FceRI-mediated Association of 6-1xm 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, RBL2H3, are normally stimulated to degranulate after aggregation of high affinity receptors for IgE (FceRI) by soluble cross-linking ligands. This cellular degranulation process requires sustained elevation of cytoplasmic Ca 2÷. In this study, we investigated the response of RBL-2H3 cells to 6-p~m beads coated with IgE-specific ligands. These ligand-coated beads cause only small, transient Ca 2÷ responses, even though the same ligands added in soluble form cause larger, more sustained Ca 2+ responses. The ligand-coated 6-p~m 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-~m 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 merebrane components from the 6-~m bead/cell interface, including p53/56 lyn and several other markers for detergent-resistant membrane domains, as well as an integrin and unliganded IgE-Fc~RI. The fluorescent lipid probe DiICx6 is a marker for the membrane domains that is excluded from the cell/bead interface, whereas a structural analogue, fast DiI, which differs from DilC16 by the presence of unsaturated acyl chains, is not substantially excluded from the interface. None of these components are excluded from the interface of RBL2H3 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. GREGATION of Fc~RI, the high affinity receptor for IgE found predominantly on mast cells and basophils, stimulates a signaling cascade that leads to exocytosis of inflammatory mediators of the allergic response (5, 33). The signaling pathways initiated by FceRI aggregation are similar to those initiated by aggregation of the T and B cell multichain immune recognition receptors (9, 23, 52). Some of the steps in these signaling pathways are phosphorylation of tyrosine residues in the immunoreceptor tyrosine activation motif (ITAM) 1 sequences of reAddress all correspondence to David Holowka and Barbara Baird, Department of Chemistry, Cornell University, Ithaca, NY 14853-1301. Tel.: (607) 255-4095. Fax: (607) 255-4137. E-mail: [email protected] 1. Abbreviat ions used in this paper: DilCI6, 3, 3'-dihexadecylindocarbocyanine; DNP-BGG, bovine gamma globulin multiply conjugated to DNP; fast DiI, 3, 3'-dilinoleylindocarbocyanine; GPI, glycosylphosphatidylinositol: IgEt, io~in, biotinylated IgE: ITAM, immunoreceptor tyrosine activation motif; PTK, protein tyrosine kinase; sAv, streptavidin; TNP, 2, 4, 6-trinitrophenyl; TX-100, Triton X-100. ceptor cytoplasmic segments by src-family protein tyrosine kinases (PTKs), binding and activation of ZAP70/syk-family PTKs, phosphatidyl inositol hydrolysis, protein kinase C activation, and mobilization of intracellular Ca 2+ (5, 43). Fc~RI is a multisubunit receptor comprising or, 13, and a disulfide-linked pair of ",/subunits (7). Whereas the c~ subunit contains the IgE binding site, the 13 and -,/ subunits contain the ITAMs and are involved in signal transduction. As the result of Fc~RI aggregation, the src-family PTK p53/56 lyn phosphorylates the tyrosines within the ITAMs, leading to recruitment of p72 ~yk to the ~ subunit (6, 26, 39, 44). The mechanism by which FceRI aggregation leads to the earliest phosphorylation events, i.e., phosphorylation of 13 and ~/by p53/56 ly", is not completely understood (41). Likewise, coupling of tyrosine kinase activation and substrate phosphorylation to more downstream signaling events, including C a 2+ mobilization and stimulated exocytosis, is only partially understood (24). We recently showed that a large fraction of the total cellular p53/56 lyn associates with low-density Triton X-100 © The Rockefeller University Press, 0021-9525196/09/1427/13 $2.00 The Journal of Cell Biology, Volume 134, Number 6, September 1996 1427-1439 1427 on July 8, 2017 jcb.rress.org D ow nladed fom (TX-100)-insoluble membrane domains isolated from the mucosal mast cell line RBL-2H3 and that the extent of this association increases after Fc~RI-aggregation (17). We also observed that the fluorescent lipid probe 3,3'-dihexadecylindocarbocyanine (DilC16) labels membrane domains on the surface of RBL cells that coredistribute with aggregated FceRI (49). In the same study, we found that these membrane domains can be similarly redistributed by aggregating c~-galactocyl derivatives of a GDlb ganglioside via a specific mAb, AA4 (4, 20). Importantly, these gangliosides, as well as the glycosyl phosphatidylinositol (GPI)linked protein Thy-1, was found to coisolate with p53/56 ly" in immunoprecipitates from TX-100-1ysed cells (14, 34) and with the detergent-resistant membrane domains containing p53/56 jyn (17), suggesting that the cell surface membrane domains observed with fluorescence microscopy are related to those structures isolated from TX-100-1ysed cells. These studies led to our hypothesis that coalescence of membrane domains with aggregated Fc~RI is functionally important because it allows communication of domain-associated signaling molecules with these aggregated receptors (17, 24). In this study, we used fluorescence flow cytometry and confocal microscopy to investigate the capacity of ligandcoated, 6-1~m-diam beads to bind IgE-Fc~RI and stimulate RBL cells. We find that the beads elicit neither degranulation nor sustained Ca 2÷ responses from RBL cells. Instead, RBL cells phagocytose these beads, and this process causes physical segregation of membrane domain components from Fc~RI at the cell/bead interface. The implications of these results for Fc~RI-mediated signaling, as well as for plasma membrane structure, and phagocytosis, are discussed. Materials and Methods Purified Antibodies and Other Reagents Mouse anti-rat Thy-1.1 mAb was obtained from PharMingen (San Diego, CA). Leu4, a mouse IgG mAb that recognizes the ~ subunit of the CD3 complex of the TCR, was obtained from Becton-Dickinson Immunocytometry Sys. (San Jose, CA). The mouse mAb, AA4, which recognizes c~-galactosyl derivatives of the ganglioside GDlb (20) was from Dr. R. Siraganian (National Institutes of Health, Bethesda, MD). The mouse mAb TA2, which recognizes the a subunit of rat VLA4 integrin (27), was from Dr. T. Issekutz (Toronto Hospital, Toronto, Ontario). Anti-2,4-dinitrophenyl mouse IgE mAb (30) was purified as described previously (25). Fluorescein 5'-isothiocyanate-labeled biotinylated IgE (FITC-IgEbiotin) was prepared by first conjugating biotin to IgE as previously described (17) and then, after extensive dialysis against PBS, labeling with FITC as previously described (16). The resulting FITC-IgEbiotin contained approximately nine fluoresceins per IgE as determined by UV-vis absorption spectroscopy. Biotinylated Leu4 was prepared similarly to biotinylated IgE (17). The multivalent antigens, 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) and 2,4-dinitrophenylated bovine gamma globulin (DNP-BGG), contained 16-18 DNP groups per BSA and 24-27 DNP groups per BGG, respectively, as determined by UV-vis spectroscopy, and were prepared as described previously (51). The amino reactive carbocyanine dye, Cy3, was conjugated to antibodies or BGG following the procedure provided by the manufacturer (Biological Detection Systems, Pittsburgh, PA). The streptavidin (sAy; Sigma Chemical Co., St. Louis, MO)-coated beads were prepared by covalently conjugating 20 Ixg streptavidin to 0.5 mL of 2.5% (wt/vo[) carboxylated microspheres (6-~m diam, carboxylated, latex-coated polystyrene; Polysciences, Inc., Warrington, PA) with a carbodiimide reaction, as previously described (22). Streptavidin-coated fluorescent beads, used for flow cytometry experiments, were prepared as above except with 6-1~m carboxylated beads that had red fluorescent dye incorporated (Polysciences Inc.). Fluorescent DNP-BGG-coated 6-i.Lm beads were prepared by conjugating 160 I~g of DNP-BGG premixed with 240 Ixg Cy3-conjugated BGG to 0.5 ml of 2.5% (wt/vol) carboxylated beads with the same procedure described above. Anti-rat lgE (IgE,~t)-coated 6-1xm beads were prepared by first conjugating 30 ~g of goat anti-mouse kappa lgG (Fisher Scientific, Pittsburgh, PA) to fluorescent carboxylated beads and then successively incubating these beads (8 × 107/ml) with 10 t~g/ml anti-rat ~ lgE mAb (A2; reference 11) and then 33 ixg/ml nonspecific mouse IgG (Cappel/ Organon Teknika, Durham, NC) for 45 min at room temperature, followed by a wash with buffered saline solution (BSS: 135 mM NaCI, 5.0 mM KCI, 1.8 mM CaCI2, 1.0 mM MgCI2, 5.6 mm glucose, 1 mg/mL BSA, and 20 mM Hepes, pH 7.4). 2,4,6-trinitrophenyl (TNP)-derivatized beads were prepared by reacting cyanogen bromide-activated 100 gm Sepharose 4B beads (Sigma Chemical Co.) with e-TNP-L-lysine hydrochloride (Research Organics Inc., Cleveland, OH). 15 g of cyanogen bromide-activated Sepharose 4B beads were swelled in 1 mM HCI for 15 min and then washed with several volumes of I mM HCI, followed by a wash with 0.1 M NaHCO3, pH 8.3.50 mg e-TNP-L-lysine hydrochloride in 75 ml of 0.1 M NaHCO3 was added to the swollen beads. After a 2 h incubation at room temperature, the beads were further incubated at 4°C overnight. The beads were then washed once with 0.2 M glycine, followed by incubation for 2 h at room temperature in 0.2 M glycine to block any remaining active sites. They were stored in 0.2 M H3BO3, 0.16 M NaCI, pH 8.5.