Formation and release of inflammatory mediators by mast cells play central roles in immediate hypersensitivity reactions. In addition, mast cell derived mediators appear to play significant roles in the evolution of certain complement-induced inflammatory mechanisms and exert regulatory influences on particular lymphocyte subclasses. Recent work has shown that alterations in lipid metabolism likely play a central role in mast cell mediator release. In addition to lipid methylation events described by others, this laboratory has shown that 32PO4 labeling of selected phospholipids and accumulation of the lipid intermediate sn-1,2-diacylglycerol (diglyceride or DG) kinetically parallel mediator release and have nearly identical pharmacologic inhibition and dose response relationships. The long range goal of this work is to understand what roles lipid metabolic pathways play in specific subcellular compartments in causing and/or regulating mast cell mediator release. DG metabolism will be of central interest. A newly developed system utilizing mast cell """"""""cytoplasts"""""""" (bodies formed from intact cells that are surrounded by intact plasma membrane and contain cytoplasm nearly devoid of granules and other organelles) will be employed to characterize lipid metabolic reactions associated with the plasma membrane. Later studies will explore lipid metabolic reactions in cytoplasmic granule membranes.
Specific aims of this proposal include: 1) characterizing physical and biochemical parameters of mast cell cytoplasts; 2) determining the fatty acid composition of the DG that accumulates during mast cell secretion; 3) investigating how in situ generation of DG or exogenous addition of a DG analogue affects mediator release; 4) determining the metabolic origin and mechanism of production of DG in stimulated mast cells; 5) investigating the pathways that remove DG and their potential importance in negative modulation of secretion. These studies of the nature and subcellular sites of the biochemical mechanisms leading to mediator release should contribute to a much clearer view of immunologic activation of mast cells. Such knowledge should facilitate the development of new pharmacologic strategies to suppress allergic disorders or to augment the contribution of mast cells to protective immunity or the resolution of infections.
