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E-prostanoid 2 receptors dampen mast cell degranulation via cAMP/PKA-mediated suppression of IgE-dependent signaling.

ABSTRACT: The experimental administration of PGE(2) for the treatment of asthma dampens clinical symptoms, and similar efficacy has been found in dust mite-induced hypersensitivity reactions in animal models. Here, we investigate the mechanism by which PGE(2) mediates suppression of MC degranulation. We find that the effect of PGE(2) on FcεRI-dependent MC degranulation varies from activating to suppressing, depending on the relative ratio of EP(2) to EP(3) expression on these cells with suppression evident only in cells having increased EP(2) to EP(3) expression. Consistent with a role for EP(2) in suppressing MC responses in vitro, we found that a selective EP(2) agonist, Butaprost, inhibited MC-mediated FcεRI-induced immediate hypersensitivity in a model of PCA. EP(2) engagement on MCs increased cAMP production and inhibited FcεRI-mediated calcium influx. In addition, it also decreased the extent of FcεRI-induced Fyn kinase activity, leading to decreased phosphorylation of key signaling molecules such as Gab2 and Akt. Treatment with an antagonist of cAMP or shRNA down-regulation of PKA (the principal intracellular target of cAMP) reversed the EP(2)-mediated inhibitory effect on MC degranulation and restored calcium influx and phosphorylation of Akt. Collectively, the findings demonstrate that EP(2) suppresses the Fyn-mediated signals that are central to FcεRI-dependent MC degranulation, suggesting that engagement of the EP(2) on MCs may be beneficial in dampening allergic responses.

SUBMITTER: Serra-Pages M

PROVIDER: S-EPMC3501892 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Rictor negatively regulates high-affinity receptors for IgE-induced mast cell degranulation.

Project description:Rictor is a regulatory component of the mammalian target of rapamycin (mTOR) complex 2 (mTORC2). We have previously demonstrated that rictor expression is substantially downregulated in terminally differentiated mast cells as compared with their immature or transformed counterparts. However, it is not known whether rictor and mTORC2 regulate mast cell activation. In this article, we show that mast cell degranulation induced by aggregation of high-affinity receptors for IgE (Fc?RI) is negatively regulated by rictor independently of mTOR. We found that inhibition of mTORC2 by the dual mTORC1/mTORC2 inhibitor Torin1 or by downregulation of mTOR by short hairpin RNA had no impact on Fc?RI-induced degranulation, whereas downregulation of rictor itself resulted in an increased sensitivity (?50-fold) of cells to Fc?RI aggregation with enhancement of degranulation. This was linked to a similar enhancement in calcium mobilization and cytoskeletal rearrangement attributable to increased phosphorylation of LAT and PLC?1. In contrast, degranulation and calcium responses elicited by the G protein-coupled receptor ligand, C3a, or by thapsigargin, which induces a receptor-independent calcium signal, was unaffected by rictor knockdown. Overexpression of rictor, in contrast with knockdown, suppressed Fc?RI-mediated degranulation. Taken together, these data provide evidence that rictor is a multifunctional signaling regulator that can regulate Fc?RI-mediated degranulation independently of mTORC2.

| S-EPMC4258480 | biostudies-literature

Co-Stimulation of Purinergic P2X4 and Prostanoid EP3 Receptors Triggers Synergistic Degranulation in Murine Mast Cells.

Project description:Mast cells (MCs) recognize antigens (Ag) via IgE-bound high affinity IgE receptors (Fc?RI) and trigger type I allergic reactions. Fc?RI-mediated MC activation is regulated by various G protein-coupled receptor (GPCR) agonists. We recently reported that ionotropic P2X4 receptor (P2X4R) stimulation enhanced Fc?RI-mediated degranulation. Since MCs are involved in Ag-independent hypersensitivity, we investigated whether co-stimulation with ATP and GPCR agonists in the absence of Ag affects MC degranulation. Prostaglandin E2 (PGE2) induced synergistic degranulation when bone marrow-derived MCs (BMMCs) were co-stimulated with ATP, while pharmacological analyses revealed that the effects of PGE2 and ATP were mediated by EP3 and P2X4R, respectively. Consistently, this response was absent in BMMCs prepared from P2X4R-deficient mice. The effects of ATP and PGE2 were reduced by PI3 kinase inhibitors but were insensitive to tyrosine kinase inhibitors which suppressed the enhanced degranulation induced by Ag and ATP. MC-dependent PGE2-triggered vascular hyperpermeability was abrogated in a P2X4R-deficient mouse ear edema model. Collectively, our results suggest that P2X4R signaling enhances EP3R-mediated MC activation via a different mechanism to that involved in enhancing Ag-induced responses. Moreover, the cooperative effects of the common inflammatory mediators ATP and PGE2 on MCs may be involved in Ag-independent hypersensitivity in vivo.

| S-EPMC6829402 | biostudies-literature

Combinatorial Genomic Biomarkers Associated with High Response in IgE-Dependent Degranulation in Human Mast Cells.

Project description:Mast cells are the major effector cells that mediate IgE-dependent allergic reactions. We sought to use integrated network analysis to identify genomic biomarkers associated with high response in IgE-mediated activation of primary human mast cells. Primary human mast cell cultures derived from 262 normal donors were categorized into High, Average and Low responder groups according to their activation response profiles. Transcriptome analysis was used to identify genes that were differentially expressed in different responder cultures in their baseline conditions, and the data were analyzed by constructing a personalized perturbed profile (PEEP). For upregulated genes, the construction of PEEP for each individual sample of all three responder groups revealed that High responders exhibited a higher percentage of "perturbed" samples whose PEEP values lay outside the normal range of expression. Moreover, the integration of PEEP of four selected upregulated genes into distinct sets of combinatorial profiles demonstrated that the specific pattern of upregulated expression of these four genes, in a tandem combination, was observed exclusively among the High responders. In conclusion, this combinatorial approach was useful in identifying a set of genomic biomarkers that are associated with high degranulation response in human mast cell cultures derived from the blood of a cohort of normal donors.

| S-EPMC11311466 | biostudies-literature

P2X7 receptors induce degranulation in human mast cells.

Project description:Mast cells play important roles in host defence against pathogens, as well as being a key effector cell in diseases with an allergic basis such as asthma and an increasing list of other chronic inflammatory conditions. Mast cells initiate immune responses through the release of newly synthesised eicosanoids and the secretion of pre-formed mediators such as histamine which they store in specialised granules. Calcium plays a key role in regulating both the synthesis and secretion of mast-cell-derived mediators, with influx across the membrane, in particular, being necessary for degranulation. This raises the possibility that calcium influx through P2X receptors may lead to antigen-independent secretion of histamine and other granule-derived mediators from human mast cells. Here we show that activation of P2X7 receptors with both ATP and BzATP induces robust calcium rises in human mast cells and triggers their degranulation; both effects are blocked by the P2X7 antagonist AZ11645373, or the removal of calcium from the extracellular medium. Activation of P2X1 receptors with αβmeATP also induces calcium influx in human mast cells, which is significantly reduced by both PPADS and NF 449. P2X1 receptor activation, however, does not trigger degranulation. The results indicate that P2X7 receptors may play a significant role in contributing to the unwanted activation of mast cells in chronic inflammatory conditions where extracellular ATP levels are elevated.

| S-EPMC4854833 | biostudies-literature

SLC10A4 regulates IgE-mediated mast cell degranulation in vitro and mast cell-mediated reactions in vivo.

Project description:Mast cells act as sensors in innate immunity and as effector cells in adaptive immune reactions. Here we demonstrate that SLC10A4, also referred to as the vesicular aminergic-associated transporter, VAAT, modifies mast cell degranulation. Strikingly, Slc10a4 -/- bone marrow-derived mast cells (BMMCs) had a significant reduction in the release of granule-associated mediators in response to IgE/antigen-mediated activation, whereas the in vitro development of mast cells, the storage of the granule-associated enzyme mouse mast cell protease 6 (mMCP-6), and the release of prostaglandin D2 and IL-6 were normal. Slc10a4-deficient mice had a strongly reduced passive cutaneous anaphylaxis reaction and a less intense itching behaviour in response to the mast cell degranulator 48/80. Live imaging of the IgE/antigen-mediated activation showed decreased degranulation and that ATP was retained to a higher degree in mast cell granules lacking SLC10A4. Furthermore, ATP was reduced by two thirds in Slc10a4 -/- BMMCs supernatants in response to IgE/antigen. We speculate that SLC10A4 affects the amount of granule-associated ATP upon IgE/antigen-induced mast cell activation, which affect the release of granule-associated mast cell mediators. In summary, SLC10A4 acts as a regulator of degranulation in vitro and of mast cell-related reactions in vivo.

| S-EPMC5430724 | biostudies-literature

Inhibition of weak-affinity epitope-IgE interactions prevents mast cell degranulation.

Project description:Development of specific inhibitors of allergy has had limited success, in part, owing to a lack of experimental models that reflect the complexity of allergen-IgE interactions. We designed a heterotetravalent allergen (HtTA) system, which reflects epitope heterogeneity, polyclonal response and number of immunodominant epitopes observed in natural allergens, thereby providing a physiologically relevant experimental model to study mast cell degranulation. The HtTA design revealed the importance of weak-affinity epitopes in allergy, particularly when presented with high-affinity epitopes. The effect of selective inhibition of weak-affinity epitope-IgE interactions was investigated with heterobivalent inhibitors (HBIs) designed to simultaneously target the antigen- and nucleotide-binding sites on the IgE Fab. HBI demonstrated enhanced avidity for the target IgE and was a potent inhibitor of degranulation in vitro and in vivo. These results demonstrate that partial inhibition of allergen-IgE interactions was sufficient to prevent mast cell degranulation, thus establishing the therapeutic potential of the HBI design.

| S-EPMC3913000 | biostudies-other

Trimellitic anhydride induces low-grade mast cell degranulation without specific IgE.

Project description:Low-molecular-weight (LMW) substances are known to be causative agents of occupational asthma (OA) and occupational rhinitis (OR). Although most LMW substances are irritants or allergens, some can cause immediate type immunoglobulin E (IgE)-mediated allergic reactions. Trimellitic anhydride (TMA) is one such LMW substance, which is known as an immunological sensitizer. However, the exact molecular biological details of the effects of TMA remain unclear.We measured the ?-hexosaminidase release from mast cells after directly exposing the cells to various LMW substances. The tyrosine phosphorylation of whole cellular molecules and the phosphorylation of extracellular signal-regulated kinase (ERK) were assessed by immunoblot assay.Among the LMW substances tested, only TMA induced ?-hexosaminidase release. However, the mast cell degranulation induced by TMA was lower than that induced by an antigen or a calcium ionophore. Moreover, the pattern of tyrosine phosphorylation of whole cellular molecules was quite different between IgE-mediated antigen stimulation and TMA exposure. The TMA effect on mast cells was independent of not only IgE but also Ca2+ influx. ERK phosphorylation was not detected in mast cells exposed to TMA.TMA induced mild degranulation of mast cells without IgE, even though the phosphorylation of ERK was not detected. This reaction suggests that TMA affects humans even upon first exposure. Therefore, it is imperative to avoid human exposure to high concentrations of TMA. In order to stop the development of severe asthma in individuals with OR, we need to be able to identify cases of OR caused by TMA as soon as possible.

| S-EPMC5616080 | biostudies-other

Small interfering RNA screen for phosphatases involved in IgE-mediated mast cell degranulation.

Project description:Mast cells play pivotal roles in the initiation of the allergic response. To gain an understanding of the functions played by phosphatases in IgE-mediated mast cell activation, a small interfering RNA (siRNA) library that targets all mouse phosphatase genes was screened in a mouse mast cell line, MMC-1. Of 198 targets, 10 enhanced and 7 inhibited FcepsilonRI-induced degranulation. For seven of the strongest hits, four different siRNAs per target were tested, and at least two out of the four single siRNA per target had similar effects as the pool suggesting that these were true hits. Bone marrow-derived mast cells from normal mice further validated these results for six definite positive targets. The mechanism of the reduced mast cell degranulation due to calcineurin B deficiency was investigated. Calcineurin B deficiency reduced the phosphorylation of MAPKs and the phosphorylation of protein kinase D/protein kinase Cmu and protein kinase Cdelta, which are involved in FcepsilonRI signaling. The screen, therefore, has identified several new molecules that are critical for FcepsilonRI-induced degranulation. Regulating the function of these proteins may be potential targets for the treatment of allergic inflammation. The result also indicates that the system used is efficient for searching molecules implicated in complex receptor-induced signaling.

| S-EPMC2882511 | biostudies-literature

Regulation of M2-type pyruvate kinase mediated by the high-affinity IgE receptors is required for mast cell degranulation.

Project description:BACKGROUND AND PURPOSE: M2-type pyruvate kinase (M2PK) was found to interact directly with the 'ITAM' region of the gamma chain of the high-affinity IgE receptor (FcvarepsilonRI). Our hypothesis was that mast cell degranulation might require the FcvarepsilonRI-mediated inhibition of M2PK activity. EXPERIMENTAL APPROACH: In rat basophilic leukaemia (RBL-2H3) cells, the effects of directly inhibiting M2PK or preventing the FcvarepsilonRI-mediated inhibition of M2PK (disinhibition) on degranulation was measured by hexosaminidase release. Effects of blocking the FcvarepsilonRI-mediated inhibition of M2PK was also assessed in vivo in a mouse model of allergen-induced airway hyper-responsiveness. KEY RESULTS: Activation of FcvarepsilonRI in RBL-2H3 cells caused the rapid phosphorylation of tyrosine residues in M2PK, associated with a decrease in M2PK enzymatic activity. There was an inverse correlation between M2PK activity and mast cell degranulation. FcvarepsilonRI-mediated inhibition of M2PK involved Src kinase, phosphatidylinositol 3-kinase, PKC and calcium. Direct inhibition of M2PK potentiated FcvarepsilonRI-mediated degranulation and prevention of the FcvarepsilonRI-mediated inhibition of M2PK attenuated mast cell degranulation. Transfection of RBL-2H3 cells with M1PK which prevents FcvarepsilonRI-induced inhibition of M2PK, markedly reduced their degranulation and exogenous M1PK (i.p.) inhibited ovalbumin-induced airway hyper-responsiveness in vivo. CONCLUSIONS AND IMPLICATIONS: We have identified a new control point and a novel biochemical pathway in the process of mast cell degranulation. Our study suggests that the FcvarepsilonRI-mediated inhibition of M2PK is a crucial step in responses to allergens. Moreover, the manipulation of glycolytic processes and intermediates could provide novel strategies for the treatment of allergic diseases.

| S-EPMC2451040 | biostudies-other

The tetraspanin CD63 is required for efficient IgE-mediated mast cell degranulation and anaphylaxis.

Project description:Mast cell (MC) activation through the high-affinity IgE receptor Fc?RI leads to the release of mediators involved in immediate-type allergic reactions. Although Abs against the tetraspanins CD63 and CD81 inhibit Fc?RI-induced MC degranulation, the intrinsic role of these molecules in Fc?RI-induced MC activation is unknown. In MCs, CD63 is expressed at the cell surface and in lysosomes (particularly secretory lysosomes that contain allergic mediators). In this study, we investigated the role of CD63 in MC using a CD63 knockout mouse model. CD63-deficiency did not affect in vivo MC numbers and tissue distribution. Bone marrow-derived MC developed normally in the absence of CD63 protein. However, CD63-deficient bone marrow-derived MC showed a significant decrease in Fc?RI-mediated degranulation, but not PMA/ionomycin-induced degranulation, as shown by ?-hexosaminidase release assays. The secretion of TNF-?, which is both released from granules and synthesized de novo upon MC activation, was also decreased. IL-6 secretion and production of the lipid mediator leukotriene C? were unaffected. There were no ultrastructural differences in granule content and morphology, late endosomal/lysosomal marker expression, Fc?RI-induced global tyrosine phosphorylation, and Akt phosphorylation. Finally, local reconstitution in genetically MC-deficient Kit(w/w-v) mice was unaffected by the absence of CD63. However, the sites reconstituted with CD63-deficient MC developed significantly attenuated cutaneous anaphylactic reactions. These findings demonstrate that the absence of CD63 results in a significant decrease of MC degranulation, which translates into a reduction of acute allergic reactions in vivo, thus identifying CD63 as an important component of allergic inflammation.

| S-EPMC3780990 | biostudies-literature

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