Recent studies from our own and other laboratories demonstrate that the number of C3b and C3bi receptors on the surface of human neutrophils increases when the cells are exposed to chemoattractants or other stimuli. We propose that this increase in complement receptor expression during neutrophil activation is an important response that is necessary for proper phagocytic activity. We plan to document the physiological significance of this phenomenon and to study the mechanism by which the number of receptors expressed on the cell surface increases. First, we will study neutrophils from inflammatory sites in vivo to demonstrate that they bear increased numbers of C3b and C3bi receptors using mnonoclonal antibodies and flow cytometry. We will correlate this with the ability of the cells to phagocytose complement coated particles. Next, we will analyze cells that may be deficient in receptor content or expression in similar studies of newborns' (cord blood) neutrophils. Both of these types of cells will be compared to control peripheral blood cells of normal donors. Our studies of the mechanisms by which the receptors are translocated from their intracellular pools to the surface wil focus on the hypothesis that receptor mobilization can occur independently from granular enzyme secretion. Teleologically, this would be advantageous as cells with optimal phagocytic activity would still contain important bactericidal proteins and enzymes in the granules. This will be studied by: 1) Correlating receptor expression with granular enzyume content of cells isolated from the sources described above as well as on normal cells incubated under conditions selected to effect one process but not the other. Receptor expression will be quantitated using monoclonal antibodies and flow cytometry as well as by radio-immunoassays. 2) Direct immunoelectron microscopic localization of receptors in permeabilized and sectioned cells before and during stimulation. 3) Localizing receptor protein pools in subcellular fractions of resting and activated cells. 4) Inducing the promyeloctic HL-60 cell line for granulocytic differentiation in vitro for studies of the order of appearance of surface C3b and C3bi receptors, intracellular receptor pools determining the fraction which will also be used for biosynthetic labelling studies to determine if protein modification accompanies receptor translocation. In addition to providing valuable information on the mechanism and significance of complement receptor expression, these studies should also yield important insights on the overall process of neutrophil activation.
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