The purpose of this research project is to elucidate the biochemical mechanisms of IgE-dependent mediator release from mast cells, which cause allergic diseases. Our efforts will be focused to analyze biochemical events which are caused by the bridging of IgE-receptors on mast cells. Recently, we established a culture system to obtain human basophilis in the culture of the mononuclear cells in umbilical cord blood. Cultured basophils could be sensitized with human IgE for anti-IgE-induced histamine release. Thus, i) we shall identify growth factor(s) for human basophils and characterize the cultured human basophils. ii) Experiments will be carried out to determine that membrane-associated enzymes, such as serine protease and methyltransferase, are involved in the activation of cultured basophils, and that this process will lead to Ca2+ uptake and mediator release. iii) The affinity of human IgE and rodent IgE for IgE-receptors on cultured human basophilis will be determined, and iv) IgE-receptors on the cells will be identified by SDS gel electrophoresis. v) We shall determine the substrate specificity of membrane-associated serine protease, which is activated upon bridging of IgE receptors. vi) Possible relationship between the proteolytic enzyme and Alpha or Beta subunits of IgE-receptors will be determined. vii) The possibility is considered that the activation of the protease results in the formation of a kinin-like substance, which in turn activates the other membrane-associated enzymes for mediator release. We shall determine whether kallikrein-like enzymes and/or bradykinin may induce the activation of methyltransferases and adenylate cyclase in rodent mast cells and human basophils. viii) The possible role of phospholipid methylation in the enhancement of turnover of phosphatidylinositol will be investigated by using rodent mast cells and human cultured basophils. ix) We shall analyze intracellular Ca2+ movement and Ca++ influx into mast cells (basophils) induced by bridging of IgE-receptors. A fluorescent quinoline Ca++ indicator, Quin 2, will be employed for the anlysis. x) Finally, biochemical mechanisms of desensitization will be investigated in mouse mast cells. We shall test the hypothesis that desensitization is due to activation of membrane-associated enzymes, and that different enzymes are involved in the activation of mast cells for mediator release and desensitization. Similar experiments on desensitization will be carried out with cultured human basophils in future years.
