HUMAN ALVEOLAR MACROPHAGE ELASTASES IN EMPHYSEMA

巨噬细胞在肺气肿和其他疾病的组织病理学中占有重要地位。 以过度结缔组织为特征的慢性炎症过程 组织周转。这项提案的总体目标是定义 肺巨噬细胞在吸烟相关性慢性肺损伤病理生物学中的作用 肺部疾病。已知的四种真核生物的最新克隆 半胱氨酸蛋白酶已经鉴定出其中之一，人组织蛋白酶S，是一种 弹性酶。组织蛋白酶S被发现具有明显的弹性蛋白酶 在酸性和中性pH下都有活性。有证据表明，肺泡 吸烟者的巨噬细胞比不吸烟者的巨噬细胞活性更高 这种巨噬细胞弹性蛋白酶活性可在 活细胞与弹性蛋白共培养及潜在调节的体外实验 由这些细胞分泌的两种内源性抑制物TIMP和TIMP 胱抑素C的实验针对的是一个中心假设，即 香烟诱导的巨噬细胞弹性蛋白酶活性失调 吸烟--是肺损伤的一个重要决定因素。为了测试 这一假说是针对重组人组织蛋白酶S的抗体 引发以协助进一步提纯和表征 酵素。吸烟者组织蛋白酶S活性升高的机制 将检查巨噬细胞以及细胞上是否存在半胱氨酸蛋白酶 用免疫学和电子显微镜确定巨噬细胞表面 技巧。细胞外基质蛋白、弹性蛋白的影响 受体、细胞因子对巨噬细胞组织蛋白S和细胞因子表达的影响 L，也是一个92 kDa的明胶酶，将被用mrna和 体外功能测定。半胱氨酸氨基转移酶C的功能重要性 将评估TIMP在调节巨噬细胞基质代谢中的作用 部分，通过部分阻断胱抑素C和TIMP的生物合成 反义寡核苷酸及其对细胞外功能的影响 巨噬细胞对基质的降解。最后，巨噬细胞表达 组织蛋白酶S和L、92 kDa明胶酶及其抑制剂TIMP和TIMP 将在从以下来源获得的肺泡巨噬细胞中检测胱抑素C 肺功能正常或降低的吸烟者(FEV1/FVC)。这些 实验应该阐明人类通过什么分子过程 巨噬细胞在体外降解弹性蛋白，确定其调节，并直接 检测巨噬细胞是否改变了酶和/或抑制物的表达 与吸烟相关肺部疾病的病理生物学有关。这个 结果应该会提供一个更清楚的观点，巨噬细胞在 肺气肿和为今后的监测和监测工作奠定了良好的基础 调节体内巨噬细胞的蛋白分解。

Macrophages are prominent in the histopathology of emphysema and other chronic inflammatory processes characterized by excessive connective tissue turnover. The overall objective of this proposal is to define the role of lung macrophages in the pathobiology of smoking-related chronic lung disease. Recent cloning of all four of the known eukaryotic cysteine proteases has identified one of these, human cathepsin S, as an elastase. Cathepsin S has been found to have appreciable elastase activity at both acidic and neutral pH. Evidence indicates that alveolar macrophages from smokers have increased activity over those of nonsmoker cells of cathepsin S. This macrophage elastase activity is inducible in vitro by co-culture of live cells with elastin and potentially regulated by two endogenous inhibitors secreted by these same cells, TIMP and cystatin C. Experiments are directed toward the central hypothesis that dysregulation of macrophage elastase activity -- inducible by cigarette smoking -- is an important determinant of lung injury. In order to test this hypothesis antibodies to recombinant human cathepsin S will be raised to assist in further purification and characterization of the enzyme. The mechanism of increased cathepsin S activity in smoker macrophages will be examined and whether cysteine proteases exist on the macrophage cell surface defined by immunologic and electron microscopic techniques. the influence of extracellular matrix proteins, elastin receptors, and cytokines on the expression of macrophage cathepsins S and L, as well a the 92-kDa gelatinase, will be studied by mRNA and functional assays in vitro. The functional importance of cystatin C and TIMP in regulating matrix metabolism by macrophages will be assessed, in part, by partially blocking cystatin C and TIMP biosynthesis with antisense oligonucleotides and measuring the effects on extracellular matrix degradation by macrophages. Finally, macrophage expression of cathepsins S and L, the 92-kDa gelatinase, and the inhibitors TIMP and cystatin C will be examined in alveolar macrophages obtained from cigarette smokers with normal or reduced lung function (FEV1/FVC). These experiments should elucidate the molecular process by which human macrophages degrade elastin, define its regulation in vitro, and directly test whether altered enzyme and/or inhibitor expression by macrophages is implicated in the pathobiology of smoking-related lung disease. The results should provide a clearer view of the role of macrophages in emphysema and a sound foundation for future efforts both to monitor and to modulate macrophage proteolysis in vivo.