Association of IL-6 with PM2.5 Components: Importance of Characterizing Filter-Based PM2.5 Following Extraction.

Filter-based toxicology studies are conducted to establish the biological plausibility of the well-established health impacts associated with fine particulate matter (PM2.5) exposure. Ambient PM2.5 collected on filters is extracted into solution for toxicology applications, but frequently, characterization is nonexistent or only performed on filter-based PM2.5, without consideration of compositional differences that occur during the extraction processes. To date, the impact of making associations to measured components in ambient instead of extracted PM2.5 has not been investigated. Filter-based PM2.5 was collected at locations (n = 5) and detailed characterization of both ambient and extracted PM2.5 was performed. Alveolar macrophages (AMJ2-C11) were exposed (3, 24, and 48 h) to PM2.5 and the pro-inflammatory cytokine interleukin (IL)-6 was measured. IL-6 release differed significantly between PM2.5 collected from different locations; surprisingly, IL-6 release was highest following treatment with PM2.5 from the lowest ambient concentration location. IL-6 was negatively correlated with the sum of ambient metals analyzed, as well as with concentrations of specific constituents which have been previously associated with respiratory health effects. However, positive correlations of IL-6 with extracted concentrations indicated that the negative associations between IL-6 and ambient concentrations do not accurately represent the relationship between inflammation and PM2.5 exposure. Additionally, seven organic compounds had significant associations with IL-6 release when considering ambient concentrations, but they were not detected in the extracted solution. Basing inflammatory associations on ambient concentrations that are not necessarily representative of in vitro exposures creates misleading results; this study highlights the importance of characterizing extraction solutions to conduct accurate health impact research.

Secreted Phosphoprotein 1 and Sex-Specific Differences in Silica-Induced Pulmonary Fibrosis in Mice.

BACKGROUND: Fibrotic lung diseases occur predominantly in males, and reports describe better survival in affected females. Male mice are more sensitive to silica-induced lung fibrosis than silica-treated female mice. Secreted phosphoprotein 1 (SPP1, also known as osteopontin) increases in pulmonary fibrosis, and Spp1 transcription may be regulated by estrogen or estrogen receptor-related receptors. OBJECTIVE: We determined whether differences in silica-induced SPP1 levels contribute to sex differences in lung fibrosis. METHODS: Male and female mice were treated with 0.2 g/kg intratracheal silica, and lung injury was assessed 1, 3, or 14 days post-exposure. Gene-targeted (Spp1-/-) mice, control Spp1+/+ (C57BL/6J) mice, ovariectomized (OVX) female mice, and estrogen-treated male mice were treated with silica, and lung injury was assessed. RESULTS: Silica-induced SPP1 in lung tissue, bronchoalveolar lavage, and serum increased more in male than in female mice. Following silica treatment, bronchoalveolar lavage cell infiltrates decreased in female Spp1-/- mice compared with female Spp1+/+ mice, and lung hydroxyproline decreased in male Spp1-/- mice compared with male Spp1+/+ mice. OVX female mice had increased lung SPP1 expression in response to silica compared with silica-treated sham female mice. Silica-induced lung collagen and hydroxyproline (markers of fibrosis), and SPP1 levels decreased in estrogen-treated males compared with untreated males. CONCLUSION: These findings suggest that sex-specific differences in SPP1 levels contribute to the differential sensitivity of male and female mice to the development of silica-induced fibrosis. CITATION: Latoche JD, Ufelle AC, Fazzi F, Ganguly K, Leikauf GD, Fattman CL. 2016. Secreted phosphoprotein 1 and sex-specific differences in silica-induced pulmonary fibrosis in mice. Environ Health Perspect 124:1199-1207; http://dx.doi.org/10.1289/ehp.1510335.

Secreted phosphoprotein 1 is a determinant of lung function development in mice.

Secreted phosphoprotein 1 (Spp1) is located within quantitative trait loci associated with lung function that was previously identified by contrasting C3H/HeJ and JF1/Msf mouse strains that have extremely divergent lung function. JF1/Msf mice with diminished lung function had reduced lung SPP1 transcript and protein during the peak stage of alveologenesis (postnatal day [P]14-P28) as compared with C3H/HeJ mice. In addition to a previously identified genetic variant that altered runt-related transcription factor 2 (RUNX2) binding in the Spp1 promoter, we identified another promoter variant in a putative RUNX2 binding site that increased the DNA protein binding. SPP1 induced dose-dependent mouse lung epithelial-15 cell proliferation. Spp1((-/-)) mice have decreased specific total lung capacity/body weight, higher specific compliance, and increased mean airspace chord length (Lm) compared with Spp1((+/+)) mice. Microarray analysis revealed enriched gene ontogeny categories, with numerous genes associated with lung development and/or respiratory disease. Insulin-like growth factor 1, Hedgehog-interacting protein, wingless-related mouse mammary tumor virus integration site 5A, and NOTCH1 transcripts decreased in the lung of P14 Spp1((-/-)) mice as determined by quantitative RT-PCR analysis. SPP1 promotes pneumocyte growth, and mice lacking SPP1 have smaller, more compliant lungs with enlarged airspace (i.e., increased Lm). Microarray analysis suggests a dysregulation of key lung developmental transcripts in gene-targeted Spp1((-/-)) mice, particularly during the peak phase of alveologenesis. In addition to its known roles in lung disease, this study supports SPP1 as a determinant of lung development in mice.

TNFR1/phox interaction and TNFR1 mitochondrial translocation Thwart silica-induced pulmonary fibrosis.

Macrophages play a fundamental role in innate immunity and the pathogenesis of silicosis. Phagocytosis of silica particles is associated with the generation of reactive oxygen species (ROS), secretion of cytokines, such as TNF, and cell death that contribute to silica-induced lung disease. In macrophages, ROS production is executed primarily by activation of the NADPH oxidase (Phox) and by generation of mitochondrial ROS (mtROS); however, the relative contribution is unclear, and the effects on macrophage function and fate are unknown. In this study, we used primary human and mouse macrophages (C57BL/6, BALB/c, and p47(phox-/-)) and macrophage cell lines (RAW 264.7 and IC21) to investigate the contribution of Phox and mtROS to silica-induced lung injury. We demonstrate that reduced p47(phox) expression in IC21 macrophages is linked to enhanced mtROS generation, cardiolipin oxidation, and accumulation of cardiolipin hydrolysis products, culminating in cell death. mtROS production is also observed in p47(phox-/-) macrophages, and p47(phox-/-) mice exhibit increased inflammation and fibrosis in the lung following silica exposure. Silica induces interaction between TNFR1 and Phox in RAW 264.7 macrophages. Moreover, TNFR1 expression in mitochondria decreased mtROS production and increased RAW 264.7 macrophage survival to silica. These results identify TNFR1/Phox interaction as a key event in the pathogenesis of silicosis that prevents mtROS formation and reduces macrophage apoptosis.

IL-22 is essential for lung epithelial repair following influenza infection.

Influenza infection is widespread in the United States and the world. Despite low mortality rates due to infection, morbidity is common and little is known about the molecular events involved in recovery. Influenza infection results in persistent distal lung remodeling, and the mechanism(s) involved are poorly understood. Recently IL-22 has been found to mediate epithelial repair. We propose that IL-22 is critical for recovery of normal lung function and architecture after influenza infection. Wild-type and IL-22(-/-) mice were infected with influenza A PR8/34 H1N1 and were followed up for up to 21 days post infection. IL-22 receptor was localized to the airway epithelium in naive mice but was expressed at the sites of parenchymal lung remodeling induced by influenza infection. IL-22(-/-) mice displayed exacerbated lung injury compared with wild-type mice, which correlated with decreased lung function 21 days post infection. Epithelial metaplasia was observed in wild-type mice but was not evident in IL-22(-/-) animals that were characterized with an increased fibrotic phenotype. Gene expression analysis revealed aberrant expression of epithelial genes involved in repair processes, among changes in several other biological processes. These data indicate that IL-22 is required for normal lung repair after influenza infection. IL-22 represents a novel pathway involved in interstitial lung disease.

Metallothionein-induced zinc partitioning exacerbates hyperoxic acute lung injury.

Hypozincemia, with hepatic zinc accumulation at the expense of other organs, occurs in infection, inflammation, and aseptic lung injury. Mechanisms underlying zinc partitioning or its impact on extrahepatic organs are unclear. Here we show that the major zinc-binding protein, metallothionein (MT), is critical for zinc transmigration from lung to liver during hyperoxia and preservation of intrapulmonary zinc during hyperoxia is associated with an injury-resistant phenotype in MT-null mice. Particularly, lung-to-liver zinc ratios decreased in wild-type (WT) and increased significantly in MT-null mice breathing 95% oxygen for 72 h. Compared with female adult WT mice, MT-null mice were significantly protected against hyperoxic lung injury indicated by reduced inflammation and interstitial edema, fewer necrotic changes to distal airway epithelium, and sustained lung function at 72 h hyperoxia. Lungs of MT-null mice showed decreased levels of immunoreactive LC3, an autophagy marker, compared with WT mice. Analysis of superoxide dismutase (SOD) activity in the lungs revealed similar levels of manganese-SOD activity between strains under normoxia and hyperoxia. Lung extracellular SOD activity decreased significantly in both strains at 72 h of hyperoxia, although there was no difference between strains. Copper-zinc-SOD activity was ~4× higher under normoxic conditions in MT-null compared with WT mice but was not affected in either group by hyperoxia. Collectively the data suggest that genetic deletion of MT-I/II in mice is associated with compensatory increase in copper-zinc-SOD activity, prevention of hyperoxia-induced zinc transmigration from lung to liver, and hyperoxia-resistant phenotype strongly associated with differences in zinc homeostasis during hyperoxic acute lung injury.

Innate immune activation by inhaled lipopolysaccharide, independent of oxidative stress, exacerbates silica-induced pulmonary fibrosis in mice.

Acute exacerbations of pulmonary fibrosis are characterized by rapid decrements in lung function. Environmental factors that may contribute to acute exacerbations remain poorly understood. We have previously demonstrated that exposure to inhaled lipopolysaccharide (LPS) induces expression of genes associated with fibrosis. To address whether exposure to LPS could exacerbate fibrosis, we exposed male C57BL/6 mice to crystalline silica, or vehicle, followed 28 days later by LPS or saline inhalation. We observed that mice receiving both silica and LPS had significantly more total inflammatory cells, more whole lung lavage MCP-1, MIP-2, KC and IL-1ß, more evidence of oxidative stress and more total lung hydroxyproline than mice receiving either LPS alone, or silica alone. Blocking oxidative stress with N-acetylcysteine attenuated whole lung inflammation but had no effect on total lung hydroxyproline. These observations suggest that exposure to innate immune stimuli, such as LPS in the environment, may exacerbate stable pulmonary fibrosis via mechanisms that are independent of inflammation and oxidative stress.

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.

Lung extracellular superoxide dismutase overexpression lessens bleomycin-induced pulmonary hypertension and vascular remodeling.

Interstitial lung disease is a devastating disease in humans that can be further complicated by the development of secondary pulmonary hypertension. Accumulating evidence indicates that the oxidant superoxide can contribute to the pathogenesis of both interstitial lung disease and pulmonary hypertension. We used a model of pulmonary hypertension secondary to bleomycin-induced pulmonary fibrosis to test the hypothesis that an imbalance in extracellular superoxide and its antioxidant defense, extracellular superoxide dismutase, will promote pulmonary vascular remodeling and pulmonary hypertension. We exposed transgenic mice overexpressing lung extracellular superoxide dismutase and wild-type littermates to a single dose of intratracheal bleomycin, and evaluated the mice weekly for up to 35 days. We assessed pulmonary vascular remodeling and the expression of several genes critical to lung fibrosis, as well as pulmonary hypertension and mortality. The overexpression of extracellular superoxide dismutase protected against late remodeling within the medial, adventitial, and intimal layers of the vessel wall after the administration of bleomycin, and attenuated pulmonary hypertension at the same late time point. The overexpression of extracellular superoxide dismutase also blocked the early up-regulation of two key genes in the lung known to be critical in pulmonary fibrosis and vascular remodeling, the transcription factor early growth response-1 and transforming growth factor-ß. The overexpression of extracellular superoxide dismutase attenuated late pulmonary hypertension and significantly improved survival after exposure to bleomycin. These data indicate an important role for an extracellular oxidant/antioxidant imbalance in the pathogenesis of pulmonary vascular remodeling associated with secondary pulmonary hypertension attributable to bleomycin-induced lung fibrosis.

Gender influences the response to experimental silica-induced lung fibrosis in mice.

Accumulating evidence suggests that gender can have a profound effect on incidence and severity of a variety of pulmonary diseases. To address the influence of gender on the development of silica-induced pulmonary fibrosis, we instilled 0.2 g/kg silica into male and female C57BL/6 mice and examined the fibrotic and inflammatory response at 14 days postexposure. Both silica-exposed male and female mice had significant increases in total lung hydroxyproline compared with saline controls. However, silica-exposed female mice had significantly less total lung hydroxyproline than silica-exposed male mice. This observation was confirmed by color thresholding image analysis. Interestingly, silica-exposed female mice had significantly more inflammatory cells, the majority of which were macrophages, as well as higher levels of the macrophage-specific chemokines MCP-1 and CCL9 in whole lung lavage compared with silica-exposed male mice. We also show that at baseline, estrogen receptor α (ERα) mRNA expression is lower in female mice than in males and that ERα mRNA expression is decreased by silica exposure. Finally, we show that the response of ovariectomized female mice to silica instillation is similar to that of male mice. These observations together show that gender influences the lung response to silica.

The role of the receptor for advanced glycation end-products in a murine model of silicosis.

BACKGROUND: The role of the receptor for advanced glycation end-products (RAGE) has been shown to differ in two different mouse models of asbestos and bleomycin induced pulmonary fibrosis. RAGE knockout (KO) mice get worse fibrosis when challenged with asbestos, whereas in the bleomycin model they are largely protected against fibrosis. In the current study the role of RAGE in a mouse model of silica induced pulmonary fibrosis was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Wild type (WT) and RAGE KO mice received a single intratracheal (i.t.) instillation of silica in saline or saline alone as vehicle control. Fourteen days after treatment mice were subjected to a lung mechanistic study and the lungs were lavaged and inflammatory cells, protein and TGF-beta levels in lavage fluid determined. Lungs were subsequently either fixed for histology or excised for biochemical assessment of fibrosis and determination of RAGE protein- and mRNA levels. There was no difference in the inflammatory response or degree of fibrosis (hydroxyproline levels) in the lungs between WT and RAGE KO mice after silica injury. However, histologically the fibrotic lesions in the RAGE KO mice had a more diffuse alveolar septal fibrosis compared to the nodular fibrosis in WT mice. Furthermore, RAGE KO mice had a significantly higher histologic score, a measure of affected areas of the lung, compared to WT silica treated mice. A lung mechanistic study revealed a significant decrease in lung function after silica compared to control, but no difference between WT and RAGE KO. While a dose response study showed similar degrees of fibrosis after silica treatment in the two strains, the RAGE KO mice had some differences in the inflammatory response compared to WT mice. CONCLUSIONS/SIGNIFICANCE: Aside from the difference in the fibrotic pattern, these studies showed no indicators of RAGE having an effect on the severity of pulmonary fibrosis following silica injury.

Iron accumulation and expression of iron-related proteins following murine exposure to crocidolite.

We tested the postulate that asbestos exposure alters iron homeostasis in the mouse lung. Crocidolite asbestos (100 microg intratracheally) was instilled into C57BL/6 mice. TiO2 served as a control exposure. Using iron staining and immunohistochemistry, concentrations of this metal and expression of several iron transport and storage proteins were evaluated at one day and one month following asbestos exposure. Iron was not stainable one day following asbestos instillation but was increased one month later. There was an elevated expression of duodenal cytochrome b (Dcytb), divalent metal transporter 1 (DMT1), and ferritin at both one day and one month after crocidolite exposure. While ferroportin (FPN1) expression was increased one day after asbestos exposure, levels of this metal exporter had returned to baseline at one month. TiO2 did not affect changes in either the iron concentration or the expression of these iron-related proteins at one day and one month. We conclude that asbestos exposure alters lung iron homeostasis with an accumulation of the metal resulting. Elevations in available iron affect changes in the expression of Dcytb, DMT1, ferritin, and FPN1, which further modify metal homeostasis in the lung.

Superoxide dismutase 3, extracellular (SOD3) variants and lung function.

Polymorphisms in Superoxide dismutase 3, extracellular (SOD3) have been associated with reduced lung function and susceptibility to chronic obstructive pulmonary disease (COPD) in adults. Previously, we identified SOD3 as a contributing factor to altered ventilation efficiency (dead space volume/total lung capacity) in mice. Because SOD3 protects the extracellular matrix of the lung, we hypothesized that SOD3 variants also may influence postnatal lung function development. In this study, SOD3 transcript and protein localization were examined in mouse strains with differing ventilation efficiency [C3H/HeJ (high), JF1/Msf (low)] during postnatal lung development. Compared with C3H/HeJ mice, JF1/Msf mice had Sod3 promoter single nucleotide polymorphisms (SNPs) that could affect transcription factor binding sites and a decline in total lung SOD3 mRNA during postnatal development. In adult JF1/Msf mice, total lung SOD3 activity as well as SOD3 transcript and protein in airway epithelial and alveolar type II cells and the associated matrix decreased. In children (n = 1,555; age 9-11 yr), two common SOD3 SNPs, one located in the promoter region [C/T affecting a predicted aryl hydrocarbon receptor-xenobiotic response element (AhR-XRE) binding motif] and the other in exon 2 (Thr/Ala missense mutation), were associated with decreased forced expiratory volume in 1 s (FEV(1)), and the promoter SNP was associated with decreased maximal expiratory flow at 25% volume (MEF(25)). In vitro, a SOD3 promoter region-derived oligonucleotide containing the C variant was more effective in competing with the nuclear protein-binding capacity of a labeled probe than that containing the T variant. Along with the previous associated risk of lung function decline in COPD, these findings support a possible role of SOD3 variants in determining lung function in children.

A role for the receptor for advanced glycation end products in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a severely debilitating disease associated with a dismal prognosis. There are currently no effective therapies for IPF, thus the identification of novel therapeutic targets is greatly needed. The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptors whose activation has been linked to various pathologies. In healthy adult animals, RAGE is expressed at the highest levels in the lung compared to other tissues. To investigate the hypothesis that RAGE is involved in IPF pathogenesis, we have examined its expression in two mouse models of pulmonary fibrosis and in human tissue from IPF patients. In each instance we observed a depletion of membrane RAGE and its soluble (decoy) isoform, sRAGE, in fibrotic lungs. In contrast to other diseases in which RAGE signaling promotes pathology, immunohistochemical and hydroxyproline quantification studies on aged RAGE-null mice indicate that these mice spontaneously develop pulmonary fibrosis-like alterations. Furthermore, when subjected to a model of pulmonary fibrosis, RAGE-null mice developed more severe fibrosis, as measured by hydroxyproline assay and histological scoring, than wild-type controls. Combined with data from other studies on mouse models of pulmonary fibrosis and human IPF tissues indicate that loss of RAGE contributes to IPF pathogenesis.

Epithelial expression of TIMP-1 does not alter sensitivity to bleomycin-induced lung injury in C57BL/6 mice.

Matrix metalloproteinases (MMPs) are mediators of lung injury, and their activity has been associated with the development of pulmonary fibrosis. To understand how MMPs regulate the development of pulmonary fibrosis, we examined MMP expression in two strains of mice with differing sensitivities to the fibrosis-inducing drug bleomycin. After a single intratracheal injection of the drug, bleomycin-sensitive C57BL/6 mice showed increased expression for MMPs (-2, -7, -9, -13) at both 7 and 14 days posttreatment compared with the bleomycin-resistant BALB/c strain. In addition, TIMP-1, an endogenous inhibitor of MMPs, was upregulated in the lungs of C57BL/6 mice but not BALB/c mice. We designed two strategies to decrease MMP expression to potentially decrease sensitivity of C57BL/6 mice: 1) we engineered C57BL/6 mice that overexpressed TIMP-1 in their lungs via surfactant protein C (SP-C) promoter; and 2) we inhibited expression of MMPs independent of TIMP-1 by knocking out metallothionein (MT), a critical zinc binding protein. SP-C-TIMP-1 mice reduced MMP expression in response to bleomycin. However, they were equally sensitive to bleomycin as their wild-type counterparts, displaying similar levels of hydroxyproline in the lung tissue. MT null mice displayed decreased lung activity of MMPs with no change in TIMP-1. Nonetheless, there was no difference between the MT null and wild-type control littermates with regards to any of the lung injury parameters measured. We conclude that although TIMP-1 expression is differentially regulated in fibrosis-sensitive and fibrosis-resistant strains, epithelial overexpression of TIMP-1 does not appear to substantially alter fibrotic lung disease in mice.

Apoptosis in pulmonary fibrosis: too much or not enough?

Apoptosis plays an important role in both normal lung homeostasis and lung remodeling associated with fibrotic lung disease. Whether apoptosis promotes or inhibits the pathogenesis of pulmonary fibrosis depends upon the cell type involved and the microenvironment of the affected lung. Undue cell loss in the alveolar epithelium may be important early in idiopathic pulmonary fibrosis (IPF) progression, while reduced fibroblast and myofibroblast apoptosis has been associated with the formation of fibrotic lesions. As such, novel therapies based on the stimulation or inhibition of apoptosis may prove beneficial to the treatment of patients with IPF.

Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury.

Mesenchymal stem cells (MSCs) have been exploited as cellular vectors to treat a wide array of diseases but the mechanisms responsible for their therapeutic effect remain indeterminate. Previously, we reported that MSCs inhibit bleomycin (BLM)-induced inflammation and fibrosis within the lungs of mice. Interrogation of the MSC transcriptome identified interleukin 1 receptor antagonist (IL1RN) as a potential mediator of this effect. Fractionation studies indicated that MSCs are the principal source of IL1RN in murine bone marrow and that its expression is restricted to a unique subpopulation of cells. Moreover, MSC-conditioned media was shown to block proliferation of an IL-1alpha-dependent T cell line and inhibit production of TNF-alpha by activated macrophages in vitro. Studies conducted in mice revealed that MSC administration was more effective than recombinant IL1RN delivered via adenoviral infection or osmotic pumps in inhibiting BLM-induced increases in TNF-alpha, IL-1alpha, and IL1RN mRNA in lung, IL1RN protein in bronchoalveolar lavage (BAL) fluid, and trafficking of lymphocytes and neutrophils into the lung. Therefore, MSCs protect lung tissue from BLM-induced injury by blocking TNF-alpha and IL-1, two fundamental proinflammatory cytokines in lung. Identification of IL1RN-expressing human MSC subpopulations may provide a novel cellular vector for treating chronic inflammatory diseases in humans.

Matrix metalloproteinases promote inflammation and fibrosis in asbestos-induced lung injury in mice.

Inhalation of asbestos fibers causes pulmonary inflammation and eventual pulmonary fibrosis (asbestosis). Although the underlying molecular events are poorly understood, protease/antiprotease and oxidant/antioxidant imbalances are believed to contribute to the disease. Implicated in other forms of pulmonary fibrosis, the matrix metalloproteinases (MMPs) have not been examined in asbestosis. We therefore hypothesized that MMPs play a pathogenic role in asbestosis development. Wild-type C57BL/6 mice were intratracheally instilled with 0.1 mg crocidolite asbestos, causing an inflammatory response at 1 d and a developing fibrotic response at 7, 14, and 28 d. Gelatin zymography demonstrated an increase in MMP-9 (gelatinase B) during the inflammatory phase, while MMP-2 (gelatinase A) was profoundly increased in the fibrotic phase. Immunohistochemistry revealed MMP-9 in and around bronchiolar and airspace neutrophils that were often associated with visible asbestos fibers. MMP-2 was found in fibrotic regions at 7, 14, and 28 d. No increases in RNA levels of MMP-2, MMP-9, or MMP-8 were found, but levels of MMP-7, MMP-12, and MMP-13 RNA did increase at 14 d. The MMP inhibitors, TIMP-1 and TIMP-2, were also increased at 7-28 d after asbestos exposure. To confirm the importance of MMP activity in disease progression, mice exposed to asbestos were given daily injections of the MMP inhibitor, GM6001. MMP inhibition reduced inflammation and fibrosis in asbestos-treated mice. Collectively, these data suggest that MMPs contribute to the pathogenesis of asbestosis through effects on inflammation and fibrosis development.

Increased sensitivity to asbestos-induced lung injury in mice lacking extracellular superoxide dismutase.

Asbestosis is a chronic form of interstitial lung disease characterized by inflammation and fibrosis that results from the inhalation of asbestos fibers. Although the pathogenesis of asbestosis is poorly understood, reactive oxygen species may mediate the progression of this disease. The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) can protect the lung against a variety of insults; however, its role in asbestosis is unknown. To determine if EC-SOD plays a direct role in protecting the lung from asbestos-induced injury, intratracheal injections of crocidolite were given to wild-type and ec-sod-null mice. Bronchoalveolar lavage fluid (BALF) from asbestos-treated ec-sod-null mice at 24 h, 14 days, or 28 days posttreatment showed increased inflammation and total BALF protein content compared to that of wild-type mice. In addition, lungs from ec-sod-null mice showed increased hydroxyproline content compared to those of wild-type mice, indicating a greater fibrotic response. Finally, lungs from ec-sod-null mice showed greater oxidative damage, as assessed by nitrotyrosine content compared to those of their wild-type counterparts. These results indicate that depletion of EC-SOD from the lung increases oxidative stress and injury in response to asbestos.