Journal of Toxicology and Environmental Health
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Surface morphology and morphometry of rat alveolar macrophages after ozone exposure J. A. M. A. Dormans , P. J. A. Rombout & H. Van Loveren To cite this article: J. A. M. A. Dormans , P. J. A. Rombout & H. Van Loveren (1990) Surface morphology and morphometry of rat alveolar macrophages after ozone exposure, Journal of Toxicology and Environmental Health, 31:1, 53-70, DOI: 10.1080/15287399009531437 To link to this article: http://dx.doi.org/10.1080/15287399009531437
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SURFACE MORPHOLOGY AND MORPHOMETRY OF RAT ALVEOLAR MACROPHAGES AFTER OZONE EXPOSURE J. A. M. A. Dormans
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Laboratory for Pathology, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands P. J. A. Rombout
Laboratory for Toxicology, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands H. Van Loveren Laboratory for Pathology, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands
As the ultrastructural data on the effects of ozone on pulmonary alveolar macrophages (PAM) are lacking, transmission (TEM) and scanning (SEM) electron microscopy were performed on rat RAM present in alveolar lavages following exposure to ozone. Rats were continuously exposed for 7 d to ozone concentrations ranging from 0.25 to 1.50 mg/m3 for 7 d followed by a 5-d recovery period. Additionally, morphometry on lung sections was performed to quantitate PAM. In a second experiment rats were continuously exposed to 1.50 mg O3/m3 for 1, 3, 5, or 7 d. To study the influence of concurrent ozone exposure and lung infection, due to Listeria monocytogenes, rats were exposed for 7 d to 1.50 mg O3/m3 after a Listeria infection. The surface area of lavaged control PAM was uniformly covered with ruffles as shown by SEM and TEM. Exposure to 0.5 mg ozone/m3 for 7 d resulted in cells partly covered with microvilli and blebs in addition to normal ruffles. The number of large size PAM increased with an increase in ozone concentration. After 1 d of exposure, normalappearing as well as many small macrophages with ruffles and scattered lymphocytes were seen. Lavage samples taken after 5 or 7 d of exposure showed an identical cell composition to that taken after 3 d of exposure. After Listeria infection alone, lavage samples consisted of mainly lymphocytes and some macrophages. Small quantitative
The authors are grateful for expert technical assistance of G. Riool-Nesselaar, J. Bos, and J. Boere. M. Marra and P. Fokkens are acknowledged for the ozone exposure of the rats. Requests for reprints should be sent to Dr. J. A. M. A. Dormans, Laboratory for Pathology, National Institute of Public Health and Environmental Protection, P.O. Box 1, 3720 BA Bilthoven, The Netherlands. 53 Journal of Toxicology and Environmental Health, 31:53-70, 1990 Copyright © 1990 by Hemisphere Publishing Corporation
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changes, such as an increase in the number of polymorphonudear neutrophils and large-size PAM, occurred in lavages after ozone exposure and infection with L. monocytogenes.
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Morphometric examination of lung sections revealed a concentration-related increase in the number of PAM, even in animals exposed to 0.25 mg ozone/m3 for 7 d. Centriacinar regions were more severely affected than other regions of lung tissue. By 5 d after termination of exposure to ozone, the number of lysozyme-positive alveolar cells was still significantly increased in centriacinar areas of the lung. The results indicate that ozone exposure causes major changes in the number, size, and surface morphology of PAM in rat lung. Furthermore, the results presented here suggest that changes in alveolar macrophage function are reflected by morphological changes.
INTRODUCTION Ozone is recognized as a highly toxic and ubiquitous air pollutant that produces various toxic effects on alveolar macrophages. Metabolic, functional (Kimura and Goldstein, 1981; Wenzel and Morgan, 1983; Goldstein et al., 1978; Van Loveren et al., 1988; Veninga and Evelyn, 1986; McAllen et al., 1981), and structural (Dowell et al., 1970) parameters of cells are influenced by ozone exposure. Dose- and time-dependent changes in the composition and number of lavaged cells have been observed. Although changes in surface morphology of human pulmonary alveolar macrophages (PAM) have been described after smoke exposure (Finch et al., 1982; Quan and Golde, 1977; Plowman, 1982), electron microscopic descriptions of the effects of ozone on human PAM are lacking. The effects of ozone on rabbit PAM by scanning electron microscopy (SEM) have been described (Aranyi et al., 1976). SEM and transmission electron microscopical (TEM) studies were performed to determine the effects of different ozone concentrations and of exposure times on the surface and interior morphology of lung alveolar cells recovered by bronchoalveolar lavage. Complementary to this, morphometry of PAM was performed to detect an effect on the number of these cells. To study the effects of ozone on a newly recruited population of PAM, rats were injected intratracheally with Listeria monocytogenes. MATERIALS AND METHODS Ozone Exposure
Groups (8 animals per group) of 8-wk-old male specific-pathogen-free (SPF) bred Wistar RIV:TOX rats from the Institute's breeding colony were continuously exposed for 7 d to 0, 0.25, 0.50,1.00, or 1.50 mg O3/m or for 7 d followed by a 5-d recovery period in 0.2-m3 rectangular stainless steel and glass inhalation chambers. In a second experiment, rats were continuously exposed to 1.50 mg O3/m3 for 1, 3, 5, or 7 d. In a third experiment,
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rats were intratracheally infected with L monocytogenes and then exposed to 1.5 mg O3/m3 for 7 d. The strain of L monocytogenes used and the method of intratracheal injection of organisms are described by Van Loveren et al. (1988). Animals were subjected to a 12-h light-12-h dark regimen, and food and water were given ad libitum. An air flow of 6 m3/h was maintained through the chambers. The air was filtered by a permanganese filter, an activated charcoal filter, and a HEPA filter, respectively, and was conditioned to 22 ± 1°C and 50 ± 5% relative humidity. Generation of ozone and measurement and calibration of ozone concentration are described elsewhere (Van Loveren et al., 1988). Downloaded by [Universite Laval] at 09:39 07 November 2015
Morphometry
Five rats per group were killed with CO2. The lungs were taken from the thoracic cavity and fixed intratracheally with a 4% neutral phosphate buffered formaldehyde solution for at least 24 h and under a continuous hydrostatic pressure of 20 cm water. The lungs were embedded in paraplast and 5-fim cross sections at the largest transversal level of the three lobes were prepared. For immunocytochemical demonstration of lysozyme-positive cells, sections were incubated in a phosphate-buffered saline (PBS) containing 0.25% trypsin and 0.01% calcium chloride for 30 min at room temperature. Sections were stained with rabbit anti-rat lysozyme serum, prepared at our institute by immunization of rabbits with purified rat lysozyme for 30 min at room temperature. After washing with PBS, the sections were incubated with goat anti-rabbit antibodies, conjugated with peroxidase for 30 min at room temperature. After subsequent washing the sections were then incubated in a solution containing 0.03% diaminobenzidine in 0.05 M Tris-HCI (pH 7.6), to which hydrogen peroxide (0.03%) was added just before use. After incubation for 5 min at room temperature and an additional cycle of washings, the slides were mounted in coverslip resin. Morphometry was performed using an automated Videoplan Morphometric System (Kontron, Munich, Federal Republic of Germany). Ten fields of vision at a magnification of 25 x were evaluated for numbers of alveolar lysozyme-positive cells. Fields were chosen either at random or selectively at the centriacinar region. Differences between individual groups were tested with Student's f-test; p values < .05 (two-tailed) were considered significant. SEM and TEM of Lavaged Cells
Three animals per group were anesthetized with CO2 and the lungs were taken from the thoracic cavity and lavaged 5 times with 8 ml PBS at 4°C. After centrifugation (2200 rpm) the pellet was resuspended in 3 ml PBS. For SEM, some drops of this cell suspension were mixed with an equal volume of 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) and centrifuged after 30 min of fixation. Some drops of the resuspended
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pellet were placed on 0.1% poly-L-lysine coated glass discs. After adherence for 10 min, the discs with cells were postfixed in 1% OsO4, dehydrated in an acetone series, and dried in a critical-point drying device (Polaron, Watford, U.K.). They were then mounted on aluminum stubs and sputter coated with a thin layer of gold in a Polaron SEM coating unit E5000. Specimens were examined in a Philips PSEM 501B scanning electron microscope (Philips, Eindhoven, The Netherlands). For TEM the resuspended cell suspension was fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 3 h at 4°C and postfixed in 1% OsO4 for 1 h. They were embedded in agar, dehydrated in alcohol, and embedded in glycydether. Ultrathin sections were cut with an LKB ultramicrotome and subsequently stained with uranyl acetate and lead citrate. The sections were examined with a Philips EM 201 transmission electron microscope. RESULTS Pulmonary lavages of control rats showed a homogeneous cell population consisting mainly of PAM. These cells were round, uniform in size (8-12 fim), and all were similar in appearance as determined by TEM (Figure 1) and by SEM (Figure 2). The entire surface area of the cells (SEM) appeared to be uniformly covered by ruffles. These ruffles were identi-
FIGURE 1. Lavage sample of control rat showing alveolar macrophages of uniform size. TEM, X1450.
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FIGURE 2. View of alveolar macrophages from control rat. Note that the entire surface of the cells appears ruffled. SEM, X5500.
'i f. 'i^.-««*••'•''
FIGURE 3. Detail of macrophage from control rat with prominent electron dense lysosomes. The intersection of the cell border shows small thin ruffles. TEM, X9800.
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FIGURE 4. Surface morphology of a macrophage covered with thin microvilli and numerous blebs; 0.5 mg O 3 /m 3 , 7 d. SEM, x 11,700.
fied by TEM as thin extensions of the plasma membrane, roughly perpendicular to the surface, that were approximately 0.2 ¡xm in width (Figure 3). The cytoplasm contained a pleomorphic nucleus, many electron-dense secondary lysosomes, and a well-developed Golgi area. Incidentally, lamellar cytoplasmic inclusions were also seen. Cells recovered by lavage from rats exposed to 0.25 mg O3/m3 for 7 d showed no differences in morphology from cells obtained from control rats. After exposure to 0.50 mg O3/m , in one out of three animals the lavage sample contained cells (7-8 /¿m in diameter) partially covered with microvilli, or with microvilli and blebs (Figure 4). In addition, large (15-25 /xm) ellipsoid macrophages were also present. After exposure to 1.0 mg O3/m3 there was an increase in the number of cells with these surface changes, and the number of large-size macrophages increased correspondingly. Some PAM were observed undergoing cell division (Figure 5). After exposure to 1.0 and 1.50 mg O3/m3 concentration-related increased numbers of small round cells (6-8 /xm) were observed with only few microvilli or small ruffles on the surface (Figure 6). These cells were identified by TEM as lymphocytes, polymorphonuclear neutrophils and eosinophils (Figure 7). In contrast, PAM were covered with a combination of blebs, villi, and ruffles (Figures 8 and 9); blebs (Figure 10); or villi or ruffles (Figure 11). No relationship could be demonstrated by TEM be-
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FIGURE 5. View of a dividing macrophage. Note the ruffles and the focal surface area of blebs; 1 mg O 3 /m 3 , 7 d. SEM, X5500.
FIGURE 6. Small cell, presumably a lymphocyte, with surface microvilli; 1.5 mg O 3 /m 3 , 7 d. SEM, x 11,000.
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FIGURE 7. View of a lavage sample from a rat, exposed to 1.5 mg O 3 /m 3 for 7 d. Note the small and large macrophages, eosinophils, erythrocytes, and abundant cell debris. TEM, X1450.
FIGURE 8. Macrophage with ruffles to one side and small microvilli and blebs on the other side. The cytoplasm is filled with phagosomes and lysosomes; 1.5 mg O 3 /m 3 , 7 d. TEM, X4600.
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FIGURE 9. Detail of the surface of a macrophage showing thin microvilli intermingled with variable size blebs; 1.5 mg O 3 /m 3 , 7 d. SEM, x22,600.
FIGURE 10. View of the relatively smooth surface of a large alveolar macrophage partially covered with blebs; 1.5 mg O 3 /m 3 , 7 d. SEM, X2800.
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FIGURE 11. At right is a large macrophage with ruffles, and at left, a macrophage with microvilli; 0.5 mg O 3 /m 3 , 7 d. SEM, X5500.
FIGURE 12. A large macrophage covered mainly with microvilli. Listeria infected and ozone exposed, 1.5 mg/m3, 7 d. SEM, X5500.
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FIGURE 13. Detail of the wavy surface of a macrophage consisting primarily of blebs of varying size. Listeria infected and ozone exposed, 7 d, 1.5 mg/m3. SEM, X5500.
cr "
FIGURE 14. Lavage sample composed of small and large macrophages, lymphocytes, erythrocytes, and cell debris. Listeria infected and ozone exposed, 7 d, 1.5 mg O 3 /m 3 . TEM, X2000.
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TABLE 1. Characteristics of Alveolar Macrophages and Lavage Cell Composition after Exposure to Ozone, Infection by Listeria, or Both Exposure and Infection
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Alveolar macrophage diameter (;tm) and surface structures Treatment
7-8
Control 0.25 mg O3/m3, 7 d 0.5 mg O 3 /m 3 ,7d 1.0 mg O3/m3, 7 d 1.5 mg O3/m3, 1 d 1.5 mg O 3 /m 3 ,3,5,7 d Listeria infection Listeria infection plus 1.5 mgO 3 /m 3 for 7 d
— + — + + + + + + +++ +++ + ++ — + + -
8-12
+
+
15-25
+
+
15-40
Ruffles
Villi
— -
+ + + + + + +
— — ++ ++ + + ++
+
+
++
++
++ —
Blebs
Lym.
Eos.
_ _ _ + _ ++ + -
_ _ _
Neu _ _ _ + _
_ + + + + — +
Note. Abbreviations: Lym., lymphocytes; Eos., eosinophilic granulocytes; Neu., neutrophilic granulocytes; - , absent; + , present, + + , present in increased number.
tween distinct surface structures of a PAM, such as microvilli or blebs, and the presence of cytoplasmic cell organelles. The size of the PAM varied from 8 to 40 fxm. The larger-size PAM contained many lysosomes, vacuoles, and lamellar inclusions. Remarkable at concentrations of 1.0 and 1.5 mg O3/m3 was that the lavages contained a substantial quantity of cell debris and free surfactant. In the second experiment after exposure to 1.5 mg O3/m3 for 1 d, lavage samples contained a large amount of cell debris and fragmented cells. Besides "normal" ruffled macrophages (8-12 (im), there were numerous smaller size cells with the same SEM appearance and small round cells with a few microvilli. Lavaged samples obtained after exposure for 3 or 5 d showed an identical composition of cells as the samples obtained after 7 d of exposure. Infection with Listeria organisms induced a population of small cells with some microvilli. These cells were identified mainly as lymphocytes as determined by TEM. The surface of the PAM was clad not only with ruffles but also with microvilli and blebs. Occasionally, large macrophages were also observed. Cell debris was not present. A combination of Listeria infection and ozone exposure resulted in a large variation of the PAM size. The number of large macrophages (15-40 firm) was increased considerably when compared to samples obtained from rats only infected with Listeria; their surface was mainly covered by blebs (Figures 12 and 13). Besides PAM and cell debris the samples showed lymphocytes, polymorphonuclear neutrophils and eosinophils (Figure 14). In all lavages an incidental ciliated cell, a type II or type III pneumocyte, or a Clara cell and usually some erythrocytes were observed.
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Changes in lavage cell composition and PAM size and surface characteristics after various ozone exposure regimens and Listeria infection are summarized in Table 1. Morphometric examination of the lungs revealed a clear concentration effect after ozone exposure as shown by the number of lysozymecontaining alveolar cells. Alveolar and septal lysozyme-positive cells were distinguished not only by their localization but also by their morphologic characteristics. In this case the alveolar cells were larger and more intensely stained than the septal type II cells (Figure 15). The number of free lysozyme-positive alveolar cells were significantly increased even in animals exposed to the lowest concentration of ozone (Figure 16). The
•*••?
rJ>-
FIGURE 15. Rat lung, immunocytochemically stained for lysozyme, x390. (a) Control: a few dark stained alveolar macrophages (arrow) and weak stained septal type II pneumocytes. (b) Ozone, exposed for 7 d to 1.5 mg O 3 /m 3 : an increased number of free alveolar macrophages (arrow).
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centriacinar regions of the lungs were more severely affected when compared to other areas of the tissue. By 5 d after termination of exposure to ozone, the number of lysozyme-positive alveolar cells was still significantly increased in centriacinar areas of lung (Figure 17).
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DISCUSSION Results here clearly demonstrate that ozone has distinct effects on number, size, and surface morphology of rat PAM. Initial morphological changes were observed at a concentration of 0.5 mg O3/m3 by SEM. However, morphometric examination of lungs revealed that the number of PAM in the centriacinar region and in aselect areas of the rat lung has been significantly increased from 0.25 mg O3/m3; at 5 d after the end of the exposure to 0.25 mg O3/m3 a significant increase was still shown in the centriacinar region. To our knowledge this ozone concentration has not been mentioned earlier to have an effect on PAM population. Furthermore, dividing PAM have been noticed in the present study. PAM are important in maintaining primary host defense mechanisms in the lung. Harmful effects of inhaled gases, particulates, or other agents can result in respiratory disorder, caused by functional alterations
0.25
0.S0
0.75
1.00
1.25 1.50 mg/m 3 OZONE
FIGURE 16. Numbers of alveolar lysozyme-positive cells in lungs of rats exposed to various concentrations of ozone for 1 wk. Ten fields at a magnification of x25 were counted in aselect areas of the lung (O), or in areas selected in the centriacinar region (•). Significance: *p < .05, **p < .01, ***p < .001.
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m g / m 3 OZONE FIGURE 17. Numbers of alveolar lysozyme-positive cells in lungs of rats 5 d after termination of 1 wk of exposure to various concentrations of ozone. Ten fields at a magnification of x25 were counted in aselect areas of the lung (O), or in areas selected in the centriacinar region (•). Significance: *p < .05, **p < .01, ***p < .001.
of PAM. Exposure of rats to 1.6 mg O3/m3 for only 6 h has been shown to induce a distinct proliferation of PAM as determined by a transient decrease in the nuclear/cytoplasmic ratio and an increased mitotic and labeling index of cells (Hotchkiss et al., 1989). In contrast, exposure to 0.25 mg O3/m3 induced no morphologic changes in cells (Hotchkiss et al., 1989), which is in agreement with our results. Further, results reported here correlated well with an increased mean cell volume after ozone exposure as reported by Hotchkiss et al. (1989) and Lum et al. (1983). Increased phagocytosis of surfactant and cell debris, resulting from pulmonary damage, were accompanied simultaneously by changes in surface morphology. Similarly, exposure to cigarette smoke resulted in an increase in secondary lysosome content in cells (Lewis et al., 1980; Matulionis, 1977; Davies et al., 1977) and distinct cell surface changes from a smooth to a cobblestone appearance (Quan and Golde, 1977; Finch et al., 1982; Davis et al., 1980). Similarly, anesthesia with tetracaine or lidocaine (Finch et al., 1982) and exposure to metals (Waters et al., 1975; Johansson et al., 1983, 1987, 1988) were shown to induce a transition in the surface morphology of PAM from sheetlike ruffles to tightly packed villous projections, further suggesting that the observed morphological changes of the surface of PAM are a nonspecific reaction of these cells to toxic agents.
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The observed surface morphology of lavaged cells after Listeria infection and ozone exposure did not differ qualitatively from the changes observed after ozone exposure alone. In both cases the same cell types were present although in different ratios. PAM of control rats as well as rat PAM evoked by a Listeria infection seem to have the same sensitivity to ozone with respect to surface changes. The most prominent shift in cellular composition of lavage samples and in surface morphology of PAM occurred during the first 3 d of ozone exposure. After 5 or 7 d of exposure no additional changes were observed, suggesting that adaption of these cells occurred. Boorman et al. (1980) described less severe lesions in the centriacinar region of the rat lung after 90 d of exposure to ozone than after 7 d of exposure, while Evans et al. (1985) have discussed a morphologic mechanism as a basis for ozone tolerance. It is obvious that morphological changes will correlate with functional changes of cells such as phagocytosis, adherence, and lysosomal enzyme activities. Remarkably, Finch et al. (1982) indicated that there are no data available to correlate macrophage morphology with cellular function. However, Quan and Golde (1977) suggested that prominent changes in surface morphology reflect functional and metabolic alterations of cells. Rasp et al. (1978) hypothesized that the decreased adherence of PAM from smokers was related to the observed differences in surface morphology. Veninga and Evelyn (1986) reported that differences in adherence of PAM between first and second lavage samples appeared to correspond to the differences in morphology of cells; the smaller-size PAM found in the second lavage samples represented a more juvenile population. The smaller-size PAM observed here after ozone exposure may have been derived from a relatively young population evoked by ozone. Variation in surface morphology may be attributed to functional and metabolic differences of cells in the early stages of the cell's life span. However, plasma membrane blebbing is a general phenomenon associated with toxic and ischémie cell injury (Orrenius et al., 1989). Ozone exerts its toxic effects on cells presumably by highly reactive free radical oxygen species, resulting in peroxidation of phospholipids in the cell membrane. A negative effect of ozone on phagocytosis and killing of bacteria by PAM has been reported (Holzman et al., 1968; Goldstein et al., 1978; Kimura and Goldstein, 1981). Also in the experiments by Van Loveren et al. (1988) at our institute, conducted with the same experimental conditions, a negative effect of ozone on phagocytosis and killing of bacteria by PAM has been reported. Wenzel and Morgan (1983) suggested that the reduced phagocytic function of macrophages evoked by exposure to ozone is caused by generation of hydrogen peroxide. These data suggest that peroxidation by oxygen radicals and reduced phagocytic activity of cells are related. Whether the observed morphological surface alterations are directly or indirectly
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linked to the forementioned biochemical and functional changes of cells remains to be established. In conclusion, changes observed in the surface and ultrastructural morphology of PAM are the result of the response of these cells to environmental exposure to ozone. Morphological data presented here and physiological data of Van Loveren et al. (1988) from the same experiments provide further indications for the important relationship between morphology and function of phagocytic cells of the lungs.
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Orrenius, S., McConkey, D. J., Bellomo, C., and Nicotera P. 1989. Role of Ca 2+ in toxic cell killing. Trends in Pharmacologic. Sci. (TIPS) 10:281. Plowman, P. N. 1982. The pulmonary macrophage population of human smokers. Ann. Occup. Hyg. 25:393. Quan, S. C., and Golde, D. W. 1977. Surface morphology of the human alveolar macrophage. Exp. Cell Res. 109:71. Rasp, F. L., Clawson, C. C., Hoidal, J. R., and Repine, J. E. 1978. Reversible impairment of the adherence of alveolar macrophages from cigarette smokers. Am. Rev. Respir. Dis. 118:979. Van Loveren, H., Rombout, P. J. A., Wagenaar, S. S., Walvoort, H. C., and Vos, J. C. 1988. Effects of ozone on the defense to a respiratory Listeria monocytogenes infection in the rat. Suppression of macrophage function and cellular immunity and aggravation of histopathology in lung and liver during infection. Toxicol. Appl. Pharmacol. 94:374. Veninga, T. S., and Evelyn, P. 1986. Activity changes of pulmonary macrophages after in vivo exposure to ozone as demonstrated by cell adherence. J. Toxicol Environ. Health 18:483. Waters, M. D., Cardner, D. E., Aranyi, C., and Coffin, D. L. 1975. Metal toxicity for rabbit alveolar macrophages in vitro. Environ. Res. 9:32. Wenzel, D., and Morgan, D. L. 1983. In vitro inhibition of alveolar macrophage phagocytosis by ozone: Absence of a role for serum or mode of ozone administration. Toxicol. Lett. 18:57. Received December 18, 1989 Accepted March 30, 1990