Cross-linkage of the high affinity receptor for IgE, FcEpsilonRl (found on mast cells and basophils), is the initial event leading to IgE-dependent degranulation and release of inflammatory mediators. This is the trigger for immediate hypersensitivity reactions and thus is the primary event underlying a number of atopic diseases such as allergic rhinitis and allergic asthma. Hydrolysis of phosphoinositides (Pls), catalyzed by phosphohpase C (PLC), is closely linked to activation of this receptor and is thought to contribute substantially to FcepsilonRI- stimulated degranulation. Using rat basophilic leukemia (RBL) cells, a mast cell analog, we have developed a novel, cell-free, cytoplasm-depleted system in which to study the mechanism whereby FcEpsilonRI activates PLC. Unlike membranes prepared by classical means, these "RBL cell ghosts" retain a functional relationship between IgE receptor cross-linkage and PI hydrolysis. Unexpectedly, FcEpsilonRImediated PI hydrolysis is greatly stimulated (4-15 fold) by 5 mM phosphoenolpyruvate (PEP) and this effect persists in ghosts that have been permeabilized by bacterial toxins to allow diffusion of small molecules. The RBL cell ghost system will be used to achieve the following aims: 1) characterize the RBL cell ghost system following permeabilization and determine if the role of PEP in FcEpsilonRI-mediated activation of PI hydrolysis is independent of its ability to regenerate ATP; 2) examine the role ATP to phosphorylate aqueous molecules, phospholipids, and proteins during FcEpsilonR1-mediated triggering; 3)enlarge our preliminary evidence that a novel phosphorylated aqueous product, peak "y", is generated during FcepsilonR1 -mediated triggering, and 4) examine available variant RBL cell lines for ones which are specifically deficient in the ATP-dependent activation of PLC and/or in the PEP-dependent enhancement of the ATP-dependent signal.

These studies will define the role of ATP and PEP in IgE receptor-mediated activation of PLC and increase our understanding of the mechanism whereby this signal is transduced. In that the molecule(s) responsible for this effect may be specific for FcepsilonRI, this information has the potential to lead to novel pharmacologic agents for the prevention of immediate hypersensitivity reactions. On the other hand, if the role of PEP is eventually found to form the basis for a new paradigm of signal transduction for those receptors requiring aggregation for the initiation of a signal, these findings may have great impact on our understanding of antigen receptors on T cells and B cells as well as Fcgamma receptors on macrophages.

R01GM045578

1991-07-01

1996-06-30