Membrane and cytoskeletal changes associated with IgE-mediated serotonin release from rat basophilic leukemia cells.
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-BSA-gold is found in coated pits and is subsequently internalized. Antigen internalization occurs in the presence and absence of extracellular Ca2+. 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 rhodamine-phalloidin. 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 F-actin changes show the same dependence on DNP-protein concentration as stimulated [3H]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 IgE-receptor cross-linking are independent of extracellular Ca2+ and are mimicked by phorbol myristate acetate, whereas ligand-dependent mediator release depends on extracellular Ca2+ and is mimicked by the Ca2+ ionophore A23187.
Pfeiffer
J R
JR
Seagrave
J C
JC
Davis
B H
BH
Deanin
G G
GG
Oliver
J M
JM
D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism; D000818:Animals; D001491:Basophils / Q000378:metabolism* / Q000648:ultrastructure; D002448:Cell Adhesion; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D003599:Cytoskeleton / Q000502:physiology / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D007073:Immunoglobulin E; D007700:Kinetics; D007938:Leukemia; D008560:Membrane Fluidity; D008854:Microscopy, Electron; D010873:Pinocytosis; D051381:Rats; D011961:Receptors, Fc / Q000378:metabolism*; D017455:Receptors, IgE; D011971:Receptors, Immunologic / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism*
D000199:Actins; D011961:Receptors, Fc; D017455:Receptors, IgE; D011971:Receptors, Immunologic; D012701:Serotonin; D007073:Immunoglobulin E