Mast Cell Cytokine Production Research Articles

Ethyl acetate extract of Psidium guajava inhibits IgE-mediated allergic responses by blocking FcεRI signaling

Psidium guajava (P. guajava) is an important food crop and medicinal plant with antioxidant, anti-inflammatory, and anti-allergic activities, supporting its traditional uses. However, its precise effects remain unknown. We investigated the effects of P. guajava ethyl acetate extract (PGEA) on IgE-mediated allergic responses in rat mast RBL-2H3 cells. PGEA reduced antigen (DNP–BSA)-induced release of β-hexosaminidase and histamine in IgE-sensitized RBL-2H3 cells. In addition, it inhibited antigen-induced IL-4 and TNF-α mRNA expression and protein production in IgE-sensitized RBL-2H3 cells. PGEA also suppressed antigen-induced COX-2 mRNA and protein expression in these cells, as well as antigen-induced activation of NFAT and reactive oxygen species. Moreover, it inhibited antigen-induced activation of NF-κB and degradation of IκB-α. To identify the mechanisms underpinning the inhibition of degranulation and cytokine production by PGEA, we examined the activation of intracellular FcεRI signaling molecules. PGEA suppressed antigen-induced phosphorylation of Syk, LAT, Gab2, and PLCγ2 but not Lyn, and inhibited antigen-induced phosphorylation of downstream signaling intermediates including MAP kinases and Akt. Collectively, the anti-allergic effects of PGEA in vitro suggest its possible therapeutic application to inflammatory allergic diseases, in which its inhibition of inflammatory cytokine production and FcεRI-dependent signaling events in mast cells may be hugely beneficial.

IL-33 Is Produced by Mast Cells and Regulates IgE-Dependent Inflammation

BackgroundIL-33 is a recently characterized IL-1 family cytokine and found to be expressed in inflammatory diseases, including severe asthma and inflammatory bowl disease. Recombinant IL-33 has been shown to enhance Th2-associated immune responses and potently increase mast cell proliferation and cytokine production. While IL-33 is constitutively expressed in endothelial and epithelial cells, where it may function as a transcriptional regulator, cellular sources of IL-33 and its role in inflammation remain unclear.Methodology/Principal FindingsHere, we identify mast cells as IL-33 producing cells. IgE/antigen activation of bone marrow-derived mast cells or a murine mast cell line (MC/9) significantly enhanced IL-33. Conversely, recombinant IL-33 directly activated mast cells to produce several cytokines including IL-4, IL-5 and IL-6 but not IL-33. We show that expression of IL-33 in response to IgE-activation required calcium and that ionomycin was sufficient to induce IL-33. In vivo, peritoneal mast cells expressed IL-33 and IL-33 levels were significantly lower within the skin of mast cell deficient mice, compared to littermate controls. Local activation of mast cells promotes edema, followed by the recruitment of inflammatory cells. We demonstrate using passive cutaneous anaphylaxis, a mast cell-dependent model, that deficiency in ST2 or antibody blockage of ST2 or IL-33 ablated the late phase inflammatory response but that the immediate phase response was unaffected. IL-33 levels in the skin were significantly elevated only during the late phase.Conclusions/SignificanceOur findings demonstrate that mast cells produce IL-33 after IgE-mediated activation and that the IL-33/ST2 pathway is critical for the progression of IgE-dependent inflammation.

A New Class of Human Mast Cell and Peripheral Blood Basophil Stabilizers that Differentially Control Allergic Mediator Release

Treatments for allergic disease block the effects of mediators released from activated mast cells and blood basophils. A panel of fullerene derivatives was synthesized and tested for their ability to preempt the release of allergic mediators in vitro and in vivo. The fullerene C(70)-tetraglycolic acid significantly inhibited degranulation and cytokine production from mast cells and basophils, while C(70)-tetrainositol blocked only cytokine production in mast cells and degranulation and cytokine production in basophils. The early phase of FcepsilonRI inhibition was dependent on the blunted release of intracellular calcium stores, elevations in reactive oxygen species, and several signaling molecules. Gene microarray studies further showed the two fullerene derivatives inhibited late phase responses in very different ways. C(70)-tetraglycolic acid was able to block mast cell-driven anaphylaxis in vivo, while C(70)-tetrainositol did not. No toxicity was observed with either compound. These findings demonstrate the biological effects of fullerenes critically depends on the moieties added to the carbon cage and suggest they act on different FcepsilonRI-specific molecules in mast cells and basophils. These next generation fullerene derivatives represent a new class of compounds that interfere with FcepsilonRI signaling pathways to stabilize mast cells and basophils. Thus, fullerene-based therapies may be a new approach for treating allergic diseases.

Effects of propolis from different areas on mast cell degranulation and identification of the effective components in propolis

Propolis is considered to down-regulate type I allergy, but the effective components of propolis remain unknown. In addition, propolis components vary depending on the area from which they are collected due to variations among wild plants in an area. Therefore, we compared the effects of water and ethanol extracts of propolis from Brazil and China on mast cell degranulation and cytokine production, thereby identifying effective components in propolis. The amount of released β-hexosaminidase via high-affinity IgE receptor I (FcεRI) from rat basophilic leukemia (RBL-2H3) cells was used as an index of degranulation. All propolis extracts inhibited degranulation from antigen-stimulated RBL-2H3 cells, but the effective doses differed according to collection areas. The ethanol extract of Chinese propolis, which was the strongest inhibitor of mast cell degranulation, was divided into compounds using normal- and reversed-phase liquid chromatography. The isolated anti-allergic components were identified as chrysin, kaempferol and its derivative, and chrysin was revealed to inhibit IL-4 and MCP-1 production from antigen-stimulated RBL-2H3 cells. HPLC quantification also revealed the Brazilian propolis extract to contain only small amounts of these flavonoids, which suggested that variation in propolis components could affect anti-allergic properties.

The receptor tyrosine kinase c-Kit controls IL-33 receptor signaling in mast cells

AbstractMembers of the Toll/interleukin-1 receptor (TIR) family are of importance for host defense and inflammation. Here we report that the TIR-family member interleukin-33R (IL-33R) cross-activates the receptor tyrosine kinase c-Kit in human and murine mast cells. The IL-33R–induced activation of signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase B (PKB), and Jun NH2-terminal kinase 1 (JNK1) depends on c-Kit and is required to elicit optimal effector functions. Costimulation with the c-Kit ligand stem cell factor (SCF) is necessary for IL-33–induced cytokine production in primary mast cells. The structural basis for this cross-activation is the complex formation between c-Kit, IL-33R, and IL-1R accessory protein (IL-1RAcP). We found that c-Kit and IL-1RAcP interact constitutively and that IL-33R joins this complex upon ligand binding. Our findings support a model in which signals from seemingly disparate receptors are integrated for full cellular responses.

Inhibition of IgE and IgE/anti-IgE mediated responses in mast cells by Omalizumab

BackgroundIgE binding via the high affinity FceRI receptor modu-lates FceRI expression and cytokine production in mastcells. Antigen crosslinking of bound IgE further activatesmast cells, inducing degranulation and inflammatorymediator release. Omalizumab (Xolair; Genentech Inc)is a recombinant human monoclonal anti-IgE antibodythat prevents IgE binding to FceRI.ObjectiveWe investigated the effects of omalizumab on IgE-mediated responses in human mast cells.MethodsLAD2 and CD34

The role of MyD88 in the synergy between FcϵR1, TLR4 and ST2 in mast cells. (91.5)

Abstract Mast cells are well established as important effector cells in allergic reactions. Cross-linking of the high affinity IgE receptor (FcϵRI) on mast cells by allergen causes degranulation leading to the release and generation of a variety of inflammatory mediators. IL-33 and lipopolysaccharide (LPS) are members of the IL-1 family that use MyD88 as an adaptor molecule for signalling. Alone, IL-33 and LPS are unable to cause mast cell degranulation but they can induce cytokine generation. In addition, IL-33 and LPS synergise with antigen/IgE for cytokine production in mast cells. IL-33 and LPS-induced cytokine production was abolished in MyD88-deficient (MyD88-/-) bone marrow-derived mast cells (BMMCs). Surprisingly, antigen/IgE-mediated cytokine production was also substantially reduced in MyD88-/- BMMCs. However, MyD88 had no effect on FcϵRI expression, calcium mobilisation or degranulation. The synergy between IL-33 and antigen/IgE in IL-13 production by BMMCs was shown to be MyD88-dependent. Similarly, MyD88 was required for cytokine production in LPS and antigen/IgE stimulated BMMCs. The synergy between LPS and antigen/IgE for cytokine generation also required p38 MAP kinase activation. These findings suggest that MyD88 signalling in mast cells could provide a novel target to modulate allergic diseases.

Antagonistic Control of IgG-mediated Mast Cell Activation by Lyn and Fyn Kinases (86.8)

Abstract Mast cells are key players of the innate immune system. In addition to their role in atopic disease, mast cell involvement is strongly suspected in inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, and atherosclerosis. Once activated, they degranulate and produce cytokines, resulting in the recruitment of other immune cells. It is well-appreciated that mast cells are activated via the high-affinity immunoglobulin E (IgE) receptor FcϵRI. In this study we focus on mast cell activation via the immunoglobulin G (IgG) receptor FcγRII/RIII and downstream signaling by Fyn and Lyn kinases. Lyn-deficient mast cells displayed increased FcγR expression, correlating with enhanced IgG-mediated histamine and leukotriene C4 secretion. The mast cells did not show a change in IgG-mediated cytokine production, suggesting a restricted and negative regulatory role for Lyn kinase in mast cell IgG signaling. In contrast, we show that Fyn kinase is rapidly activated by IgG crosslinkage, and is required for both IgG-mediated histamine release and cytokine production in mast cells. We obtained similar results when studying Lyn- or Fyn-deficient basophils and macrophages. Finally, we found that Lyn-deficient mice had exacerbated responses to passive systemic anaphylactic shock elicited by IgG challenge in vivo. These data illustrate the importance of Src family kinases in IgG signaling, a means by which mast cells may be activated in chronic inflammatory diseases.

Contrasting requirements for DAP12 in receptor-mediated mast cell activation (86.4)

Abstract By inducing FcϵRI aggregation, antigen/IgE promotes mast cell cytokine production; a response markedly enhanced by specific TLR agonists. Critical to the ability of FcϵRI to activate mast cells is the recruitment of the tyrosine kinase Syk via the FcϵRIγ chains. Recent reports in other cell types have indicated that TLR’s may alternatively signal via the adaptor molecule DAP12 which also has the ability to bind Syk following tyrosine phosphorylation. We therefore examined whether DAP12 similarly regulates TLR-mediated responses in mast cells. DAP12 was confirmed to be expressed in both human and mouse mast cells (BMMCs) and, following its phosphorylation by pervanadate treatment, DAP12 recruited Syk. Although the TLR agonists LPS, Pam3Cys, and MALP2 induced cytokine production and synergistically enhanced this response mediated by FcϵRI, they failed to increase basal DAP12 phosphorylation. Furthermore, no up-regulation or down-regulation of TLR-mediated responses, nor FcϵRI-mediated responses, was observed in DAP12-/- BMMCs. In contrast, DAP12 phosphorylation and Syk recruitment were observed in mast cells following concanavalin A (Con A)-induced aggregation of mannose-glycosylated receptors, and these responses, together with ConA-induced degranulation, were substantially reduced in the DAP12-/- BMMCs. These data show that in mast cells DAP12 does not regulate TLR- or FcϵRI-mediated responses, but is required for responses elicited by mannose-glycosylated receptors.

Inhibition of degranulation and cytokine production in bone marrow-derived mast cells by hydrolyzed rice bran

We investigated the effects of hydrolyzed rice bran (HRB), an arabinoxylan extracted from rice bran, on mast cell degranulation and cytokine production. HRB was obtained by treating rice bran with an extract obtained from shiitake mushrooms. Bone marrow-derived mast cells (BMMCs) were prepared by culturing bone marrow cells from BALB/c mice in the presence of interleukin-3 and stem cell factor for 4 weeks. BMMCs were pretreated with HRB (0-3 mg/ml) for 30 min and were then antigen activated. Pretreatment of BMMCs with HRB significantly inhibited antigen-induced degranulation and cytokine production (tumor necrosis factor-alpha and interleukin-4) in a dose-dependent manner. HRB also diminished membrane fusion between liposomes in which soluble N-ethyl maleimide-sensitive factor attachment protein receptors were reconstituted. Phosphorylation of RelA and mitogen-activated kinases after antigen stimulation was suppressed by pretreatment of BMMCs with HRB. These findings suggest that HRB may have an anti-inflammatory effect by inhibiting mast cell degranulation and cytokine production.

A Minor Catalytic Activity of Src Family Kinases Is Sufficient for Maximal Activation of Mast Cells via the High-Affinity IgE Receptor

Src family kinases (SFK) are critical for initiating and regulating the response of mast cells activated by engagement of the high-affinity IgE receptor, FcepsilonRI. Lyn is the predominant SFK in mast cells and has been ascribed both positive and negative roles in regulating mast cell activation. We analyzed the mast cell phenotype of WeeB, a recently described mouse mutant that expresses a Lyn protein with profoundly reduced catalytic activity. Surprisingly, we found that this residual activity is sufficient for wild-type levels of cytokine production and degranulation in bone marrow-derived mast cells after low-intensity stimulation with anti-IgE. High-intensity stimulation of lyn(-/-) bone marrow-derived mast cells with highly multivalent Ag resulted in enhanced cytokine production as previously reported, and WeeB cells displayed an intermediate phenotype. Under this latter condition, SFK inhibition using PP2 increased cytokine production in wild-type and WeeB but not lyn(-/-) cells, resulting in substantially higher levels in the PP2-treated WeeB than in lyn(-/-) cells. Restoration of wild-type and WeeB lyn alleles in lyn(-/-) cells generated activation phenotypes similar to those in nontransduced wild-type and WeeB cells, respectively, whereas a kinase-dead allele resulted in a phenotype similar to that of empty-vector-transduced cells. These data indicate that inhibition of Lyn and/or SFK activity can result in higher levels of mast cell activation than simple deletion of lyn and that only near-complete inhibition of Lyn can impair its positive regulatory functions. Furthermore, the data suggest that both positive and negative regulatory functions of Lyn are predominantly carried out by its catalytic activity and not an adaptor function.

High affinity receptor for IgE stimulation activates protein kinase D augmenting activator protein-1 activity for cytokine producing in mast cells

Protein kinase D (PKD) is a serine–threonine kinase involved in the activation of a variety of cells. In mast cells, activation of PKD by cross-linking of high affinity receptor for IgE (FcεRI) has been reported, but little is known for its effects on cytokine production. We investigated the roles of PKD on FcεRI-induced activator protein-1 (AP-1) activation and proinflammatory cytokine productions in mast cells. Pharmacological inhibition of PKD strongly inhibited production of interleukin (IL)-13 and tumor necrosis factor (TNF)-α induced by FcεRI stimulation, and the overexpression of PKD significantly increased the IL-13 and TNF-α production. Reporter assay revealed that the overexpression of PKD enhanced FcεRI-induced IL-13 promoter activation, and that the 5′-flanking region of IL-13 gene from positions − 110 to − 52 was under the regulation of PKD. The overexpression of PKD enhanced the induction of AP-1 luciferase activity by FcεRI stimulation, while it had no effect on luciferase activities dependent upon NF-κB and NF-AT activated by FcεRI stimulation. In EMSA, c-Jun and c-Fos appear to be the major components of AP-1 complexes activated by FcεRI stimulation. Moreover the overexpression of PKD strongly enhanced the phosphorylation of both c-Jun and c-Fos following FcεRI stimulation. Although stress-activated protein kinase/c-Jun N-terminal kinase (JNK) is known to be an important regulator for c-Jun phosphorylation and AP-1 activation, overexpression and inhibition of PKD had no effects on JNK phosphorylation. These results suggest that PKD may play a pivotal role in FcεRI-induced cytokine production in mast cells through the activation of c-Jun, c-Fos, and AP-1.

Ca2+ Waves Initiate Antigen-Stimulated Ca2+ Responses in Mast Cells

Ca(2+) mobilization is central to many cellular processes, including stimulated exocytosis and cytokine production in mast cells. Using single cell stimulation by IgE-specific Ag and high-speed imaging of conventional or genetically encoded Ca(2+) sensors in rat basophilic leukemia and bone marrow-derived rat mast cells, we observe Ca(2+) waves that originate most frequently from the tips of extended cell protrusions, as well as Ca(2+) oscillations throughout the cell that usually follow the initiating Ca(2+) wave. In contrast, Ag conjugated to the tip of a micropipette stimulates local, repetitive Ca(2+) puffs at the region of cell contact. Initiating Ca(2+) waves are observed in most rat basophilic leukemia cells stimulated with soluble Ag and are sensitive to inhibitors of Ca(2+) release from endoplasmic reticulum stores and to extracellular Ca(2+), but they do not depend on store-operated Ca(2+) entry. Knockdown of transient receptor potential channel (TRPC)1 and TRPC3 channel proteins by short hairpin RNA reduces the sensitivity of these cells to Ag and shifts the wave initiation site from protrusions to the cell body. Our results reveal spatially encoded Ca(2+) signaling in response to immunoreceptor activation that utilizes TRPC channels to specify the initiation site of the Ca(2+) response.

Downregulation of protease activated receptor expression and cytokine production in P815 cells by RNA interference

BackgroundProtease-activated receptors (PAR) are seven transmembrane G-coupled receptors comprising four genes (PAR-1 ~ PAR-4). Mast cell has been identified to be able to express PARs and release an array of cytokines upon activation. Recently, it was reported that interleukin (IL)-12 could regulate the expression of PARs in mast cells, and tryptase could induce IL-4 and IL-6 release from mast cells. In order to further investigate the issues, RNA interference (RNAi) technique was employed and small interfering RNAs (siRNA) of PARs were transfected in P815 cells.ResultsThe results showed that siRNAs for PAR-1, PAR-2 and PAR-4 significantly downregulated expression of PAR-1, PAR-2 and PAR-4 mRNAs and proteins in P815 cells at 24, 48 and 72 h following transfection. siRNA PAR-1.2 and siRNA PAR-4.2 significantly reduced IL-12 induced upregulation of PAR-1 and PAR-4 expression, respectively when P815 cells were transfected with them for 48 h. siRNA PAR-2.3 blocked IL-12 induced downregulation of PAR-2 expression on both mRNA and protein levels. It was also observed that siRNA PAR-2.3 and siRNA PAR-1.2 reduced trypsin induced IL-4 release by approximately 92.6% and 65.3%, and SLIGKV-NH2 induced IL-4 release by 82.1% and 60.1%, respectively. Similarly, siRNA PAR-2.3 eliminated tryptase-induced IL-4 release by 75.3%, and siRNA PAR-1.2 diminished SFLLR-NH2 induced IL-4 release by 79.3%. However, siRNA PAR-1.2, siRNA PAR-2.3 and siRNA PAR-4.3 at 10 nM did not show any effect on tryptase-induced IL-6 release from P815 cells.ConclusionIn conclusion, siRNAs of PARs can modulate PAR expression and PAR related cytokine production in mast cells, confirming that PARs are likely to play a role in allergic reactions.

An Activating and Inhibitory Signal from an Inhibitory Receptor LMIR3/CLM-1: LMIR3 Augments Lipopolysaccharide Response through Association with FcRγ in Mast Cells

Leukocyte mono-Ig-like receptor 3 (LMIR3) is an inhibitory receptor mainly expressed in myeloid cells. Coengagement of Fc epsilonRI and LMIR3 impaired cytokine production in bone marrow-derived mast cells (BMMCs) induced by Fc epsilonRI crosslinking alone. Mouse LMIR3 possesses five cytoplasmic tyrosine residues (Y241, Y276, Y289, Y303, Y325), among which Y241 and Y289 (Y241/289) or Y325 fit the consensus sequence of ITIM or immunotyrosine-based switch motif (ITSM), respectively. The inhibitory effect was abolished by the replacement of Y325 in addition to Y241/289 with phenylalanine (Y241/189/325/F) in accordance with the potential of Y241/289/325 to cooperatively recruit Src homology region 2 domain-containing phosphatase 1 (SHP)-1 or SHP-2. Intriguingly, LMIR3 crosslinking alone induced cytokine production in BMMCs expressing LMIR3 (Y241/276/289/303/325F) mutant as well as LMIR3 (Y241/289/325F). Moreover, coimmunoprecipitation experiments revealed that LMIR3 associated with ITAM-containing FcRgamma. Analysis of FcRgamma-deficient BMMCs demonstrated that both Y276/303 and FcRgamma played a critical role in the activating function of this inhibitory receptor. Importantly, LMIR3 crosslinking enhanced cytokine production of BMMCs stimulated by LPS, while suppressing production stimulated by other TLR agonists or stem cell factor. Thus, an inhibitory receptor LMIR3 has a unique property to associate with FcRgamma and thereby functions as an activating receptor in concert with TLR4 stimulation.

SHIP1 Is a Repressor of Mast Cell Hyperplasia, Cytokine Production, and Allergic Inflammation In Vivo

SHIP1 inhibits immune receptor signaling through hydrolysis of the PI3K product phosphatidylinositol 3,4,5-trisphosphate, forming phosphatidylinositol 3,4-bisphosphate. In mast cells, SHIP1 represses FcepsilonRI- and cytokine-mediated activation in vitro, but little is known regarding the function of SHIP1 in mast cells in vivo or the susceptibility of Ship1(-/-) mice to mast cell-associated diseases. In this study, we found that Ship1(-/-) mice have systemic mast cell hyperplasia, increased serum levels of IL-6, TNF, and IL-5, and heightened anaphylactic response. Further, by reconstituting mast cell-deficient mice with Ship1(+/+) or Ship1(-/-) mast cells, we found that the above defects were due to loss of SHIP1 in mast cells. Additionally, we found that mice reconstituted with Ship1(-/-) mast cells suffered worse allergic asthma pathology than those reconstituted with Ship1(+/+) mast cells. In summary, our data show that SHIP1 represses allergic inflammation and mast cell hyperplasia in vivo and exerts these effects specifically in mast cells.

The cytokine interleukin-33 mediates anaphylactic shock.

Anaphylactic shock is characterized by elevated immunoglobulin-E (IgE) antibodies that signal via the high affinity Fc epsilon receptor (Fc epsilonRI) to release inflammatory mediators. Here we report that the novel cytokine interleukin-33 (IL-33) potently induces anaphylactic shock in mice and is associated with the symptom in humans. IL-33 is a new member of the IL-1 family and the ligand for the orphan receptor ST2. In humans, the levels of IL-33 are substantially elevated in the blood of atopic patients during anaphylactic shock, and in inflamed skin tissue of atopic dermatitis patients. In murine experimental atopic models, IL-33 induced antigen-independent passive cutaneous and systemic anaphylaxis, in a T cell-independent, mast cell-dependent manner. In vitro, IL-33 directly induced degranulation, strong eicosanoid and cytokine production in IgE-sensitized mast cells. The molecular mechanisms triggering these responses include the activation of phospholipase D1 and sphingosine kinase1 to mediate calcium mobilization, Nuclear factor-kappaB activation, cytokine and eicosanoid secretion, and degranulation. This report therefore reveals a hitherto unrecognized pathophysiological role of IL-33 and suggests that IL-33 may be a potential therapeutic target for anaphylaxis, a disease of considerable unmet medical need.

Lipoteichoic acid improves the capability of mast cells in the host defense system against bacteria

We investigated the effects of microbial components on the uptake of microbes by mast cells (MCs), and studied the change in cytokine production in MCs after bacterial uptake. LAD2 human mast cells, cord-blood and peripheral-blood derived MCs were employed to analyze their surface molecule expression and cytokine generation by flow cytometry. Bacterial internalization in these MCs was observed by confocal microscopy and flow cytometry. Complement receptor 3 expression was augmented by LTA but not by PGN or 3CpG-oligodeoxynucleotide. LTA also enhanced the uptake of opsonized bacteria (over twofold augmentation). After bacterial uptake, MCs augmented the production of chemoattractant cytokines for neutrophils, while Th1 and Th2 cytokine production showed little or no change. LTA increases the capability of the MC as a sentinel in the host immune response, and some bacterial components direct human MC function towards innate immunity after pathogen infection.

Upregulation of Toll-like receptor (TLR) expression and release of cytokines from P815 mast cells by GM-CSF

BackgroudRecently, mast cells have been recognized to express several Toll-like receptors (TLRs) on their membrane surfaces, and granulocyte-macrophage colony-stimulating factor (GM-CSF) was reported to be able to alter expression of TLRs and cytokine production in neutrophils. However, whether GM-CSF modulates the expression of TLR and cytokine production in mast cells is not clear.ResultsUsing flow cytometry and real time PCR techniques, we found that GM-CSF upregulated expression of TLR3 and TLR7 in P815 cells in a concentration dependent manner. GM-CSF also provoked approximately up to 2.4 and 2.3 fold increase in IL-13 and IL-6 release from P815 cells, respectively following 16 h incubation. GM-CSF induced IL-13 secretion, TLR3 and TLR7 expression appeared to be through activation of mitogen-activated protein kinase (MAPK) and phosphotidylinositol 3-kinase (PI3K)/Akt signaling pathways, whereas GM-CSF elicited IL-6 release seemed via Akt signaling pathway. At 10 ng/ml, GM-CSF significantly enhanced R-848-induced IL-6 release from P815 cells.ConclusionThe ability of GM-CSF in modulation of expression of TLR3 and TLR7 in P815 mast cells and in stimulation of IL-13 and IL-6 release from P815 mast cells in vitro suggests that GM-CSF might play an important role in enhancing the innate immune responses of mast cell to viral infection

Opposing Roles for Lyn and Fyn Kinases in Mast Cell IgG Receptor Signaling (139.4)

Abstract Mast cells are key players of the innate immune system. In addition to their role in atopic disease, mast cell involvement is strongly suspected in inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, and atherosclerosis. Once activated, they degranulate and produce cytokines, resulting in the recruitment of other immune cells. It is well-appreciated that mast cells are activated via the high-affinity immunoglobulin E (IgE) receptor FceRI. In this study we focus on mast cell activation via the immunoglobulin G (IgG) receptor FcγRII/RIII and downstream signaling by Fyn and Lyn kinases. Lyn-deficient mast cells displayed increased histamine and leukotriene C4 secretion, but no change in cytokine production, suggesting a restricted and negative regulatory role for Lyn kinase in mast cell IgG signaling. In contrast, we show that Fyn kinase is required for IgG-mediated histamine, LTC4, and cytokine production in mast cells, and has a similar role in basophils and macrophages. These data illustrate the importance of Src family kinases in IgG signaling, a means by which mast cells may be activated in chronic inflammatory diseases. Supported by NIH grants 1R01 AI59638 and U19A1077435.