Involvement of filamentous actin in setting the threshold for degranulation in mast cells

Previous studies using cytochalasins and latrunculin B, inhibitors of actin polymerization, showed that filamentous (F)-actin had a negative regulatory role in Fc epsilon receptor I (Fc epsilon RI) signaling. How F-actin is involved in regulating the activation of mast cells is unknown. In this study we investigated the role of F-actin in mast cell activation induced by aggregation of the glycosylphosphatidylinositol (GPI)-anchored proteins Thy-1 and TEC-21, and compared it to activation via Fc epsilon RI. Pretreatment of rat basophilic leukemia cells with latrunculin B inhibited the Thy-1-induced actin polymerization and elevated the Thy-1-mediated secretory and calcium responses. Inhibition of actin polymerization followed by Thy-1 aggregation resulted in an increased tyrosine phosphorylation of Syk, phospholipase C gamma (PLC gamma), Gab2 and linker for activation of T cells (LAT) adapters, and some other signaling molecules. Enzymatic activities of phosphatidylinositol 3-kinase, PLC gamma, and phosphatase SHP-2 were also up-regulated, but tyrosine phosphorylation of ezrin was inhibited. Similar changes were observed in Fc epsilon RI-activated cells. Significant changes in intracellular distribution, tyrosine phosphorylation, and/or enzymatic activities of signaling molecules occurred in latrunculin-pretreated cells before cell triggering. The combined data suggest that actin polymerization is critical for setting the thresholds for mast cell signaling via aggregation of both Fc epsilon RI and GPI-anchored proteins.     

Src family-selective tyrosine kinase inhibitor,PP1, inhibits both FcεRI- and Thy-1-mediated activation of rat basophilic leukemia cells

Cross-linking of the surface receptor with high affinity for IgE (FcεRI) by multivalent antigen/immunoglobulin E complexes, as well as aggregation of Thy-1 glycoprotein by monoclonal antibodies lead in rat basophilic leukemia cells, clone RBL-2H3, to tyrosine phosphorylation of several cellular proteins, followed by a release of secretory components. To investigate the molecular mechanisms of FcεRI- and Thy-1-mediated transmembrane signaling and to map a step at which they converge into a common secretory pathway, we used a novel Src family-selective tyrosine kinase inhibitory, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), and analyzed its inhibitory activity on cell activation. Here we show that in RBL-2H3 cells PP1 demonstrates substrate specificity for a Src family kinase Lyn. In immunocomplex kinase assays in vitro, PP1 inhibited the Lyn kinase activity at nanomolar levels without any effect on Syk kinase activity. However, in RBL cells activated via aggregation of FcεRI, phosphorylation of both Syk and Lyn kinases was inhibited. FcεRI- and Thy-1-mediated early (protein-tyrosine phosphorylation) and late (release of β-hexosaminidase) activation events were similarly affected by PP1. The inhibition was specific for membrane receptor-mediated signaling and was not observed in cells activated by an exposure to pervanadate. The combined data suggest that activation of Lyn is the early activation step at which the FcεRI- and Thy-1-mediated activation pathways of mast cells and basophils may converge.     

Tyrosine phosphorylation of phospholipase C gamma 1 couples the Fc epsilon receptor mediated signal to mast cells secretion.

Mast cells respond to clustering of the type I Fc epsilon receptor (Fc epsilon RI) on their membranes by mediator secretion. Recently, a marked enhancement of tyrosine phosphorylation on several proteins has been observed as a result of antigen-induced Fc epsilon RI aggregation on these cells. We report here that the phosphatidyl inositide specific phospholipase C gamma 1 (PLC gamma 1) is one of the prime proteins that undergoes tyrosine phosphorylation as a result of this stimulus. This was determined by immunoprecipitation of phosphotyrosine containing proteins from detergent lysates of rat mucosal mast cells (rat basophilic leukemia cells, subline 2H3; RBL-2H3) and Western blotting analysis of the separated components. A fast appearance of phosphorylated tyrosine residues on PLC gamma 1 was observed, reaching its maximal intensity at approximately 1-3 min after stimulation and declined afterwards to basal levels. Moreover, the phosphorylation depended on maintaining the aggregated Fc epsilon RI as did other cellular responses (e.g. phosphatidyl inositides hydrolysis and secretion). The time course of both Fc epsilon RI induced phospholipase C gamma 1 activation, as monitored by the formation of inositol phosphates, and of the secretory response of the cells followed that of the PLC gamma 1 phosphorylation. Furthermore, the tyrphostin AG490, a protein tyrosine kinase inhibitor, caused similar inhibition of the Fc epsilon RI-induced PLC gamma 1 phosphorylation, inositol phosphates formation, and mediator secretion. Significantly, no tyrosine phosphorylation of PLC gamma 1 was induced by the Ca2+ ionophore, ionomycin, even at doses that cause optimal secretory response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Negative regulation of Lyn protein-tyrosine kinase by c-Cbl ubiquitin-protein ligase in Fc epsilon RI-mediated mast cell activation

BACKGROUND: Recent studies have demonstrated that c-Cbl functions as a ubiquitin-protein ligase toward immune receptors and non-receptor protein-tyrosine kinase Syk by facilitating their ubiquitination and subsequent targeting to proteasomes. However, it was not clear whether Src family kinase Lyn is regulated by the Cbl family of ubiquitin-protein ligases. RESULTS: Aggregation of the high affinity IgE receptor (Fc epsilon RI) induces the rapid ubiquitination of Lyn in rat basophilic leukaemia RBL-2H3 cells. Treatment of cells with a proteasome inhibitor enhances the ubiquitination of Lyn. Stimulation of Fc epsilon RI results in the association of Lyn with c-Cbl and Cbl-b, both of which then become tyrosine phosphorylated. Co-transfection study shows that both c-Cbl and Cbl-b could induce the ubiquitination of activated Lyn in COS cells. Furthermore, over-expression of membrane-anchored form of c-Cbl inhibits the Fc epsilon RI-mediated degranulation and cytokine gene production in RBL-2H3 cells by the down-regulation of the kinase activity of Lyn through the enhanced ubiquitination. CONCLUSIONS: These results demonstrate that Lyn is down-regulated by c-Cbl-mediated ubiquitination and subsequent degradation in proteasome after Fc epsilon RI stimulation in mast cells. Targeting of c-Cbl in the lipid raft results in the inhibition of Fc epsilon RI-mediated mast cell activation.     

Comparison of Fc epsilon RI- and Fc gamma RI-mediated degranulation and TNF-alpha synthesis in human mast cells: selective utilization of phosphatidylinositol-3-kinase for Fc gamma RI-induced degranulation

We have demonstrated that CD34(+) IFN-gamma-treated human mast cells (HuMC) express functional Fc gamma RI and that aggregation of these receptors leads to mediator release. As the signaling pathways linking Fc gamma RI aggregation to mediator release are unknown, we examined Fc gamma RI-dependent activation of specific signal transduction molecules and determined the relative involvement of these events in HuMC degranulation and TNF-alpha production following both Fc gamma RI and Fc epsilon RI aggregation. Fc gamma RI aggregation resulted in the phosphorylation/activation of src kinases and p72(syk) and subsequent tyrosine phosphorylation of multiple substrates. Inhibitor studies revealed that these responses were required for degranulation and TNF-alpha synthesis. Both Fc gamma RI and Fc epsilon RI aggregation also activated the MAP kinases ERK 1/2, JNK and p38 and this was necessary for TNF-alpha synthesis, but not degranulation for both receptors. Thus, signaling events in HuMC following aggregation of Fc gamma RI were generally similar to those observed following Fc epsilon RI aggregation. The one exception was that, although phosphatidylinositol-3-kinase was activated after both Fc epsilon RI and Fc gamma RI aggregation, only the Fc gamma RI appeared to require this molecule for degranulation.     

The leukocyte receptor CD84 inhibits Fc epsilon RI-mediated signaling through homophilic interaction in transfected RBL-2H3 cells

Signaling through the high-affinity receptor for immunoglobulin E (Fc epsilon RI) results in the coordinated activation of tyrosine kinases, thus leading to calcium mobilization, degranulation, and leukotriene and cytokine synthesis. Here, we show that CD84, a member of the CD150 family of leukocyte receptors, inhibits Fc epsilon RI-mediated mast cell degranulation in CD84-transfected rat basophilic leukaemia-2H3 mast cell line cells (RBL-2H3) through homophilic interaction. There was no reduction in overall protein phosphorylation following IgE triggering in CD84 RBL-2H3 cells. Indeed, phosphorylation of Dok-1 and c-Cbl increased in CD84 RBL-2H3, suggesting that inhibition is mediated by these molecules. MAP kinase phosphorylation (ERK1/2, JNK and p38) and cytokine synthesis were impaired in CD84 RBL-2H3. This inhibitory mechanism was independent of SAP and SHP-2 recruitment. Interestingly, CD84 mutants in tyrosines (Y279F and DeltaY324) reversed this inhibitory profile. These data suggest that CD84 may play a role in modulating Fc epsilon RI-mediated signaling in mast cells. Thus, CD84 could play a protective role against undesired allergic and inflammatory responses.     

The effect of a naphthalene derivative,TEI-6472, on histamine release and tyrosine phosphorylation in rat basophilic leukemia cells

It is well known that rat basophilic leukemia cells (RBL-2H3) express high-affinity IgE receptors (FcεRI) and that the aggregation of these receptors causes the release of chemical mediators. When RBL-2H3 cells are sensitized with IgE antibody and subsequently stimulated by an antigen, significant histamine release and the tyrosine phosphorylation of several proteins are observed. In this study, we examined the effects of a synthetic naphthalene derivative, (7E)-N-(2-carboxyphenyl)-8-(2-naphthyl)-5,6-trans-5,6-methano-7-octenamide (TEI-6472), on the FceRI-mediated histamine release from RBL-2H3 cells. Preincubation for 10 min with 100 μM TEI-6472 caused significant inhibition of FceRI-mediated histamine release from RBL-2H3 cells. Furthermore, Western blotting analysis using anti-phosphotyrosine antibody showed that FcεRI-mediated tyrosine phosphorylation of 78 and 92 kDa proteins in RBL-2H3 cells was also significantly inhibited. Tyrosine phosphorylation of these 78 and 92 kDa proteins was not induced by direct activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) and the calcium ionophore A23187. However, the inhibition of histamine release from TEI-6472-treated RBL-2H3 cells was restored by direct activation of PKC. Taken together, these results suggest that tyrosine phosphorylation of the 78 and 92 kDa proteins in RBL-2H3 cells is involved in a signal transduction system for histamine secretion, and that these tyrosine phosphorylations may occur upstream of PKC activation.     

Nonreceptor protein tyrosine and lipid phosphatases in type I fc(epsilon) receptor-mediated activation of mast cells and basophils

Protein tyrosine and lipid phosphorylations are early and critical events in type 1 Fc(epsilon) receptor (Fc(epsilon)RI)-mediated activation of mast cells and basophils. Tyrosine phosphorylation of Fc(epsilon)RI subunits as well as other signal transduction molecules reflects the balance between the action of protein tyrosine kinases and phosphatases. Similarly, the phosphate content of inositol phospholipids, involved in the recruitment of signalling molecules to the plasma membrane and the generation of secondary messengers, is the net result of the opposing effects of phosphoinositide kinases and lipid phosphatases. This review summarizes the current understanding of the structural and functional aspects of nonreceptor protein tyrosine phosphatases (SHP-1, SHP-2, HePTP, PTP20, PRL1, PRL2, PTP-MEG1 and PTP-MEG2) and lipid phosphatases (SHIP and SHIP2) in the activation of mast cells and basophils after Fc(epsilon)RI aggregation. New approaches towards a deeper understanding of the role of phosphatases in mast cell physiology are also discussed.     

Protein tyrosine kinase Syk is involved in Thy-1 signaling in rat basophilic leukemia cells

Thy-1, a glycosyl-phosphatidylinositol-anchored surface glycoprotein, has been shown to possess transmembrane signaling capacity. In rat mast cells and rat basophilic leukemia cells (RBL) aggregation of surface Thy-1 with antibodies triggers a series of intracellular events, resembling those induced by aggregation of the high-affinity receptor for IgE (FcεRI), including tyrosine phosphorylation of multiple proteins and release of secretory components. Unlike the FcεRI-mediated activation, where both the membrane-associated protein tyrosine kinase (PTK) Lyn and the cytoplasmic PTK Syk are responsible for initiating the signaling cascade, only Lyn has been implicated in Thy-1-mediated activation in RBL cells. Here we report that Syk is also rapidly tyrosine phosphorylated upon Thy-1 cross-linking. Increased Syk tyrosine phosphorylation is observed only in cells in which extensive aggregation of Thy-1 is induced by two layers of cross-linking reagents. RBL-derived mutant cells deficient in the expression of surface Thy-1 and transfectants re-expressing surface Thy-1 were used to exclude the possibility that Syk activation reflects an interaction of the cross-linking reagents with surface molecules other than Thy-1. As FcεRI γ subunits are well known to promote activation of Syk and its recruitment to membrane complexes, we also investigated the role of these subunits in Thy-1-mediated Syk activation, using RBL-derived mutant cells deficient in the expression of FcεRI γ subunits and their revertants. Consistent with the lack of FcεRI expression, no IgE-induced response could be elicited, while Thy-1-inducible Syk phosphorylation was preserved. Our results suggest that Syk might be one of the kinases responsible for signal propagation upon Thy-1 cross-linking in a FcεRI-independent pathway.     

Positive and negative regulation of Fc epsilon receptor I-mediated signaling events by Lyn kinase C-terminal tyrosine phosphorylation

Although it is known that the Src family tyrosine kinase Lyn initiates Fc epsilon receptor I (Fc epsilon RI) signaling by phosphorylation of the receptor subunits, regulation of Lyn kinase activity and its consequences for receptor signaling are incompletely understood. Using a phospho-Lyn-specific antiserum, we show an increased phosphorylation of the Lyn C-terminal regulatory tyrosine and decreased Lyn kinase activity during Fc epsilon RI-mediated mast cell activation. Mutant Lyn, defective in the C-terminal tyrosine, constitutively phosphorylated several substrates in resting cells, but did not cause Fc epsilon RI internalization or spontaneous degranulation. Fc epsilon RI-induced signaling in the presence of constitutively active Lyn exhibited enhanced phosphorylation of the receptor subunits, Syk, LAT, Gab2, phospholipase C (PLC)gamma 1 and PLC gamma 2, and production of phosphatidylinositol 3,4,5-trisphosphate. Although enzymatic activities of PLC gamma 1 and PLC gamma 2 were also up-regulated, amounts of inositol 1,4,5-trisphosphate, mobilization of intracellular calcium and degranulation were suppressed. Additionally, constitutively active Lyn was strikingly less efficient than wild-type Lyn in restoring the receptor-mediated calcium responses in bone marrow mast cells derived from Lyn(-/-) mice. These findings pinpoint the tight regulation of Lyn kinase activity as a critical event in mast cell degranulation.     

Protein-tyrosine phosphorylation: an essential component of Fc epsilon RI signaling.

Mast cell and basophil activation can be achieved by antigen-mediated aggregation of cell surface Fc epsilon RI molecules. At least two signaling pathways are triggered by this activation. Both involve tyrosine phosphorylation. This aspect of Fc epsilon RI signaling is examined here in detail and its position in a complex network of post-binding events assessed.     

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.     

The characterization of tyrosine phosphorylation of 78 and 92 kDa proteins in rat basophilic RBL-2H3 cells

Previously, we have demonstrated that tyrosine phosphorylation of 78 and 92 kDa proteins in rat basophilic leukemia cells (RBL-2H3) is involved in a signal transduction system for high-affinity IgE receptor (FcRI)-mediated histamine secretion. However, it is not clarified whether the tyrosine phosphorylation of 78 and 92 kDa proteins in RBL-2H3 cells is regulated by activation of protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI3-kinase). In this study, therefore, the effect of depletion of PKC in RBL-2H3 cells, or the influence of PKC, PI3-kinase and tyrosine kinase inhibitors on histamine release from RBL-2H3 cells was examined. The elimination of PKC in RBL-2H3 cells induced significant suppression of histamine release, although the tyrosine phosphorylation of 78 and 92 kDa proteins was not inhibited. The inhibition of histamine release was also observed by the treatment with a PKC inhibitor such as H-7, calphostin C, a PI3-kinase inhibitor such as wortmannin or a tyrosine kinase inhibitor such as ST638, genistein, hervimycin A, although the tyrosine phosphorylation of both proteins was inhibited by only ST638. These results suggest that the 78 kDa protein in RBL-2H3 cells is not identical to the protein-tyrosine kinase PTK72 and the tyrosine phosphorylation of 78 and 92 kDa proteins in RBL-2H3 cells occurs upstream of PKC and PI3-kinase activation or is regulated independently of the PKC- and PI3-kinase-dependent signaling pathway.accepted by M. Katori     

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).     

Association of 3BP2 with SHP-1 regulates SHP-1-mediated production of TNF-α in RBL-2H3 cells

Adaptor protein 3BP2, a c-Abl Src homology 3 (SH3) domain-binding protein, is tyrosine phosphorylated and positively regulates mast cell signal transduction after the aggregation of the high affinity IgE receptor (FcεRI). Overexpression of the Src homology 2 (SH2) domain of 3BP2 results in the dramatic suppression of antigen-induced degranulation in rat basophilic leukemia RBL-2H3 cells. Previously, a linker for activation of T cells (LAT) was identified as one of the 3BP2 SH2 domain-binding protein. In this report, to further understand the functions of 3BP2 in FcεRI-mediated activation of mast cell, we explored the protein that associates with the SH2 domain of 3BP2 and found that SH2 domain-containing phosphatase-1 (SHP-1) inducibly interacts with the SH2 domain of 3BP2 after the aggregation of FcεRI. The phosphorylation of Tyr(564) in the carboxy (C)-terminal tail region of SHP-1 is required for the direct interaction of SHP-1 to the SH2 domain of 3BP2. The expression of the mutant form of SHP-1 which was unable to interact with 3BP2 resulted in the significant reduction in SHP-1-mediated tumor necrosis factor-α (TNF-α) production without any effects on the degranulation in antigen-stimulated RBL-2H3 cells. These findings suggest that 3BP2 directly interacts with Tyr(564) -phosphorylated form of SHP-1 and positively regulates the function of SHP-1 in FcεRI-mediated signaling in mast cells.     

Lipid rafts in mast cell signaling

Lipid rafts are defined as plasma membrane microdomains enriched with glycosphingolipids and cholesterol which render them insoluble in non-ionic detergents. Many surface receptors are constitutively or inducibly associated with lipid rafts, and it has been suggested that the rafts function as platforms regulating the induction of signaling pathways. The signaling capacity of lipid rafts has been extensively studied in rat basophilic leukemia cells. An aggregation of lipid raft components, such as glycosylphosphatidylinositol (GPI)-anchored glycoproteins (Thy-1 or TEC-21), triggers cell activation events which are similar to, but not identical with activation via the high-affinity IgE receptor (FcepsilonRI). Although FcepsilonRI in resting cells is not associated with lipid rafts, its aggregation induces a weak association with rafts and subsequent activation events. The properties of lipid rafts as well as the molecular mechanisms of their involvement in signal transduction are poorly understood. This review presents a critical analysis of recent results on structure-function relationship of lipid rafts and their regulatory role in signal transduction in mast cells.     

Proximal signaling events in Fc epsilon RI-mediated mast cell activation

Mast cells are central mediators of allergic diseases. Their involvement in allergic reactions is largely dependent on activation through the specific receptor for IgE (Fc epsilon RI). Cross-linking of Fc epsilon RI on mast cells initiates a cascade of signaling events that eventually results in degranulation, cytokine/chemokine production, and leukotriene release, contributing to allergic symptomology. Because of the importance of IgE in allergy, much focus has been placed on deciphering the signaling events that take place downstream of Fc epsilon RI. Studies have identified spleen tyrosine kinase as a key proximal regulator of Fc epsilon RI-mediated signaling. In this review, we discuss the multiple pathways that diverge from spleen tyrosine kinase with emphasis on the role of adapter molecules to orchestrate these signaling events. Understanding the molecular mechanisms underlying mast cell activation ideally will provide insights into the development of novel therapeutics to control allergic disease.     

Isolation and characterization of IgE receptors from rat intestinal mucosal mast cells

High-(Fc epsilon RI) and low-(Fc epsilon RII) affinity IgE receptors were isolated from surface radioiodinated, Nonidet-P40-solubilized rat intestinal mucosal mast cells (IMMC) and compared with those on rat peritoneal mast cells (PMC) and rat basophilic leukemia (RBL) cells. Fc epsilon RII were isolated by affinity chromatography using IgE-Sepharose or by anti-Fc epsilon RII antisera and protein A-Sepharose. The surface-exposed, IgE-binding alpha subunits of Fc epsilon RI [Fc epsilon RI alpha] were isolated by affinity chromatography using IgE and anti-IgE-Sepharose. Fc epsilon RI alpha on IMMC had an apparent molecular mass of 59 kDa, somewhat larger than that of PMC (51 kDa), RBL-2H3 cells (51 kDa) or RBL-CA10.7 cells (46 kDa). Brief (45 s) incubation of IMMC or PMC in glycine-HCl, pH 3, prior to iodination removed much of the surface-bound IgE. This permitted more thorough labeling of the receptors, but had no affect on the estimate of receptor size. Surprisingly and in contrast to acid-treated PMC, upon anti-IgE-Sepharose isolation acid-treated IMMC yielded an intensely radioactive Fc epsilon RI alpha band in the absence of added IgE. Such a finding suggests that IMMC, more so than PMC, may have an intracellular store of IgE, as has been suggested by many others. IMMC also differed from PMC in the number of forms of Fc epsilon RII isolated; 50-kDa and 58-kDa forms of Fc epsilon RII were obtained from IMMC, whereas PMC yielded most often a single 56-kDa Fc epsilon RII band. These results were mimicked by the two RBL cell sublines: RBL-2H3 cells yielded two Fc epsilon RII (46 kDa and 55 kDa), but only one form of Fc epsilon RII (54-kDa) was obtained from RBL-CA10.7 cells. Thus, the two subtypes of rat mast cells, which have previously been shown to differ in mediator profile and responsiveness to secretagogues and antiallergic drugs, are also distinguished by differences in IgER profile.     

RabGEF1 is a negative regulator of mast cell activation and skin inflammation

Mast cell activation induced by aggregation of Fc epsilon RI receptors with immunoglobulin E and antigen is mediated through the activation of multiple protein kinase cascades. Here we report that the regulatory protein RabGEF1 bound to Ras and negatively regulated Ras activation and its 'downstream' effector pathways in Fc epsilon RI-dependent mast cell activation. RabGEF1-deficient mast cells showed enhanced degranulation and release of lipid mediators and cytokines in response to Fc epsilon RI aggregation. RabGEF1-deficient mice developed severe skin inflammation and had increased numbers of mast cells. Thus, RabGEF1 is a negative regulator of Fc epsilon RI-dependent mast cell activation, and a lack of RabGEF1 results in the development of skin inflammation in vivo.