Mononuclear phagocytes are recruited to sites of pulmonary inflammation. These sites can be associated with infectious, fibrosing and granulomatous lung diseases. Recruitment is accomplished by directed migration (chemotaxis). CD87 (the urokinase plasminogen activator (uPA) receptor) is required for mononuclear phagocyte chemotaxis. The central hypothesis of this proposal is that the essential role CD87 plays in mononuclear phagocyte chemotaxis is dependent upon its expression, its localization to specific plasma membrane micro-domains called caveolae, and its sequential interaction with signaling proteins and other cell surface receptors. The overall objective of this proposal is to delineate the mechanism by which CD87 mediates mononuclear phagocytes chemotaxis. Our specific objectives are: l) Determine a) whether aggregation of CD87 within caveolae is required, and b) determine whether caveolin, the protein lining caveolae, participates in signal transduction in during chemotaxis. 2) Determine whether CD87 associates with complement receptor 3 (CR3) via carbohydrate- lectin interactions to effect chemotaxis. 3) Determine if a glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) mediated mechanism contributes importantly to CD87 shedding. 4) Using transgenic uPA deficient mice, determine if binding of uPA participates importantly in CD87 function during chemotaxis in vitro, and cellular recruitment in response to pulmonary inflammation induced by C. neoformans infection in vivo. l) Visual localization of CD87 to caveolae and colocalization of CD87 and caveolin will be accomplished by immunolabeling and electron microscopy and immunofluorescent quantitative confocal microscopy. Physical binding of CD87 and caveolin will be assessed by immunoprecipitation and Western blotting. Signal transduction via caveolin will be assessed by immunoprecipitation and Western blotting with anti- phosphorotyrosine antibodies. 2) Carbohydrate-lectin interactions between CD87 and CR3 will be assessed by colocalization studies in the presence and absence of specific saccharides. Studies evaluating chemotaxis will be done in parallel. Direct interaction between CR3 and CD87 and the specific relevant domains of CD87 will be assessed using murine cells transfected with CR3 and soluble human recombinant CD87. 3) PMA-and cytokine-induced CD87 shedding will quantitated by ELISA and the mechanism determined by analysis of the molecular weight of shed CD87 compared with known CD87 variants. Specific inhibitors of candidate mechanisms will be used to assess the relative contribution of each to agonist-specific CD87 shedding. 4) Using uPA deficient mice, we will determine whether uPA plays a role in mononuclear phagocytes chemotaxis in vitro, or in leukocyte recruitment in response to pulmonary C. neoformans infection in vivo. These studies will elucidate the molecular mechanism of CD87 dependent mononuclear phagocyte chemotaxis. Through this knowledge, specific therapeutic interventions may be developed to control and modulate inflammatory responses.
| Knott, Andrew W; Juno, Russell J; Jarboe, Marcus D et al. (2004) Smooth muscle overexpression of IGF-I induces a novel adaptive response to small bowel resection. Am J Physiol Gastrointest Liver Physiol 287:G562-70 |
| Gyetko, M R; Sud, S; Kendall, T et al. (2000) Urokinase receptor-deficient mice have impaired neutrophil recruitment in response to pulmonary Pseudomonas aeruginosa infection. J Immunol 165:1513-9 |
| Gyetko, M R; Libre, E A; Fuller, J A et al. (1999) Urokinase is required for T lymphocyte proliferation and activation in vitro. J Lab Clin Med 133:274-88 |
| Hiraoka, N; Allen, E; Apel, I J et al. (1998) Matrix metalloproteinases regulate neovascularization by acting as pericellular fibrinolysins. Cell 95:365-77 |
| Gyetko, M R; Chen, G H; McDonald, R A et al. (1996) Urokinase is required for the pulmonary inflammatory response to Cryptococcus neoformans. A murine transgenic model. J Clin Invest 97:1818-26 |
