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C C CHARACTERIZATION OF LIPID INCLUSIONS IN ALVEOLAR MACROPHAGES AFTER TOBACCO SMOKE EXPOSURE AMERICAN COLLEGE OF CHEST PHYSICIANS LAS VEGAS, NEVADA NOVEMBER 1, 1977 5:00 P,M, MGM GRAND HOTEL (PAUL DAVIES) IN DEALING WITH PARTICULATE DEPOSITION IN THE DISTAL -LUNG, THE ALVEOLAR MACROPHAGE IS THE MAJOR DEFENSE CELL AND IS LARGELY RESPONSIBLE FOR THE MAINTENANCE OF STERILITY WITHIN THE LUNG PARENCHYMA. IN ADDlTION, IT HAS BEEN SUGGESTED~ THAT IT PLAYS A ROLE IN THE TURNOVER OF THE SURFACE LINING MATERIAL. THUS, CHANGES IN ITS FUNCTIONAL CAPABILITY MAY HAVE MAJOR CONSEQUENCES AND MAY ELICIT, WITHIN THE LUNG, FUNCTIONAL AND MORPHOLOGIC CHANGES INDICATIVE OF THE FIRST STAGES OF LUNG DISEASE. TOBACCOiSMOKE MAY REPRESENT A POTENTIALLY SIGNIFICANT AND CONTINUING AEROSOL AND GASEOUS INSULT WHICH HAS BEEN SHOWN TO PRODUCE MORPHOLOGIC AND METABOLIC CHANGES IN THE ALVEOLAR MACROPHAGES. IN ORDER TO STUDY THESE CHANGES FURTHER, MALE CD STRAIN RATS WERE EXPOSED TO CIGARETTE SMOKE UND R EXPERIMENTAL CONDITIONS. MAY WE HAVE THE FIRST SLIDE, PLEASE? q

SLIDE 1 ~THE ANIMALS WERE EXPOSED TO SMOKE WITH THE AID OF THE MACHINE SHOWN IN THE SLIDE. THE MACHINE AUTOMATICALLY LOADS AND LIGHTS THE CIGARETTES, A 35 MILLILITER, 2 SECOND PUFF OF WHOLE SMOKE IS GENERATED AND IMMEDIATELY DILUTED ONE TO NINE WITH FRESH ROOM AIR, THE DILUTED SMOKE IS DISTRIBUTED TO~THE ANIMALS IN PLASTIC TUBES WITHIN FOUR SECONDS OF GENERAT1ON, THE- RATS WERE RESTRAINED IN PLASTIC HOLDERS WHICH ALLOW ONLY THEIR SNOUTS TO PROJECT INTO THE PATH OF THE SMOKE, IN THIS WAY, THE ANIMALS WERE EXPOSED TO THE SMOKE OF 2R1 KENTUCKY REFERENCE CIGARETTES FOR TEN MINUTE PERIODS, THREE TIMES A DAY. THIS REGIMEN HAS BEEN'SHOWN TO BE VERY ROUGHLY EQUIVALENT .TO ONE AND ONE-HALF PACKS OF "HIGH" TAR CIGARETTES PER DAY IN MAN', THE EXPOSURE PERIOD EXTENDED UP TO 9D CONSECUTIVE DAYS. NEXT SLIDE, PLEASE, ,. SLIDE 2 ANIMALS WERE ANESTHETIZED WITH PENTOBARBITAL AND THEIR LUNGS LAVAGED WITH ISOTONIC SALINE AT ROOM TEMPERATURE. AFTER CENTRIFUGATION, THE PELLET OF LUNG CELLS WAS TREATED IN ONE OF TWO WAYS, FIRSTLY, THE CELLS WERE RESUSPENDED IN A GLUTARALDEHYDE-OSMIUM MIXTURE AND COLLECTED BY A FILTRATION TECHNIQUE FOR ELECTRON MICROSCOPIC STEREOLOGY. ALTERNATELY, THE CELLS WERE RESUSPENDED IN HANK'S BALANCED SALT SOLUTION AND PLATED ONTO GLASS COVER SLIPS. AFTER ONE HOUR, THE ALVEOLAR MACROPHAGES HAD.ADHERED TO THE GLASS AND COULD BE FIXED JA SITU FOR HISTOCHEMISTRY. NEXT SLIDE, PLEASE.

SLIDE 3 ALVEOLAR MACROPHAGES FROM ANIMALS EXPOSED TO SMOKE FOR UP Th 90 DAYS WERE CHARACTERIZED AS POSSESSING LARGE NUMBERS OF CYTOP'LASMIC INCLUSIONS, GIVING THEM A FOAMY APPEARANCE. IN HISTOCHEMICAL PREPARATIONS, THESE INCLUSIONS STAINED POSITIVELY WITH OIL RED 0, INDICATING THAT THEY MAY BE LIPID IN NATURE (1). UNDER THE ELECTRON MJCROSCOPE, THEY APPEAR AS VARIABLY OSMIOPHILIC, SOMETIMES OCCURRING WITHIN STRUCTURES LINED BY A UNIT MEMBRANE AND SOMETIMES LYING FREE WITHIN THE CYTOPLASM. CAN WE HAVE THE NEXT SLIDE, PLEASE? SLIDE 4 WE USED STEREOLOGIC POINT COUNTING ON A LARGE .NUMBf R OF RANDOMLY SELECTED ELECTRON MICROGRAPHS TO DETERMINE THE RELATIVE PROPORTION OF LIPID INCLUSIONS WITHIN THE CELLS, SPECIFICALLY, THE RATIO OF THE MEAN NUMBER OF POINTS ON LIPID AND THE MEAN'NUMBER OF POINTS ON TOTAL CYTOPLASM GIVES A DIRECT, UNB'IASED ESTIMATE OF THE VOLUME OF LIPID INCLUSIONS PER UNIT VOLUME OF TOTAL CYTOPLASM. THIS FACTOR IS GENERALLY CALLED THE VOLUME DENSITY. OUR RESULTS ARE PRESENTED IN THE NEXT SLIDE. 03748;os SLIDE 5 AFTER 30 DAYS OF SMOKE EXPOSURE, THERE WAS A 10-FOLD INCREASE IN THE VOLUME OF LIPID OVER THE VALUES FROM AGE-MATCHED CONTROLS. AFTER 60 DAYS THERE WAS A 16-FOLD INCREASE, BUT AFTER 90 DAYS THERE WAS LITTLE INCREASE BEYOND THIS LEVEL. THE CONTROL CELL POPULATION MAINTAINS A LOW, FAIRLY CONSTANT VOLUME OF CYTOPLASMIC LIPID INCLUSIONS, WHETHER THE ALVEOLAR MACROPHAGE POPU- LATION IS APPROACHING A PLATEAU-LEVEL FOR CYTOPLASMIC LIPID INCLUSIONS AFTER 90 DAYS IS UNKNOWN AT PRESENT. MAY WE HAVE THE NEXT SLIDE, PLEASE'

SLIDE 6 AT HIGHER MAGNIFICATIONS, THE SMALLER PROFILES OF LIPID WERE OBSERVED WITHIN MEMBRANE-LINED STRUCTURES, WHICH RESEMBLED DENSE BODY LYSOSOMES. WE ATTEMPTED TO DETERMNE IF THE LIPID WAS ASSOCIATED WITH LYSOSOMAL ACTIVITY BY ULTRASTRUCTURAL LOCALIZATION OF ACID PHOSPHATASE, WHICH IS GENERALLY CONSIDERED TO BE AN EXCLUSIVELY LYSOSOMAL ENZYME (2), CAN WE HAVE THE NEXT SLIDE, PLEASE? SLIDE 7 THE RESULTS INDICATED THAT THERE WAS INDEED SUCH AN ASSOCIATION, WITH THE LEAD REACTION PRODUCT INDICATING SITES OF ENZYME ACTIVITY PREDOMINANTLY AROUN'D THiE LIPID .INCLUSIONS. WE CAN, THEREFORE, CONSIDERSUCH INCLUSIONS AS LIPOLYSOSOMES. THE INCLUSIONS EXHIBITED A YELLOWISH`GREEN AUTOFLUORESCENCE, WHICH COULD BE ERADICATED BY TREATMENT WITH PYRIDENE AND WAS, THEREFORE, ASSOCIATED WITH THE LIPID. FURTHER HISTOCHEMICAL TESTS INDICATED A POSITIVE REACTION AT THE PERIPHERY OF THE INCLUSIONS FOR LIPOFUSCIN, THE SO-CALLED AGE PIGMENT. NEXT SLIDE, PLEASE. 4

SLIDE,8 IN ELECTRON MICROSCOPIC PREPARATIONS, THE LIGHT ~. STAINING LIPID~'INCLUSIONS WERE SOMETIMES SURROUNDED BY INTENSELY OSMIOPHILIC MATERIAL WHICH MAY CORRESPOND TO THE LIPOFUSCIN- POSITIVE MATERIAL UNDER THE LIGHT MIlCROSCOPE, LIPOFUSCIN IS AN INSOLUBLE COMPOUND WHICH REPRESENTS THE END STAGE OF LIPID OXIDATION, IT HAS BEEN OBSERVED, FOR EXAMPLE, IN CELLS OF MICE AND RATS FED DIETS HIGH IN CHOLESTEROL AND DEFICIENT IN VITAMIN E. ELONGATED, CLEFT-LIKE STRUCTURES BOUNDED BY MEMBRANE OF THE ENDOPLASMIC RETICULUM WERE ALSO1PRESENT IN THE CYTOPLASM. THESE HAVE ALSO BEEN DESCRIBED IN HYPERCHOLESTEROLEMIA, .AS WELL AS IN CELLS CULTURED IN MEDIA SUPPLEMENTED WITH HIGH CONCENTRATIONS OF FATTY ACIDS. POSITIVE STAINING OF SOME CYTOPLASMIC INCLUSIONS WITH ACID HEMATEIN INDICATED THE PRESENCE OF PHOSPHOLIPID, NEXT SLIDE, PLEASE. SLIDE 9 IN ELECTRON MICROSCOPIC MATERIAL, STRUCTURES WERE FOUND IN THE CYTOPLASM WHICH CONTAINED PHOSPHOLIPID MICELLES EITHER IN AN AMORPHOUS FORM OR IN THE CONFIGURATION OF TUBULAR MYELIN AND MAY, THEREFORE, REPRESENT LUNG SURFACE LINING MATERIAL, WHICH HAD PRESUMABLY BEEN PHAGOCYTIZED BY THE MACROPHAGE. IN ANiEFFORT TO QUANTITATE THESE MYELIN0CO~NTAIN~ING STRUCTURES, WE APPLIED STEREOLOGIC METHODS TO CELLS FROM ANIMALS EXPOSED T0 TOBACCO SMOKE FOR A TOTAL OF 90 CONSECUTIVE DAYS. NEXT SLIDE, PLEASE.

SLIDE 10 JHE SLIDE INDICATES THAT THE VOLUME PROPORTION Y OF MYELIN BODIES IN CYTOPLASM IN MACROPHAGES FROM SMOKE- EXPOSED ANIMALS WAS DECREASED BY A RELATIVE FACTOR OF 53% FROM AGE-MATCHED CONTROL ANIMALS. WHILE WE CAN ONLY SPECULATE ABOUT THE SIGNIFICANCE OF THIS FINDING, IT DOES INDI'CATE THAT THE TURNOVER OF PHOSPHOLIPID AND POSSIBLY OF INGESTED SURFACE LINING MATERIAL MAY BE ALTERED AFTER EXPOSURE TO TOBACCO SMOKE. TOGETHER WITH OUR FINDINGS ON THE MARKED INCREASE IN CYTOPLASMIC LIPID INCLUSIONS, THESE RESULTS INDICATE A DRASTIC ALTERATION IN THE HANDLING OF LIPID BY ALVfOLAR',MACROPHAGES FROM SMOKE-EXPOSED ANIMALS. THE PREVALENCE -OF INSOLUBLE COMPLEXES OF OXIDIZED LIPID AND THE POSSIBLE PRESENCE OF TOBACCO COMPONENTS WITHIN HETEROLYSOSOMES MAY SERVE AS A FACTOR IN THE INCREASED EXTRACELLULAR PROTEASE RELEASE RECENTLY REPORTED FOR ALVEOLAR MACROPHAGES FROM HUMAN SMOKERS AND MAY LEAD TO LUNG TISSUE DAMAGE (3), d

C RENC S ~ 1. PEARSE, A,G,E, 1960. HISTOCHEMISTRY. WILLIAMS AN'D~ I"IILKINS, BALTIMORE. 2. ESSNER, E, 1973, PHOSPHATASES. IN ELECTRON' MICROSCOPY OF ENZYMES, VOL. 1(ED, M.A, H'AYAT), VAN NOSTRAND REINHOLD, N,Y „ P. 44, 3. R'ODR I GUEZ, R, J, EI gL, 1977. ELASTASE RELEASE FROM HUMAN ALVEOLAR MACROPHAGES: COMPARISON BETWEEN SMOKERS AND NON SMOKERS. SCIENCE 198:313, d

C 1 SLUE 1 i

C FILTRATION : PLATING : Glutaraldehyde/Osmium Fixation Formalin Fixation Electron Microscopy Stereology Histochemistry SLIDE 2 i

C SLIDE 3 EL~.CTRON MICROGRAPH OF AN ALVEOLAR MACROPHAGE FROM A SMOKE-EXPOSED ANIMAL, SLIDE 4 EXPLANATION OF THE POINT-COUNTING PRINCIPLE. 4