MACROPHAGE NEUTRAL PROTEINASES

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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.