Production of Tumor Necrosis Factor-a and Interleukin-6 by Human Alveolar Macrophages Exposed In Vitro to Coal Mine Dust Philippe Gosset, Philippe Lassalle, Dominique Vanhee, Benoit Wallaert, Colette Aerts, Cyr Voisin, and Andre-Bernard Tonnel CIBP, INSERM Contrat Jeune Formation no. 90-06, and the Laboratoire de pathologie respiratoire experimentale et de pollution atmospherique, Institut Pasteur, Lille, France

Following our previous demonstration of cytokine secretion by alveolar macrophages (AM) from coal miners and from patients with coal workers' pneumoconiosis, we investigated the effectof in vitro exposure to coal dust and to its silica content on tumor necrosis factor-a (TNF) , interleukin (IL)-I,B, and IL-6 production by normal human AM. TNF and IL-l,B concentrations were estimated by a specific radioimmunoassay, while IL-6levels were evaluated by the proliferation of 7TDI cells. After 24-h culture, coal dust triggered a significant release of TNF and IL-6 at the dose of 0.1 mg/ml and more obviously at 1 mg/ml in comparison with titanium dioxide (TiOz) , used as a biologically inert control dust (with 1 mg/ml of dust: 3,526 ± 3,509 versus 330 ± 138 pg TNF/ml and 224 ± 74 versus 72 ± 34 U IL-6/ml, respectively; P < 0.01 in both cases). After 3-h culture, a significant TNF secretion as well as an increased TNF mRNA expression were also detected for AM stimulated by coal dust at variance with TiOz. In contrast, no modification ofIL-I/J concentration could be evidenced in AM exposed to coal dust, although we detected an increased expression of specific mRNA expression. In order to define the role of silica among the main components of coal dust in AM activation, we evaluated the effect of silica (a-quartz, 30 ,ug/ml, which is the concentration and the type of silica present in our coal dust) alone or mixed with TiOz (l mg/ml) on monokine production. Both preparations had no effect on TNF, IL-l/J, or IL-6 secretion by normal AM, whereas a higher dose of silica (0.1 mg/ml) induced a significant production of TNF (1,223 ± 354 versus 315± 158 pg/ml for AM exposed to TiOz; n = 3). In conclusion, we demonstrated that coal dusts induced the release of AM-derived mediators possibly involved in lung damage occurring in pneumoconiosis. However, this cytokine secretion was induced by compounds from our coal mine dust that are not directly related to the simple presence of silica, an additional argument supporting that besides silica other compounds or a complex interaction between the different particles exert a significant role in the development of coal workers' pneumoconiosis.

Coal workers' pneumoconiosis (CWP) is one of the most widespread fibrotic lung diseases. Caused by inhalation of coal dust, CWP is usually divided into two groups: simple pneumoconiosis (SP), in which opacities smaller than 1 em are observed, and progressive massive fibrosis (PMF) , characterized by the development of extensive radiologic abnormalities and severe alterations in pulmonary functions (1-6). At present, the relationship between the severity of CWP and the composition of inhaled mine dust remains a (Received in original form December 28, 1990 and in revised form April 16, 1991) Address correspondence to: Dr. P. Gosset, CIBP, Institut Pasteur, BP 245, 59019, Lille, France. Abbreviations: alveolar macrophage(s), AM; bronchoalveolar lavage, BAL; coal workers' pneumoconiosis, CWP; progressive massive fibrosis, PMF; interleukin, IL; lipopolysaccharide, LPS; radioimmunoassay, RIA; sodium dodecyl sulfate,'SDS; titanium dioxide, Ti(h; tumor necrosis factor-a, TNF. Am. J. Respir. Cell Mol. BioI. Vol. S. pp. 431-436, 1991

subject of controversy: there is no conclusive evidence for a direct relationship between the occurrence of PMF lesions and either the total quantity of inhaleu' dust during the occupational exposure or the respective content in silica and/or other mineral component of various dusts to which coal workers were exposed (7). Moreover, PMF has also been reported to occur after inhalation of silica-free carbon (8). Some pathologic studies have demonstrated in the lungs of coal miners with PMF the presence of only small quantities or even the absence of silica (9). Although the physiopathology of pneumoconiosis remains incompletely understood, several lines of evidence suggest the participation of the alveolar macrophage (AM), at least in the initiation of the alveolitis: (1) phagocytosis of mineral dusts induces the secretion of inflammatory and immune products that could be relevant to the development of interstitial lung disorders (9-12, 41); (2) human bronchoalveolar lavage (BAL) studies have demonstrated that in the lower respiratory tract the total cellular count was increased

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several-fold, essentially related to the local influx of mononuclear phagocytes (13); and (3) AM from pneumoconiotic patients were shown to spontaneously produce large amounts of oxidants, neutrophil chemotactic factor, fibronectin, interleukin (lL)-l, and tumor necrosis factor-a (TNF) (14-19). Among several AM-derived fibrogenic factors now identified, TNF appears to playa key role in that a single instillation of silica in mice leads to a marked increase in the level of lung TNF production. In addition, silica-induced lung fibrosis is almost completely prevented by anti-TNF antibodies (16). In order to better characterize the mechanism of coal dust-dependent AM activation, we have investigated the effects of silica (a component present in the coal mine dust) on the production of various cytokines (TNF, IL-ll3, and IL-6) by normal human AM. Our results indicate that TNF release induced by our coal mine dust is not exclusively linked to the presence of silica, suggestingthat the initial AM activation was due to other as yet unidentified compounds or a complex interaction between the different particles (i.e., a matrix effect).

Materials and Methods Patients and BAL Procedure Informed consent for BAL was obtained from 10 normal donors. BAL was performed as described previously (13) by instillation of saline into the bronchoalveolar tree under fiberoptic bronchoscopy. AM Isolation and Culture AM were isolated as previously described (20). Briefly, the lavage fluid was filtered through sterile surgical gauze and centrifuged at 400 x g for 10 min at 4° C. After three washings, the pellet was resuspended at a cell concentration of 1.5 X lQ6/ml in RPMI 1640 containing 5 % of heat-inactivated fetal calf serum and 2 mM L-glutamine (GIBCO, Flobio, Courbevoie, France). Endotoxin contamination of medium was controlled by limulus amoebocyte test (Coatest; Kabevitrum, Vienna, Austria) and was less than 50 pg/ml. This quantity was insufficient to trigger TNF mRNA expression and secretion as previously demonstrated (21). Cells were allowed to adhere to plastic petri dishes (2 ml in a 35-mmdiameter well) for 2 hat 37° C. The nonadherent cells were removed by three washings with RPMI. Adherent cells contained more than 95 % AM and less than 1% lymphocytes. AM Exposure to Mineral Dusts Coal mine dust samples were obtained by air filtration from the coal basin of the north of France. They were representative of this basin and contained 55.4% mineral components, of which 3.2 % was silica of the a-quartz type. Pure silica, an a-quartz from Madagascar, and titanium dioxide (Ti02) (Kronos, Neuilly, France), known as an example of inert dust, were provided by the Cerchar (Verneuil en Halatte, France). All particles had a diameter smaller than 5 jtm and had similar size distributions. The mineral dusts were successively autoclaved for 90 min at 180° C, incubated 3 h with 1% E-Toxa clean (Sigma Chemical Co., St. Louis, MO), and washed extensively with endotoxin-free RPMI 1640. These

preparations contain less than 50 pg/ml of endotoxins (Coatest). After 24-h incubation at 37° C in humidified air with 5% CO 2 , culture supernatants were harvested and filtered through a 0.45-mm filter (Millipore, Molsheim, France). AM were also stimulated with lipopolysaccharide (LPS) (10 jtg/ml, type 055B5; Difco, Detroit, MI), supernatants of which were used as positive controls of monokine production. TNF Assay TNF concentration was evaluated sequentially by the L929 cytotoxic test and by radioimmunoassay (RIA). Biologic activity was estimated from a standard curve as the amount necessary to kill 50 % of actinomycin-treated L929 mouse fibroblasts after an 18-h culture. Subsequently, TNF level was confirmed by competitive RIA (IRE-Medgenix, Fleurus, Belgium) after adequate dilution. A highly significant correlation was found between both methods (r = 0.9, P < 0.001). TNF concentration was finally expressed in ng/ml as defined by RIA. IL-l Assay We have recently demonstrated the presence of an IL-I inhibitory factor in the supernatant of AM unstimulated or activated by anti-IgE (22). Consequently, the use of the IL-1 bioassay was inappropriate in this study and IL-1{j levels were evaluated by an immunoradiometric assay (IRE-Medgenix). Results were expressed in ng/ml. IL-6 Analysis IL-6 was assayed as described by Van Snick and associates (23) with some modifications. Briefly, the hybridoma cells 7TD1 (a generous gift of Dr. Van Snick, Institut Ludwig, Brussels, Belgium) were incubated with serial dilutions of cell supernatants in microtiter plates. After 4 days of incubation, the number of cells was evaluated by a colorimetric method. A 4 mg/ml solution of3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT; Sigma) was added at 10%. After a 4-h incubation, the supernatants were discarded and 0.2 ml of 0.04 N HCI-isopropanol solution was added to each well to solubilize the reduced MTT precipitate. After homogenization, the optical density was read in a multiwell spectrophotometer at 570 nm with a reference at 650 nm. All analyses were performed in duplicate, and concentrations were calculated by a probit analysis in comparison with a standard of recombinant human IL-6 (Boehringer Mannheim, Meylan, France). Results are expressed in V/ml. Specificity of the test was controlled by inhibition of cell proliferation with addition of a neutralizing anti-IL-6 rabbit antibody (Genzyme, Boston, MA). In all cases, specific antibody addition to AM supernatants induced an inhibition greater than 90 %. Northern Blot Analysis of TNF, IL-I13, and IL-6 mRNA Mononuclear phagocyte stimulation was performed as outlined above. After 3-h culture, total cellular RNA was isolated by a guanidium isothiocyanate method with cesium chloride modification (24). Equal amounts of RNA, determined by adsorption at 260 nm, were denatured at 50° C for

Gosset, Lassalle, Vanhee et al.: TNF-a and IL-6 Production by Human AM Exposed In Vitro to Coal Mine Dust

1 h in sample buffer containing glyoxal and then fractionated by electrophoresis through agarose gel. RNA transfer to nitrocellulose membranes was accomplished by capillary blotting for 18 h. After transfer, membranes were dried, baked at 80° C in vacuum, and RNA was visualized by methylene blue staining to determine the position of 18S and 28S rRNA bands, to assess the integrity of RNA, and to check that equal amounts had been loaded in all wells after transfer to nitrocellulose, Nucleic acid hybridization was carried out to quantify the expression of IL-l{3 and TNF mRNA. Prehybridization was performed at 63° C for 16 to 24 h in buffer containing 50 % formamide, 50 mM phosphate buffer, 5 X SSC, 2 mM EDTA, 0.1% sodium dodecyI sulfate (SDS), and 2.5 x Denhardt solution (all from Sigma) . Labeled RNA probes were obtained by transcription of the Pst 1 linear fragment (0.3-kb TNF fragment length in pRSl plasmid) and of the Eco RI linear fragment (0.3-kb IL-l{3 fragment length in pSP 1 plasmid, a generous gift ofP. Vassali, Geneva). The probes were labeled with a specific activity of 5 X 106 cpm/ng by using (3zP]dUTP (Amersham International, Amersham, UK) . Hybridization was performed for 18 h at 65° C in prehybridization buffer with 2 X 106 cpm/ml of labeled probe. The membrane was successively washed with 2x SSC, 0.1% SDS (2 times for 15 min at 65° C), and then with 0.2x SSC and 0.1% SDS for 15 min at 73° C. To estimate the IL-6 mRNA expression, we used a probe of 0.8 kb (kindly provided by D. Koffman, DNAX, Palo Alto, CA) . The insert was labeled by the hexamer technique using the Klenow enzyme (Boehringer-Mannheim). Hybridization was performed in the same buffer as described previously at 60° C. The membrane was washed 2 times with 0.2x SSC and 0.1% SDS and then with O.1x SSC and 0.1% SDS for 15 min at 60° C. The blots were then dried and exposed to Kodak X-Omat X-ray film (Eastman Kodak, Rochester, NY). Statistical Analysis Statistical analysis was performed by the Wilcoxon rank-sum test, and results are expressed as mean ± SEM .

Results Effects of Coal Dusts on TNF Production by AM: Dose Response and Kinetics Increasing concentrations of TiOz and coal dust were added to AM preparations, and TNF was measured after 24-h culture. TNF assay was performed concomitantly by the L929 cytotoxic test and by an RIA . Both tests gave equivalent results with a significant correlation (r = 0.9; P < 0.001). Whereas TiOz, even at the highest concentration (1 mg/ml) induced only a slight increase (P = NS) of TNF production, coal dusts triggered a significant release of TNF at the dose of 0.1 mg/ml and more obviously at 1 mg/ml (Figure 1). In contrast, culture medium incubated for 24 h with coal dust or TiO z contained no detectable amounts of TNF. As shown in Figure J, a significant TNF amount was already detected in supernatants collected at 3 h from AM stimulated with coal dusts at variance with TiOz (n = 3, P < 0.05). The values obtained after 3-h culture corresponded to 30%

433

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of the amount obtained after 24-h culture. This ratio was equivalent to that obtained from LPS-activated AM (23 %, TNF from LPS-activated AM after 3-h culture and 24-h culture: 59,846 ± 14,978 and 271,450 ± 52,255 pg/ml, respectively). Levels of TNF Release by AM Exposed to Optimal Concentrations of Coal Dusts and Role of Silica Content We then evaluated the effect of coal dust addition (at the dose of 1 mg/ml) on cultured AM isolated from nine different healthy subjects. As shown in Table 1, coal dust exposure induced a 12-fold increase ofTNF release (P< 0.01), whereas TiO z had no significant effect (P = NS). The level obtained in the presence of coal dust represented less than 2 % of that detected in LPS-stimulated AM (271,450 ± 52,255 pg/ml). Microscopic observation of AM exposed to particles during 24 h revealed that all AM had phagocytized a large number of TiO z particles or coal dusts, but these cells were still 95 % viable as revealed by trypan blue exlusion test. In order to evaluate the involvement of silica content in the TNF production by AM treated by coal dust, the effect of the exposure to an amount of silica equivalent to that contained in coal dust was measured. The coal dust used in our study contained 3 % silica. The addition of the same type of silica (a-quartz, 0.03 mg/rnl) alone or simultaneously with TiOz induced no significant TNF release (Table 1). However, as demonstrated with rat AM (25), a higher concentration of silica (0.1 mg/ml) increased TNF production (1,223 ± 354 versus 315 ± 158 pg/ml for AM exposed to TiOz; n = 3). Evaluation of IL-l,6 Concentrations in AM Exposed to Mineral Dusts AM exposed to mineral dusts (coal dust, silica, and TiOz with or without addition of 0.03 mg/ml silica) released no significant amounts ofIL-IJj (Table 1). Moreover, evaluation on supernatants collected after 3- or 24-h cultures from AM stimulated with different concentrations of coal dusts or with 0.1 mg/ml of silica revealed no modification ofIL-l,6 concentrations (data not shown), In contrast, LPS addition (10 I-tg/ml) on the same AM preparations induced IL-IJj production (13,372 ± 4,112 pg/ml) .

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TABLE 1

Production of monokines by human alveolar macrophages exposed in vitro to mineral dusts during 24 h* Conditions of Culture

Monokine

TNF (pg/ml) IL-1{3 (pg/ml) IL-6 (U/ml)

Medium 284 149 61

± 139 ± 81 ± 35

Ti0 2 (l mg/ml)

330 196 72

± 138 ± 80 ± 34

(1

Ti0 2 + Silica + 0.03 mg/ml)

423 195 67

Coal Dustt (l mg/ml)

Silica (0.03 mg/ml)

± 209t ± 147 ± 39

488 169 60

± 395t ± 95 ± 36

3,526 274 224

± 3,509§ ± 220:1: ± 74§

Definition of abbreviations: Ti0 2 = titanium dioxide; IL = interleukin. * All values are mean ± SEM. t Coal dust at 1 mg/ml contains 0.03 mg/rnl of silica. t P = NS. § P < 0.01 versus results from alveolar macrophages exposed to Ti0 2 (Wilcoxon test).

Evaluation of IL-6 Concentrations in AM Exposed to Mineral Dusts Exposure of AM to TiOz, to silica (0.03 mg/ml), or to the combination of both, did not increase the IL-6 concentration in AM supernatants compared with supernatants from unstimulated AM (Table 1; P = NS). Moreover, no significant increase of IL-6 secretion was observed after addition of 0.1 mg/ml of silica (117 ± 17 compared with AM exposed to TiOz: 112 ± 16 V/ml; n = 3). In contrast, addition of coal dusts (1 mg/ml) triggered a significant release of IL-6 (P < 0.01). Moreover, a significant correlation was observed between TNF and IL-6 amounts measured in these AM supernatants (Figure 2; P < 0.001). The concentration obtained in the presence of coal dust represented 0.7% of that released by AM stimulated with LPS (32,850 ± 4,350 V/ml). TNF, IL-l/3, and IL-6 mRNA Expression by AM Exposed In Vitro to Coal Dust Ten million unstimulated AM or AM exposed to 1 mg/ml of coal dust were collected after 3-h culture, and RNA was isolated. TNF and IL-l/3 mRNA expression was analyzed by Northern hybridization with labeled RNA probes, whereas IL-6 mRNA expression was evaluated using a labeled DNA probe. Exposure of AM to coal dust but not to TiOz, induced a net augmentation of TNF, IL-lt3, and IL-6 mRNA expression (Figure 3), whereas any increase of the IL-l secretion was observed after addition of coal dust compared with medium alone or to TiOz in this representative experiment. As previously. demonstrated (21), adherent AM expressed spontaneously a low level of monokine mRNA except for IL-6, which was nearly undetectable.

Discussion In the investigation of the mechanisms of coal dust-induced lung fibrosis, it has been shown that, either in vitro or in vivo, following silica exposure AM from various animal models produced IL-l, TNF, and chemoattractant factors that regulated fibroblast connective tissue biosynthesis. Whether the release of these factors depends in part on the known cytotoxic effect of these dusts remains unclear. In this report, we show that coal dust exposure of AM induced successively the mRNA expression and the secretion of TNF and IL-6. These results demonstrate evidence of cellular activation rather than a deleterious effect on exposed AM. In addition, no

cytotoxic effect was noted after 24-h culture, although longer incubation could probably trigger AM lysis. However, the fact that we used human AM revealed some differences in comparison with previous reports. Borm and associates demonstrated that silica (0.5 mg/ml) and coal mine dust induced an increase of TNF concentration in monocyte supernatants from controls or dust-exposed miners (18). Moreover, rat AM cultured with silica (min-U-Sil particles, which is also a quartz) produced TNF at the 100 jlg/ml dose as in the presence of chrysotile A (25). The variations observed between these and our data concerning the level of TNF production certainly reflect the different abilities of these mononuclear phagocytes to synthesize this cytokine. In addition, silica-stimulated monocytes released fibroblast proliferation factors identical to IL-l (17), whereas it was not secreted by AM. These results suggest that the monokine production induced by exposure to a-quartz varied according to the cell type used. Moreover, the concentration of silica present in our coal dust was too low to induce monokine production.

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Gosset, Lassalle, Vanhee et al. : TNF-o: and IL-6 Production by Human AM Exposed In Vitro to Coal Mine Dust

MEDIUM ALONE

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Figure 3. Northern blot analysis of AM mRNA obtained from unstimulated cells and from mononuclear phagocytes exposed to titanium dioxide (Ti Ox) or to coal dust (1 mg/rnl in both cases). The first panel represents the methylene blue staining of the membrane before hybridization with TNF-labeled RNA probe (second panel), with IL-l{3-labeled RNA probe (third panel) , and with IL-6-labeled DNA probe (fourth panel). Technical conditions are described in MATERIALS AND METHODS.

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Piguet and co-workers recently demonstrated that silicainduced lung fibrosis in mice was mediated by AM-derived TNF because the use of neutralizing anti-TNF antibodies could prevent the increase of lung collagen content (16). At the level of gene expression, it has been found that mRNA encoding for TNF was highly expressed in the lung, even several weeks after silica exposure. IL-6 was also detectable in most cells from the silicotic nodule, but not in the serum. As with these animal models, human monocytes have been shown to produce IL-l and TNF in response to silica exposure (17, 18), indicating that these cytokines may be also involved in the pathogenesis of human silicosis. We have reported recently that AM from patients with CWP were activated to release various mediators such as superoxide anion, IL-l, and TNF (14, 17-19). This spontaneous high cytokine release was also recovered with AM from coal miners still working and exposed to mine dust, indicating that TNF may participate in the early phases of lung inflammation and fibrosis observed in CWP (19). The absence of IL-llS secretion by stimulated AM observed in this study contrasts with the release of IL-l activity by AM from pneumoconiotic patients (19). However, this latter result was obtained by the use of a nonspecific test, the C3H/HeJ thymocyte proliferation assay, which is also positive in the presence of IL-6. Additional experiments performed by the use ofIL-llS RIA and by a biologic test specific for IL-6 demonstrated that AM from pneumoconiotic patients secrete no increased concentrations of IL-llS but

435

released significant amounts of IL-6 (data not shown). In fact, IL-6 production by AM exposed to coal dust in vitro or in vivo suggests the involvement oflL-6 with TNF in lung inflammation. Moreover, recombinant human TNF was able to stimulate IL-6 synthesis by chondrocytes (26). These data and the correlation observed in this study between TNF and IL-6 concentrations suggest that production of TNF by AM could then induce IL-6 secretion. On the other hand, the discrepancy between IL-11S mRNA expression and the absence of monokine secretion has been previously demonstrated with AM stimulated by an IgE-dependent mechanism (21). Under these conditions, AM released a specific IL-I inhibitory factor (22), which was also detected in supernatants from AM exposed to coal dust (data not shown). In the pathophysiology of pneumoconiosis, the monokine involvement deserves discussion. In vitro, TNF induces the recruitment and activation of several inflammatory cells, including lymphocytes, eosinophils, and particularly neutrophils, potentially involved in interstitial lung injury (27-30). In vivo, TNF infusion induces pulmonary hyperpermeability and edema (31), whereas TNF inhalation provokes a limited pulmonary inflammatory response and enhances mononuclear phagocyte functions (32). Furthermore, monokines are known to alternatively stimulate or inhibit fibroblast growth: TNF alone stimulates fibroblast proliferation (33), whereas interaction of TNF with IL-I or interferon-v inhibits fibroblast proliferation at least partly via a prostaglandin E-mediated mechanism (34). With regard to IL-6, TNF induces the final maturation ofB cells, enhancing several-fold the secretion of immunoglobulins, and participates in the activation of resting T cells (reviewed in reference 35). In vivo, IL-6 seems to be implicated in hyper-gamma globulinemic syndrome and to contribute to some autoimmune diseases such as rheumatoid arthritis. Although requirements of TNF and IL-6 for development of CWP remain unproved, it can be postulated that these cytokines may play an important role in that: (I) TNF-induced fibroblast proliferation and collagen synthesis might account for lung fibrosis and (2) IL-6-induced B cell maturation, immunoglobulin secretion, and Tcell activation might explain the frequency of hyper-gamma globulinemia and dysimmune anomalies occurring in CWP (36-40). A number of distinctive features emerge from the pathologic comparison of lung fibrosis lesions between pure silicosis and CWP. Pathogenetic mechanisms involved in pure silicosis and CWP are not the same, as attested by a different pattern of histologic, radiologic, and functional parameters. In our study, we have demonstrated that coal dusts induced the release of AM-derived mediators. This cytokine secretion was triggered by compounds from coal mine dust that are not directly or exclusively related to silica , representing an additional reason to suggest that besides silica other compounds may exert a significant role in the development of CWP. This study suggests that the use of drugs such as anticytokines to inhibit monokine production might be effective in preventing the lung fibrosis observed in pneumoconiosis. Acknowledgments: This work was supported by Grant 89-38965 from the commission of European Communities, by Groupe d'Etudes et de Recherches sur les Pneumoconioses (GERP), and by a grant from the University of Lille II.

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References 1. Parkes, W. R. 1982. Occupational Lung Disorders. 2nd ed. Butterworths, London. 2. Soutar, C. A., and H. P. R. Collins. 1984. Classification of progressive massive fibrosis of coal miners by type of radiographic appearance. Br. J. Ind. Med. 41:334-339. 3. Morgan, W. K. C., L. Handelsman, J. Kilbelstis, N. L. Lapp, and R. B. Reger. 1974. Ventilatory capacity and lung volumes of U.S. coal miners. Arch. Environ. Health 28:182-189. 4. Cochrane, A. L. 1973. Relation between radiographic categories of pneumoconiosis and expectation of life. BMJ 2:532-534. 5. Ortmeyer, C. E., J. Costello, W. K. C. Morgan, S. Swecker, and M. Peterson. 1974. The mortality of Appalachian coal miners, 1963 to 1971. Arch. Environ. Health 29:67-72. 6. Davis, J. M. G., J. Chapman, P. Collings et al. 1983. Variations in the histological patterns of the lesions of coal workers' pneumoconiosis in Britain and their relationship to lung dust content. Am. Rev. Respir. Dis. 128:118-124. 7. Nagelschmidt, G., D. Rivers, E. J. King, and W. Trevella. 1963. Dust and collagen content oflungs of coal-workers with progressive massive fibrosis. Br. J. Ind. Med. 20:181-191. 8. Warson, A. J., J. Black, A. T. Doig, and G. Nagelschmidt. 1959. Pneumoconiosis in carbon electrode workers. Br. J. Ind. Med. 16:274-285. 9. Naeye, R. L. 1972. Types of fibrosis in coal worker's pneumoconiosis. Ann. NY Acad. Sci. 200:381-400. 10. Lugano, E. M., J. H. Dauber, and R. P. Daniele. 1982. Acute experimental silicosis. Lung morphology, histology and macrophage chemotaxis secretion. Am. J. Pathol. 109:27-36. 11. Donaldson, K., and R. T. Cullen. 1984. Chemiluminescence of asbestos activated macrophages. Br. J. Exp. Pathol. 65:81-90. 12. Davies, P., G. Ergodu, R. J. Hill, and J. H. Edward. 1985. Secretion of fibronectin by dust induced alveolar macrophages. In In Vitro Effects of Mineral Dusts. NATO A.S.1. Series. Vol. 63. E. G. Beck and J. Bignon, editors. Springer Verlag, Berlin. 353-358. 13. Voisin, C., B. Gosselin, P. Ramon, B. Wallaert, C. Aerts, and L. Lenoir. 1983. Le lavage bronchoalveolaire dans la pneumoconiose des mineurs de charbon. Aspects cytologiques. Rev. Fr. Mal. Respir. 11:455-466. 14. Wallaert, B., P. Lassalle, F. Fortin et al. 1990. Superoxide anion generation by inflammatory cells in simple pneumoconiosis and in progressive massive fibrosis of nonsmoking coal workers. Am. Rev. Respir. Dis. 141:129-133. 15. Gritter, H. L., I. Y. R. Adamson, and G. M. King. 1986. Modulation of fibroblast activity by normal and silica exposed alveolar macrophages. J. Pathol. 148:263-271. 16. Piguet, P. F., M. A. Collart, G. E. Grau, A. P. Sappinot, and P. Vassalli. 1990. Requirement of tumour necrosis factor for development of silicainduced pulmonary fibrosis. Nature 344:245-247. 17. Schmidt, J. A., C. N. Oliver, J. L. Lepe-Zuniga, I. Green, and I. Grey. 1984. Silica-stimulated monocytes release fibroblast proliferation factors identical to interleukin-I : potential role for interleukin-l in the pathogenesis of silicosis. J. CUn. Invest. 72:1462-1472. 18. Borm, P. J. A., N. Palmen, J. J. M. Engelen, and W. A. Buurman. 1988. Spontaneous and stimulated release of tumor necrosis factor-alpha (TNF) from blood monocytes of miners with coal workers' pneumoconiosis. Am. Rev. Respir. Dis. 138:1589-1594. 19. Lassalle, P., P. Gosset, C. Aerts et al. 1990. Abnormal secretion of interleukin-I and tumor necrosis factor alpha by alveolar macrophages in coal workers pneumoconiosis: comparison between simple pneumoconiosis and progressive massive fibrosis. Exp. Lung Res. 16:73-80. 20. Gosset, P., A. B. Tonnel, M. Josephetal. 1984. Secretionofachemotactic factor for neutrophils and eosinophils by alveolar macrophages from asthmatic patients. J. Allergy CUn. Immunol. 74:827-834. 21. Gosset, P., P. Lassalle, J. Pestel, A. B. Tonnel, and A. Capron. 1989.

22. 23. 24. 25.

26. 27.

28. 29.

30.

31.

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