Proteolysis of lung extracellular matrix during inflammation is a pivotal event in pathogenesis of pulmonary emphysema and other disabling lung diseases. We have several lines of preliminary evidence which indicate that catalytic activity of human leukocyte elastase (HLE) and cathepsin G (CG) is focused at the cell surface of human polymorphonuclear neutrophils, even in an inhibitor-rich environment such as exists in the extracellular space in vivo. Our working hypothesis is that active cell- surface-bound proteinases on inflammatory cells are brought into contact with potential matrix substrates by specific adherence molecules, permitting the cells to penetrate tissue barriers and creating the potential for matrix injury. Moreover, recent evidence indicates that another serine proteinase of neutrophils (proteinase 3; PR3) is the antigen recognized by one class of """"""""anti-neutrophil cytoplasmic antibodies"""""""" (ANCA) in individuals with Wegener's granulomatosis. Our working hypothesis is that when PR3 is expressed on the cell surface of neutrophils and mononuclear phagocytes, it becomes the primary target of the autoimmune process. Ensuing cellular events then both activate and attract inflammatory cells, leading to a vicious cycle which culminates in necrotizing vasculitis. We will address the following Specific Aims. 1) Study of serine proteinases (HLE, CG, and PR3) bound to the cell surface of human neutrophils and monocytes, with regard to: a) quantification on resting and stimulated cells; b) catalytic activity and resistance to inhibition; c) mechanism(s) of binding to the cell surface; and d) inflammatory cell activation by autoantibodies. 2) Study of proteolysis in zones of close contact between cells and subjacent substrates, with regard to: a) co- distribution of cell-surface-bound serine proteinases and selected adherence molecules on polarized cells; and b) importance of adhesion patches in permitting inhibitor-resistant proteolysis. Inflammatory cells both degrade biologically important matrix proteins in vivo and also provide excellent models for study of inhibitor-resistant matrix proteolysis in vitro. We anticipate that the proposed studies of serine proteinase activity at the cell surface will provide insights into mechanisms of tightly controlled extracellular proteolytic activity occurring at the cell surface, as well as into potential basic mechanisms of tissue injury during inflammation.
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