Lymphocytes, lymphokines, and silicosis.

Silicosis is characterized by mononuclear cell aggregation with mineral particles and fibrosis. Lymphocytes are abundant in these lesions. We exposed inbred strains of mice to a respirable aerosol of cristobalite silica (70 mg/m3, 5 h/d, 12 d) or shamair. Silicosis evolved over months after exposure. The silica-exposed mice showed the accumulation of lymphocytes in alveolar spaces (seen in bronchoalveolar lavage), in lung parenchymal lesions and nodules, and in enlarged bronchial-associated lymphoid tissues and thoracic lymph nodes. The lung lymphocytes were predominantly CD4+ T cells, but numerous CD8+ T cells, natural killer cells, and CD4- gammadelta-TCR+ T cells were present as well. Interferon-gamma (IFN-gamma) production was upregulated, suggesting a THelper-1-like response in silicosis. In silicotic lung tissue, mRNA transcripts for the macrophage-derived cytokines IL-12 and -18 were increased. IFN-gamma gene-deleted mice (C57Bl/6-Ifngtm1 Ts) exposed to silica developed less extensive silicosis and less lung collagen accumulation than wild-type mice. We hypothesize that there is a reiterative amplification cycle in which macrophages with silica may produce cytokines, such as IL-12 and -18, that attract and activate lymphocytes. These activated lymphocytes may then produce additional mediators that in turn attract and activate an expanded secondary population of macrophages. IFN-gamma would be a likely cause of macrophage activation in this cycle. More work is needed to understand the biological events that lead from the inhaled dust to the scarred lung, and to clarify the role of lymphocytes in this process.

Asbestos exposure induces MCP-1 secretion by pleural mesothelial cells.

We showed previously that both crocidolite and chrysotile asbestos inhalation induced a persistent macrophage inflammatory response within the pleural space of the rat. We postulated that the stimulus for pleural macrophage recruitment after asbestos exposure was the induction of monocyte chemoattractant protein-1 (MCP-1) synthesis by pleural mesothelial cells. To test this hypothesis, rat pleural mesothelial cells (RPMC) were cultured with or without chrysotile or crocidolite asbestos fibers (8 micrograms/cm2) in the presence (50 ng/mL) or absence of either tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta). MCP-1 mRNA expression was assessed by RT-PCR in RPMC cultured for 2 to 24 hours, and MCP-1 protein secretion was measured by ELISA in conditioned medium from 24-hour and 48-hour cultures. Crocidolite and chrysotile fibers induced MCP-1 mRNA expression in RPMC which was maximal after 12 hours in the absence of cytokines, but which peaked after 2 hours when RPMC were challenged with asbestos + TNF-alpha or IL-1 beta. Both types of asbestos also significantly increased MCP-1 protein secretion after 24 and 48 hours (P < .0001), an effect that was potentiated by cytokine stimulation. Rats exposed by inhalation to either chrysotile or crocidolite asbestos fibers also had greater amounts of MCP-1 protein in their pleural lavage fluid than did sham-exposed rats. These findings suggest that MCP-1 secretion by RPMC may have a role in the initiation and/or potentiation of asbestos-induced pleural injury.

Interferon-gamma production by specific lung lymphocyte phenotypes in silicosis in mice.

We recently described overproduction of interferon (IFN)-gamma by lung lymphocytes in mice with silicosis (11% of cells in air-control versus 19% of cells from silica-exposed mice; Davis and colleagues, Am. J. Respir. Cell Mol. Biol. 1999;20:813-824). We hypothesized that the increased IFN-gamma production might be due to selective enrichment of one lymphocyte phenotype. To test this hypothesis, small mononuclear cells from lung digest preparations of mice exposed 4 mo previously to cristobalite silica (70 mg/m(3), 12 d, 5 h/d) or to sham-air were stained for intracellular cytokines and surface antigen phenotypes, and examined by flow cytometry. Air-sham mouse lung digests included CD4(+) (16%) and CD8(+) (6%) T cells, gammadelta T-cell antigen receptor (TCR)(+) CD4(-)CD8(-) T cells (3%), natural killer (NK) cells (15%), B cells (6%), and macrophages (12%). The total number of lung lymphocytes was increased 1.7-fold in silicosis, but the phenotype frequencies did not change significantly. In the control lungs IFN-gamma was produced by three major phenotypes of lymphocytes: 5% of CD4(+) T cells, 5% of gammadelta-TCR(+) CD4(-)CD8(-) T cells, and 2% of NK cells. The percentage of each type producing IFN-gamma was increased 2- to 3-fold in silicosis. When multiplied by cell number, the increased percentages yielded a 3- to 5-fold increase in the total number of each IFN- gamma-producing phenotype in the lung. Our results demonstrate no selective phenotype enrichment but upregulated IFN-gamma production by at least three lymphocyte phenotypes. IFN-gamma may be an important signal driving lymphocyte differentiation and macrophage activation in silicosis.

Asbestos exposure upregulates the adhesion of pleural leukocytes to pleural mesothelial cells via VCAM-1.

This study was designed to assess the effects of in vitro and in vivo asbestos exposure on the adhesion of rat pleural leukocytes (RPLs) labeled with the fluorochrome calcein AM to rat pleural mesothelial cells (RPMCs). Exposure of RPMCs for 24 h to either crocidolite or chrysotile fibers (1.25-10 microgram/cm(2)) increased the adhesion of RPLs to RPMCs in a dose-dependent fashion, an effect that was potentiated by interleukin-1beta. These findings were not observed with nonfibrogenic carbonyl iron particles. Crocidolite and chrysotile plus interleukin-1beta also upregulated vascular cell adhesion molecule-1 mRNA and protein expression in RPMCs, and the binding of RPL to asbestos-treated RPMCs was abrogated by anti-vascular cell adhesion molecule-1 antibody. PRLs exposed by intermittent inhalation to crocidolite for 2 wk manifested significantly greater binding to RPMCs than did RPLs from sham-exposed animals. The ability of asbestos fibers to upregulate RPL adhesion to RPMCs may play a role in the induction and/or potentiation of asbestos-induced pleural injury.

Asbestos inhalation induces reactive nitrogen species and nitrotyrosine formation in the lungs and pleura of the rat.

To determine whether asbestos inhalation induces the formation of reactive nitrogen species, three groups of rats were exposed intermittently over 2 wk to either filtered room air (sham-exposed) or to chrysotile or crocidolite asbestos fibers. The rats were killed at 1 or 6 wk after exposure. At 1 wk, significantly greater numbers of alveolar and pleural macrophages from asbestos-exposed rats than from sham-exposed rats demonstrated inducible nitric oxide synthase protein immunoreactivity. Alveolar macrophages from asbestos-exposed rats also generated significantly greater nitrite formation than did macrophages from sham-exposed rats. Strong immunoreactivity for nitrotyrosine, a marker of peroxynitrite formation, was evident in lungs from chrysotile- and crocidolite-exposed rats at 1 and 6 wk. Staining was most evident at alveolar duct bifurcations and within bronchiolar epithelium, alveolar macrophages, and the visceral and parietal pleural mesothelium. Lungs from sham-exposed rats demonstrated minimal immunoreactivity for nitrotyrosine. Significantly greater quantities of nitrotyrosine were detected by ELISA in lung extracts from asbestos-exposed rats than from sham-exposed rats. These findings suggest that asbestos inhalation can induce inducible nitric oxide synthase activation and peroxynitrite formation in vivo, and provide evidence of a possible alternative mechanism of asbestos-induced injury to that thought to be induced by Fenton reactions.

Silicosis in mice: effects of dose, time, and genetic strain.

Experimental silicosis allows study of the mechanisms of lung injury, inflammation, and fibrosis. Inbred mice are an attractive species in which to study these mechanisms because of recent progress in murine immunology, molecular biology, and genetics. We exposed mice to an aerosol of silica and examined the effects of exposure dose, the evolution of disease features over time, and the variation in responses among four inbred strains. In C3H/HeN mice incremental cumulative exposure doses of cristobalite silica caused increased initial lung dust burden 12 to 16 weeks post-exposure, progressively intense pathological responses, and increased total lung collagen (hydroxyproline). The histopathological changes and total lung collagen increased progressively over time after exposure. We compared the features of silicosis in four strains of inbred mice selected for common use or immunologic reactivity 16 weeks after aerosol inhalation exposure to crystalline cristobalite silica (70 mg/m3, 5 hours/day, 12 days). C3H/HeN mice demonstrated histopathological silicotic lesions and enlarged intrapulmonary lymphoid tissue, and increased lung wet weight, bronchoalveolar lavage (BAL) recovery of macrophages, lymphocytes, and neutrophils, and total lung collagen (hydroxyproline). BALB/c mice developed slight pulmonary lesions; MRL/MpJ mice demonstrated prominent pulmonary infiltrates with lymphocytes; New Zealand Black mice developed extensive alveolar proteinaceous deposits, inflammation, and fibrosis. Our findings demonstrate orderly dose-time-response relationships, and a substantial variation of responses among inbred strains of mice. This model should prove valuable for future experimental interventions into the mechanisms of silicosis.

Persistent overexpression of interleukin-1beta and tumor necrosis factor-alpha in murine silicosis.

The cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNFalpha), derived from macrophages and other cells, may promote mononuclear cell inflammation and fibrosis in pulmonary silicosis. C3H/HeN mice were exposed to control air or to an aerosol of 70 mg/m3 cristobalite silica for 5 h/d for 12 days and examined at 2 and 16 weeks after exposure. This exposure resulted in murine silicosis, as manifested by focal mononuclear cell accumulations, diffuse interstitial fibrosis, lymphoid tissue enlargement, recruitment of inflammatory cells into BAL fluid, and increased total lung collagen. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) with designed primers and membrane hybridization with biotinylated cDNA probes were used to assess the abundance of IL-1beta and TNFalpha mRNA. In situ hybridization with digoxigenin-labeled cDNA probes was used to localize gene expression. Persistent overexpression of both IL-1beta and TNFalpha were found at 2 and 16 weeks in the lungs of silica-exposed mice compared with air-sham control mice. IL-1beta and TNFalpha expression localized to individual mononuclear cells in the alveolar spaces, groups of cells within the aggregate lesions, and scattered mononuclear cells in BALT and lymphoid nodules. Thus, cells producing IL-1beta and TNFalpha appear to be intimately associated with the evolving lesions of silicosis, and the lymphoid tissue of the lung may be important in driving the pathogenesis of this disease.

Pleural macrophage recruitment and activation in asbestos-induced pleural injury.

The pathogenesis of asbestos-induced pleural fibrosis is poorly understood. Moreover, there has been a long-standing controversy regarding the relative potential of different commercial types of asbestos to cause pleural disease. We postulated that inhaled asbestos fibers translocate to the pleural space where they stimulate the recruitment and activation of pleural macrophages. To test this hypothesis, and to determine whether there are differences between inhaled amphibole and serpentine asbestos, Fischer 344 rats were exposed by intermittent inhalation (6 hr/day for 5 days/week over 2 weeks) to either National Institute of Environmental Health Sciences (NIEHS) crocidolite (average concentration 7.55 mg/m3) or NIEHS chrysotile fibers (average concentration 8.51 mg/m3). Comparisons were made with sham-exposed rats. The rats were sacrificed at 1 and 6 weeks after the cessation of exposure. More pleural macrophages were recovered at 1 and 6 weeks after crocidolite and chrysotile exposure than after sham exposure. Small numbers of crocidolite fibers (approximately 1 per 4000 cells) were detected in the pleural cell pellet of one crocidolite-exposed rat by scanning electron microscopy. Pleural macrophage supernatants were assayed for production of nitric oxide (NO) (by the Griess reaction) and tumor necrosis factor alpha (TNF-alpha) (by an enzyme-linked immunosorbent assay method). Significantly greater amounts of NO as well as TNF-alpha were generated by pleural macrophages at 1 and 6 weeks after either crocidolite or chrysotile inhalation than after sham exposure. Conceivably, translocation of asbestos fibers to the pleural space may provide a stimulus for persistent pleural space inflammation, cytokine production, and the generation of toxic oxygen and nitrogen radicals. Enhanced cytokine secretion within the pleural space may in turn upregulate adhesion molecule expression and the synthesis of extracellular matrix constituents by pleural mesothelial cells. Thus, our findings may have significance for the development of asbestos-induced pleural injury.

T-lymphocyte activation in the enlarged thoracic lymph nodes of rats with silicosis.

Silicosis is primarily a mononuclear cell inflammatory and fibrotic disease of the pulmonary parenchyma. It is known that lung-associated lymph nodes are also affected. To study the involvement of lymphocytes in silicosis, we examined lymph nodes of rats 12 months after an 8-day silica aerosol exposure. We found that 2 thoracic lymph nodes close to the thymus were enormously enlarged in silicotic rats and contained a 49-fold higher cell number than control lymph nodes. The higher cell number was caused by parallel increases in T- and B-lymphocytes, natural killer (NK) cells, and macrophages without change in the relative proportions when compared with control thoracic lymph nodes. By examining interleukin-2 (IL-2) receptor and intercellular adhesion molecule-1 expression, we detected a significantly higher percentage of activated CD8+ T cells and, to a lower degree, of CD4+ T cells in thoracic lymph nodes of silicotic animals. In contrast, no differences in the activation state were found in T cells obtained from cervical or mesenteric lymph nodes of silicotic and control rats. The occurrence of activated T cells in thoracic lymph nodes of silicotic rats was documented further by selectively enhanced interferon-gamma (IFN-gamma) mRNA expression in the absence of IL-2 and IL-4 mRNA changes. These data show that T-lymphocytes of thoracic lymph nodes have become activated with an enhanced IFN-gamma gene transcription which may be an important cause of macrophage activation during silicosis.

Respiratory aflatoxicosis: suppression of pulmonary and systemic host defenses in rats and mice.

Dietary aflatoxin B1 (AFB1) exposure impairs innate and acquired host defenses resulting in increased susceptibility to infections in domesticated animals. Experimental studies have confirmed this observation by demonstrating the immunosuppressive effects of AFB1 ingestion. In addition to being present in dietary components, AFB1 is also found in significant amounts in respirable particles of grain dust. To determine the effect of respiratory tract exposure to AFB1 on host defenses, rats and mice were exposed either by aerosol inhalation or intratracheal instillation to AFB1. Nose-only inhalation exposure of rats to AFB1 aerosols suppressed alveolar macrophage (AM) phagocytosis at an estimated dose of 16.8 micrograms/kg with the effect persisting for approximately 2 weeks. To determine whether another mode of respiratory tract exposure, intratracheal instillation, reflected inhalation exposure, animals were treated with increasing concentrations of AFB1 which also suppressed AM phagocytosis in a dose-related manner albeit at doses at least an order of magnitude more than that obtained by aerosol inhalation. Intratracheal administration of AFB1 also suppressed the release of tumor necrosis factor-alpha from AMs and impaired systemic innate and acquired immune defenses as shown, respectively, by suppression of peritoneal macrophage phagocytosis and the primary splenic antibody response. These findings demonstrate that experimental respiratory tract exposure to AFB1 suppresses pulmonary and systemic host defenses and indicates that inhalation exposure to AFB1 is an occupational hazard where exposure to AFB1-laden dust is common.

Asbestos exposure stimulates pleural mesothelial cells to secrete the fibroblast chemoattractant, fibronectin.

Parietal pleural plaques and visceral pleural fibrosis are well-recognized stigmata of occupational asbestos exposure. However, their pathogenesis is poorly understood. Conceivably, phagocytosis of asbestos fibers by pleural mesothelial cells may stimulate the recruitment of fibroblasts to sites of asbestos-induced pleural injury. To test this hypothesis, rat parietal pleural mesothelial cells were cultured for 6 to 96 h with or without crocidolite or chrysotile asbestos fibers (concentration range, 2 to 100 micrograms/cm2). Asbestos fibers were actively phagocytosed by pleural mesothelial cells and were incorporated within phagosomes. Conditioned medium was assayed for chemotactic activity toward RL-87 rat lung fibroblasts and for fibronectin immunoreactivity. The effects of asbestos were compared with those of alpha-cristobalite (which is strongly fibrogenic), alpha-quartz (a less fibrogenic particulate), and carbonyl iron (a nonfibrogenic agent). Both types of asbestos stimulated the secretion of fibroblast chemoattractant activity by pleural mesothelial cells in a time-dependent manner. This effect peaked at 96 h in cultures containing 4 micrograms/cm2 of asbestos (P < 0.001). alpha-Cristobalite also enhanced the secretion of the mesothelial cell-derived chemoattractant, an effect that was maximal at a concentration of 20 micrograms/cm2 (P < 0.001). Furthermore, crocidolite, chrysotile, and alpha-cristobalite stimulated pleural mesothelial cell fibronectin synthesis. In contrast, alpha-quartz and carbonyl iron particles had no noticeable effect on either immunoreactive fibronectin secretion or chemoattractant release by pleural mesothelial cells. The ability of asbestos fibers and alpha-cristobalite particles to stimulate the secretion of the fibroblast chemoattractant, fibronectin, by pleural mesothelial cells may have relevance to the induction of pleural injury by fibrogenic particulates.

Reduced release of leukotrienes B4 and C4 from alveolar macrophages of rats with silicosis.

Silicosis leads to altered release of fibrogenic and immunomodulating mediators from alveolar macrophages (AM). Since 5-lipoxygenase metabolites have been shown to possess proinflammatory effects and to promote the release of cytokines such as tumor necrosis factor-alpha (TNF-alpha) from mononuclear phagocytes, we determined leukotriene secretion from silica-exposed AM. Rats were exposed to an aerosol of silica particles for 8 days and AM were harvested by bronchoalveolar lavage 5 to 7 mo after exposure. AM from both air-sham control and silica-exposed rats displayed minimal spontaneous leukotriene release upon in vitro culture. Stimulation with opsonized zymosan particles induced leukotriene B4 (LTB4) and leukotriene C4 (LTC4) secretion, which was much greater in control AM than in AM from silica-dusted rats. The reverse was found for zymosan-induced TNF-alpha production, which was higher in AM from silica-exposed than from control rats. To study the interrelation between leukotriene and TNF-alpha release, we incubated zymosan-stimulated AM with the 5-lipoxygenase inhibitor VZ 65. VZ 65 suppressed zymosan-induced TNF-alpha release from AM in a dose-dependent manner, and TNF-alpha production could be restored almost completely by addition of LTB4. These experiments demonstrate that silica exposure resulted in a decreased LTB4 and LTC4 production from AM, which may represent a regulatory mechanism to counterbalance enhanced TNF-alpha production during silicosis.

Use of physical chemistry and in vivo exposure to investigate the toxicity of formaldehyde bound to carbonaceous particles in the murine lung.

Knowledge about the health effects of exposure to formaldehyde associated with automotive emissions is of pivotal importance in the risk assessment of this agent. Mobile sources emit many combustion-derived pollutants, including formaldehyde, in association with respirable carbon particles. Because it is hydrophilic, most of the inhaled formaldehyde is absorbed in the upper respiratory tract. However, if the organic vapor is adsorbed on respirable particles, formaldehyde may be deposited in the deep lung with the inhaled particles and may be available to interact adversely with cells along the lung parenchyma. On the respiratory surface, the alveolar macrophage phagocytic system plays the pivotal role in defending the lung against infectious agents. Susceptibility to respiratory infections is a relevant and sensitive indicator of the adverse effects of air pollution because acute and chronic exposures to a variety of air pollutants have been shown to decrease pulmonary antibacterial defenses. The goal of this research was to investigate whether exposure to formaldehyde decreases resistance to respiratory infections through dysfunctions of the alveolar macrophage phagocytic system. The study also explored whether interactions between formaldehyde and respirable carbon black particles alter susceptibility to respiratory infections and impairment of alveolar macrophage phagocytosis by delivering adsorbed formaldehyde to the deep lung with the inhaled particles. A carbon black, Regal GR, was used in these studies as a surrogate for the carbonaceous core of Diesel particulate matter. This material was selected to represent the worst-case scenario because the carbon black was expected to adsorb formaldehyde strongly. To accomplish this goal, mice were exposed to formaldehyde and to carbon black and formaldehyde combinations; increased susceptibility to respiratory infections was quantified by alveolar macrophage-dependent intrapulmonary killing of Staphylococcus aureus after an inhalation challenge with the bacterium. The salient findings of the bactericidal studies are as follows: Fifteen parts per million (ppm)* formaldehyde impaired the intrapulmonary killing of S. aureus when exposure followed the bacterial challenge. One ppm formaldehyde impaired the intrapulmonary killing of S. aureus when exposure preceded and was continued after the bacterial challenge. Coexposures to target concentrations of 3.5 mg/m3 carbon black and 2.5 ppm formaldehyde, or 10 mg/m3 carbon black and 5 ppm formaldehyde after the bacterial challenge had no effect on the intrapulmonary killing of S. aureus. Preexposure for four hours per day for four days to target concentrations of 3.5 mg/m3 carbon black and 2.5 ppm formaldehyde had no effect on the intrapulmonary killing of S. aureus when the assay was performed one day after the cessation of exposure.(ABSTRACT TRUNCATED AT 400 WORDS)

Aflatoxin B1--DNA adduct formation in rat liver following exposure by aerosol inhalation.

There is growing concern that human exposure to respirable grain dust contaminated with aflatoxin B1 (AFB1), a potent hepatocarcinogen, may be a risk factor for a number of human diseases. The objective of this study was to determine if liver DNA adduct formation occurs in rats following either intratracheal injection or nose-only aerosol inhalation exposure to AFB1. Male Fischer 344 rats were exposed by both routes of administration, and in preliminary data using intratracheal instillation, up to 2% of the administered dose became bound to liver DNA. In the nose-only aerosol inhalation experiments, rats were exposed for up to 120 min. Immediately after exposure, four animals were killed at each time point and their livers removed, DNA isolated and purified and analyzed for aflatoxin-DNA adducts by HPLC. A linear dose-response relationship was observed with a correlation coefficient of 0.96 between increasing length of exposure, and the amount of aflatoxin-N7-guanine adducts formed per mg DNA, the mean values and standard errors were 4.2 +/- 0.18, 15.3 +/- 4.3, 21.6 +/- 2.8 and 56.8 +/- 4.6 pmol aflatoxin-DNA adducts per mg DNA for the 20, 40, 60 and 120 min exposures respectively. The amounts of aflatoxin-DNA adducts formed were statistically significantly different (P less than 0.01) among the treated groups. These results indicate that aerosol inhalation is an effective route of exposure to AFB1 in rats that results in genotoxic damage in the liver.

Enhanced release of prostaglandin E2 from macrophages of rats with silicosis.

The pathogenesis of silicosis results, in part, from interactions between silica particles and alveolar macrophages (AM) with release of cytokines and other mediators. Different arachidonic acid metabolites have been shown to promote or to suppress inflammation and fibrosis. We designed experiments to study the production of cyclooxygenase metabolites and tumor necrosis factor-alpha (TNF-alpha) from macrophages during active silicosis. Macrophages were harvested from rats 5 to 7 mo after an 8-day silica aerosol exposure. Upon in vitro culture of AM, the spontaneous release of prostaglandin E2 (PGE2), thromboxane B2 (TXB2), and prostaglandin D2 (PGD2) of silica-exposed animals was higher than that of sham-exposed animals. Moreover, AM from silicotic rats displayed an increased sensitivity to low concentrations of lipopolysaccharide (LPS, 10 ng/ml) and released copious amounts of PGE2 and TXB2. When compared with similarly enhanced release of TNF-alpha from AM of silica-exposed rats, PGE2 production occurred later and started to increase when TNF-alpha production declined. Addition of the cyclooxygenase blocker indomethacin augmented TNF-alpha production, whereas the addition of PGE2 counteracted TNF-alpha release. Also peritoneal macrophages, which did not have direct contact with silica particles, released enhanced levels of PGE2 in response to low LPS doses. We conclude that AM and other macrophages from silica-exposed rats are preactivated and display an enhanced prostanoid production that could serve anti-inflammatory or immunomodulating roles in silicosis.

Effects of work exposure, retirement, and smoking on bronchoalveolar lavage measurements of lung dust in Vermont granite workers.

Estimation of the exposure to respirable dust in the workplace is an important aspect of industrial hygiene. We performed bronchoalveolar lavage (BAL) on 42 healthy nonindustrial control subjects and 44 workers in the Barre, Vermont granite industry to determine whether BAL materials reflected occupational exposure. The granite workers held jobs with a range of dust exposure intensities and had employment histories from 1 to 43 yr; 12 workers were retired. None of the workers had radiologic evidence of silicosis. The granite dust content of BAL materials was measured by enumerating the percentage of cells positive for particulates by polarized light microscopy, by analysis for silicon by scanning electron microscopy with energy-dispersive X-ray spectrometry, and by chemical analysis. Minerals were present in greater quantity in the BAL cells from granite workers than from nonindustrial control subjects. Polarized light microscopy was comparable in sensitivity, specificity, and overall accuracy to electron microscopy; chemical analysis was less accurate than either microscopic technique. Tobacco smoking did not interfere with the detection of the occupationally related minerals or influence the quantity of dust present. The mineral in BAL cells was partially related to the intensity of exposure within the granite industry and to the duration of employment, but there was great individual variation among subjects. Retirement led to a decrease in BAL mineral content, but substantial dust remained for many years.

Systemic macrophage stimulation in rats with silicosis: enhanced release of tumor necrosis factor-alpha from alveolar and peritoneal macrophages.

In silicosis, alveolar macrophages (AM) are thought to induce chronic inflammation and fibrosis by release of cytokines. Rats were exposed to aerosols of alpha-quartz and examined 4 to 9 mo later for persistence of silica particles and release of tumor necrosis factor-alpha (TNF-alpha) from macrophages. Silica particles were detected in AM, lung parenchyma, and thoracic lymphoid organs, whereas extrathoracic lymphoid tissues and organs were free of the mineral. When AM were tested functionally, no spontaneous release of TNF-alpha was observed. However, upon in vitro stimulation of AM from silicotic rats with a low concentration of lipopolysaccharide (10 ng/ml), abundant TNF-alpha production was found that was higher and occurred more rapidly than with AM from sham-exposed animals. Peritoneal macrophages, which did not have contact with silica particles, displayed a similarly enhanced TNF-alpha release in response to low doses of lipopolysaccharide. These data demonstrate a state of systemic preactivation ("priming") of macrophages that supports the notion that silicosis is associated with a general immunostimulation.

Comparison of lung alveolar and tissue cells in silica-induced inflammation.

The silicon dioxide mineral, cristobalite (CRS) induces inflammation involving both alveolar cells and connective tissue compartments. In this study, we compared lung cells recovered by whole lung lavage and by digestion of lung tissue from rats at varying times after 8 days of exposure to aerosolized CRS. Control and exposed rats were examined between 2 and 36 wk after exposure. Lavaged cells were obtained by bronchoalveolar lavage with phosphate-buffered saline. Lung wall cells were prepared via collagenase digestion of lung tissue slices. Cells from lavage and lung wall were separated by Percoll density centrifugation. The three upper fractions, containing mostly macrophages, were cultured, and the conditioned medium was assayed for effect on lung fibroblast growth and for activity of the lysosomal enzyme, N-acetyl-beta-D-glucosaminidase. Results demonstrated that the cells separated from the lung walls exhibited different reaction patterns compared with those cells recovered by lavage. The lung wall cells exhibited a progressive increase in the number of macrophages and lymphocytes compared with a steady state in cells of the lung lavage. This increase in macrophages apparently was due to low density cells, which showed features of silica exposure. Secretion of a fibroblast-stimulating factor was consistently high by lung wall macrophages, whereas lung lavage macrophages showed inconsistent variations. The secretion of NAG was increased in lung lavage macrophages, but decreased at most observation times in lung wall macrophages. No differences were found among cells in the different density fractions regarding fibroblast stimulation and enzyme secretion.(ABSTRACT TRUNCATED AT 250 WORDS)