Cross-linking of Thy-1 glycoproteins or high-affinity IgE receptors induces mast cell activation via different mechanisms.

Rat peritoneal and pleural mast cells and rat basophilic leukemia cells, RBL-2H3, have been previously shown to be activated by Thy-1-specific monoclonal antibodies (mAb). In the present study we investigated the mechanism of Thy-1-mediated activation and compared it with activation induced by cross-linking of the high-affinity IgE receptor. Binding of an IgG Thy-1 x 1-specific mAb, MRCOX7 (OX7), to RBL-2H3 cells and mast cells, and activation of RBL-2H3 by the OX7 were abrogated by pretreatment of the cells with phosphatidyl inositol-specific phospholipase C (PI-PLC). The F(ab')2 fragment of OX7, in contrast to the Fab' fragment, induced cell activation as well as intact OX7 mAb. Cells sensitized with IgE exhibited an increased responsiveness to anti-Thy-1 antibodies suggesting formation of functional complexes of IgE receptor/IgE/Thy-1/anti-Thy-1. Pretreatment of RBL-2H3 cells with cholera toxin potentiated activation induced by IgE+antigen (Ag) and IgE+OX7, but had no effect on activation induced by OX7 antibody alone. Similarly, dexamethasone had no effect on OX7-induced activation but inhibited IgE+Ag- and IgE+OX7-induced activation. Analysis of phosphotyrosine-containing proteins in RBL-2H3 cell lysates revealed that IgE+Ag and IgE+OX7 induced a marked increase in tyrosine phosphorylation of several proteins that were not tyrosine phosphorylated in cells exposed to OX7 mAb alone. Similar results were obtained when RBL-2H3-derived cells, expressing transfected mouse Thy-1.2, were activated with Thy-1.2-specific IgM antibody. The combined data suggest that Thy-1-specific antibodies activate cells by a mechanism that is different from activation induced by cross-linking of high-affinity IgE receptor.