THE INTERACTION OF IgE WITH RAT BASOPHILIC LEUKEMIA CELLS

Kulczycki, Anthony; Metzger, Henry

doi:10.1084/jem.140.6.1676

Cited by 306 publications

(117 citation statements)

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“…When cells that had been incubated with any of the labeled fractions were then exposed to an excess of unlabeled IgE, no significant release of cell-bound radioactivity occurred over several hours. This indicates that the bound IgE dissociated very slowly (5).…”

Section: Characterization Of Ige Oligomersmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

“…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).…”

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confidence: 99%

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

Segal

Taurog

Metzger

1977

Proc. Natl. Acad. Sci. U.S.A.

325

153

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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...

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Section: Characterization Of Ige Oligomersmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Segal

Taurog

Metzger

1977

Proc. Natl. Acad. Sci. U.S.A.

325

153

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“…less than tenfold that of FceRI (3Â10 5 -5Â10 5 ) [10,11]. Therefore the question arose how this comparatively small number of MAFA molecules is capable of inhibiting the cells' response to the FceRI stimulus and was rationalized by the assumption that the MAFA recruited enzymes have the capacity of amplifying the inhibitory signal.…”

Section: Introductionmentioning

confidence: 99%

Regulation of mast cells’ secretory response by co‐clustering the Type 1 Fcϵ receptor with the mast cell function‐associated antigen

2005

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The mast cell function-associated antigen (MAFA) is a type II membrane glycoprotein first identified on rat mast cells and basophils. Clustering MAFA inhibits these cells' secretory response to the type 1 Fce receptor (FceRI) stimulus. To quantitatively characterize this inhibition and its dependence on MAFA-FceRI co-clustering, we investigated the secretory response of rat mucosal-type mast cells of the RBL 2H3 line carrying an IgE class, 2,4 dinitrophenyl (DNP) specific monoclonal antibody to DNPconjugated Fab and F(ab 0 ) 2 fragments of (1) mouse IgG, and (2) of the MAFA-specific, monoclonal antibody G63. The first reagent clusters FceRI-IgE complexes into oligomers by reacting with the DNP residues. The DNP conjugated G63 Fab and F(ab 0 ) 2 fragments, additionally aggregate MAFA and form FceRI-IgE-MAFA co-clusters. All experiments using these ligands were performed in the absence or presence of an excess of intact mAb G63, which clusters MAFA molecules. Empirical Hill functions were used to relate the secretory response of mast cells to the equilibrium concentrations of FceRI-IgE or MAFA clusters and co-clusters calculated as function of the employed ligands concentrations. This analysis of the experimental results indicates that coclustered MAFA molecules have a markedly higher inhibitory capacity than MAFAclusters alone. The molecular basis of the enhanced inhibition observed upon coclustering MAFA with the FceRI is most probably the increased concentration of the inhibitory cell components in the immediate proximity of the activation coupling elements.

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“…It is well known that IgE molecules bind with high affinity to the Fc receptors on these cells (1,2). The bridging of the cell-bound IgE molecules with multivalent ligand initiates a cascade of biochemical events (3) resulting in the release of a variety of preformed and newly generated chemical mediators (4).…”

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confidence: 99%

Electron Microscopic Studies on the Inhibition of Degranulation of Rat Mast Cells by a Novel Anti-Allergic Agent, PTPC

Ohashi

Watanabe

Nishigaki

et al. 1994

Japanese Journal of Pharmacology

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ABSTRACT-When rat mast cells sensitized by IgE antibody were exposed to antigen, transmission elec tron microscopy revealed alteration of the granules, cavity formation by fusion of the perigranular mem brane and granule release by the fusion of the cavity membrane with the mast cell membrane. Scanning elec tron microscopy disclosed the extrusion of smooth and round bodies from pores formed on the cell surface. These changes were accompanied by the release of histamine. The inhibition of this degranulation by a novel anti-allergic agent, 6-(1-pyrrolidinyl)-N-(1H-tetrazol-5-yl)-2-pyrazinecarboxamide (PTPC), was evalu ated quantitatively as an inhibition of the granule alteration and cavity formation. At a concentration of 100 nM, PTPC inhibited the granule alteration and cavity formation as well as histamine release. In the same concentration, PTPC significantly increased the cyclic AMP content in the mast cells. These results sug gest that the inhibition of the morphological changes in mast cells by PTPC might be due to the increased cyclic AMP caused by the agent and plays an important role in the suppression of chemical mediators re lease.

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THE INTERACTION OF IgE WITH RAT BASOPHILIC LEUKEMIA CELLS

Cited by 306 publications

References 10 publications

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

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

Regulation of mast cells’ secretory response by co‐clustering the Type 1 Fcϵ receptor with the mast cell function‐associated antigen

Electron Microscopic Studies on the Inhibition of Degranulation of Rat Mast Cells by a Novel Anti-Allergic Agent, PTPC

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