FcεRI-mediated Mast Cell Activation Research Articles

Role of CCL7 in Type I Hypersensitivity Reactions in Murine Experimental Allergic Conjunctivitis.

Molecules that are necessary for ocular hypersensitivity reactions include the receptors CCR1 and CCR3; CCL7 is a ligand for these receptors. Therefore, we explored the role of CCL7 in mast cell activity and motility in vitro and investigated the requirement for CCL7 in a murine model of IgE-mediated allergic conjunctivitis. For mast cells treated with IgE and Ag, the presence of CCL7 synergistically enhanced degranulation and calcium influx. CCL7 also induced chemotaxis in mast cells. CCL7-deficient bone marrow-derived mast cells showed decreased degranulation following IgE and Ag treatment compared with wild-type bone marrow-derived mast cells, but there was no difference in degranulation when cells were activated via an IgE-independent pathway. In vivo, CCL7 was upregulated in conjunctival tissue during an OVA-induced allergic response. Notably, the early-phase clinical symptoms in the conjunctiva after OVA challenge were significantly higher in OVA-sensitized wild-type mice than in control challenged wild-type mice; the increase was suppressed in CCL7-deficient mice. In the OVA-induced allergic response, the numbers of conjunctival mast cells were lower in CCL7-deficient mice than in wild-type mice. Our results demonstrate that CCL7 is required for maximal OVA-induced ocular anaphylaxis, mast cell recruitment in vivo, and maximal FcεRI-mediated mast cell activation in vitro. A better understanding of the role of CCL7 in mediating ocular hypersensitivity reactions will provide insights into mast cell function and novel treatments for allergic ocular diseases.

Tetraspanin CD151 Is a Negative Regulator of FcεRI-Mediated Mast Cell Activation.

Mast cells are critical in the pathogenesis of allergic disease due to the release of preformed and newly synthesized mediators, yet the mechanisms controlling mast cell activation are not well understood. Members of the tetraspanin family are recently emerging as modulators of FcεRI-mediated mast cell activation; however, mechanistic understanding of their function is currently lacking. The tetraspanin CD151 is a poorly understood member of this family and is specifically induced on mouse and human mast cells upon FcεRI aggregation but its functional effects are unknown. In this study, we show that CD151 deficiency significantly exacerbates the IgE-mediated late phase inflammation in a murine model of passive cutaneous anaphylaxis. Ex vivo, FcεRI stimulation of bone marrow-derived mast cells from CD151(-/-) mice resulted in significantly enhanced expression of proinflammatory cytokines IL-4, IL-13, and TNF-α compared with wild-type controls. However, FcεRI-induced mast cell degranulation was unaffected. At the molecular signaling level, CD151 selectively regulated IgE-induced activation of ERK1/2 and PI3K, associated with cytokine production, but had no effect on the phospholipase Cγ1 signaling, associated with degranulation. Collectively, our data indicate that CD151 exerts negative regulation over IgE-induced late phase responses and cytokine production in mast cells.

ERK1/2 antagonize AMPK-dependent regulation of FcεRI-mediated mast cell activation and anaphylaxis

Extracellular signal-regulated kinases 1/2 (ERK1/2) make important contributions to allergic responses via their regulation of degranulation, eicosanoid production, and cytokine expression by mast cells, yet the mechanisms underlying their positive effects on FcεRI-dependent signaling are not fully understood. Recently, we reported that mast cell activation and anaphylaxis are negatively regulated by AMP-activated protein kinase (AMPK). However, little is known about the relationship between ERK1/2-mediated positive and the AMPK-mediated negative regulation of FcεRI signaling in mast cells. We investigated possible interactions between ERK1/2 and AMPK in the modulation of mast cell signaling and anaphylaxis. Wild-type or AMPKα2(-/-) mice, or bone marrow-derived mast cells obtained from these mice, were treated with either chemical agents or small interfering RNAs that modulated the activity or expression of ERK1/2 or AMPK to evaluate the functional interplay between ERK1/2 and AMPK in FcεRI-dependent signaling. The ERK1/2 pathway inhibitor U0126 and the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside similarly inhibited FcεRI-mediated mast cell signals in vitro and anaphylaxis in vivo. ERK1/2-specific small interfering RNA also mimicked this effect on FcεRI signals. Moreover, AMPKα2 knockdown or deficiency led to increased FcεRI-mediated mast cell activation and anaphylaxis that were insensitive to U0126 or activator 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside, suggesting that the suppression of FcεRI signals by the inhibition of the ERK1/2 pathway relies largely on AMPK activation. ERK1/2 controlled AMPK activity by regulating its subcellular translocation. ERK1/2 ablated the AMPK-dependent negative regulatory axis, thereby activating FcεRI signals in mast cells.

Protein tyrosine phosphatase-PEST (PTP-PEST) regulates mast cell-activating signals in PTP activity-dependent and -independent manners

Aggregation of the high-affinity IgE receptor (FcεRI) in mast cells leads to degranulation and production of numerous cytokines and lipid mediators that promote allergic inflammation. Tyrosine phosphorylation of proteins in response to FcεRI aggregation has been implicated in mast cell activation. Here, we determined the role of PTP-PEST (encoded by PTPN12) in the regulation of mast cell activation using the RBL-2H3 rat basophilic leukemia cell line as a model. PTP-PEST expression was significantly induced upon FcεRI-crosslinking, and aggregation of FcεRI induced the phosphorylation of PTP-PEST at Ser39, thus resulting in the suppression of PTP activity. By overexpressing a phosphatase-dead mutant (PTP-PEST CS) and a constitutively active mutant (PTP-PEST SA) in RBL-2H3 cells, we showed that PTP-PEST decreased degranulation and enhanced IL-4 and IL-13 transcription in FcεRI-crosslinked RBL-2H3 cells, but PTP activity of PTP-PEST was not necessary for this regulation. However, FcεRI-induced TNF-α transcription was increased by the overexpression of PTP-PEST SA and suppressed by the overexpression of PTP-PEST CS. Taken together, these results suggest that PTP-PEST is involved in the regulation of FcεRI-mediated mast cell activation through at least two different processes represented by PTP activity-dependent and -independent pathways.

Highly expressed cytoplasmic FcεRIβ in human mast cells functions as a negative regulator of the FcRγ‐mediated cell activation signal

We recently reported that the interaction between Lyn and FcεRIβ is indispensable for FcεRI-mediated human mast cell (MC) activation and that FcεRIβ functions as an amplifier of FcεRI-mediated activation signal. Some of FcεRIβ in cytoplasm appeared not to be co-localized with FcεRIα. The function of FcεRIβ in the cytoplasm remains unknown. The localization of FcεRIβ and FcεRIα in giant papillae specimens from patients with allergic keratoconjunctivitis and of FcεRIβ, FcεRIα, and Lyn in cultured human MCs was examined using confocal microscopy. FcεRIβ was overexpressed using an adenovirus vector system. Mediators were measured by enzyme immunoassays or enzyme-linked immunosorbent assays. In the subepithelial region, FcεRIβ was mainly localized in the cell membrane of MCs. In the perivascular region, FcεRIβ expression was scattered throughout the cytoplasm and in the cell membrane of MCs. Overexpression of FcεRIβ in MCs mainly increased its cytoplasmic expression and slightly up-regulated cell surface FcεRI expression. However, overexpression of FcεRIβ in MCs resulted in down-regulation of the tyrosine phosphorylation levels of FcεRIβ and Syk and down-regulation of the Ca(2+) influx soon after FcεRI aggregation and then resulted in down-regulation of degranulation, PGD2 synthesis, and production of a set of cytokines. This negative regulatory effect may be due to inhibition of the redistribution of Lyn to small patches within the plasma membrane. Cytoplasmic FcεRIβ, which is not co-localized with FcεRIα, may function as a negative regulator, as it can capture important signalling molecules such as Lyn.

Sphingomyelin and ceramide are physiological ligands for human LMIR3/CD300f, inhibiting FcεRI-mediated mast cell activation

Leukocyte mono-immunoglobulin-like receptor (LMIR) belongs to a paired activating and inhibitory receptor family.1-3 LMIR is also known to be a CMRF-35-like molecule, a myeloid-associated immunoglobulin-like receptor, an immune receptor expressed on myeloid cells, or CD300.4-7 Mouse LMIR3 (mLMIR3)/CD300f is an inhibitory receptor containing 2 immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a single immunoreceptor tyrosine-based switch motif (ITSM).1,2 We have recently demonstrated that extracellular ceramide is a ligand for mLMIR3/CD300f, which is a negative regulator of high-affinity IgE receptor (FcεRI)-mediated activation of mast cells (MCs).

Microfilaments make mast cells migrate (rather than degranulate)

Expression of the high-affinity receptor for IgE (FcεRI) provides mast cells with the ability to react in a proinflammatory manner to antigens (Ags). In particular, the immediate secretion of preformed mediators from secretory lysosomes (degranulation) is typical for FcεRI-mediated mast cell activation. In addition to the FcεRI, the stem cell factor receptor, KIT, is expressed at high levels on the surface of mast cells. KIT activation controls mast cell differentiation and survival in vivo and potently stimulates the chemotaxis of these cells. Although FcεRI and KIT initiate many of the same early signaling events in mast cells, FcεRI activation results in potent degranulation and a poor chemotactic response while KIT activation triggers very little degranulation and a strong chemotactic response. Novel data published in this issue of the European Journal of Immunology [Smrž et al. Eur. J. Immunol. 2013. 43: 1873-1882] demonstrate that actin de- and repolymerization, involved in both degranulation and chemotaxis, make all the difference: Pharmacological suppression of F-actin formation converts activated KIT into a strong degranulator. The possible implications for mast cell physiology and pathophysiology are discussed in this Commentary.

Regulation of FcϵRI-mediated signaling and mast cell activation by adaptor protein Tespa1 (P1068)

Abstract Tespa1 (thymocyte-expressed, positive selection-associated 1) is a newly identified adaptor protein that is expressed in T cells, B cells and mast cells. Our previous study revealed its critical role in mediating TCR signaling and thymocyte positive selection. Tespa1-deficient mice have fewer mature CD4+ and CD8+ SP cells in thymus, reflecting the impairment of T cell development in these mice. We further revealed that Tespa1 interacts with Lat signolosome components Grb2 and PLC-γ1 and is essential for the assembly of this signolosome and mediating of downstream signals. Tespa1 is also highly expressed in Mast cells. Here we further explored the role of Tespa1 in Mast cell activation. We found that disruption of Tespa1 causes mast cells to be hyperresponsive upon the stimulation of FceRI receptor, evidenced by enhanced degranulation, cytokine production, and their ability to mediate anaphylaxis. We also found that more Grb2 and PLC-γ1 were recruited to the LAT1 signolosome rather than LAT2 signolosome, suggesting Tespa1 might regulate the balance of LAT1 and LAT2 signolosome assembly and downstream distal signaling transduction in mast cells.

Tetraspanin CD151 is a negative regulator of FcϵRI-mediated mast cell activation (P6018)

Abstract Mast cells are critical in the pathogenesis of allergic disease due to the release of preformed and newly synthesized mediators, but the mechanisms controlling mast cell activation are not well understood. The tetraspanin family has recently emerged as novel signaling molecules and negative regulators of “outside-in” signals for mast cell activation. Specifically, antibodies against tetraspanins CD63 or CD81 can inhibit FcϵRI-mediated mast cells degranulation. CD151, a poorly understood member of this famility, is specifically induced in human mast cells upon FcϵRI aggregation, however, the functional significance of this response is not yet known. In this study, we found that CD151 deficiency significantly exacerbated the IgE-mediated late phase inflammation in a murine model of passive cutaneous anaphylaxis. Bone marrow-derived mast cells from CD151-deficient mice exhibited significantly enhanced de novo synthesis of pro-inflammatory cytokines IL-3, IL-4, IL-13 and TNF-α compared to controls. However, FcϵRI-induced mast cell degranulation from these mice was unaffected. At the molecular signaling level, CD151 appeared to selectively regulate IgE-induced activation of Syk and ERK1/2 associated with cytokine production but had no effect on the PLCγ1 pathway associated with degranulation. Collectively, our data indicate that tetraspanin CD151 exerts a selective negative regulation over IgE-induced late phase anaphylactic reaction and cytokine production in mast cells.

IgE production in CD40/CD40L cross-talk of B and mast cells and mediator release via TGase 2 in mouse allergic asthma

TGase 2 is over-expressed in a variety of inflammatory diseases including allergic asthma. This study aimed to investigate the role of TGase 2 on IgE production and signaling pathways in mast cell activation related to OVA-induced allergic asthma. Bone marrow-derived mast cells (BMMCs) isolated from WT or TGase 2−/− mice were activated with Ag/Ab (refer to act-WT-BMMCs and act-KO-BMMCs, respectively). B cells isolated from splenocytes were activated with anti-mouse IgM (act-B cells), and B cells were co-cultured with BMMCs. WT and TGase 2−/− mice were sensitized and challenged with OVA adsorbed in alum hydroxide. Intracellular Ca2+ ([Ca2+]i) levels were determined by fluorescence intensity; IgE, mediators and TGase 2 activity by ELISA; the CD138 expression by FACS analyzer; cell surface markers and signal molecules by Western blot; NF-κB by EMSA; co-localization of mast cells and B cells by immunohistochemistry; Fcε RI-mediated mast cell activation by PCA test; expression of cytokines, MMPs, TIMPs, TLR2 and FcɛRI by RT-PCR. In vitro, act-KO-BMMCs reduced the [Ca2+]i levels, NF-κB activity, expression of CD40/CD40L, plasma cells, total IgE levels and TGase 2 activity in act-B cells co-cultured with act-BMMCs, expression of inflammatory cytokines and MMPs2/9, release of mediators (TNF-α, LTs and cytokines), and activities of signal molecules (PKCs, MAP kinases, I-κB and PLA2), which were all increased in act-WT-BMMCs. TGase 2 siRNA transfected/activated-BMMCs reduced all responses as same as those in act-KO-BMMCs. In allergic asthma model, TGase 2−/− mice protected against PCA reaction, OVA-specific IgE production and AHR, and they reduced co-localization of mast cells and B cells or IgE in lung tissues, expression and co-localization of surface molecules in mast cells (c-kit and CD40L) and B cells (CD23 and CD40), inflammatory cells including mast cells, goblet cells, amounts of collagen and mediator release in BAL fluid and/or lung tissues, which were all increased in WT mice. TLR expression in TGase 2−/− mice did not differ from those in WT mice. Our data suggest that TGase 2 expression and Ca2+ influx required by bidirectional events in mast cell activation facilitate IgE production in B cells via up-regulating mast cell CD40L expression, and induce the expression of numerous signaling molecules associated with airway inflammation and remodeling in allergic asthma.

The Receptor LMIR3 Negatively Regulates Mast Cell Activation and Allergic Responses by Binding to Extracellular Ceramide

Mast cells (MCs) are key effector cells in allergic reactions. However, the inhibitory mechanism that prevents excessive activation of MCs remains elusive. Here we show that leukocyte mono-immunoglobulin-like receptor 3 (LMIR3; also called CD300f) is a negative regulator of MC activation in vivo. LMIR3 deficiency exacerbated MC-dependent allergic responses in mice, including anaphylaxis, airway inflammation, and dermatitis. Both physical binding and functional reporter assays via an extracellular domain of LMIR3 showed that several extracellular lipids (including ceramide) and lipoproteins were possible ligands for LMIR3. Importantly, MCs were frequently surrounded by extracellular ceramide in vivo. Upon engagement of high-affinity immunoglobulin E receptor, extracellular ceramide-LMIR3 binding inhibited MC activation via immunoreceptor tyrosine-based inhibitory and switch motifs of LMIR3. Moreover, pretreatment with LMIR3-Fc fusion protein or antibody against either ceramide or LMIR3 interfered with this binding in vivo, thereby exacerbating passive cutaneous anaphylaxis. Thus, the interaction between extracellular ceramide and LMIR3 suppressed MC-dependent allergic responses.

The interaction between Lyn and FcεRIβ is indispensable for FcεRI-mediated human mast cell activation.

FcεRIβ reportedly functions as an amplifier of the FcεRIγ-mediated activation signal using a reconstitution system. However, the amplification mechanisms in human mast cells (MCs) are poorly understood. We previously reported the hyperexpression of FcεRIβ of MCs in giant papillae from vernal keratoconjunctivitis patients, compared with that in conjunctivae from nonallergic conjunctivitis patients. Elucidation of the molecular mechanisms of the amplification induced by FcεRIβ should provide new targets for novel therapeutic interventions. The aim is to understand in greater details the function of FcεRIβ in human MC FcεRI expression and signaling.FcεRIβ and Lyn expression was reduced using a lentiviral shRNA silencing technique. Localization of Lyn and FcεRIβ in cultured MCs was examined by confocal microscopic analysis. Mediators were measured by ELISAs.The diminution of FcεRIβ significantly downregulated cell surface FcεRI expression and FcεRI-mediated mediator release/production. The downregulation of FcεRI-mediated degranulation was not only due to the decrease in FcεRI expression. The diminution of FcεRIβ inhibited the redistribution of Lyn within the cell membrane following IgE sensitization. The diminution of Lyn in MCs significantly downregulated FcεRI-mediated degranulation. The recombinant cell-penetrating forms of phosphorylated FcεRIβ immunoreceptor tyrosine-based activation motif (ITAM) for intracellular delivery disturbed the interaction between Lyn and phosphorylated endogenous FcεRIβ ITAM, resulted in inhibiting IgE-dependent histamine release from MCs in vitro and from giant papillae specimens ex vivo.The interaction between Lyn and FcεRIβ is indispensable for FcεRI-mediated human MC activation, and specific inhibition of the interaction may represent a new therapeutic strategy for the treatment of human allergic diseases.

MAPK Kinase 3 Specifically Regulates FcεRI-Mediated IL-4 Production by Mast Cells

Mast cells play a central role in allergic inflammation and are activated through cross-linking of FcεRI receptor-bound IgE, initiating a signaling cascade resulting in production of biologically potent mediators. Signaling pathways in the regulation of specific mediators remain incompletely defined. In this study, we examined the role of MAPK kinase 3 (MKK3) in IgE-dependent mast cell activation. In an in vivo model of passive cutaneous anaphylaxis, MKK3-deficient mice showed a deficit in late-phase IgE-dependent inflammation. To characterize the mechanism of this deficiency, we cultured bone marrow-derived mast cells (BMMCs) from wild-type and MKK3-deficient mice. We found that FcεRI-mediated mast cell activation induced rapid MKK3 phosphorylation by 5 min, diminishing slowly after 6 h. In MKK3-deficient BMMCs, phosphorylation of p38 was reduced at early and later time points. Among 40 cytokines tested using a protein array, IL-4 was the only cytokine specifically downregulated in MKK3-deficient BMMCs. Reduced IL-4 expression was seen in the local skin of MKK3-deficient mice following passive cutaneous allergic reaction. Furthermore, early growth response-1 (Egr1) bound to the promoter of IL-4 in FcεRI-activated mast cells, and Egr1 transcription factor activity was diminished in MKK3-deficient BMMCs. Finally, mast cell-deficient mice reconstituted with MKK3-deficient BMMCs displayed a significantly impaired late-phase allergic inflammatory response. Thus, mast cell MKK3 signaling contributes to IgE-dependent allergic inflammation and is a specific regulator of FcεRI-induced IL-4 production.

PTPα Activates Lyn and Fyn and Suppresses Hck to Negatively Regulate FcεRI-Dependent Mast Cell Activation and Allergic Responses

Mast cell activation via FcεRI involves activation of the Src family kinases (SFKs) Lyn, Fyn, and Hck that positively or, in the case of Lyn, negatively regulate cellular responses. Little is known of upstream activators of these SFKs in FcεRI-dependent signaling. We investigated the role of receptor protein tyrosine phosphatase (PTP)α, a well-known activator of SFKs in diverse signaling systems, FcεRI-mediated mast cell activation, and IgE-dependent allergic responses in mice. PTPα(-/-) bone marrow-derived mast cells hyperdegranulate and exhibit increased cytokine and cysteinyl leukotriene secretion, and PTPα(-/-) mice display enhanced IgE-dependent anaphylaxis. At or proximal to FcεRI, PTPα(-/-) cells have reduced IgE-dependent activation of Lyn and Fyn, as well as reduced FcεRI and SHIP phosphorylation. In contrast, Hck and Syk activation is enhanced. Syk hyperactivation correlated with its increased phosphorylation at positive regulatory sites and defective phosphorylation at a negative regulatory site. Distal to FcεRI, we observed increased activation of PI3K and MAPK pathways. These findings demonstrate that PTPα activates the FcεRI-coupled kinases Lyn and Fyn and suppresses Hck activity. Furthermore, the findings indicate that hyperactivation of PTPα(-/-) mast cells and enhanced IgE-dependent allergic responses of PTPα(-/-) mice are due to the ablated function of PTPα as a critical regulator of Lyn negative signaling.

Independent and Cooperative Roles of Adaptor Molecules in Proximal Signaling during FcεRI-Mediated Mast Cell Activation

Activation through FcepsilonRI, a high-affinity IgE-binding receptor, is critical for mast cell function during allergy. The formation of a multimolecular proximal signaling complex nucleated by the adaptor molecules SLP-76 and LAT1 is required for activation through this receptor. Based on previous T-cell studies, current dogma dictates that LAT1 is required for plasma membrane recruitment and function of SLP-76. Unexpectedly, we found that the recruitment and phosphorylation of SLP-76 were preserved in LAT1(-/-) mast cells and that SLP-76(-/-) and LAT1(-/-) mast cells harbored distinct functional and biochemical defects. The LAT1-like molecule LAT2 was responsible for the preserved membrane localization and phosphorylation of SLP-76 in LAT1(-/-) mast cells. Although LAT2 supported SLP-76 phosphorylation and recruitment to the plasma membrane, LAT2 only partially compensated for LAT1-mediated cell signaling due to its decreased ability to stabilize interactions with phospholipase Cgamma (PLCgamma). Comparison of SLP-76(-/-) LAT1(-/-) and SLP-76(-/-) mast cells revealed that some functions of LAT1 could occur independently of SLP-76. We propose that while SLP-76 and LAT1 depend on each other for many of their functions, LAT2/SLP-76 interactions and SLP-76-independent LAT1 functions also mediate a positive signaling pathway downstream of FcepsilonRI in mast cells.

Egr1-Rcan1 axis serves as a molecular switch from activation to inhibition in IgE-mediated signalling (139.3)

Abstract Mast cells play a central role in allergy. Aggregation of FcεRI activates a cascade of signaling events leading to mast cell activation. Subsequently, inhibitory signals are switched on for turning off activated signals. Here we identified that Rcan1 serves as a negative regulator for turning off FcεRI-mediated mast cell activation. FcεRI-induced Rcan1 expression was identified by suppression subtractive hybridization and verified by real-time quantitative PCR and Western blotting. Deficiency of Rcan1 led to increased calcineurin activity, increased NFAT and NF-κB activation, increased cytokine production and enhanced IgE-mediated late phase cutaneous reactions. Forced expression of Rcan1 in wild type or Rcan1 deficient mast cells reduced FcεRI-mediated cytokine production in vitro. Rcan1 deficiency also led to increased FcεRI-mediated mast cell degranulation and enhanced passive cutaneous anaphylaxis. Analysis of the Rcan1 promoter identified a functional Egr1 binding site. Rcan1 expression is preceded by Egr1 expression. Egr1 is a positive regulator of FcεRI-induced cytokine production. Biochemical and genetic evidence suggested that Egr1 controls Rcan1 expression. Our results identified Rcan1 as a novel inhibitory signal in FcεRI-induced mast cell activation and established a new link of Egr1 and Rcan1 in FcεRI signaling.

TRAF6 Specifically Contributes to FcϵRI-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.

Procyanidin C1 from Apple Extracts Inhibits FcεRI-Mediated Mast Cell Activation

Background: Polyphenol-enriched fractions, which are extracted from unripe apples (Rosaceae, Malus spp.), consisting of procyanidins (polymers of catechins) are known to have an anti-allergenic effect on patients with various allergic diseases. Although it has been reported that apple extracts inhibit histamine release from mast cells, the molecular mechanisms for this anti-allergenic effect are not well understood. To elucidate the molecular mechanisms by which apple extracts induce their anti-allergenic effects, the effects of purified apple extract components on high-affinity receptors for IgE (FcΕRI)-mediated mast cell activation were investigated. Methods: The anti-allergic effect of oral administration of apple procyanidin extracts on passive cutaneous anaphylactic responses of BALB/c mice was assessed. We evaluated the effects of procyanidin C1 (PC1) [epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin], a component of the procyanidin fraction, on mouse bone-marrow-derived mast cell degranulation, cytokine production, protein tyrosine phosphorylation and on the generation of intracellular reactive oxygen species (ROS) of cells stimulated by FcΕRI cross-linking in vitro. Results: In an in vivo study, oral administration of the procyanidin fraction suppressed the mast-cell-dependent allergic reaction. In in vitro studies, PC1 dose-dependently decreased FcΕRI-mediated degranulation and cytokine production of mast cells. Furthermore, PC1 inhibited tyrosine phosphorylation of Syk and linker for activation of T cells, and the ROS generation in stimulated mast cells. Conclusions: PC1 suppresses FcΕRI-mediated mast cell activation by inhibiting intracellular signaling pathways. These observations provide evidence for the anti-allergenic effects of the procyanidin-enriched apple extract.

PICOT, protein kinase C θ-interacting protein, is a novel regulator of FcεRI-mediated mast cell activation

PICOT (PKC-interacting cousin of thioredoxin) consists of one thioredoxin homology domain in the N-terminal and two tandem PICOT homology domains in the C-terminal. PICOT specifically interacts with protein kinase C θ (PKC-θ) via its thioredoxin homology domain and acts as an important modulator of T cell receptor (TCR)-signaling. Using PICOT overexpressing rat basophilic leukemia cells (RBL-2H3), we evaluated the effect of PICOT overexpression on the FcεRI-mediated signaling. In comparison to the control cells, introduction of PICOT to RBL-2H3 cells induced increased degranulation and the activation of NFAT and in the expression of IL-4 and TNF-α transcripts by FcεRI-crosslinking, whereas no significant change was observed with the elevation of ERK1/2 and p38 MAP kinase phosphorylation and NF-κB activation by FcεRI aggregation. More interesting was the exogenous PICOT overexpression in RBL-2H3 cells causing a large decrease in the elevation of JNK phosphorylation. PICOT-regulated FcεRI-mediated signals in RBL-2H3 cells and acted as a positive regulator on IL-4 and TNF-α expression, NFAT and degranulation signal pathways and a negative regulator on a JNK signal pathway. Considering that PICOT has no enzymatic activity, the regulation of PICOT on FcεRI-signaling may depend on PICOT-associated molecule(s).

Zinc Is Required for FcεRI-Mediated Mast Cell Activation

Zinc (Zn) is an essential nutrient, and its deficiency causes growth retardation, immunodeficiency, and neuronal degeneration. However, the precise roles and molecular mechanism(s) of Zn function in immune response have not been clarified. Mast cells (MCs) are granulated cells that play a pivotal role in allergic reactions and inflammation. The granules of MCs contain various chemical mediators and inflammatory cytokines that are released upon FcepsilonRI cross-linking. In this study, we report that Zn is essential for MC activation both in vitro and in vivo. We showed that a Zn chelator, N,N,N,N-tetrakis (2-pyridylmethyl) ethylenediamine, inhibited in vivo allergic reactions such as PCA and PSA. Consistent with this, N,N,N,N-tetrakis (2-pyridylmethyl) ethylenediamine significantly inhibited the FcepsilonRI-induced degranulation and cytokine production. We found that Zn was required for FcepsilonRI-induced translocation of granules to the plasma membrane, a process that we have shown to be important for MC degranulation. In addition, we showed that Zn was essential for plasma membrane translocation of protein kinase C and subsequent nuclear translocation of NF-kappaB, leading to cytokine production, such as IL-6 and TNF-alpha. These results revealed that Zn was involved in multiple steps of FcepsilonRI-induced MC activation and required for degranulation and cytokine production.