Peripheral neutrophils play important roles as the first line of defense against infection and as mediators of inflammation. Neutrophils cause damage to the connective tissue ground substance and collagen fibers due to release of lytic enzymes during degranulation. Understanding the physiological mechanism of control of neutrophil degranulation may lead to strategies for local control of inflammation and treatment of neutrophil disorders that cause arthritis, periodontitis and other diseases. Degranulation involves intracellular events leading to fusion of neutrophil granules with the plasma membrane or phagosomes. Several intracellular signal processing and membrane fusion. As Ca2+ and phospholipid binding proteins, they form a completely new class whose mechanism of membrane binding is unknown. Some of these proteins are physiological targets of protein kinase C and other receptor-activated membrane-bound kinases. Like protein kinase C, they appear to translocate from the cytoplasm to membrane-bound locales in response to agonist-induced increases in intracellular Ca2+. Many of the annexins also mediate Ca2+-dependent vesicle aggregation, suggesting they may assist membrane fusion. Members of this class of proteins have been found in neutrophils, and our own experiments indicate their possible mediation of Ca2+-dependent fusion of neutrophil granules. The proposed project will involve study of the interactions of annexins with model membranes and human neutrophil membranes. The goal of this project is to understand the mechanism of annexin binding to membranes and the possible role of annexins in the regulation of neutrophil function. A reconstituted system will be developed to study the role of annexins and Ca2+ in neutrophil degranulation. In this system, the fusion of isolated granules with isolated phagosomes or plasma membrane will be studied by fluorescence and flow cytometry. This will allow the effect of individual soluble components to be evaluated kinetically in a system less complex than whole cells. The structural basis of the interaction of annexins with model membranes will be studied as well. NMR and fluorescence methods are chosen because of the difficulty of crystallization of the membrane-bound system for diffraction experiments. Immunological methods will also be used to determine functionally important parts of the proteins. Using this data, a model for the structure of the bound protein will be developed. Structural features will be correlated to function as monitored by binding and fusion assays. The results of these studies will help to elucidate the role of annexins in neutrophil activation, the factors controlling their binding to membranes and strategies for controlling neutrophil function in disease.
