Neutrophils are the most numerous of the white blood cells in man. They respond to specific ligands which have specific receptors on the membrane surface. In response, these cells migrate, phagocytize particles, generate oxidants, and release proteases. All of these responses are fundamental facets of inflammation and contribute to host defense and tissue injury in inflammatory diseases. Cytoskeletal rearrangement is rapidly elicited upon stimulation and dramatic morphological changes occur in these cells. F-actin polymerization occurs as a biphasic response with an initial transient rise to a maximum within 10 seconds followed by a slower sustained elevation. These two phases appear to be regulated differently in that they have different occupancy requirements and calcium dependencies. The rise in F-actin is tightly correlated with a decrease in right angle light scattering of the cells. The second phase also seems to correlate with membrane ruffling (as observed microscopically) and cell aggregation. The goal of the proposed research is 1) to understand the quantitative relationship between receptor occupancy and various facets of this cytoskeletal response by analyzing the relationship between F-actin polymerization (as determined by a fluorescent phallacidin staining method), right angle light scattering (which appears to correlate with F-actin formation), and membrane ruffling (observed microscopically), and determining their involvement in neutrophil aggregation, and 2) to begin delineating the biochemistry involved in the cytoskeletal response by studying the role of protein phosphorylation events in initiating and regulating F-actin polymerization. These studies rely on an instrumental set-up and assays that allow simultaneous measurement of the responses. The various responses will be correlated based on their calcium requirement using the intracellular calcium chelator Quin 2 to deplete intracellular calcium. In addition, the responses will be characterized based on their behavior under conditions where receptor occupancy is manipulated: using a """"""""pulse"""""""" protocol, the number of occupied receptors can be varied, and using an """"""""infusion"""""""" protocol the rate of receptor occupancy can be controlled. These studies should provide unique information about the role of actin polymerization in cytoskeletal rearrangement and the role of protein phosphorylation in initiating and regulating these events.
