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Philip Morris

The Effect of Tobacco Smoke on the Metabolism and Function of Rat Alveolar Macrophages

Date: 19780805/P
Length: 8 pages
2083039185-2083039192
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Author
Drath, D.B.
Gharibian, J.
Harper, A.
Huber, G.L.
Karnovsky, M.L.
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PSCI, PUBLICATION SCIENTIFIC
ABST, ABSTRACT
BIBL, BIBLIOGRAPHY
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CORPORATE SECRETARY/FILE ROOM
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2083038652/2083039227/Smoking & Health Scientific Research 700000 to 790000 Published Literature Charles R. Wall Shb, 961100
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Feda/Produced
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EXTR, EXTRA
MARG, MARGINALIA
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N2
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American Society for Clinical Investigat
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Asca
Harvard
Journal of Cellular Phys
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W, B.
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2083038653/9226

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Page 1: vso92c00
Journal o£ Ce11.u;ar Phya logy y7/l;yC7_13 9 The Effect of Tobacco Smoke on the Met~aolism and Function of Rat A!veolar Macrophages 1 NOTICE This materi>tl ruY bt protadrd by apyrght law (lftle 17 U.S. Code). I.AI'll) R UIL\l'Ii. A\'S:SHF,I.II:U(i9FJt. J:1?F'::~!1:1411iIAS _ Vt?.NFRELI I. R:1RNOCChY t1Ak5 I. Hi'ItPa( fh~prrmp.rt~nj8wl.,yrcolChrw,.;r.nndSfi+ir~mr.lktnanl.lL,:~• iin,,. ?,GShn(TVi A 8trrer, linnton..t(n..n; h n v; t.vl11! l S 0 0 ABS'TRA("P Alveolar macrnphak*e> harca-;tvd h5' hr,nchoptrlnior:.rn from rats oxpoeed to tobacco smoke fnr 30 dnca 1-smnko r.-t ehuvced .rl ~'r, tron. in oxid.ruc:• metub,)lism, lnclatc productlon ;Ind ph:q;.., t trsi. od ~r.,.. - n particles when compared with control mucrophagen I'Iwl;nrvtu.v:: of cmble Staph' vlaenccus aureu,s was unaffecled !n' tubacco snwke ra,icnse nxidation ma:r sured by conversion of gluco.rt-"C to "CO, wn,; mndh•r.rio :r ufte'•ted «hilc os:dation of glucose-6"C to "C0. was nnt. Smnkers rouunt•ly yielded fcwer rclla than controls, Lhnugh thesecellscontainerl:rppruximuteiy 17'cn mnre prutrin than did contrnls. Gpsomzation of p.)rtiolc. wan not neccv~ary I„r macrnphuqcs in,n: either smoker or control animals to m.tnifr.;t n respirawrc burxt and :nrrousi•tl superoxide and hydrogen pernxtde release doriny; phaput,cli„is. Thc {;htuly tic rm hibitors, sodium fluoride and indoacetamtie, while efiectIvcly hlr,cking ;;lycolysis, did not inhibit phagocyto.,;s hy macrophat;es frnm e:ther group. Tnv result, reported clearly dr,tinguish nlccoiar macruphages front ntl:er phap,c~ttc cull, Iperitoneal macrophnResand pohcmorphnnucle.v Ieukucytesr nnd su{;gc: a stati~ of non~specific activation cauced by expnxure to tohaccn snnke. Sterility of the lower respiratory truct is maintained largely by the pulmnary alveolar macrophnge iGreen, '6.1,, ,t cell which mu.,t ingest /phagocytizel and inactivnte lit inl; nnd non-living particulate material to ochich thr lung is exposed. Potential duma;;e a tP,e mac rophage by an dgent such as tobacco vn:oke, which contains both particulate and nomparv ticulate material, could presumably lead to microbial infection. Further, impaired pulmnary function. parenchyrnul damage. or air way pathology could result from excessive release of reactive entities by these macrophages, Such release is usually augmented during phagocytosis by leukocytes. :Root et al., '74: Babior et at. '73:. We have examined the effect of ':bucco smoke on the functional ability or elvcolar macrophages and on the release of superoxide and hydrogen peroxide'1'he experimrntal :rnimals used in the studies described bt•low werc rats which were subjected to 30 consecutive days of smoke inhalation. Previous studies of the effect uf tobacco J. CELL. PHYSIUL i19781 95r Ivu 114. ASCA 5 MaY 1978 xnioke on phat;ocytosis by ult'eui',tr macro- phages have protlucud dt:crepant results. Creen and colluburcuara r'ri7haoo 1hown .~n i:r.pnirment in - h:,, pruce.,s in rah6a ulvokrLtr m:,rrophal;e, • t:, ected to yrnt,ke challenRe in vu n.r. tchilr uthrrr have fnund ph¢f;ncvtoe,is o}y al%,o~or macropnages Ihnn hum:ms tu he unaffected by rzpnsure to c:E' aette smok, in ea„ rHarris .. al .'70: Keynurd.< et al., '75:. With respect to the metabalism i,f these cells, Hnrris and collcagues (70: havc vhown th:rt smuke inhalatrnn caused an increase in gtu. cose oxidation by resting :nnn-phagncytizingl huwnan alveolur macrophages.-Additional smdies by Grern et aL i'71/. wrth cells exposed tu smoke in ztua rncf(cated I hat cotnpnnents of tho filtered ga., phase of tnoacco moke. such as acrolein, inhibited "CO, prroduction from "('kluco"e whrle whole filtt-red gits phase strmulatud thc production ,f r1CUi from ghlcasr6-"C and glucose-I-'^C. These meto- r., u.dmi,...,im,-rs-rrm,4 un1 ,.iu~..n,,,,o us,1 l'linirnl inrrA'.Y'mm-
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106 I)N,\l'IL IlAltl'I:It, GV ' ItIIUAV, KAHSUC. NP ASII flt'nElt holic studlee relet r lon,m-phagurvtruig ~ir,t- ing) cells. During phnttocytnst~. ~uhsbintial changes in meteibulism are known touccur and these are -ometimc~ subject tn the htstory of . the cells fKarnovskv et al., ' 7i) To our knnwledge, there have not as yet been studie; on the effect of' cigurette ~moke un the metabolism of alveolar tnacrophuges during phagucytosi~. \lcasurcmwnts of the cffects of exposurc to tubacco +muke on ntetabolte aetivities of resUnp and phagocytizing cells are a major part of this report. V[ATEIt1AtS AND ,UE1'llO[)S Anunnts mtd .smoking conditiaes Male rats IIN0-250 gl strain f.'D were used in all expertntents li'harles Itiver Breeding Laboratories, Inc., lStlmingtnn, Mascnchu- settsL Rats were subjected to I30 cnnsccutivc days of s(nnkr inhalntwn on Multipurtal smoking machines (Schultz and 6Vagner,'75). The animals were placed in conical containers with an openim; for the sm~ut secured there and then put into carouscls or racks which held 50 animal-laden containers. '['he car- ousels were connected to the smoking ma- chine, which delivered to the animals fresh whole smoke from 10 2HI reference cigarettes (University of Kentucky, Lexington. Ken- tucky) appropriately diluted with 10 volumes of air, three times a day. This amount, as determined by means of a particulate matter tracer material, roughly corresponds to between 1-1.,5 packs of unfiltered cigarettes/day in man ~Huber et al.,'771. Rats not subjected to smoke-inhalaticn via the smoking machine, as well as sham-treated animals, served as controls for most experiments. fsolatmn of cells Rats were injected intraperitoneally with sodium pentobarbitol (30 mg/rat), on the day following the last smoke exposure. The abdom- inal aorta was severed and the trachea was cannulated followed by pulmonary lavage (Ylvrvik et aL, '61) of the intact lung (7 ml saline, 6 x i. This yielded a suspension of cells which was routinely better than 90% alveolar macrophages, as determined by differential counts (lVrights stain) performed on an nll- quotof lavage fluid. and examined under light microscapy. Cells other than pulmonary mac rophages present in this suspension, were small lymphocytes fZS."J and polymorphonu- clear leukocytes (2.59"J. Exfoliated epithelial cells were not present. Cells- were centrifuged I100 x pi, wushn•d tcvicv with Hanks h,danced salt, solut.,on I f 113SS/ a nd bruupht tu the neces- ;ary volume for the purUcular experimcnt to be pcrfr,rtned. Aliquots were taken from this suspenSiun fnr cell cnubts and protein deter- mination, ILuwry et nl.,'.i11. The suspensions wcre used either ae such. ot' were studied as munelaycrr, 'scc belnw, on tissue culture dishcs , Fulcon Plastics 1)xfnrd, Cali6.rnia. 3.5 cm diameteri ,ts described by Jiitchell et al. "691. f.ptake of Iabeled partzclce The moihod of Mitchell et at 1'69I for fol. lowing ingestion of "'C-labeled particles by cells in monola' vers was used for uptake of both "Cstarch and "(Sfnphylneorrmcaurr¢.a tFU:1 stramn 209 P1. Tho tinsuc cultum disltes were cut (uwn to fit into a Nuclear Chicago gas f7ot, ' cuuuter. 11uth9 447 lNuclcur-Chicaf;o Corp-. Dus Plaines, Illinnisl. Cells were al- lowed to adhere tu the_dish surface for 45 minutes after which non-adherent cells were remnved bv swirling through solutions of Iif35S nr aaline. The mnnolayers were preincu- bated for tun minutes in fresh HBSS contain. ing 0.1'G glucose. Particles istarch or bacteria) were next added and kept in contact with the • monnlayers for periods of 5 to 120 minutes. All timepoints were run in triplicate. Zero time controls were used to assess background which waa then subtracted from each subse- quent timepoint. Monolayers were washed through nine beakers nf saline, dried and counted (n the gas flow counter. A multiplic- ity of approximately ten_bacteria was used per macrophage. Starch particles were added in considerable excess of this figure (Michell et al., '69). Oxygen measurements Oxygen consumption was determined polar graphically with the oxygen electrode (Yellow Springs Instrument Co., Yellow Springs, Ohio) on cell suspensions containing between 24 x 10" alveolar macrophages both at rest and dur- ing phagocytosis. The particles used as the phagocytic stimulus in these experiments were polystyrene or zymosan, opsonized and unnpsunized. Superoxide release was measured on mono. layers as previously described I Drath and Kar- novsky, '75f. Superoxide-dependent cyto- chrome C reduction was determined spectrophotometrically at 550 nm in the presence or absence of superoxide dismutase (Truett Lab., Dallas, Texas). ~
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iuuArt't, ;•~Inlfl•. t,VU " cr.ul.•Vt uA, la 'IIArI:n Nydro9en pero.rtdc ralc•as'e Peroxide releace was determined bv the method of Root et a1 C75i, where the oxida- tion of scopoletin tI\hi Pharmuccuticnl~, Plainsview, New Yorkl by excess horseradiah peroxidase causes an extinction of fluorescence of scopoletin, The stnichiumetry Is di- rectLy proportional to the peroxide cnnce•nlra- tion in the medium. Suspensions n( cclls contained 1.1 x 10^ alveolar macrophagea. Oxidation oj spert/rrnlf,v-lnbeled glucosc The method of blichcll et al. C1i9), was modified so that 4 kmoles (0.5 ucl of t;luco.vr 1-"C or 4 µmoles ('2.5 µcl of glucose 6-"C was used. Incubation with labeled glucose was for 30 minutes after which time the reaction was ended by addition of 0.1 ntl of I N I{,50,. Alkali-soaked filter paper conteining the evolved CO, was transferred to 15 ml of Buhler's solution (13uhlcr, -fi2) in a counting vial. The radioactivity was assayed with an Ansitron II automatic liquid-scintillation counter (Picker Nuclear, New Ilaven, Connecticut). Lactate production Lactate was determined on the supernatants of monol;ryers which were incuhated with and without particles in the presence or absence of inhibitors for varying periods of time. The enzymatic technique of Holzer and Sohling ('62) was used, which employs the ace- tyl-pyridine analogue of NAU' ipL Hiochem- icals, Milwaukee, Wisconsuv. Inhibitor studies Sodium fluoride and ioduacetamide were used at final concentrations of 2 x 10-' ai and 1 x 10-' x respecticely. The cell monolnyers were incubated with the inhibitor for ten minutes before addition of particle. Results, where appropriate, are expressed as means -!- one s.e.m. Statistical significance between data was determined by two tailed t-tests. RBSUL'rS General characleri.stic,s n(atuenlar macrophages from smoker rats Lavage fluid and the isolated cell pellets from smoker animals were readily distin- guished by their brownish color frum the lavage fluid and greyish pellets of controls. Macrophages from smoker animals appeared by light microscopy to contain more granules lu' Ihanc'nntrol crll.l althuu};h thi. sen.n,rt quun. tiGcd. Cunlrury to previnus obsarvatlona 'm httmun als't•ul:,r macruphuyn•s tllarris ut al., .70. Reynnlds rl tl..'75r, we obtninrd aur re11.. m no grcatrr numher frnm na,kor anin;nls than from c,mtral.:and in fuct control amm.ils rnutinely sleld-d nl,kht.l}' more crlls (fi-4 - 1.2 >< 10^ cclls/rntU than smnker anlmalz 15.2 t L.Y .r IIl" rrll<'rat'. Smoker cclh, hou'cs'er, enniu,ncd approximately 17'1, mnre protein par 1(I•' c,•IL 12J0 mR t 341 than did control cellv ''L04 mg - 621. •1'he differvnce, in cell cuuntn and prntcm content were, huwecer, not .uttistically .,if;ndicant, P/taE:ocryto.vt.y _ In order tn determine %c'.rthcr expna.ro to >mnkc fnr 3U days uffoctod lito ubility uf alvem lar mncrophul;te lo phat;noytir,,•, we expnaed mmmluycrn of thc.c ccllv, as wull us control cells, to either "CInbclcd stureh ur Stu- plrvlorucrus aurru.c. Rosults of these experimenls can be ~ccn in fi{;ure, I nnd 2. Several nbnervations ure immedit+tcdy appurent from fi;;ure 1, which ,Ilustrates uptake of .tarch purticles 6vbulh rxpormn•ntal und control cells. 'I'he Inittal rate of' particle uptake was not significantly altered by smoke exposure, but the capucit~was approximateLy doubled at all time periods from 5 tn 60 rnittutes. Fur- thertnnre, it was .;hown that pha({ocytasis of this inert particle was essentially complete by 15 minutes. The information presented in figure 1 may bc compared with that of figure 2, where uptake of a viable particle. Sfaphyloc•orcus aurcus, is shnwn. It can be seen that the kinetics of particle ingestion are dil'ferent from those reported for starch. •I'wo further ohse,rvatiuns can be made: First tobacco smoke was without effect on uptake of the labeled bacterium and, second, phagocytosis was not complete by 60 rninutes. In another ex. pe•riment Inot reported here) it was shown that particle uptake tS. arrreu,s/ leveled off after the first huur and fell somewhat by the third hour. Orvgen con.sumption was measured at rest and during phagocytosis by macrophages from both control and smokeexpo.,ed animals using three particles. Results of this series of experiments can be seen in figure 3, panel A. Several uhscrvations can be made. First control alveo- lar macrophaKes had approximutely a 40-100% increase in oxygen consumption during phago- cytosis, depending on the particle used. The response for polystyrene, while in agreement 10
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108 4GOr - - ~... 0 5 15 30 45 MINUTES 60 Pn;. I Timernar..•.~fph,~.u<v~ny.bg~Jvr~~Inrm..:rnphnCesGinnc.,. ~tr.dirrats Phng.~retrmi+vv,s qoantdled on mun.dayvr. cnntaumnp olrvular murrnpha¢rs nrcunimc tn i ho mettmJvf phtvhell.i uI ''fi4~ whcre "Cauctyiatrd starch vvan u,rd as thr~ peruc!e. Eevh (ime pomt iv tho meAn I•f tnpiLc~ti~ Azeermmations. Duta shown are from a repre,ntat,e expenment frnm a=rries of three u'hmh w.a'e perfnrmed. 80- MINUTES F1F2 71me eou, if phegne)tn.as hy olvroler ninernphagus from couLrol nnd mm.. krr ratz Condtinns uro smnlar to those <icscnhed fnr fipure I with the xole exception uf the purucle. In this experiment viable Staphvlococcus oureus was urnd.
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'rtlltAl('U .q\L16P. AVII 'I.A6uI.:VI Q B .OXYGEN ' I SUPE RO,tiOE ~ CONSUMPTIGN REL EASE 1500 >t:v lu„. , nr,, Ull C HYDRG~EV PE, rCX/OE '. RELEASE , ; ? Bcst ~ F:^a, crrasis Po!rsqrere 7S- f ~n~~~e-„Z0^.. ZrTnSnn , Opsonaed Zymosan . ,T_ Smoker Ftg. 3 Theeffnvuoflobecrosmnp,..pruolLns.nndn~z,.tu,n„nI ,ndut,".meln6.,L.m.,l'r.rtollurmnr rophagaa. A I I data presenlud nre us meuna LL s r of e mnima:n nf three vxDer,n',, nt+, e:mh a~mv, um; of mulnplc delermmations. All v;dues are evpres.+ed as nananud,r mg protem m 311 mmutrs Panel A 9xygen n,m sumptton by alxeolar macrophage+ frnm vnntnd anrl . mnker ndw in re.pm-e Li ~eilrrtcrono pnrI mlr<und ~,prum ued and ¢nopon¢ed zym'~san. Ponel B Svperax:de nd•-,-r ;n rx~p~'~nrc to +o~nkmf_ and partwlc npnn,r.nuon. Panel Ct Hydrogen peroxide release in response to xmnkmg and pncCle opr-on¢nunn • wlth values obtained for guinea pig af.'eular macrophages, is somewhat less marked than has been reported for other phagocytuc crli., from several species IKarnocsky,'7}1, Secund, alveolar macrophages from both exper ime,ital and control groups were considerably more re- sponsive in this regard to zymosan th"n bi polystyrene (p < 0.05). Third, opsonizatiun nf . the particle was not crucial to manll'eutation of a respiratory burst in either Rroup. Fuwth, smoke inhalation did not significantlc ul'fuc[ oxygen consumption by resting macrophuR<•a whereas the rate during phagocytnsls was etp. proximately double that for control cells, Superoxide rrlrn.se The effects of phagocytosis, npsunizatiun and smoke inhalation on superoxide rcle.,.c by alveolar macrophages is shown in fi;;ure 3I panel B. As can be seen, there is approxintutr ly a two and one half-fold increase in supernx- ide release with phagocytosis Ip < fl,0al,'fhls release is unaffected bv either smoke inhala. tion or particle opsonization. The increase due to pha{{oaytusl; oF both epson¢ect and unopp sunized zymoaan hy experimentnl and control groups t., not slgmfictlntly altered. It ~cus dctarmined front the infarmatiun In 'Igure 3 panels A and H. that apprnximuloh' 5~'5 nf the respiratarc hura that ucc'nmpanic; phagucy- lusis is accututted for by supernxide release in cnntrnl macrnphaRrex. The value for macrophaties I'rom smoker unimuln was 3`,. Ilydrohe¢ prraridr Hclea,e ut' hqdroRen perr,,eidr under conditiuns simllar to those descrihcd in patnel B is shown in pancl C As can be seen, there is a dramatic increase in peroxide relcnxc with p6,ll;acylnsis hy contrul marruphal;cs Ip < O.U(1.51. Qp,strnizeLiun waa without affect on peroxide rnlease, since the apparent incre- ment of apprnximately ".51Y, upon opsonizatinn uf the zymnsan was shown to bc not statisti- cstlly sienific:mt. Simiiar obsurvations a'ere mude on macrophages from sntoker animals, where a 25°.: increment in peroxide release oc. curred with particle opsonization (notstatisti- 0
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• 110 J HATII, ILNIPIQtt c A ~!-C GLUCO OX/OAFION SE a 4 [-~.7ResI a E WA Phagocylos !s \ ~ 40 E ~ 30 Tf ~ T ~ 0 20 ~ ta Control Smoker ~ iI_ P% 1 .~I nl- L:i Smoker Control Smoker f1K. 4 Oxldntlon nf speufic:dl. iahrled Xluro1e. und lnrmte Prnduruun by •dcnnlal' mncrnphuAra'. The efl'net of tobaccn smnke and pnnu~layb,an. Itemits exprc .ed are means ~ e for mm,mom orlhrne uxpenments eaeh contmnurq multlple determnmUt lnc Thc prnrln^ u=ed tu Imttaf c• phapncvroe/s uore ol•n~lnlmd zpmosun Lor 4•lu cnse measuremenla and.vtzrrh fur the Ipctate determmatluns A I I data expreand as nanmmO le=mX-` pruteln in 30 minutes. :1\Ol.h I Ef7"et olklyan(,vrtu mBthlt"ns,.n Pnrrmfe upmko hy alrenfnr „Irlrr nr,nuKrsRm, ront-l and.arnuker rats Ad•LU.am lbne Jnd/um Ouonde •Z , IO - >I l ' Indnucutumldr'I < lU'tU ' I'hahm'HUnx . -._ r,Fnd Irr.drr yfA'1't] rrnantme hrut.kdled,cJpurrns lux.n upmg cell prntr,n , been eyVlu•II ~nAmtwn 24 1 tiN Itl 4a tc - pA'n N1.9'nult. rtll Idan'. Yllt .aln+ ., exPee.rru r. mmueo m.nim6n, . l~mrorLrn L.r mru bllre munu hnee ' Luatum pmdv<lmn Inea.urad m drr.nLed undor, p1AT}'IfIALS AFU ]IliTllbl>,5 nnd u e.prn.red ax n.annmolesnl' Ivctnre pr,duerd per .lo n. mtrv 'Inh,bitor9wcrru•id+dlnlh.lunn,imvrslrnmmute+p.wrwpertlrlr.uldnrmt- -, a •.hnwnzrxl'maIlnlMrexu mn lreln tvlnreCunmdrnihlr IuLIW n•dlnthun mba•rofplvl ll pNIXnr tllrln,ryur r'~rnttnexperlmenttLm,gh br llon6hlphelwet'nrnntrnl9ndsm1~ha`r , nedPlnvlnnlavdld\~nn•x eFlnrlllttatl`prndemmF~t?thlxrrngnnlhldutadlnatYlnlhl, tnhle Is rrmm nnr, repre.enlallve t'xpe rlment eVnrlRLlnX pf trlPlll`Ille dCletr111nAtlan+. I he nlf.ln nf u hlfh ly [hUwn, A .erlea Vf thre! were prrforlnrdd cally significantl. Of primary interest, how. ever, was the finding that macrophages from smoker animals released twice as much perox, ide as control cells during phagocytosis tp < 0.02). Values for cells at rest were identical in both groups and it appears as though rat alveolar macrophay;es rclease almost no pln- oxide in the unstimulated state. Glucose oxidafiou Figure 4 presents information on the oxida. tion of specifically labelled glucose and pro tllld.tlWAp. IiAft.~,tt'elU' A.4U IR'IrtEit B C ~ 6-C GLUCOSE 300~AC;ATE CX/0A770N I PPC'DU'CT/ON- 2001- - duction of Iactate. Oxidation of glucose carv bon-1, an indication of hexose monophosphate shunt activity, is shown in panel A. The in- crease in glucose oxidation with phagocytosis bv control cells is not dranmtie, at least when une compares the values shown with those ob- lained for uther macrophages /Karnovsky, '74). iVtacrophagcs obtained frum smoker ani. ntals, on the other hand, show an apparent though only marginally significant, decrease in "COi formation from glucose-l.l `C both at rest and during phagocytosis Ip < 0.05). Oxi- •
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Tutj*.vCCO SMUKF. Af` dation of glucose carbnn~6 was not significn itly affected by either phahocytosis or smoke inhalation although there appeared to be it ten- dency toward an increase in smoker animat<. Lactate praducteun is shown in figure 4, panel C. As can be soen there is no increase in lactate production with phagocytosis by muc. rophages from either control or smoker ani~ mals. The particle used in the experiments shown in this figure was starch, though vir- tually identical resultc were obtained using opsonized zymosan. Alveolar macrophages from smoker animals were shown, however, to produce significantly greater quantities of lactate than control cells (p < 0.01). We ini- tially thought this might indicate an increased reliance on glycolytic energy by these cells. That this was most probably not the case can be seen in table I. Sodium fluoride and iodoacetamide, both inhibitors of glycolysis, were shown not to inhibit phagocytosis of heat•killedS. aureus while effectively block- ing lactate production. Similar results have also been obtained using "Glabeled starch particles (not shown here). This particle was used to establish more securely the observa- tion that the monolayer assay used, was indeed measuring phagocytosis rather than adherence to the surface of the macrophage. It is not probahle that these cells would have membrane receptors that bind starch pmrti- cles avidly enough to resist the vigorous and repeated washings to which the plates were subjected. The increase seen in phagocytic ability in both experimental and control cells treated with sodium fluoride is presently um der investigation. DISCUSSION We have shown that alveolar macrophages obtained by lavage from the intact lung of rats subjected to 30 consecutive days of smoke in. halation differed from those obtained from control animals, in terms of physical appear- ance and biochemical characteristics. Phagocytosis of a viable bacterium, S. aureus, was unaffected by tobacco smoke, in agreement with observations previously made by Reynolds et al. t751 and Harris et al. ('70) for Pseudomonas aerugin osa and Staphylococ- cus albus, respectively. It is difficult to com- pare our work with that of Green and Carolin ('67) where a decrease in phagocytic el'ficiency was shown towards Slaphyiococcus a(bus. These investigators used a system in which they exposed cells to smoke in vitro, rather than the animals per se. I.cr:ul.,ut ytariu„^,htul~a I 11 On the other hand, we hucr ~hutcn al~r,dur macrophngod from +muker an imnls to hc ntarc ufficicnt than cuntruls in the up[uk~• of rnnviable purLicler. Perhaps tluu is u rvllocton if ,an adaptation to the concentration of ptrti- c'les with which the cellsarc cunfrontvd-u far greator p:u'tiile'cnll multiplicitv is used with the nun-viable ptrticlus than with hnctcrla. With respect to mclabolism our findmqa of an increase above cnntrol values in oxygen coasumplion and hydrogen peroxide rulcuse by alveolar macrophages from smokerexposed animals clearlp separated the:r crlls from those obtained from controls. Stimuluted oxv- gen uptake and peroxide production have re- puatedly been shown to accnmpuuy the -ngalf ment process in virtually all phaqocytic , ells IKurnovsky,'74; Knrnovsky et al.,'761. It is conceivahle, therefore that a ccll already exposed to an environment laden with tubucco particles, would be further stimulated to cnn. sume more oxygen and release more peroxide during phogocytosis in vitro. It is .+omewhaL surprising, however, that superoxide release did not follow a similar pattern. The fact that less than 5"L of the respiratore hurst was accounted for in terms of superoxide release in either group indicated that only small amounts of superoxide were actually pro- duced. or that this product was being con- stmied by various reacTinns and pathways within the cell. Alternatlcely, conversion of superoxidc to hydrngen peroxide intracellularly may be so rapid that the increased release of the latter substance was the major end result. Particle npsonization was not found to he a critical factor or phagocytnsis of starch by ah'eolat' macrophages. The increases in oxy- gen consumption, superoxide and hydrogen peroxide release when zymosan served as the phagocytic stimulus was also not affected by opsonization. This is in contradiction to what has been shown for other phagocytic cells iDrath and Karnovsky, '75; Curnutte and Babior, '74; Goldstein et al., '751. Root ('75) has previously shown that without openiza- tion there was no release of peroxide by human granulocytes. We have had similar findings with all phagocytic cells we exam. ined from rat, mouse and guinea pig, with the exception of rat alveolar maernphages, (Kar- novsky et-al.,'76). Pnrhaps this indicates that only a small portion ot'the peroxide release in this cell type is triggered by a complement-re- lated membrane reaction. The oxidation of specifically labeled glucose is
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0 'remarCv JD1uKI . v\ i ,vAP.~:L;.it )1ni Ib, l uACb:S Reynolda. H Y.. J A. Nnznurrnwrk~ nnd Fl H N.,•:11 1975 Specdlur, 'o(nP'~~nm:mbbuA:estaenn::nvc cytosLS uf Psrudnninnn, arrvKUmsu hy hmn:m uh',."I:,r mncmphnpes..I, t'hn, Invea.5ti 11;6.8sG HnnpR K,.J.11c«v1L'.,A Il.h:nnunrltl C'hunrc 1974 11.1). n•Ieuee (rmn humnn grnnc.locytrx durmp ph,~gnrreI f1/1cllmCnt.lllVn. ~IuAnll[aLlun nnd 3JmL rv,U1:Ilm1: 1tnrs-J. Cl:n tnvc..L,.5i: 945 955 Schultz. E. J., and J. R, lVagnur 1975 A thirty purt . n:u:c:n g IIJ ll.:ch:m. (or rnnunw~or-:nrnk.. u..orr.:::.n p,. m.nrduck ~.::. Ut ~urnErmr1'uhnkdsahunu U.m~ln 5f.5:: T ll. Pullnrd .:nd NI Ca~::hun J L~~Inln:n end pr„poruva.~l' Vh.~A~,ra:r _~ . II ~11vr:.laru:an,qd,.a:r..l l'im pn,~.L.s! ~.::.;:11a St.l'. I' i'h"Pma. C I.. Cn..,:ilis Ii li Il:"i~in:d,inA.l II L IL~e I'J71 t;lw.~lhumc Jcprnden; prrnWat:rr nm- t.:h~ ..m m thc elvuubtr mucrnphapu. J. Chn In': sL..50' b:i-01:1 0

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