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Asbestos effects on superoxide production
ENVIRONMENTALRESEARCH39, 299--306 (1986)
Asbestos Effects on Superoxide Production An in Vitro Study of Hamster Alveolar Macrophages BRUCE W. CASE, MICHAEL P. C. lP, MARIA PADILLA, AND JEROME KLEINERMAN Lillian and Hemw M. Stratton-Hans Popper Department qf Patlzology, The Mo,nt Sinai School of, Medicine, One Gustave Levy Pl~lc'e, New York, New York 10029; and Department of Pathology, McGill Unit'ersiO', 3775 Unit'ersio' Street, Montrc;al, Quebec H3A 2B4, Canada
Received May 13, 1984 Inhaled asbestos induces accumulation of alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) in lung. Asbestos-enhanced production of superoxide anion (O ~) by AM and/or PMN may be involved in the pathogenesis of asbestos-induced fibrosis, either through direct effects on collagen synthesis or via mediation of tissue injury and repair. In in vivo experiments, broncboalveolar lavage (BAL) 3 to 8 weeks following intratracheal asbestos injections showed increases in both PMN and AM, with AM representing 78 to 82% of cells recovered. Inhalation models, generally regarded as more analagous to human exposures, have confirmed AM as the predominant component of the cellular response to inhaled asbestos. In this study, the in vitro effects of asbestos fiber on 02 production by AM have been determined in cell populations derived from the Syrian golden hamster. AM for in vitro study were obtained by BAL. 02 production was monitored as superoxide dismutase (SOD) - inhibitable cytochrome c reduction. Significant rises in O ~ release by AM were noted in the presence of 0.4 mg/ml crocidolite (2.53 _+ 0.33 nmole cytochrome c reduced/106 cells/30 rain, 37°C: controls 1.13 -+ 0.18 nmole; P < 0.02). Chrysotile induced levels of O2 release in AM which were similar to those evoked by crocidolite. © 1986AcademicPress, Inc.
INTRODUCTION In m o s t a n i m a l s y s t e m s , p u l m o n a r y i n t e r s t i t i a l f i b r o s i s d e v e l o p s w e e k s to m o n t h s f o l l o w i n g a s b e s t o s f i b e r i n h a l a t i o n ( W a g n e r et al., 1974; D a v i s et al., 1980; L e e et al., 1981) o r i n t r a t r a c h e a l i n j e c t i o n ( R o l a - P l e s z c z y n s k i et al., 1984). I n h a l e d o r i n j e c t e d f i b e r s elicit an i n f l a m m a t o r y r e s p o n s e w h i c h i n c l u d e s a c c u mulation of polymorphonuclear leukocytes (PMN) and alveolar macrophages ( A M ) . A n i m m e d i a t e e x u d a t e o f P M N is e v i d e n t 2 to 4 hr f o l l o w i n g i n t r a t r a c h e a l i n j e c t i o n in b r o n c h i ( D o d s o n et al., 1980). T h e P M N r e s p o n s e p e a k s at 12 hr but P M N r e m a i n p r e s e n t in s m a l l e r n u m b e r s for w e e k s to m o n t h s w h e r e v e r f i b e r s a n d f i b e r a g g r e g a t e s p e r s i s t ( R o l a - P l e s z c z y n s k i et al., 1984). T h r o u g h o u t the first w e e k f o l l o w i n g i n t r a t r a c h e a l i n j e c t i o n s , A M i n c r e a s e i n n u m b e r s a n d d~stribution to b e c o m e t h e p r e d o m i n a n t cell t y p e p r e s e n t . E x p e r i m e n t a l m o d e l s o f asb e s t o s e x p o s u r e w h i c h e m p l o y i n h a l a t i o n c h a m b e r s s u g g e s t that the a l v e o l a r m a c r o p h a g e is t h e m o s t i m p o r t a n t c o m p o n e n t o f the i n f l a m m a t o r y r e s p o n s e f r o m its e a r l i e s t s t a g e s o n w a r d ( B r o d y et al., 1981). R e c e n t w o r k b y B a t e m a n et al. (1982) s u g g e s t s that m a c r o p h a g e - a s b e s t o s f i b e r i n t e r a c t i o n s in vivo l e a d to t h e r e l e a s e o f an as y e t u n i d e n t i f i e d diffusible fibrog e n i c factor. M e c h a n i s m s s u g g e s t e d for t h e d e v e l o p m e n t o f a s b e s t o s - r e l a t e d pul299 0013-9351/86 $3.00 Copyright © 1986 by Academic Press, inc. All rights of reproduction in any form reserved
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vaonary interstitial fibrosis through interactions with inflammatory cells have included production of catalytic enzymes (Dean et al., 1979; Jaurand et al., 1980), chemotactic factors (Miller, 1978), fibroblast-stimulating factors (Aalto et al., 1979), and oxygen radicals (Hatch et al., 1980). Phagocytosing AM and PMN release a reactive oxygen intermediate, superoxide anion (Or), into the extracellular milieu (Salin and McCord 1975; Drath and Karnovsky 1975; Stokes et al., 1978; Papermeister-Bender et al., 1980; Sweeney et al., 1981). This release appears to be related to surface membrane interactions preceding and during phagocytosis (Williams and Cole, 1981). Increased net production of O~ could represent an important consequence of asbestos-induced accumulation of AM and PMN. Recent work by Rola-Pleszczynski et al. (1984) has established PMN-asbestos fiber interaction as a generator of O~7. Increased O_~ production by AM and PMN could contribute to fibrosis, either directly through effects on collagen metabolism (Bhatnagar, 1977), or through its highly reactive derivative, hydroxyl radical ( O H - ) (Drath et al., 1979). The latter oxidant causes tissue injury (Salin and McCord 1975; Drath et al., 1979) which may be followed by repair. The current study was designed to test the first part of our hypothesis: that asbestos fibers can induce increased O7 production by hamster AM in vitro using ferricytochrovae c reduction as an indicator. METHODS Materials
Animals used in all experiments were male Syrian golden hamsters, weight 100-140 g. Asbestos samples used were standard UICC crocidolite (Timbrell, 1970) and short-fiber Coalinga chrysotile (Langer et al., 1978; Yeager et aI., 1983). Superoxide disvautase (SOD) Type I and cytochrome c Type VI were purchased from Sigma. Asbestos, SOD, cytochrome c, and cell preparations were suspended in Hepes-buffered Hanks' balanced salt solution (HBSS), with calcium, magnesium, and penicillin/streptomycin and without phenol red (GIBCO), pH 7.40 at 4°C. Sodium azide (10-3M) was added to HBSS to prevent reoxidation of cytochrovae c during incubations (Rister and Baehner, 1976). Degree of reduction of cytochrovae c was established spectrophotometrically prior to each experiment from the extinction coefficient (Massey, 1959). Derivation o f A M f o r in Vitro Study
AM were derived by bronchoalveolar lavage (BAL) of six to nine hamsters per experiment, as described previously (Kleinerman et al., 1982). Following intraperitoneal injection of 0.3 ml 1:1 pentobarbital:saline, hamsters were exsanguihated from the abdominal aorta and their lungs collapsed for 15 vain. BAL was performed six times for each animal with 3.5 ml warmed saline. Lavage fluid was drained by gravity and centrifuged for 10 vain at 150g. Final cell suspensions were diluted to a concentration of 1 _+ 0.2 × l06 cells per 0.45-mi aliquot. Cell counts were determined in duplicate in a hemacytometer, cell viability was established by trypan blue exclusion, and differential counts were obtained from smears (Wright- Gievasa).
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Assay Procedures Aliquots (0.45 ml) of cell suspensions were added to plastic cuvettes (Evergreen) containing 126 nmole unreduced cytochrome c in HBSS, with 10% heatinactivated fetal calf serum added. "Asbestos" assays contained crocidolite or chrysotile suspensions in a final concentration of 0.4 mg/ml. "Control" assays contained an equal volume of HBSS without asbestos. Additional "asbestos" and "control" assays contained 50 txg/ml SOD. In each experiment, three to seven separate assays were prepared for each condition. Reference blanks contained cytochrome c, serum, and HBSS without cells, asbestos, or SOD. All assays were prepared at 4°C and transferred immediately to the incubator. Final assay volume was 1.26 ml and cell suspensions, prepared at 4°C, were added last. Cuvettes were inverted three times and incubated at 37°C, 3% CO2, for 30 rain. Cuvettes were then centrifuged at 1500g for 10 rain at 4°C. For each assay, 20 optical density (OD) readings were taken at 550 nm in a Beckman DU8 spectrophotometer. Mean OD readings were expressed vs reference blanks kept at 4°C. OD increases in assays containing SOD were subtracted from the larger increases in "control" and "asbestos" assays lacking SOD to obtain SOD-inhibitable cytochrome c reduction as an index of 0 2 generation. Statistical Procedures In each experiment, three to seven separate assay determinations were performed with AM alone, with asbestos, and with asbestos plus superoxide dismutase. Values for superoxide production within each experiment were then recorded as mean SOD-inhibitable cytochrome c reduction -- SD,,_I. Values for crocidolite-exposed AM compared mean results in all experiments, +_SE, using the paired t test for independent samples (Colton, 1974). All cell counts were recorded as mean number +_ SD,, 1 per assay. In Vivo Asbestos Exposures A series of intratracheal asbestos injection experiments was performed in order to quantify the relative importance of alveolar macrophages and polymorphonuclear leukocytes in the pulmonary response of the Syrian golden hamster. In the first experiment, two groups of pentobarbital-anesthetized hamsters (N = 4, each group) received single 0.2-ml aliquots of HBSS with or without 1 mg Coalinga chrysotile asbestos. Injections were administered under direct fiberoptic illumination via gas-sterilized PE-50 polyethylene catheters. Animals were sacrificed at 3 weeks and BAL was performed as described above, with duplicate determinations of cell count, viability, and differential. Methods in the second experiment were identical, but animals received a second asbestos/HBSS or HBSS injection 6 weeks after the first. Hamsters were sacrificed 2 weeks following the second intratracheal injection. RESULTS
Results of the in vivo intratracheal asbestos injection experiments are summarized in Table 1. Three weeks following a single exposure, asbestos-treated an-
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CASE ET AL. TABLE 1 AM ANDPMN RESPONSETO INTRATRACHEALASBESTOS INJECTIONS Experiment 1
Total cells recovered % Alveolar macrophages Total PMN Total AM
Experiment 2
Control
Asbestos
Control
Asbestos
(N = 4)
(N = 4)
(N = 2)
(N = 2)
3.0 × 106 97% 0.1 × 106 2.9 x 106
4.1 )< 106 78% 0.9 x 106 3.2 × 106
2.8 × 106 96% 0.1 x 106 2.7 x 106
9.2 x 106 82% 1.7 × 106 7.5 x l06
Note. In Experiment 1 cells were obtained by BAL 3 weeks following an injection of 1 mg chrysotile suspended in HBSS, or HBSS alone in controls. In Experiment 2, animals received a second injection at 6 weeks and were sacrificed at 8 weeks. Duplicate hemacytometer counts and WrightGiemsa differential counts are expressed as mean totals recovered " p e r animal." Cell viability exceeded 95% at all times.
imals show a ninefold increase in PMN with a small increase in AM. Following two exposures 6 weeks apart, the PMN count increases 17-fold, with a concomitant threefold increase of AM. In both experiments, however, AM represent approximately 80% of all cells recovered from asbestos-treated animals. In in vitro experiments, using the cell preparation methods and assay conditions specified, alveolar macrophages produce increased levels of superoxide anion when stimulated by exposure to crocidolite or chrysotile asbestos fiber (Fig. 1). The increase with crocidolite is from 1.13 _+ 0.18 nmole SOD - inhibitable cytochrome c reduction/106 cells/30 rain in controls to 2.53 _+ 0.33 nmole in asbestos-treated cells. A nearly identical result was recorded in the single experiment using short-fiber chrysotile, performed for comparison purposes (Fig. 1). AM constituted 96 + 3% of the cell populations. AM viability exceeded 90%. DISCUSSION Our values for O~ production by resting hamster AM as indicated by SOD-inhibitable cytochrome c reduction are similar to those reported by Hoidal et al. 4
~2 2E
Con- CrecidoNe
trots
Con- Chryso!ite
trois
FIG. I. (*)Results of four experiments to determine effects of 0.4 mg crocidolite asbestos/ml on O : production by AM (P < 0.02; paired t). Height of bars indicates mean; vertical uprights are standard error. In each experiment, three to five separate assays were prepared and cells lavaged from six to nine hamsters (total, 31). Cell viability was 96 + 3%; more than 90%. of cells were AM. ~-Results of one experiment with 0.4 mg/ml Coalinga RO-144 chrysotile, included for comparison purposes. Cells lavaged from nine hamsters.
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(1981). Hatch et al. (1980) have previously noted a large difference in the O5 evoking potential of chrysotile and crocidolite in guinea pig AM, with the amphibole mineral generating much higher values. The methods used in their work, however, differ from our own in species selection, incubation time, assay temperature, and use of adherent cells; all factors which can influence O7 production. Most important, as indicated by the authors, the use of chemiluminescence as the O 7 detection system may have produced an artificial elevation due to catalyzed oxidation of luminol by metals complexed with crocidolite (Hatch et al., 1980). Our study establishes that asbestos fiber induces superoxide anion production by AM, as indicated by SOD-inhibitable cytochrome c reduction. It is important to note that other components of the inflammatory response, particularly PMN, produce 0 5 when challenged with asbestos fibers in vitro (Rola-Pleszczynski et al., 1984). There has been some debate regarding the relative importance of AM and PMN in the intrapulmonary response to asbestos. In general, intratracheal injection models such as our own result in a large influx of PMN, although AM remain numerically predominant after the initial stages (Table 1). Inhalation models generally confirm AM as the chief component of the cellular response to asbestos (Brody et al., 1981). Bronchoalveolar lavage of human asbestotic subjects indicates that both cell types may be increased (Bignon et al., 1978). Work in progress in our laboratory indicates that the AM response of fiber-exposed miners may be quantitatively correlated to lung asbestos content (E Sebastien et al., unpublished observations). The concomitant increase in intrapulmonary PMN may be nonspecifically related to smoking and to the increased susceptibility to infection of patients with asbestosis. Regardless of the divergent stimuli to their accumulation, it is now clear that both AM and PMN may serve as generators of superoxide anion in asbestos-exposed individuals. Superoxide anion has been implicated in the pathogenesis of a number of other conditions in lung which can progress to fibrotic change. It is believed to contribute to the development of pulmonary oxygen toxicity (Crapo and Tierney, 1974) which can lead to interstitial fibroblastic proliferation (Crapo et al., 1980). O 5 is also generated in paraquat-induced lung injury, which produces subsequent fibroblastic proliferation in areas most affected (Smith et al., 1979). Other pulmonary conditions possibly involving 0 5 and characterized by interstitial fibrosis include long-term ozone exposure (Last and Greenberg, 1980) and idiopathic pulmonary fibrosis (McCormick et al., 1981). Superoxide anion generated by AM exposed to asbestos could contribute to pulmonary fibrosis either directly, through effects on collagen metabolism, or indirectly, through modulation of inflammation and repair. Reported O 7 effects on collagen metabolism have included displacement of vitamin C as a cofactor in collagen synthesis and induction of prolyl hydroxylase (Bhatnager, 1977). Increased prolyl hydroxylase activity has been noted 7 days following intratracheal asbestos injections (Davis and Reeves, 1971). Modulation of inflammation by O 2 was suggested by McCord et aI. (1980), who observed that exposure of human plasma to 0 2 and its reaction products resulted in the creation of a heat-labile, high-molecular-weight chemotactic factor for PMN. Complementary evidence is offered by studies in which hyperoxia induces increased numbers of PMN (Crapo et al., 1980; Fox et al., 1981).
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An example of the kind of mechanism possibly involved in asbestosis may be offered by the oxygen toxicity model of pulmonary fibrosis. Hyperoxia leads in experimental models to selective killing of pneumocytes, with subsequent overgrowth of interstitial cells and collagen deposition (Crapo et al., 1980). Asbestos may act in a similar fashion; the initial injury to the alveolar epithelium (Brody et al., 1981) evolving into ongoing interstitial cell proliferation and fibrosis. O 2 may play an early role in the development of this cycle of attraction of inflammatory cells, tissue destruction, and eventual interstitial scarring. Amphibole asbestos fibers such as crocidolite, amosite, and tremolite can remain in lung parenchyma for many years following inhalation exposures (Rowlands et al., 1982). The increased AM O~7 generation observed in vitro in our experiments may also occur in vivo in the continued presence of fibers. Net increases in O ~7 release in lung could occur either through an increased production efficiency by AM similar to that observed in vitro, and/or through the continuous recruitment of increased numbers of AM. In summary, crocidolite and short-fiber chrysotile fibers induced increased levels of superoxide anion generation by AM in vitro, as indicated by superoxide dismutase-inhibitable cytochrome c reduction. Future work in this area should concentrate on the in vivo replication of the observed effect following controlled inhalation exposures to asbestos. The effects of the specific O5 inhibitor SOD on the development of asbestos-induced bronchioloalveolitis and subsequent fibrosis should also be assessed.
ACKNOWLEDGMENTS The writers thank Dr, Arthur Langer for providing the short-fiber Coalinga asbestos samples, and Alex Collins and Joan Sorensen for technical and administrative assistance. This work was supported by a generous grant from the American Lung Association. Dr. Case is a fellow of the Conseil de la recherche en santo du Qu6bec.
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Asbestos Effects on Superoxide Production An in Vitro Study of Hamster Alveolar Macrophages BRUCE W. CASE, MICHAEL P. C. lP, MARIA PADILLA, AND JEROME KLEINERMAN Lillian and Hemw M. Stratton-Hans Popper Department qf Patlzology, The Mo,nt Sinai School of, Medicine, One Gustave Levy Pl~lc'e, New York, New York 10029; and Department of Pathology, McGill Unit'ersiO', 3775 Unit'ersio' Street, Montrc;al, Quebec H3A 2B4, Canada
Received May 13, 1984 Inhaled asbestos induces accumulation of alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) in lung. Asbestos-enhanced production of superoxide anion (O ~) by AM and/or PMN may be involved in the pathogenesis of asbestos-induced fibrosis, either through direct effects on collagen synthesis or via mediation of tissue injury and repair. In in vivo experiments, broncboalveolar lavage (BAL) 3 to 8 weeks following intratracheal asbestos injections showed increases in both PMN and AM, with AM representing 78 to 82% of cells recovered. Inhalation models, generally regarded as more analagous to human exposures, have confirmed AM as the predominant component of the cellular response to inhaled asbestos. In this study, the in vitro effects of asbestos fiber on 02 production by AM have been determined in cell populations derived from the Syrian golden hamster. AM for in vitro study were obtained by BAL. 02 production was monitored as superoxide dismutase (SOD) - inhibitable cytochrome c reduction. Significant rises in O ~ release by AM were noted in the presence of 0.4 mg/ml crocidolite (2.53 _+ 0.33 nmole cytochrome c reduced/106 cells/30 rain, 37°C: controls 1.13 -+ 0.18 nmole; P < 0.02). Chrysotile induced levels of O2 release in AM which were similar to those evoked by crocidolite. © 1986AcademicPress, Inc.
INTRODUCTION In m o s t a n i m a l s y s t e m s , p u l m o n a r y i n t e r s t i t i a l f i b r o s i s d e v e l o p s w e e k s to m o n t h s f o l l o w i n g a s b e s t o s f i b e r i n h a l a t i o n ( W a g n e r et al., 1974; D a v i s et al., 1980; L e e et al., 1981) o r i n t r a t r a c h e a l i n j e c t i o n ( R o l a - P l e s z c z y n s k i et al., 1984). I n h a l e d o r i n j e c t e d f i b e r s elicit an i n f l a m m a t o r y r e s p o n s e w h i c h i n c l u d e s a c c u mulation of polymorphonuclear leukocytes (PMN) and alveolar macrophages ( A M ) . A n i m m e d i a t e e x u d a t e o f P M N is e v i d e n t 2 to 4 hr f o l l o w i n g i n t r a t r a c h e a l i n j e c t i o n in b r o n c h i ( D o d s o n et al., 1980). T h e P M N r e s p o n s e p e a k s at 12 hr but P M N r e m a i n p r e s e n t in s m a l l e r n u m b e r s for w e e k s to m o n t h s w h e r e v e r f i b e r s a n d f i b e r a g g r e g a t e s p e r s i s t ( R o l a - P l e s z c z y n s k i et al., 1984). T h r o u g h o u t the first w e e k f o l l o w i n g i n t r a t r a c h e a l i n j e c t i o n s , A M i n c r e a s e i n n u m b e r s a n d d~stribution to b e c o m e t h e p r e d o m i n a n t cell t y p e p r e s e n t . E x p e r i m e n t a l m o d e l s o f asb e s t o s e x p o s u r e w h i c h e m p l o y i n h a l a t i o n c h a m b e r s s u g g e s t that the a l v e o l a r m a c r o p h a g e is t h e m o s t i m p o r t a n t c o m p o n e n t o f the i n f l a m m a t o r y r e s p o n s e f r o m its e a r l i e s t s t a g e s o n w a r d ( B r o d y et al., 1981). R e c e n t w o r k b y B a t e m a n et al. (1982) s u g g e s t s that m a c r o p h a g e - a s b e s t o s f i b e r i n t e r a c t i o n s in vivo l e a d to t h e r e l e a s e o f an as y e t u n i d e n t i f i e d diffusible fibrog e n i c factor. M e c h a n i s m s s u g g e s t e d for t h e d e v e l o p m e n t o f a s b e s t o s - r e l a t e d pul299 0013-9351/86 $3.00 Copyright © 1986 by Academic Press, inc. All rights of reproduction in any form reserved
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vaonary interstitial fibrosis through interactions with inflammatory cells have included production of catalytic enzymes (Dean et al., 1979; Jaurand et al., 1980), chemotactic factors (Miller, 1978), fibroblast-stimulating factors (Aalto et al., 1979), and oxygen radicals (Hatch et al., 1980). Phagocytosing AM and PMN release a reactive oxygen intermediate, superoxide anion (Or), into the extracellular milieu (Salin and McCord 1975; Drath and Karnovsky 1975; Stokes et al., 1978; Papermeister-Bender et al., 1980; Sweeney et al., 1981). This release appears to be related to surface membrane interactions preceding and during phagocytosis (Williams and Cole, 1981). Increased net production of O~ could represent an important consequence of asbestos-induced accumulation of AM and PMN. Recent work by Rola-Pleszczynski et al. (1984) has established PMN-asbestos fiber interaction as a generator of O~7. Increased O_~ production by AM and PMN could contribute to fibrosis, either directly through effects on collagen metabolism (Bhatnagar, 1977), or through its highly reactive derivative, hydroxyl radical ( O H - ) (Drath et al., 1979). The latter oxidant causes tissue injury (Salin and McCord 1975; Drath et al., 1979) which may be followed by repair. The current study was designed to test the first part of our hypothesis: that asbestos fibers can induce increased O7 production by hamster AM in vitro using ferricytochrovae c reduction as an indicator. METHODS Materials
Animals used in all experiments were male Syrian golden hamsters, weight 100-140 g. Asbestos samples used were standard UICC crocidolite (Timbrell, 1970) and short-fiber Coalinga chrysotile (Langer et al., 1978; Yeager et aI., 1983). Superoxide disvautase (SOD) Type I and cytochrome c Type VI were purchased from Sigma. Asbestos, SOD, cytochrome c, and cell preparations were suspended in Hepes-buffered Hanks' balanced salt solution (HBSS), with calcium, magnesium, and penicillin/streptomycin and without phenol red (GIBCO), pH 7.40 at 4°C. Sodium azide (10-3M) was added to HBSS to prevent reoxidation of cytochrovae c during incubations (Rister and Baehner, 1976). Degree of reduction of cytochrovae c was established spectrophotometrically prior to each experiment from the extinction coefficient (Massey, 1959). Derivation o f A M f o r in Vitro Study
AM were derived by bronchoalveolar lavage (BAL) of six to nine hamsters per experiment, as described previously (Kleinerman et al., 1982). Following intraperitoneal injection of 0.3 ml 1:1 pentobarbital:saline, hamsters were exsanguihated from the abdominal aorta and their lungs collapsed for 15 vain. BAL was performed six times for each animal with 3.5 ml warmed saline. Lavage fluid was drained by gravity and centrifuged for 10 vain at 150g. Final cell suspensions were diluted to a concentration of 1 _+ 0.2 × l06 cells per 0.45-mi aliquot. Cell counts were determined in duplicate in a hemacytometer, cell viability was established by trypan blue exclusion, and differential counts were obtained from smears (Wright- Gievasa).
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Assay Procedures Aliquots (0.45 ml) of cell suspensions were added to plastic cuvettes (Evergreen) containing 126 nmole unreduced cytochrome c in HBSS, with 10% heatinactivated fetal calf serum added. "Asbestos" assays contained crocidolite or chrysotile suspensions in a final concentration of 0.4 mg/ml. "Control" assays contained an equal volume of HBSS without asbestos. Additional "asbestos" and "control" assays contained 50 txg/ml SOD. In each experiment, three to seven separate assays were prepared for each condition. Reference blanks contained cytochrome c, serum, and HBSS without cells, asbestos, or SOD. All assays were prepared at 4°C and transferred immediately to the incubator. Final assay volume was 1.26 ml and cell suspensions, prepared at 4°C, were added last. Cuvettes were inverted three times and incubated at 37°C, 3% CO2, for 30 rain. Cuvettes were then centrifuged at 1500g for 10 rain at 4°C. For each assay, 20 optical density (OD) readings were taken at 550 nm in a Beckman DU8 spectrophotometer. Mean OD readings were expressed vs reference blanks kept at 4°C. OD increases in assays containing SOD were subtracted from the larger increases in "control" and "asbestos" assays lacking SOD to obtain SOD-inhibitable cytochrome c reduction as an index of 0 2 generation. Statistical Procedures In each experiment, three to seven separate assay determinations were performed with AM alone, with asbestos, and with asbestos plus superoxide dismutase. Values for superoxide production within each experiment were then recorded as mean SOD-inhibitable cytochrome c reduction -- SD,,_I. Values for crocidolite-exposed AM compared mean results in all experiments, +_SE, using the paired t test for independent samples (Colton, 1974). All cell counts were recorded as mean number +_ SD,, 1 per assay. In Vivo Asbestos Exposures A series of intratracheal asbestos injection experiments was performed in order to quantify the relative importance of alveolar macrophages and polymorphonuclear leukocytes in the pulmonary response of the Syrian golden hamster. In the first experiment, two groups of pentobarbital-anesthetized hamsters (N = 4, each group) received single 0.2-ml aliquots of HBSS with or without 1 mg Coalinga chrysotile asbestos. Injections were administered under direct fiberoptic illumination via gas-sterilized PE-50 polyethylene catheters. Animals were sacrificed at 3 weeks and BAL was performed as described above, with duplicate determinations of cell count, viability, and differential. Methods in the second experiment were identical, but animals received a second asbestos/HBSS or HBSS injection 6 weeks after the first. Hamsters were sacrificed 2 weeks following the second intratracheal injection. RESULTS
Results of the in vivo intratracheal asbestos injection experiments are summarized in Table 1. Three weeks following a single exposure, asbestos-treated an-
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CASE ET AL. TABLE 1 AM ANDPMN RESPONSETO INTRATRACHEALASBESTOS INJECTIONS Experiment 1
Total cells recovered % Alveolar macrophages Total PMN Total AM
Experiment 2
Control
Asbestos
Control
Asbestos
(N = 4)
(N = 4)
(N = 2)
(N = 2)
3.0 × 106 97% 0.1 × 106 2.9 x 106
4.1 )< 106 78% 0.9 x 106 3.2 × 106
2.8 × 106 96% 0.1 x 106 2.7 x 106
9.2 x 106 82% 1.7 × 106 7.5 x l06
Note. In Experiment 1 cells were obtained by BAL 3 weeks following an injection of 1 mg chrysotile suspended in HBSS, or HBSS alone in controls. In Experiment 2, animals received a second injection at 6 weeks and were sacrificed at 8 weeks. Duplicate hemacytometer counts and WrightGiemsa differential counts are expressed as mean totals recovered " p e r animal." Cell viability exceeded 95% at all times.
imals show a ninefold increase in PMN with a small increase in AM. Following two exposures 6 weeks apart, the PMN count increases 17-fold, with a concomitant threefold increase of AM. In both experiments, however, AM represent approximately 80% of all cells recovered from asbestos-treated animals. In in vitro experiments, using the cell preparation methods and assay conditions specified, alveolar macrophages produce increased levels of superoxide anion when stimulated by exposure to crocidolite or chrysotile asbestos fiber (Fig. 1). The increase with crocidolite is from 1.13 _+ 0.18 nmole SOD - inhibitable cytochrome c reduction/106 cells/30 rain in controls to 2.53 _+ 0.33 nmole in asbestos-treated cells. A nearly identical result was recorded in the single experiment using short-fiber chrysotile, performed for comparison purposes (Fig. 1). AM constituted 96 + 3% of the cell populations. AM viability exceeded 90%. DISCUSSION Our values for O~ production by resting hamster AM as indicated by SOD-inhibitable cytochrome c reduction are similar to those reported by Hoidal et al. 4
~2 2E
Con- CrecidoNe
trots
Con- Chryso!ite
trois
FIG. I. (*)Results of four experiments to determine effects of 0.4 mg crocidolite asbestos/ml on O : production by AM (P < 0.02; paired t). Height of bars indicates mean; vertical uprights are standard error. In each experiment, three to five separate assays were prepared and cells lavaged from six to nine hamsters (total, 31). Cell viability was 96 + 3%; more than 90%. of cells were AM. ~-Results of one experiment with 0.4 mg/ml Coalinga RO-144 chrysotile, included for comparison purposes. Cells lavaged from nine hamsters.
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(1981). Hatch et al. (1980) have previously noted a large difference in the O5 evoking potential of chrysotile and crocidolite in guinea pig AM, with the amphibole mineral generating much higher values. The methods used in their work, however, differ from our own in species selection, incubation time, assay temperature, and use of adherent cells; all factors which can influence O7 production. Most important, as indicated by the authors, the use of chemiluminescence as the O 7 detection system may have produced an artificial elevation due to catalyzed oxidation of luminol by metals complexed with crocidolite (Hatch et al., 1980). Our study establishes that asbestos fiber induces superoxide anion production by AM, as indicated by SOD-inhibitable cytochrome c reduction. It is important to note that other components of the inflammatory response, particularly PMN, produce 0 5 when challenged with asbestos fibers in vitro (Rola-Pleszczynski et al., 1984). There has been some debate regarding the relative importance of AM and PMN in the intrapulmonary response to asbestos. In general, intratracheal injection models such as our own result in a large influx of PMN, although AM remain numerically predominant after the initial stages (Table 1). Inhalation models generally confirm AM as the chief component of the cellular response to asbestos (Brody et al., 1981). Bronchoalveolar lavage of human asbestotic subjects indicates that both cell types may be increased (Bignon et al., 1978). Work in progress in our laboratory indicates that the AM response of fiber-exposed miners may be quantitatively correlated to lung asbestos content (E Sebastien et al., unpublished observations). The concomitant increase in intrapulmonary PMN may be nonspecifically related to smoking and to the increased susceptibility to infection of patients with asbestosis. Regardless of the divergent stimuli to their accumulation, it is now clear that both AM and PMN may serve as generators of superoxide anion in asbestos-exposed individuals. Superoxide anion has been implicated in the pathogenesis of a number of other conditions in lung which can progress to fibrotic change. It is believed to contribute to the development of pulmonary oxygen toxicity (Crapo and Tierney, 1974) which can lead to interstitial fibroblastic proliferation (Crapo et al., 1980). O 5 is also generated in paraquat-induced lung injury, which produces subsequent fibroblastic proliferation in areas most affected (Smith et al., 1979). Other pulmonary conditions possibly involving 0 5 and characterized by interstitial fibrosis include long-term ozone exposure (Last and Greenberg, 1980) and idiopathic pulmonary fibrosis (McCormick et al., 1981). Superoxide anion generated by AM exposed to asbestos could contribute to pulmonary fibrosis either directly, through effects on collagen metabolism, or indirectly, through modulation of inflammation and repair. Reported O 7 effects on collagen metabolism have included displacement of vitamin C as a cofactor in collagen synthesis and induction of prolyl hydroxylase (Bhatnager, 1977). Increased prolyl hydroxylase activity has been noted 7 days following intratracheal asbestos injections (Davis and Reeves, 1971). Modulation of inflammation by O 2 was suggested by McCord et aI. (1980), who observed that exposure of human plasma to 0 2 and its reaction products resulted in the creation of a heat-labile, high-molecular-weight chemotactic factor for PMN. Complementary evidence is offered by studies in which hyperoxia induces increased numbers of PMN (Crapo et al., 1980; Fox et al., 1981).
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An example of the kind of mechanism possibly involved in asbestosis may be offered by the oxygen toxicity model of pulmonary fibrosis. Hyperoxia leads in experimental models to selective killing of pneumocytes, with subsequent overgrowth of interstitial cells and collagen deposition (Crapo et al., 1980). Asbestos may act in a similar fashion; the initial injury to the alveolar epithelium (Brody et al., 1981) evolving into ongoing interstitial cell proliferation and fibrosis. O 2 may play an early role in the development of this cycle of attraction of inflammatory cells, tissue destruction, and eventual interstitial scarring. Amphibole asbestos fibers such as crocidolite, amosite, and tremolite can remain in lung parenchyma for many years following inhalation exposures (Rowlands et al., 1982). The increased AM O~7 generation observed in vitro in our experiments may also occur in vivo in the continued presence of fibers. Net increases in O ~7 release in lung could occur either through an increased production efficiency by AM similar to that observed in vitro, and/or through the continuous recruitment of increased numbers of AM. In summary, crocidolite and short-fiber chrysotile fibers induced increased levels of superoxide anion generation by AM in vitro, as indicated by superoxide dismutase-inhibitable cytochrome c reduction. Future work in this area should concentrate on the in vivo replication of the observed effect following controlled inhalation exposures to asbestos. The effects of the specific O5 inhibitor SOD on the development of asbestos-induced bronchioloalveolitis and subsequent fibrosis should also be assessed.
ACKNOWLEDGMENTS The writers thank Dr, Arthur Langer for providing the short-fiber Coalinga asbestos samples, and Alex Collins and Joan Sorensen for technical and administrative assistance. This work was supported by a generous grant from the American Lung Association. Dr. Case is a fellow of the Conseil de la recherche en santo du Qu6bec.
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