These findings suggest that PBSC-derived basophils expanded in vitro are morphologically and functionally mature and will be a useful tool for the analysis of basophil functions.
A BSTRACTBackground: We previously reported that crosslinking of IgG Fc receptor II (Fc γ RII) induces intracellular calcium mobilization, but not histamine release in human basophils. To clarify functional activities of Fc γ RII on human basophils, we analyzed the Fc γ RIImediated signaling events in the human basophilic leukemia cell line KU812F. Methods: Flow cytometric methods were used to investigate the effect on intracellular calcium mobilization of cross-linking of Fc γ RII. KU812F cells were preincubated with anti-Fc γ RII monoclonal antibody (IV.3). After the addition of various concentrations of the tyrosine kinase inhibitor genistein or buffer alone, cells were stimulated with goat antimouse IgG F(ab ′ ) 2 (GAM) and analyzed with the flow cytometer. Next, in order to test the signaling events after cross-linking of Fc γ RII, we examined tyrosine kinase activity. The timecourse of tyrosine phosphorylation after cross-linking of Fc γ RII and the effect of genistein on this tyrosine phosphorylation were tested by immunoblotting. Immunoprecipitation was also performed to identify the type of tyrosine kinase associated with signal transduction of Fc γ RII. Results: The tyrosine kinase inhibitor genistein inhibited intracellular calcium mobilization caused by cross-linking of Fc γ RII in a dose-dependent manner. Rapid tyrosine phosphorylation after Fc γ RII cross-linking was shown by immunoblot analysis and this phosphorylation was inhibited by genistein. Furthermore, tyrosine phosphorylation of Lyn and Syk was observed upon crosslinking of Fc γ RII. Conclusions: Tyrosine phosphorylation is necessary for the signaling pathway through Fc γ RII and tyrosine phosphorylation of Lyn and Syk, at least, is actively involved in this signal transduction.
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