Dimeric immunoglobulin E serves as a unit signal for mast cell degranulation.

Abstract

Rat immunoglobulin E (IgE) was treated with a crosslinking reagent, dimethyl suberimidate, and fractionated by gel filtration into monomers, dimers, trimers, and higher polymers. The fractions retained substantial ability to bind specifically to mast cells. About one-third of the cell-bound dimers appeared to bind bivalently. The fractions were assayed in vivo by passive cutaneous anaphylaxis in rats, and for histamine or serotonin release in vitro using normal or The purpose of the present study was to determine the minimal degree of crosslinking required. Toward this end, we treated IgE with the crosslinking reagent dimethyl suberimidate and separated the mixture into monomers, dimers, trimers, and higher polymers. We found that preformed dimers were capable of triggering exocytosis when added to mast cells.MATERIALS AND METHODS Materials. Rat IgE was isolated from the ascitic fluid of Lou/M rats carrying the immunocytoma IR162 (4) by methods previously described (5). It was iodinated with 1311 or 125I by the chloramine-T method (6). The preparations of fluoresceinated IgE and rabbit antibody against fluorescein have also been described (7). Rat basophilic leukemia cells (8) were grown to the stationary stage of growth (9). Unfractionated mouse peritoneal cells were used as a source of normal mast cells. For a few experiments, cells from the mouse mastocytoma AB-CBFI-MCT-1 (10) were used.Preparation of IgE Oligomers. IgE (27 mg/ml, 52 mg) was covalently crosslinked in 0.2 M Tris.HCl, pH 8.5, using a 30-fold molar excess (2.1 mg) of dimethyl suberimidate (Pierce Chemicals). The solution was incubated for 2 hr at 300, and the Abbreviations: IgE, immunoglobulin E; PCA, passive cutaneous anaphylaxis.oligomers were separated by fractionation on sequential Sephadex 0-200 (Pharmacia) and Ultrogel AcA22 (LKB) columns as described (11). Elution was carried out in 0.15 M NaCl/0.01 M Tris-HCl, pH 7.5.Oligomers that were trace labeled with 1251 were prepared from the radio-labeled native protein. In this case, approximately 3 mg of l25-Ilabeled IgE in 1 ml of phosphate-buffered saline was precipitated by addition of an equal volume of 30% polyethylene glycol 6000. After centrifugation the precipitate was redissolved in 0.25 ml of 0.2 M Tris-HCl, pH 8.5, and a 30-fold molar excess of dimethyl suberimidate in 30,ul of H20 was added. After 2 hr at 300, the oligomers were fractionated as described above.Fractions were analyzed by polyacrylamide gel electrophoresis using a Pharmacia GE-4 gel electrophoresis apparatus and PAA 2/16 gradient slab gels. Electrophoresis was carried out for 8 hr in Tris-borate buffer, pH 8.4, at 200. Gels were stained with 0.05% Coomassie blue in aqueous 25% isopropanol/10% acetic acid, and were destained in 10% acetic acid.Binding Studies. The binding of derivatized IgE was studied indirectly by its capacity to inhibit the binding of untreated radioiodinated IgE in preliminary tests, but principally by direct studies using iodinated derivatized IgE (5).Assays In Vivo. The capacity of deri...

The exogenous lipid requirement for histamine release from rat peritoneal mast cells stimulated by concanavalin A

“…The mechanism of stimulation involves the crosslinking of the IgE molecules bound to F, receptors on the cell surface [3]. Dimerisation of the receptors appears to generate a sufficient signal, since stimulation by dimeric IgE is as effective as polymeric forms of the antibody [4]. The stimulation of the cells in vitro occurs with a wide range of agents which interact with the plasma membrane, including the plant lectin concanavalin A (con A) [5,6].…”

Negative Signaling in Fc Receptor Complexes

“…Dimerization was, long ago, shown to be the minimal degree of FcεRI aggregation capable of generating activation signals sufficient for triggering mediator release by mast cells (Siraganian et al, 1975); (Segal et al, 1977). Intracellular signals are generated within juxtamembrane signaling complexes that assemble under FcR aggregates and form signalosomes.…”

Intramembrane particles and the organization of lymphocyte membrane proteins

“…Apparently, the majority of E-face IMP and virtually all P-face IMP do not present accessible antigenic sites on the lymphocyte surface and do not associate in a stable manner with surface protein antigens . This finding suggests that IMP, as observed in freeze-fracture analysis, may not comprise a representative reflection of lymphocyte transmembrane protein molecules and complexes because other evidence establishes : (a) that at least some common lymphocyte surface antigens are indeed exposed portions of transmembrane proteins and (b) that the aggregation of molecules of any surface antigen results in altered organization of contractile proteins at the cytoplasmic face of the membrane .The role of the plasma membrane in the transmission of information can be seen in a number of systems where cells have been shown to be activated by an event at the cell membrane leading to proliferation and/or differentiation (5,8,23,26). Although the mechanism of lymphocyte activation is not known, there is evidence to suggest that the binding of insolubilized antigen or mitogen to membrane receptors may, under some conditions, be sufficient to initiate the cellular activation process (8) .…”

“…The role of the plasma membrane in the transmission of information can be seen in a number of systems where cells have been shown to be activated by an event at the cell membrane leading to proliferation and/or differentiation (5,8,23,26). Although the mechanism of lymphocyte activation is not known, there is evidence to suggest that the binding of insolubilized antigen or mitogen to membrane receptors may, under some conditions, be sufficient to initiate the cellular activation process (8) .…”