Negative regulation of FceRI signaling by FcgRII costimulation in human blood basophils

Background: Signaling through the antigen receptors of human B and T cells and the high-affinity IgE receptor Fc-epsilonRI of rodent mast cells is decreased by cross-linking these receptors to the low-affinity IgG receptor Fc-gammaRIIB. The inhibition is thought to involve the tyrosine phosphorylationof immunoreceptor tyrosine–based inhibitory motifs (ITIMs) in the Fc-gammaRII cytoplasmic tail, creating binding sites for SH2-containing protein (Src homology domain containing protein tyrosine phosphatase 1 and 2 [SHP-1, SHP-2]) and/or lipid (SH2 domain-containing polyphosphatidyl-inositol 5-phosphatase) phosphatases that oppose activating signals from the costimulated antigen receptors. Objective: In human basophils and mast cells Fc-epsilonRI signaling generates mediators and cytokines responsible for allergic inflammation. We proposed to determine whether Fc-epsilonRI signaling is inhibited by Fc-gammaRII costimulation in human basophils and to explore the underlying mechanism as an approach to improving the treatment of allergic inflammation. Methods: Fc-gammaR expression on human basophils was examined using flow cytometryand RT-PCR analysis. Fc-gammaRII/Fc-epsilonRI costimulation was typically accomplished by priming cells with anti-dinitrophenol (DNP) IgE and anti-DNP IgG and stimulating with DNP-BSA. Phosphatases were identified by Western blotting, and their partitioning between membrane and cytosol was determined by cell fractionation. Biotinylated synthetic peptides and phosphopeptides corresponding to the Fc-gammaRIIB ITIM sequence were used for adsorption assays. Results: We report that peripheral blood basophils express Fc-gammaRII (in both the ITIM-containing Fc-gammaRIIB and the immunoreceptor tyrosine–based activation motif–containing Fc-gammaRIIA forms) and that costimulating Fc-gammaRII and Fc-epsilonRI inhibits basophil Fc-epsilonRI-mediated histamine release, IL-4 production, and Ca2+ mobilization. The inhibition of basophil Fc-epsilonRI signaling by Fc-gammaRII/Fc-epsilonRI costimulation is linked to a significant decrease in Syk tyrosine phosphorylation. Human basophils express all 3 SH2-containing phosphatases. Conclusions: Evidence that Fc-gammaRII/Fc-epsilonRI costimulation induces SHP-1 translocation from the cytosolic to membrane fractions of basophils and that biotinylated synthetic peptides corresponding to the phosphorylated Fc-gammaRIIB ITIM sequence specifically recruit SHP-1 from basophil lysates particularly implicates this protein phosphatase in the negative regulation of Fc-epsilonRI signaling by costimulated Fc-gammaRII