Inhibition of Antigen-Induced Mediator Release from IgE-Sensitized Cells by a Monoclonal Anti-FcεRI α-Chain Receptor Antibody: Implications for the Involvement of the Membrane-Proximal α-Chain Region in FcεRI-Mediated Cell Activation1

The interaction between human IgE and its high affinity receptor, FcεRI, is a critical event in mediating the allergic response. Aggregation of the α-chain of FcεRI (FcεRIα) occurs via cross-linking of receptor-bound IgE by Ag, resulting in cell activation and the release of mediators of hypersensitivity. Recently, we mapped the epitopes of two anti-FcεRIα mAbs, 15/1 and 5H5F8. In contrast to 15/1, mAb 5H5F8 does not inhibit IgE binding to FcεRIα. Here we demonstrate both 5H5F8 binding to FcεRI+ cells as well as a high level of IgE binding to 5H5F8-saturated cells. At the same time 5H5F8 strongly inhibits hexosaminidase release and Ca2+ flux after Ag triggering from human IgE-sensitized RBL-2H3 cells stably transfected with human FcεRIα. Further, 5H5F8 and its Fab inhibit sulfidoleukotriene and histamine release from primary human peripheral blood leukocytes, including cells bearing endogenous IgE. Furthermore, we confirm that 5H5F8 maps to a linear peptide sequence in close proximity to the cell membrane. Two chemically synthesized peptides containing the 5H5F8 epitope sequence PREKY were selected for detailed analysis of 5H5F8 and 5H5F8 Fab binding and were found to produce Kd values of similar magnitude to that observed for binding to recombinant FcεRIα. These peptides may prove useful as targets for the identification of antagonists of FcεRIα-mediated biological activity. Moreover, our data indicate that FcεRIα-mediated activation may involve a novel α-chain epitope in an early step of the cell-triggering pathway leading to cellular activation.