It has been established that IgE molecules bind to Fc delta receptors on mast cells with high affinity, and that cross linking of the cell bound IgE antibodies by antigen trigger the cells for the release of mediators which cause allergic diseases. The purpose of this research project is to elucidate the immunological and biochemical mechanisms of IgE-dependent mediator release. Previous work indicated that cross-linking of cell-bound IgE antibody molecules by multivalent ligand induced activation of phospholipase C (PLC) for hydrolysis of inositol phospholipids, and that this process may play a major role in the transduction of triggering signals for mediator release. In this proposal, i) the applicant shall study whether guanidine- nucleotide binding protein(s) is involved in the IgE-mediated activation of PLC and histamine release. As the investigators have established the method to permeabilize rat mast cells and bone marrow-derived mouse mast cells (BMMC), the investigators shall introduce non-hydrolyzable GTP analogue, such as GTP gammaS or GDPbetaS, into the permeabilized cells and determine the effect of the GTP analogue, on antigen-induced hydrolysis of inositol phospholipids and mediator release. ii) The investigator's recent experiment indicated that a newly synthesized inhibitor of phospholipase A2 (PLA2) inhibited the antigen-induced histamine release and arachidonic acid release from sensitized BMMC without affecting the formation of inositol phosphates. The investigators shall confirm that the inhibitor does not affect the antigen-induced activation of PLC, and determine whether the inhibitor may affect other enzymes involved in phosphatidylinositol turnover. The investigators expect that the experiments will establish a possible role of PLA2 in IgE-dependent mediator release. iii) The investigators shall try to establish culture conditions for selective differentiation and growth of human basophil granulocytes and mast cells from umbilical cord blood cells and bone marrow cells. Recombinant interleukins from human T-cells or non-T-cells will be employed. Fibroblast monolayers will also be employed for the differentiation of mast cells. iv) The investigators shall also continue their collaboration with molecular biologists to determine the binding site on human IgE molecules to Fc delta receptors on basophils. Recombinant peptides and synthetic peptides representing small segments of the Fc portion of delta chain will be tested for the ability to block passive sensitization of human basophils with IgE antibodies, and for the ability to block the binding of 125 I-labeled IgE to Fc delta receptors on cultured human basophils.
