HUMAN ALVEOLAR MACROPHAGE ELASTASES IN EMPHYSEMA

肺气肿中的人肺泡巨噬细胞弹性蛋白酶

• 批准号: 2224331
• 负责人: Harold A Chapman
• 金额: $ 25.75万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2224331
• 关键词: alveolar macrophages; antisense nucleic acid; collagenase; elastases; elastin; electron microscopy; emphysema; enzyme activity; enzyme induction /repression; enzyme mechanism; extracellular matrix proteins; fusion gene; human subject; immunofluorescence technique; lung injury; pathologic process; protease inhibitor; protein degradation; protein purification; receptor; tissue /cell culture; tissue inhibitor of metalloproteinases; tobacco abuse

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.

在肺气肿和其他疾病的组织病理学中， 以过度结缔组织为特征的慢性炎症过程 组织周转 本提案的总体目标是界定 肺巨噬细胞在吸烟相关慢性肺损伤病理生物学中的作用 肺病。 最近克隆了所有四种已知的真核生物 半胱氨酸蛋白酶已经鉴定了其中之一，人组织蛋白酶S，作为一种蛋白酶。 弹性蛋白酶 已发现组织蛋白酶S具有可观的弹性蛋白酶 活性在酸性和中性pH值。证据表明，肺泡 吸烟者的巨噬细胞比非吸烟者的活性高 组织蛋白酶S细胞。 这种巨噬细胞弹性蛋白酶活性是可诱导的， 通过活细胞与弹性蛋白的共培养进行体外研究， 由这些相同的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。 实验是针对中心假设， 香烟诱导的巨噬细胞弹性蛋白酶活性失调 吸烟是肺损伤的重要决定因素。 为了测试 这一假设将重组人组织蛋白酶S的抗体 以帮助进一步纯化和表征 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，以及半胱氨酸蛋白酶是否存在于巨噬细胞上。 免疫学和电子显微镜确定的巨噬细胞表面 技术. 细胞外基质蛋白、弹性蛋白 受体和细胞因子对巨噬细胞组织蛋白酶S和 L，以及92-kDa明胶酶，将通过mRNA和 体外功能测定。 半胱氨酸蛋白酶抑制剂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，通过部分阻断半胱氨酸蛋白酶抑制剂C和TIMP的生物合成， 反义寡核苷酸和测量对细胞外 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 半胱氨酸蛋白酶抑制剂C将在肺泡巨噬细胞中进行检查， 吸烟者肺功能正常或降低（FEV 1/FVC）。 这些 这些实验应该阐明人类 巨噬细胞降解弹性蛋白，在体外定义其调节，并直接 测试巨噬细胞是否改变酶和/或抑制剂表达 与吸烟相关的肺部疾病的病理学有关。 的 结果应该提供一个更清楚的观点，巨噬细胞的作用， 肺气肿和良好的基础，为今后的努力， 以调节体内巨噬细胞蛋白水解。