Membrane and cytoskeletal changes associated with IgE-mediated serotonin release from rat basophilic leukemia cells.

Abstract

Binding of antigen to IgE-receptor complexes on the surface of RBL-2H3 rat basophilic leukemia cells is the first event leading to the release of cellular serotonin, histamine, and other mediators of allergic, asthmatic, and inflammatory responses. We have used dinitrophenol-conjugated bovine serum albumin (DNP-BSA) as well as the fluorescent antigen, DNP-B-phycoerythrin, and the electron-dense antigen, DNP-BSA-gold, to investigate dynamic membrane and cytoskeletal events associated with the release of [3H]serotonin from anti-DNP-IgE-primed RBL-2H3 cells. These multivalent antigens bind rapidly to cell surface IgE-receptor complexes. Their distribution is initially uniform, but within 2 min DNP-BSAgold is found in coated pits and is subsequently internalized. Antigen internalization occurs in the presence and absence of extracellular Ca 2÷. The F-actin content of the detergent-extracted cell matrices analyzed by SDS PAGE decreases during the first 10-30 s of antigen binding and then increases by 1 min to almost double the control levels. A rapid and sustained increase is also observed when total F-actin is quantified by flow cytometry after binding of rhodaminephalloidin. The antigen-stimulated increase in F-actin coincides with (and may cause) the transformation of the cell surface from a finely microvillous to a highly folded or plicated topography. Other early membrane responses include increased cell spreading and a 2-3-fold increase in the uptake of fluorescein-dextran by fluid pinocytosis. The surface and Factin changes show the same dependence on DNP-protein concentration as stimulated [3HI serotonin release; and both the membrane responses and the release of mediators are terminated by the addition of the non-cross-linking monovalent ligand, DNP-lysine. These data indicate that the same antigen-stimulated transduction pathway controls both the membrane/cytoskeletal and secretory events. However, the membrane and actin responses to IgEreceptor cross-linking are independent of extracellular Ca 2+ and are mimicked by phorbol myristate acetate, whereas ligand-dependent mediator release depends on extracellular Ca 2+ and is mimicked by the Ca 2+ ionophore A23187.The release of histamine, serotonin, and other inflammatory mediators from mast cells and basophils is the precipitating event in a variety of acute allergic, asthmatic, and inflammatory reactions (38). This release occurs by the fusion of cytoplasmic granules with each other and with the plasma membrane, leading to the discharge of granule matrix and soluble mediators (13). Current studies of the sequence of biochemical and morphological events leading to degranulation are focused on the rat basophil-like cell line, . In this cell line degranulation is measured by the release of [3H]serotonin or histamine after cross-linking of surface IgE-receptor complexes with multivalent antigen. Biochemical studies have shown that antigen binding stimulates phosphatidylinositol turnover and generates two second messengers, diacylglycerol to activate pro...

Changes in actin network during calcium-induced exocytosis in permeabilized GH3 cells: calcium directly regulates F-actin disassembly

“…The changes in the cytoskeleton during cell stimulation have been studied using a variety of cells, such as adrenal chromaffin cells (Zhang et al 1995), mast cells (Norman et al 1994), leukemia cells (Pfeiffer et al 1985) and pancreatic acinar cells (Muallem et al 1995). F-actin network disassembly has been observed in these cells upon stimulation.…”

Multiple Defects in FcεRI Signaling in Syk-Deficient Nonreleaser Basophils and IL-3-Induced Recovery of Syk Expression and Secretion

“…Ruffling is a well-known response of RBL-2H3 mast cells to Fc⑀RI cross-linking and has been implicated in the stimulation of macropinocytosis that also occurs when these cells are activated (9,13). In contrast to releaser basophils, nonreleaser basophils do not form membrane ruffles in response to Fc⑀RI cross-linking.…”

“…In some experiments, cells were incubated in IL-3 as above. Cells were collected by centrifugation, RBC added back as described above to increase pellet volume, and pellets were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for 30 min at room temperature, rinsed with cacodylate, and processed, as described before (9). Thin sections were observed using a Hitachi 600 transmission electron microscope.…”

p110β and p110δ Phosphatidylinositol 3-Kinases Up-regulate FcεRI-activated Ca2+ Influx by Enhancing Inositol 1,4,5-Trisphosphate Production

“…In the RBL-2H3 cell line, wortmanninsensitive PI 3-kinase activity is required for the formation of large, actin-based, plasma membrane ruffles at the cell surface after Fc⑀RI cross-linking (6). Although antigen-stimulated ruffling does not require extracellular Ca 2ϩ , it is mimicked by phorbol ester treatment (24) and is thus potentially dependent on PLC␥ activation.…”