title: Inhibition of antigen-induced mediator release from IgE-sensitized cells by a monoclonal anti-Fc epsilon RI alpha-chain receptor antibody: implications for the involvement of the membrane-proximal alpha-chain region in Fc epsilon RI-mediated cell activation.
abstract: The interaction between human IgE and its high affinity receptor, FcepsilonRI, is a critical event in mediating the allergic response. Aggregation of the alpha-chain of FcepsilonRI (FcepsilonRIalpha) 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-FcepsilonRIalpha mAbs, 15/1 and 5H5F8. In contrast to 15/1, mAb 5H5F8 does not inhibit IgE binding to FcepsilonRIalpha. Here we demonstrate both 5H5F8 binding to FcepsilonRI(+) cells as well as a high level of IgE binding to 5H5F8-saturated cells. At the same time 5H5F8 strongly inhibits hexosaminidase release and Ca(2+) flux after Ag triggering from human IgE-sensitized RBL-2H3 cells stably transfected with human FcepsilonRIalpha. 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 K(d) values of similar magnitude to that observed for binding to recombinant FcepsilonRIalpha. These peptides may prove useful as targets for the identification of antagonists of FcepsilonRIalpha-mediated biological activity. Moreover, our data indicate that FcepsilonRIalpha-mediated activation may involve a novel alpha-chain epitope in an early step of the cell-triggering pathway leading to cellular activation.
authors.affiliation: Novartis Forschungsinstitut GmbH, Vienna, Austria.
authors.forename: A
authors.initials: A
authors.lastname: Nechansky
authors.forename: M W
authors.initials: MW
authors.lastname: Robertson
authors.forename: B A
authors.initials: BA
authors.lastname: Albrecht
authors.forename: J R
authors.initials: JR
authors.lastname: Apgar
authors.forename: F
authors.initials: F
authors.lastname: Kricek
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology*; D000941:Antigens / Q000276:immunology*; D001491:Basophils / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism*; D001666:Binding Sites, Antibody / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D006224:Cricetinae; D000939:Epitopes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D006633:Histamine Antagonists / Q000378:metabolism / Q000494:pharmacology; D006636:Histamine Release / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism; D020024:Leukotriene Antagonists; D015289:Leukotrienes / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000276:immunology; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology* / Q000378:metabolism / Q000502:physiology; D010648:Phenylacetates; D051381:Rats; D017455:Receptors, IgE / Q000276:immunology* / Q000378:metabolism / Q000502:physiology; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D001619:beta-N-Acetylhexosaminidases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D000939:Epitopes; D006633:Histamine Antagonists; D007140:Immunoglobulin Fab Fragments; D020024:Leukotriene Antagonists; D015289:Leukotrienes; D009596:Nitrophenols; D010446:Peptide Fragments; D010648:Phenylacetates; D017455:Receptors, IgE; D011994:Recombinant Proteins; C026242:4-hydroxy-5-nitrophenyl acetic acid; D007073:Immunoglobulin E; D001619:beta-N-Acetylhexosaminidases; D002118:Calcium