MACROPHAGE NEUTRAL PROTEINASES

巨噬细胞常见于慢性炎症部位。 与组织破坏和纤维化有关的情况。 尽管有这种联系，但人们对酶的成分知之甚少。 可能介导细胞外降解的巨噬细胞 矩阵组件。此前的研究表明，这些细胞 分泌几种不同的蛋白酶，这些酶能够降解 基质中的胶原成分以一种特定类型的方式。模型 该方案中使用的是体内激活的兔肺泡巨噬细胞。 该提案的第一个目标是提纯和表征 这些细胞分泌的胶原酶。包括：1)a) 特异性降解天然类型(细胞外)V型的胶原酶 胶原蛋白；2)间质胶原酶；以及3)一种降解酶 IV型(基底膜)胶原。这些酶将用来提纯 标准的层析方法及其分子特征 组成、底物专一性、激活机制和 由蛋白水解酶抑制剂抑制。单特异性多价抗体 会被用来对抗这些酶。 胶原蛋白的裂解产物是由 我们将对这些酶进行表征。初步研究将集中在 特异性巨噬细胞蛋白水解酶降解V型胶原的研究 中性粒细胞酶弹性蛋白酶对IV型胶原的降解。 所使用的方法将包括胶原片段的肽图 用聚丙烯酰胺凝胶电泳法和柱层析法分离 以及氨基酸分析和片段的氨基末端测序 用后一种方法分离。 这些酶的合成和释放的调节将是 调查过了。各种调节剂(皮质类固醇， 环氧合酶抑制剂、细菌产品等)将被确定为 在体外通过用不同的试剂孵化细胞，在体内通过 给动物注射毒剂。酶的分泌将会是 通过酶活性测定和免疫化学技术进行定量。 这些研究将有助于我们理解 巨噬细胞在破坏性和纤维性肺部疾病中的作用

Macrophages are frequently found at sites of chronic inflammatory conditions which are associated with tissue destruction and fibrosis. Despite this association, little is known about the enzymatic constituents of macrophages which might mediate the degradation of the extracellular matrix components. Previous studies have indicated that these cells secrete several different proteinases which are capable of degrading the collagenous components of the matrix in a type-specific manner. The model used in this proposal is the "in vivo" activated rabbit alveolar macrophage. The first objective of this proposal is to purify and characterize the collagenolytic proteinases secreted by these cells. Included are: 1) a collagenase which specifically degrades native type (pericellular) V collagen; 2) an interstitial collagenase; and 3) an enzyme which degrades type IV (basement membrane) collagen. These enzymes will be purified using standard methods of chromatography and characterized by their molecular composition, substrate specificity, mechanisms of activation, and inhibition by proteinase inhibitors. Monospecific polyvalent antibodies will be raised against these enzymes. The cleavage products of the collagens which are generated by the action of these enzymes will be characterized. Initial studies will focus on the degradation of type V collagen by the specific macrophage proteinase and the degradation of type IV collagen by the neutrophil enzyme, elastase. Methods employed will include peptide mapping of collagen fragments isolated by polyacrylamide gel electrophoresis and by column chromatography as well as amino acid analysis and amino terminal sequencing of fragments isolated by the latter method. The regulation of synthesis and release of these enzymes will be investigated. The effect of various modulating agents (corticosteroids, cyclo-oxygenase inhibitors, bacterial products, etc.) will be determined in vitro by incubating the cells with the various agents, and in vivo by administering the agents to the animals. Enzyme secretion will be quantitated by assay of enzyme activity and by immunochemical techniques. These studies will contribute to our understanding of the role of macrophages in the destructive and fibrotic pulmonary diseases.