FcεRI-mediated association of 6-μm beads with RBL-2H3 mast cells results in exclusion of signaling proteins from the forming phagosome and abrogation of normal downstream signaling

Abstract

Cells of the mucosal mast cell line, RBL-2H3, are normally stimulated to degranulate after aggregation of high affinity receptors for IgE (FcεRI) by soluble cross-linking ligands. This cellular degranulation process requires sustained elevation of cytoplasmic Ca2+. In this study, we investigated the response of RBL-2H3 cells to 6-μm beads coated with IgE-specific ligands. These ligand-coated beads cause only small, transient Ca2+ responses, even though the same ligands added in soluble form cause larger, more sustained Ca2+ responses. The ligand-coated 6-μm beads also fail to stimulate significant degranulation of RBL-2H3 cells, whereas much larger ligand- coated Sepharose beads stimulate ample degranulation. Confocal fluorescence microscopy shows that the 6-μm beads (but not the Sepharose beads) are phagocytosed by RBL-2H3 cells and that, beginning with the initial stages of bead engulfment, there is exclusion of many plasma membrane components from the 6-μm bead/cell interface, including p53/56(lyn) and several other markers for detergent-resistant membrane domains, as well as an integrin and unliganded IgE-FcεRI. The fluorescent lipid probe DiIC16 is a marker for the membrane domains that is excluded from the cell/bead interface, whereas a structural analogue, fast DiI, which differs from DiIC16 by the presence of unsaturated acyl chains, is not substantially excluded from the interface. None of these components are excluded from the interface of RBL-2H3 cells and the large Sepharose beads. Additional confocal microscopy analysis indicates that microfilaments are involved in the exclusion of plasma membrane components from the cell/bead interface. These results suggest that initiation of phagocytosis diverts normal signaling pathways in a cytoskeleton-driven membrane clearance process that alters the physiological response of the cells.