In chronic inflammatory disease states ranging from rheumatoid arthritis to ulcerative colitis, blood monocytes infiltrate affected tissues and differentiate into tissue-destructive populations of macrophages. Unlike neutrophils or eosinophils which predominantly rely on their ability to generate halogenated oxidants and release serine proteinases to mediate tissue damage, the destructive systems mobilized by human macrophages remain undefined. In order to identify and characterize the mechanisms by which macrophages mediate tissue-destructive effects in chronic inflammatory disease states, human monocytes will be cultured in vitro and induced to mature into a macrophage population capable of expressing an extracellular matrix-degrading phenotype hundreds of times greater than that of any other leukocyte population described previously. Based on preliminary data, the macrophage's unique destructive activity is mediated by the exocytosis/secretion of active cysteine proteinases, a class of acid proteases normally categorized as intracellular, lysosomal catabolic enzymes. To date, little is known with regard to the function of these enzymes in human macrophages since many members of this proteinase family have only recently been identified and few molecular or biochemical tools have been developed for their analysis in intact cell systems. Furthermore, the mechanisms by which lysosomal enzymes could be routed to an extracellular compartment in which conditions permissive for cysteine proteinase activity could be generated and maintained remain unknown. Thus, in an attempt to identify a novel role for cysteine proteinases in macrophage effector functions, the following five aims will be addressed. First, to characterize the intracellular and extracellular expression of the cysteine proteinases, cathepsins B, L and S, as well as the cysteine proteinase inhibitor, cystatin C, in monocyte-derived macrophages. Second, to determine the role of the mannose-6-phosphate receptor recognition systems in directing cysteine proteinase traffic from the lysosomal to the extracellular compartment. Third, to characterize the role of the macrophage vacuolar-type H+-ATPase and L-cystine transport systems in generating an acidic and reducing extracellular environment permissive for cysteine proteinase activity. Fourth, to assess the role of the cysteine proteinase system in the macrophage-mediated degradation of the extracellular matrix via the selective """"""""knockout"""""""" of individual cathepsins, the vacuolar-type H+-- ATPase or the L-cystine transport system. Fifth, and finally, to determine the response and function of the macrophage cysteine proteinase system during chemotactic factor-induced tissue invasion. The characterization of the cysteine proteinase system mobilized by human macrophages at inflamed sites should not only provide new insights into the pathogenesis and treatment of chronic inflammatory disease states, but also into the regulation of the tissue-invasive phenotype in vivo.
