FC-EPSILON-RI-MEDIATED ASSOCIATION OF 6-MU-M BEADS WITH RBL-2H3 MAST-CELLS RESULTS IN EXCLUSION OF SIGNALING PROTEINS FROM THE FORMING PHAGOSOME AND ABROGATION OF NORMAL DOWNSTREAM SIGNALING

Cells of the mucosal mast cell line, RBL-2H3, are normally stimulated to degranulate after aggregation of high affinity receptors for IgE (F c epsilon RI) by soluble cross-linking ligands. This cellular degranul ation process requires sustained elevation of cytoplasmic Ca2+, In thi s study, we investigated the response of RBL-2H3 cells to 6-mu m beads coated with IgE-specific ligands, These ligand-coated beads cause onl y small, transient Ca2+ responses, even though the same ligands added in soluble form cause larger, more sustained Ca2+ responses, The ligan d-coated 6-mu m beads also fail to stimulate significant degranulation of RBL-2H3 cells, whereas much larger ligand-coated Sepharose beads s timulate ample degranulation. Confocal fluorescence microscopy shows t hat the 6-mu m beads (but not the Sepharose beads) are phagocytosed by RBL-2H3 cells and that beginning With the initial stages of bead engu lfment, there is exclusion of many plasma membrane components from the 6-mu m bead/cell interface, including p53/56(lyn) and several other m arkers for detergent-resistant membrane domains, as well as an integri n and unliganded IgE-Fc epsilon RI. The fluorescent lipid probe DiIC(1 6) is a marker for the membrane domains that is excluded from the cell /bead interface, whereas a structural analogue, fast DiI, which differ s from DiIC(16) by the presence of unsaturated acyl chains, is not sub stantially excluded from the interface, None of these components are e xcluded from the interface of RBL-2H3 cells and the large Sepharose be ads, Additional confocal microscopy analysis indicates that microfilam ents are involved in the exclusion of plasma mem brane components from the cell/bead interface, These results suggest that initiation of pha gocytosis diverts normal signaling pathways in a cytoskeleton-driven m embrane clearance process that alters the physiological response of th e cells.