Genetic loss of Gas6/Mer pathway attenuates silica-induced lung inflammation and fibrosis in mice.

Long-term inhalation of crystalline silica particles leads to silicosis characterized by pulmonary inflammation and interstitial fibrosis. The growth arrest-specific protein 6 (Gas6) and its tyrosine receptor Mer have been implicated to involve in the regulation of inflammation, innate immunity and tissue repair. However, the role of Gas6 or Mer in silica-induced lung inflammation and fibrosis has not been investigated previously. In this study, we observed a remarkable increase of Gas6 in bronchoalveolar lavage fluid (BALF) from wild-type C57BL/6 mice after silica intratracheal administration. Then, we investigated whether genetic loss of Gas6 or Mer could attenuate silica-induced lung inflammation and fibrosis. Our results showed that Gas6-/- and Mer-/- mice exhibited reduced lung inflammation response from days 7 to 84 after silica exposure. We also uncovered an overexpression of the suppressor of cytokine signaling protein 1 in silica-treated deficient mice. Moreover, Gas6 or Mer deficiency attenuated silica-induced collagen deposition by inhibiting the expression of transforming growth factor-ß. We conclude that gene absence of Gas6 or Mer is protective against silica-induced lung inflammation and fibrosis in mice. Targeting Gas6/Mer pathway may be a potential therapeutic approach to treat pulmonary fibrosis in patients with silicosis.

Ac-SDKP increases α-TAT 1 and promotes the apoptosis in lung fibroblasts and epithelial cells double-stimulated with TGF-ß1 and silica.

Crystalline silica (SiO2) particles have very strong toxicity to the lungs, and silicosis is an excessive pulmonary interstitial remodeling disease that follows persistent SiO2 injury. We showed here that DNA double strand breaks (DSBs) and apoptosis were aggravated during rat silicosis induced by SiO2 exposure. Ac-SDKP attenuates lung parenchymal distortion and collagen deposition, and decreases the expression of γH2AX, p21, and cleaved caspase-3, as well as improves the reduction of pulmonary function caused by silicosis. In vitro, we found an evolution of smooth muscle actin α (α-SMA), collagen type I (Col I) in both A549 and MRC-5 cells in response to transforming growth factor-beta 1 (TGF-ß1) + SiO2. Only A549 cells showed any reduction in the rate of apoptosis induced by the double stimulation, because of the anti-apoptotic effects of TGF-ß1. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is an anti-fibrotic tetrapeptide. It also has the ability to promote the apoptosis of leukemia cells. However its role in promoting cell apoptosis in silicosis is still unknown. We here found that Ac-SDKP could induce cell apoptosis and inhibit fibrotic response in A549 and MRC-5 cells treated with TGF-ß1 + SiO2, and these effects depended on regulation of α-tubulin acetyltransferase 1 (α-TAT1). These findings suggest that Ac-SDKP may have therapeutic value in the treatment of silicotic fibrosis.

Crystalline silica alters Sulfatase-1 expression in rat lungs which influences hyper-proliferative and fibrogenic effects in human lung epithelial cells.

Lung epithelial cells are the first cell-type to come in contact with hazardous dust materials. Upon deposition, they invoke complex reactions in attempt to eradicate particles from the airways, and repair damage. The cell surface is composed of a heterogeneous network of matrix proteins and proteoglycans, which act as scaffold and control cell-signaling networks. These functions are controlled, in part, by the sulfation patterns of heparin-sulfate proteoglycans (HSPGs), which are enzymatically regulated. Although there is evidence of altered HSPG-sulfation in idiopathic pulmonary fibrosis (IPF), this is not investigated in silicosis. Our previous studies revealed down-regulation of Sulfatase-1 (SULF1) in human bronchial epithelial cells (BECs) by crystalline silica (CS). In this study, CS-induced down-regulation of SULF1, and increases in Sulfated-HSPGs, were determined in human BECs, and in rat lungs. By siRNA and plasmid transfection techniques the effects of SULF1 expression on silica-induced fibrogenic and proliferative gene expression were determined. These studies confirmed down-regulation of SULF1 and subsequent increases in sulfated-HSPGs in vitro. Moreover, short-term exposure of rats to CS resulted in similar changes in vivo. Conversely, effects were reversed after long term CS exposure of rats. SULF1 knockdown, and overexpression alleviated and exacerbated silica-induced decrease in cell viability, respectively. Furthermore, overexpression of SULF1 promoted silica-induced proliferative and fibrogenic gene expression, and collagen production. These findings demonstrate that the HSPG modification enzyme SULF1 and HSPG sulfation are altered by CS in vitro and in vivo. Furthermore, these changes may contribute to CS-induced lung pathogenicity by affecting injury tolerance, hyperproliferation, and fibrotic effects.

The induction of lipid peroxidation during the acute oxidative stress response induced by intratracheal instillation of fine crystalline silica particles in rats.

Crystalline silica (SiO2) is an important material for industry but is considered potentially carcinogenic. Inhalation of a crystalline SiO2 aerosol may contribute to serious lung diseases. Crystalline SiO2 particles are commonly used as a positive control in toxicity assays of particulate materials (e.g. nanoparticles). Crystalline SiO2 induces oxidative stress resulting in lipid peroxidation, but the acute oxidative stress response in the lung is not well understood. Lipid peroxidation during the acute stage of oxidative stress after instillation of crystalline SiO2 into rats was examined by bronchoalveolar lavage fluid (BALF) analysis. The levels of 8-iso-prostaglandin F2α and hydroxyoctadecadienoic acid (HODE) in the BALF were measured using liquid chromatography coupled to quadrupole mass spectrometry. The concentration of the antioxidant protein heme oxygenase-1 (HO-1) in the BALF was determined using enzyme-linked immunosorbent assay. Intratracheal instillation of crystalline SiO2 increased the level of HODE and HO-1 in BALF at 24 h after administration. The levels of HODE and HO-1 returned to baseline at 72 h after instillation. Lactate dehydrogenase leakage was observed only after 1 h instillation. These results suggest that the contribution of oxidative stress to the pulmonary toxicity of crystalline SiO2 is minimal in the early acute stage after exposure.

[Changes in serum protease and cytokine in patients with silicosis, tuberculosis, and lung cancer].

OBJECTIVE: To investigate the changes in serum protease and cytokine in patients with silicosis, tuberculosis, and lung cancer. METHODS: Serum samples of patients with silicosis, tuberculosis, and lung cancer were collected. The variation trends of the expression of granzyme A, cathepsin G, apolipoprotein A, and interferon-ß (IFN-ß) were analyzed using enzyme-linked immunosorbent assay. RESULTS: The concentration of apolipoprotein A of the silicosis group was 200 µg/ml, significantly higher than those of the tuberculosis and lung cancer groups (P < 0.05), and the lung cancer group had a significantly higher concentration of apolipoprotein A compared with the tuberculosis group (P < 0.05). The silicosis group had significantly higher expression of cathepsin G compared with the tuberculosis and lung cancer groups (P < 0.05), and the tuberculosis group and lung cancer group showed no significant difference in the concentration of cathepsin G (P > 0.05). The tuberculosis group had a significantly higher concentration of granzyme A than the silicosis and lung cancer groups (P < 0.05), and the silicosis group and lung cancer group had similar protein concentration trends (P > 0.05). The tuberculosis group and lung cancer group had significantly higher concentration of IFN-ß compared with the silicosis group (P < 0.05), and the tuberculosis group and lung cancer group showed no significant difference in IFN-ß concentration (P > 0.05). CONCLUSION: This study may offer diagnostic markers for the clinical diagnosis of silicosis, tuberculosis, and lung cancer, and could provide a basis for the research, as well as potential molecular targets for the diagnosis and treatment of these diseases.

[Serum levels of matrix metalloproteinase 9 and 19 in the patients with pneumoconiosis].

OBJECTIVE: To measure peripheral serum levels of matrix metalloproteinase 9 (MMP9) and matrix metalloproteinase 19 (MMP19) in patients with pneumoconiosis, and to investigate their feasibility as potential biomarkers for pneumoconiosis. METHODS: Ninety-eight male patients with pneumoconiosis (49 patients in phase I, 36 patients in phase II, and 13 patients in phase III) were enrolled as subjects, which included 41 patients with silicosis and 57 patients with coal workers' pneumoconiosis. Ninety-eight healthy male physical examinees were used as controls. A fasting blood sample (3 ml) was collected from the peripheral venous blood of each patient or control, and the serum was separated from the blood sample. The expression levels of MMP9 and MMP19 in serum were measured by enzyme-linked immunosorbent assay. RESULTS: Serum levels of MMP9 and MMP19 in patients with silicosis or coal workers' pneumoconiosis were significantly lower than those in the control group (P < 0.05). Serum levels of MMP19 in patients with silicosis were significantly higher than those in patients with coal workers' pneumoconiosis (P < 0.05). Serum levels of MMP19 in patients exposed to dust for less than 7 years were significantly higher than those in patients exposed to dust for more than 20 years (P < 0.05). There were no significant differences in serum levels of MMP9 and MMP19 between patients with different levels of pulmonary function impairment (P > 0.05). Serum expression levels of MMP9 and MMP19 were positively correlated with each other in both patients with pneumoconiosis and those in the control group (P < 0.05). The serum expression level of MMP9 was negatively correlated with the stage of pneumoconiosis (P < 0.05). CONCLUSION: Serum MMP9 and MMP19 may be used as potential biomarkers for pneumoconiosis.

[Changes in activities of SOD and GSH-Px in induced sputum and their significance among silicosis patients].

OBJECTIVE: To observe the changes in activities of superoxide dismutase (SOD) and glutathione peroxide (GSH-Px) in the induced sputum of silicosis patients, and to investigate the roles of SOD and GSH-Px in the development and progression of silicosis and the significance of measuring activities of SOD and GSH-Px in induced sputum among silicosis patients. METHODS: Fifty hotel attendants were chosen as control group, 50 workers with more than one year of silica dust exposure as dust exposure group, 32 silica dust-exposed workers as observation subject group, and 52 silicosis patients as silicosis group. The activities of SOD and GSH-Px in their induced sputum were measured by enzyme-linked immunosorbent assay. RESULTS: Compared with the control group, the observation subject group and silicosis group had significantly decreased SOD activity (68.16 ± 30.17 and 66.38 ± 47.32 U/ml vs 75.81 ± 11.92 U/ml, P < 0.05); compared with the dust exposure group, the silicosis group had significantly decreased SOD activity (66.38 ± 47.32 U/ml vs 70.12 ± 14.31 U/ml, P < 0.05). Compared with the control group and dust exposure group, the observation subject group and silicosis group had significantly increased GSH-Px activity (268.21 ± 15.45 and 279.34 ± 29.26 U/ml vs 224.22 ± 12.64 and 236.41 ± 14.54 U/ml, P < 0.05 or P < 0.01). CONCLUSION: The SOD activity in dust exposure group and silicosis group decreased, but there were no significant differences between patients with different stages of silicosis. The GSH-Px activity in dust exposure group and silicosis group was significantly higher than that in control group, and there were significant differences between patients with different stages of silicosis. These suggest that the imbalance of oxidative/antioxidant systems is associated with the development and progression of silicosis.

[Expression of peroxiredoxin I in the rats exposed to silica].

OBJECTIVE: To evaluate the change in protein expression of peroxiredoxin I (Prx I) during pulmonary fibrosis among rats exposed to silica dust and to investigate the role of Prx I in pulmonary fibrosis. METHODS: Ninety male Wistar rats were randomly divided into control group (n = 60) and experimental group (n = 30). The control group received intratracheal perfusion of saline (1 ml), while the experimental group received intratracheal perfusion of suspension of silica dust (50 mg/ml) to establish a rat model of silicosis. At 1, 2, 3, 4, 6, or 8 weeks after treatment, 10 rats in control group and 5 rats in experimental group were sacrificed. The lung tissues were collected for conventional pathological observation. The protein expression of Prx I at each time point was measured by immunohistochemistry and Western blot. RESULTS: Among the rats exposed to silica dust, Prx I was seen in the form of brown particles that were mainly distributed in the alveolar septa and the cytoplasm of alveolar epithelial cells, macrophages, vascular endothelial cells, and smooth muscle cells around the blood vessels and tracheae. The control group showed weak protein expression of Prx I, and the experimental group had significantly higher protein expression of Prx I than the control group at all time points (P < 0.05). In the experimental group, the protein expression of Prx I was upregulated significantly at 1 and 2 weeks and decreased at 3∼8 weeks. CONCLUSION: The change in protein expression of Prx I may be one of the important causes of the onset and development of pulmonary fibrosis in rats exposed to free silica.

Serum heme oxygenase-1 as a marker of lung function decline in patients with chronic silicosis.

OBJECTIVE: To identify predictive factors of excess decline in forced expiratory volume in one second (FEV1) in patients with chronic silicosis. METHODS: Forty-six male patients enrolled in 2004 were screened and received pulmonary function tests. RESULTS: Among the 33 included patients, 12 were categorized as rapid decliners (reduction in FEV1 > 60 mL/yr). The mean level of serum heme oxygenase-1 (HO-1), a marker of oxidative stress, was significantly lower in rapid decliners than in normal decliners (P = 0.002). Logistic regression analysis revealed that serum HO-1 was a factor affecting clinically important decline in FEV1 (odds ratio = 0.52; 95% confidence interval, 0.31 to 0.88) independent of the effects of age, height, weight, smoking, exposure status, and C-reactive protein. CONCLUSIONS: Serum HO-1 may be a predictor of lung function decline in silicosis patients.

Fas/FasL pathway-mediated alveolar macrophage apoptosis involved in human silicosis.

In vitro and in vivo studies have demonstrated that lung cell apoptosis is associated with lung fibrosis; however the relationship between apoptosis of alveolar macrophages (AMs) and human silicosis has not been addressed. In the present study, AM apoptosis was determined in whole-lung lavage fluid from 48 male silicosis patients, 13 male observers, and 13 male healthy volunteers. The relationships between apoptosis index (AI) and silica exposure history, soluble Fas (sFas)/membrane-bound Fas (mFas), and caspase-3/caspase-8 were analyzed. AI, mFas, and caspase-3 were significantly higher in lung lavage fluids from silicosis patients than those of observers or healthy volunteers, but the level of sFas demonstrated a decreasing trend. AI was related to silica exposure, upregulation of mFas, and activation of caspase-3 and -8, as well as influenced by smoking status after adjusting for confounding factors. These results indicate that AM apoptosis could be used as a potential biomarker for human silicosis, and the Fas/FasL pathway may regulate this process. The present data from human lung lavage samples may help to understand the mechanism of silicosis and in turn lead to strategies for preventing or treating this disease.

Silica-induced TNF-alpha and TGF-beta1 expression in RAW264.7 cells are dependent on Src-ERK/AP-1 pathways.

The cytokines secreted by lung macrophages have been shown to play a critical role in the pathogenesis of silicosis, tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta1 (TGF-beta1) are prominent cytokines in silicosis, but the underlying mechanism remains to be determined. The aim of the present study was to investigate the roles of Src-mitogen-activated protein kinase (MAPKs)/activator protein-1 (AP-1) signaling pathways in silica-induced TNF-alpha and TGF-beta1 expression in macrophage cells (RAW264.7). It was found that silica activated Src, p38 kinase, and extracellular signal-regulated kinase (ERK) in RAW264.7 cells. The induction of TNF-alpha and TGF-beta1 by silica was suppressed by Src inhibitor (PP1), ERK inhibitor (PD98059), but not by p38 kinase inhibitor (SB203580). Dominant negative mutant c-Jun (TAM67) inhibited silica-induced AP-1 DNA binding activity and downregulated the TNF-alpha and TGF-beta1 expression. In addition, PD98059 but not SB203580 inhibited the AP-1 DNA binding activity induced by silica. Based on these findings, it was conclude that Src-ERK/AP-1 signaling pathways are involved in the TNF-alpha and TGF-beta1 expression induced by silica in macrophages.

Cloning of a rat lung fibrogenic factor.

In a previous study a specific single polypeptide has been purified and characterized that it was capable of promoting human embryonic lung 2BS fibroblasts proliferation in vitro, whose N-terminal 15 amino acid have high sequence homology with members of the mammalian chitinase-like protein family. Here the cloning of the gene is reported. Its cDNA contains an open reading frame 1421 bp long and encodes a protein with a characteristic N-terminal 21 amino acid endoplasmic reticulum signal peptide and the putative protein is highly homologous to acidic mammalian chitinase (AMCase) precursor of mouse and human. Recombinant proteins demonstrate chitinolytic activity, therefore the gene is termed as rat AMCase. Sequence analysis indicates that the gene spanned a 46.2 kb region in rat chromosome 2. Its expression in several tissues other than alveolar macrophages suggests that it might play multiple biological roles in vivo. Our findings will facilitate studies on its roles in physiological and pathological processes.

Cysteine cathepsins and caspases in silicosis.

Silicosis is an occupational pneumoconiosis caused by inhalation of crystalline silica. It leads to the formation of fibrohyalin nodes that result in progressive fibrosis. Alternatively, emphysema may occur, with abnormal destruction of collagen fibres in the advanced stages. Although the pathophysiological mechanisms remain unclear, it has been established that the lung responds to silica by massive enrollment of alveolar macrophages, triggering an inflammatory cascade of reactions. An imbalance in the expression of lung proteases and their inhibitors is implicated in extracellular matrix remodelling and basement membrane disruption. Moreover, exposure to silica can initiate apoptotic cell death of macrophages. This review summarises the current knowledge on cysteine cathepsins that have been ignored so far during silicosis and outlines the recent progress on cellular pathways leading to silica-induced caspase activation, which have been partly delineated.

Heme oxygenase-1, a potential biomarker of chronic silicosis, attenuates silica-induced lung injury.

RATIONALE: Heme oxygenase-1 (HO-1), a rate-limiting enzyme in heme catabolism, has antioxidative, antiapoptotic, and antiinflammatory activities. We examined whether HO-1 might be involved in silicosis. OBJECTIVES: To investigate whether HO-1 can reduce silicosis in mice and humans. METHODS AND MEASUREMENTS: Silicosis was studied using a murine model, and in 46 male patients. Serum HO-1 and 8-hydroxydeoxyguanosine (a marker of oxidative stress) were measured by enzyme-linked immunosorbent assay. Levels of HO-1 were measured by immunohistochemistry and immunoblotting. MAIN RESULTS: Serum HO-1 levels were significantly elevated in patients with silicosis compared with age-matched control subjects or patients with chronic obstructive pulmonary disease. Serum HO-1 levels also correlated inversely with serum 8-hydroxydeoxyguanosine levels and positively with vital capacity and forced expiratory volume in one second in patients with silicosis. HO-1 was present in the lungs of humans and mice with silicosis, especially at sites of silica particle deposition. In mice, silica exposure was associated with acute leukocyte infiltration, leading to development of silicotic lung lesions. The inflammation was suppressed by treatment with hemin, an inducer of HO-1, and enhanced by zinc protoporphyrin, an inhibitor of HO-1. CONCLUSIONS: Pulmonary HO-1 expression is increased in silicosis. HO-1 suppresses reactive oxygen species activity, and subsequent pathologic changes, thereby attenuating disease progression.

[Apoptosis and caspase-3 in the model of rat silicosis].

OBJECTIVE: To explore the changes of apoptosis of cells in the lung tissue of rats with silica instillation and to its significance in silicosis, and to clarify the role of caspase-3 in the apoptosis progress. METHODS: Forty-eight rats were randomly divided into saline control groups and silica instillation groups, and the silicosis model was established in rats. Flow cytometry was used for detecting the rate of apoptosis at various stages. Immunohistochemistry for the expression of cleaved caspase-3. RESULTS: The model of rat silicosis was established successfully. The apoptosis rate in the experimental group was significantly higher than that in the control group, and was increased with time. Caspase-3 was mainly expressed in alveolar epithelium cells, pulmonary macrophages and infiltrated inflammation cells. The expression of caspase-3 in the experimental group was stronger than that in the control group, but its expression intensity was not related to the cell apoptosis (r = 0.215, P > 0.05). CONCLUSION: The apoptosis of the lung cells plays an important role during rat silicosis genesis. Caspase-3 plays an important role in regulating cell apoptosis during rat silicosis genesis.

Matrix metalloproteinase induction in fibrosis and fibrotic nodule formation due to silica inhalation.

Matrix metalloproteinases (MMPs) are the principle enzymes that initiate degradation of collagen. We examined the role of MMPs during alveolar wall fibrosis and fibrotic nodule formation from silica exposure. Rats were exposed to filtered air or 15 mg/m(3) silica by inhalation for 5 days/wk, 6 h/day. Lungs were preserved by intratracheal instillation of fixative at 20, 40, 60, 79, and 116 days of exposure. Additional groups were fixed after 20, 40, and 60 days of exposure followed by 36 days of recovery. The number of nodules, defined by a collagenous core and a bounding cell layer detached from the alveolar wall, was determined by morphometry. Lungs showed increased alveolar wall collagen and fibrotic nodules at 79 and 116 days of exposure with increased collagenase and gelatinase activity. The number of nodules per lung in exposed groups increased from 619 +/- 447 at 40 days to 13,221 +/- 1,096 at 116 days (means +/- SE, n = 5). No nodules were seen in control lungs. Silica-exposed rats with a 36-day recovery in filtered air showed enhanced MMP activity over exposure to silica for the same duration with no recovery. MMP-2 and MMP-9 were significantly elevated in alveolar macrophages after 40-day exposure. Stromelysin expression was demonstrated in alveolar macrophages and cells within fibrotic nodules. TIMP-1 expression was not significantly altered. In summary, MMP activity was upregulated at 40 days of silica exposure and progressively increased during ensuing fibrotic responses. Early expression of stromelysin was found in fibrosing alveolar walls and fibrotic nodules.

Resistance to acute silicosis in senescent rats: role of alveolar macrophages.

We have previously demonstrated in alveolar macrophages that aging is associated with a decline in lipopolysaccharide-induced tumor necrosis factor-alpha production. The purpose of the present study was to investigate the immunotoxicological consequences of this defective activation in an experimental model of acute silicosis. Young (3 months old) and old (>18 months old) rats were intratracheally instilled with silica or saline as control. In young animals, as expected, silica induced a significant increase in bronchoalveolar lavage fluid of tumor necrosis factor-alpha, lactate dehydrogenase, and cell numbers, which correlated with increased collagen deposition and silicotic nodule formations. On the contrary, in old rats, no changes in bronchoalveolar lavage fluid or lung parameters were observed, indicating that senescent rats are resistant to the acute effects of silica. These in vivo results were confirmed in vitro, where silica-induced tumor necrosis factor-alpha release was drastically reduced in alveolar macrophages obtained from old animals. This could be explained with a defective protein kinase C betaII translocation in aged macrophages, due to decreased expression of its anchoring protein RACK-1. Furthermore, a decrease in FAS-L expression and silica-induced apoptosis in old macrophages was observed, supporting the idea that age-associated alterations in signal transduction pathways contribute to decreased sensitivity to silica-induced acute lung fibrosis in old animals.

Expression of matrix metalloproteinase, tissue inhibitors of metalloproteinase and adhesion molecules in silicotic mice with lung tumor metastasis.

The number of metastatic foci in silicotic mice is approximately 1.5-fold that in normal mice and in mice treated with TiO2 as inert particles. Expression of matrix metalloproteinases (MMPs) tissue inhibitors of metalloproteinases (TIMPs) and selectins was investigated in silicotic mice with lung tumor metastasis. Expression of MMP-9 and P-selectin mRNA, but not MMP-2 and E-selectin, increased significantly, showing decreases of the ratio of expression in TIMPs/MMP-9 in tumor-bearing silicotic mice compared with the tumor-bearing normal mice and mice treated with TiO2. Pretreatment with anti-P-selectin antibody inhibited number of metastatic foci significantly in silicotic mice, while pretreatment of animals with anti MMP-9 antibody showed slight decrease of metastatic foci. This evidence indicated that up-regulation of P-selectin expression contributed to enhanced rate of tumor metastasis in lung with silicosis.