Targeting Janus kinase 3 in mast cells prevents immediate hypersensitivity reactions and anaphylaxis.

Janus kinase 3 (JAK3), a member of the Janus family protein-tyrosine kinases, is expressed in mast cells, and its enzymatic activity is enhanced by IgE receptor/FcepsilonRI cross-linking. Selective inhibition of JAK3 in mast cells with 4-(4'-hydroxylphenyl)-amino-6, 7-dimethoxyquinazoline) (WHI-P131) blocked the phospholipase C activation, calcium mobilization, and activation of microtubule-associated protein kinase after lgE receptor/FcepsilonRI cross-linking. Treatment of IgE-sensitized rodent as well as human mast cells with WHI-P131 effectively inhibited the activation-associated morphological changes, degranulation, and proinflammatory mediator release after specific antigen challenge without affecting the functional integrity of the distal secretory machinery. In vivo administration of the JAK3 inhibitor WHI-P131 prevented mast cell degranulation and development of cutaneous as well as systemic fatal anaphylaxis in mice at nontoxic dose levels. Thus, JAK3 plays a pivotal role in IgE receptor/FcepsilonRI-mediated mast cell responses, and targeting JAK3 with a specific inhibitor, such as WHI-P131, may provide the basis for new and effective treatment as well as prevention programs for mast cell-mediated allergic reactions.     

Co-aggregation of FcgammaRII with FcepsilonRI on human mast cells inhibits antigen-induced secretion and involves SHIP-Grb2-Dok complexes

Signaling through the high affinity IgE receptor FcepsilonRI on human basophils and rodent mast cells is decreased by co-aggregating these receptors to the low affinity IgG receptor FcgammaRII. We used a recently described fusion protein, GE2, which is composed of key portions of the human gamma1 and the human epsilon heavy chains, to dissect the mechanisms that lead to human mast cell and basophil inhibition through co-aggregation of FcgammaRII and FcepsilonRI. Unstimulated human mast cells derived from umbilical cord blood express the immunoreceptor tyrosine-based inhibitory motif-containing receptor FcgammaRII but not FcgammaRI or FcgammaRIII. Interaction of the mast cells with GE2 alone did not cause degranulation. Co-aggregating FcepsilonRI and FcgammaRII with GE2 1) significantly inhibited IgE-mediated histamine release, cytokine production, and Ca(2+) mobilization, 2) reduced the antigen-induced morphological changes associated with mast cell degranulation, 3) reduced the tyrosine phosphorylation of several cellular substrates, and 4) increased the tyrosine phosphorylation of the adapter protein downstream of kinase 1 (p62(dok); Dok), growth factor receptor-bound protein 2 (Grb2), and SH2 domain containing inositol 5-phosphatase (SHIP). Tyrosine phosphorylation of Dok was associated with increased binding to Grb2. Surprisingly, in non-stimulated cells, there were complexes of phosphorylated SHIP-Grb2-Dok that were lost upon IgE receptor activation but retained under conditions of Fcepsilon-Fcgamma co-aggregation. Finally, studies using mast cells from Dok-1 knock-out mice showed that IgE alone triggers degranulation supporting an inhibitory role for Dok degranulation. Our results demonstrate how human FcepsilonRI-mediated responses can be inhibited by co-aggregation with FcgammaRIIB and implicate Dok, SHIP, and Grb2 as key intermediates in regulating antigen-induced mediator release.     

Role of Janus kinase-2 in IgE receptor-mediated leukotriene C4 production by mast cells

We have previously shown that Janus kinase 3, a member of the family of non-receptor protein tyrosine kinases, plays a critical role in the regulation of FcεRI-mediated mast cell responses. In the current study, we investigated the role of another JAK family member, JAK2, in these responses. Our results show that the treatment of IgE-sensitized mouse mast cells with an inhibitor of JAK2 (AG490) blocked the release of leukotriene C₄ in a dose-dependent fashion after antigen challenge. However, prostaglandin PG D₂ production and degranulation were not affected under identical experimental conditions. Transfection of RBL-2H3 mast cells with JAK-2 specific small interfering RNA resulted in a 50% reduction of LTC₄ release in response to FcεRI crosslinking, but did not inhibit mast cell degranulation or calcium ionophore-induced LTC₄ release, indicating involvement of JAK2 in IgE receptor-mediated leukotriene release. Taken together, these data suggest that JAK2 is a critical regulator of IgE/antigen-induced production of LTC₄ in mast cells.     

Effects of arachidonic acid analogs on FcepsilonRI-mediated activation of mast cells

Polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA) have been shown to modulate a number of inflammatory disorders. Mast cells play a critical role in the initiation and maintenance of inflammatory responses. However, the effects of PUFAs on mast cell functions have not been fully addressed. We here-in examined the effects of PUFAs on the high affinity IgE receptor (FcepsilonRI)-mediated mast cell activation using RBL-2H3 cells, a rat mast cell line, that were cultured in the medium containing palmitic acid (PA), AA, or the AA analogs mead acid (MA) and eicosapentaenoic acid (EPA). In AA-supplemented cells, the FcepsilonRI-mediated beta-hexosamidase and TNF-alpha release, calcium (Ca(2+)) influx, and some protein tyrosine phosphorylations including Syk and linker for activation of T cells (LAT) were enhanced, whereas, in MA- or PA-supplemented cells, they were not changed when compared with cells cultured in control medium. In EPA-supplemented cells, the enhancements of beta-hexosamidase release and protein tyrosine phosphorylations were observed. Furthermore, in AA- or EPA-supplemented cells, FcepsilonRI-mediated intracellular production of reactive oxygen species (ROS) that is required for the tyrosine phosphorylation of LAT and Ca(2+) influx were enhanced when compared with the other cells. Thus, preincubation of AA or EPA augmented FcepsilonRI-mediated degranulation in mast cells by affecting early events of FcepsilonRI signal transduction, which might be associated with the change of fatty acid composition of the cell membrane and enhanced production of ROS. The results suggest that some PUFAs can modulate FcepsilonRI-mediated mast cell activation and might affect FcepsilonRI/mast cell-mediated inflammation, such as allergic reaction.     

Fc epsilon RI gamma-ITAM is differentially required for mast cell function in vivo

The cross-linking of IgE-bound FcepsilonRI by Ags triggers mast cell activation leading to allergic reactions. The in vivo contribution of FcepsilonRIgamma signaling to IgE/FcepsilonRI-mediated mast cell responses has not yet been elucidated. In this study FcepsilonRIgamma(-/-) mast cells were reconstituted with either wild-type or mutant FcepsilonRIgamma in transgenic mice and transfected mast cells in vitro. We demonstrate that FcepsilonRIgamma-immunoreceptor tyrosine-based activation motif is essential for degranulation, cytokine production, and PG synthesis as well as for passive systemic anaphylaxis. Recent reports have suggested that cell surface FcepsilonRI expression and mast cell survival are regulated by IgE in the absence of Ag, although the molecular mechanism is largely unknown. We also found that the promotion of mast cell survival by IgE without Ags is mediated by signals through the FcepsilonRIgamma-immunoreceptor tyrosine-based activation motif. In contrast, the IgE-mediated up-regulation of FcepsilonRI is independent of FcepsilonRIgamma signaling. These results indicate that FcepsilonRIgamma-mediated signals differentially regulate the receptor expression, activation, and survival of mast cells and systemic anaphylaxis.     

Prostaglandin E2 selectively enhances the IgE-mediated production of IL-6 and granulocyte-macrophage colony-stimulating factor by mast cells through an EP1/EP3-dependent mechanism

PGE(2) is an endogenously synthesized inflammatory mediator that is over-produced in chronic inflammatory disorders such as allergic asthma. In this study, we investigated the regulatory effects of PGE(2) on mast cell degranulation and the production of cytokines relevant to allergic disease. Murine bone marrow-derived mast cells (BMMC) were treated with PGE(2) alone or in the context of IgE-mediated activation. PGE(2) treatment alone specifically enhanced IL-6 production, and neither induced nor inhibited degranulation and the release of other mast cell cytokines, including IL-4, IL-10, IFN-gamma, and GM-CSF. IgE/Ag-mediated activation of BMMC induced the secretion of IL-4, IL-6, and GM-CSF, and concurrent PGE(2) stimulation synergistically increased mast cell degranulation and IL-6 and GM-CSF, but not IL-4, production. A similar potentiation of degranulation and IL-6 production by PGE(2), in the context of IgE-directed activation, was observed in the well-established IL-3-dependent murine mast cell line, MC/9. RT-PCR analysis of unstimulated MC/9 cells revealed the expression of EP(1), EP(3), and EP(4) PGE receptor subtypes, including a novel splice variant of the EP(1) receptor. Pharmacological studies using PGE receptor subtype-selective analogs showed that the potentiation of IgE/Ag-induced degranulation and IL-6 production by PGE(2) is mediated through EP(1) and/or EP(3) receptors. Our results suggest that PGE(2) may profoundly alter the nature of the mast cell degranulation and cytokine responses at sites of allergic inflammation through an EP(1)/EP(3)-dependent mechanism.     

TRAF6 specifically contributes to FcepsilonRI-mediated cytokine production but not mast cell degranulation

TRAF6 (tumor necrosis factor-associated factor 6) is an essential adaptor downstream from the tumor necrosis factor (TNF) receptor and Toll-like receptor superfamily members. This molecule is critical for dendritic cell maturation and T cell homeostasis. Here we show that TRAF6 is important in high affinity IgE receptor, FcepsilonRI-mediated mast cell activation. In contrast to dendritic cells and T cells, TRAF6-deficient mast cells matured normally and showed normal IgE-dependent degranulation. Importantly, TRAF6-deficient mast cells showed impaired production of cytokine interleukin-6, CCL-9, interleukin-13, and TNF following FcepsilonRI aggregation. Chromatin immunoprecipitation assay showed decreased NF-kappaB p65 binding to CCL-9 and TNF promoters in TRAF6-deficient mast cells. Antigen and IgE-induced IkappaB phosphorylation and NF-kappaB p65 translocation to the nucleus were diminished in TRAF6-deficient mast cells. NF-kappaB luciferase activity in response to antigen and IgE stimulation was severely impaired in TRAF6-deficient mast cells. In addition, antigen and IgE-induced phosphorylation of mitogen-activated protein kinase p38 and JNK, but not ERK1/2, was significantly reduced in TRAF6-deficient mast cells. These results identified TRAF6 as an important signal transducer in FcepsilonRI-mediated signaling in mast cells. Our findings implicate TRAF6 as a new adaptor/regulator molecule for allergen-mediated inflammation in allergy.     

The adaptor molecule CIN85 regulates Syk tyrosine kinase level by activating the ubiquitin-proteasome degradation pathway

Triggering of mast cells and basophils by IgE and Ag initiates a cascade of biochemical events that lead to cell degranulation and the release of allergic mediators. Receptor aggregation also induces a series of biochemical events capable of limiting FcepsilonRI-triggered signals and functional responses. Relevant to this, we have recently demonstrated that Cbl-interacting 85-kDa protein (CIN85), a multiadaptor protein mainly involved in the process of endocytosis and vesicle trafficking, regulates the Ag-dependent endocytosis of the IgE receptor, with consequent impairment of FcepsilonRI-mediated cell degranulation. The purpose of this study was to further investigate whether CIN85 could alter the FcepsilonRI-mediated signaling by affecting the activity and/or expression of molecules directly implicated in signal propagation. We found that CIN85 overexpression inhibits the FcepsilonRI-induced tyrosine phosphorylation of phospholipase Cgamma, thus altering calcium mobilization. This functional defect is associated with a substantial decrease of Syk protein levels, which are restored by the use of selective proteasome inhibitors, and it is mainly due to the action of the ubiquitin ligase c-Cbl. Furthermore, coimmunoprecipitation experiments demonstrate that CIN85 overexpression limits the ability of Cbl to bind suppressor of TCR signaling 1 (Sts1), a negative regulator of Cbl functions, while CIN85 knockdown favors the formation of Cbl/Sts1 complexes. Altogether, our findings support a new role for CIN85 in regulating Syk protein levels in RBL-2H3 cells through the activation of the ubiquitin-proteasome pathway and provide a mechanism for this regulation involving c-Cbl ligase activity.     

Redundant and opposing functions of two tyrosine kinases, Btk and Lyn, in mast cell activation

Protein-tyrosine kinases play crucial roles in mast cell activation through the high-affinity IgE receptor (FcepsilonRI). In this study, we have made the following observations on growth properties and FcepsilonRI-mediated signal transduction of primary cultured mast cells from Btk-, Lyn-, and Btk/Lyn-deficient mice. First, Lyn deficiency partially reversed the survival effect of Btk deficiency. Second, FcepsilonRI-induced degranulation and leukotriene release were almost abrogated in Btk/Lyn doubly deficient mast cells while singly deficient cells exhibited normal responses. Tyrosine phosphorylation of cellular proteins including phospholipases C-gamma1 and C-gamma2 was reduced in Btk/Lyn-deficient mast cells. Accordingly, FcepsilonRI-induced elevation of intracellular Ca2+ concentrations and activation of protein kinase Cs were blunted in the doubly deficient cells. Third, in contrast, Btk and Lyn demonstrated opposing roles in cytokine secretion and mitogen-activated protein kinase activation. Lyn-deficient cells exhibited enhanced secretion of TNF-alpha and IL-2 apparently through the prolonged activation of extracellular signal-related kinases and c-Jun N-terminal kinase. Potentially accounting for this phenomenon and robust degranulation in Lyn-deficient cells, the activities of protein kinase Calpha and protein kinase CbetaII, low at basal levels, were enhanced in these cells. Fourth, cytokine secretion was severely reduced and c-Jun N-terminal kinase activation was completely abrogated in Btk/Lyn-deficient mast cells. The data together demonstrate that Btk and Lyn are involved in mast cell signaling pathways in distinctly different ways, emphasizing that multiple signal outcomes must be evaluated to fully understand the functional interactions of individual signaling components.     

Activation of murine lung mast cells by the adenosine A3 receptor

Adenosine has been implicated to play a role in asthma in part through its ability to influence mediator release from mast cells. Most physiological roles of adenosine are mediated through adenosine receptors; however, the mechanisms by which adenosine influences mediator release from lung mast cells are not understood. We established primary murine lung mast cell cultures and used real-time RT-PCR and immunofluorescence to demonstrate that the A(2A), A(2B), and A(3) adenosine receptors are expressed on murine lung mast cells. Studies using selective adenosine receptor agonists and antagonists suggested that activation of A(3) receptors could induce mast cell histamine release in association with increases in intracellular Ca(2+) that were mediated through G(i) and phosphoinositide 3-kinase signaling pathways. The function of A(3) receptors in vivo was tested by exposing mice to the A(3) receptor agonist, IB-MECA. Nebulized IB-MECA directly induced lung mast cell degranulation in wild-type mice while having no effect in A(3) receptor knockout mice. Furthermore, studies using adenosine deaminase knockout mice suggested that elevated endogenous adenosine induced lung mast cell degranulation by engaging A(3) receptors. These results demonstrate that the A(3) adenosine receptor plays an important role in adenosine-mediated murine lung mast cell degranulation.     

Interaction of a monoclonal IgE-specific antibody with cell-surface IgE-Fc epsilon RI: characterization of equilibrium binding and secretory response

Aggregation of FcepsilonRI, the high-affinity cell-surface receptor for IgE antibody, is required for degranulation of basophils and mast cells, but not all receptor aggregates elicit this cellular response. The stereochemical constraints on clusters of FcepsilonRI that are able to signal cellular responses, such as degranulation, have yet to be fully defined. To improve our understanding of the properties of FcepsilonRI aggregates that influence receptor signaling, we have studied the interaction of 23G3, a rat IgG(1)(kappa) IgE-specific monoclonal antibody, with IgE-FcepsilonRI complexes on rat mucosal-type mast cells (RBL-2H3 line). We find that 23G3 is a potent secretagogue. This property and the structural features of 23G3 (two symmetrically arrayed IgE-specific binding sites) make 23G3 a potentially valuable reagent for investigating the relationship between FcepsilonRI clustering and FcepsilonRI-mediated signaling events. To develop a mathematical model of 23G3-induced aggregation of FcepsilonRI, we used fluorimetry and flow cytometry to quantitatively monitor equilibrium binding of FITC-labeled 23G3 intact Ab and its Fab' fragment to cell-surface IgE. The results indicate that IgE bound to FcepsilonRI expresses two epitopes for 23G3 binding; that 23G3 binds IgE resident on the cell surface with negative cooperativity; and that 23G3 appears to induce mostly but not exclusively noncyclic dimeric aggregates of FcepsilonRI. There is no simple relationship between receptor aggregation at equilibrium and the degranulation response. Further studies are needed to establish how 23G3-induced aggregation of IgE-FcepsilonRI correlates with cellular responses.     

Role of STAT6 in IgE receptor/FcepsilonRI-mediated late phase allergic responses of mast cells.

In this study we show that IgE receptor engagement triggers activation of STAT6 in mast cells. We sought to determine the role of STAT6 activation in IgE receptor-mediated mast cell responses using STAT6 knockout mice. After IgE receptor engagement, bone marrow mast cells from STAT6(-/-) mice exhibited normal histamine and leukotriene C(4) release, but their cytokine release was markedly reduced. In accordance with these in vitro data, IgE/Ag-challenged STAT6(-/-) mice showed normal early phase, but severely impaired late phase, allergic reactions. These findings provide unprecedented evidence that STAT6 plays a pivotal role in mast cell responses to IgE/Ag stimulation.     

Isoform-specific functions of phosphoinositide 3-kinases: p110 delta but not p110 gamma promotes optimal allergic responses in vivo

The leukocyte-enriched p110gamma and p110delta isoforms of PI3K have been shown to control in vitro degranulation of mast cells induced by cross-linking of the high affinity receptor of IgE (FcepsilonRI). However, the relative contribution of these PI3K isoforms in IgE-dependent allergic responses in vivo is controversial. A side-by-side comparative analysis of the role of p110gamma and p110delta in mast cell function, using genetic approaches and newly developed isoform-selective pharmacologic inhibitors, confirms that both PI3K isoforms play an important role in FcepsilonRI-activated mast cell degranulation in vitro. In vivo, however, only p110delta was found to be required for optimal IgE/Ag-dependent hypersensitivity responses in mice. These observations identify p110delta as a key therapeutic target among PI3K isoforms for allergy- and mast cell-related diseases.     

c-Fos as a regulator of degranulation and cytokine production in FcepsilonRI-activated mast cells

The AP-1 complex is composed of c-Jun and c-Fos and is a key component in the regulation of proinflammatory genes. Mast cells play a significant role in the initiation of many inflammatory responses, such as allergy and allergy-associated diseases. In the present work, we characterized the role of c-Fos in mast cell function by investigating IL-3-dependent cell proliferation, degranulation capability, and cytokine expression in c-Fos-deficient mice. In c-Fos-deficient mast cells, we found that FcepsilonRI-mediated degranulation was significantly inhibited, which correlates with the reduced expression of SWAP-70, VAMP-7, and Synaptotagmin I genes, which are involved directly in the degranulation process. These findings show that c-Fos plays an important role in FcepsilonRI-mediated regulation of mast cell function.     

Differential requirement for adapter proteins Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa and adhesion- and degranulation-promoting adapter protein in FcepsilonRI signaling and mast cell function

The adapter molecule Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is essential for FcepsilonRI-mediated signaling, degranulation and IL-6 production in mast cells. To test the structural requirements of SLP-76 in mast cell signaling and function, we have studied the functional responses of murine bone marrow-derived mast cells (BMMCs) expressing mutant forms of SLP-76. We found that the N-terminal tyrosines as well as the central proline-rich region of SLP-76 are required for participation of SLP-76 in FcepsilonRI-mediated signaling and function. The C-terminal SH2 domain of SLP-76 also contributes to optimal function of SLP-76 in mast cells. Another adapter molecule, adhesion- and degranulation-promoting adapter protein (ADAP), is known to bind the SH2 domain of SLP-76, and cell line studies have implicated ADAP in mast cell adhesion and FcepsilonRI-induced degranulation. Surprisingly, we found that mast cells lacking ADAP expression demonstrate no defects in FcepsilonRI-induced adhesion, granule release, or IL-6 production, and that ADAP-deficient mice produce a normal passive systemic anaphylactic response. Thus, failure to bind ADAP does not underlie the functional defects exhibited by SLP-76 SH2 domain mutant-expressing mast cells.     

SHIP down-regulates FcepsilonR1-induced degranulation at supraoptimal IgE or antigen levels

Cross-linking of the IgE-loaded high-affinity IgE receptor (FcepsilonR1) by multivalent Ags results in mast cell activation and subsequent release of multiple proinflammatory mediators. The dose-response curve for FcepsilonR1-mediated degranulation is bell-shaped, regardless of whether the IgE or the Ag concentration is varied. Although overall calcium influx follows this bell-shaped curve, intracellular calcium release continues to increase at supraoptimal IgE or Ag concentrations. As well, overall calcium mobilization adopts more transient kinetics when stimulations are conducted with supraoptimal instead of optimal Ag concentrations. Moreover, certain early signaling events continue to increase whereas degranulation drops under supraoptimal conditions. We identified SHIP, possibly in association with the FcepsilonR1 beta-chain, as a critical negative regulator acting within the inhibitory (supraoptimal) region of the dose-response curve that shifts the kinetics of calcium mobilization from a sustained to a transient response. Consistent with this, we found that degranulation of SHIP-deficient murine bone marrow-derived mast cells was not significantly reduced at supraoptimal Ag levels. A potential mediator of SHIP action, Bruton's tyrosine kinase, did not seem to play a role within the supraoptimal suppression of degranulation. Interestingly, SHIP was found to colocalize with the actin cytoskeleton (which has been shown previously to mediate the inhibition of degranulation at supraoptimal Ag doses). These results suggest that SHIP, together with other negative regulators, restrains bone marrow-derived mast cell activation at supraoptimal IgE or Ag concentrations in concert with the actin cytoskeleton.     

Down-Regulation of MicroRNA-223 Promotes Degranulation via the PI3K/Akt Pathway by Targeting IGF-1R in Mast Cells

Background

Mast cells play a central role in allergic and inflammatory disorders by inducing degranulation and inflammatory mediator release. Recent reports have shown that miRNAs play an important role in inflammatory response regulation. Therefore, the role of miR-223 in mast cells was investigated.

Methods

The expression of miR-223 was quantified by quantitative real-time polymerase chain reaction (qRT-PCR) in immunoglobulin E (IgE)-mediated mast cells. After successful miR-223 inhibition by transfection, degranulation was detected in IgE-mediated mast cells. The phosphorylation of IκB-α and Akt were examined using western blotting. NF-κB was tested using electrophoretic mobility shift assay. PI3K-inhibitor (LY294002) was used to investigate whether the PI3K/Akt pathway was essential for mast cell activation. The TargetScan database and a luciferase reporter system were used to identify whether insulin-like growth factor 1 receptor (IGF-1R) is a direct target of miR-223.

Results

MiR-223 expression was up-regulated in IgE-mediated mast cells, whereas its down-regulation promoted mast cell degranulation. Levels of IκB-α and Akt phosphorylation as well as NF-κB were increased in miR-223 inhibitor cells. LY294002 could block the PI3K/Akt signaling pathway and rescue the promotion caused by suppressing miR-223 in mast cells. IGF-1R was identified as a direct target of miR-223.

Conclusions

These findings suggest that down-regulation of miR-223 promotes degranulation via the PI3K/Akt pathway by targeting IGF-1R in mast cells.     

Systemic effects of ingested Lactobacillus Rhamnosus: inhibition of mast cell membrane potassium (IKCa) current and degranulation

Exposure of the intestine to certain strains lactobacillus can have systemic immune effects that include the attenuation of allergic responses. Despite the central role of mast cells in allergic disease little is known about the effect of lactobacilli on the function of these cells. To address this we assessed changes in rat mast cell activation following oral treatment with a strain of Lactobacillus known to attenuate allergic responses in animal models. Sprague Dawley rats were fed with L. rhamnosus JB-1 (1×10(9)) or vehicle control for 9 days. Mediator release from peritoneal mast cells (RPMC) was determined in response to a range of stimuli. Passive cutaneous anaphylaxis (PCA) was used to assess mast cell responses in vivo. The Ca(2+) activated K(+) channel (KCa3.1) current, identified as critical to mast cell degranulation, was monitored by whole cell patch-clamp. L. rhamnosus JB-1 treatment lead to significant inhibition of mast cell mediator release in response to a range of stimuli including IgE mediated activation. Furthermore, the PCA response was significantly reduced in treated rats. Patch-clamp studies revealed that RPMC from treated animals were much less responsive to the KCa3.1 opener, DCEBIO. These studies demonstrate that Ingestion of L. rhamnosus JB-1 leads to mast cell stabilization in rats and identify KCa3.1 as an immunomodulatory target for certain lactobacilli. Thus the systemic effects of certain candidate probiotics may include mast cell stabilization and such actions could contribute to the beneficial effect of these organisms in allergic and other inflammatory disorders.     

Exposure of RBL-2H3 mast cells to Ag(+) induces cell degranulation and mediator release

There is a growing need to understand the impact of environmental sulfhydryl group-reactive heavy metals on the immune system. Here we show that Ag(+) induces mast cell degranulation, as does the aggregation of the high affinity immunoglobulin E receptor (FcepsilonRI). Micromolar quantities of Ag(+) specifically induced degranulation of mast cell model rat basophilic leukemia (RBL-2H3) cells without showing cytotoxicity. The Ag(+)-mediated degranulation could be observed as rapidly as 5 min after the addition of the ions. Ag(+) also induced a rapid change in tyrosine phosphorylation of multiple cellular proteins including the focal adhesion kinase but not Syk kinase. The Syk-selective inhibitor piceatannol and the Src family-selective tyrosine kinase inhibitor PP1 dose-dependently inhibited FcepsilonRI-mediated degranulation, whereas neither compound inhibited the Ag(+)-mediated degranulation. Furthermore, likewise FcepsilonRI aggregation, Ag(+) also induced leukotriene secretion. These results show that Ag(+) activates RBL-2H3 mast cells through a tyrosine phosphorylation-linked mechanism, which is distinct from that involved in FcepsilonRI-mediated activation.     

Regulation of rat basophilic leukemia-2H3 mast cell secretion by a constitutive Lyn kinase interaction with the high affinity IgE receptor (Fc epsilon RI)

Signaling through the high affinity IgE receptor is initiated by noncovalently associated Lyn kinase, resulting in the secretion of inflammatory mediators from mast cells. A fraction of the total cellular Lyn is associated via its N-terminal unique domain with the cytoplasmic domain of the Fc epsilonRI beta subunit before receptor aggregation. In the current study, we stably transfected the unique domain of Lyn into rat basophilic leukemia-2H3 mast cells and examined the consequences on Fc epsilonRI-induced signal transduction and mediator secretion to further define the role of the unique domain of Lyn in mast cell secretion. Tyrosine phosphorylation of Fc epsilonRI beta and gamma subunits was partially inhibited in the Lyn unique domain transfectants after Ag stimulation. Ag stimulation of Lyn unique domain transfectants was accompanied by enhanced phosphorylation of MEK and ERK-2, which are required for leukotriene C4 (LTC4) release, and production of LTC4 was increased 3- to 5-fold, compared with cells transfected with vector alone. Conversely, tyrosine phosphorylation of the adaptor protein Gab2, which is essential for mast cell degranulation, was inhibited after Ag stimulation of Lyn unique domain transfectants, and Ag-induced release of histamine was inhibited up to 48%. In rat basophilic leukemia-2H3 cells, Lyn thus plays a dual role by positively regulating Fc epsilonRI phosphorylation and degranulation while negatively regulating LTC4 production. This study provides further evidence that the constitutive interaction between the unique domain of Lyn and the Fc epsilonRI beta subunit is a crucial step in the initiation of Fc epsilonRI signaling and that Lyn is limiting for Fc epsilonRI-induced secretion of inflammatory mediators.