Co-aggregation of FcγRII with FcϵRI on Human Mast Cells Inhibits Antigen-induced Secretion and Involves SHIP-Grb2-Dok Complexes*

Abstract

Signaling through the high affinity IgE receptor FcϵRI on human basophils and rodent mast cells is decreased by co-aggregating these receptors to the low affinity IgG receptor FcγRII. We used a recently described fusion protein, GE2, which is composed of key portions of the human γ1 and the human ϵ heavy chains, to dissect the mechanisms that lead to human mast cell and basophil inhibition through co-aggregation of FcγRII and FcϵRI. Unstimulated human mast cells derived from umbilical cord blood express the immunoreceptor tyrosine-based inhibitory motif-containing receptor FcγRII but not FcγRI or FcγRIII. Interaction of the mast cells with GE2 alone did not cause degranulation. Co-aggregating FcϵRI and FcγRII with GE2 1) significantly inhibited IgE-mediated histamine release, cytokine production, and Ca²⁺ mobilization, 2) reduced the antigen-induced morphological changes associated with mast cell degranulation, 3) reduced the tyrosine phosphorylation of several cellular substrates, and 4) increased the tyrosine phosphorylation of the adapter protein downstream of kinase 1 (p62^dok; Dok), growth factor receptor-bound protein 2 (Grb2), and SH2 domain containing inositol 5-phosphatase (SHIP). Tyrosine phosphorylation of Dok was associated with increased binding to Grb2. Surprisingly, in non-stimulated cells, there were complexes of phosphorylated SHIP-Grb2-Dok that were lost upon IgE receptor activation but retained under conditions of Fcϵ-Fcγ co-aggregation. Finally, studies using mast cells from Dok-1 knock-out mice showed that IgE alone triggers degranulation supporting an inhibitory role for Dok degranulation. Our results demonstrate how human FcϵRI-mediated responses can be inhibited by co-aggregation with FcγRIIB and implicate Dok, SHIP, and Grb2 as key intermediates in regulating antigen-induced mediator release.