Activation of plasminogen to plasmin, a broad spectrum protease, is believed to be integral to extracellular matrix remodeling and leukocyte migration in inflammation and tissue repair. Mononuclear phagocytes regulate plasminogen activation by the combined expression of urokinase- type plasminogen activator (uPA), plasma membrane receptors for uPA (uPAR), and a potent inhibitor of uPA (PAI-2). This is evidence that these effects are agonist-specific and influenced by the maturational state of the macrophage. Moreover, we hypothesize that cytokines and lymphocytes will induce a heterogeneous profile of parallel and divergent responses in macrophage expression of uPA, PAI-2, and uPAR. This proposal is designed to determine the mechanisms by which cytokines and stimulated lymphocytes modulate macrophage-mediated plasminogen activation. We will approach this problem by a comprehensive examination of the synthesis and functional activity of uPA, PAI-2, and uPAR, using macrophage-like U937 cells and human peripheral blood monocytes. Cells will be stimulated in vitro with a select group of cytokines, including: interferon-gamma, tumor necrosis factor-alpha, interleukin-1beta, interleukin-2, granulocyte-macrophage colony stimulating factor, and macrophage-colony stimulating factor. The effects on expression of uPA and PAI-2 will be examined by i) measuring secreted and cell-associated PA and PA inhibitor activities, using a plasminogen-dependent esterolytic assay, and ii) Northern blot analysis of uPA and PAI-2 mRNA levels. To examine effects on the uPAR, we will examine i) uPAR mRNA levels, using Northern blot analysis ii) surface expression of uPAR by binding studies, using a radiolabeled synthetic peptide ligand representing the receptor binding region of the uPA molecule, and iii) occupancy of uPAR, by measuring the PA activity dissociable from the cell surface by brief treatment with weak acid. The regulatory effects of antigen-stimulated lymphocytes will be examined, using these same techniques. These studies will be pursued further by comparing the effects of CD4+ and CD8+ cells on macrophage expression of uPA, PAI-2, and uPAR. Monocytes will be co-incubated with lymphocytes and neutralizing anti- cytokine antibodies to determine which cytokines are required for periods of culture to determine if in vitro maturational differentiation alters expression of uPA, PAI-2 and uPAR under basal or stimulated conditions. These studies should provide new insights into the mechanisms by which macrophage PA activity is modulated in the inflammatory milieu. Hopefully, this information will foster new strategies for further elucidating the role of macrophage-derived PA activity in inflammatory tissue injury and repair.
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