Hamster Alveolar Macrophages Research Articles

Lack of iNOS expression and NO formation by mononuclear phagocytes from hamsters upon stimulation in vitro

In previous investigations, we demonstrated that hamster alveolar macrophages failed to induce the iNOS and produce NO upon stimulation in vitro. The subject of this study was to examine whether the lack of the iNOS pathway in hamster mononuclear phagocytes is tissue-specific. We investigated iNOS expression and NO production in isolated hamster (Lak:LVG(SYR)BR) monocytes (MO), pleural macrophages (PLM), and peritoneal macrophages (PM) in comparison to isolated rat (Crl:CD®(SN)BR) cells. NO release was measured as nitrite with the Griess reaction upon stimulation with lipopolysaccharide (LPS) and/or interferon-γ (INF-γ). The expression of the iNOS protein was detected with western blot-technique and iNOS mRNA was analyzed by RT-PCR. As expected, rat MO, PLM, and PM released NO upon stimulation with LPS and/or IFN-γ. However, hamster cells were not able to generate detectable amounts of NO. Expression of the iNOS protein and iNOS mRNA was only detected in rat, but not in hamster MO, PLM, and PM. Our data indicate that NO may not belong to the effector system of hamster mononuclear phagocytes.

Role of the scavenger receptor MARCO in alveolar macrophage binding of unopsonized environmental particles.

Alveolar macrophages (AMs) avidly bind and ingest unopsonized environmental particles and bacteria through scavenger-type receptors (SRs). AMs from mice with a genetic deletion of the major macrophage SR (types AI and AII; SR-/-) showed no decrease in particle binding compared with SR+/+ mice, suggesting that other SRs are involved. To identify these receptors, we generated a monoclonal antibody (mAb), PAL-1, that inhibits hamster AM binding of unopsonized particles (TiO2, Fe2O3, and latex beads; 66 +/- 5, 77 +/- 2, and 85 +/- 2% inhibition, respectively, measured by flow cytometry). This antibody identifies a protein of approximately 70 kD on the AM surface (immunoprecipitation) that is expressed by AMs and other macrophages in situ. A cDNA clone encoding the mAb PAL-1-reactive protein isolated by means of COS cell expression was found to be 84 and 77% homologous to mouse and human scavenger receptor MARCO mRNA, respectively. Transfection of COS cells with MARCO cDNA conferred mAb-inhibitable TiO2 binding. Hamster MARCO also mediates AM binding of unopsonized bacteria (67 +/- 5 and 47 +/- 4% inhibition of Escherichia coli and Staphylococcus aureus binding by mAb PAL-1). A polyclonal antibody to human MARCO identified the expected approximately 70-kD band on Western blots of lysates of normal bronchoalveolar lavage (BAL) cells (>90% AMs) and showed strong immunolabeling of human AMs in BAL cytocentrifuge preparations and within lung tissue specimens. In normal mouse AMs, the anti-MARCO mAb ED31 also showed immunoreactivity and inhibited binding of unopsonized particles (e.g., TiO2 approximately 40%) and bacteria. The novel function of binding unopsonized environmental dusts and pathogens suggests an important role for MARCO in the lungs' response to inhaled particles.

Formation of nitric oxide by rat and hamster alveolar macrophages: an interstrain and interspecies comparison

Nitric oxide (NO) plays an important role in non-specific host defense, which can be recognized by its antimicrobial and cytotoxic activity against pathogens. However, there appear to exist interspecies differences in the ability of macrophages to generate NO. The object of this study was to determine whether there exist intraspecies differences in the production of NO. We compared NO formation by alveolar macrophages (AM) from five different rat strains (Sprague–Dawley, Wistar, Lewis, Fisher, and Brown Norway), two different stocks of Syrian Golden hamsters, and one stock of Chinese hamsters. The AM were harvested by bronchoalveolar lavage and stimulated in vitro with various concentrations of LPS and/or IFN- γ. The oxidation product of NO, nitrite, was measured in the AM supernatant by the Griess reaction. Upon stimulation with LPS and/or IFN- γ, AM from all five rat strains were able to release NO, but the amount of NO produced differed significantly among the rat strains. However, none of the stimuli was able to induce AM from the two stocks of Syrian Golden hamsters as well as AM from the stock of Chinese hamsters to release measurable amounts of NO. These findings point to distinct regulatory mechanisms of the NO pathway in AM from different species and to variations of this mechanism in the AM from the investigated rat strains.

ANALYSIS OF AIR POLLUTION PARTICULATE-MEDIATED OXIDANT STRESS IN ALVEOLAR MACROPHAGES

Adverse health effects of urban air pollution particulates may be attributable to particle mediated oxidant stress and inflammation. Intracellular oxidant production in normal hamster alveolar macrophages (AMs) was measured upon exposure to concentrated ambient particulates (CAPs), residual oil fly ash (ROFA), and their water-soluble and particulate fractions. ROFA and CAPs caused increases in dichlorofluorescin (DCFH) oxidation, a fluorescent measure of intracellular reactive oxygen species (ROS) production, comparable to the positive control, phorbol myristate acetate (PMA). The water-soluble component of both CAPs and ROFA (CAPs, S and ROFA, S) significantly increased AM oxidant production over negative control. CAPs samples and components showed substantial day-to-day variability in their oxidant effects. Metal chelation by desferrioxamine (DF, 1 mM) caused significant inhibition of particulate-induced AM oxidant production. ROFA exposure resulted in increased macrophage inflammatory protein-2 (MIP-2) message in AMs and in increased tumor necrosis factor alpha(TNF-alpha) production by the monocyte-macrophage cell line, RAW 264.7. TNF- production was inhibitable by the antioxidant N -acetylcysteine (NAC). The data suggest that metal components adsorbed to urban air pollution particulates can significantly contribute to particulate ability to cause oxidant stress and cytokine production in AMs.

Alveolar macrophage interaction with air pollution particulates.

We applied flow cytometric analysis to characterize the in vitro response of alveolar macrophages (AM) to air pollution particulates. Normal hamster AM were incubated with varying concentrations of residual oil fly ash (ROFA) or concentrated ambient air particulates (CAP). We found a dose-dependent increase in AM-associated right angle light scatter (RAS) after uptake of ROFA (e.g., mean channel number 149.4 +/- 6.5, 102.5 +/- 4.1, 75.8 +/- 3.5, and 61.0 +/- 4.6 at 200, 100, 50, and 25 mg/ml, respectively) or CAP. A role for scavenger-type receptors (SR) in AM uptake of components of ROFA and CAP was identified by marked inhibition of RAS increases in AM pretreated with the specific SR inhibitor polyinosinic acid. We combined measurement of particle uptake (RAS) with flow cytometric analysis of intracellular oxidation of dichlorofluorescin. Both ROFA and CAP caused a dose-related intracellular oxidant stress within AM, comparable to that seen with phorbol myristate acetate (PMA) (e.g., fold increase over control, 6.6 +/- 0.4, 3.6 +/- 0.4, 4.6 +/- 0.5, 200 mg/ml ROFA, 100 mg/ml ROFA, and 10(-7) M PMA, respectively). We conclude that flow cytometry of RAS increases provides a useful relative measurement of AM uptake of complex particulates within ROFA and CAP. Both ROFA and CAP cause substantial intracellular oxidant stress within AM, which may contribute to subsequent cell activation and production of proinflammatory mediators.

Species differences in NO formation by rat and hamster alveolar macrophages in vitro.

Nitric oxide (NO) is a cellular mediator and regulator of multiple biologic functions. NO released by alveolar macrophages (AM) is suggested to play a role in mediating pulmonary injury. In murine and rat macrophages, the expression of inducible NO synthase (iNOS) and the release of NO are well established. However, the existence of such a pathway in other species remains controversial. In this study, we examined NO production and iNOS expression by AM from rats and hamsters, two laboratory animal species that are characterized by their disparate pulmonary responses to various inhaled irritants/toxicants. AM were treated with lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), or tumor necrosis factor-alpha (TNF-alpha) in vitro, and nitrite, the stable oxidation product of NO, was assayed by the Griess reaction. Rat AM produced NO in a dose- and time-dependent manner upon stimulation with LPS and/or IFN-gamma, but not with TNF-alpha. Surprisingly, hamster AM did not release detectable levels of NO after the same treatment. Although iNOS expression was demonstrated in rat AM by immunocytochemical and Western blot analyses, no induction of iNOS expression could be found in hamster AM. Using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, we found that rat and hamster AM could be induced to express iNOS mRNA after treatment with LPS and IFN-gamma. The results presented here indicate that hamster AM, in contrast to rat AM, lack the ability to express iNOS protein and to generate NO in response to LPS, IFN-gamma, or TNF-alpha in vitro. In conclusion, our data suggest striking differences in iNOS regulation and NO production by AM from rats and hamsters, two rodent species that are commonly used in biomedical research and well-known for their disparate responses to pulmonary irritants/toxicants.

Mechanisms regulating macrophage-induced nitric oxide production by spontaneously transformed hamster fibroblasts

Nitric oxide has been implicated as an important effector molecule involved in tumor cell growth and cytotoxicity. In these studies we examined mechanisms regulating nitric oxide production by hamster tumor cells. Cocultures of hamster alveolar macrophages (HAM) and spontaneously transformed hamster embryonic fibroblasts (STHE cells) produced significant quantities of nitric oxide in response to lipopolysaccharide (LPS). Culture supernatants from HAM treated with LPS also stimulated nitric oxide production by STHE cells, whereas tumor cell culture supernatants had no effect on HAM. These data, together with the findings that paraformaldehyde treatment of STHE cells, but not macrophages, completely abrogated nitric oxide production in the cocultures demonstrate that the tumor cells were the source of this mediator. In contrast to STHE cells, STHE-83/20 cells, a highly malignant variant, did not produce nitric oxide in response to HAM or HAM culture supernatants even in the presence of LPS. Both anti-tumor necrosis factor-alpha (TNF-alpha) and anti-interleukin-1alpha (IL-1alpha) antibodies inhibited HAM-induced nitric oxide production by STHE cells. However, the kinetics of their effects were different. Moreover, although the nitric oxide stimulating activity in HAM culture supernatants was abrogated by anti-TNF-alpha antibody, it was only minimally reduced by anti-IL-1alpha antibody. These data demonstrate that TNF-alpha and IL-1alpha play distinct roles in induction of nitric oxide synthesis in STHE cells. HAM were also found to suppress proliferation of STHE cells, an effect that was inhibited by anti-TNF-alpha antibody, but not NG-monomethyl-L-arginine, which blocks nitric oxide synthase. Abrogation of macrophage-induced cytostasis in STHE cells by anti-TNF-alpha antibody was associated with decreased nitric oxide production. Thus TNF-alpha released by macrophages may indirectly activate STHE cells for nitric oxide synthesis by suppressing tumor cell proliferation.

Chronic inhalation and biopersistence of refractory ceramic fiber in rats and hamsters.

Lifetime "nose-only" inhalation studies were conducted in rats using four types of refractory ceramic fibers (FCF), 1 micron in diameter x 22 to 26 microns length: High Purity, Kaolin, Zirconia, and After-Service; and on hamsters using Kaolin RCF. For comparison, animals also were exposed to chrysotile fibers. Rats were exposed 6 hr/day, 5 days/week for 24 months to concentrations ranging between 3 and 30 mg/m3. Time- and dose-dependent lesions in the rat included the development of interstitial fibrosis, pleural fibrosis, pulmonary tumors, and mesothelioma. Exposure to 3, 9 or 16 mg/m3 produced no excess lung tumors; no fibrosis was seen at 3 mg/m3. A significant increase in lung tumors and interstitial fibrosis was observed at 30 mg/m3. A single mesothelioma was observed in rats exposed to 9 mg/m3, while two occurred at 30 mg/m3. Hamsters were similarly exposed to 30 mg/m3 Kaolin RCF for 18 months; no lung tumors were induced, but pulmonary and pleural fibrosis were observed and there was a 42% incidence of mesothelioma. Multiple interim sacrifices together with recovery animals allowed detailed assessment of the lung burden of RCF, which was found to be dose related and, at the high doses, exceeded 10(5) fibers/mg of dry lung. During the various recovery periods there was a clear reduction in fiber burden. Mathematical modeling of these data for deposition, clearance, and retention and for species is currently underway.

Influence of particle dose on the cytotoxicity of hamster and rat pulmonary alveolar macrophage in vitro.

Silica and ferric oxide are common industrial exposures. Studies have indicated that all commonly occurring forms of crystalline silica can cause fibrotic lung disease. There is evidence to indicate that crystalline silica is carcinogenic in humans who have not developed silicosis, while amorphous silica is not carcinogenic in humans. An important biological response to particles deposited deep in the lung is their engulfment by pulmonary alveolar macrophages (AM). To assess the role of AM in silica-induced lung disease, particle size distribution and surface area of crystalline, gelled, precipitated, and fumed silica, ferric oxide, and aluminum oxide were characterized; the cytotoxicity of the particles to hamster and rat AM in vitro was measured at 0.0-0.5 mg/1 x 10(6) cells at 24 and 48 h using dye exclusion procedures. The count medium diameter for aluminum oxide, ferric oxide, and amorphous silica was equal to or less than 0.38 microns, while for crystalline silica the value was 0.83 microns. The surface areas for the amorphous silicas and the aluminum oxide ranged from 253 to 125 m2/g with gelled silica having the highest value; the values for crystalline silica and ferric oxide were 4.3 and 10.8 m2/g, respectively. Crystalline silica (1.6%) was detected in the fumed silica, while none was detected in precipitated or gelled silica. With gelled silica, based on the dose of the particle, the viability of the hamster AM decreased to 27% at 0.05 mg and to zero at 0.1 mg at 24 h. At doses of 0.05 and 0.1 mg of crystalline, precipitated, or fumed silica, the percent viability decreased significantly to 76-67% and 51-42%, respectively, and to zero at 0.5 mg. Macrophages viable at 24 h decreased further at 48 h compared with the control culture. The ferric oxide and the aluminum oxide showed minimal to no changes in viability. Similar results for the particles were obtained with rat AM. The results indicate that precipitated and fumed amorphous silica tested at equivalent doses are equally as toxic to AM lavaged from two species of rodents as crystalline silica; gelled silica is more toxic than crystalline. Ferric oxide and aluminum oxide are noncytotoxic in this system. The results of this study indicate that the dose as well as the surface area and surface characterization are important determinants in the cytotoxicity of hamster and rat AM to these particles.

Contribution of osmium tetroxide to the image quality and detectability of iron in cells studied by electron spectroscopic imaging and electron energy loss spectroscopy.

The detection of elemental distributions within ultrastructural cellular components presents a number of challenges. There are many technical questions that need to be resolved including optimal fixation protocols. Another is the impact of heavy metals, such as osmium tetroxide (OsO4), on the detectability of other elements when OsO4 is used in chemical fixation protocols for biological samples. OsO4 was examined by varying its concentrations from 0% to 1% and time of fixation from 5 to 30 minutes with hamster alveolar macrophages. The morphological quality of cellular images observed and the detectability of iron using electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) were evaluated. One percent OsO4 for 30 minutes in the chemical fixation protocol enhances the quality of the ESI and does not interfere with the ESI or EELS signal of iron. Positive results from both methods indicate the presence of the specific element. The loss of 59Fe during the chemical fixation procedure was also studied. Less than 10% was lost during the primary fixation step, but minimal losses occurred through dehydration, embedding, and sectioning. Careful technical assessment of the presence of an element as well as factors which might interfere with its detection is an important step in the application of any analytical microscopic technique.

The contribution of osmium tetroxide to the image quality and detectability of elements in cells studies by electron spectroscopic imaging

Osmium tetroxide has been recognized as an excellent fixative which also contributes to the quality of TEM micrographs (1). In the preparation of biological materials for electron spectroscopic imaging (ESI) of 30 run sections with the Zeiss CEM902 electron microscope, OsO4 is usually used in fixation, but poststaining with heavy metals is not needed. However, the extent of the contribution of OsO4, to the images obtained had not been defined clearly, nor was it clear the extent to which the presence of OsO, might obscure detection of elements by ESI or EELS. To establish optimal liquid fixation for the preservation of soluble ions we monitored the loss of Fe ions during preparation. To determine the role of Os4O, in ESI imaging, hamster alveolar macrophages (AMS) were incubated with 0.ImM FeCLo for 15 minutes, then fixed with 2.5% glutaraldehyde in 0.IM K phosphate buffer in 0.01% CaCLo, then grouped into 6 samples varying the times and percentages of OsO, . One sample was not exposed to any OsO4.

Dynamics of uptake and channeling of arachidonic acid in hamster alveolar macrophages and the effect of bleomycin on arachidonyl distribution

The uptake and distribution of [ 14C]-arachidonic acid (AA) by primary culture hamster alveolar macrophages (AM) were examined. The macrophages were incubated for 2 to 18 hrs in RPMI-1640 medium containing 0.1% BSA and [ 14C]-AA. The uptake of [ 14C]-AA by AM was rapid with 71% and 83% of exogenous [ 14C]-AA taken up after 2 and 4 hrs of incubation, respectively. Initially, the uptaken [ 14C]-AA was equally distributed between phospholipids (PL) and neutral lipids (NL). However, by 8 hrs, 86% and 14% of [ 14C]-AA was found in PL and NL, respectively. This distribution pattern remained constant through 18 hrs. Within the PL pool, most of the [ 14C]-AA was initially incorporated into phosphatidylcholine (PC). However, with time, as the percent of [ 14C]-AA incorporated in PC declined, the percent incorporated in phosphatidylethanolamine increased. The incorporation of [ 14C]-AA into sphingomyelin, phosphatidylinositol, and phosphatidylserine remained constant with time. Within the NL, most of the [ 14C]-AA was initially found incorporated into triacylglycerols (TG). After 4 hrs, the percent of [ 14C]-AA found in TG decreased markedly, while the percent found in cholesterol esters markedly increased. Incubation of AM prelabeled with [ 14C]-AA with bleomycin at 0.5, 5, 50 and 500 μM for 2 and 8 hrs failed to have any effect on the distribution of [ 14C]-AA in PL and NL pools.

Selective down-regulation of alveolar macrophage oxidative response to opsonin-independent phagocytosis.

We have compared the oxidative response of alveolar macrophages (AM) during opsonin-dependent and independent phagocytosis by using multiparameter flow cytometry. The respiratory burst of AM during phagocytosis was quantitated by the intracellular oxidation of the nonfluorescent precursors dichlorofluorescin diacetate (DCFH) or hydroethidine (HE, a reduced precursor of ethidium) to their fluorescent (oxidized) counterparts. After loading freshly isolated normal hamster AM with DCFH or HE, red or green fluorescent beads, respectively, were added to the shaking cell suspensions. Ingestion of opsonized particles by AM caused a marked increase in oxidation of both DCFH and HE proportional to the number of beads ingested. In contrast, uptake of one to three unopsonized particles per cell led to inhibition of oxidative activity compared to control cells incubated without particles. AM ingesting four or more unopsonized particles showed some increase in oxidative metabolism, but far less than that with identical numbers of particles in opsonin-dependent ingestion. Similar results were obtained using fluorescent labeled staphylococcal bacteria. Using three-color flow cytometry to study cells ingesting both types of particles, cells first ingesting unopsonized beads were also found to have an inhibited oxidative response to subsequently ingested opsonized particles. The mitochondrial poison antimycin inhibited most of the intracellular oxidative response to either type of phagocytosis. The remaining antimycin-insensitive, membrane derived respiratory burst of AM was also substantially diminished after phagocytosis of unopsonized particles vs similar numbers of opsonized particles. The greatly increased mitochondrial respiration in AM during phagocytosis of opsonized particles may be related to bactericidal mechanisms. Killing of ingested Staphylococcus by AM was markedly impaired in the presence of antimycin. The results suggest that AM may ingest the numerous, unopsonized inert particles that are inhaled without generation of potentially toxic oxygen metabolites, while retaining the capacity to undergo a respiratory burst after ingesting opsonized particles and bacteria. The mechanism(s) for this distinct response may include generation of an inhibitor of intracellular oxidative metabolism.

Oxidative Metabolism in the Alveolar Macrophage: Analysis by Flow Cytometry

We evaluated the reagents dichlorofluorescin (DCFH) and hydroethidine (HE) for use in flow cytometric analysis of the respiratory burst of alveolar macrophages and monocytes. DCFH and HE are non-fluorescent precursors which can be oxidized intracellularly to the fluorescent compounds dichlorofluorescein and ethidium. Alveolar macrophages (AMs) loaded with either DCFH or HE were analyzed after phorbol myristate acetate (PMA) stimulation. The results, expressed as fmol/cell oxidation product (DCF or ethidium) after fluorometric standardization of the flow cytometer, show that both DCFH (273 +/- 48, mean increase over control +/- SE, fmol/cell, N = 9) and HE (416 +/- 54, N = 11) detected the substantial respiratory burst of hamster AMs. Similar results were obtained with normal human AMs. By using multiparameter analyses, the oxidative response of AMs ingesting opsonized fluorescent latex beads was measured in subpopulations ingesting increasing numbers of particles. A graded increase in oxidation of both DCFH and HE was found in response to increasing phagocytosis. Ingestion of fluoresceinated staphylyococcal bacteria caused similar changes in HE-loaded AMs. Inhibition of respiration with antimycin showed that approximately 95% of the increased oxidative metabolism of hamster AMs ingesting opsonized beads or bacteria was mitochondrial. The remaining 5% (10-40 fmol/cell) is membrane-derived oxidative activity quantitatively similar to that measured in assays of extracellular release of H2O2. Monocytes loaded with either DCFH or HE showed substantial increases in fluorescence after PMA stimulation (mean % increase over control +/- SE at 30 min: 464 +/- 104, DCFH, 505 +/- 156, HE). While DCHF is known to measure H2O2, HE is less well characterized. Exposure of cells to an extracellular source of both superoxide anion (O2-) and H2O2, xanthine oxidase-xanthine, resulted in marked oxidation of intracellular HE. Addition of both superoxide dismutase and catalase blocked this oxidation, indicating that HE can detect both O2- and H2O2. These agents can be useful probes for precise analysis of oxidative metabolism during phagocytosis in AMs and other mononuclear phagocytes.

Isolation and antigenic identification of hamster lung interstitial macrophages.

Lung interstitial macrophages (IMs) are a large, distinctive population of cells with important proliferative capacities. Characterization of their role in health and disease has been hampered by inadequate methods to separate interstitial from residual alveolar macrophages (AMs) in preparations of individual mononuclear cells from lung tissue. In this study, a specific cell-surface antigen (HAM1) present on more than 90% of hamster AMs, but not expressed by hamster IMs, was used to distinguish these populations. After collagenase digestion of lung tissue slices from exhaustively lavaged and perfused hamster lungs, mononuclear phagocytes were isolated by density gradient centrifugation. The mean yield of lung digest macrophages (3.9 +/- 1.9 (SD) x 10(6] was comparable to the yield of lavaged AMs (4.2 +/- 1.9 x 10(6]. The proliferative capacity of lavaged AMs, blood monocytes, and lung digest macrophages was compared using a soft-agar colony-forming unit (CFU) assay. Both lung digest macrophages and blood monocytes had significantly more CFUs (68.7 +/- 2.6 and 53.5 +/- 8.4 CFU/10(3) cells [mean +/- SEM], respectively) than did AMs (16.5 +/- 1.7) (p less than 0.01). To further define the composition of the lung digest macrophage population, flow cytometric analysis of fixed cells from six experiments was performed using a mouse monoclonal antibody specific for the HAM1 antigen found only on AMs. The lung digest macrophage population consisted of both antigen-negative IMs (78.2% +/- 3.7% [SEM]; n = 6) and antigen-positive, residual AMs (21.8% +/- 3.7%). Morphometric counts confirmed that substantial numbers of AMs are left behind after lavage and contribute to macrophages obtained from lung tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Asbestos effects on superoxide production: An in Vitro study of hamster alveolar macrophages

Inhaled asbestos induces accumulation of alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) in lung. Asbestos-enhanced production of superoxide anion (O2-) 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, bronchoalveolar 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 O2- production by AM have been determined in cell populations derived from the Syrian golden hamster. AM for in vitro study were obtained by BAL. O2- production was monitored as superoxide dismutase (SOD) - inhibitable cytochrome c reduction. Significant rises in O2- release by AM were noted in the presence of 0.4 mg/ml crocidolite (2.53 +/- 0.33 nmole cytochrome c reduced/10(6) cells/30 min, 37 degrees C; controls 1.13 +/- 0.18 nmole; P less than 0.02). Chrysotile induced levels of O2- release in AM which were similar to those evoked by crocidolite.