Human polymorphonuclear leukocytes (PMN) are important mediators of the inflammatory process and are essential for the maintenance of normal host defenses against infection. Central to these functions is the process of degranulation, whereby azurophil and specific granules fuse with the plasma membrane, releasing their contents into the phagocytic vacuole. Among the early steps following PMN stimulation is the activation of various phospholipases which alter membrane phospholipid composition. We propose that these changes in membrane phospholipid composition are essential for the membrane fusing events observed during degranulation. Specifically we propose that PMN degranulation is regulated by the local activation and metabolism of phospholipase A2 and D in conjunction with the annexins and the rise in intracellular calcium. Our hypothesis is that the localized conversion of plasma membrane and/or granule membrane phosphatidylcholine to phosphatidic acid (PA) by PLD directs Ca+2- dependent apposition of PMN granules with the plasma membrane mediated by one or more of the annexins. Ca+2- dependent fusion is then further facilitated by the activation of PLA2 and the generation of arachidonic acid. The objective of the proposed project is to determine the location and significance of phospholipid remodeling. We will develop an in vitro model of PMN degranulation, examining the relationship between phospholipid remodeling, the annexins, and Ca+2- dependent fusion. We will begin by examining the effect of PMN granules with plasma membrane vesicles. Once the relationships between phospholipid remodeling, the annexins, and Ca+2- dependent fusion have been established, we will attempt to reconstitute degranulation in permeabilized PMN.
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