The overall goal of this project are to document the physiologic importance of the increased surface membrane expression of the types 1 and 3 complement receptors (CP1 and CR3) during activation of human neutrophils (PMN), and to determine the mechanisms of this process, specifically focusing on the hypothesis that receptor upregulation may occur without degranulation. Using immunoelectron microscopy, we have shown that CR1 in resting PMN is stored in unique vesicular structures that are clearly distinct from primary and secondary granules. In addition, quantitative immunofluorescence studies of fixed, permeabilized PMN suggest that upon activation by chemoattractants there is ligand- dependent internalization and degradation of CR1. Results of immunoassays of total CR1 in cell extracts are consistent with this proposal, and EM studies show that in activated cells the internal pool is in large multivesicular bodies. These findings can be integrated into an overall model for trafficking of CR1 and other proteins of interest during neutrophil activation. We will now test several hypotheses predicted by this model: 1> We will identify characterize a common storage pool for CR1, decay accelerating factor (DAF) and alkaline phosphatase in resting PMN. 2. We will determine if CR1 remains clustered and in association with the membrane segments with which it is brought to the cell surface while DAF and alkaline phosphatase, which are anchored by glycolipids, diffuse laterally. 3. We will study how CR1 and DAF are selectively packaged into their storage vesicles during in vitro differentiation of myeloid precursors. 4. We will document that CR1 that was expressed on the surface actually becomes internalized and characterize its degradation. 5. We will map the pathway of internalization of CR1 during ligand-independent recycling and compare this to endocytosis after crosslinking and ligand-dependent phagocytosis. The roles of clathrin and Ca++ in each of these processes will be defined. To accomplish these goals we will use the immunoelectron microscopic approach and other immunologic assays we have developed during the initial period of this grant. We will also study directly isolate the vesicles in which CR1 is stored by the use of antibodies against its cytoplasmic tail. These studies should yield much valuable information about the dynamics of receptor trafficking that allow phagocytes such as neutrophils to perform their vital roles in host defense.
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