SIMULTANEOUS CROSS-LINKING BY 2 NONTRIGGERING BIVALENT LIGANDS CAUSESSYNERGISTIC SIGNALING OF IGE FC-EPSILON-RI COMPLEXES

We have used two bivalent ligands that bind IgE to study the relations hip between the aggregation of receptors with high affinity for IgE (F c epsilon RI) and the responses (receptor immobilization, Ca2+ influx, and degranulation) of rat basophilic leukemia (RBL-2H3) cells. One of these is a symmetric bivalent ligand, -dinitrophenyl)amino]caproyl]-L -tyrosyl]-L-cystine ((DCT)(2)-cys), which binds specifically to the co mbining sites of a mAb anti-DNP IgE and efficiently cross-links cell s urface IgE, but does not trigger significant degranulation or increase s in intracellular Ca2+. Several lines of evidence, including lateral mobility measurements, indicate that this ligand preferentially forms stable cyclic complexes containing two (DCT)(2)-cys and two IgE. The s econd ligand is a mAb anti-IgE, B1E3, which causes lateral mobility ch anges consistent with dimerized IgE-Fc epsilon RI and also does not tr igger increases in intracellular Ca2+ or degranulation. The two ligand s together trigger robust responses. In the presence of B1E3, (DCT)(2) -cys causes immobilization of IgE-Fc epsilon RI in a broad concentrati on range; in a more narrow concentration range, it is a potent stimula nt of changes in both degranulation and Ca2+. We have compared the dos e-response curves for cellular activation to simulated IgE aggregation curves, i.e., curves that predict the equilibrium IgE aggregate size distribution as a function of the (DCT)(2)-cys concentration. Our resu lts indicate that maximal cellular activation occurs at a much higher (DCT)(2)-cys concentration than maximal IgE aggregation. When IgE aggr egation is maximal, almost all aggregated IgE is in cyclic dimers. Thu s, cyclic dimers appear to be functionally ineffective, even after the y have been cross-linked by B1E3. Aggregated IgE-Fc epsilon RI that is effective in stimulating a cellular response may have particular stru ctural or dynamic properties that allow critical interactions for init iating the signaling cascade.