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很突出 以过度结缔组织为特征的慢性炎症过程 组织周转。 该提案的总体目标是定义 肺巨噬细胞在吸烟相关慢性病病理学中的作用 肺部疾病。 最近克隆了所有四种已知的真核生物 半胱氨酸蛋白酶已鉴定出其中一种，人组织蛋白酶 S，作为一种 弹性蛋白酶。 已发现组织蛋白酶 S 具有明显的弹性蛋白酶 在酸性和中性 pH 值下均具有活性。 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 组织蛋白酶 S 细胞。这种巨噬细胞弹性蛋白酶活性可在 通过活细胞与弹性蛋白共培养进行体外实验，并具有潜在的调节作用 由这些相同细胞分泌的两种内源性抑制剂 TIMP 和 半胱氨酸蛋白酶抑制剂 C。实验针对中心假设： 巨噬细胞弹性蛋白酶活性失调——由香烟诱导 吸烟——是肺损伤的一个重要决定因素。 为了测试 该假设重组人组织蛋白酶 S 的抗体将是 提出来协助进一步纯化和表征 酶。 吸烟者组织蛋白酶S活性增加的机制 将检查巨噬细胞以及巨噬细胞上是否存在半胱氨酸蛋白酶 通过免疫学和电子显微镜定义的巨噬细胞表面 技术。 细胞外基质蛋白、弹性蛋白的影响 受体和细胞因子对巨噬细胞组织蛋白酶S和表达的影响 L 以及 92-kDa 明胶酶将通过 mRNA 进行研究 体外功能测定。 胱抑素 C 和胱抑素 C 的功能重要性 将评估 TIMP 在巨噬细胞调节基质代谢中的作用， 部分，通过部分阻断胱抑素 C 和 TIMP 生物合成 反义寡核苷酸并测量对细胞外的影响 巨噬细胞的基质降解。 最后，巨噬细胞表达 组织蛋白酶 S 和 L、92 kDa 明胶酶以及抑制剂 TIMP 和 胱抑素 C 将在肺泡巨噬细胞中进行检查 肺功能正常或降低的吸烟者 (FEV1/FVC)。 这些 实验应该阐明人类的分子过程 巨噬细胞降解弹性蛋白，在体外确定其调节，并直接 测试巨噬细胞是否改变了酶和/或抑制剂的表达 与吸烟相关肺部疾病的病理学有关。 的 结果应该可以更清楚地了解巨噬细胞在 肺气肿并为未来监测和预防肺气肿奠定了良好的基础 调节体内巨噬细胞的蛋白水解作用。

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.