ftc. 3. Epithelial lesion of ferret's tnrbina'te 24 hours after administration of zinc sulfate solution in'tranasall}-. -Note loss of epithelial cell.s. Ft~:. ~}. l~;t,itli~liat ir,ll rr<n•nrratinn siti ~la~~~ .~ft~~r ~n rumrni u~iih zin~~ ~uliatr -ulirnion tntruia~allv. h'tc. 5. Normal ferret lung, showing ciliated epi- thelium of bronchiolc. Frc. 6. liront•hiolar clnlhelium of ft-rret lunv 4S hours aft(•r infection with 1'Il, S virus intranasallv under ether anes'thesia. Aoie destruction of ciliated opitheIial rrlk, but prr,~zorvation of basal ]a.•er. Note also fornt:tiion of exudahe of ri, s(tuamated cells and b-nkorN tr.4. 1nrnt~e tll(, R', \~ini~ li:l.< heen fotund ill tnultipl.v in iht, irrrt•t,' re,piratorv tract (L°), Lut ihe t u-bin:uc~ :ire not, in niv own exherience, uec(-:,irilY tli-torhed. Adc•novirus tylx.e 5 will uronw in tlic- ~itrklinm liam_ter t131, hnt it pro~l~irr.~ luclr;ltiti>: other t\~l,(- _ of ~;irn~-!_'. ]~. ~. :untt :;1-],rndttcr ;mapla.~tic tiintor.~ in tlti- :utimal 114-M. l;ltinovirnses
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154 C. H. ~'rCART I3AItR1S have not been transmitted to animals so far; and the respiratory enteroviruses, such as vari- ous Coxsackie A or B viruses, produce in suckling animals either general ilamage to Vj b i ' Ar ~Y s ~ . s kw4 iS i'Ipo FIG. 7. Regeneration of bronchiolar epithelium seven days after PR 8 virus intranasallN• under anesthesia. Note the stratified form of epithelium which has temporarily replaced the original ciliated cells. INFLUENZA AND FEBRILE ACUTE RESPIRATORY ILLNESS RESEMBLING INFLUENZA FEBRILE SORE THROAT (PHARYNGITIS AND TONSILLITIS) COMMON COLD CROUP IN INFANTS OBSTRUCTIVE TRACHEO- LARYNGO BRONCHITIS ACUTE BRONCHITIS IN CHILDREN ACUTE BRONCHIOLITIS IN INFANTS ATYPICAL PNEUMONIA PNEUMONIA striated muscles or nervous tissue or no effects at all. One can therefore only apologize for the fact that the pathologist is at present gravely handicapped in attempting to interpret the cytopathology of respiratory viral infections. RESPIRATORY SYSDRONSES AND THE RESPIRATORY 1IRUSES A final look at the various clinical syndromes of respiratory tract illnesses and of present known relationships to the various respiratory viruses is sltown in fi,ure S. Each fi_ured block represents very approximately the pro- portion of the whole syndrome caused by partic- ular ,igents. It is seen that each syndrome is cau,cil bly a epectrtun of a_ents, btrt that partic- ular viruses appear to favor particular sy-n- dromes. This is exemplified by the febrile char- acter of inHuenza an<l by the propensity for parainfluenza viruses to cause croup and for the RS viruses to cause bronchiolitis in babies. In adults, the rhinoviruses, respiratory entero- viruses, and influenza viruses seem to be rela- HAEMOLYT II ~~~~~~II S PTOCOCCI I I imifl ADENO .' . . T , INFL i ~ .3,4,7, 14, 21 A, iiii I i II I ~ RHINOVIRUSES ARa x o j I~IIII~IIIII IIII'ill~llillllllill ~~ R S W y=j ~ . J ' I(incl ECHO 28) FL ~ Q Ni OWN I!I IIII I I < o ~ ~ PARA NFLUENZA 1,2,3,4 ~= AU r 1[ 11 RHINOVIRUSESII (incl. ECHOIh28)I~ I R S PAR 9 FLU a a R. S PARA FLj MYCOPL. PNEUMONIAE EATON PPL.O. ~ SECONDARY aA2TEHIAL t =~nwiumillllllluuuuunhudlllluul ADENO 1 771 ~ ~ 770 z az , FI ( ; . ~. I;I~~Ilir;tll) r }- :.\ - T Ilirutrn:'s anll thh rlI :IliraUorv \ irn~-. .,. ...~. _
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VIRUS:S, CILIATED EPITHELIUM, AND BRONCHITIS !ffects Ir the •avelv ; the romes iesent •ttorv -ured pro- ,.trtic- IIe is ..Irtic- `\-n- ,har- - nsity tl for I ibies. t.tero- rela- J I 1 f tively more active in cautiin, colds and febrile illnesses than are thc adenoviru<es and so on. If bronchitis is considered, then there is a clinical distinction between acute bronchitis in previously well persons and the exacerbation of s, ymptoms which occur so frequently in those with chronic chest diseases, including chronic bronchitis and emphysema. Inflttenza and rhino- viruses are both kno«•n to cause bronchitic illnesses in healthy persons or in per~ons with chest disease. The participation of RS and parainfluenza viruses in acute exacerbations of bronchitis in adults has been sug_ge~ted by Sommerville ('1S) in Scotland and bv Carilli and associates (19) in the L-nited States. All th,tt can be said at the moment is that all four groups of viruses appear able to prodace in man the symptoms and signs of irritation, fluid exu- dation, and the increased mucus proclnction by the lower respiratory tract seen in bronchjtis. None of these viruses, however, are likely to he persistent causes of sputuni production so characteristic of the patient with chronic bron- chitis. The acti.-ee collaboration of viral and bacterial infection and a syroerristic effect of noxious atmospheric inflttence~ mtist he postu- htted to account for the chronic stnoldering phases of this distressin,n, complaint. REh'1~;RI:\CES (1) Enders, F. J., Weller. T. H., and Robbins, F. C.: Cultivation of thc, Lansing ,train of poliom.vclitis virus in ciiltttt•es of various human cmbr}-onice tissues, Sc•ience, 1949, 1109. S5. (2) Jackson, G. G., Dowling• H. F., and i13o- gabgab, Il-. J.: Infcctit•ilY and r:•lation- ships of 2060 and JH viru.,es in tolimleers, J Lab Clin 1led, 1960, 55. 331. (3) Taclor-Robinson, D., and B}noe, M. L.: In- oculation of volunteers with H rhinoviruses, Brit 'Med ,I, 1964. 1, 540. (4) Johnson, K. M., Chanoc•k, R. M., Rifl:ind, D., Kravetz, H. bl.. and Iiniait, Z".: Resplra- tory svncN-tial airns infection in adult vol- untcers: 11. Corrclation ut tirus <hoclding, scrologic response and illnc,,s in adult vol- unteers• J.1mer AIed _1~s, 1961, 1;i:. 663. (5) Chanock, P.. M., Parrott. R. H., Johnson, Ii. M., Kapikian. A. Z., and B~ 11. J. _1.: 14Ysoviruses: Parainfluenza, :',mer J Resp Dis, 1063. 5S (Supplement, p. 152). (6) Allison, A. C.. and Sandelin. B. _11.: lctica- tion of 1.-sosotnal enzvmes in virus-infected colls and its possible relation to cytopathic rffc cts. J Exp AIed• 1963, 11 -,, 879. (7) Picrcc, C. H., and Hirsch, J. G.: Cilioc' vto- phoria: Relationship to viral respiratorc infec•tions of humans, Proc Soc Exp Biol Jlcd, 1958. 1i:5', 489. (8) Pierce, C. H., and Knox, A. W.: Ciliocyto- plioria in sputum from patients with ad- (.novirns infections, Proc Soc Exp Biol 1ied,1960.10.i.41)2. (9) Hc•I;~, J. F. Ph: Fluorescent antibody tech- (10) (11) niyue in re.spirator' v viral discases, Amer Rev Resp Dis. 1963, SS (Supplement, p. 316). Francis, T., Jr., and ~tnart-Harris, C. H.: Studies on the na.'al histolox}- of the epi- clenliv influenza virus infection in the fer- rc,t : L The det-elopment and repair of the nasal lesion, J Exp _Med, 1938, 789. Sntart-Harris, C. H., and Franci~. T., Jr.: titudics on tlte na,_al histolop- of epidemic influenza virus infection in the ferret: II• The resisht.ncc of regenerating respiratory epithclitun to reinfection and to phV sico- c•hemic•;d injury. J Exp \Ieci. 193S. 68, 803. (12) Coater, H. V., and Chanock, R. Al.: Experi- mental infection with resoiratorY sYncYtial % irns in se% c•ral spe_cies of animals, Amer .I It ' ~g. 1962, 302. (13) 1', reira, H. G., Allison. A. C., and Niven, J. S. F.: Fatal infection of newhorn ham- Ac-rs bc an ailenovirus of human origin, \ ature ( London), 1962,1(trJ, 244. (14) Trrntin. J. J., Yabe, T., and TaYlor. G.: The (15) que..t for htunan rancer viruse~:. Science, 1962, 1-1;. 835. Hnebncr. R. J.. Rocce. W. I'.. and Lane, 1C. T.: Onc•ogenic effects in hamsters of hunlan adenot-irn< tvpes 12 ,tucl lb. Proc \at. Acud S:•i, 1932, .rS, 2051. (16) Giraurdi. ~k. ,I., Hillcman, AI. II.. and Zwicke.•, (17) (18) I1. 1:.: Tests in hamstet•s for onc•ogcnic qunlitly of ordin.u;Y \ iru~r~ including ad- enoviru.ws t' vpe 7• 1'roc• Soc I:xp liiol \Ied, 1964, 11,5• 1141. Pcnvira. AI. S.. Pereirt. H. (:.. nnd Clark, S . . Ii. I1.: Human adenotirii< tYpe 31: A nww serot}•pe with onco._enic properties, Lanc•(•t. 1965, 1. 21. S S unlmc rt ille, R. C~.: R(spiratur~ s~-nc}•tial tIPtls in aCnt^ f`xa(•('rhatlonr ~)I chronlc I,ronchiti~z, L:uu•et. 1063. .'. 12-1I. (19) Carilli. _1. I)_ (;ohd, R. S.. ancl Gonlon, W.: _k ~-irolo;ie studc• of chronic l,rondiitis, \ow F.n; J Med. 1J6-I. ?(0, 123. t t
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EFFECTS OF SOME VIRUSES ON CILIATED CELLS'• 2 B. HOOR\ AND D. A. J. TYRRELL I\THODliC'TIOS \Iany previously unknown viruses have been cultivated in the laboratory in recent years, and this has greatly increased our understanding of the cause of acute respiratory infection,~. The advances have all been achieved bv esploitin,-, the techniques of tissue culture. For example, Enders and his colleagues showed that cul- tures of human fibroblasts sul;ported the growth of polioviruses; it was later shown that cultures of monkeY kidney cells dispersed with tr}•psin were equally suitable for this purpose. Then it was found that both human and monkey renal celk would support the growth of influenza .-iruses (1, 2) which pro- duced hema„lutinin, ~uid eventuallt- d:un- aged the cells. Vogel and Shelokov (3) later showed that if red blood cells were added to the tissue culture, they would ~tick to the surface of cultures which were producing virus -the method of hemadsorption. This method was then applied to monkey kidne' v cultures wllich had been inoculaed with respiratory se- cretions from children with acute respiratory infections; this showed the presence of several viruses, now known as parainfluenza viruses (4). Four serotypes have been reco(_,nized so far. They are usually isolated onh- in primary cultures maintained in a mediuln free of sertun, which contains inhibitorv substances. A related virus, called respiratory .~.-ncYtial lPS) virus is an important cause of bronchiolitis and pneumonia of infants. This virus is isolated most readily in tissue cultures of a susceptible line of htun~n malianant epitheli:~l cells in which it cau~es cellular de;eneration. and often larre s' vnc~-tia. (5). On the other hanti, human atleno- viruse~, which occur particularl}- in cases of fe- veri~lt ~ore throat and eve tlisen~c, will ;•row rendil, v in malimn:lnt cell lines ,nid primar}- ]nt- m.tn kidneY, but not well in monkeY lciilneV tiil. Fin,llly, rhinovirtt<c.~, which r1re rcloted to en- teroviru~4es but inh:thit tlte rnpper re.~pirator,v 'From the Cotnmon Cohl Research L%nit. Fl:tr- v;tr'l Hosl,ital. Sali?bun-. 1\ ilt'. -Tl e ti-ork on ehich this revirw i!~ ha~(,t La~ heru publi.Ahcd ek,whwre:>.,, t( frrcnc"s 9. 10. and 11. tract, are ,rown readilY onlY in human primary kidneY cells or diploid strains of fibroblasts; thev need a temperature of about 33°C., a pH near neutrality, and constant rolling of the cul- ture, if thev are to uow freely and cause cellular dc_eneration (i). With the present batterY of test systems, we can study the growth and cytopathic effect of many viruses in detail, but such studies have two defects: Fir~4t, the cells infected are morpholo;icallY unlike cells involved in a natural infection. Second, the techniques are inadequate for the growth of all respiratory ~iru,~es; this is proved by the fact that certain nasal secretions from which no viruses can be i~olated produce colds when inoculated into volunteers (8). These colds develop after the inoculation of material which has been filtered through a bacteria-tight filter into volunteers Who have been _iven full doses of a broad-spectrum antimicrobial, and it is there- fore apparent that the illness is due to a virus and not to a bacterium or Jlycoplasma. Thus there are several reasons for wantin; to infect normal ciliated cells of the human or animal respiratory tract maintained in vitro with a variety of respiratory viruses, and it seemed to us that this might be possible ttsing organ cultures. OIICAN Cri.•rt-ttL The tcchniques of organ culture enable one to retain the various constituent~ of a tissue in norm:tl relationship to onc another and func- tioniDr in an npharentlY norntal way, while tlley are outside the bodY in an artificial culture nierlium. One of us (Hoorn, in 1964) ddeveloped a method b' v which organ cultures cru1 be prepared from the nasal and tracheal epitlteliunl of human fetul~es aud of several vlimals. Tis,~ue i, di,_sccted asehticalh- -cithin one hour oc op,ration, an i cut into ronglilc square pieces 1 to 3 nnn. acros?. Very ~hatp knives are n.~ed, an1t t•are is iaken not to touclt tho ciliated suriace. 'I'ho ~,,uare:. ;tre place<1 on the bottoni of a 60 by 1:i mun. pl:tstic p(•tri di<li whFlre it. has been ~,tatnccd with a srrilpel hladr. It :ulheres to this ~uriace tisithout the a(idition of pla~ma or other 156 0009'7513
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.,re ts; pH ul- ]ar we ect ( lies ire a are ort• 1ain ,In a ted ter -en iato ta .re- a na. to or ~t~ro .1 it -ible One e in unc- :hile icial )6:}) ures -heal ~-ra1 hour Ce9 lce.. 11 hv ~men this ~ther I EFFECTS OF VIRUSES ON CILIATED CELLS adhesive. Four to six fragments are placed in each dish, and about 1.25 ml. of Parhers' nledium 199, nith a reduced concentration of bicarbonate, is added (fieure 1). The cultures are inc•uhated in a humidified box at 33'C., and the medium is changed each da}-, and stored if required for virus a~sav. The di~hes are examined dailv b}" reflected light, and the presence of ciliar}• activity in each fragment is recorded. It ma' v persist for at least a month. At the end of the experiment, the tissue is fixed in situ with Bouin's fluid and stained with hematoxYlin and cosin. A11 cells are usualh- well preserved although the cut ends of the fra;- Fic. 1. Photograph of a typical organ culture dish, 60 mm. in diameter, containing small pieces of ferret trachea epithelium. 6 157 ment are u,,ualh• covered h}- flattened or cuboidal (,pitheliuni apparentl}° dericed from the edge of t hc ettrfac•cc'pithc'lium (figure 2). Tfll? (..RO,TH OF VIRC~,E~ IS ORGAN ClLTtiRE, Having, found a technique «"hich would allow at least one physioloeic activity-namely, cili- ar}• inovement-to per4st and which main- tained the structural integrity of the cultured cells, we wondered whether ~ueh cells would support the arowth of respiratory viruses as they would have done in the intact host. It was thon;ht that a«"ide ran-re of viruses might be cultivated because not onlY were the cells apparently unchanged by cultivatioll, but ako a number of special requirements could be met-serum and plasma, w]Iich might in- hibit tn' vxoviruse~., could be excluded from the medium, the temperature could be kept at 33°C. in order to avoid inhibition of rhinovirus multiplication, and the pH could to some ex- tent he controlled ne,ir the phY~ioloEfic neutral level bY adiustin_ the amonnt of bicarbonate iu the nteditun. Repeated attemptS were then made to grow viruses representative of all the groups which attack ihe human respiratorN" n•act; but, owing to the ~4hortare of lmmau embrYonic material, :mimal tissues were used for certain experi- ments. ~~. r V F !. -1& ' - as~'s^ r~ Vr ~ Z" AL . _. .- Ptc.2. ~4, (-tion of human nasal epithelium maintaincd in organ culture for 1-1 ~IaYa. (Ilema- toxylin ;m,i (.u<in ctain: niaerniticalion, ::9S0).
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158 TABLE I tiRO1C'rIt IN nR(',dV CCLTi"RES OF i`OA]E ""TABLE VIRt"IE5 HOORN AND TYRRELL 1'.T[IER- Respiratory F.pithelium Human Rhesus ~ Virus 'i Aasal Tra- cheal , Sasal Tra- cheal I -_ Enteroviruses Poliovirus ~ ~ Vaccine Virulent 1/1$ 1/1 ~~~ ~ - Ecao virus 11 2/2 2/2 0/1 Coxsackie virus A-21~ 1/2 1/1 0/1 Phinuviruses M tYpe 5/6 3/5 ~ 0/1 0/4 H type ~~ 1/1 ~ :3/:3 Adenoviruses ~~ tiimian - - I 1/1 7/7 * In this and succeeding tahles the fr,rctions show the pr,)prrtion of growth curve e.perinrents iu which virus rnttltiplicatiou was detected. Vumeratot• = irurnher of experiments iii n-hiclt virus growth «-as detected. llenomiuator = nunr- ber of experiments performed. f Dash indieates that experintent uc,t doue. $ In experiments marked in italics there N%-,ts well-marked reduction in ciliary activity, or extensive d.uuage to cells was seen on sec.tiou. In table 1 are summarized the results ob- tained utiin_- ether-stclble viruses-enteroti-i- ruses, rhinoviruses, and adenoviruses. Viral growth was assessed by titrating the inoctilum, the culture medium, and also the medium from parallel dumm' v dishe:s in which there was no tissue. Viral multiplication «-as usually obvious, because more virus «-as recovered front the di,~h than was inoculated. In some instances the virus disappeared from the meditun soon after inoculation, as it, did in the dumm' v dishes, and then returned and remained at a low titer for a week or more. Thia was interpreted as a low ;rade infec- tion. On a number of occ;t~ions Virus Nr;is passed serially in organ cultures. Whenever viral -~rowth occurred, it «-as f,tirl' y certain that it wa< takin; place in the ciliated epithr•- littm, beenu~e, apart from thc• dedifferentiatecl cells coverin~ the cut edmes of the fraE~ment, there were no others to which the virus could become ;~ttachcd. In some iustance-, as tn;iv be seen in t;ihle~s 1.iud '_', the %irit.s c.litsccl de_eneritne cham*es in the cells, ,,, ~hown bY loss of ciliar' v activity and morphologic changes in -ections. ECHO virus 11 and virulent polio- viruses caused marked dezeneration of the epithclial cells, whereas the Coxsackie virus A- 21 an(l rllinol•iru.se.s produced only focal necrosis of the epithelium. Adenoviru~e~ also caused degeneration of epithelial cel]s (figures :~ ;ulrl -I). There was evidence that when Cos- ~.rckic virus A_'1, rhinoviruses, and adenovi- ruses were passed serially in organ cultures, the degeueration produced was greater in later passa;es. The de~~enerative c1lanzes seen were similar cytologically to those seen in cultures of tr' ypsin-dispersed cells and in the intact host. ~intilar experiment.< were performed with the totally unrelated myxo.-iruses. In this case, inflttenza A and B multiplied and changed the cells; however, the parainfiuenza viruses, inHuenza C, and retipiratory- syncytial virus lu•o,luced no obvious dama;e although some tuultiplied to high titer. In one seriess of experi- ments, however, the parainfluenza 1 and 3 .-irus(~,, which had been passed several times in standard tissue cultures, were passed serially in human orzan cultures and then prodnced a marked degenerative chan:_c in the ciliated cells. It was concluded that all the upper respira- torv tract viruse.~ tested would multiply in or,-an cultures of human ciliated respiratory epithelium and sometimes in that of other ani- mals. The muitiplication was often ac•com- T_aI3LL 2 GRO1C'rIt IN ORGA] CL'LTL'RES OF SOAIE LTHFR- L:ABILE \"tRr.Es -ASD I''AILURE uB- nTHERS TO (IROW* irus Human Emb-o Trachea Ferret Trachea Influc•nzta A ?0/~0 Inflrtcnza 13 2 Influenza C 1/1 1 1 Ibnrainfluenz:r, 1 1/1 :3r4 P:u•uiiiHueuz,t 2 1 /1 2 2 P;u-aiiifluenza :i '/'' .5,.i 1';u•;tiiifiuc•nza 4 1/1 2 2 ~c~url;ii )/I i ~ I;c•spirat0 inrncytial t/1 3 } .1dc•novirus 1.4 0:1 I'-diuviru's 1 I: n(virus 11 11 1 ' tiee fnnt noter t~~ t;ililp 1. ooos'751S
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EFFECIS OF VIRtiaEa ON CILIATI1) CELLS Fta. 3. Organ culture of rhesus monkey trachea inoculated ten days previoush- Ncith adeno- virus (serotype SV 17). Note the extensive destruction of the ciliated epithelium and nuclear changes typical of those seen in cells infected with adenoviruses. (The rather thick 1>asement membrane is a feature of the normal structure of rhesus monkey trachea.) (Hemato.Ylin and (,osin stain; nlagnification, y480). t h,luied by damane to cilia, particularly if the virus had been adapted to growth in this.t}-pe of culture THE hROP.aG.1TION OF VIRU3ES IN ORGA\ CtiLTURES OF DIFFERENT TISSUES It has often been noted that species speci- ficit- v i~ lost during the chanves which nccom- pan y the formation of a culture of, say, kidney c•ells from a suspension of trypsin-dispersed kidney tissue. For example, polioviruses ma,y ,~row in cultured cells althou-,h they will not -row in the intact tissue from whicll these cells :ire derived. ( )n the whole, in our studies, the ti~zsue ~hec- ificitv of or--an cttltttre? appeared to bc ver' v ~imilar to that of the whole animal. For exnulhle, ferret trachea Nras su~ceptible to lmman myloviruses but not to poliovirus or :tdenovirus. Furthermore, there was indication i hat rhinovint:<e~ and Colz~acl;ie virus _1=21 uiniltiplied onl~- iu cultures of tllo~;e tissues iu which theY "took" when itlocttlated exlmrimen- tull~~ in intact hulnan stbjects (table 31, aI- tllun,jl fnrtlter e.periment~ hn%c showu that it i: po.~-ible to overcome thi~ z~hecificit}• to ~ome rment l,Y one l~:t~~ane of ('()s~ackie virns. A-21 on tissue cultures and inoculation of lar,e doses . of viruses. EcHo virus and polio- virus, on the other hand, were able to grow in organ cultures of the alimentary tract, such as the esophagus and the palate. There were eome odd findin~s, however, lor iniiuenza A virus q:rew in culture, ~ of llmn:ul fet:ll esoph- r;nus, ~rllicll is, however, lined t~•ith ciliated o1lithelirtnl. It Nva, concluded that or,_nan cultures showed many of' the specific charncteristics of the int:lct host, in that ciliated epithelia from different species are Inorpholo;icalh- rather ~iluil,tr but susceptiblv to ditierent viruses, tt•hereas sqttanlou~, epithelinm mi,ht resist infection with viru.;es which would attack the riliated epithelium from ille suue species. This a>pec•t ol'the ivork needs ttlrther sttld}-. Disct-s~ro_,~ We :tre only hc,inuin~, to explore all the po~silllc nseS of orran cultnres in virolo_}• and i>atholu_Y, althou'-h increasinn tt.~e li:is been tnade nf thenl in ph.N,;iuloaY and emin•vroloL~t' u%er the txo (12). It is obvions that <nch culcnres can he it~ed to ~tnrlv the ;rowth of virn~('s in differernti,lted vrll's which are
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160 HOORN A\D TYRRELL Trachea Uninocutated ~^. 3 Trachea 2 ~ •-------- Adenovirus 1 1 neg ~ Trachea Adenovirus neg. Trac hea Intluenza A2 Nose Intluenza A2 I Frc. 4. A representative growth curve experiment shoWingr the multiplication of adenovirtts tYpe SV' 17 in rhesus monkeY tissues. The crosshatcheti hnrs shocc the ciliar' v actitit" v. >`n- sluttletl bars indicate absence of ciliarv ,tictiaitY, and h;ttched areas indicate retlucctl ciliarv actieit.•. The arrow shows the calculated titer of the ntetiium at the time of inocuL•ltion, tuttl the curves show the titer of c-irus in the mediutn harT-csted from the ~li<lter. The top cur\c- shows the result of inoculating adenovirus grown in monkev kiclneY cultures into or;;an cul- tnres ohtaineli from rhesus monkev tt.`ichca. There was a marl:etl tlrop in titr,r in the tlnmmv culture. The curve second from the top shows a definite increa,:e in tittT attrr inoctilation oi virus pas'~ecl three times in oruan cultures. Influenza virus spparenth- grcw poorlY in tracLea tissue but Lnttltiplied freel' v in na~zal epithelitun. (Pef~~i~+terl with pc~nri.,siutz (,om Tht Rritish .Iwnwrl of Expcrimcil(al PatbologiJ, 1965, .i(3, 514). TABLE 3 Fnr:c2tr:M v (,t V-lat-s Grtu\\ rrt 1-1 ('t-L'rrnr:S t>>: A-_Mit>tS HrNtAN OltoANS* Culture Inoculated ~cith T-sue Culture hluid l'ontainin_: Culture Prepared from M Rhinocirus licHOCirus 11 Nose 3/3 2'_ Tracltca 3/:; ~~;2 F.snpharus ll!a 2/2 Palate u; 1 1,_° * See ti~utlintes to ~ tahle 1. vrorv much like those in intact anim:rls while controllinafactora such a.~ thc emironment anci the mode of exposure to virus as can be done when olher ti~aue clllturc techniques are u~ed. There have been few hreT-iolt.~ Studies, r.(,;., the Incthotl uI-ccl b.- I3,tnu, anci \ i~clt (131. Ilowever, their method for ;rowiur influenza virn~z in orman culttur~z of icrret trachea -rown on plasmaa clots haS a number of dra«•- hacks for virolo-,ic work. Aevcrtllele.". we do not believe tha't our nTethocl i; perfect. It -eclt,~ tll:1i rhe techni<llte mi~ht I,e improved 0009'751'7
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EFFECTS OF VIRUSES ON CILIATED CELLS 161 7 hile ient i be are ,nza hea ran•- do It ved 3 f urtlier by modifying the conditions to resem- 1ile more clo~elv those of the intact host. It has been remarkable to discover that !he~:e cultures can at times produce quite large i;umberS of viruses while the cilia continue to I,cat normally. This may have occurred because eulY a small proportion of cells was infected and ,iama,ed, but it is equally possible that viral cro`rth can continue without upsetting the cells <eriously. It has been observed in recent experi.- nients that cultures which show normall ciliarv activit}- while ~zupporting the nrowth of one virus may be able to produce only one per cent of he normal yiehl of a second virus. This miLdht be due to viral infection of 99 per cent of the cells or to the effect of interferon on the cultures. It is known that this substance is produced in response to many viral infections and that it can protect differentiated cells, and, althou,h we failed to detectt it in the medium of some of our infected cultures, this is not a conclusive argument. In fact, infec- tion with rhinoviruses often sub'~ides in the absence of antibodies and while the epithelium is morphologically intact. The simplest explana- tion for this is that the cells adjacent to those infected with the virus are exposed to inter- feron and become resistant to infection. Finally, although not closely relevant to the sub,ject of this meeting, we believe that we have now proved our original hypothesis that ciliated epithelial cells in culture are likelv to be excellent hosts for the so-called "difficult" viruses, for we believe tve have cultivated and passed seriallY an ether-labile common cold virns which is distinct from all hitherto described viruses of the re,~piratory tract (14). 1t"1I\L-1[2I" Organ cultures of ciliated respiratory epi- t helium -upportcd the rrowth of a~reat of viruses which could infect the re.~pir,Itor}- tract of the intact host. In some in~tance~; ciliar}- activitv was halted and the c rll~: de_,eiierated. REFERENCES (1) Nlogabnab, W. J.. Green, I. J., and Dierk- hi.sing, O. C.: Primary isolation and prop- aration of influenza virus in cultures of human embryonic renal tissue, Science, 1954, 120, 320. (2) 11Togabgab, W. J., Green, I. J., Dierkhising, 0. C., and Phillips, I. A.: Isolation and cytopathogenic effect of influenza B viruses in monkey kidney cultures, Proc Soc Exp Biol AIed, 1955. ,ti. 654. (3) Voge1, .I., and :-4helokov, A.: Adsorption-he- magglutination test for influenza virus in nionke y kidney tissue culture, Science, 1957, 1?6, 358. (4) Chanock, R. Af., Parrott, R. H., Cook, M. K., Andrews, B. E., Bell, J. A., Reichelderfer, T., Kapikian. A. Z., D1aStrota, F. M., and Huebncr. R. J.: \ewlv recognized viruses from children with respiratory disease, New Eng J 1led, 1958. °.5S, 207. (5) Chanock, R. 1M., Roizman, B., and 'Myers, R.: Recovery from infants with respiratory ill- ness of virus related to chimpanzee coryza agent (CCA). I. Isolation, properties and characterization, Amer J Hyg, 1957, 66, 281. (6) Pereira, H. G., Huebner, R. J., Ginsberg, H. S., and van der Veen, J.: A short de- scription of the adenovirus group, Virology, 1963, 20, 613. (7) Tyrrell, D. A. J., and Chanock, R. M.: Rhino- viruses: A description, Science, 1963, 141, 152. (8) Tyrrell, D. A. J.: Cellular Biology of 11yxo- Nlrus Infections. J. & A. Churchill Ltd., London, England, 1964, pp. 182-184. (9) Hoorn, B.: Respiratory viruses in model ex- periments, Acta Otolaryng (Stockholm), 196l.1SS (Supplemcnt, p. 13S). (10) Hoorn, B., and Tyrrell, D. A. J.: On the rroNrtll of certain "newer" respiratory vi- ruses in or,an culture, Brit J Exp Path, 19( 5. ,i6. 514. (11) Tyrrell. D. A. J., and Hoorn, B.: The growth of ,orne myloviruses in organ cultures, Brit .1 P;xp Path, 1965 (in press). (12) Fell, II. B.: The future of tissue culture in relation to morphololw, J\at Cancer Inst, 1!1,57. 19. 643. (13) Ban„ F. B., and \ivcn, J. S. F.: A etud_v of infection in organized tissue cultures, Brit .1 1';sh Path, 195ti. ,3.9, 317, 14) TYrrell. D. A. J., and Bynor,, JI. L.: Cultiva- tion of a novol type of common-cold virus in orVan rulture-. Brit 1965. 1. 1467. .~, N 'I~
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DISTURBANCES OF THE CILIATED EPITHELIti1T DUE TO INFLUENZA V-IRUS' J. F. PH. HERS I.TxoDucTtoN the ciliated epithelium of the trachea and Influenza is an infectious disease of the I>ronchi, occurs in the acutc ~ta~,e of influenza. respiratory tract in man caused bv a viru~. Oite or two rows ot a flattened la}-cr of cells From clinicnl and epideiuioloEne csperience~, 'ometimes remain coverina the confirmed virolo,_icallv , it ha~; been shown that. except for a subclinical t.pe of influenza due to a mild infection, fatal infections occur fre- quently in all age groups. In patients with fatal infections, the disease is often complicated by secondar' v bacterial im°aders. Although Heinop{eilxs influenzae and pneumococci are commonkinvolved, the pyogenic cocci, par- ticularlv hemolytic staphylococci, are the most feared pathol-ens and are responsible for the high death rate durinr influenza outbreak.< (1-5). Clinicopathololgic reports showed o~ early as the pandemic of N1S or before that ,c high percentage of deaths occurred in patient.~ suffering from pre-existinol; disease~. Chronic hmg diseases (4) and heart diseases sectned to be the important predisposing conditions (5). Virolo~nicallY proved fatal cases in the p,tn- demic of 195i extended these ohservations and confirmed that death can occur frotn the virus itself, and fatal viral pnenmonia, eVen in the ab~er,ce of secondary bacterial patltonen~, has been described (5-0). Histopatholo!sic studies on t ltc, epithclial lininIg of tlte respira- torv tract infected bY inflnenza virus are ham- pered ln- our fraffmentarY kno«-1ed~_,e of th~e normal structure of thia epithelium, the life- cycle of it- component celk, ~cnd the proce-,4 of a6mV. Studies are further complicated ln- the presence of terminal ~:t;t,v~es of ;ccitte or chronic inti;tmmatory hroce~ses in the bron- c]iial tree .cuel the pitlmonsr' v circnl:aory tems. Im-c-li<ration of inflttenza require- a multicli,ciplin.ir' v :cppro;cch, with clo<e colla- boration between investilffators in clinical and experimcntul ineciicine, viroloaY, ond bacteri- c 10,.~y. Ttll, Itt _Nr.+N LL.,ro_-\~ A more or le:~ estensive de,~trttction, c•on- ol cle<,eneration and desqttuntation c~t ' From thc f)epar'tiuent of Internal \Ieclicine. Vniv(~r~itc Hu~pital of L.riden, and the Instituu, ui Preventivr AIcdicine, L( iclc,n, ilie Aetherland". ba-ement mem- brane. In the later stau< of the disease this epithcliurn chan~e~ into a stratificd-like or undifferentiated epithelium in which the nor- mal structure of the cpithclial linint can rarclY be found. Both this delueneration of the epithelium and thc appearance of a stratified-like epithelial lininr in the later sta;es have been described b1v many authors since the pandemic of 1915. .><lthough the monoaraph of 1920 by Winter- nitz, Wason, and JIc\amara on the patholoL~y on influenza (10) can be reffarded as a classic stud.v, the first occurrence of a stratified-like epithclinm Nvas mported by Widal in 18'J9 (11). Since the dis-covery of the influenza virtt~es ill 19:i'3, these hi.~topatholo;ic findings have been confirmed in virolo' zicaily proved fatal and it is known with certaintv that cpithelird de-truction induced by inflttenza .-iru~4 maY be di~ztributed focally and that it is indcltendent of coexistent bacterial tracheo- brotichiti~. These concepts of the human le~ion, first ~tudied in fntal ca~es n-ith coexistent h;ic- teri:cl infection, Ivcre ba~zed mainlY upon in- clirect cviclence ( 12, 15). Support e~tme from thr comparable ef'fects of induced inflttenza viral infection.~ oil tlte ciliaterl epitheliutn of the air pas~.tees of ferret~: and mice. _><lthou,_~h inflnenz.c in labor;ctorv anintal; Nvas cle'crihed -itice 1!I:i7 (11i-19). the fin;il evidence of the Ie:ion in man Nvn~; ~u1111ieci clurin-, ,md at'ter the influcnza pvidemic of 1957. Ttrc Crrot,.vrt[tc 1•FFr•.cT OF THE Hr-M_tN Lr.slo~ The inAitcnza-vinc~ induced dam;cRe to ilue c•iliated epifhclial rrlk of rhe tr,tchea ancl hronchi in thc lutman has been <tudied pri- nt:irilY ill lti-tulw_ir -c•ction4. ('N-t0lu<ric ~ntearS ul' <I1uttmt ;in,l czuciate an(l iniprc- -wn preparitionti uf the tr:cclteal aud bronclii:1] c•pithelium havc° cAtendcd c>>cr kno«-lcd~_1e of nc(I Variocs oi c•clI dt-tritction. 1;I•lined mc1thodk of fiXatinn ;und trccze-ctrVin; rcnd thc• 0009'7519
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EFFECTS OF INFLUENZA VIRUS ON EPITHELIL'Vi 163 and ttza. cells rem- this ior nor- can and helial ribed ~ 918. iter- logy I t~~ic 1-like ll). uses iave :atal that r^nza ; t it I heo- ,sion, hac- I in- f rom Ienza rt of ough ribed ' the r the \N ihe und pri- e,trs I J r('s- hial rhe ;ined rhe inodern pror_•cdltres of staining have revealed ul:u1 lIc•r.iil, c f tlli~ eYtop:rthic efFect. Wett r•vtologic preparations stained in the unfixed =tate Nvith methy-lene blue and cryostat sec- t ions have .ilso been used successfulhY. 1Chen deoth occurs early in the disease, vari- uits cYro1>athic changes can be found in very re~tricte-l are:c of thc ciliated epithelium of tlle trachea and bronchi. An earlp lesion ap- pears to he a swelling of the cell, with a ~wollen ot•al nucleus situated transtiersely in the cytoplasm (figure 1). The normal palisade structure of the epithelium, due to the tall cell, with their basal nuclei, vanishes. allthough milcu~z production bv these affected cells i~; considerably- diminished and mucus can scarcely be detected by special staining procedures, cilia are ofren vi.>ible (finure 2). Structures re- <emblin; b,rophilic and eosinophilic inclu=ion bodies are -olnctimes found in the cytoplasm (figure :')). These inclu~ions are mainly phago- cytosed c_elhilar and nuclear debris derived from unknown necrotic cells, and even phagocytosed ery-throcy-tes can be seen in the cytoplasm. However, real eosinophilic inclusion bodies ma,y- also be demonstrated in the cytoplasm. blore or less at the same time the structure of the epithelium is altered by the development of intercellular ~paces (fi,ure 2). This is probably ilne to rele:ase and shrinka,e of cells, vacuoliza- tion, and edema. The small shrunken cells show irre-,ular cell bodies with pyknosis and some- times fragmentation of the nucleus (figure 4). The cytoplasm is often intensely eosinopllilic. This last type of degeneration, representing com- plete cell necrosis, is not seen frequently. It can be found nt each level of the epithelium and is often located in the midst of swollen cells or in he intercellular spaces. Both swollen and -hninken ce1L desquamate. 1'Ire swollen t' ype rounds up and the lonn c}-toplasmic tail cli.~ap- pears, probabl' v bv retraction into the cell bodv Ifirure,~ 1, 3j. In contrast to the (lesqnamated u1a11 ~;hrunken cell, this type of cell m:ny ~till hoar cilia (5. S, 1:i). Similar cytopathic chan~*es crtn he found in influenza in laboratorv animals 1 fi~rure :i 1 and in monolal-ers of calf and mouse h-iclnev tissue ciilture4 infected with inLluenz:I ~it•us I lli-'?01. The ~i;nificance of re:1l eo.~inopllilic inclll~ion hodirs and the small shrunken cells has aroused nuleh rlis.cir~~sion in the literature (21, 22). It lia~ hel~n that the former represents a specific cellular reaction due to influenza virus particles or virus clones and that the latter indicate complete cell death caused by the influenza virus. Neither is pathognomonic for this disea~4c. The eosinophilic inclusion bodies have been described in Inany- other respirator}- viral infections, but have also been recorded in the absence of such infections (22, 23). Shrunken, necrotic cells, even when abundant in tracheal and bronchial epithelium of mice infected with influenza virus, could be a toxic viral reaction or even a nonspecific toxic reac- tion (16, 1S, 2-1). Viral multiplication may occur in the absence of visible morphologic change` in cells growing in tissue culture. Furthermore, cell de4rnction i~; not related to viral nmltiplica- tion or to toxic products of the virus. Cell de- strnetion in tissue cultures depends equally upon the cell sy-stem and it.s pllY-iologic state. The small necrotic cells in uninfected tissue cultures are not morphologicalh• different from those mentioned above (20, 25). It is evident, therefore, that the appearance of these cells in man could represent phYsiologic death of the cell also. To date, ultr.IStructure research on the cell~ has not been performed in man, although the earlier stane,~ of the cell lesion have been beautifulh- descrihed in the ferretnose bY Hotz and Bang (?B). In contrast, immunofluorescence tech- niqnes have contributed much to this problem. TPII: I-NI\ICSOFLCORE~('I]SCE OF THE HUMAN LESio-, Although Coons, the originator of the im- munofluorescence technique pointed out its value for the study of antigens and antibodies in microbiologic, virolotric, and immunologic sYstems as early as 1941 ('?I, 28), Liu was the first to apply the method to influenza in man .nld in laboratorv anima6. In 1955 he found that the S or soluble complement-fixinn ribonuc•leic acid antigen of influenza virus :ippears diffu.~el}- first in the nucleus and rtfterw;ird in the cytoplasm of the epithelial linin-, cells of the air p:IS~a~e> of ferrets (29, :;0). Ilis ob~ervations o1i experimcntal inl3tt- enza in ferrets were luter confirmed in mice (:31, :1''), in pi_s and in tissue cultures (:3-1, Dctailed studies have dcmonstrated that the two types of subunits of influenza virus are synihesized at different time< and in differ- ent re,ions of the susceptible cell. In contrast ~ 0009'7520
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FIG. 1. Female, age 39. Fatal A2 influenza virus pneumonia. NIitral stenosis and pregnancy, about third da}• of illness. Wett methyl- ene blue preparation of smear of tracheal epithelium. Cytopathic change in ciliated cell, showing swelling of cell body and nucleus and dis- appearance of cy toplasmic tail. Cilia are well preserved; irregular condensation of rhromatin in nucheus. (Oil- immersion, X100). FIG. 2. Female, age 45. Fatal A2 influenza virus pneumonia. 'Mitral stenosis about third clay of illness. Main bronchus. Cytopathic changes in ciliated epithe- lium. Cilia partly preserved, mucus production absent. Edema and vacuolization in the epithelittm. Intercellular spaces probably caused by shrinkage of ciliated cells. (Hematoxylin-phloxin-tar- trazin stain ; oil-immersion. x100). Ftc. 3. Sanie case as in fig- nm 1. Fornialdehx-dc fixed prep:uation of smear of tr.u ht•al epitltelium. Cyto- puthic chanLes in ttico cril- iated cells. Auclei cituated transversol v in the cvto- plasm; disappearance of c}•- topLi~mic tsil. Brush zone and cilia hf%come blurred. P)ia(_~oc•vtosis of shrunken rwll, 'cith pyknotir nuclei and r(.ll d"l,ris. (HetnatotI- lin and ~tain oil im- ~n~r~iun. - 10O). 00097521
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I EFFECTS OF INFLUENZA VIRi;S ON EPITHELIUM Ftc. 4. Same case as in figures 1 and 3. Wett methylene blue preparation of smear of tracheal epithelium. Shrinkage of ciliated cell with pyknotic nucleus; disappearance of cytoplasmic tail and cilia. (Oil-immersion, X100). > b Ftc. 5. Section of freeze-rlried and Carbowax-embedded mouse lung 24 hours after intra- n,isal infection with sicine influenza A virus (10-- hrmagghrtinin ttnits). Bronchial ehithelium --howin:: all ~t;te<s of cvtopathic chanre<, e.g., swelling and shrinkage of cell hody and nucleus, pha<xocyto~;i.~ ot cell an(t nuclear debris, and real (-o,inophilic incln,ion bor3ies. Cilia partlY tnrserv( 11. (13(•in;irosYlin and co:~iu -tain; oil-intmct:~zion, x100). to the S-;tnti~,cu, the hcmn_,dutinin or V-anti- L,:ru i.~ located in the evtol,la.-mm near the cell mrtnthr;tne. Furthcrnlore, :In exce~s of ribo- nucleic acid iu the ;ire,i~ of the nttcletts and thc cYtohla~ul, contatinin; S-:1ntiuen, has been (i(,tuonstrated, ,md there i~ <tron; evirlence tlt:[t tlte ~-anti~r~n Wliich i~ >Vnthe-Zize(t in the intch-u< move- _ irnnt lhe nucteu~ to the ct-to- pl:i<nt r:tther th:ut h~•in~ i~~rnterl in the <~to- 1>l:t<nt (:))ti, :i-dI. In ,tccor(I:utce \cith the cVto- pathie chan'_~es, the ~Nvollen epitltelial cells in ~cctioits nnd spuntnt =mcar.~ from human sub- jec_t~ ~hotr ~pecific fitnore~cence either in the mtclcu~, in the c.vtoplet<m, or in the nucleus an1t thc cytoltla~tn (fivnn- ti, 7, and S). At- thouvh thc cvtopl:t~tnic :ut(l nuclezr flttores- ciucr;ih• tn;iinlv ~liffu~~. :t more irreRttlarh- loc;ited, luttchv flnorc=cence can l,e observed in
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FIG. 6. Female, age 39. Fatal A2 influenza virus pneumonia. Mitral stenosis and pregnancy, about third dav of illness. Immuno- fluorescent preparation of smear of tracheal epithelium. Cytopathic change in cil- iated cell, showing specific fluorescence in cytoplasm. luclear fluorescence absent. Cilia partly preserved. Small vacuoles in the cytoplasm. Swollen nucleus and disap- pearance of cytoplasmic tail. (Indirect staining method, according to Shepard and Goldwasser; oil-immersion, X100). FIG. 7. Same case as in fig- ure 6. Immunofluorescent preparation of smear of tra- cheal epithelium. Severely damaged ciliated cell, show- ing an irregularly patchy fluorescence in the cvto- plasm. Nuclear fluorescence weakly present. Complete disappearance of cilia. (Indi- rect staining method accord- ing to Shepard and Gold«•as- ser; oil-immersion, X100). Frc.8. Female, age 15. Previouslv healthy. Fatal A2 inflnenzal staphylococcal pneumonia. Immunofluores- cent preparation of smear of tracheal epithelium. Shrunken, probabl.y ciliated cell, showing specific nuclear and cytoplasmic fluorescence. Absence of cilia. (Indirect staining method according to Shepard and Goldwasser; oil-immersion, X100). .00097523
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39. ~,irus nosis hird 1 1I10- i of i unl. cil- cific asm. 1~ent. mall lem. sap- t ail. iod, and ion, EFFECTS OF I\FLUENZA VIRUS ON EPITHELIUM FIC. 9. Mouse-kidney tissue culture 24 hours after infection with A2 (Japan 305/57) influ- enza virus (0.05 hemagglutinin units). Immunofluorescent preparation showing various stages of virus multiplication. Two :everely shrunken cells show nuclear and cytoplasmic fluorescence. (Indirect staining method according to Shepard and Goldn•asser; oil-immersion, X40). I ftg- ent aa- ely ~ow- chv to- nce Aete _1di- ord- was- )). 15. t A2 Fccal >res- aear .ilm. ~ted Icar tice. roct ~Z to er; r P'ic. 10. Same tissue culture as in figure 9. Immunofluorescent preparation showing vari- ous stages of virus multiplication. One small shrunken cell, showing retractation and specific cytoplasmic fluorescence. Small pyknotic nucleus does not show fluorescenc(•. Two small cytoplasmic vacuoles. (Indirect staining method according to Shepard and Gold«-asser; oil-imFncr:4ion. X40). the c, vtopLasm. This fluore-cence is not localized to the real eosinophilic inclusion bodic" but is di~per~:ed difttt.sclY iu th(, c}tohla~;nt. The l)ri'dhtest and most dwinct ~pecific nuclear tinore~cence is found in cclJs ~ltotring little or no de,_~cuerativc cltanre. _1ltllon_li some of the ~n1,111 : hrunken cells do show a specific im- Inunoflttorescent reaction, most of these cells 167 do not ~t:litl at all or ~how a tnott~liecitic reac- tion. This ob.~crv,ttion is ;ituiLu' to the tindings in ti~~nw cilltitre of spcci[ic llnore,celu•c in only :I f(,\Nanall necrotic relk lfi~,urc~ ~I and 10). "1'hc-c ~fuclie.~ pro~-it11, ntnlde ev;dencc that the c}-topatltic efi'cet orn the ciliated epithclial cclls i~4 ctu~ed b,v the intluenza ~irtts and that the death of the ccll can rwcitr in all 00097524
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Fie. 11. Male, age 30. Cooley's erythroblastic anemia. Influenza B tracheobronchitis. Trachea. Epithelial cell lining consisting of one row of partly swollen, partly flattened cells. (Hematotylin and eosin stain; oil-immersion, X40). I ti. li; rt ei 1: Frc. 12. Frm:dt,. a,~r 49. Proviotislc b(,ultliy. InHnenza _11 stit,hclocorr,il 1,114 uuionia: dura- tion of illncss, 14 dLtYc RiL):lit ni<tin broacLu~;. nonker;itinizcd 1n,uitic(l c'l6tlifliuui. Pali~adf- like structure iu tlw lo«~or 1>art of thP ri•Il4 ;u tlu -nriauo. -Nlucu~ production uu(l Uilia :u'(' l lb ~iuatos}~lin and ("l.sin -t;iiu: oil-inttnp ~r<6,n. , 40).
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u 4 EFFECTS OF INFLtiENZA VIRUS ON EI'ITIIELItiJI vtages of the virus growth cvcle. Because a complete growth cycle of influenza virus i~ very dhort, about eight hours, the death and desquamation of the affected cells occur very earlv in the disease. Itt is clear, therefore, that staining smears from sputum or nasal esudate by the immunofluorescence method may ac- celerate the diagnosis of the disease. PreliminarY reports of the clinical applications of this method are highly promising (3S-=12) . Fi- nallY, althou,h there may be totic effects of viru~es, detailed studies on this problem have uot been accomplished. THE REPAIR OF THE IIti\LAN LESION In contrast to the acute sta;;e of degeneration and desquamation, very little is known about the actual process of restoration of an epithe- lial wound in the trachea and bronchi. The remaining cell layer (figure 11) consists of either a swollen or a flattened type of cell (5, 13-15). Convincing specific fluorescence has not been demonstrated in either, although some cells bear cilia. This indicates that these cells probabh- escape the viral infection. Further- more, this final cm•ering of the basement membrane is often very irregular because both types of cells are often lined up in one or two rows at the same time. Their precise origins are still unknown, although basal and replace- ment cells may contribute to it. It is obvious that regeneration of the epithelium starts here, but that cellular activity is low and varies considerably as measured by the nuin- ber of mitoses. In later sta,es of the disease, between the sixth and eighteenth day after on et, an undifferentiated to nonkeratinized stratified epithelium (figure 12) can be found (5, 1:3-1:i). T1Ie proliferation of this epithe- lium is ob~-ious, but its structure is not uni- form. Mucus production is often completel}- lackin; and cilia cannot be detected. The sur- face consi~t; of slightlv degenerate cells swx- ;e~tin; that the process of regeneration and dezeneration alternates. _llorem-er, re,_,enera- tion mav start in the verv eark- st~iges of de,eneration before desquamation occurs. Truc metaplasia of the epithelium is never found. A c()mptetel}- normal ciliated mttcns-prodnUinU 1'pithelium has been described two xoors :iud rhree rniontlis after the onset of intluenza wliich wn~ confirme l virolosically ( 1:;). 169 Sti 1iSiAR] There is convincinz eN-idence th,itt influenza virus attacks the ciliated epithelium of the respiratory tract in man. The cytopathic effect and immunofluorescence of this epitllelium closely resemble the findin;_s iu influenza in laboratory animals and in tissue cultures in- fected with an influenza virus. Vira1 multiplica- tion is probably one of the major causes of cell death. The degenerated cells desquamate, and there is a strong sug:;estion that .i normal cili- ated epitlielituu is restored con'~i(ierabl)• under undifferentiated and stratified epithelium after the disease. I3o«-ever, more extensive and de- tailed studies of this phenomenon :1re needed. REFERENCES (1) Stuart-Harris, C. H., Laird. J., Tyrrell, D. A. J., Kelsall, M. H., Franks, Z. C., and Pownall, M.: The relationship between influenza and pneumonia, J H}-g (Camb), 1949, 4, 434. (2) Hers, J. F. Ph., Masurel, N., and Mulder, J.: Bacteriology and histopathology of the re- spiratory tract and lungs in fatal Asian influenza, Lancet, 1958. 2, 1141. (3) Louria. D. B., Blumenfeld, T. H. L., Ellis, J. T., Iiilbourne, E. D., and Rogers, D. E.: Studies on influenza in the pandemic of 1957-58: II. Pulmonar _v complications of influenza, J Clin Invest, 1959. JS, 213. (4) Martin, C. M.. Kunin, C. M., Gottlieb, L. S., Barnes, M. W., Liu, C., and Finland. M.: Asian influenza A in Boston. 1957-55: I. Observations in thirty-two influenza as- sociated fatal cases, Arch Intern Med ( Chi- cano), 1959, 103, 515. (5) Nluldcr, J., Hers, J. F. Ph., and AIasurel, A.: In Influenza, a monoaraph to be published in 1966. (6) Netacombe, C. P.. Nixon, P. G., and Thomp- son, IL: Influenzal pneumonia in mitral stcnosis, Acta Med Scand, 1955, 162, 441. (7) Rock, J. A., Braude, A. I., and \loran, T. J.: Asian influenza and mitral ~tenosis: Re- port of a case with autopsy, JAMA, 1958, 166. 1467. (8) Hers. .J. F. Ph., and Mulder, J.: Broad as- prcts of the pathology and pathogenesis of human influenza. Amer Rev Resp Dis, 1961, S,3 (Supplement, p. 84). (9) Straub, i1f.: Tho influwnza epi(iemics of 1918 and 1957, Nederl T(;eneesk, 1959, 103, v ~ 1205. (10) Winhurnitz, M. C., Wason, I. JI., and JIe- ~ \':unara, F. P.: Chapters IA and IB. In Thc P,itholopq of hzlircenza Yale Univer- ~ , sitc- Press, Nr,R- Haven. Comiecticut. 1920. C.7 (11) jCidal. F.: Tniite de Jledecine, Masson et Cic.. Paris, 1S99. voL 2, p. 223. ~
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170 J. F. PH. HERS (12) Mulder, J., and Verdonk, G. J.: Pathogenesis of case of influenza A pneumonia of three days' dttration, J Path Bact, 1949, 81, 55. (13) Hers, J. F. Ph: The Histopathology of the Rcspiratory Tract in Human Influenza, Monograph of the Institute of Preventive Medicine, Stenfert Iiroese, Leiden, Nether- lands, 1954. (14) Hers, J. F. Ph., and Alulder, J.: Rapid tentative postmortem diagnosis of influ- enza with the aid of cytological smears of the tracheal epithelium, J Path Bact, 1951, 6,3, 329. (15) Hers, J. F. Ph., and Mulder, J.: Changes in the respiratory mucosa resulting from in- fection with influenza virus B, J Path Bact, 1957, -,3, 565. (16) Francis, T., Jr., and Stuart-Harris, C. H.: \asal histology of epidemic influenza virus infection in ferret: Development and re- pair of nasal lesion, J Exp i\-'Ied, 1938, 68, 789. (17) Straub, M.: The microscopical changes in the lungs of mice infected with influenza virus, J Path Bact, 1937, q'5, 75. (18) Straub, M.: Metaplasia of bronchial epithe- lium in mice infected with virus, Cong Internaz Pat Compar, 1939, 2, 197. (19) Harford, C. G., and Hamlin, A.: Effect of influenza virus on cilia and epithelial cells in the bronchi of mice, J Exp '_4led, 1952, 95, 173. (20) Heath. R. B.. and TYrrcll, D. A. J.: The be- haviour of some influenza viruses in tissue cultures of kidney cells of various species, Arch Gcs Virusforach, 1958, 8, 577. (21) Zhdanov, V. M., and Soloviev, V. D.: Some results of the vtudy of Asian influenza, Amer Rex- Bcsp Dis, 1961, 83 (Supplement, p. 178). (22) Pierce, C. H., and Hirsch, J. G.: Cilio- cytophthoria: Relationship to viral re- apiratory, infections of human, Proc Soc Exp Biol Jlod, 1958, S'~, 489. (23) Pierce, C. H., and Knox, A. W.: Cilio- eytopihthnria in sputuru froni patients with adeno Nirus infections, Proc Soc Exp Biol Mcd, 1960, 10.f, 492. (24) Loosli. C. G.: The pathogcne.=is and pathol- ogy of experimental airborne influenza virus A infcctions in mice, ,I Infect Dis, 1949, S r, 153. (25) \egroni, U., and T}-rrcll, D. A.: Morpho- logical obscrvations on ti~,:sue cultures of epithelial ccI1s infected with influenza A tirn~cs, J Path Bact, 1959. ~~ . 497. (26) Hot•r„ G., and Ban-, F. B.: Electron nuicro- ~4cope studi(•s of ferret rcspirator}• ce1k in- ierted with influenza, Bull 13opl:ins Hosp, 1957.1u1. 175. (27) Coons, A. H., Creech, H. J., and Jones, R. N.: Immunological properties of an antibody containing a fluorescent group, Proc Soc Exp Biol Med, 1941, 1,7, 200. (28) Coons, A. H., and Kaplan, M. H.: Localiza- tion of antigen in tissue cells: II. Improve- ments in a method for the detection of antigen by means of fluorescent antibody, J Exp Mcd, 1950, 91, 1. (29) Liu, Ch.: Studies on influenza infection in ferrets by means of fluorescein-labelled antibody: I. The pathogene~4s and diag- nosis of the disease, J Exp _Nlcd, 1955, 101, 605. (30) Liu, Ch.: Studies on influenza infection in ferrets by means of fluorescein-labelled antibody: II. The role of "soluble antigen" in nuclear fluorescence and cross-reactions, J Exp AIed. 1955, 101, 677. (31) Hers, J. F. Ph., Mulder, J., Masurel, N., Van der Kuip, L., and Tyrrell, D. A. J.: Studies on the pathogenesis of influenza virus pneu- monia in mice. J Path Bact, 1962, 83, 207. (32) Albrecht, P., Blaskovic, D., Styk, B., and Iioller, i\L: Course of A_ influenza in intranasally infected mice examined b}• fluorescent antibody technique, Acta Virol (Praha) [Eng], 1963, 7, 405. (33) BlaskoviC•, D., Szanto, Jr., Albrecht, P., S;i- derskv. E., and Lackovi~, V.: Demonstra- tion of swine influenza virus in pigs by the fluorescent antibody method, Acta Virol, (Praha) [Eng], 1964, 8, 401. (34) Breitenfeld, P. M., and Schafer, W.: The formation of fowl plague virus antigens in infected cells, as studied with fluorescent antibodies, Virology, 1957, f, 328. (35) Hultcrman, 0. A., Hillis, W. D., and Moffat. M. A. J.: The development of soluble (S) and viral (V) antigens of Influenza A virus in tissue culture as studied by the fluores- cont antibody technique: I. Studies em- ploying a low nniltiplic•ity of infection in beef embryo I:idney cells. Acta Path lIi- crobiol Scand, 1960, 50, 395. (36) \iven, J. S. F., _lrmstron(_~, J. a., Balfour, B. M., hlemperer, H. (;., and T.•rrell, 1). A. J.: Cellular changes accompany-ing the growth of influenza virus in bovine cell cultures. J Path Bact. 1962, S.t, 1. (37) Ziinuiermann, T., and Sch'iYer, AV.: l:ffect of h-tluoropheny-l.danine on fowl plague virus iniiltiplication, Virology, 1960, 11, 676. (38) Liu, Ch.: Rapid diaenocis of hum.in influenza infection from nasal smears Ln- mcans of fluorcscein labelled antibody-, Proc Soc Exp I3ici1 1956, 92, 883. (39) Liu, Ch.: Diagnosis of influenzal infection 1)r means of fluorescent :mtibodY ~:tainine. Amer Rev Resp Dis, 1961. ,ti3 (Supplement. p. 130). 0 o09 / 5-,(V7
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EFFECTS OF INFLUENZA VIRUS ON EE'ITHELII'\I 171 (40) Hers, J. F. Ph.: Fluorescent antibodt' tech- b' v tneans of the fluorescent antibod' v tech- ni,im• in respiratorc viral disea~ws, Amcr nique, Jap J I;sp lled, 1962, 32, 531. HF•t- Resp Dis, 1963, SS (Supplement, p. (42) Tatc,no, L, Suzuki, S., Nakamura, S., and :~16). Iiit;iuloto, O.: SensitiN-it~ of con~~entional (41) Tateno. L. Suzuki, S., haccamura, A., Ka- serological methods in the diagno~4s of in- cvn~liima. H., husano, A., Aovama, Y., fluenza virus inf(-ction in the livht of the Sa ~'iiua. A., <~kao. ~.. Oikawa. K., Homina, fittor~~cc~~nt antibodc technique, Jap J Exp N.. auA Naito, M.: Diagnosis of influenza AIed, 1962,..).2, 561.
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DISCUSSION Dr. Boren: Virologists, more than anyone else, have emphasized the concept that the physiologic competence of the cell determines its reaction to injury. I hope we will see concepts related to cellular function included in the discussions this aftrrnoon. Dr. Kotin: Dr. Laurenzi, what is the natural historv of the infection of the mice that -,-ou have not sacrificed? Have y-ou let any live on to see whether these staph_ylococci set up abscesses in the lung in mice with and without alcohol and without smoke? Dr. Laurenzi: Yes, we have. It is very hard to find organisms after two days. A majority of them are gone within 24 hours. We need a nonpathogen because these studies were started with pneu- mococci which produced too many unpredictable deaths. I also felt that if it could be shown that an animal has trouble handling or getting rid of a nonpathogen, due to some induced ~-ariable, this means even more difficulty handling a patho- gen. Dr. 1'yrrell: You said there was postponement of the ciliary clearance curve in the presence of alcohol. To tne, your curve looked as though it was going to flatten out and become asymptotic at a much higher level in the presence of alcohol. Is this an error in the way the curve was drawn, or have you continued to count for 24 hours and shown that levels eventuallv get as low in alcohol- treated animals as in normal ones'.' Dr. Laurenzi: Yes, we have, and they do. The postponement was purely artistic interpretation. Dr. Pates: Have you studied the effects of hulnidity and temperature in the environment of the mice'.' Dr. Lracrenzi :'_Vo, these conditions have been kept constant. Dr. Slaub: Dr. Bang, do viruses adhere to the cilicLted cells or the mucus chlls? Dr. Bang: I do not know, for we have both of these in the combination. We are scraping the cells l(f the tturbinate. 1)r. ISorcn: Dr. Brinkman, is y-our pellicular material regenerating cells'? Dr. 13•rinkman: I don't know. Dr. Rrctcs: Dr. Stuart-Harris, is the effect of N iral infection at the alveolar level the same as at the hronchial level:' Is the process at the alveolar level desq namation or is it h' yperplasia? Dr. b'triarl-Harris: Tltis (lucstion must bc di- rccted to Dr. Hers becnu,e lte and Professor \[tdd^r are two of the fc,N%• people in the world who havr acttlall}- studiell this at the alveolar level. [ don't think it would he ri;dht for me to silnplY ~ uow their work. ll,•, II'right: Do all ot the Nirushs invariably ~;toh short of irnolt-ing the basal Llver :utd, if so, whr.° 1)i..~l~lrUt-fl~lr~'7,: I clOll t know. Dr. .1rr rhese virnshs all tlie <,uue ~ize' Or isn't that known? Are the shapes different? \1'hat is the range of particle size'' Dr. Sttcart-Harris: These groups of viruses are citremeh- different. Their only common character- istic is that they are inhabitants of the human respiratory tract. They range from about 15 milli- microns tlp to about 120 to 150 millimicrons. Dr. Hilrling: Dr. Tyrrell, when ciliary activity- ceasesy are the ciliated cells gone? Dr. Tiirrell: It is very diflicult to be precise about this. On the whole, there is a good correla- tion between complete absence of ciliary activity and the presence of noticeable degenerative changes, in virus-infected cultttres; but some- times we see apparenth- normal-looking cilia de- void of activity on fragments. Perhaps our method is too crude to pick up a light reflex from the frag- ment. However, ciliary activity usually disappears because thc cell has been damaged and cilia have been lost. Dr. Gang: Dr. Tyrrell, have ~-ou noticed any pathologic changes attributable to respiratory syn- cYtial virus in this system? Dr. Tyrrell: Ao, in all cultures of respiratory wnc' vtictl virus, growth looked normal. Dr. lilcinerinan: What happens to mucous glands in the organ culture? Dr. 'L'1 yrrell: In ferrets' epithelium there are occasional mucous glands. Sometimes in an in- fected culture there are little spaces and dotlike balloon cclls «•hich may contain mucus, but no secretion that can be definitely identified as mucus over the cells. It is pretty rapidly diluted in 1.5 ml. of ,aline with only little bits of tissue on top of it. Dr. hang: We did demonstrate good mucous glands with secretion of mucus which stained metaehromatically with methylene blue. Dr. 1'in•rell: Our system was a bit different in that we were using a poor nutritional medium wllicll was not as good for the culture of mucous glands as is vour sy-stem. Dr. .1;taah: Dr. Hers. what are the difference- hetweeu S- and Z'-antigcus' Dr. llcrs: S-antigen is an R\A complement- fixing anti;,•en which is specific for a group of virus-iu this case for influenza A. The hemag- -lutinin tvllich is called the V-antigen is strain- xt-citic. From biologic, Ilectron-microscopic, .utd immnnolluorpscent studies in tissue cultures, we I,olievr tllat when viral harticles infect stlsceptible cr.lls. thw , v disintegrate on the top of the cc•lls. Sub- ~ncuH} . they lnove throuQh the cc-topllsm and into the nucleus. S-antiern is scnsitized there but nlo-es ihrough the cytoplaz~m and becomes a spe- cific inlertive protein l)article, the C-antigen in I liat n,iun ot the ce11 w;tll. Ur. Slrncb: It is ahsc,luhely impossiblc to dizz- rs rr>pirator}- trart rilinr}- action without dis- 111111'lls. This atti'rnoon the mam topic was xiruses, htlt in the fir.~t hr,)sentation, Dr. Laurenzi 00097529
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rt- ,is us ed in m us it- of u- nd R"e ed it in DISCUSSION ,li~zcussed the clearance of bacteria, and I was espe- iially impressed by the effect of alcohol on the Lcarance from the lung. My understanding of viruses is that they are completely inert particles itntil they attack and penetrate a cell. Maybe tltat's wrong, I don't know. But that is why I isked the questions about the size of these par- ticles. What do viruses do to mucus? Are they mucolytic or are they completely inert? Is it possible that a general viremia develops after viruses are inoculated into the respiratory tract and that the route of infection is via the capillaries into specific tissues where they grow and not on the surface. Dr. Hers' discussion of tissue cultures in the mouse suggested that at one hour there was specific immunofluorescence on the surface of cells and that four hours later it was present within the nucleus, but I don't know what the process is in between. After the cells are infected there may bo desquamation and death. Do viruses have any ,,pecijic efject on ciliated cells before they pene- trate them' Dr. Lattrenzi's data suggested that the effect of alcohol was on the very earliest phase of clearance. He said it was a shift in time of a parallel curve, suggesting that alcohol effects are clarlv. This was supported by the fact that alcohol did not affect macrophages. 4ti'hat does alcohol, in the concentration used, do to mucus? And what does this dose of alcohol do to cilia? If we could obtain answers, or even suggestions of how to answer these questions, I would be satisfied. Dr. Ilers: It is not known whether mucus- producing goblet cells are ciliated cells or a sep- arate type. Beautiful electron microscopic studies suggested the latter view. \obody knows if ciliated cells are precursors of mucus. Viruses are very complicated things, as we have heard from Dr. Stttart-Harris, and what we know is very super- ficial. The best model is the influenza virus. It is rhe oldest virus known and the one most carefull}- investivatcd by virologists. In a serirs of 300 pa- tient., with proved influenza, from 1948 to now, we have been impressed by the lack of mucus pro- duction in affected respiratory epithelium ciurine the earlv stages of infection. '(irus infection of the rc,spiratorv tract reduces mucus transport within 21 hours before cilia disappear. Often cilia can be detected after mucus production has ceased. Mu- cus itself has an enormous capacity for cle_aring substances off of the epithelium. Perhaps the ability of influenza virus to defy or abolish mucus pro- dnction is its most dangerous effect because this leaves the epitheliurn unprotected from the ~cIcondcu.v invtidet~s. Patients usually die from snb- <rctuent bacterial infection durine influonza epi- for in:~tance from staphylococcal pneu- tnonia. 1)r. Stnnrt-Ilarris: In answc,r to Dr. St:tiub's oiuestion. "What happens to a virn.s when it meets mucus?". ml-loviruses were so named becattse the~- hnc-e ;in at}iniiy for muropolvsacchnridc and for nincins and combim~ with them in the sputum, the itrine, or wherever the~, are found. Thov are not iurrt, httt theY have ruzyme.s to free thwmselvc, 173 from combination with red blood cells or respira- tory mucus. How this aids them in gettinz through the mucus blanket we don't know. Other viruses which are not myxoviruses penetrate mucus. How tlteY do it, is not known. However, their passage into cells is not necessarily active, involving com- hination with a receptor site. They presumably enter by pinocytosis so they are engulfed by the c•ell which thereby brings about its own undoing. In the close which we receive. in an ordinary contagion, the virus is virtually inert. Inactivated viruses in large amount~ have damaging effects on cells. Dr. Bang should discuss this because in Vewcastle disease virus this propertc is better developed than in any of the human viruses. Dr. Rang: We think that the viruses go through t he mucus blanket, because when 10° virus particles are innoculated upon mucus, only about 100 or 1,000 get through into susceptible mucosa. If hy- persecretion is produced, larger numbers of virus particles enter the mucosa. We are approaching an answer to the very fundamental but simple ques- tion asked years ago, "How do viruses get into cells?" This simple concept has been very difficult to evahtate. Viruses in large dosey kill cells, but it is unlikely that this is importcmt in infection except dtrim„ the abundant growth of adenoviruses which produce a toxic material. Dr. 1'urrell: Some of the rhinoviruses do not have this enzYme bttt still infect cells. Ciliated epithelial cells have a pectdiar and efficient way of pickinl_~ np some respiratory viruses which are not m}-vovirttnes. One tissue culture infectious dose of rhinovirus in a small drop put into the nasal c•avit}- directlv is infective when it lands on ciliated epithclium. Ten times the dose of virus placed on t he outside of the nose does not ptroduce infec- tion. In thc conjtmctiva. about 16 doses are re- quirel, whereas on the posterior phar' yngeal wall, it, takes 200 dose.s. Deposition of virus on stratified ~4quamous epithelia rarel.• produces infec•tion. Why i-~ a riliafed ehitltelial ccll particularly attractive to Ihc~4e agents? M y guess is that viruses may ;cctunllY attach to cili,i. Viruses are not motile I heucsoh, c~, but they ha\ e a powerful aflinity to the ('itia Whic•h are wiggline .ticvay like fitry near by. 'I'hc , v stic•Ic onto cilia, and once there they are not -oin~ to e,~et washed awa.•. The}- must be ver}- efftcic~nt. The half-life of one ti,sue culture in- f~•ctiotts dose of virus in the nose may be only a frw minutes so they must get stuck on cells qnicl:lv, tnuctts or no tnttcus, to initiate infection. If there were wa}-s of sop,lratin, cilia from cells. the ;ittachment of ~irnse~ to them could be ,-heclc(,d. I),•. I:c,tin: I don't ltnder-tand th, ~e meta- physical attributes of ciliated _cells. C.'.crcinogenic li}-ch-ec,u•bons fiuoresce. We have do monstrated fluorr,,crttce in viable cc]ls of the respiratory tract ver}• xoon after elposttro to hydroearbon aerosols. I woulcl assumr that Rct into ecils inde- 1,r•ndc nt of the iact that theY arr s_o-called "viable n"rents. 'Sc.conclh-, viru>es may or ma% - not attach tu the rc Ik quiel;l}-. I'tn surt~ri~,,d that thev at-
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174 DISCL"SSloN tach to the ciliated epithelium, because it seems to be designed to get them off and away and not allow it. However, if in vitro, viruses are put onto cells which look much like susceptible cells but are not, the viruses attach to the cells and then fail to replicate properly, although the cells have in fact taken them in. Dr. II'ynder: For two days we have been dis- cussing ciliary motility and mucus flow and have described methods for measuring them. Chemical, ph.-sical, viral, nutritional, and even climatic fac- tors are ciliotoxic. One may conclude that every- thing that is toxic to cells may adversely affect cilia motility and mucus flow. I3'e should, however, limit conclusions from our particular measure- ments to the specific study that we are doing. For instance, in the studies on clams done by Dr. David Goodman and me 1 we measured gaseous components primarily and perhaps got positive results because they were water-soluble. There- fore, our conclusions can be only for these com- ponents. As was demonstrated, smoke which has passed through a Cambridge filter has almost the same effects as whole smoke. Ciliary toxicity should be differentiated from tumorogenic activity, because smoke pulled through an activated cellu- lose filter and a charcoal filter produces as many tumors gram for gram as unfiltered smoke. Dr. Bang's observations about vitamin A deficiency confirm Dr. Wolbach's studies' of many years ago, and I have wondered whether deficiency of vita- min A might play a role in various lung diseases. Vitamin deficiency is increased by infection, and perhaps an experiment could be designed to make an animal vitamin A deficient and then expose it to different inhalants. The cumulative effects of repeated exposures to pollutants and viruses need to be identified. Is it possible to repeatedly in- fect animals to different types of influenza viruses which produce cross immunity to see whether repeated stimulation with influenza virus produces different results from a single exposure? Studies to date show that the effects of influenza virus and the effects of tobacco smoke are, up to a cer- tain point, reversible. Although a normal appear- ing epithelium returns, the question remains whether initiated cells are left which could be ac- tivated later. Finally, what re]ationship does in- fluenza virus have to lung cancer or to chronic bronc•hitis? This question was first asked by «'inter- nitZ many years ago. Our data suggest that in- fluenza infection has no clear-cut relationship to the development of hmr cancer, nor does it have any relationship to the devrlopment of simple bronchitis. Simple bronchitis and lun; cancer seem to be related to environmental factors to the same extent. The patient with simple bronchitis who has dhe s'ame environment as one n-ho does not ~ IF, x-nder, 1:. L.. rt cil.: C.cncer. 1965. 15, 50. - =1Folbacl:, S. B., and Brs<elv, 0. _~.: Phy.Siol Jdt~v. 1942.." 233. ~1Cinternitz. M. C-. , t n/.: Thw Patholovv of In- fluenza, Yale L'nivoi::it.v Pro:~z, Ilaven. ('on- not_cut. 1cJ20, p. 61. have simple bronchiti.s does not have an increased risk of lung cancer. Our studies on simple bron- chitis in \'ew York, Caliiornia, tmd England show that with the same smoking history the rate is the same. These data are confirmed by Fletcher and co-authors' and by Anderson and Ferris' ° who showed that other factors are not very important in chronic and simple bronchitis, with thee pos- sible exception of a postnasal drip. How important is simple bronchitis, and can short-term studies in patients yield useful information? We have shown that if persons with simple bronchitis who smoke heavily shift to filter cigarettes the cough improves in 20 per cent of them. The cough im- proved and disappeared in from two to three weeks in almost all of 200 such patients who stopped smoking. Whether the cough was produc- tive or nonproductive, when they stopped smoking it disappeared within four weeks unless it was due to postnasal drip and sinusitis. Admittedly, the chronic cough or simple cough is very subjective; however, its disappearance does provide a human reph- as to whether an environmental change was beneficial. I would like to agree with Dr. Kotin that ciliai;v stasis and damage is an important step in the pathogene.zis of lung cancer. And perhaps one could coin from Dr. Bereblum' the concept of a three-stage mechanism of lung carcinogenesis: First, the destruction of the defensive barrier; second, the absorption of initiators; and third, the absorption of promotors. This, I think, is a useful concept of lung cancer development. Finally, after identif,ving the agents in our environment that lead to destruction of this defensive barrier, it is our job to eliminate or at least reduce them. Dr. T yrrell: I think that the mechanical damage is another very important way of upsetting cilia, and we have noticed that if .rou touch the cellular surface with an instrument in preparing organ cultures. the cells die. Dr. Spoclc: Most of the patients I encounter as a pediatrician do not smoke, so this does not contribute to their respiratory disease. However, chronic respiratory disease occurs in children. Ap- proximately 50 per cent of chronic disease in those imder age 17 is respiratory, including bron- chial asthma, the pneumonias, and cystic fibrosis of the pancreas. The latter, which includes dys- Ginctioi of mucous glands, destruction of cilia, and, fina.llv, secondary bacterial infection of the lung, is related to the problems we are discussing. The bacterial infection is due to species of staphy- lowcci ancl Pmudomonas. The latter is impossible fo cro-ldicate. What does mucus dysiunriion contrib- ute to the presence of Pseudomonas? It was re- ported mnmtly that mucus or some type of slime 'uhstance was actualh- produced bv this thickly ' FLotchc r. C. JI., rt ~,1.: :1mcv Ii;ev Resp Dis, 1!)fil, Df i. 1. '_1ndei:son, 1). O., c/ nl.: _1nuer R6v v o=p Dis. 196-1. 'J0, S77. " Perri:. It. G.. .Jr.. and .1nder-on. D. O.: _1mer Hct-Ilc rlr 1)i~:. 1J62..tiG, 1615. Bc,rel,lnni, I.: ('anc•er Hesearch, 1954, 1q: 471. 00097531
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DISCUSSIOS i;e ia, lar an . r rncapsttlated Pseudomonas which has reduced its ;,nthogenicitY. In cYstic fibrosis of the p:mc•reas, rnucus completely fills the tracheobronchial tree. I,ung sections resemble a sponge, and when the, y :o•e scttteezed, mucus passes from the bronchi and trachea. Mucus is abundant despite frequent sttc- tioning through a tracheostomy. In this disease patients usuall}• die within three days after tra- (•heostomy because their cough reflex i:~ thereby ~iiminished. Aow I wish to comment about the organ cultures which Dr. T}•rrell described. In urran culttur, ciliated cclls grow out from an ex- plant of respirator.v epithelium in medium 199. \Iucus-producing cells cannot be identified in our outgt•owths. However, both mucus-producing cells ,utd ciliatecl cells are present in the explant. If tlte tissuc dies, cilia stop moti•ing; however, cilia usu- alh- remain on thw cell, at least for a time. Serum irom one patient with cystic fibrosis of the pan- c•reas producrd asYmmetrieal beating when put upon cilia of an explant, but serum froni patients with bronchiecta.,is and hN -persensitivity states had no effect. The reason for this is unknown. Al- though Nm should consider the cilia, mucus, and t he reapirator,c cell separately, I think studti• of cystic fibrosis of the pancreas in which all three are involced ma' v gice us c•lues to disturbed lnnn clearance. _lgammarlobnlinemia gave us such a clue with regard to the pathogenesis of imniunologic dcficiencv states. Dr. Stuarl-/Iatri.,: I think that Dr. Wvnder and I could probabl.- argue for a cer.• long time about what is meant b}• simple ht•oncltitis. With regard to the role of influenza virus in lung cancer or c•lu•onic bronchitis. I would agree that there is no .specific relationship hetwcen the two. Influr•nza has nr•ver been Ahown to have an.v relationAhip to lunh c.uicer. ;So far as chronic bronchitis is c•on- corned, the papers that were published in the United -States :tftcr the influenza A2 epidemic in 1957 reiraled that a high proportion of adtdt (lcatlts lcere in two groups of p;uit•nts: those with ln•o-cxi~zting, chronic chest disease such as bron- chitis attd emphYscma and those with Itoart dis- asc. It. Scems inescapable that influenza is a partivulat•lY letlral ini'ection to those Who ;u•e con- 'lanll}• oeerproduc•ing mucus and hax-o a dis- ordercd respirator}- trat•t. I am interested to hear of Dr. R-cnder's rtperirnents Ncith 200 paticnts with so-t•a11ed ~impho bronchitis which Nc-as rc- ~cr~ed b}• _icint[ up cigar•e[te smol:inr. This is the ~ort of ,tttdc that need~ to be done in ditfc•rent ( m-ironmcnts oil a vot.t• large sc•alr- bet'ore wi< will licnvr• tLo facts %chirh we hndlv necd. If :~tnokina' Inthit,4 ;tre st:urdardized, therc is a dif(erencc in hr I'r"valr•ncr• of hronrhiti, in Enllland cornpart•d %ci'rlt ilte comparahke areas in tite Lnited Statc's. 1),•. It ,r~t~Jrr: ACo ~ta}•ed acva}• front the term ~itnpke hroncltitis and rtlled it porri~4tcnt cougIt, producrive and ttnproihtctic-e. ICe rhink tlrat the incidr•ncc• of ~implr~ hronc•hiti., i, ~~intilar in tlie two "ounn•ics. 'Phc• tuulor dit'ferenr(• lirs in clironic• l,rrntchitis Nvliic•li i.; ~o mucli IiiRhrrr in Grrat ltrit;iin than in the LTnit(A Sr:ues <urd whic•h i~: - I~,nk noi rAat~ d to ttr ~nrul:ini,. Dr. 13uren: Dr. Bang will describe the urn cell. :t model for stud}-ina mucus production and ciliarY activitY. :17ucIts llyperseeretioia in Ciliated Unz Ce1l,s" "•'° nr. Bang: Urn cr•lls" are introduced to this nudience hrcau~e the' v have too lonn rernainecl out- sicle the literature of medical biology, and their Fic. 1. Longitudinal-section diagram of Szpun- c•ulas nmcdu•s, details omitted, showing large coe- lomic fluid cavitY. e Prepared b}• B. G. Bang and F. B. Bang. From t he ~!;tation Biologique de Roscot7. Roscori. hinititcre, and the Department of Patltobioloe' v, Johns Hoplcins IIniver>it.v School of II1vaiene and Public Health• Baltimore. Mar.•land. "Thc>e studies were carried out during ille o-ntne of a John Simon Gug,enheini AIe•ntori.al Foundation Fellowship. "'Thr authors are indcb'ted to Dr. Gcazes Teissier, Director of the Station Biologiquc (lc, l:oscoff, Finisterc, Brittany; and to th(, \Iarins and the Admini~tration of the Station for makin, u nilahle to u: the full rc•sources of the area. "(':utut nzi n°. .1.: .1rrh Ko-unru Yntli F:,~p \Iicrohiol, 1925. 1, 7.
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DISCUSSION FIG. 2. Phase microscope photograph of living urn cell. V-vesicle: C-cilia: S->ccretor~. portion of base cell; 'AI-Dlncous tail with small hurden of debris. Smaller binrred cells are living blood cells. experimmntal value should be exploited. These remarks lean heavily- on two previously published report-;. "1'rn cells" are free-swimming, ciliated, mucus- producin.1"-" bicelhtlar organelles which scavenge debris from the coelomic cavity of the imerte- brate marine worm SipruacOu.c nudu.e." The Ncornt looks like a bloated angle Ncorm. It is about four to six inches long and about an inch in diatueter and has been aptly described" as a nuiscular bag full of fluid. Found in tidal sands alternatelY cx- posed or covered by some 20 feet of w;tter. ihcsr worms live in shallow burrows and ingest s;tnd. utilizing the organic bits and casting the rest. The ;ut and gonads lie in a vast open coelom ( ti,aure 1) which is lined with epithelial cells, soute of whiclt become mucociliatecl. Once dift'erenti;tted, these cells, in this species, liroak l"os r, from tho bod}- wall and beconre free-sicimmin?. The definitive urn is mmpo<c~d of two oolls. e;tch nucleated: an anterior halloon-lil.e vesicl(~, "13ang, F. B., and 13:utR, B. (;.: C'ult Biol NInrine, 1S)62, ,3, 363. " Bang, B. G., and Bane, F. B.: Cah Biol \Iarine, 1965 (in press). "Hc-man, L.: Thc Invertebrates, vol. 5. AIc- t4;tW-I1ill. Aew lork, 1959. ,tnd a ciliated base cell which produces astickv t ail of mucus (fivure 2). The organelle darts swiftly tlu•ough masres of living t•ed :md white blood cells, Icecping tltem chnrnint* in circulation but not stickin-, to t.h em. Howerer. foreign material and inot:eanic dehri., in thc fluid are caught in the ciliar, v cnrrent and are flnn, firtnly- onto the ~4ticlcv t:til. Cantncuz~~nc" empha-sized the importance of tntts fr•om lhe patholo;:ic point of view and pointed out I he differences iu size and arglutiuat- in` capacitie,, of thc, secreted tails during and after inlections of host worms with differcnt substances. lCe Iriecl io anah•ze these dillerences by atternpt- in; to induce ,=ontc of thc changes ex perimentall}°. 1ctn:d rales of secretion could he followed in in- dicidual urns tmdrr high power (phase I00X) nticrosrot,v, usinl-r a tnicronueti,r and a stopwatch. Lt a seril•.s of <uch attent},ts, the most a.stonish- iug cAttecis followed introdtution of ma>ses of live I;tchoria into Ncortus or into fre,<h cultures of roelotnir 1Inid. :111 \i,ible nrns immediatelv he,an to h}•pcr«err,h, at rates front ten to thirtv titnes the norntal: Ihc}- tnaintainE•d such rah•s for Well over vt honr :tnd, despite vital stainine. continued to :i•crrtt, ;it 1hr .:une ratr. in ~onu• c;tses for over tlireo honrs. Tlt(, induced ~ecreticun was unlike th:tt of thw normnl mucott~ tail, it w:c exception- 0009'7533
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DICCtiSSIO\ Fia. 3. Drawing of bacterially stimulated hyper- secretion of an urn base cell as seen in the Phase II microscope; live shimmering bacteria are, of course, stuck to the entire circumference of the tail but show only along the periphery of a given focus. 1;v fly ells, nott and the the 3 all.y efficient in trapping, bacteria (figure 3), and it was src•retr,d first at a rate too rapid for re- cordinv. L`rn ci•1ls are particularly important for the studv of mucus formation, for they remain alive and swinnininn in the original coelomic fluid for several weeks at room temprraturc= without ~ 11anae of fluid. W(irms are ca~z.v to collect from known breedin- <ites in Pini,tcre, and theY are easily maintained lii for Weel:s or months in running sea «-ater. «'ith- rlrawal of coclomic fliiid and maintenance of urn volls in cultures are uncomplicated, and the urns rc:;pon(l to experimental factors eqttall.• well in ricn or in 2•itro. We know of no othcr normallc frec-living cell capable of sumztained s' ynthesis of great volumes of nmucus while -self-propelled by ciliarY action.
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PERSPECTIVE AND HISTORY OF INVESTIGATION OF CILIA IN HUMAN DISEASEI, Z A. C. HILDING A few months ago there came a very kind invitation from Dr. Salzano, asking me to speak at this banquet, saying, "1j'e would enjoy hearinr• how you got involved with cilia,... since you are one of the pioneers in this area....° Recalling the work of Heide and von Leemvenhoek in the seventeenth century (1, 2), I had never pictured myself as a pioneer. In any- c,i.Se, the immediate effect of the in1-itation in tny- laboratory was to change my title from °~lave drivcr" to "Grandfather Cilia." _lccording to the advance program notes, my talk , ~hould be about Perspectives and Ili~tory. Ilowever, especially after today's pro- gratn, I am convinced that vou are thoroughly- familiar with the history, so I shall take the liberty- of reverting to the sttgtestion on the oririn;tl ilivitation. Over 100 y-eat:4 ago, extensive studies of ciliary- action in reptiles, birds, and mammals were tnade 1-,y Purkinje and Valentin (3), Shancoy (4) and others (5, 6). 11T. Gosselin, in 1S51, found that ciGart- activity continued for two to five days after death (5). (I think that ('Tos~~elin was not a Dartmouth man-neither did he have any-thinr to sav about the action of ;erotonin on cilia! ) Gray, in ]ti~4 book publi=hed in 1928 ((7), described ciliarv action in ,•reat detail, even including the dYnamic" and the mathematics thereof in tuticellttlar, a< trell a~ tnttlticcllular, lower anitunl form~. Sperm c_e1k caune in for vet.v extensive study, includin-1 the traveliua, Nvavc~ in the tail and the ltvdro- d, vnamic; resultinr in propttlsion-and other Ciliary- action in ihe finibriated end of the fallopian tube wa~ also described. Even the ,eotnerric p,tttern~; traced b}, the tips of the cilia itt the variou~ low formc were plotted. 13ttt oi that area mo~zt intport:ntt to mam, the respir;uorY tt.`let, almo~t nothini_was Saicl. I:arlY in nt}- own ~tttdie~-not tt ith the , Prnin th-~ l1w,e:crrh Laboratorc-, St. Litke:s Ilos- pil,ll.Diiliitli.lIinne~otn. -Tliis %corlc wascuphnrted bv t;. S. PuLli'• llr,tltli Gf,n-icr, (;r:uit (;\I-0J6U3-03. \:rtional ln=tititt (,f 17~; 116. seventeenth century pioneer;-I worked at the \Iayo Clinic. There I borrowed Gray's book from Dr. Alvarez so often that finally he it to nte, writing on the f3.v-leaf, "To Andy, who needed it most, with kinde-t regards, 1Ca1- ter C. Mvarez." How clid I become interested in cilian• ac- tion? In this way. In my first few years: of practice (opltthalmologry and otalary-ngoluRY -may-lie I am a pioneer!), it seemed that most of' 1 he ilhtesscs with n-ltich we dealt clini- callv were secondarv to the common coid: otitis media, mastoidizis, sinu~iti~:, peritonSillar absce~~ses, and so on. Consequentl}-, I tried to learn sontethini:~ about the comuton cold, and it Nva~:n't lonE~ until, in the ft'e.dt, watcrY sccretion from my- own cold, I found livin~, ehfoliated epitltelial cells. -Not knowin; what thc' v were, I~zketched a few and took the :~ketehes to E. T. Bell, profes~~or of patltolo;}-. in}- former chief in graduate school at the I nivrr,~it' v of \Iinne- ~ota. IIe clid nor know wthot tlie v ivere either, and woiidered if they might hc ~,unic kind ot a para,4ite that invacLed the epithelium. He su_- (rested that I take mv ~ketclte- to the histolozv profe.~~:or for ]ti., opinion. Thi~ I clid, and thc histolo-,t- profe~or s.iid, `'\o, those are not para:~ite~4 , they- are ?inlhly- fra,ment.~ of epithe- lial ccll~z, :ntd ever, v , vcar in ntY ela<~zc~, onc or niore i'rer'hmen come to mc in gre:tt excitement. h:tvin~;' nt:tdc u ;.rre:tt di~covcry-, and thev ,ltow tne these epitholial t'ra;ments." From that il~ tt•a~ jn,t astep to c•iliarY = action, and I madc a moVie film ~hon-inr the .-ariou~ facets of this important ph~~.~ioloric fnnction. I1:n-e copie~ to a tntntber of ph, v~iolo,-,y- de- prtrtntc-nts in medic:1l .<chool~:, bttt at that time no ()ne ~eemetl to be e-lwcizillY ittterested. \nt. lun(-, afier thnt_ po.~toperatiVc atclecta~i? F,e_an to votmnand a~*reat deal of attetttion and pro~okcd untch cli~cti--ion, hnt ciliary :w- tion w:i.~ never mentioned. I maclc ~oute stndic, and fontnd th.rt :t Nerv ~ub~_r.tuti.tl nc~cui~c pressurc could lm prndueed in the trachea front n 1'rc<hlY killc•d c•hickcn (Sl, :ts cilia a(l- 1-ancecl :ut ot•cludin~,, oi <oft mrncit~. I~ttc- _-ted 1h:tt tlti~ ntecli:uti~m tuimltt e.pl;iin <.nute ]Ts 00097535
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:ic- of hat iini- nld : llar to lI it ion ~ted re, T. liiot cr, ia bll~'- I. )P~_ the not the- I or cnt, heY rotn Id I ious ion. de- ime PER,PECI'IVE :1SD IIISTORT in,, tances of rapidly developing po.-topera- ti%-e atelectasis. Thus, under certain circum- .<tances, normal ciliary action could be an im- 1?ortant factor in producing atelectasis. A o one else was impressed. I heard a professor of surgery lecturing some time later, and he stated that a theory had been advanced that postoperative atelectasis could he produced by normal ciliary action but that the theory had not been accepted and that undoubtedly atelectasis was produced b}• ab,~orption of air behind the cork of inspissated mucu~. This, despite the fact that such corks are usually not found, and that, because the nitrogen ten- sion of the blood is very nearly that of the at- mosphere, nitrogen absorb,s exceedingly slowly. Four or five days are required for such absorption from the subcutaneous tissues, the anterior chamber of the eye, or the pleural space. Some postoperative atelectasis, on the other hand, develops right on the operating table (9). Lucas (10), Proetz (11) and a few others, were -working at that time trving to stress the importance of the ciliary mechanism in the htmiau respiratory tract and its relation to disease. We all had the same experience- teachers and clinicians paid little, if anY, atten- tion. Proetz, in some of his writings, mourned that the ciliart- mechanism did not receive a tithe of the attention to which it was entitled. In my opinion, cilian• insufficiency is a real clinical entity that played a large role in death from tN-ar ;ases, in the high mortalitY rate of the 191S influenza epidemie, and in fatal asthma (1'3), and that may be playing a major role in the increasing~ mortality from chronic bronchitis. It would be of interest to know what part, if any, it plaY.s in lobar- and bronchopncnmonia. The relation of sinusitis to the ciliary mech- auism was not clear 30 vear, aro; but, after :ntd}-in; the intra~inus ciliarv draina,e, the relation to poor surgical result wuc dcprc :in~l~ clear (1:3). The theorY went that =ince creamy pu'~ drtiucd ~luggishl,v by gravit}- thronRlt holes, if the}- were suliicientlY large, therefore one mu~t make such larve ]iole.• in the floor of tlic ~initscs, or enlarae tlte ostia. Such snr.gery w:Is plt.y,,ielo,_~icall}, wrnn~_,. This consideration of thc sinuses :t', boxes thatt could be drained hY openin; ihc hottnm didn't take into ac- 179 count the fact that in certain sinuses drainagc normallY is directed awav from the bottom. Making a hole in the bottom didn't make them drain that way, nor did enlarting the ostium ensure better drainage, as expected, because this was the equivalent of blastin; aa-ay one end of an escalator. For some vears then I«-orked on other things and forgot about c.iliary action until the concept of a causative relation between cigarette smoking and lung cancer catne to the fore-a most unlikely concept it appeared to me then. As I perused the literature, it ~eemed that no one «-as follo«•inE~ the smoke below the pharyns. So, to see what would happen, I blew some smoke into co«-.-' lungs. Two thinrs became apparent almost immedi- ateh-: Fir~t, that the lun, is a very effective filter; in other words, that deposition of tar takes place there ver' v rapidh-. Second, that the smoke stopped ciliary action both in the intact trachea and in tint- bits of livin; epi- thelial tissue suspended in a hanging drop under the microscope. I published these find- ings (14) and it wasn't long until Ballenger (15), Dalhamn (16), Falk and Kotin (17), and others were workin, on the problem and c.arried it much further than I had. And now the lowly cigarette has accomplished more by way of bringing the importance of the ciliary mechanism to the attention of doc- tors and patients than have all the research workers for the last 150 years.-Even the ctoudk of cigarette smoke have a~i1ver lining! IIigh school youngsters are now familiar with cili.ir.v action, and the ciliostatic effect of ci-a- ret tc <moke i~ kno«•in_•1Y discussed in the liter- ature, includin; Tbne and The T1'all Street Journal, and I presume it is the stimulus of cigarette smoke that has broulglrt us together here in this sympo~ium. I wonder if there has ever before been a sympo,~ium on iust thia sub- iect. I think not. In this regard ard it, is admirable tltat :t membcr of the tobacco industry should -pon~;or a symposium ~uch as this and en- cour.cre free and ttninhibitcd di-cu;~ion of the eliect~ of cigarette smoke. ~Chere do we :o from here" There iS still a L,~reat deal to be done on both the phy.-iolo~_,y riud tlte relation of the mttcociliary mechanism to di~!ease. What we have heard toda, v empha- ~ize~: what nceds to be done in ph.ysiolo,y. As
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ISO A. C. IIILDIVG far as disease is concerned, we do not yet understand at all well the role of the muco- ciliary mechanism in such common diseases as asthma, bronchitis, emphysema, broncho- pneumonia, and carcinoma. It is easy to be an armchair scientist and, from the known facts, weave attractive hypotheses and theories. But extrapolation is a dangerous game, especialh- so because one does not always realize that he is extrapolating. I have done my share and have been burned. One must be constantly on guard to be completely objective. It would perhaps not be amiss to tell a story, which some of you have heard me tell before. A lady wearing a long dress that dragged in the dust got on a bus and paid a fare of 25 cents. A little later another lady got on wearing a dress that reached mid- way between her ankles and her knees; she paid 20 cents. Then came a young woman wear- ing a skirt of 1964 style, at knee level ; her fare was 15 cents. Some time later, a beautiful young woman wearing Bermuda shorts got on; she paid 10 cents. Finally, a sweet y-oun~ fhing boarded the bus and paid no fare at all.... Now you're extrapolating!-She had a transfer. I once proposed a theory of hearing and was just as sure that I was right as any other theorists for the past 400 years. lIy theory might have been correct had it not been for one 400-year-old, unw•arranted assumption, namely, that sound waves within the cochlea stimulate the sense cells of hearing. But, the lady had a- transfer-the sound waves, if thev do actually enter the cochlea, have no physio- logic function there. I"on Bekesv has demon- Arated traveling waves in the basilar mem- brane, which actually are surface waves; these seem to do tlte stimulatin;. They resemble sound waves like a jack rabbit resembles an owl. Thus, it is easy to sit in your armchair and reason that, since to produce cancer, a cir- cinog_ enic arent mnst be applied over avery° long period of time, and that, since the car- cinogenic_ compounds from ci~arette smoke are deposited in the mucus blanket, and this is re- moved b.v ciliary- netion, when cili,lrY action is slowed or stopped entirel}-, carcinogenesis will he prontoted hccamte tlie renlova1 nf these compc iuids is dela.ved. I think that in all probability this is true, hut %N-F, actnally do not know t.he steps or how much of a factor ciliary action mai• be. Perhaps the lady has a transfer. Perhaps the carcinomata begin onhin large squamous areas devoid of cilia, where drainage is accomplished only by coughin,. In such a case, it might not matter to that spot whether or not the cilia were beating. It would he eas.y to reason that, if we could get rid of the ciliostatic effect in citzarette smoke, the incidence of carcinoma would be greatly reduced. Perhnps it would, but I wouldn't want to bet tlic lives of hundreds of thousands of smokers on the concept. How much of a factor it would be, we actuall}- do not know, do w•e? This has been a most ~timtilatinv day. For me it contained a special challenge Nchen R, vlander pointed out that old, crude methods are now insuificient and that we must have neiv, refined methods to advance. Of course, re- fined method.; will tive us much additional information, hut perhaps there still remains much to be determined by simple means. It might be well to keep in mind the story of the scicntificnllN, inclined re~ident. He came in one nilht to the residents' lounge where some of liis fellow residents Nrere pla}-ing cards and inquired, "Does anyone have an ether mask tltat will fit a ca't?" "Yes," some- one did. "i1Tav I borrow it ?" "I es." "Does an, vone have a mouth gag tha.t will fit a cat?" "Yes." "AZay' I borrow that, too?u r.Oh, \'es." "And does anvone have some catheters that can be nsed as stomach tubes for a cat?" ";I'es" someone had those too. After a few more items he was equipped, and one of those present asked, "Wcll, Ceot',e, what are ' vott ~oing_ to do?" "Well," lie replied, "Till intere.-ted in the dizes- tion of fats. I'tn going to anesthetize and in- tubate a series of cats and then iniect cream into the strnnachs of all oi them and then sacrifice them at intervals to see what has happenetl to the fat in the cream." "Well, Geou•,c," one of them ~aid. "did yeu ever try puttin, ilic crenm iu a~:ntccr.°" _1ti I,.-lander ~pokc, I~_mt ,nt idea, and I'm goin; to trv to pitt tlte crctm in a sancer. This is tthere the cilitttn aud the ci<.:arette h,ive hrou~dht n;. I f(,(,1 thtit it wa~ ,t ~_,re,tt priv- ilcer to h:n o Leen iuvite(l to this ~ympositnn and to take part in it, and I teant to thank 00097537
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factor ly has in only where in;. In It spot ~g It could =moke, _ reltlv n ildn't t~ands nf a know, For when -rhods have '=e, re- Itional maina It rv of i ne in where tv'ing e an ~ome- 'Does cat ?" that ye,,, items asked, do ?" di;_ es- 1l in- cream then t has ::Well, "' tri' iinder ry to irette p riti•- ium l:ank PERSPECTIVE AND HISTORY Doctors Parmalee, Salzano, Kilburn, Dalhamn, and the others responsible. REFERENCES (1) de Heide, A.: Anatome Mytuli Belgice Mos- sel (in Latin), 1684. (2) von Leeuwenhoek, A.: Concerning little ani- mals observed in rain, well-, sea-, and snow-water: As also in water wherein pep- per had lain infused (translated by Olden- berg), Philos Trans London, 1677.1?, 821. (3) Purkinje, J. E., and Valentin, G.: Com- mentatio Phz-siologica de Phenomeno Motus Vibratorii Continui, et cetera, Wra- tislav, 1835. (See Sharpey, reference 4 below). (4) Sharpey, W.: Cilia. In Cyclopedia of Anat- omy and Physiology, volume 1, edited by R. B. Todd, Longman, Brown, Green, Long- man and Roberts, London, 1835, pp. 606- 638. (5) Gosselin. M.: Sur la duree des mouvements vibratiles ciliares chez un supplicie, C R Soc Biol (Paris), 1851, 3,, 57. (6) Grant, R. E.: On the nervous svstem of Beroe pileets Lam, and on the structure of its cilia, Trans Zool Soc London, 1835, 1, 9. (7) Gray, J.: Ciliary Dlovement, Cambridge Uni- versity Press, Cambridge, England, 1928. (8) Hilding, A. C.: Production of negative pres- sure in the trachea of the hen by ciliary action, Amer J YhNsiol, 1951, 16 , 108. (9) Hilding, A. C.: The removal of air from the 181 respiratory tract and certain other body spaces under normal and abnormal condi- tions, Ann Otol, 1950, 59, 309. (10) Lucas, A. M. (with Douglas, L. C.) : Prin- ciples underl}•ing ciliary activity in the respirator' v tract: II. A comparison of nasal clearance in man, monkey and other mam- mals, Arch Otolaryng (Chicago), 1934, 20, 518. (11) Proetz, _~. «.: Essays on the Applied Phys- iology of the Nose, ed. 2, Annals Publish- ing Company, St. Louis, Missouri, 1953. (12) Hilding, A. C.: Relation of ciliary insuffi- cienca to death from asthma and other respiratory diseases, Ann Otol, 1943, 52, 5. (13) Hilding, A. C.: Experimental surgery of nose and sinuses: I1'. Effects of operative win- dows on normal sinuses, Ann Otol, 1941, 50, 379. (14) Hilding, A. C.: On smoking, bronchial carci- noma and ciliary action: II. Experimental study on the filtering action of cow's lungs. The deposition of tar in the bronchial tree and removal by ciliary action, A'ew Eng J Med, 1956, 2olF, 1155. (15) Ballenger, J. J.: Experimental effect of ciga- rette smoke on human respiratorly cilia, \ew Eng J _l~Ied, 1960, 263. 832. (16) Dalhamn, T.: The effect of cigarette smoke on the tipper respiratory tract, Arch Oto- laryng (Chicago), 1959, ',0, 166. (17) Falk, H. L., Tremer, H. M., and Kotin, P.: Effect of cigarette smoke and its constit- uents on ciliated mucus-secreting epithe- lium, J Nat Cancer Inst, 1959, 23, 999. GD
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SUMMARY OF CONFERENCE ON CILIARY FUNCTION D. V. BATESI I might start by reminding you that we have listened during the past two days to sixteen formal presentations of excellent qllality, and there has been a good deal of discussion, both in and out of school. I suspect the more revealing bits of this were not held in public, judging by some conversation I overheard in the tavern last night! I want to start by telling about two statisticians who were out hunting. They were standing close to each other when a (leer went by, 50 yards away. The first one fired and his shot was a foot to the left; and the second one fired and his shot was a foot to the right. He turned to the first statistician and said, "A direct hit, I think." We have to be very careful that, when experiments "bracket" a topic, we are not misled into assuming we have made a direct hit. The first thing one obviously has to stre.,zs about this symposium is that we have wrcAled through- out with the old probleni of trying to establish the meaning of differing conclusions drawn from different experimental preparations. One is, of course, aware that in biology this is a common problem, but it has come very clearly to the fore in many of the discussions and questions. Never- theless, it is remarkable that so many different preparations developed to study ciliary activity have, in general, shown unanimity. There have been some differences; but when you consider the differences of niethod and technique that many of these scientists have used, the measure of agree- ment is, I think, more remarkable than the points of difference. We have all been struck by several remarkable demonstrations of ingenuity. We are dealing with a most complex system with pe- culiar difficulties of its own from the experimental point of view, and I think we could agree that manv of the approaches adopted, including the remarkable "chicken in the basket" front Balti- more, have demonstrated estraordinary ingenuity and persistence in tackling very diflicult problems. I'd like to emphasize what seems to me the key to some of otu• difl^iculties. This is, that we don't know very much about the normal environment of the cilia. We have to remind ourselves that the clertron microscopy preparations, bcautiful as they arc, do not tell us very much of this aspect of structures. What is the fluid in wliich the cilia are normallY lir,ating and having their rxistence? Furthcrtuore, when you move on to the next rtucstion. namely, what is the ..tructure, r•ontititu- tion, thirl:ne~s. anrl so on, of tile nntcotts blanket wc haie talked about, again we are blocked bv ' From the Department of Experimental NIed- icine, ~L•Gill Univct:sity, and the 1{cspiratory Di- vision. Joint Cardio-respiratorv Service, RoVal Victoria Hospit_al unct \lontroal Chilclrcn's Ilos- !,ital. Jlontreal, P.Q. Canada. real difficulties of experimentation and handling. It is our inability to be confident that we under- stand the normal environment of these remark- able structures that underlies many of our diffi- culties. 1t'e cannot yet have much confidence that we are able to draw a really complete picture of the way these structures normally survive, where their nutrition comes from, and the problems of altered vi,rosity of mucus which maY determine the effectivettess of their function. Ever.vone can agree that cilia have, at least in the human respitatory tract, a remarkable number of enetnies! For me, one of the most revealing aspects of this conference has been the clear demonstration of the complexity of the clearance process. I now know that I came to this sym- po.sium with a very superficial knowledge of par- ticle clcarunce and transport of mucus. I had a be- ginner's view of this phenomenon, and I have cortainly been educated. I have some problem~ to add to those which we have discussed. We have becn concerned with ciliary action and the prob- lem of how fast these things can beat, and what will stop t.hem. We have been shown that, for somc reason quite unetiplained, their integrity is dependent on vitatnin A: I might add that it may also be dependent on thyroid extract or the pres- ence of normal thyroid hornione, since it has long been known that the disease of myxedema is associated with a persistent form of chronic bron- clritis without the influence of cigarettes. We have a good dcal to learn here and, although vitamin A seems essential, we haven't been told very much about why this is a critical nutrient. However «•e cannot; confine our attention soleh- to whether cilia oun or cannot beat individually. We have been shown some remarkable photographs of "rotating explants" of human ciliary cells in a spherical form, and it was clearly demonstrated that these would stop rotating under certain cir- cumstanres although the cilia were still beating. What is it that co-ordinates such a sheet of cells.° 13ecause whatever it i~z, it is clearly onee of the factor., on which the successful transport of a ntucous blanket would depend. We have to con- fcss th;tlwe are lart;eh- i,_,norant of the factors which tnirlit interfere with the synchron, v of beat- ing on whir h effr•rtive function tdtimately depends. 11 e thwn mmr a sta,c further out, passinr the cnvironnicnt wliich, as I pointed ont, wo don't tmderstand, ancl cume to the problr~m of viseosity of the mucotrs blanket. I ha~•e Ncondr•rerl whether some of ihc more erndite matheniaticians could be ,c.:kerL wluothcr it is possible to approac•h, theo- rr~tic,dl.v, the mlationshin brtwcen ntovement ,ilone ,t ~* v•tctu like this and the visco,itv of what han to be niovrl. We have hcard( o]' rour:•r. that c'is- cn-ity will iudccri affect movrment. and wc I:now this to bw trtte, hut we cinn't tuite nnrirt~stand hov I I 0009'750"3
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SL'SI\LART OF CONFERENCE ~ritical a factor this is in quantitative terms. It would be '.•er}• interesting to know. When you go un from there and consider other factors related to clearance, the difficulties become perhaps even ,rectter. We have been introduced to the problem of the function of alveolar macrophages. We were shown that you can influence the macrophage activity independently of ciliary activity. We cnight also consider other factors rather simpler in nature. What do we do when we cough? Do we roll up the normal mucous blanket and spit it out? When you go on to consider the dynamic human bronchial tree as it exists in life, it is not enough to think of it in static terms. As yott breathe in and out, your bronchial tree is lengthening with each inspiration. It is also altering in diameter, all the way down, probably; and if you take deep breaths, these alterations in diameter may be considerable. We are looking at a system which is continuously altering its linear dimension and its caliber. What do you suppose is the effect of these alterations on the surface cells? It mi2ht be quite important. We do know, of eoursed and nobody has mentioned this, so I will-that pa- tients with a lot of sputum production can greatly influence the amount of sputum they produce by sy-stematic alterations of posture. In other words, tc-hen vou have a well-localized region that is pro- ducing a lot of secretion, yott can influence the amount produced by assisting drainage by gravity. Some parts of the lung are at a distinct disad- .antage in relation to gra.-ity as compared to uthcrs. Perhaps the ciliary mechanism is best developed in relation to the lower part ;ind less necessary, or primitive, in relation to clearance from the tipper lobe. Lastly, in this problem of factors affecting clearance, we have the difficulty that some substanees, of which alcohol is one, are cnstomarily etichanged across the alveolar mem- br.me, and so come up the bronchial tree from the bottom. This is true of other hydrocarbons, iucluding kerosene. In this way the whole bron- chial tract can become exposed to a substance which is not part of the normal environment. Thus tit-<, can see, rou-dhly, where many uncertainties lie without vet bein~t' able to pin down the influence ui these different factors. 11-hen y'ou add to this complex situation the problem of the nature of cigarette smoke and ,Liscuss the effect of this on the ciliary system, you oi,t-iouslv are aetting into avery difficult field indced. As we went alon; in this area. Iwas re- niinded of the remark attributed to Voltaire. It wmuld be 100 vears a2~o since he described the inwdical pruff-ion, Yott remember, as concerned ,,%-itu thr -:ulnmnistr:ttion of drul-,s of the effects of u0hich thwY laww little, to diseases about which l-.• l:now nothinu,." 1Vhrn I sr- a man ~ntokinc: ~i_arri+~iu ihw futnre. I-hall 1'( (•I tluit I:uu louk- ine ,it thr adnuinistratiun of amaterial about liich I kuow littt(1, to aSN'St"nt nhuitt Nchicli I <nnw nothin'_'. In ?liis nr~ a. we have <ntnr tm- -rt:tnt ~oualitic:itions that pwrhaps didn't fnterl~e 183 as clearly as they might have. In the case of cigarette smoke, the experimentation so far has been larrely concerned with short-term effects, and as Dr. tiCrit;ht, I believe, pointed out, that is not realh- a pattern that concerns us very much. One .peaker mentioned that the absorption of noxious -aaes onto the smoke particles of tobacco smoke didn't look like an important factor in carcino- genosis, but in relation to atmospheric pollution, it may be critical. The problem of chronic lung disease in industrial countries is almost always a problcm when both of these effects exist together. I do think that in this general area we are facing major difficulties in discussing the effects of acute versus chronic exposure. One figure, for instance, chowed us the effect of 25 parts per million of u•r,one on a ciliary preparation. If an adult man breathes nine parts per million of this gas, he develops pulnionary edema. Twenty-five parts per million is a collosal dose and, really, one is not terribh- interested in coneentrations at that level, any more than one is interested in concentrations of oxides of sulfur a hundred times greater than occur in any environment. No doubt all in- vestigators feel constrained to choose a model or preparation which they think will give them a positive result. That is satisf' ving from one point of view; but when you come to discuss whether the findings are conceivablY relevant to the ozone concentration in Los Angeles, you obviotu_lY have designed the wrong etperiment. This 1e:tds me on to another area which we have .~kirted around (and in this summary I am more concerned to look at areas we have touched on rather than those that have been exhaustiveh- dis- cussed). It concerns the general problem of dif- ferences between acute and chronic esposure. I do think that we arc going to be concerned over the next tcn years with this problem and we just have to resi;n ourselves. I believe, to getting into the area ut lung-term chronic exposure. I often wonder how f;n• some of the ;rantin, policies of our agencws have tended to encoura,re investigators to do tlue acute, short-term experiment, as op- posed to the much longer range and ntuch less quicl:h• rewarding lons-term experiment. I think that we must examine our polic.- in this regard, hecause, if we arc serious abotrt lone-tcrm cnviron- utent.d exposure. then we just have to ;et down and z4tudy long-term emironmental cxposttre to vory low eoncentrations of thin-s ovor a period of .-eai:s. _1nd that's the kind of research which looks, irum soutc points of :iwfnllc- tutattrac- titr :utd has to have secure, lunur-term tinancing, for ann- investia,uor to bc• willin<_ to embark on it. Thrre arc exceptions to this _~eneral rttle, but 1 wotdd vrrv ntuch lilce to s,~e anituall work with lonr-nrnt (.xt,o~uns to .-erv low concen'trations of I:nawn ]"nllutants ovor ,t lunt; period of tinte. We know that the mnin effcet (if ri~~:irette smoke and t)osrihl}• of alrnosphe,rit- pnllutiun is on the bron- (hi:t1 niucous _Lmds. 1-ct wh,i~ runnin~* an ex- ~wrinn•nt '.t-ith clos Icrpt for tlinr snd four ccars in a <ttitnhlr em-ironnunt aud makin-, comparison
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184 D. V. BATES between Reid Index counts of mucous gland thick- ness between these animals and others? I really want to point a finger at this problem because I know of no way of getting at long-term, chronic effects, without studying the long-term, chronic effects. That is a hard conclusion but one that has to be emphasized. Lastly, I question whether our continuous em- phasis on lung cancer is wise at this particular juncture. I have been told at this meeting that the latest data on mortality- in the United States shows that the recorded deaths from chronic bron- chitis and emphysema are increasing more rapidly than are those from lung cancer in the Continental Lnited States. It may be true that chronic bron- chitis, as such, does not predispose to lunr cancer; but it certainly has something to do with emphy- sema, and this is not onh• important as a cause of death, but is responsible for a tremendous amount of incapacity. Lung cancer is; very im- portant, but I wonder whether we shouldn't be looking as carefully at the less spectacular, less publicized diseases which may be doing really more harm within the community on a long-term basis. In this regard, we have an interesting point that we know that the striking feature of the patient with chronic bronchitis, as far as I am aware in everY countrY in which it has been studied, is the bronchial mucous gland hypertro- phy. But, as somebody pointed out. we don't know whether these large glands, which may be twice the normal thickness in relation to the bronchial wall, are secreting normal material. If theY are not secreting normal material, then wo are right back at the clearance problem, because we know we c•an't fiddle very much with the constitution of normal mucus without interfering with clear- ance. We have been shown, for instance, that de- hYdratins an animal alters the viscosity of this normal mucus and interferes with clearance. Well, what material are these hypertrophied bronchial glands putting out in such rjuantit.- in the chronic bronchitic? Do we need first to look at the ciliarv mechanism? We know that the bronchial mucous glands are very large, and surely the next thing we want to know is whether they are producing a normal or abnormal mucous blanket. So I como to my last point, and that is that those of us who have attended this meeting have been privileged to view a problem of enormous contemporary interest and importance in great detail. I would like to conclude by expressing the thanla of Nour guests for the excellent arrange- ments and hospitality you have given us. There are some here who, like myself, have been privi- leged to attend this conierence. and thus get a view of contemporary work in a field in which we are not actic•ely engaged. We have all found it an exceptionally interesting and rewarding et- perience, and I wouhl like to express the thanks of all of us for the initiative, resource, and care with which you have arran_ed it. We are indeed indebted to Duke 1Jniversir_y and the P. Lorillard Tobacco Company who have made it possible for the program to be organized.

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