Kegg Pathway: Fc epsilon RI signaling pathway

J Allergy Clin Immunol. 2009 Oct; 124(4): 639-46; quiz 647-8
Metcalfe DD, Peavy RD, Gilfillan AM

The recent development of a consensus definition and proposed diagnostic cRIteRIa for anaphylaxis offers promise for research efforts and a better understanding of the epidemiology and pathogenesis of this enigmatic and life-threatening disease. This review examines basic pRInciples and recent research advances in the mechanisms of mast cell signaling believed to underlie anaphylaxis. The unfolding complexity of mast cell signaling suggests that the system is sensitive to regulation by any of several individual signaling pathways and intermediates and that complementary pathways regulate mast cell activation by amplified signals. The signaling events underlying anaphylactic reactions have largely been identified through expeRIments in genetically modified mice and supported by biochemical studies of mast cells deRIved from these mice. These studies have revealed that signaling pathways exist to both upregulate and downregulate mast cell responses. In this review we will thus descRIbe the key molecular players in these pathways in the context of anaphylaxis.

J Immunol. 2009 Oct 15; 183(8): 5104-12
Lisboa FA, Peng Z, Combs CA, Beaven MA

Initial IgE-dependent signaling events are associated with detergent-resistant membrane microdomains. Following Ag stimulation, the IgE-receptor (Fc(epsilon)RI ) accumulates within these domains. This facilitates the phosphorylation of Fc(epsilon)RI subunits by the Src kinase, Lyn, and the interaction with adaptor proteins, such as the linker for activation of T cells. Among the phospholipases (PL) subsequently activated, PLD is of interest because of its presence in lipid microdomains and the possibility that its product, phosphatidic acid, may regulate signal transduction and membrane trafficking. We find that in Ag-stimulated RBL-2H3 mast cells, the association of Fc(epsilon)RI with detergent-resistant membrane fractions is inhibited by 1-butanol, which subverts production of phosphatidic acid to the biologically inert phosphatidylbutanol. Furthermore, the knockdown of PLD2, and to a lesser extent PLD1 with small inhibitory RNAs, also suppressed the accumulation of Fc(epsilon)RI and Lyn in these fractions as well as the phosphorylation of Src kinases, Fc(epsilon)RI , linker for activation of T cells, and degranulation. These effects were accompanied by changes in distRIbution of the lipid microdomain component, ganglioside 1, in the plasma membrane as determined by binding of fluorescent-tagged cholera toxin B subunit and confocal microscopy in live cells. Collectively, these findings suggest that PLD activity plays an important role in promoting IgE-dependent signaling events within lipid microdomains in mast cells.

J Immunol. 2009 Oct 15; 183(8): 4940-7
McPherson VA, Sharma N, EveRIngham S, Smith J, Zhu HH, Feng GS, Craig AW

ClusteRIng of the high affinity IgE receptor (Fc(epsilon)RI) in mast cells leads to degranulation and production of numerous cytokines and lipid mediators that promote allergic inflammation. Initiation of FFc(epsilon)RI signaling involves rapid tyrosine phosphorylation of Fc(epsilon)RI and membrane-localized adaptor proteins that recruit additional SH2 domain-containing proteins that dynamically regulate downstream signaling. SH2 domain-containing phosphatase-2 (SHP2) is a protein-tyrosine phosphatase implicated in Fc(epsilon)RI signaling, but whose function is not well defined. In this study, using a mouse model allowing temporal shp2 inactivation in bone marrow-deRIved mast cells (BMMCs), we provide insights into SHP2 functions in the Fc(epsilon)RI pathway. Although no overt defects in Fc(epsilon)RI-induced tyrosine phosphorylation were observed in SHP2 knock-out (KO) BMMCs, several proteins including Lyn and Syk kinases displayed extended phosphorylation kinetics compared with wild-type BMMCs. SHP2 was dispensable for Fc(epsilon)RI-induced degranulation of BMMCs, but was required for maximal activation of Erk and Jnk mitogen-activated protein kinases. SHP2 KO BMMCs displayed several phenotypes associated with reduced Fyn activity, including elevated phosphorylation of the inhibitory pY531 site in Fyn, impaired signaling to Grb2-associated binder 2, Akt/PKB, and IkappaB kinase, and decreased TNF-alpha release compared with control cells. This is likely due to elevated Lyn activity in SHP2 KO BMMCs, and the ability of Lyn to antagonize Fyn activity. Overall, our study identifies SHP2 as a positive effector of Fc(epsilon)RI-induced activation of Fyn/Grb2-associated binder 2/Akt and Ras/Erk pathways leading to TNF-alpha release from mast cells.

Biochem Biophys Res Commun. 2009 Nov 27; 389(4): 651-6
Itoh T, Fujita Y, Ito M, Masuda A, Ohno K, Ichihara M, Kojima T, Nozawa Y, Ito M

Molecular hydrogen ameliorates oxidative stress-associated diseases in animal models. We found that oral intake of hydrogen-RIch water abolishes an immediate-type allergic reaction in mice. Using rat RBL-2H3 mast cells, we demonstrated that hydrogen attenuates phosphorylation of the FcepsilonRI-associated Lyn and its downstream signal transduction, which subsequently inhibits the NADPH oxidase activity and reduces the generation of hydrogen peroxide. We also found that inhibition of NADPH oxidase attenuates phosphorylation of Lyn in mast cells, indicating the presence of a feed-forward loop that potentiates the allergic responses. Hydrogen accordingly inhibits all tested signaling molecule(s) in the loop. Hydrogen effects have been solely ascRIbed to exclusive removal of hydroxyl radical. In the immediate-type allergic reaction, hydrogen exerts its beneficial effect not by its radical scavenging activity but by modulating a specific signaling pathway. Effects of hydrogen in other diseases are possibly mediated by modulation of yet unidentified signaling pathways. Our studies also suggest that hydrogen is a gaseous signaling molecule like nitRIc oxide.

Immunity. 2009 Sep 18; 31(3): 452-4
Varma R

In this issue of Immunity, Andrews et al. (2009) used single-molecule fluorescence microscopy to demonstrate that the IgE receptor FcepsilonRI in the plasma membrane can signal in a mobile state.

Immunity. 2009 Sep 18; 31(3): 469-79
Andrews NL, Pfeiffer JR, Martinez AM, Haaland DM, Davis RW, Kawakami T, Oliver JM, Wilson BS, Lidke DS

Crosslinking of IgE-bound FcepsilonRI tRIggers mast cell degranulation. Previous fluorescence recovery after photobleaching (FRAP) and phosphorescent anisotropy studies suggested that FcepsilonRI must immobilize to signal. Here, single quantum dot (QD) tracking and hyperspectral microscopy methods were used for defining the relationship between receptor mobility and signaling. QD-IgE-FcepsilonRI aggregates of at least three receptors remained highly mobile over extended times at low concentrations of antigen that induced Syk kinase activation and near-maximal secretion. Multivalent antigen, presented as DNP-QD, also remained mobile at low doses that supported secretion. FcepsilonRI immobilization was marked at intermediate and high antigen concentrations, correlating with increases in cluster size and rates of receptor internalization. The kinase inhibitor PP2 blocked secretion without affecting immobilization or internalization. We propose that immobility is a feature of highly crosslinked immunoreceptor aggregates and a tRIgger for receptor internalization, but is not required for tyrosine kinase activation leading to secretion.

J Biol Chem. 2009 Oct 9; 284(41): 28172-9
Sakai S, Sugawara T, Matsubara K, Hirata T

Carotenoids have been demonstrated to possess antioxidative and anti-inflammatory effects. However, there is no report that the effects of carotenoids on degranulation of mast cell is cRItical for type I allergy. In this study, we focused on the effect of carotenoids on antigen-induced degranulation of mast cells. Fucoxanthin, astaxanthin, zeaxanthin, and beta-carotene significantly inhibited the antigen-induced release of beta-hexosaminidase in rat basophilic leukemia 2H3 cells and mouse bone marrow-deRIved mast cells. Those carotenoids also inhibited antigen-induced aggregation of the high affinity IgE receptor (Fc epsilonRI), which is the most upstream of the degranulating signals of mast cells. Furthermore, carotenoids inhibited Fc epsilonRI-mediated intracellular signaling, such as phosphorylation of Lyn kinase and Fyn kinase. It suggests that the inhibitory effect of carotenoids on the degranulation of mast cells were mainly due to suppressing the aggregation of Fc epsilonRI followed by intracellular signaling. In addition, those carotenoids inhibited antigen-induced translocation of Fc epsilonRI to lipid rafts, which are known as platforms of the aggregation of Fc epsilonRI. We assume that carotenoids may modulate the function of lipid rafts and inhibit the translocation of Fc epsilonRI to lipid rafts. This is the first report that focused on the aggregation of Fc epsilonRI to investigate the mechanism of the inhibitory effects on the degranulation of mast cells and evaluated the functional activity of carotenoids associated with lipid rafts.

Curr Allergy Asthma Rep. 2009 Sep; 9(5): 353-9
Ryan JJ, Fernando JF

Mast cells are present in nearly all vasculaRIzed tissues, but not the blood. They are best known for the prominent role they play in atopic disease. However, our current understanding of their direct and indirect roles in the immune response offers a more nuanced picture of both villain and hero. Although they are implicated in many inflammatory disorders, they also defend us from bacteRIal pathogens, prevent dangerous overreactions by the immune system, and even protect us from snake venom. Perhaps there is more to these maligned cells than we thought.

Nat Immunol. 2009 Sep; 10(9): 1018-25
Engels N, König LM, Heemann C, Lutz J, Tsubata T, GRIep S, Schrader V, Wienands J

The improved antibody responses of class-switched memory B cells depend on enhanced signaling from their B cell antigen receptors (BCRs). However, BCRs on both naive and antigen-expeRIenced B cells use the canonical immunoglobulin-associated alpha and beta-protein signaling subunits. Here we identified a BCR isotype-specific signal-amplification mechanism. Whereas immunoglobulin M (IgM)-containing BCRs initiated intracellular signals exclusively through immunoglobulin-associated alpha- and beta-proteins, IgG- and IgE-containing BCRs also used a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. When phosphorylated, this tyrosine recruited the adaptor Grb2, resulting in sustained protein kinase activation and prolonged generation of second messengers, which together culminated in enhanced B cell proliferation. Hence, membrane-bound IgG and IgE exert antigen recognition as well as costimulatory functions, thereby rendeRIng memory B cells less dependent on T cell help.

Eur J Immunol. 2009 Aug; 39(8): 2293-301
Lind SM, Kuylenstierna C, Moll M, D Jordö E, Winqvist O, Lundeberg L, Karlsson MA, T Linder M, Johansson C, Scheynius A, Sandberg JK, Karlsson MC

Atopic eczema (AE) is a chronic relapsing inflammatory skin disease where the commensal yeast Malassezia can act as a microbial tRIgger factor. Malassezia activates human DC to produce IL-18, an innate cytokine that is elevated in serum of AE patients; however, the precise role of IL-18 in human AE etiology is unknown. Herein, we investigated the effect of IL-18 on the human invaRIant NKT (iNKT) cell compartment in AE. We found that IL-18 was a potent activator of human iNKT-cells and promoted a pro-inflammatory CD1d-dependent response, even in the absence of exogenous ligands. Chronic activation via IL-18 on the other hand was inhibitory and skewed the iNKT-cell pool by selectively suppressing CD4(+) iNKT-cells. This was mimicked in AE patients where the proportion of CD4(+) iNKT-cells was reduced in peRIpheral blood and coincided with elevated plasma levels of IL-18. Furthermore, a reduced CD4(+) iNKT-cell pool was associated with elevated IgE levels in plasma, and the plasma levels of IL-18 correlated with both total IgE and disease seveRIty in the AE patients. Based on these findings, we propose that IL-18-mediated activation and subsequent dysregulation of the CD1d-restRIcted iNKT-cells plays a role in the pathogenesis of human AE.

J Immunol. 2009 Aug 15; 183(4): 2585-92
Jackson L, Cady CT, Cambier JC

IgE production is inversely regulated by circulating and B cell surface levels of the low affinity IgE receptor, CD23. To begin to understand physiologic determinants of CD23 expression, we analyzed effects of BCR and TLR stimulation on CD23 levels. BCR and TLR 2, 3, 4, 6, and 9 agonists induced CD23 down-modulation from the cell surface. However, among the ligands only TLR4 agonists induced transcRIptional activation of CD23 and generation of significant soluble CD23. These responses were induced by LPS both in vitro and in vivo, and were seen in both muRIne and human B cells. LPS also induced expression of matRIx metalloprotease 9 (MMP9) and failed to induce CD23 cleaving activity in MMP9(-/-) cells, thus implicating MMP9 in the LPS-induced release of CD23 from the cell surface. Finally, type 1 transitional B cells uniquely produce MMP9 in response to LPS, suggesting a mechanism wherein endotoxin induces T1 cell expression of MMP9, which mediates cleavage of CD23 on distinct, mature B cells.

Cell Signal. 2009 Nov; 21(11): 1698-705
Jung ID, Lee HS, Lee HY, Choi OH

IgE-sensitized rat basophilic leukemia (RBL)-2H3 mast cells have been shown to migrate towards antigen. In the present study we tRIed to identify the mechanism by which antigen causes mast cell migration. Antigen caused migration of RBL-2H3 cells at the concentration ranges of 1000-fold lower than those required for degranulation and the dose response was biphasic. This suggests that mast cells can detect very low concentration gradients of antigen (pg/ml ranges), which initiate migration until they degranulate near the oRIgin of antigen, of which concentration is in the ng/ml ranges. Similar phenomenon was observed in human mast cells (HMCs) deRIved from CD34(+) progenitors. As one mechanism of mast cell migration, we tested the involvement of sphingosine 1-phosphate (S1P). Fc epsilon RI-mediated cell migration was dependent on the production of S1P but independent of a S1P receptor or its signaling pathways as determined with S1P receptor antagonist VPC23019 and Gi protein inhibitor pertussis toxin (PTX). This indicated that the site of action of S1P produced by antigen stimulation was intracellular. However, S1P-induced mast cell migration was dependent on S1P receptor activation and inhibited by both VPC23019 and PTX. Cell migration towards antigen or extracellular S1P was dependent on the activation of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, while only migration towards antigen was inhibited by the inhibitors of sphingosine kinase and phospholipase C (PLC) and intracellular calcium chelator BAPTA. In summary, our data suggest that the high affinity receptor for IgE (Fc epsilon RI)-mediated mast cell migration is dependent on the production of S1P but independent of S1P receptors. Cell migration mediated by either Fc epsilon RI or S1P receptors involves activation of both PI3K and MAPK.

Leukemia. 2009 Oct; 23(10): 1807-17
Acharya M, Edkins AL, Ozanne BW, Cushley W

CD23 acts through the alphavbeta5 integRIn to promote growth of human pre-B cell lines in an adhesion-independent manner. alphavbeta5 is expressed on normal B-cell precursors in the bone marrow. Soluble CD23 (sCD23), short CD23-deRIved peptides containing the arg-lys-cys (RKC) motif recognized by alphavbeta5 and anti-alphavbeta5 monoclonal antibodies (MAbs) all sustain growth of pre-B cell lines. The chemokine stromal cell-deRIved factor-1 (SDF-1) regulates key processes duRIng B-cell development. SDF-1 enhanced the growth-sustaining effect dRIven by ligation of alphavbeta5 with anti-alphavbeta5 MAb 15F-11, sCD23 or CD23-deRIved RKC-containing peptides. This effect was restRIcted to B-cell precursors and was specific to SDF-1. The enhancement in growth was associated with the activation of extracellular signal-regulated kinase (ERK) and both these responses were attenuated by the MEK inhibitor U0126. Finally, platelet-deRIved growth factor also enhanced both alphavbeta5-mediated cell growth and ERK activation. The data suggest that adhesion-independent growth-promoting signals delivered to B-cell precursors through the alphavbeta5 integRIn can be modulated by cross-talk with receptors linked to both G-protein and tyrosine kinase-coupled signalling pathways.

Bioorg Med Chem. 2009 Aug 1; 17(15): 5374-9
Itoh T, Ninomiya M, Yasuda M, Koshikawa K, Deyashiki Y, Nozawa Y, Akao Y, Koketsu M

We isolated the 4 kinds of flavonoids from strawberry 'Nohime' and examined the effect of these flavonoids on the degranulation in RBL-2H3 cells. The flavonoids were found to suppress the degranulation from Ag-stimulated RBL-2H3 cells to different extents. To disclose the inhibitory mechanism of degranulation by flavonoids, we examined their effects on the intracellular free Ca(2+) concentration ([Ca(2+)]i) and the intracellular signaling pathway such as Lyn, Syk, and PLCgammas. The intracellular free Ca(2+) concentration ([Ca(2+)]i) was elevated by Fc epsilonRI activation, but these flavonoid treatments reduced the elevation of [Ca(2+)]i by suppressing Ca(2+) influx. Kaempferol strongly suppressed the activation of Syk and PLCgammas. It was thus suggested that suppression of Ag-stimulated degranulation by the flavonoids is mainly due to suppression of [Ca(2+)]i elevation and Syk activation. These results suggested that strawberry would be of some ameliorative benefit for the allergic symptoms.

Int Immunol. 2009 Aug; 21(8): 991-1001
Fifadara NH, Aye CC, Raghuwanshi SK, RIchardson RM, Ono SJ

Chemokine receptors (CCRs) are important co-stimulatory molecules found on many blood cells and associated with vaRIous diseases. The expression and function of CCRs on mast cells has been quite controversial. In this study, we report for the first time that muRIne bone marrow-deRIved mast cells (BMMC) express messenger RNA and protein for CCR1. BMMC cultured in the presence of muRIne recombinant stem cell factor and muRIne IL-3 expressed CCR1 after 5-6 weeks. We also report for the first time that mBMMC(CCR1+) cells endogenously express neurokinin receptor-1 and intercellular adhesion molecule-1. To examine the activity of CCR1 on these BMMC, we simultaneously stimulated two receptors: CCR1 by its ligand macrophage inflammatory protein-1alpha and the IgE receptor FcepsilonRI by antigen cross-linking. We found that co-stimulation enhanced BMMC degranulation compared with FcepsilonRI stimulation alone, as assessed by beta-hexosaminidase activity (85 versus 54%, P < 0.0001) and Ca(2+) influx (223 versus 183 nM, P < 0.05). We also observed significant increases in mast cell secretion of key growth factors, cytokines and chemokine mediators upon CCR1-FcepsilonRI co-stimulation. These factors include transforming growth factor beta-1, tumor necrosis factor-alpha and the cytokine IL-6. Taken together, our data indicate that CCR1 plays a key role in BMMC function. These findings contRIbute to our understanding of mechanisms for immune cell trafficking duRIng inflammation.

J Cell Sci. 2009 Jul 15; 122(Pt 14): 2567-74
Vasudevan L, Jeromin A, Volpicelli-Daley L, De Camilli P, Holowka D, Baird B

Crosslinking of IgE receptors by antigen initiates Ca2+ mobilization in mast cells by activating phospholipase-C gamma-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. The resulting inositol 1,4,5-tRIsphosphate-mediated Ca2+ release from the endoplasmic reticulum (ER) activates store-operated Ca2+ entry, which is necessary for exocytotic release of inflammatory mediators. To investigate roles for PtdIns(4,5)P2-synthesizing isozymes of the type I phosphatidylinositol 4-phosphate 5-kinase family (PIP5K-I) in mast cell signaling, we compared the ectopic expression of wild-type and catalytically inactive PIP5K-I beta in RBL-2H3 mast cells. SurpRIsingly, both antigen and thapsigargin-stimulated Ca2+ influx were reduced by overexpression of active PIP5K-I beta, whereas antigen-stimulated Ca2+ release from ER stores was unaffected. Consistent with these results, Ca2+ entry stimulated by antigen or thapsigargin was enhanced by expression of a plasma-membrane-associated inositol polyphosphate 5'-phosphatase, whereas antigen-stimulated Ca2+ release from stores was reduced. To investigate the role of PIP5K-I gamma in antigen-stimulated Ca2+ mobilization, we used bone-marrow-deRIved mast cells from PIP5K-I gamma(-/-) mice. Antigen-stimulated Ca2+ release from ER stores was substantially reduced in the absence of PIP5K-I gamma, but thapsigargin-mediated Ca2+ entry was unaffected. In summary, PIP5K-I gamma positively regulates antigen-stimulated Ca2+ release from ER stores, whereas PIP5K-I beta negatively regulates store-operated Ca2+ entry, suggesting that these different PIP5K-I isoforms synthesize functionally distinct pools of PtdIns(4,5)P2 at the plasma membrane.

J Immunol. 2009 Jul 1; 183(1): 221-7
Pushparaj PN, Manikandan J, Tay HK, H'ng SC, Kumar SD, Pfeilschifter J, Huwiler A, Melendez AJ

Mast cell degranulation is pivotal to allergic diseases; investigating novel pathways tRIggeRIng mast cell degranulation would undoubtedly have important therapeutic potential. FcepsilonRI-mediated degranulation has contradictoRIly been shown to require SphK1 or SphK2, depending on the reports. We investigated the in vitro and in vivo specific role(s) of SphK1 and SphK2 in FcepsilonRI-mediated responses, using specific small interfeRIng RNA-gene silencing. The small interfeRIng RNA-knockdown of SphK1 in mast cells inhibited several signaling mechanisms and effector functions, tRIggered by FcepsilonRI stimulation including: Ca(2+) signals, NFkappaB activation, degranulation, cytokine/chemokine, and eicosanoid production, whereas silencing SphK2 had no effect at all. Moreover, silencing SPHK1 in vivo, in different strains of mice, strongly inhibited mast cell-mediated anaphylaxis, including inhibition of vascular permeability, tissue mast cell degranulation, changes in temperature, and serum histamine and cytokine levels, whereas silencing SPHK2 had no effect and the mice developed anaphylaxis. Our data differ from a recent report using SPHK1(-/-) and SPHK2(-/-) mice, which showed that SphK2 was required for FcepsilonRI-mediated mast cell responses. We performed expeRIments in mast cells deRIved from SPHK1(-/-) and SPHK2(-/-) mice and show that the calcium response and degranulation, tRIggered by FcepsilonRI-cross-linking, is not different from that tRIggered in wild-type cells. Moreover, IgE-mediated anaphylaxis in the knockout mice showed similar levels in temperature changes and serum histamine to that from wild-type mice, indicating that there was no protection from anaphylaxis for either knockout mice. Thus, our data strongly suggest a previously unrecognized compensatory mechanism in the knockout mice, and establishes a role for SphK1 in IgE-mediated mast cell responses.

Mol Immunol. 2009 Aug; 46(13): 2539-47
Grodzki AC, Moon KD, Berenstein EH, Siraganian RP

High affinity IgE receptor (FcvarepsilonRI)-induced activation of mast cells results in degranulation and generation of leukotRIenes and cytokines. FcvarepsilonRI-induced mast cell activation was analyzed at a single cell basis using a rat basophilic leukemia (RBL-2H3) cell line transfected with a reporter plasmid containing three tandem NFAT (nuclear factor of activated T cells) binding sites fused to enhanced green fluorescent protein (GFP). SurpRIsingly, with this sensitive detection system, there is activation of IgE sensitized cells at concentrations of antigen as low as 10pg/ml, which was 10-fold lower than was detected by degranulation. There were differences in signaling pathways leading to degranulation compared to NFAT-mediated gene activation. Both signaling to NFAT activation and degranulation required Syk and calcineuRIn. However inhibitors of the phosphatidylinositol 3-kinase pathway blocked degranulation but did not NFAT activation. The results also indicate that NFAT was activated at lower intracellular signals compared to degranulation. Therefore, FcvarepsilonRI activation can result in nuclear signals in the absence of the release of mediators.

Int Arch Allergy Immunol. 2009; 149 Suppl 1: 73-6
Yamaoka K, Okayama Y, Kaminuma O, Katayama K, MoRI A, Tatsumi H, Nemoto S, Hiroi T

BACKGROUND: Mast cells (MCs) play a central role in allergic reactions through high-affinity IgE receptor (FcepsilonRI)-mediated responses. Many attempts have been performed to investigate MC functions, though molecular bases of the intracellular signaling cascade through FcepsilonRI, especially in human MCs, remain scant and unexplored. METHODS: Human MCs were differentiated from CD34+ cells by culture with stem cell factor, IL-6 and IL-3. The differential phosphorylation profiles of protein tyrosine residues in the resulting MCs with or without FcepsilonRI aggregation were examined by two-dimensional gel electrophoresis. The candidate phosphoproteins of interest were picked, in-gel digested and mass spectrometry fingerpRInted. RESULTS: Approximately 40 proteins in MCs were phosphorylated on their tyrosine residues in response to activation and some of them were identified. Particularly IL-31 receptor alpha, solute carRIer family 39, syntaxin 5 and heterogeneous nuclear RIbonucleoprotein are newly identified as phosphoproteins that are potentially involved in the MC signaling cascade through FcepsilonRI. CONCLUSION: Our present phosphoproteome data may provide the clue to understand the molecular mechanisms for the activation of human MCs.

Int Arch Allergy Immunol. 2009; 149 Suppl 1: 66-72
Endo D, Gon Y, Nunomura S, Yamashita K, Hashimoto S, Ra C

Phosphatidylinositol 3-kinase (PI3K) has been recognized as an important downstream effector of high-affinity receptor for IgE (FcepsilonRI) signaling in mast cells, but little is known about the isoform specificity of PI3Ks on the FcepsilonRI-mediated migration toward the antigen (Ag). In the present study, we explored the role of PI3Kgamma on mast cell migration. The treatment of bone marrow-deRIved mast cells (BMMCs) with a PI3Kgamma inhibitor, AS605240, significantly repressed FcepsilonRI-induced degranulation and migration. The culture supernatants of wild-type mast cells stimulated with IgE and Ag attracted FcepsilonRIbeta(-/-) mast cells which did not express FcepsilonRI on their cell surface, indicating that the migration appears to be dependent on an autocRIne/paracRIne secretion of soluble factors from the mast cells. Adenosine, which is produced by mast cells, showed a strong activity to attract mast cells. Pertussis toxin (PTX) significantly inhibited the migration toward both the supernatant and adenosine. The supernatant from mast cells pretreated with wortmannin (Wort) and stimulated with IgE and Ag still exhibited the activity as chemoattractant, while the BMMCs pretreated with Wort did not migrate toward the supernatant. Although PTX significantly reduced the activation of AKT/PKB and migration, PTX had no effects on degranulation. These results suggest that PI3Kgamma activation through PTX-sensitive G-protein-coupled receptor as a secondary response of FcepsilonRI cross-linking regulates FcepsilonRI-mediated mast cell migration toward the Ag, while simultaneously activated PI3Kgamma through a PTX-insensitive pathway might have an effect on degranulation.