A glycolipid-specific monoclonal antibody modulates Fc receptor stimulation of mast cells

In an effort to identify membrane components participating in coupling stimulus to secretion in mast cells, monoclonal antibodies were produced from spleen cells of mice immunized with plasma membranes isolated from rat mast cells of the RBL-2H3 line. The resultant mAbs were screened by their capacity to modulate the secretory response of these cells to crosslinking of their type 1 Fc receptor (FcRI). Following this scheme, we obtained a hybridoma designated B17, which secretes an IgM-class mAb (B17) that binds to and modulates secretion from RBL-2H3 cells. By immunoblotting, B17 was shown to bind to a membrane component of low molecular weight, later identified as a glycolipid. While B17 partially inhibits IgE binding to RBL-2H3 cells, no noticeable inhibition of B17 binding by IgE was observed. mAb B17 does not cause any secretory response on its own, and its modulatory effect on FcRI-mediated secretion is bimodal: it either enhances or inhibits secretion, depending on the B17 dose and also on the nature and dose of the agent used for crosslinking the FcRI. When secretion was induced by IgE and suboptimal or optimal doses of multivalent antigen, B17 (2–80 nM) caused an increase in secretion. However, higher doses of B17 (>150nM) inhibited secretion. Secretion induced by supraoptimal doses of antigen, or by the FcRI-specific mAb F4 was inhibited by B17 at all the dose range tested (2–200 nM). In contrast, B17 had no effect on secretion induced by Ca²⁺ ionophores. These results demonstrate that FcRI function is modulated by a mAb binding to a membrane glycolipid.     

An IgE-dependent secretory response of mast cells can be induced by a glycosphingolipid-specific monoclonal antibody

The signal transduction pathway of the type 1 Fcepsilon receptor (FcepsilonRI) has been proposed to be spatially constrained to plasma membrane microdomains enriched in glycosphingolipids and cholesterol. These domains are proposed to serve as platforms that enhance the efficiency of the antigen-receptor stimulus-response coupling process. Here we describe a monoclonal antibody (mAb) designated 2B5, raised by immunizing mice with rat mucosal-type mast cell (line RBL-2H3) membranes, which binds to glycosphingolipids and causes a dose-dependent secretory response of these cells. This secretory response to mAb 2B5 requires binding of IgE to the FcepsilonRI on these cells, although direct interactions between IgE and mAb 2B5 are excluded. The bound IgE- or FcepsilonRI-specific mAb did not affect binding of mAb 2B5 or its Fab fragments to the RBL-2H3 cells and only a limited interference with the binding of IgE to the FcepsilonRI by mAb 2B5 was observed. Binding of mAb 2B5 to the RBL-2H3 cells induced a distribution of fluorescently labeled IgE similar to that produced by antigen-induced aggregation of the IgE-FcepsilonRI. Thus we suggest that mAb 2B5 binds to cell surface glycosphingolipids that are probably associated with the FcepsilonRI-IgE complexes and causes their aggregation, thereby initiating the cascade leading to the cell's secretory response.     

Cross-linking of Thy-1 glycoproteins or high-affinity IgE receptors induces mast cell activation via different mechanisms.

Rat peritoneal and pleural mast cells and rat basophilic leukemia cells, RBL-2H3, have been previously shown to be activated by Thy-1-specific monoclonal antibodies (mAb). In the present study we investigated the mechanism of Thy-1-mediated activation and compared it with activation induced by cross-linking of the high-affinity IgE receptor. Binding of an IgG Thy-1 x 1-specific mAb, MRCOX7 (OX7), to RBL-2H3 cells and mast cells, and activation of RBL-2H3 by the OX7 were abrogated by pretreatment of the cells with phosphatidyl inositol-specific phospholipase C (PI-PLC). The F(ab')2 fragment of OX7, in contrast to the Fab' fragment, induced cell activation as well as intact OX7 mAb. Cells sensitized with IgE exhibited an increased responsiveness to anti-Thy-1 antibodies suggesting formation of functional complexes of IgE receptor/IgE/Thy-1/anti-Thy-1. Pretreatment of RBL-2H3 cells with cholera toxin potentiated activation induced by IgE+antigen (Ag) and IgE+OX7, but had no effect on activation induced by OX7 antibody alone. Similarly, dexamethasone had no effect on OX7-induced activation but inhibited IgE+Ag- and IgE+OX7-induced activation. Analysis of phosphotyrosine-containing proteins in RBL-2H3 cell lysates revealed that IgE+Ag and IgE+OX7 induced a marked increase in tyrosine phosphorylation of several proteins that were not tyrosine phosphorylated in cells exposed to OX7 mAb alone. Similar results were obtained when RBL-2H3-derived cells, expressing transfected mouse Thy-1.2, were activated with Thy-1.2-specific IgM antibody. The combined data suggest that Thy-1-specific antibodies activate cells by a mechanism that is different from activation induced by cross-linking of high-affinity IgE receptor.     

Characterization of monoclonal antibodies produced by immunization with partially purified IgE receptor complexes

A series of monoclonal antibodies (mAb) were produced following the immunization of mice with partially purified IgE receptors from the rat basophilic leukemia cell line (RBL-2H3). Twelve hybridoma cell lines were selected that secreted monoclonal antibodies capable of binding RBL-2H3 plasma membranes. These antibodies were all of the IgG1, or IgG2a subclass. All 12 antibodies bound to either intact or glutaraldehyde-fixed RBL-2H3 cells. Only one monoclonal (mAb 2AC3) inhibited 125I-labeled IgE binding (IC50 = 65 micrograms/ml compared to 1.0 microgram/ml for unlabeled IgE). This same mAb weakly precipitated the alpha component of the receptor from 125I-surface-labeled cells and directly triggered histamine secretion when incubated with RBL-2H3 cells. Therefore, this hybridoma most likely represents a low affinity anti-receptor antibody. Among the other 11 monoclonals, two caused direct histamine secretion from RBL-2H3 cells. These same two, as well as four others, released greater than 10% of total cellular histamine when rabbit anti-mouse antibody was added to cross-link mAb bound to the cell surface. One monoclonal (mA 1AD3) did not trigger histamine secretion but did inhibit IgE-mediated histamine release when incubated with pre-sensitized RBL-2H3 cells. Except for mAb 2AC3, none of the other monoclonal antibodies that caused or inhibited histamine secretion immunoprecipitated receptor or other protein components from either 125I-surface-labeled or intrinsically-labeled cells. However, two monoclonal antibodies (mAb 1CC4 and 1CD1) immunoprecipitated 45,000 and 55,000 proteins from 125I-surface-labeled cells. One hybridoma (mAb 2AA2) that failed to immunoprecipitate surface-labeled proteins did precipitate a 20,000 band from intrinsically-labeled cells. This band increased slightly in apparent mol. wt after reduction and, therefore, was not the previously described gamma component of the receptor. Because several mAb were capable of modulating histamine secretion, it appeared that some of the present monoclonal antibodies bound to undefined membrane components that are crucial to the secretory process of rat basophilic leukemia cells.     

Possible interactions between the Fc epsilon receptor and a novel mast cell function-associated antigen.

We have recently described a monoclonal antibody, mAb G63, which identifies a novel membrane component of mast cells. This antigen is a glycoprotein with an apparent molecular mass of 28-40 kd, and is present on the surface of rat mucosal and serosal mast cells. Its density on cells of the mucosal mast cell line RBL-2H3 is 1 - 2 x 10(4) copies per cell. Crosslinking of this membrane protein by the intact mAb G63 results in a pronounced inhibition of the Fc epsilon RI-mediated secretion of RBL-2H3 cells. Here we show that crosslinking this novel membrane component inhibits biochemical processes initiated by Fc epsilon RI aggregation, such as the hydrolysis of phosphatidylinositides, the influx of Ca2+ ions, and the synthesis and release of de novo formed inflammatory mediators. Furthermore, by fluorescence microscopy, we show that crosslinking of Fc epsilon RI-IgE complexes by multivalent antigen results in redistribution of the membrane component recognized by G63, leading to its co-localization with the aggregated Fc epsilon RI. This localization is inhibited by NaN3, but not by colchicine or cytochalasin D. Fc epsilon RI crosslinking also promotes internalization of this novel membrane component. Taken together these data suggest that the mast cell membrane component recognized by mAb G63 is involved in the Fc epsilon RI-mediated stimulation of these cells, and thus can be considered a mast cell function-associated antigen (MAFA).     

Mast cell stimulation by monoclonal antibodies specific for the Fc epsilon receptor yields distinct responses of arachidonic acid and leukotriene C4 secretion

The release of arachidonic acid ([3H]AA) and leukotriene C4 (LTC4) from the rat mucosal mast cells of the RBL-2H3 line stimulated by Fc epsilon receptor-specific monoclonal antibodies (mAb), by IgE and multivalent antigen, or by Ca2+ ionophores, was investigated. In parallel, secretion of the granular enzyme beta-hexosaminidase was also assayed. The release of [3H]AA and LTC4 in response to stimulation by three Fc epsilon RI-specific mAb shows similar quantitative differences to those observed for the secretion of granule-stored mediators. The mAb F4 induced a substantial release of both [3H]AA and LTC4, which is as high as that induced by IgE and multivalent antigen. mAb J17 and H10 were found to induce an insignificant release of [3H]AA, but while J17 did induce release of LTC4, H10 failed to induce it, even though both J17 and H10 caused substantial release of beta-hexosaminidase. Ca2+ ionophores were found to be relatively more effective in inducing release of [3H]AA and LTC4 than in causing the secretion of granular mediators, as compared to cell stimulation by Fc epsilon RI aggregation. These results illustrate that the cell responses of degranulation and de novo synthesis and release of mediators have different sensitivities to stimulation by (a) configurationally distinct Fc epsilon RI dimers, (b) Fc epsilon RI aggregates induced by IgE and multivalent antigen, and (c) Ca2+ ionophores.     

Inflammatory signal transduction from the Fc epsilon RI to NF-kappa B

Mast cells are essential effector cells in IgE-associated immune responses. The major receptor for mast cell activation is the high affinity IgE receptor FcεRI. FcεRI crosslinking induces mast cell degranulation and de novo synthesis of potent proinflammatory mediators. Recent work identified Bcl10 and Malt1 as central regulators of a specific signaling pathway that controls NF-κB activation and proinflammatory cytokine production upon FcεRI ligation on mast cells. Bcl10 and Malt1 cooperate for the activation of this signaling cascade and selectively function downstream of PKC isoforms. However, Bcl10 and Malt1 are not involved in FcεRI- or PKC-induced signaling events that control degranulation or leukotriene synthesis. Thus, the Bcl10/Malt1 complex specifically uncouples the pathway for cytokine production from degranulation events. This review will summarize our current knowledge of the regulation of FcεRI-induced NF-κB activation in mast cells and discuss potential implications for allergic inflammatory diseases.     

银杏叶提取物对肥大细胞活化的影响

目的： 观察银杏叶提取物（EGB）对肥大细胞活化脱颗粒的影响。方法：动物实验分为6个组,分别是正常对照组、空白对照组、EGB高（30 mg/kg）、中（10 mg/kg）、低（3 mg/kg）剂量组及阳性药物对照组（氮卓斯汀,5 mg/kg）,通过大鼠被动皮肤过敏反应（PCA）实验,用比色测定法检测EGB在体内对肥大细胞(MC)影响;细胞实验分为6个组,分别是正常对照组、空白对照组、EGB高（200 mg/L）、中（100 mg/L）、低（50 mg/L）剂量组及阳性药物对照组（氮卓斯汀,30 mg/L）,然后分别将其加入抗原致敏的（DNP-HSA, 200 μg/L 培养过夜）RBL-2H3细胞中,观察EGB对RBL-2H3细胞脱颗粒、炎症介质释放及Akt、p38磷酸化的影响。结果： 动物实验显示,EGB高（30 mg/kg）、中（10 mg/kg）剂量组能明显抑制大鼠PCA。细胞实验显示,EGB高（200 mg/L）、中（100 mg/L）、低（50 mg/L）剂量组均能抑制RBL-2H3细胞脱颗粒;EGB高（200 mg/L）、中（100 mg/L）剂量组能抑制RBL-2H3细胞释放组胺、白细胞三烯C4（LTC4）、白细胞介素4（IL-4）及肿瘤坏死因子α(TNF-α);EGB高（200 mg/L）、中（100 mg/L）剂量组能抑制Akt和p38的磷酸化。结论： EGB的抗过敏作用与其能抑制肥大细胞的脱颗粒以及抑制组胺、白细胞三烯C4、肿瘤坏死因子α、白细胞介素4炎症介质释放有关;EGB抑制肥大细胞的活化可能与其抑制Akt和p38的活性相关。     

The low affinity IgG receptor Fc gamma RIIB contributes to the binding of the mast cell specific antibody, mAb BGD6

The mast cell specific monoclonal antibody, mAb BGD6, is a mast cell lineage marker [Jamur, M.C., Grodzki, A.C., Berenstein, E.H., Hamawy, M.M., Siraganian, R.P., Oliver, C., 2005. Identification and characterization of undifferentiated mast cells in mouse bone marrow. Blood 105, 4282-4289]. In rat basophilic leukemia (RBL-2H3) cells, mAb BGD6 precipitates cell-surface proteins of approximately 110 and 40-60 kDa. An expression cloning strategy was used to identify proteins that interact with mAb BGD6. A RBL-2H3 cDNA library in plasmids was transfected into PEAK cells, which do not bind mAb BGD6, and positive cells were selected with mAb BGD6. The plasmids recovered from the positive cells were amplified; retransfected into PEAK cells and after several screening cycles a positive clone was identified. This clone showed almost complete identity to Fc gamma RIIB (CD32), the low affinity IgG receptor. However, in contrast to the sequence in GenBank, this clone had an insert of 141 bp which codes for a longer isoform of this molecule with an extra 47 aa in its cytoplasmic domain. In RBL-2H3 cells both isoforms were expressed, with higher expression of the shorter form. The mechanism of binding of mAB BGD6 on both RBL-2H3 and CD32 transfected PEAK cells was then examined. Intact mAb BGD6 bound to both RBL-2H3 and CD32 expressing PEAK cells, but F(ab')(2) fragments bound only to RBL-2H3 cells demonstrating that mAb BGD6 binds to Fc gamma RIIB only through its Fc portion. On RBL-2H3 cells, the Fab of an anti-CD32 mAb partially inhibited the binding of intact mAb BGD6. The binding pattern of mAb BGD6 inhibited with anti-CD32 resembled that of the F(ab')(2) fragment of the antibody suggesting that the Fc portion of mAb BGD6 contributes to its binding on cells that have Fc gamma RIIB. These results are consistent with a model where mAb BGD6 binds through its Fab portion to a approximately 110 kDa protein and the Fc tail interacts with Fc gamma RIIB (CD32).     

Desensitization of mast cells' secretory response to an immuno-receptor stimulus

Knowledge of the desensitization process of responses to the type I receptor for IgE (FcepsilonRI) is rather limited. We investigated whether mast cells' secretory response to this receptor's stimulus can be subjected to desensitization under protocols usually employed for hormonal or neural receptors, i.e. by excessive, prolonged or repetitive exposure to the stimulus. To study this we have employed the rat mucosal-type mast cells of the RBL-2H3 line, which enables a rigorous examination of the response to the FcepsilonRI stimulus. These cells exhibited a marked decrease of both, secretion of granule-stored and de novo synthesized mediators to an optimal stimulation, when first exposed to prolonged FcepsilonRI-IgE clustering by specific antigen (DNP(11)-BSA) or by the IgE specific mAb 95.3 at concentrations that are below the threshold of inducing secretion. The extent of desensitization depended on the employed concentrations of IgE and on the clustering agents, as well as on the length of the desensitization period. The levels of cell surface FcepsilonRI expression and of cell-bound IgE were determined following the desensitization period and no significant correlation has been observed between the extent of endocytosis and the observed desensitization. Thus, a different process, which interferes with FcepsilonRI stimulus-response coupling network, is responsible for the observed desensitization.     

Correlation between cytosolic calcium concentration and degranulation in RBL-2H3 cells in the presence of various concentrations of antigen-specific IgEs

We studied the dependence of beta-hexosaminidase release from RBL-2H3 cells on the antigen-specific IgE concentrations. The cells were sensitized with DNP-specific IgE (0.5-5000 ng/ml) or OVA-specific IgE (5-50 ng/ml) and stimulated with DNP(35)-HSA (10(-2)-100 ng/ml) or OVA (10(-1) ng/ml-10 microg/ml). It was found that the beta-hexosaminidase release increased in a dose-dependent manner with the concentration of the IgEs and antigens added to the mast-cell suspension. We also studied the correlation between the intracellular calcium concentration ([Ca(2+)]i) and degranulation in RBL-2H3 cells. The percentage of beta-hexosaminidase release from the cells was well correlated with [Ca(2+)](i) increase, and the correlation coefficient was 0.88 for DNP-specific IgE and 0.99 for OVA-specific IgE. The minimum [Ca(2+)](i) required to induce the beta-hexosaminidase release was 100 nM for DNP-specific IgE, and 70 nM for OVA-specific IgE. Therefore, the [Ca(2+)](i) monitoring system is a sensitive marker of degranulation from RBL-2H3 cells and can be used to measure even low amounts of antigen-specific IgE.     

Clustering the mast cell function-associated antigen (MAFA) leads to tyrosine phosphorylation of p62Dok and SHIP and affects RBL-2H3 cell cycle

The mast cell function-associated antigen (MAFA) is a type II membranal glycoprotein expressed by rat mast cells and basophils. MAFA clustering by its specific monoclonal antibody, (mAb) G63, efficiently inhibits the FcvarepsilonRI induced secretory response of mucosal-type mast cells of the RBL-2H3 line, as well as bone marrow-derived mast cells. Here we present results which suggest that MAFA has also a capacity of modulating the cell cycle of the RBL-2H3 line. We found that MAFA clustering, by mAb G63 or by its F(ab')2 fragments, reduces the cell proliferation rate. Cell cycle analysis by flow cytometry revealed that the number of cells in sub-G phase is considerably higher for cells on which MAFA was clustered. Results of biochemical experiments established that MAFA clustering leads to a marked increase in the transient tyrosine phosphorylation of the adaptor protein p62(Dok) and the inositol phosphatase SHIP. Concomitantly, their respective binding to RasGAP and Shc was increased. Furthermore, the GTP binding protein Sos1 was found to dissociate from Shc upon MAFA clustering, suggesting that SHIP and Sos1 compete for Shc binding. We therefore suggest that MAFA has also a role in regulating RBL-2H3 cell proliferation rate by inhibiting RasGTP formation in the Ras signaling pathway.     

Thy-1-mediated activation of rat basophilic leukemia cells does not require co-expression of the high-affinity IgE receptor

The glycosylphosphatidylinositol (GPI)-anchored protein Thy-1 is one of the most abundant molecules expressed on the surface of rat mast cells and rat basophilic leukemia cells, RBL-2H3. Antibody-mediated aggregation of Thy-1 induces in these cells release of secretory components; so does aggregation of the receptor with high affinity for IgE (Fc?RI). To examine whether there is any relationship between Thy-1- and Fc?RI-mediated activation, we have isolated from mutagenized RBL-2H3 cells a variant cell line deficient in the expression of surface Fc?RI, and analyzed its ability to be activated by an antibody to Thy-1. Northern and immuno-blot analyses revealed that the variant cells were deficient in the expression of a structural or a regulatory gene for Fc?RI γ subunit. The cells did not respond by release of secretagogues and protein-tyrosine phosphorylation to IgE and antigen and anti-Fc?RI monoclonal antibody (mAb) but their response to anti-Thy-1.1 mAb and calcium ionophore A23187 was retained. Transfection of the cloned Fc?RI γ subunit into the variant cells restored the surface expression of Fc?RI and responsiveness to both the antigen and anti-Fc?RI mAb but had no effect on responsiveness to anti-Thy-1 mAb. The combined data indicate that aggregation of surface Thy-1 glycoproteins activates a metabolic pathway which is independent of the presence of Fc?RI γ subunit and surface expression of Fc?RI.     

Mast cell degranulation induced by lectins: effect on neutrophil recruitment

The mammalian lectin macrophage-derived neutrophil chemotactic factor (MNCF) and the plant lectin KM+ were characterized for their ability to activate and degranulate mast cells. The association between mast cell activation and the induction of neutrophil migration was also investigated. Incubation of rat peritoneal mast cells with these lectins resulted in degranulation and mediator release. By confocal microscopy, both lectins were evenly distributed on the cell surface. MNCF activated RBL-2H3 mast cells only if the cells had been sensitized with IgE. KM+ was able to activate either unsensitized or IgE sensitized RBL-2H3 cells. In microplate assays MNCF, but not KM+, bound to rat IgE. In rats that were depleted of mast cells, neutrophil recruitment by MNCF and KM+ were significantly reduced indicating that mast cell activation provides an amplification loop for the neutrophil recruitment induced by these lectins. The present study supports the concept that mammalian lectins play a fundamental role in innate immunity.     

β-galactosylceramide alters invariant natural killer T cell function and is effective treatment for lupus

NZB/W female mice spontaneously develop systemic lupus, an autoantibody mediated disease associated with immune complex glomerulonephritis. Natural killer (NK) T cells augment anti-dsDNA antibody secretion by NZB/W B cells in vitro, and blocking NKT cell activation in vivo with anti-CD1 mAb ameliorates lupus disease activity. In the current study, we show that β-galactosylceramide reduces the in vivo induction of serum IFN-γ and/or IL-4 by the potent NKT cell agonist α-galactosylceramide and reduces NKT cell helper activity for IgG secretion. Treatment of NZB/W mice with the β-galactosylceramide ameliorated lupus disease activity as judged by improvement in proteinuria, renal histopathology, IgG anti-dsDNA antibody formation, and survival. In conclusion, β-galactosylceramide, a glycolipid that reduces the cytokine secretion induced by a potent NKT cell agonist ameliorates lupus in NZB/W mice.     

CD45-Deficient RBL-2H3 Cells Cellular Response to FCER -and Ionophore-Induced Stimulation

The CD45 protein is a transmembrane tyrosine phosphatase that is required for normal T and B cell receptor-mediated signaling. In order to study the function of this phosphatase in mast cells, we have isolated a CD45-deficient variant from the rat basophilic leukemia cell line (RBL-2H3), a tumor analog of mucosal mast cells. The secretory response as well as the inositol 1,4,5-triphosphate (InsP₃) formation to FcRI and ionophore stimuli were similar in the RBL-2H3 cell line and its derived CD45-deficient sub-population. However, pretreatment with the phorbol ester TPA, which directly activates protein kinase C (PKC), caused a marked increase in mediator release and InsP₃ production in the CD45-deficient variant compared to the parental RBL-2H3 cells. These findings suggest that CD45 might directly or indirectly modify the activity of PKC or the InsP₃-dephosphorylating phosphatase.