Antibody cross-linking of human CD9 and the high-affinity immunoglobulin Ereceptor stimulates secretion from transfected rat basophilic leukaemia cells

Previous studies have shown that antibody cross-linking of the tetraspanin protein CD9 stimulates the degranulation of platelets and eosinophils, alth ough the mechanism of activation is unclear. In this work we transfected hu man CD9 into the rat basophilic leukaemia (RBL-2H3) cell line and studied t he stimulation of secretion from these cells in response to a panel of anti -CD9 antibodies. Intact immunoglobulin G1 (IgG1) antibodies activated trans fected cells whereas F(ab')(2) fragments of antibody and an intact IgG2a di d not. Stimulation of secretion was inhibited by co-incubation with monomer murine immunoglobulin E (IgE) but not with an IgG1 isotype control, indica ting that the response involves the endogenous high-affinity IgE receptor ( Fc epsilon RI). The anti-CD9 antibody activation curve was biphasic, and su praoptimal antibody concentrations stimulated little or no degranulation, i ndicating that multivalent binding of human CD9 molecules is necessary for the formation of an active complex with rat Fc epsilon RI. Immunoprecipitat ion of Fc epsilon RI under mild detergent conditions co-precipitated CD9, s uggesting the presence of pre-existing complexes of CD9 and Fc epsilon RI t hat could be activated by antibody cross-linking. These data are further ev idence that tetraspanins are involved in Fc epsilon RI signalling and may r eflect the participation of tetraspanins in the formation of complexes with other membrane proteins that use components of Fc receptors for signal tra nsduction.