stereology of lavaged populations of alveolar macrophages: effects of in vivo exposure to tobacco...

EXPERIMENTAL AND MOLECULAR PATHOLOGY 29, 170-182 ( 1978)

Stereology of Lavaged Populations of Alveolar Mocrophages: Effects of in Vivo Exposure to Tobacco Smoke

PAUL DAVIES, G. CLINTON SORKREKGEK, En~-ilnr, E. EKGEL, AND GARY L. HUBEH.

Depurtrnmt of Jledirine, Beth Isruel Hospital, Thorndike Memorial l,aboratory, Hurvarrl Metlicul School, Boston, llussachusetts

I<rceivrd danuarg 18, 1975, and in revised jorm Nnrch~ 15, 197’8

A multi-level morphomrtric and stercologic st,udy was performed on alveolar macro,- phages obtained by bronrhopullnonary lavage of unexposed control rats and rats cxperi- mentally rsposcld to whole tobacco cigxclttc smoke for 00 consecutive days. Measure- ments of macrophage profile diameters made at the light microscope were used to derive cell size distribution curves, with a mc‘an rell vol~7mr: in the control group of 623 pnl”. At the electron microscope level, the volume density and numerical density of mitochondria in unexposed control cells were lower than the valrles reported for peritoneal macrophages, but the mean volume of individual mitochondria was five times higher in the alveolar than in the peritoneal macrophage. The volume density of lyaosomes was slightly higher in alveolar than in peritoneal macrophages. Esposurc to smoke re- sulted in changes in several subcellular compartments, including a Z-fold inrrease in mean cell volume, a l%fold increase in the volume density of lipid inclusions, an increase in the surface density of mitochondrial inner membrane and a decrease in the surface density of rough endoplasmic retic~drun. The significance of these morphologic changes in terms of ccl1 function is disrnsscd.

As an active phagocyte, the alveolar macrophagc is vital in maintaining the sterility and functional capability of the distal lung. Should its activities be com- promised in any way t’hrn disease may ensue (Huber et nl., 1977a). The monitor- ing of changes in the subcellular st,ructure of t#he alveolar macrophage is one means by which early functional changes may be detected. We have previously described by quantitative stereology the morphology of alveolar macrophages harvested from the lungs of male rats and have applied the same techniques to macrophages obtained from rats experimentally exposed to tobacco cigar&e smoke for up to 60 days (Davies et al., 1977b). The quant*itat>ive dat#a largely confirmed earlier, more qualitative, studies on the structure of alveolar macrophages recovered, by bronchopulmonary lavage, from the lungs of humans and animals after smoke exposure (Harris et al., 1970; Mann et al., 1971; Martin, 1973; Pratt et al., 1971). Our study clearly indicated that nft#cr prolonged smoke inhalation, major struc- t,ural changes, such as an increase in mean cell volume and in intracellular lipid accumulat,ion, occurred in the alveolar macrophage population. The study used only a low level of magnification and was consequent,ly unable t#o evaluate those

00144800/78/0292-0170$02.00/0 Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved

ALVEOLAR MACROPHAGE STEREOLOGY 171

functionally important structures which can only be resolved at higher levels of magnification. Despite numerous biochemical investigations, no data exist in the literature on the stereology of major subcellular organelles in the alveolar macrophage such as mitochondrial membranes, rough and smoot8h endoplasmic reticulum and the Golgi complex. The quantitative effects of tobacco smoke inhalation on these parameters have, similarly, never been described. The present paper describes a comprehensive morphometric and stereologic study performed at several progressively higher magnification levels on alveolar macrophages recovered from control rats and from ram after exposure to tobacco smoke for a total of 90 consecutive days.

MATERIALS AXD METHODS

Thirty male CD st.rain pathogen-free rats (Charles River Breeding Labora- t,ories, Wilmington, Mass.) with an initial body weight of 125 g were exposed three t’imes a day in an automatic smoking machine to whole smoke from ten 2Rl Kentucky reference tobacco cigarettes in the regimen previously described (Davies et al., 197713) for a tot’al of 90 consecutive days. In our exposure system, decachlorobiphenyl (DCBP) has been used as a t’racer to det’ermine the total particulate phase delivery to the animal and it,s deposition within the lung. Daily exposures have been correspondingly adjusted to an equivalent of approximately 14 packs per day of unfiltered tobacco cigarette consumption in man (Huber et al., 197710). The same number of age-matched, non-exposed animals were kept as controls. The animals were housed in wire mesh cages with food (Rat-Mouse- Hamster Formula, Agway, Inc., Syracuse, N.Y.) and water provided ad Zibitum. At the end of 90 days, the animals were weighed, anesthet#ized with intraperitoneal sodium pentobarbital, and exsanguinat’ed by aortic transection. The trachea was cannulated and the lungs washed seven t,imes wit.h 6 ml aliquots of heparinized, isotonic saline at room temperature (Myrvik et al., 1961). The lung washings were centrifuged at 600g for 10 min and the individual lung cell pellets fixed using a filtration t’echnique which collects the cells into a flat pellicle in which the size distribution is uniform (Davies et al., 1977c). Fixation and processing details have been described elsewhere (Davies et al., 197713). For light microscopy, 1 pm Epon sections were cut and st’ained in alkaline toluidene blue. For electron microscopy, silver-gold sections were cut with a diamond knife, picked up on 200-mesh copper grids with a collodion-carbon support film and stained in uranyl acetate and lead citrate. The material was viewed in a Philips 300 EM operating at 60 kV.

Selection of MntericrZ

From a t’otal of 30 individual pellicles (one pellicle per animal) in both the exposed and non-exposed control groups, nine pellicles were randomly chosen for analysis. Each pellicle was cut into six blocks, of which two to three were randomly selected to provide sections for light and electron microscopy. In each case, one section per block was randomly selected for analysis.

Light Microscope Morphometry

One field per s&ion was viewed on a Wild (Wild, Heerbrugg, Switzerland) automated sampling stage microscope using the 100X oil immersion objective

and the projecGon head fitted with a latt’ice which defined the test’ area. The maximum diamet’ers of all macrophage profiles lying within the lattice were measured from the projection screen with a pair of calipers and the values con- verted to microns after calibration of the system. The number of profiles measured was 954 and 914 in t,he control group and exposed group, respect#ively. The distribution of cell diameters and det’ermination of mean cell volume were made using the Schwartz-Saltykov diameter analysis (Fndcrwood, 1970) with up to 15 size classes in each group.

Subcellular Stereology

One section per grid was selected at t)hc electron microscope and all macrophagc profiles lying within one grid square were individually photographed onto 35 mm film. A 35 mm contact posit’ive was made for use in a project’ion t’able (Weibel et nl., 1966). Three magnification levels were used in t,he study. At Level I, t,he entire profile of each cell was photographed; final magnification was 12,400X. The profiles were analyzed using a square test lat)ticr incorporating 64 syst’ematic test points. at8 t,his level, 155 and 136 profiles were analyzed in control and smoke-exposed groups, respectively. At I,evel II, the major part of the cell profile excluding the nuclear profile was photographed; the final magnification was 24,000X. A multipurpose coherent, lat’tice (Wcibel, 1973) with l&S test points was used, and 17s and 168 profiles were analyzed in control and exposed groups, respectively. At Level III, each cell profile was divided into three possible areas for phot,ography, t)op, middle left or right (depending on the position of the nuclear profile, if present) and bottom. One of the three areas was photographed in each cell by random selection at the microscope; the final magnification was 63,400X. In order to compensate for the rest)rictcd profile area, the sample size was increased to 324 and 302 profiles in the control and exposed groups, rrspec- Gvely. The same test lat’tice was used for analysis at t,his level as at Level II In this report8, the intcrnat,ional notat,ions for volume densit’y, Vv, and surface density, Sv, have been adopted and were calculated by point and inters&ion counts, respectively (Weibcl, 1973).

Point count’s were performed at Levol I on profiles of nucleus and cyt’oplasm. Intersection counts were made on the nuclear membrane and plasma membrane. These data enabled calculations to be made of

VivNUe = volume density of nucleus in totSal cytoplasm, t’he nucleocytoplasmic ratio ;

S/\‘Nuo = surface to volume rat’io of the nucleus; and

S/VCEI,L = surface to volume ratio of the cell.

At l,evel II, point counts were mndr on profiles of mitlochondria, lysosomcs, lipid inclusions, myelin bodies and remaining cytoplasm. Total cytoplasm was defined as the sum of these compartments. The crit’eria used in defining the structures have been described (Davies et al., 1973b), wit’h t’hc exception of myelin bodies which were characterized as variable sized structures lined by a single iinil. tt~f~tttlnttte a nrl ~nlainitig phospliolipi~l rtiicc~llf~s, f*iI hr 3s :trtrr~rphous

myelin whorls or in the contigiirntion of I u1~11ar my(Llin, 1 hc alveolar surl’:lce lining m:ctcrial, which had pr~uni:~bly bc~~n pliagocylixcd by 1110 i~~acwpl~:gcs

ALVEOLAR XlACROl’HAGE STEREOI,OGY 173

FIG. 1. Myelin bodies in an alveolar macrophage from a control animal. One (tmb) contains .ubular myelin in a similar configlwation to that of the lnng surface lining material. The other [mh) contains more amorpholls myelin figures. X35,625.

IFig. 1). The data collected at Level II enabled calculations to be made of

VVMIT = volume density of mitochondria in total cytoplasm; VvLys = volume density of lysosomes in total cytoplasm ; VvLIp = volume density of lipid inclusions in total cytoplasm ; VVM~ = volume density of myelin bodies in total cytoplasm ; and

VVncYr = volume dcnsit,y of remaining cytoplasm in total cytoplasm.

174 DAVIES ET AL.

In addition, counts were made of the mean number (K) of mitochondrial proliles per macrophage profile. This was expressed as mean number per unit area (NA) by applying the formula derived from Weibel (1973) :

CT

where 1’Toy~ is the mean number of points on total cyt’oplasm and z is the length of one test line on the coherent lattice. The value obtained was then substituted in t,he formula for numerical density (Weibel and Gomez, 1962) :

where Ii is a size constant and was taken as 1.00 (Wcibel, 1973). /3 is a shape constant and was determined by measuring the long and short axes of SO mitochondrial profiles in cells from control and smoke-exposed animals, respectively. In each group the profile axial ratio was 1.5. Assuming cylindrical shape, this gave a true axial ratio of 3 t’o 1 (Mayhew and Williams, 1974) and a ,B value of 2.25 (Weibel, 1973).

At Level III, point count’s were made of total cytoplasm. Int,ersection counts were made of the outer mit’ochondrial membrane, inner mitochondrial membrane, smoot8h endoplasmic reticulum, rough endoplasmic reticulum and Golgi complex membrane. These data enabled calcula,tions to be made of

SVUMERI = surface density of outer mitochondrial mcmbranc in to&l cytoplasm ;

Svihrebr = surface densit’y of inner mitochondrial membrane in total cytoplasm ;

Svsea = surface densit,y of smooth endoplasmic ret’iculum in total cytoplasm; SVR~R = surface density of rough endoplasmic reticulum in total cytoplasm;

and SvooL = surface density of Golgi membrane in tot’al cytoplasm.

The calculation of volume density is given by the ratio of the mean point counts per profile for each compartment t’o that of the reference compart#ment. The surface density is calculated from t’he rat’io bet’ween the mean number of intersections made by a boundary membrane wit’h a test line system and the mean point count per reference compart’ment profile and mult,iplied by a factor which depends on the test lattice used (Weibel, 1973). The standard error of each ratio was derived using a formula for the standard deviation of a ratio estimator and utilized the correlation coefficient between the numerator and denominator (Davies et nl., 197713).

RESULTS

The mortality during the exposure period was OyO in control animals. Four animals from the exposed group died at one time due to machine dysfunction; the remainder, representing 1% of the exposed group, died of unknown causes. ,4t the time of sacrifice, the mean body weights were 4S4 and 257 g in control and exposed groups, respectively, Differential cell count’s, made at electron microscope level on all the material analyzed, indicated that 93.5cy0 of the lung cell

ALVEOLAR MACROPHAGE STEREOLOGY 17.5

0 4 8 12 16 20 24 28

FIG. 2. Distribution of the diameters of alveolar macrophages from control animals and from animals exposed to smoke.

sample harvested from control animals consisted of macrophages and 2.2% of polymorphonuclear leukocyt.es, the remainder being made up of lymphocytes and exfoliated epithelial cells. In the exposed group, the corresponding figures were 92.0 and 4.6%.

The profile diameter distribution studies made at light microscope level and the subsequent Schwart’z-Saltykov analysis enabled size distribution curves to be constructed (Fig. 2). The mean cell diamet’er was 9.57 f 3.29 pm in the control group and 12.18 f 2.62 pm in the exposed group.

The qualitative morphologic characteristics of the cells under t#he light and electron microscope were very similar to those exhibited in cells from controls and from animals after 30 and 60 days of smoke exposure. The most distinctive change in the cell profiles from smoke-exposed animals was the cytoplasmic accumulation of lipid, both within secondary lysosomes and free in the cytoplasm. The lipid was oil red 0 positive in light microscope preparations, and in electron microscope material it exhibited a low electron density characteristic of neutral lipid and was often surrounded by an intensely osmiophilic rim (Fig. 3). The results of the stereologic evaluations are presented in Tables I-IV, with the percentage composition of the whole cell and the percent changes in stereologic values displayed in Figs. 4 and 5, respectively. Compartmental volume densities are given in Table I. VvNUC was decreased in t’he exposed group from a value of 26 to 12%. VVRCYT exhibited a very significant decrease falling from 81 to 64%. VVMIT underwent no change with smoke exposure remaining at 5%. Similarly, the numerical density of mitochondria, NvMIT, showed no statistically significant change. VVLYS displayed a small, but nonsignificant decrease in the exposed group. In VVMB, however, there was a very significant decrease, from 3y0 in the control group to 1.4y0 in the exposed group. The major detectable morphologic change was clearly in VvLIp which underwent a 14fold increase with smoke exposure.

The surface to volume ratio of the cell decreased relative to controls by a fact’or of 17%, but the surface to volume ratio of the nucleus underwent no significant change (Table II). The surface density of mitochondrial outer membrane underwent a relative increase with smoke exposure by a factor of 22%. Similarly, the surface density of mitochondrial inner membrane, SvIMEM, was increased by a

176 DAVIES ET AL.

FIG. 3. Portion of the cytoplasm of a macrophage from a smoke-espcwd animal. A large lipid inclusion (li) is surrounded by an osmiophilic rim (arrows). A lysc~somc (ly) contains a lipid inclusion and a narrow lipid cleft. X34,200.

fact,or of 27% in the exposed group. SVRER exhibited a significant decrease, but neither SvsER nor SvGoL displa,yed a significant change at’ the 5%) level (Table II).

The mean cell volumes, derived from the Schwartz-Saltykov analysis of t’he profile size distributions, were 62.3 and 116s pm3 in control and exposed groups respectively. These values were used to derive ahsolut8e volumes for cellular


60

%

40 -Lipid Inclusions

20

0

FIG. 4. The proportions of the whole cell occupied by subcellular compartments in alveolar macrophages from control animals and from animals exposed to smoke.

compartments and surface areas for boundary membranes and these are presented in Tables III and IV, respectively. The 88% relative increase in cell volume was contributed entirely by the total cytoplasm and, in particular, by an accumu- Iat.ion of almost 200 pm3 of lipid inclusions. The abso1ut.e volumes of nucleus and myelin bodies showed no change after smoke exposure.

DISCUSSION

Stereology is particularly valuable in establishing the relative importance of subcellular organelles in the function of a normal cell population and of a population adapt,ing to alterations in its tissue environment. In the latter case, stereology is capable of detecting morphologic changes which might be undeterminable by other means. An extension of our previous measurements was necessary, firstly, to establish a cornplebe stereology for the alveolar macrophage population in normal adult male rats and, secondly, to determine t’he changes in functionally and metabolically important subcellular compartments after exposure of the animals to tobacco smoke.

The value for VV~UC in alveolar macrophages from control animals was similar to that reported by Mayhew and Williams (1974) in rat peritoneal macrophages

FIG. 5. The percent changes relative to control values in volume and surface density measurements of alveolar macrophages from smoke-exposed animals.

178 DAVIES ET AL.

TABLE I

Volumetric Composition of Subcellular Compartments Relative to Total Cytoplasm

(mean i SEM)

Nucleus, Vvxnc

Mitochondria, VVMIT (z) Lysosomes, Vvi,vs (%) Myelin bodies, Vvbrre (yc) Lipid inclusions, VVLW (%) Remaining cytoplasm, VVRCYT (70)

Numerical density of mitochondria, NVMIT (No./km3)

Control Smoke exposed

25.69 rk 2.07 12.11 zt 1.42=

5.0s I!z 0.24 5.08 f 0.22 (NS) 10.41 f 0.53 9.38 f 0.57 (NS) 2.96 f 0.45 1.39 f 0.23= 1.03 f 0.22 19.52 f 1.36U

80.53 f 0.73 64.41 f. 1.17=

0.18 & 0.001 0.16 zt 0.010 (NS)

a P < 0.01.

where a value of 25.31% relative to the total cell was obtained. If we recalculate our data to derive the volume density of nucleus per unit volume of alveolar macrophage we obtain a value of 20.43%. The value of VvhliT was lower t.han that obtained by Mayhew and Williams (1974) in peritoneal macrophages (8.550j0). This comes as a surprise when we consider that the alveolar macrophage is more dependent than the peritoneal macrophage on aerobic respiration (Simon et al., 1977). The value given by Blouin and coworkers (1977) for VV&~~T in the Kupffer cells of the rat liver (4.5270 of total cell) is closer to our finding, particularly if we adjust our value to unit volume of total cell (4.047,). Mayhew and Williams (1974) gave a value for Nv~HT in rat peritoneal macrophages of 1.43 per pm3 of total cytoplasm. This compares wit,h our value of 0.1s for alveolar macrophages. These figures indicate that the mean volume of a mit’ochondrion in the peritoneal macrophage is 0.0598 pm3 and in the alveolar macrophage 0.2822 pm3, or nearly five times as large. Major differences in mitochondrial activity, however, are likely to be reflect,ed in the surface areas of the inner and outer mitochondrial membranes (Lehninger, 1970) for which no data have been reported for the peritoneal macrophage.

TABLE II

Relative Surface Areas

(mean f SEM)


S/VCELL (Mm"/Pm") 0.95 f 0.038 0.79 f 0.031a S/VNUCLEUS (rmz/rm3) 1.17 f 0.050 1.28 f 0.102 (NS)

Surface densities (pm”/,urn” of total cytoplasm)

Mitochondrial membrane

Outer Svonen 0.28 f 0.001 0.34 f 0.001” Inner Sv1ME.II 0.64 f 0.003 0.81 f 0.003a

Rough ER, Svnnn 0.43 f 0.023 0.33 It 0.0216 Smooth ER, Svs~a 1.55 f 0.060 1.41 f 0.067 (NS) Golgi, Svoo~ 0.36 f 0.048 0.39 f 0.060 (NS)

a P < 0.01.

ALVEOLAR MACROPHAGE STEREOLOCY 179

TABLE 111

Derived Compartmental Volumes in (pm)”


Cell

Nucleus Mitochondria Lysosomes Myelin bodies Lipid inclusions Remaining cytoplasm

No. of mitochondria per cell

623 1168

127 126 25 53 52 9s 15 15

5 204 399 672

89 169

Mayhew and Williams (1974) reported that lysosomal granules constituted 6.317, of the t.otal cytoplasm of normal, unstimulated peritoneal macrophages. Our results for alveolar macrophages show a higher proportion of these organelles (10.41%), with an additional 2.96% contributed by hetero- and telo-lysosomes containing myelin bodies. Both normal alveolar and normal peritoneal macrophages possessed similar proportions of remaining cytoplasm @O-81%).

The differences in mean body weights between control and smoke-exposed animals have been noted in many reports of experimental smoke inhalation and may be due, in part, to a decreased food consumption in the exposed group. Other factors, such as the potentially lipolytic actions of nicotine-induced cate- cholamine release in the exposed animals, may also have played a role (Schievel- bein, 1976). Limitations in the number of personnel available and the very considerable cost of preparing an additional group of animals did not allow us to use a “sham” exposed control group. Previously reported results, however, showed that “sham” exposure for 30 days did not induce any demonstrable changes in alveolar macrophage morphology (Davies et al., 1977b).

The decrease in S/V om,L after smoke exposure represents a rounding of the cell which has been shown in our previous study to be due mainly to an increase in cell volume, although there may also be a loss of plasma membrane convolu- tions (Davies et al., 197713). The lowered value for S/Vonnn may have occurred secondary to an increased amount of phagocytosis with its associated plasma

TABLE IV

Derived Surface Areas in (pm)”


Cell membrane 592 924 Nuclear membrane 149 162 Mitochondrial outer membrane 139 355 Mitochondrial inner membrane 317 845 Rough ER 213 344 Smooth ER 768 1471 Golgi 178 407

180 DAVIES ET AL.

niembranr ut,ilization and,‘or lo :t dt*crr!:lsc in plabma IIICIII~JI'LLII~~ IV~J~~WIII~JI~~.

The lack of change in S/V,,W, on the other hand suggrxt,s thaf c~sposurc~ to tobacco smoke produced little change in nuclrocytoplasmic exchange. The change in VvNUp after smoke exposure was largely due to an increase in the volume of the total cyt’oplasm compartment. The remaining cytoplasm, which mainly consisted of the cyboplasmic matrix, however, cont’ributed a smaller proportion to the total cyt,oplasm compartment’ in the smoke-exposed group t’han in t,he control group.

The increase in mean cell volume aft’cr smoke exposure was accompanied by an increase in the absolute number of mitochondria, though the mechanism for this increase is unknown. The surface density of out)er mitochondrial membrane increased after smoke exposure indicating a more ruffled membrane. The 27% increase in the surface density of mitochondrial inner membrane provides a greater surface area for a potenCially increased level of oxidative phosphorylation in cells from smoke-exposed animals. Metabolic st,udics by other workers have found that alveolar macrophages from human smokers have increased levels of glucose oxidation (Harris et al., 1970).

The lysosomal compart,mcnt in control cells was predominantly made up of dense body granules. A small, but’ non-significant’ decrease occurred in this compartment after smoke exposure. However, a considerable proportion of the secondary lysosomes in cells from esposed a)nimals may be filled with lipid and would therefore be scored as lipid inclusions. The values for VVLI~ were similar to those after 60 days of smoke exposure (15..57y0), an observation which may indicate a maximum level for cytoplasmic lipid st,ornge in these cells. The lipid was seen to accumulate in membrane-bound structures which are positive for acid phosphatase activity. Larger lipid inclusion profiles were non-membranc- bound under t,he elect’ron microscope and in hist’ochcmical preparations they have been shown to be autofluorescent with peripheries posit’ive for lipofuscin (Davies et al., 1977a). The lower value for Yvnfu in the exposed group compared with controls implies a decrease in t’he uptake of phospholipid, at least’ in a bulk and morphologically recognizable phase. Since the in situ alveolar mncrophage is bat)hed by the alveolar surface lining material, it has been suggested that it’ is responsible for the removal of this material from the lung surface (Clements, 1970). Thus, any change in the rat)e of this removal could have a profound effect on the functional capabilit#y of t’he distal lung. In an analysis of lipid distribution in the lavage fluid obtained from eight smokers and eight nonsmokers, Pratt and coworkers (1969) found t,hat dipalmitoyl lecithin, a surface- active constituent of the alveolar lining material, predominated in the cellular phase of the sample from smokers and in the non-cellular phase in that from nonsmokers. This is difficult to reconcile with our results unless we assume that t,he phospholipid loses its membranous structure very quickly after upt,ake.

The reduction in SVRER suggest’s a decrease in protein synthesis in the alveolar macrophages from smoke-exposed animals. A small decrease occurred in SvsEn and an increase in SvcoL. Both however, were statistically non-significant at the 5y0 level. The Golgi complex is considered important in the packaging of lysosomal enzymes (Cook, 1973), and, in mouse alveolar macrophages, is con- nected with a GERL system (Essner and Haimes, 1977). The comparatively small changes in the surface densities of Golgi and SER membranes, therefore, may be directly related to the relative stability in the value of Vvl,us. Neverthe-


less, the increased absolute areas of those membranes and increased absolute volumes of lysosomes indicate that the alveolar macrophages from smoke- exposed animals still have considerable potential for synthesis.

In summary, our results have established detailed stereologic parameters for the normal rat alveolar macrophage population recoverable by bronchopulmonary lavage. Since lavage is the easiest and most widely used method for obtaining a fairly pure sample of these cells, we hope that the results will provide useful information for additional morphologic and biochemical studies. In addition, these studies define the morpholbgic alterations in this key host defense cell that are induced by repeated exposure to tobacco smoke. The “leveling off” of stereologic values in cells from animals after 60 and 90 days of smoke exposure may indicate an upper limit for change in the cell population. We cannot exclude, however, the possible “diluting” effects by populations of younger, and morphologically unaltered, cells even though no direct evidence of such sub-populations has been found in our results.

ACKNOWLEDGMENTS

Supported in part by Grant RR01032 from the General Clinical Research Center Program of the Division of Research Resources, National Institutes of Health. Data organization and analysis were performed on the PROPHET system, a national computer resource sponsored by the Chemical/Biological Information Handling Program, National Institutes of Health.

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