A monoclonal antibody that inhibits secretion from rat basophilic leukemia cells and binds to a novel membrane component

Rat basophilic leukemia (RBL-2H3) cells, like mast cells and basophils, carry monovalent membrane receptors with high affinity for IgE (Fc epsilon R). Cross-linking of these receptors provides the immunologic stimulus which initiates a series of biochemical events, culminating in secretion of inflammatory mediators. In an attempt to identify membrane components involved in the stimulus-secretion coupling of these cells, hybridomas were produced from splenocytes of mice immunized with intact RBL-2H3 cells. Here we report the production of a mAb (designated G63) that inhibits the Fc epsilon R-mediated secretion from RBL cells. At low degrees of Fc epsilon R aggregation, the mAb G63-induced inhibition may be complete, whereas at the maximum of secretion the inhibition is in the range of 30 to 40%. The relative degree of inhibition of secretion is dependent on the dose of mAb G63. Furthermore, inhibition requires the bivalency of G63, as the Fab fragments are inactive. The number of antigenic epitopes recognized by G63 per RBL-2H3 cell is 1.8 x 10(4) epitopes/cell, as determined by direct binding studies of 125I-labeled Fab fragments of G63. This number is 20 to 30 times smaller than that of Fc epsilon R on the same cells. The membrane component to which G63 binds has been identified by immunoprecipitation as a glycoprotein with an apparent Mr of 58 to 70 kDa. All of these results, and the fact that no competition for binding to RBL cells between mAb G63 and IgE can be resolved, indicate that mAb G63 binds to a membrane component which is distinct from the Fc epsilon R. mAb G63 suppresses the Fc epsilon R-mediated rise in cytoplasmic concentration of free Ca2+ ions, known to be one of the biochemical signals involved in the stimulus-secretion coupling in RBL-2H3 cells. G63 does not affect, however, the degranulation induced by the Ca2+ ionophore A23187. Therefore, mAb G63 probably exerts its inhibitory effect on a step preceding the rise in cytoplasmic free Ca2+. Thus, mAb G63 defines a previously unidentified membrane component that is involved in one of the early steps of the RBL-2H3 activation mediated by their Fc epsilon R.