Neutrophils (PMN) are primary phagocytic cells in the blood and tissue spaces. These terminally differentiated cells have a 1-2 day half-life and are considered capable of little if any protein synthesis and their functional ability has been presumed to last only as long as pools of preformed molecules lasted. However, it has been shown by the applicant that circulating PMN are capable of synthesizing mRNA transcripts and protein and that the levels of protein synthesis are increased when cells are treated with the cytokine GM-CSF. Since PMN are important phagocytic cells, the contribution of de novo protein synthesis to function and lifespan of these cells is of great interest. In this proposal, the complement receptor type 1 (CR1), which is important in the phagocytic function of PMN will be used as a model protein. The objectives of this proposal will study: I. constitutive levels and modulation of mRNA and protein synthesis in peripheral blood PMN using Northern blot and mRNA synthesis assays and pulse-chase experients analyzed by immunoprecipitation, electrophoresis and autoradiography, II. the site of the rapidly upregulatable intracellular pool of CR1 by imunocytochemical, biochemical and immunological techniques and, III. phagocytic pathways of CR1 induced by ligand-dependent or independent stimuli from the time of CR1 plasma membrane expression to intern- alization and subsequent fate. These studies are technically feasible because of the development by the applicant of systems to analyze synthesis of MRNA transcripts and proteins by peripheral blood PMN, development of antibodies to CR1 which are necessary for measurement of cell surface expression of these proteins by flow cytometry, and for quantitation of these molecules by radioimmunoassays capable of detecting 5 x 10-14 moles of CR1 and the development of immunocytochemical electron micrographic methodology for localizing CR1.
