The role of pro- and anti-inflammatory responses in silica-induced lung fibrosis

BACKGROUND: It has been generally well accepted that chronic inflammation is a necessary component of lung fibrosis but this concept has recently been challenged. METHODS: Using biochemical, histological, immunohistochemistry, and cellular analyses, we compared the lung responses (inflammation and fibrosis) to fibrogenic silica particles (2.5 and 25 mg/g lung) in Sprague-Dawley rats and NMRI mice. RESULTS: Rats treated with silica particles developed chronic and progressive inflammation accompanied by an overproduction of TNF-α as well as an intense lung fibrosis. Dexamethasone or pioglitazone limited the amplitude of the lung fibrotic reaction to silica in rats, supporting the paradigm that inflammation drives lung fibrosis. In striking contrast, in mice, silica induced only a limited and transient inflammation without TNF-α overproduction. However, mice developed lung fibrosis of a similar intensity than rats. The fibrotic response in mice was accompanied by a high expression of the anti-inflammatory and fibrotic cytokine IL-10 by silica-activated lung macrophages. In mice, IL-10 was induced only by fibrotic particles and significantly expressed in the lung of silica-sensitive but not silica-resistant strains of mice. Anti-inflammatory treatments did not control lung fibrosis in mice. CONCLUSION: These results indicate that, beside chronic lung inflammation, a pronounced anti-inflammatory reaction may also contribute to the extension of silica-induced lung fibrosis and represents an alternative pathway leading to lung fibrosis

Upregulation of urokinase in alveolar macrophages and lung tissue in response to silica particles.

Impaired fibrinolytic activity and persistent fibrin deposits in lung tissue have been associated with lung fibrotic disorders. The present study examined the sources of plaminogen activator (PA) changes induced by a single intratracheal administration of silica particles (5 mg) in the mouse lung. We found in both control and silica-treated animals that amiloride almost totally abolished PA activity in bronchoalveolar lavage (BAL) fluid (BALF), indicating that initial upregulation (from day 1) as well as sustained PA activity (up to day 30) observed in response to silica is related to changes in urokinase-type PA (uPA). The upregulation of BALF uPA activity was associated with a marked and persistent increase in uPA mRNA levels in lung tissue. Changes in uPA expression were also reflected in the BAL cell fraction. A maximal and constant increase in cell uPA activity was associated with the early response to silica, whereas significant but lower upregulation was still noted at the fibrotic stage. From days 3 to 30, a progressive increase in uPA mRNA levels was noted in BAL inflammatory cells elicited by silica. Because the number of BAL neutrophils was strongly correlated with BALF and BAL cell-associated uPA activity, their involvement in uPA upregulation was addressed by inducing neutropenia with cyclophosphamide (200 mg/kg ip) before administration of the silica. Neutrophilic depletion did not, however, reduce, and even increased, the BAL cell-associated uPA activity. At the BALF level, neutropenia did not change PA activity in silica-treated mice, pointing to alveolar macrophages as the principal source of uPA in response to silica. Immunohistochemical stainings identified alveolar macrophages and pneumocytes as uPA-expressing cells in silica-treated animals (day 30). Intense and heterogenous staining was observed in silicotic nodules. These findings indicate that urokinase produced by alveolar macrophages is operative not only at the alveolitis stage but also later in the fibrotic process, produced by silica particles, supporting the role of uPA in fibrogenesis

Depressed urokinase activity in bronchoalveolar lavage fluid from patients with sarcoidosis, silicosis or idiopathic pulmonary fibrosis: relationship to disease severity

Intraalveolar fibrinolysis, is regulated by the concerted actions of plasmin, plasminogen activators (PAs), and their specific inhibitors (PAIs). This event is considered as a critical step in the pathogenesis of pulmonary fibrosis. The aim of this study was to evaluate whether local PA activity can be held as a marker of fibrosis in chronic interstitial lung disorders (ILD). Changes in both PA activity and PA-related proteins (urokinase-type PA (uPA), tissue-type PX (tPA), PAI-1 and PAI-2) were assessed in bronchoalveolar fluid (BALF) of 60 subjects: 18 healthy controls, 18 non-fibrotic sarcoidosis patients, 16 patients with idiopathic pulmonary fibrosis (IPF) and eight silicotic patients with established fibrosis. We observed a significant decrease of BALF PA activity in the three groups of patients as compared with controls. Reduction in BALF PA activity was compatible with lower uPA protein levels associated, especially in IPF patients, with an increased occurrence of PAI-1 and PAI-2 antigens. Soluble tPA antigen was never detected either in control subjects or in patients. Most importantly, the reduction in BALF PA activity and uPA protein levels was found to be most severe in patients with advanced fibrotic disease, namely IPF, while moderate and only weak alterations were found in silicosis and non-fibrotic sarcoidosis, respectively. In addition, significant positive correlations were found between BALF PA activity and functional impairment as assessed by TLC % and DLCO%. Finally, the reduction in uPA and PA activity levels observed in BALF from sarcoidosis patients was found to be proportional to the degree of BAL lymphocytosis. These findings indicate that an intense reduction in BALF PA activity is associated with severe stages of the parenchymal disease, possibly reflecting the degree of the fibrotic process

The delayed lung responses to single and repeated intratracheal administration of pure cobalt and hard metal powder in the rat.

Epidemiological and clinical studies suggest that inhalation of cobalt metal dust (Co) mixed with tungsten carbide particles (WC), but not of cobalt dust alone, may cause interstitial pulmonary lesions (hard metal disease). In previous experimental studies in the rat, we have demonstrated the greater acute pulmonary toxicity of a WC-Co mixture compared to Co or WC alone. The present study was undertaken to compare in the same animal model the delayed lung response after intratracheal administration of Co or WC-Co particles (cobalt particle 6.3 wt%). The responses were also compared with those obtained after treatment with arsenic trioxide and crystalline silica used a reference materials producing an acute toxic insult and a progressive fibrogenic response, respectively. Cellular (total and differential counts) and biochemical parameters (LDH, N-acetyl-beta-D-glucosaminidase, total protein, albumin, fibronectin, and hyaluronic acid) were measured in bronchoalveolar lavage fluid following single and repeated intratracheal instillations. The results indicate that the delayed lung response observed after WC-Co is different from that after cobalt metal alone. A single intratracheal dose of WC-Co (1, 5, or 10 mg/100 g body wt) induced an acute alveolitis which persisted for at least 1 month. Four months after a single instillation of WC-Co, no clear histological lung fibrosis could however be evidenced, indicating a reversibility of the lesions. The effects of cobalt (0.06, 0.3, or 0.6 mg/100 g body wt) or tungsten carbide alone (1, 5, 10 mg/ 100 g body wt) were very modest, if any. Following repeated intratracheal instillations (four administrations at 1-month interval), increased lung hydroxyproline content and histopathological evidence of interstitial fibrosis were observed after WC-Co (4 x 1 mg/100 g body wt), but not after administration of each component separately, i.e., Co (4 x 0.06 mg/100 g body wt) or WC (4 x 1 mg/100 g body wt). The mechanism of the fibrotic reaction induced by WC-Co seems different from the progressive inflammatory reaction induced by crystalline silica. We suggest that it might result from a scarring reaction elicited by repeated acute insults as observed after repeated administration of arsenic trioxide

Expression of plasminogen activator inhibitors type-1 and type-2 in the mouse lung after administration of crystalline silica.

Altered expression of plasminogen activator inhibitors (PAIs) is of potential relevance to the process of lung fibrosis. To clarify the involvement of PAIs in interstitial lung diseases, we examined whether alterations in PAI-1 and PAI-2 were induced in response to a single intratracheal administration of a fibrosing dose of crystalline silica in mice (5 mg x animal(-1)). The time course of changes in PAI activity and PAI-1 protein were characterized in bronchoalveolar lavage fluid (BALF) and changes in PAI-1 and PAI-2 messenger ribonucleic acid (mRNAs) were monitored by reverse transcriptase polymerase chain reaction (RT-PCR) in BALF cells and lung tissue up to the fibrotic stage of the disease. Substantial levels of PAI activity were found in BALF of control animals, whereas no PAI-1 protein was detected. In response to silica treatment, we observed an acute increase of PAI activity and PAI-1 protein levels in BALF (day 1), associated with an induction of PAI-1 and PAI-2 mRNA levels in lung tissue. In alveolar macrophages, silica treatment induced a persistent upregulation of PAI-2 mRNA only. One month after silica treatment, PAI activity was undetectable in BALF while substantial PAI activity was still present in controls. At the same time point, sustained upregulation of PAI-1 and PAI- 2 mRNAs was, however, noted in lung tissue of animals treated with silica. These findings support the possible implication of PAIs in the remodelling process induced by silica in the lung

Role of urokinase in the activation of macrophage-associated TGF-beta in silica-induced lung fibrosis.

Since tumor growth factor beta (TGF-beta) and its receptor are ubiquitously expressed and because latent TGF-beta cannot bind to the cell surface receptor, the ability of a cell to activate latent TGF-beta upon secretion represents an important regulatory mechanism of TGF-beta action. In vivo, the protease plasmin is considered to be one of the main enzymes operative in the proteolytic cleavage of the latency-associated peptide moiety from TGF-beta, which converts it into the biologically active form. The TGF-beta response was characterized in alveolar macrophages during pulmonary inflammation (d 3) and fibrosis (d 120) induced by a single intratracheal instillation of silica particles (5 mg/mouse). To appreciate the role of urokinase-type plasminogen activator (uPA) in the activation of TGF-beta, the production of total, active and latent TGF-beta by explanted alveolar macrophages was compared in uPA-deficient (uPA-/-) mice and their normal counterparts (uPA+/+). At d 3 and 120 after silica treatment, a significant increase in cell-associated PA activity was found in uPA+/+ mice compared to that of saline controls. As expected, this response was almost totally absent in uPA-/- mice. Alveolar macrophages from uPA+/+ controls were found to release TGF-beta mainly expressed in a biologically active form. In response to silica treatment, inflammatory cells were found to upregulate, especially at the fibrotic stage, their secretion of total and bioactive TGF-beta. No significant difference was found between uPA-/- and uPA+/+ silica-treated animals for the expression of total, active, or latent TGF-beta. Although it has previously been reported that macrophage surface activation of TGF-beta is dependent on both plasmin generation and uPA cell surface receptor, no evidence was found to support this hypothesis in the present study