This project is a detailed analysis of specialized receptors for the activated third component of complement (C3b receptors) on the surface of cells of the human macrophage cell line U937. The project owes its feasibility to a recent advance in our laboratory whereby radiolabeled C3131I is activated and incorporated as C3b into stable, soluble aggregates of IgG125I (A-IgG) and IgM125I (A-IgM). More recently, we have used these probes to study Fc receptors and C3b receptors on U937 cells. We have demonstrated the presence of C3b receptors on U937 cells and have shown that C3b receptors alone, do not mediate the endocytosis of ligands containing C3b. We now propose to extend these studies with the following specific aims. (1) To determine the function of C3b receptors on U937 cells in the binding and endocytosis of soluble ligands bearing C3b molecules. (2) To elucidate in a similar fashion the function of C3bi receptors on U937 cells in the binding of soluble ligands bearing C3bi. (3) To determine whether the number of C3b and C3bi receptors can be modulated as is the case for Fc receptors, and if so, whether the modulated complement receptors gain the ability to mediate endocytosis. We will utilize fluorochrome labeled monoclonal anti C3b, C3bi, and Fc receptor antibodies to quantitate the surface expression, density, intracellular pool and mobility of the C3b, C3bi and Fc receptors on U937 cells, by fluorescence activated cell sorter (FACS) analysis. The functional characteristics of these receptors separately and in interaction with each other would be determined by reacting U937 cells with C3b and C3bi dimers, AlgM.C3b, AlgM.C3bi, and AlgG.C3bi. The mechanisms of these receptor interactions would be studied by simultaneous incubation of cells with soluble ligands and anti receptor antibodies and FACS analysis. These experiments would be repeated using cells treated with potential modulators of Fc and C3b and C3bi receptor number and function, and compared with untreated cells. These studies should reveal important new information concerning the function of C3b and C3bi receptors and how different types of receptors can interact with """"""""positive cooperativity."""""""" Ultimately this information may lead to a better understanding of how immune complexes may escape normal defense mechanisms and deposit in tissues to cause such diseases as glomerulonephritis, rheumatoid arthritis and vasculitis.

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University of Rochester
Schools of Medicine
United States
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