Mechanisms of a Novel Chemotactic Cofactor for C5a

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DESCRIPTION (provided by applicant): Chemotaxis of leukocytes into various tissues is known to be a critical step in the pathogenesis of several inflammatory disorders. Complement pro-inflammatory peptides C5a and C5a des Arg also have been implicated in disease pathogenesis. C5-derived peptides are very potent chemoattractants for a wide variety of cell types. Much is known about the bioactivities of C5-derived peptides but the regulation of these functions is poorly understood. Previously, we were the first of several groups to demonstrate that the vitamin D binding protein (DBP), also known as Gc-globulin, can enhance the chemotactic activity of C5a and C5a des Arg, i.e., function as a co-chemotaxin. Moreover, the co-chemotactic activity of DBP is specific for the C5-derived peptides. Although DBP appears to be a physiologically important regulator of the chemotactic activity for activated complement, the mechanism of chemotaxis enhancement by DBP is not known. Recently, we have reported several important observations that should help define the mechanism by which DBP acts as a co-chemotactic factor for C5a. (1) DBP needs to be bound to the cell surface in order to function as a co-chemotaxin for C5a. (2) The neutrophil DBP binding site is a chondroitin sulfate proteoglycan. (3) Expression of the DBP binding site is regulated by cell surface-bound neutrophil elastase, which cleaves and sheds the proteoglycan. (4) Preliminary studies have shown that activated platelets modify DBP to an active co-chemotactic form. (5) Clinical samples from patients with inflammatory disorder (ARDS) contain the modified co-chemotactic form of DBP. It is our hypothesis that a modified form of DBP binds to the cell surface and initiates an enhanced chemotactic response to C5a. In this proposal, we endeavor to investigate the mechanism by which DBP augments the leukocyte chemotactic activity of C5a by utilizing human neutrophils and the U937 cell line transfected with the C5a receptor (U937-C5aR). The process of co-chemotaxis will be divided into component parts and examined individually. First, determine how activated platelets modify DBP by focusing on the most likely alteration: extracellular phosphorylation by ubiquitous protein kinases. Second, investigate how modified DBP interacts with cells by examining binding and shedding on the cell surface. Third, determine how cells terminate the co-chemotactic signal by focusing on dephosphorylation by phosphatases. Finally, clinical samples from patients with inflammatory disorders will be analyzed using a proteomic approach to determine if modified DBP is correlated with disease outcome. This study will bridge basic knowledge derived from in vitro biochemical approaches and apply it to examine samples obtained from patients with inflammatory disorders. Results of this study will demonstrate a novel mechanism for a chemotactic cofactor and could serve as a prototype for other cofactors yet to be discovered.