The anaphylatoxins mediate spasmogenic, chemotactic and immunoregulatory responses primarily through specific receptor interactions with granulocytes and monocytes. The primary targets of the ligand C5a in man are believed to be the neutrophil and the monocyte (macrophage). Involvement of other cell types such as platelets, lymphocytes, endothelial cells and mast cells may occur as an indirect consequence of granulocyte or monocyte activation and secondary mediator release. Evidence exists that a C5a receptor of approximately 40,000 kDa occurs on the human neutrophil surface and that this membrane component has an affinity for the ligand of 1-3 nM Kd. The human neutrophil has no receptor for the C3a anaphylatoxin. This proposal concerns both C5a and C3a receptors on monocytes (macrophages). Preliminary evidence indicates that monocytes and tissue macrophages from human and animal sources have C3a and C5a receptors. Using chemical cross-linking techniques to visualize these membrane components, a C3a binding protein of approximately 1000,000 kDa and a C5a binding protein of approximately 40,000 kDa were observed. We plan to isolate these receptors, characterize them in terms of carbohydrate content and solubility. Monospecific immune reagents will be prepared to study functional attributes of the C5a receptor on macrophages and for use in purification of the receptor in quantities adequate for partial sequence analysis. Probes designed from protein sequence analysis will be used for complete molecular analysis of the membrane component. We have observed that transformed murine macrophage cell lines express C5a receptors. When these cell lines are injected intraperitoneally into mice, tumors are grown that contain large quantities of the macrophages having markedly enhanced number of C5a receptors (i.e. upregulated from membrane components desired). We plan to use this discovery to advantage in isolation and characterization of the macrophage chemotactic (C5a) receptor. A biologic, biochemical and molecular analysis of the anaphylatoxic receptors on monocytes and macrophages should lead to a better understanding of the role that humoral factors play in modulating immune and inflammatory responses. Circulating monocytes and tissue macrophages appear to play a pivotal role in host defense mechanisms. Early events in target cell activation are elicited by factors such as anaphylatoxins and bacterial products indicating that they represent activators of major biologic consequence.
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