Negative regulation of Fc epsilon RI signaling by Fc gamma RII costimulation in human blood basophils

Background: Signaling through the antigen receptors of human B and T cells and the high-affinity IgE receptor Fc epsilon RI of rodent mast cells is de creased by cross-linking these receptors to the low-affinity Ige receptor F c gamma RII, The inhibition is thought to involve the tyrosine phosphorylat ion of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the Fc ga mma RIIB cytoplasmic tail, creating binding sites For SH2-containing protei n (Src homology domain containing protein tyrosine phosphatase 1 and 2 [SHP -1, SHP-2]) and/or lipid (SH2 domain-containing polyphosphatidylinositol 5- phosphatase) phosphatases that oppose activating signals from the costimula ted antigen receptors, Objective: In human basophils and mast cells Fc epsilon RI signaling genera tes mediators and cytokines responsible for allergic inflammation. We propo sed to determine whether Fc epsilon RI signaling is inhibited by Fc gamma R II costimulation in human basophils and to explore the underlying mechanism as an approach to improving the treatment of allergic inflammation. Methods: Fc gamma R expression on human basophils was examined using flow c ytometry and RT-PCR analysis. Fc gamma RII/Fc epsilon RI costimulation was typically accomplished by priming cells with anti-dinitrophenol (DNP) IgE a nd anti-DNP Ige and stimulating with DNP-BSA. Phosphatases were identified by Western blotting, and their partitioning between membrane and cytosol wa s determined by cell fractionation, Biotinylated synthetic peptides and pho sphopeptides corresponding to the Fc gamma RIIB ITIM sequence were used for adsorption assays. Results: We report that peripheral blood basophils express Fc gamma RII (in both the ITIM-containing Fc gamma RIIB and the immunoreceptor tyrosine-bas ed activation motif-containing Fc gamma RIIA forms) and that costimulating Fc gamma RII and Fc epsilon RI inhibits basophil Fc epsilon RI-mediated his tamine release, IL-4 production, and Ca2+ mobilization, The inhibition of b asophil Fc epsilon RI signaling by Fc gamma RII/Fc epsilon RI costimulation is linked to a significant decrease in Syk tyrosine phosphorylation, Human basophils express all 3 SH2-containing phosphatases. Conclusions: Evidence that Fc gamma RII/Fc epsilon RI costimulation induces SHP-1 translocation from the cytosolic to membrane fractions of basophils and that biotinylated synthetic peptides corresponding to the phosphorylate d Fc gamma RIIB ITIM sequence specifically recruit SHP-1 from basophil Lysa tes particularly implicates this protein phosphatase in the negative regula tion of Fc epsilon RI signaling by costimulated Fc gamma RII.