Asbestos-induced Pulmonary Fibrosis Research Articles

SIRT3 Overexpression Ameliorates Asbestos-Induced Pulmonary Fibrosis, mt-DNA Damage, and Lung Fibrogenic Monocyte Recruitment.

Alveolar epithelial cell (AEC) mitochondrial (mt) DNA damage and fibrotic monocyte-derived alveolar macrophages (Mo-AMs) are implicated in the pathobiology of pulmonary fibrosis. We showed that sirtuin 3 (SIRT3), a mitochondrial protein regulating cell fate and aging, is deficient in the AECs of idiopathic pulmonary fibrosis (IPF) patients and that asbestos- and bleomycin-induced lung fibrosis is augmented in Sirt3 knockout (Sirt3−/−) mice associated with AEC mtDNA damage and intrinsic apoptosis. We determined whether whole body transgenic SIRT3 overexpression (Sirt3Tg) protects mice from asbestos-induced pulmonary fibrosis by mitigating lung mtDNA damage and Mo-AM recruitment. Crocidolite asbestos (100 µg/50 µL) or control was instilled intratracheally in C57Bl6 (Wild-Type) mice or Sirt3Tg mice, and at 21 d lung fibrosis (histology, fibrosis score, Sircol assay) and lung Mo-AMs (flow cytometry) were assessed. Compared to controls, Sirt3Tg mice were protected from asbestos-induced pulmonary fibrosis and had diminished lung mtDNA damage and Mo-AM recruitment. Further, pharmacologic SIRT3 inducers (i.e., resveratrol, viniferin, and honokiol) each diminish oxidant-induced AEC mtDNA damage in vitro and, in the case of honokiol, protection occurs in a SIRT3-dependent manner. We reason that SIRT3 preservation of AEC mtDNA is a novel therapeutic focus for managing patients with IPF and other types of pulmonary fibrosis.

The Sphingosine Kinase 1 Inhibitor, PF543, Mitigates Pulmonary Fibrosis by Reducing Lung Epithelial Cell mtDNA Damage and Recruitment of Fibrogenic Monocytes.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease for which novel approaches are urgently required. We reported increased sphingosine kinase 1 (SPHK1) in IPF lungs and that SPHK1 inhibition using genetic and pharmacologic approaches reduces murine bleomycin-induced pulmonary fibrosis. We determined whether PF543, a specific SPHK1 inhibitor post bleomycin or asbestos challenge mitigates lung fibrosis by reducing mitochondrial (mt) DNA damage and pro-fibrotic monocyte recruitment—both are implicated in the pathobiology of pulmonary fibrosis. Bleomycin (1.5 U/kg), crocidolite asbestos (100 µg/50 µL) or controls was intratracheally instilled in Wild-Type (C57Bl6) mice. PF543 (1 mg/kg) or vehicle was intraperitoneally injected once every two days from day 7−21 following bleomycin and day 14−21 or day 30−60 following asbestos. PF543 reduced bleomycin- and asbestos-induced pulmonary fibrosis at both time points as well as lung expression of profibrotic markers, lung mtDNA damage, and fibrogenic monocyte recruitment. In contrast to human lung fibroblasts, asbestos augmented lung epithelial cell (MLE) mtDNA damage and PF543 was protective. Post-exposure PF543 mitigates pulmonary fibrosis in part by reducing lung epithelial cell mtDNA damage and monocyte recruitment. We reason that SPHK1 signaling may be an innovative therapeutic target for managing patients with IPF and other forms of lung fibrosis.

NOX4 modulates macrophage phenotype and mitochondrial biogenesis in asbestosis.

Macrophage activation is implicated in the development of pulmonary fibrosis by generation of profibrotic molecules. Although NADPH oxidase 4 (NOX4) is known to contribute to pulmonary fibrosis, its effects on macrophage activation and mitochondrial redox signaling are unclear. Here, we show that NOX4 is crucial for lung macrophage profibrotic polarization and fibrotic repair after asbestos exposure. NOX4 was elevated in lung macrophages from subjects with asbestosis, and mice harboring a deletion of NOX4 in lung macrophages were protected from asbestos-induced fibrosis. NOX4 promoted lung macrophage profibrotic polarization and increased production of profibrotic molecules that induce collagen deposition. Mechanistically, NOX4 further augmented mitochondrial ROS production and induced mitochondrial biogenesis. Targeting redox signaling and mitochondrial biogenesis prevented the profibrotic polarization of lung macrophages by reducing the production of profibrotic molecules. These observations provide evidence that macrophage NOX4 is a potentially novel therapeutic target to halt the development of asbestos-induced pulmonary fibrosis.

Similar and Differential Canonical Pathways and Biological Processes Associated With Multiwalled Carbon Nanotube and Asbestos-Induced Pulmonary Fibrosis: A 1-Year Postexposure Study.

Respiratory exposure to multiwalled carbon nanotubes (MWCNT) or asbestos results in fibrosis; however, the mechanisms to reach this end point may be different. A previous study by our group identified pulmonary effects and significantly altered messenger RNA (mRNA) signaling pathways following exposure to 1, 10, 40, and 80 µg MWCNT and 120 µg crocidolite asbestos on mouse lungs over time at 1-month, 6-month, and 1-year postexposure following pulmonary aspiration. As a continuation to the above study, this current study took an in-depth look at the signaling pathways involved in fibrosis development at a single time point, 1 year, and exposure, 40 µg MWCNT, the lowest exposure at which fibrosis was pathologically evident. The 120 µg asbestos exposure was included to compare MWCNT-induced fibrosis with asbestos-induced fibrosis. A previously validated computational model was used to identify mRNAs with expression profiles matching the fibrosis pathology patterns from exposed mouse lungs. mRNAs that matched the pathology patterns were then input into ingenuity pathway analysis to determine potential signaling pathways and physiological disease functions inherent to MWCNT and asbestos exposure. Both MWCNT and asbestos exposure induced changes in mouse lungs regarding gene expression, cell proliferation, and survival, while MWCNT uniquely induced alterations in pathways involved in oxidative phosphorylation, mitochondrial dysfunction, and transcription. Asbestos exposure produced unique alterations in pathways involved in sustained inflammation. Although typically considered similar due to scale and fiber-like appearance, the different compositional properties inherent to either MWCNT or asbestos may play a role in their ability to induce fibrosis after pulmonary exposure.

Cu,Zn-Superoxide Dismutase-Mediated Redox Regulation of Jumonji Domain Containing 3 Modulates Macrophage Polarization and Pulmonary Fibrosis.

M2 macrophages are implicated in the development of pulmonary fibrosis as they generate profibrotic signals. The polarization process, at least in part, is regulated by epigenetic modulation. Because Cu,Zn-superoxide dismutase-induced H2O2 can polarize macrophages to a profibrotic M2 phenotype, we hypothesized that modulation of the redox state of the cell is involved in the epigenetic modulation of the macrophage phenotype. In this study, we show that signal transducer and activator of transcription 6 (STAT6) regulates Jumonji domain containing (Jmjd) 3, a histone H3 lysine 27 demethylase, and mutation of a redox-sensitive cysteine in STAT6 attenuates jmjd3 expression. Moreover, Jmjd3 deficiency abrogates profibrotic M2 gene expression. Treatment with leflunomide, which reduces mitochondrial reactive oxygen species production and tyrosine phosphorylation, inhibits jmjd3 expression and M2 polarization, as well as development of a fibrotic phenotype. Taken together, these observations provide evidence that the redox regulation of Jmjd3 is a unique regulatory mechanism for Cu,Zn-superoxide dismutase-mediated profibrotic M2 polarization. Furthermore, leflunomide, which reduces reactive oxygen species production and tyrosine phosphorylation, may prove to be therapeutic in the treatment of asbestos-induced pulmonary fibrosis.

The Role of Mitochondrial DNA in Mediating Alveolar Epithelial Cell Apoptosis and Pulmonary Fibrosis.

Convincing evidence has emerged demonstrating that impairment of mitochondrial function is critically important in regulating alveolar epithelial cell (AEC) programmed cell death (apoptosis) that may contribute to aging-related lung diseases, such as idiopathic pulmonary fibrosis (IPF) and asbestosis (pulmonary fibrosis following asbestos exposure). The mammalian mitochondrial DNA (mtDNA) encodes for 13 proteins, including several essential for oxidative phosphorylation. We review the evidence implicating that oxidative stress-induced mtDNA damage promotes AEC apoptosis and pulmonary fibrosis. We focus on the emerging role for AEC mtDNA damage repair by 8-oxoguanine DNA glycosylase (OGG1) and mitochondrial aconitase (ACO-2) in maintaining mtDNA integrity which is important in preventing AEC apoptosis and asbestos-induced pulmonary fibrosis in a murine model. We then review recent studies linking the sirtuin (SIRT) family members, especially SIRT3, to mitochondrial integrity and mtDNA damage repair and aging. We present a conceptual model of how SIRTs modulate reactive oxygen species (ROS)-driven mitochondrial metabolism that may be important for their tumor suppressor function. The emerging insights into the pathobiology underlying AEC mtDNA damage and apoptosis is suggesting novel therapeutic targets that may prove useful for the management of age-related diseases, including pulmonary fibrosis and lung cancer.

Asbestos-induced pulmonary fibrosis is augmented in 8-oxoguanine DNA glycosylase knockout mice.

Asbestos causes asbestosis and malignancies by mechanisms that are not fully established. Alveolar epithelial cell (AEC) injury and repair are crucial determinants of the fibrogenic potential of noxious agents such as asbestos. We previously showed that mitochondrial reactive oxygen species mediate asbestos-induced AEC intrinsic apoptosis and that mitochondrial human 8-oxoguanine-DNA glycosylase 1 (OGG1), a DNA repair enzyme, prevents oxidant-induced AEC apoptosis. We reasoned that OGG1 deficiency augments asbestos-induced pulmonary fibrosis. Compared with intratracheal instillation of PBS (50 μl) or titanium dioxide (100 μg/50 μl), crocidolite or Libby amphibole asbestos (100 μg/50 μl) each augmented pulmonary fibrosis in wild-type C57BL/6J (WT) mice after 3 weeks as assessed by histology, fibrosis score, lung collagen via Sircol, and type 1 collagen expression; these effects persisted at 2 months. Compared with WT mice, Ogg1 homozygous knockout (Ogg1(-/-)) mice exhibit increased pulmonary fibrosis after crocidolite exposure and apoptosis in cells at the bronchoalveolar duct junctions as assessed via cleaved caspase-3 immunostaining. AEC involvement was verified by colocalization studies using surfactant protein C. Asbestos increased endoplasmic reticulum stress in the lungs of WT and Ogg1(-/-) mice. Compared with WT, alveolar type 2 cells isolated from Ogg1(-/-) mice have increased mtDNA damage, reduced mitochondrial aconitase expression, and increased P53 and cleaved caspase-9 expression, and these changes were enhanced 3 weeks after crocidolite exposure. These findings suggest an important role for AEC mtDNA integrity maintained by OGG1 in the pathogenesis of pulmonary fibrosis that may represent a novel therapeutic target.

Asbestos-induced Disruption of Calcium Homeostasis Induces Endoplasmic Reticulum Stress in Macrophages

Although the mechanisms for fibrosis development remain largely unknown, recent evidence indicates that endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) may act as an important fibrotic stimulus in diseased lungs. ER stress is observed in lungs of patients with idiopathic pulmonary fibrosis. In this study we evaluated if ER stress and the UPR was present in macrophages exposed to chrysotile asbestos and if ER stress in macrophages was associated with asbestos-induced pulmonary fibrosis. Macrophages exposed to chrysotile had elevated transcript levels of several ER stress genes. Macrophages loaded with the Ca(2+)-sensitive dye Fura2-AM showed that cytosolic Ca(2+) increased significantly within minutes after chrysotile exposure and remained elevated for a prolonged time. Chrysotile-induced increases in cytosolic Ca(2+) were partially inhibited by either anisomycin, an inhibitor of passive Ca(2+) leak from the ER, or 1,2-bis(2-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), an intracellular Ca(2+) chelator known to deplete ER Ca(2+) stores. Anisomycin inhibited X-box-binding protein 1 (XBP1) mRNA splicing and reduced immunoglobulin-binding protein (BiP) levels, whereas BAPTA-AM increased XBP1 splicing and BiP expression, suggesting that ER calcium depletion may be one factor contributing to ER stress in cells exposed to chrysotile. To evaluate ER stress in vivo, asbestos-exposed mice showed fibrosis development, and alveolar macrophages from fibrotic mice showed increased expression of BiP. Bronchoalveolar macrophages from asbestosis patients showed increased expression of several ER stress genes compared with normal subjects. These findings suggest that alveolar macrophages undergo ER stress, which is associated with fibrosis development.

Allele-specific effects of ecSOD on asbestos-induced fibroproliferative lung disease in mice

Previous work by others suggests that there is a strain-dependent variation in the susceptibility to inflammatory lung injury in mice. Specifically, the 129/J mice appear to be more resistant to asbestos-induced pulmonary fibrosis than the C57BL/6 strain. A separate line of evidence suggests that extracellular superoxide dismutase (ecSOD) may play an important role in protecting the lung from such injuries. We have recently reported that the 129/J strain of mice has an ecSOD genotype and phenotype distinctly different from those of the C57BL/6 mice. In order to identify ecSOD as a potential “asbestos-injury resistance” gene, we bred congenic mice, on the C57BL/6 background, carrying the wild type (sod3wt) or the 129/J (sod3129) allele for ecSOD. This allowed us to examine the role of ecSOD polymorphism in susceptibility to lung injury in an otherwise identical genetic background. Interestingly, asbestos treatment induces a significant (~40%) increase in plasma ecSOD activity in the sod3129 mice, but not in the sod3wt mice. Asbestos administration results in a loss of ecSOD activity and protein from lung tissue of both congenic strains, but the lung ecSOD activity remains significantly higher in sod3129 mice. As expected, asbestos treatment results in a significant recovery of ecSOD protein in bronchoalveolar lavage fluid (BALF). The BALF of sod3129 mice also have significantly lower levels of proteins and inflammatory cells, especially neutrophils, accompanied by a significantly lower extent of lung injury, as measured by a pathology index score or hydroxyproline content. Immunohistochemistry reveals a significant loss of ecSOD from the tips of the respiratory epithelial cells in response to asbestos treatment and that the loss of immunodetectable ecSOD is compensated for by enzyme expression by infiltrating cells, especially in the sod3wt mice. Our studies thus identify ecSOD as an important anti-inflammatory gene, responsible for most, if not all of the resistance to asbestos-induced lung injury reported for the 129/J strain of mice. The data further suggest allele-specific differences in the regulation of ecSOD expression. These congenic mice therefore represent a very useful model to study the role of this enzyme in all inflammatory diseases. Polymorphisms in human ecSOD have also been reported and it appears logical to assume that such variations may have a profound effect on disease susceptibility.

Rac1-mediated Mitochondrial H2O2 Generation Regulates MMP-9 Gene Expression in Macrophages via Inhibition of SP-1 and AP-1

Aberrant matrix deposition is a hallmark of pulmonary fibrosis and is characterized by an imbalance between matrix deposition and degradation. We have previously shown that mice harboring a conditional deletion of the GTP-binding protein, Rac1, in macrophages are protected from asbestos-induced pulmonary fibrosis. To investigate the contribution of aberrant matrix degradation, we addressed the role of Rac1 in regulating expression of macrophage-specific MMP-9 (matrix metalloproteinase-9). We found that MMP-9 gene transcription was significantly greater in Rac1 null macrophages. Deletion and mutational analysis of the MMP-9 promoter revealed that both SP-1 and AP-1 are essential for MMP-9 transcription. Overexpression of constitutive active Rac1 (V12) revealed that H(2)O(2) was derived from the mitochondria. Rac1-induced H(2)O(2) generation down-regulated MMP-9 gene transcription, whereas catalase overexpression in WT cells enhanced MMP-9 expression. SP-1 interacted directly with both c-Jun and c-Fos, and H(2)O(2) decreased this binding, suggesting that SP-1 and AP-1 function cooperatively to regulate MMP-9 transcription. Rac1-mediated H(2)O(2) inhibited the ERK MAPK, which was essential for activation of SP-1 and AP-1. ERK activation and MMP-9 expression were recovered by overexpressing catalase or transfecting siRNA for the mitochondrial iron-sulfur protein, Rieske. These observations were recapitulated in vivo. MMP-9 mRNA was higher in alveolar macrophages isolated from Rac1 null mice and wild type mice given catalase. Rac1 regulates MMP-9 transcription via mitochondrial H(2)O(2) generation, providing a potential mechanism by which Rac1 null mice fail to develop pulmonary fibrosis.

Rac1‐mediated H2O2 generation suppresses MMP‐9 expression via ERK

Our previous observations demonstrate that deletion of Rac1 from cells of the myeloid lineage protects mice from asbestos‐induced pulmonary fibrosis due to attenuated H2O2 generation. To address whether this was due to increased matrix degradation, we investigated the expression of matrix metalloproteinase (MMP‐9) in Rac1 null macrophages. Compared to wild type (WT) macrophages, MMP‐9 gene expression was significantly greater in Rac1 null cells. Site directed mutagenesis of the MMP‐9 promoter revealed that SP‐1 and AP‐1 were necessary for MMP‐9 expression. As these transcription factors are activated by MAP kinases, we examined the role of ERK in Rac1‐mediated MMP‐9 gene transcription. ERK activation was dramatically increased in Rac1 null cells and transfection with dominant‐negative ERK significantly decreased MMP‐9 promoter activity. Exogenous H2O2 inhibited MMP‐9 promoter activity in Rac1 null cells whereas over expressed catalase in WT cells enhanced ERK activation and MMP‐9 expression. Thus Rac1‐mediated H2O2 production suppresses MMP‐9 expression via inhibition of ERK. In vivo, MMP‐9 mRNA was enhanced in lung macrophages of Rac1 null mice and in WT mice administered catalase after asbestos exposure. These data suggest that enhanced MMP‐9 expression attenuates, in part, the development of asbestos‐induced pulmonary fibrosis. This work was supported by NIH grant ES‐015981.

Modulation of reactive oxygen species by Rac1 or catalase prevents asbestos-induced pulmonary fibrosis

The release of reactive oxygen species (ROS) and cytokines by alveolar macrophages has been demonstrated in asbestos-induced pulmonary fibrosis, but the mechanism linking alveolar macrophages to the pathogenesis is not known. The GTPase Rac1 is a second messenger that plays an important role in host defense. In this study, we demonstrate that Rac1 null mice are protected from asbestos-induced pulmonary fibrosis, as determined by histological and biochemical analysis. We hypothesized that Rac1 induced pulmonary fibrosis via generation of ROS. Asbestos increased TNF-alpha and ROS in a Rac1-dependent manner. TNF-alpha was elevated only 1 day after exposure, whereas ROS generation progressively increased in bronchoalveolar lavage cells obtained from wild-type (WT) mice. To determine whether ROS generation contributed to pulmonary fibrosis, we overexpressed catalase in WT monocytes and observed a decrease in ROS generation in vitro. More importantly, administration of catalase to WT mice attenuated the development of fibrosis in vivo. For the first time, these results demonstrate that Rac1 plays a crucial role in asbestos-induced pulmonary fibrosis. Moreover, it suggests that a simple intervention may be useful to prevent progression of the disease.

Modulation of reactive oxygen species and MMP9 by Rac1 prevents asbestos‐induced pulmonary fibrosis

The release of reactive oxygen species (ROS) and cytokines by alveolar macrophages has been demonstrated in asbestos‐induced pulmonary fibrosis, but the mechanism linking alveolar macrophages to the pathogenesis is not known. The GTPase Rac1 is a second messenger that plays an important role in host defense. In this study, we demonstrate that Rac1 null mice are protected from asbestos‐induced pulmonary fibrosis. We hypothesized that Rac1 induced pulmonary fibrosis via generation of ROS. Asbestos increased ROS in a Rac1‐dependent manner with ROS being significantly greater in wild type (WT) mice compared to Rac1 null mice. Extracellular matrix degradation was dependent on Rac1 activity and ROS. Expression of the macrophage specific metalloproteinase, MMP9, was significantly less in WT cells compared to Rac1 null cells. In addition, Rac1 null cells exhibited enhanced gene expression driven by AP1, an important transcription factor in MMP9 expression. Over expression of catalase decreased ROS generation and increased MMP9 expression in vitro. Importantly, administration of catalase to WT mice attenuated the development of fibrosis in vivo. For the first time, these results demonstrate that Rac1 plays a crucial role in asbestos‐induced pulmonary fibrosis. Moreover, it suggests that a simple intervention may be useful to prevent progression of the disease. This work was supported by NIH grant ES‐015981.

Engraftment of Bone Marrow Progenitor Cells in a Rat Model of Asbestos-Induced Pulmonary Fibrosis

Bone marrow-derived cells have been shown to engraft during lung fibrosis. However, it is not known if similar cells engraft consequent to inhalation of asbestos fibers that cause pulmonary fibrosis, or if the cells proliferate and differentiate at sites of injury. We examined whether bone marrow-derived cells participate in the pulmonary fibrosis that is produced by exposure to chrysotile asbestos fibers. Adult female rats were lethally irradiated and rescued by bone marrow transplant from male transgenic rats ubiquitously expressing green fluorescent protein (GFP). Three weeks later, the rats were exposed to an asbestos aerosol for 5 hours on three consecutive days. Controls were bone marrow-transplanted but not exposed to asbestos. One day and 2.5 weeks after exposure, significant numbers of GFP-labeled male cells had preferentially migrated to the bronchiolar-alveolar duct bifurcations, the specific anatomic site at which asbestos produces the initial fibrogenic lesions. GFP-positive cells were present at the lesions as monocytes and macrophages, fibroblasts, and myofibroblasts or smooth muscle cells. Staining with antibodies to PCNA demonstrated that some of the engrafted cells were proliferating in the lesions and along the bronchioles. Negative results for TUNEL at the lesions confirmed that both PCNA-positive endogenous pulmonary cells and bone marrow-derived cells were proliferating rather than undergoing apoptosis, necrosis, or DNA repair. Bone marrow-derived cells migrated into developing fibrogenic lesions, differentiated into multiple cell types, and persisted for at least 2.5 weeks after the animals were exposed to aerosolized chrysotile asbestos fibers.

IMATINIB MESYLATE INHIBITS FIBROGENESIS IN ASBESTOS-INDUCED INTERSTITIAL PNEUMONIA

Profibrogeneic cytokines contribute to the accumulation of myofibroblasts in the lung interstitium in idiopathic pulmonary fibrosis (IPF). Imatinib mesylate, a tyrosine kinase inhibitor specific for Abl, platelet-derived growth factor receptor (PDGFR) and c-Kit tyrosine kinases, has been shown to inhibit fibrosis and profibrotic signaling in mouse models of inflammation-mediated lung reactions. The authors tested imatinib mesylate in vivo in a mouse model of crocidolite asbestos-induced progressive fibrosis. The ability of imatinib mesylate to inhibit profibrogeneic cytokine-induced human pulmonary fibroblast migration was tested in vitro and the expression of its target protein tyrosine kinases was assessed with immunofluorescence. In vivo, 10 mg/kg/day imatinib mesylate inhibited histological parenchymal fibrosis and led to a decrease in collagen deposition, but had no significant effect on asbestos-induced neutrophilia. However, 50 mg/kg/day imatinib mesylate did not inhibit collagen deposition. In vitro, IPF fibroblasts expressed Abl, PDGFR-α, PDGF-β, but not c-Kit, and 1 µM imatinib mesylate inhibited profibrogeneic cytokine-induced IPF fibroblast migration. These results suggest that imatinib mesylate is a potential and specific inhibitor of fibroblast accumulation in asbestos-induced pulmonary fibrosis.

Role of transforming growth factor-β1in down-regulating TNF production by alveolar macrophages during asbestos-induced pulmonary fibrosis

Activation of alveolar macrophages (AM) for tumour necrosis factor production is suppressed initially during the inflammatory response to fibrogenic dusts. We investigated the mechanisms involved in TNF suppression, notably the role of other AM-derived mediators including prostaglandin E2 (PGE2), transforming growth factor-β1 (TGF-β1), and interleukin 6 (IL-6). The action of PGE2 and TGF-β1, on AM was different. At physiologically relevant doses (25–300 pg/ml), PGE2 did not cause significant inhibition of Hpopolysaccharide (Lps)-induced TNF release by AM in vitro but stimulated IL-6 (up to six fold), an inhibitor of AM-derived TNT. In contrast, TGF-β1 (0.5–50 ng/ml) inhibited both LPS-induced TNT and IL-6 release by 50% but had no effect on PGE2 production by AM. To determine the respective contribution of these different inhibitors in TNF suppression, AM from rats exposed to fibrogenic asbestos for weeks were treated with neutralizing antibody against TGF-β1 or indomethacin, an inhibitor of PGE2 synthesis. Treatment of rat AM with anti-TGF-β1 but not indomethacin, abrogated the observed TNT suppression. These results suggest that an autocrine, TGF-β1-dependent mechanism is involved in the down-regulation of TNF production by rat AM from animals with lung fibrosis.

Incorporation of Bromodeoxyuridine as a Method to Quantify Cell Proliferation in Bronchiolar-Alveolar Duct Regions of Asbestos-Exposed Mice

Inhalation of asbestos fibers results in progressive interstitial pulmonary fibrosis in the bronchiolar-alveolar regions of humans and experimental animals. The fibrogenic lesions were described previously by morphometry in a rodent model of asbestos-induced pulmonary fibrosis in our laboratory. Our prior studies also showed that incorporation of tri-tiated thymidine (3H-TdR) into nuclei of epithelial and interstitial cells of bronchiolar-alveolar regions correlates with this histopathologic response. The study presented here describes the early proliferative response in the bronchiolar-alveolar regions in mice after a brief exposure to chrysotile asbestos. Mice exposed to asbestos had significantly increased incorporation of bromodeoxyuridine (BrdU) in interstitial and epithelial cells of first alveolar duct bifurcations and terminal bronchioles at 48 h after exposure. The BrdU incorporation was similar at 24 h after exposure. This study confirms that BrdU provides information on asbestos-induced cell proliferation similar to that of 3H-TdR, but the technology is far safer and less time-consuming.

Asbestos stimulates IL-8 production from human lung epithelial cells.

Studies have indicated that soluble products, including chemotactic factors, released by activated lung macrophages and fibroblasts are critical mediators in the pathogenesis of asbestos-induced pulmonary fibrosis. We provide evidence that mediators produced by lung epithelial cells in response to asbestos may also contribute to lung disease. In the present study, the carcinogenic and fibrogenic fibers, chrysotile and crocidolite asbestos, were shown to directly stimulate the human pulmonary type-II epithelial cell line, A549, and to a lesser degree primary human bronchial epithelial cells, to elicit the chemotactic cytokine IL-8 in the absence of endogenous stimuli such as IL-1 and TNF. That the membrane signaling events responsible for asbestos-induced IL-8 production are distinct from those responsible for IL-8 induction by cytokines was confirmed by using membrane-stabilizing agents and protein synthesis inhibitors. Stimulation was not observed with nonfibrogenic fibers, wollastonite and titanium dioxide, and was the direct result of asbestos-induced initiation of transcription. Asbestos failed to stimulate the release of TNF, IL-1 beta, or monocyte chemoattractant protein-1 in A549 or primary bronchial epithelial cells, indicating that cytokine secretion by asbestos is highly selective. However, a slight release of IL-1 alpha, probably preformed, was released in human bronchial epithelial cells. These data suggest that epithelial cells may, in addition to macrophages and fibroblasts, be an important effector cell in the immunopathogenesis of asbestos-associated diseases and in particular, in the neutrophilic infiltration that is commonly observed after asbestos exposure.

Distribution of transforming growth factor-beta 1, fibronectin, and smooth muscle actin in asbestos-induced pulmonary fibrosis in rats.

We are studying the development of fibrogenic lesions in the lungs of rats exposed briefly to an aerosol of chrysotile asbestos fibers. This model of asbestosis has enabled us to establish very early cellular events at the specific locations where interstitial fibrosis will develop. These sites, the first alveolar duct bifurcations, are where the fibers are initially deposited and where macrophages first accumulate. In the studies presented here, we used immunohistochemical techniques to show that these macrophages exhibit strong localization of transforming growth factor-beta. In the adjacent developing fibrogenic lesions a clear increase in fibronectin staining was demonstrated and morphological analysis indicated a significant increase in amounts of smooth muscle actin. Such studies are essential in furthering our understanding of the distribution of potential mediators of the fibrogenic process and the cellular responses they elicit during the pathogenesis of disease.

The Relationship Between HLA-A, B, DQ, and DR Antigens and Asbestos-induced Lung Disease

To evaluate the relationship between human leukocyte antigen (HLA) and both asbestos-induced pulmonary fibrosis and pleural fibrosis, we obtained HLA-A, B, C, DQ, and DR phenotypes in 42 long-term asbestos-exposed workers. Among these exposed workers, 15 had asbestosis (ILO > or = 1/0) on the chest radiograph and 18 had asbestos-induced pleural fibrosis. We found that there was an increased percentage of HLA-A29, HLA-B44, and HLA-Bw4 in the subjects with asbestosis. In addition, we observed a marginally positive relationship between HLA-A29 and the severity of pulmonary fibrosis. Similarly, there was a higher prevalence of HLA-DRw53 and DQ2 in the subjects with asbestos-induced pleural fibrosis. The presence of HLA-DQ2 was significantly related to the extent and type of asbestos-induced pleural fibrosis while HLA-DRw53 was not consistently related to the type or extent of pleural disease. Importantly, we observed that HLA-A29, HLA-B44, HLA-Bw4, HLA-DRw53, and HLA-DQ2 do not have a significantly shorter duration or latency of asbestos exposure. Moreover, none of the HLA haplotypes (A29, B44, Bw4, DRw53, and DQ2) that we found to be associated with radiographic manifestations of asbestos-induced lung disease were associated with the physiologic abnormalities that have been traditionally associated with asbestos-induced lung disease. The only exception was an isolated decrease in the residual volume associated with the presence of HLA-A29. These results suggest that the HLA-A29 phenotype may be associated with the development of asbestosis and the HLA-DQ2 phenotype may be associated with the development of asbestos-induced pleural fibrosis. However, these associations are not particularly strong, physiologic correlation is lacking, and previous studies do not support our findings.