C5a, an 11,000 dalton glycopeptide produced by specific enzymatic cleavage of native C5, is the most potent complement-derived soluble mediator of inflammation. C5a plays a key role in the pathogenesis of a number of diseases in man and also makes important direct contributions to all inflammatory reactions associated with the activation of complement. Recent in vivo studies have demonstrated that peripheral blood leukocytes (especially neutrophils) make a significant and an immediate contribution to C5a-mediated inflammation in human skin. Because these in vivo studies in man have demonstrated that C5a-induced inflammation is in large part mediated by anaphylatoxin-stimulated leukocytes, studies in this proposal will directly characterize the interactions of purified human C5a and its less potent physiologic derivative, C5a des Arg, with different types and subpopulations of human leukocytes. Specific experiments with fluoresceinated and radiolabeled C5a and C5a des Arg will determine the expression, number, and affinity of neutrophil, monocyte, and lymphocyte plasma membrane binding sites for these complement cleavage fragments. Cytometric studies with double-stained leukocytes will precisely identify the phenotype of cells which bind these fluoresceinated ligands. Additional cytometric studies will be conducted with processed and unprocessed peripheral blood leukocytes to directly assess alterations in C5a receptor expression during purification procedures. Other studies will characterize the specific C5a receptor site on human neutrophils and monocytes using both ultrastructural and biochemical approaches. These studies, which will assess C5a receptor sites on both leukocyte intracellular organelles and plasma membranes, will provide novel information regarding the homology of human neutrophil and monocyte C5a receptors and also directly address important considerations regarding mechanisms of receptor internalization, processing, renewal, and signal transduction. Additional studies will employ an in vitro chemotaxis bioassay to define and isolate subpopulations of human neutrophils and monocytes which are responsive or nonresponsive to C5a. These subpopulations of leukocytes will be further characterized phenotypically and functionally in order to define the specific population of neutrophils and monocytes responsible for inflammation and tissue damage at sites of complement activation.
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