MECHANISMS OF LUNG FIBROSIS

肺对单核细胞的募集是一种常见的反应 损伤，并可能在肺纤维化的发病机制中起重要作用 由于它们有能力阐述重要的介体和细胞因子 成纤维细胞增殖、趋化募集和 细胞外基质基因表达。在本授权期内 纤维连接蛋白片段已被证明是主要的单核细胞 第一周后肺泡巨噬细胞产生趋化作用 博莱霉素性肺损伤。不过，最近的初步结果是 数据还表明单核细胞趋化蛋白-1表达上调。 (MCP-1)基因在肺组织中的表达，与体外数据一致 这表明其他肺细胞也能产生这种物质 单核细胞趋化作用对人类免疫功能的潜在同等或重大影响 单核细胞在肺中的募集。原位杂交分析 提示嗜酸性粒细胞可能是这种趋化作用的关键来源。 博莱霉素性肺纤维化。初步研究还表明 单核细胞趋化蛋白-1可刺激成纤维细胞胶原合成。因此， 该细胞因子在肺纤维化发病机制中的作用 潜在地超越了仅仅将单核细胞重新招募到 阿龙。鉴于这些发现，提出了以下假设 进一步探讨单核细胞趋化蛋白-1在肺纤维化中的作用。跟随 博莱霉素肺损伤，博莱霉素直接作用和 那些由内源性调节性细胞因子引起的基因上调， MCP-1基因在不同的肺细胞中的表达；并且，这增加了 单核细胞趋化蛋白-1基因的表达在肿瘤的发生发展中起重要作用 纤维化，至少部分是由于它有能力间接刺激 胶原蛋白的合成。成纤维细胞胶原合成的促进作用 似乎是间接的，并可能通过伴随的刺激 转化生长因子(TGFb)的产生及其自分泌 对胶原合成的影响。为了验证这一假设，这个项目有 四个主要目标：1)应用免疫组织化学方法检测肺组织单核细胞趋化蛋白-1基因表达 原位杂交和免疫组织化学分析与此相关 前胶原α1(I)和转化生长因子β1、β2和β3的表达上调 基因表达，并确定导致 增加MCP-1基因表达，2)分析细胞因子和博莱霉素 单核细胞趋化蛋白-1基因在肺成纤维细胞中表达的调节 正常和博莱霉素诱导的纤维化肺)、内皮细胞和 嗜酸性粒细胞，3)探讨单核细胞趋化蛋白-1刺激肺的机制 成纤维细胞胶原合成。这些研究应该提供进一步的 关于单核细胞趋化蛋白-1在肺纤维化中的潜在作用的洞察(S)， 对嗜酸性粒细胞作为重要来源的作用的具体见解 这种细胞因子在纤维化病变中的作用。最后，第四个目标是 通过观察特定药物的影响来确认这些研究 单核细胞趋化蛋白-1中和对肺组织转化生长因子β和胶原表达的影响 博莱霉素性肺纤维化过程中的基因表达。这个 体内和体外方法的结合，加上应用 分子生物学和免疫学工具应提供补充 数据，以便做出更明确的结论，这将是 单独使用任何一种方法都是不可能的。

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.