(taken directly from the application) 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. Although inhibition of these events inhibit degranulation to varying degrees, their precise role remain unclear. This research proposes that PMN degranulation is regulated by the localized activation and metabolism of phospholipase A2 and D in conjunction with the annexins and the rise in intracellular calcium (Ca2+). The hypothesis is that the localized conversion of plasma membrane and/or granule membrane phosphatidylcholine to phosphatidic acid by phospholipase D directs Ca2+ -dependent apposition of PMN granules with the plasma membrane mediated by one or more of the annexins. Ca2+ -dependent fusion is then further facilitated by the activation of phospholipase A2 and the generation of arachidonic acid. The objective of the proposed project is to determine the location and significance of phospholipid remodeling. Using radiolabeled phospholipids and subcellular fractionation, the investigator will localize the pool of phosphatidylcholine sensitive to hydrolysis by phospholipase D. The investigators have developed an in vitro model of membrane fusion. Using this model system they will examining the relationship between phospholipid remodeling, the annexins, and Ca2+ - dependent membrane fusion. They will begin by analyzing the effect of phospholipid composition and metabolism on liposome-liposome fusion. They will progress and measure the role of phospholipid metabolism and various annexins on the fusion of granules with plasma membrane vesicles. Once the effects of individual annexins, phospholipid metabolism, and Ca2+ are established on membrane fusion, the investigators will attempt to reconstitute degranulation in permeabolized PMN.
| Hessler, R J; Blackwood, R A; Brock, T G et al. (1998) Identification of glyceraldehyde-3-phosphate dehydrogenase as a Ca2+-dependent fusogen in human neutrophil cytosol. J Leukoc Biol 63:331-6 |
