MECHANISMS OF LUNG FIBROSIS

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Recruitment of monocytes into the lung is a common response to lung injury, and may be important in the pathogenesis of pulmonary fibrosis due to their ability to elaborate mediators and cytokines important in the upregulation of fibroblast proliferation, chemotactic recruitment and extracellular matrix gene expression. During the current granting period fibronectin fragments have been shown to be the predominant monocyte chemotactic produced by alveolar macrophage after the first week following lung injury induced by bleomycin. However recent preliminary data also suggest an upregulation in monocyte chemoattractant protein-1 (MCP-1) gene expression in lung tissue, consistent with in vitro data indicating that other lung cells are also capable of producing this monocyte chemotactic of potentially equal or great import to the recruitment of monocytes into the lung. In situ hybridization analysis suggest that the eosinophil may be a key source of this chemotactic in bleomycin-induced pulmonary fibrosis. Preliminary studies also indicate the MCP-1 can stimulate fibroblast collagen synthesis. Hence the importance of this cytokine in the pathogenesis of pulmonary fibrosis potentially extends beyond the mere recruitment of monocytes into the lung. In view of these findings, the following hypothesis is proposed to further evaluate the role of MCP-1 in lung fibrosis. Following bleomycin lung injury, the combination of direct bleomycin effects and those due to endogenous regulatory cytokines, causes the upregulation of MCP-1 gene expression in diverse lung cells; and, that this increased MCP-1 gene expression is important in the development of subsequent fibrosis, at least partly due to its ability to indirectly stimulate collagen synthesis. The stimulation of fibroblast collagen synthesis appears to be indirect and may be mediated by concomitant stimulation of transforming growth factor (TGFB) production with consequent autocrine effects on collagen synthesis. To test this hypothesis, this project has four primary objectives: 1) to evaluate lung MCP-1 gene expression by in situ hybridization and immunohistochemical analyses to relate this to upregulation in procollagen alpha1(I) and TGFs Beta1, Beta2 and Beta3 gene expression, and to identify the cell type responsible for the increased MCP-1 gene expression, 2) to analyze cytokine and bleomycin modulation of MCP-1 gene expression in isolated lung fibroblasts (from normal and bleomycin-induced fibrotic lungs), endothelial cells and eosinophils, 3) to examine the mechanism of MCP-1 stimulation of lung fibroblast collagen synthesis. These studies should provide further insight as to the potential role(s) of MCP-1 in lung fibrosis, with specific insights into the role of the eosinophil as an important source of this cytokine in fibrotic lesions. Finally, a fourth aim is to confirm these studies by including observation on the effects of specific MCP-1 neutralization on the upregulation in lung TGFBeta and collagen gene expression during bleomycin-induced pulmonary fibrosis. The combination of in vivo and in vitro approaches, plus the application of molecular biological and immunological tools should provide complementary data to allow more definitive conclusion to be made, which would be impossible with any one approach used in isolation.