IgE-mediated Passive Systemic Anaphylaxis Research Articles

GRK2 differentially regulates FcεRI and MRGPRB2-mediated responses in mast cells.

In addition to high-affinity IgE receptor (FcεRI), a subtype of mouse mast cells (MCs) expresses a G protein-coupled receptor known as Mas-related G protein-coupled receptor (GPCR)-B2 (MRGPRB2; human ortholog MRGPRX2). GPCR kinase 2 (GRK2) is a Serine/Threonine kinase that phosphorylates GPCRs to promote their desensitization and internalization. We previously showed that silencing GRK2 expression in mouse bone marrow-derived MCs (BMMCs) blocks IgE-mediated degranulation. Compound 48/80 (C48/80), substance P (SP) and LL-37 cause degranulation in human and mouse MCs via MRGPRX2 and MRGPRB2, respectively. We also reported that C48/80 and SP cause desensitization and internalization of MRGPRX2, but LL-37 does not. Here, we generated mice with MC-specific deletion of Grk2 (Cpa3Cre+/Grk2fl/fl ) to determine its role on IgE-mediated responses and to assess whether it differentially regulates degranulation in response to LL-37, C48/80 and SP. Absence of GRK2 substantially inhibited IgE-mediated tyrosine phosphorylation of STAT5, calcium mobilization, and degranulation in mouse primary lung-derived MCs (PLMCs). By contrast, peritoneal MCs (PMCs) from Cpa3Cre+/Grk2fl/fl mice demonstrated significant enhancement of degranulation in response to C48/80 and SP, but not LL-37. Deletion of Grk2 in MCs attenuated IgE-mediated passive cutaneous anaphylaxis (PCA) and itch but not passive systemic anaphylaxis (PSA). Surprisingly, PSA was significantly reduced in Mrgprb2-/- mice. These findings suggest that GRK2 contributes to PCA and itch but not PSA. By contrast, GRK2 desensitizes MRGPRX2/B2-mediated responses to C48/80 and SP but not LL-37. However, IgE-mediated PSA likely involves the activation of MRGPRB2 by LL-37 or a similar agonist, whose function is resistant to modulation by GRK2.

STAT5b dimers and tetramers are critical for Mast Cell Function

Abstract Signal transducers and activators of transcription (STATs) are latent transcription factors vital for lymphoid, myeloid, and erythroid cell development, survival, proliferation, and function. Activated STATs form dimers that translocate to the nucleus and bind high affinity STAT DNA motifs to modulate gene expression. We previously found that Stat5b is required for IgE-mediated mast cell cytokine production in vitro. In this study, we found that STAT5b KO mice demonstrate decreased sensitivity to IgE-mediated passive systemic anaphylaxis (PSA) and reduced cytokine production in vivo. This is not due to differences in mast cell numbers or distribution. Interestingly, STAT5b KO mice exhibit elevated levels of serum IgE that could diminish PSA by preventing binding of antigen-specific IgE. Increased serum IgE is not tied to changes in B cell number or their ability to produce IgE in vitro. In addition to dimers, activated STAT proteins can also form tetramers through an oligomerization process once dimers bind to tandem low-affinity STAT DNA motifs. Similar to STAT5bKO, in vitro IgE-mediated cytokine production is decreased in Stat5a-Stat5b double knock-in (DKI) mice in which STAT5 proteins can form dimers but not tetramers. In vivo, DKI mice exhibit decreased PSA sensitivity, with no difference in mast cell numbers or distribution, and a modest increase in serum IgE. Collectively, these data illuminate the unique and specific roles of STAT5 dimers and tetramers in mast cell function and allergic disease.

Mast cell corticotropin-releasing factor subtype 2 suppresses mast cell degranulation and limits the severity of anaphylaxis and stress-induced intestinal permeability

Psychological stress and heightened mast cell (MC) activation are linked with important immunologic disorders, including allergy, anaphylaxis, asthma, and functional bowel diseases, but the mechanisms remain poorly defined. We have previously demonstrated that activation of the corticotropin-releasing factor (CRF) system potentiates MC degranulation responses during IgE-mediated anaphylaxis and psychological stress through corticotropin-releasing factor receptor subtype 1 (CRF1) expressed on MCs. In this study we investigated the role of corticotropin-releasing factor receptor subtype 2 (CRF2) as a modulator of stress-induced MC degranulation and associated disease pathophysiology. Invitro MC degranulation assays were performed with bone marrow-derived mast cells (BMMCs) derived from wild-type (WT) and CRF2-deficient (CRF2-/-) mice and RBL-2H3 MCs transfected with CRF2-overexpressing plasmid or CRF2 small interfering RNA. Invivo MC responses and associated pathophysiology in IgE-mediated passive systemic anaphylaxis and acute psychological restraint stress were measured in WT, CRF2-/-, and MC-deficient KitW-sh/W-sh knock-in mice. Compared with WT mice, CRF2-/- mice exhibited greater serum histamine levels and exacerbated IgE-mediated anaphylaxis and colonic permeability. In addition, CRF2-/- mice exhibited increased serum histamine levels and colonic permeability after acute restraint stress. Experiments with BMMCs and RBL-2H3 MCs demonstrated that CRF2 expressed on MCs suppresses store-operated Ca2+ entry signaling and MC degranulation induced by diverse MC stimuli. Experiments with MC-deficient KitW-sh/W-sh mice systemically engrafted with WT and CRF2-/- BMMCs demonstrated the functional importance of MC CRF2 in modulating stress-induced pathophysiology. MC CRF2 is a negative global modulator of stimuli-induced MC degranulation and limits the severity of IgE-mediated anaphylaxis and stress-related disease pathogenesis.

ADAM17-deficient mast cells exhibit decreased IgE-mediated activation due to impaired Lyn phosphorylation.

Abstract Allergies and asthma are common ailments that are on the rise globally. Mast cells play a direct role in the signs and symptoms commonly displayed by allergic patients. The family of A Disintegrin And Metalloproteinases (ADAMs) are involved in regulating many cellular processes by cleaving surface receptors, ligands, and signaling molecules. We sought to determine the role of ADAM17 in mast cell activity. Using inhibitor-based and genetic knockout studies, loss of ADAM17 reduced mast cell degranulation. This resulted in decreased cytokine and histamine release. ADAM17MC−/− mice exhibited less severe IgE-mediated passive systemic anaphylaxis, while no difference was seen when initiated by exogenous histamine. We previously showed that ADAM17 forms a protein complex with the signaling molecule Lyn. Upon cross-linking, ADAM17−/− mast cells have reduced Lyn phosphorylation which affects downstream signaling events. ADAM17 may play a role in apical FcɛRI signaling through interactions with Lyn.

Influence of glucocorticoids on time-of-day-dependent variations in IgE-, histamine-, and platelet-activating factor-mediated systemic anaphylaxis in different mouse strains

A time-of-day-dependent variation in IgE-mediated passive systemic anaphylaxis was previously reported in ICR mice. In the present study, we investigated time-of-day-dependent variations in IgE-, histamine-, and platelet-activating factor (PAF)-mediated systemic anaphylaxis in C57BL/6, BALB/c, and NC/Nga mice at 9:00 h and 21:00 h, and evaluated the potential influence of glucocorticoids (GCs) on these variations. We found significant time-of-day-dependent variations in IgE-mediated systemic anaphylaxis in C57BL/6 mice, and in histamine- and PAF-mediated systemic anaphylaxis in BALB/c mice. Significant daily variations in IgE-, histamine-, and PAF-mediated systemic anaphylaxis were not observed in NC/Nga mice. Pretreatment with dexamethasone and adrenalectomy abolished the daily variations in IgE-mediated systemic anaphylaxis in C57BL/6 mice and in PAF-mediated systemic anaphylaxis in BALB/c mice, suggesting that GCs from adrenal glands are pivotal in regulating these variations. In contrast, pretreatment with dexamethasone and adrenalectomy did not abolish the daily variation in histamine-mediated systemic anaphylaxis in BALB/c mice, suggesting that GC-independent and adrenal gland-independent mechanisms are important for the variation. The present study demonstrated that time-of-day-dependent variations in systemic anaphylaxis differed among inbred mouse strains and with anaphylaxis-inducing substances. Thus, mouse strains, time of experiment, and anaphylaxis-inducing substances used must be considered to obtain appropriate experimental results.

Didox (3,4-dihydroxybenzohydroxamic acid) suppresses IgE-mediated mast cell activation through attenuation of NFκB and AP-1 transcription

Mast cell activation via the high-affinity IgE receptor (FcεRI) elicits production of inflammatory mediators central to allergic disease. As a synthetic antioxidant and a potent ribonucleotide reductase (RNR) inhibitor, Didox (3,4-dihyroxybenzohydroxamic acid) has been tested in clinical trials for cancer and is an attractive therapeutic for inflammatory disease. We found that Didox treatment of mouse bone marrow-derived mast cells (BMMC) reduced IgE-stimulated degranulation and cytokine production, including IL-6, IL-13, TNF and MIP-1a (CCL3). These effects were consistent using BMMC of different genetic backgrounds and peritoneal mast cells. While the RNR inhibitor hydroxyurea had little or no effect on IgE-mediated function, high concentrations of the antioxidant N-acetylcysteine mimicked Didox-mediated suppression. Furthermore, Didox increased expression of the antioxidant genes superoxide dismutase and catalase, and suppressed DCFH-DA fluorescence, indicating reduced reactive oxygen species production. Didox effects were not due to changes in FcεRI expression or cell viability, suggesting it inhibits signaling required for inflammatory cytokine production. In support of this, we found that Didox reduced FcεRI-mediated AP-1 and NFκB transcriptional activity. Finally, Didox suppressed mast cell-dependent, IgE-mediated passive systemic anaphylaxis in vivo. These data demonstrate the potential use for Didox asa means of antagonizing mast cell responses in allergic disease.

Frontline Science: Corticotropin-releasing factor receptor subtype 1 is a critical modulator of mast cell degranulation and stress-induced pathophysiology.

Life stress is a major risk factor in the onset and exacerbation of mast cell-associated diseases, including allergy/anaphylaxis, asthma, and irritable bowel syndrome. Although it is known that mast cells are highly activated upon stressful events, the mechanisms by which stress modulates mast cell function and disease pathophysiology remains poorly understood. Here, we investigated the role of corticotropin-releasing factor receptor subtype 1 (CRF1) in mast cell degranulation and associated disease pathophysiology. In a mast cell-dependent model of IgE-mediated passive systemic anaphylaxis (PSA), prophylactic administration of the CRF1-antagonist antalarmin attenuated mast cell degranulation and hypothermia. Mast cell-deficient KitW-sh/W-sh mice engrafted with CRF1-/- bone marrow-derived mast cells (BMMCs) exhibited attenuated PSA-induced serum histamine, hypothermia, and clinical scores compared with wild-type BMMC-engrafted KitW-sh/W-sh mice. KitW-sh/W-sh mice engrafted with CRF1-/- BMMCs also exhibited suppressed in vivo mast cell degranulation and intestinal permeability in response to acute restraint stress. Genetic and pharmacologic experiments with murine BMMCs, rat RBL-2H3, and human LAD2 mast cells demonstrated that although CRF1 activation did not directly induce MC degranulation, CRF1 signaling potentiated the degranulation responses triggered by diverse mast cell stimuli and was associated with enhanced release of Ca2+ from intracellular stores. Taken together, our results revealed a prominent role for CRF1 signaling in mast cells as a positive modulator of stimuli-induced degranulation and in vivo pathophysiologic responses to immunologic and psychologic stress.

Corticotropin releasing factor receptor 2 exerts global suppression of mast cell degranulation and associated pathophysiology.

Abstract Psychological stress triggers mast cell (MC) activation and is a major risk factor in the onset and exacerbation of MC-associated disorders including allergy/anaphylaxis, irritable bowel syndrome, and autoimmune diseases. The mechanisms by which stress modulates MC function and disease susceptibility is poorly understood. Our previous studies showed that corticotropin releasing factor receptor 1 (CRF1) signaling potentiated stress-induced MC degranulation. Here we utilized CRF2 deficient (CRF2 −/−) mice and MCs to define the in vivo and in vitro biological importance of CRF receptor subtype 2, CRF2. Compared with WT mice, CRF2 −/− mice exhibited greater serum histamine levels following restraint stress (RS) (by 143%) and IgE-mediated passive systemic anaphylaxis (PSA) (by 415%). The heightened response was greater in CRF2 −/− females compared with males. CRF2 −/− mice exhibited greater RS-induced and PSA-induced elevations in intestinal permeability compared to WT mice. Bone marrow derived MCs (BMMCs) derived from CRF2 −/− mice exhibited greater release of β-hexosaminidase and histamine and heightened Ca2+ mobilization from intracellular stores in response to diverse MC stimuli including IgE/DNP, c48/80, and A23187. MC-deficient mice engrafted with CRF2 −/− BMMCs exhibited greater RS-induced intestinal permeability and PSA-induced hypothermia compared with WT BMMC-engrafted mice. Together, these results demonstrate that MC-CRF2 receptor signaling exerts a global suppression of MC degranulation and pathophysiology. Elucidation of the mechanisms by which CRF2 dampens MC degranulation could reveal novel therapeutic targets for hyperactive MC disorders.

STAT5b Controls IgE-mediated Mast Cell Functions In Vitro and In Vivo

Abstract Signal Transducers and Activators of Transcription (STATs) are latent transcription factors that mediate a number of cellular responses including but not limited to cell survival, proliferation and differentiation. This protein family consists of seven members, STATs 1 – 6 including two closely related molecules, STAT5a and STAT5b, that show 96% amino acid sequence homology and are critical for lymphoid, myeloid, and erythroid cell development and function. We recently identified Stat5b as the critical regulator of IgE-mediated cytokine production in mast cells. STAT5b knockout (KO) cells show decreased cytokine production in vitro. In vivo STAT5b KO mice demonstrate decreased sensitivity to IgE-mediated passive systemic anaphylaxis accompanied with decreased production of IL-6 and IL-13 compared to wildtype counterparts. There was no difference in MAR-1-mediated passive systemic anaphylaxis or mast cell number and distribution. Interestingly STAT5b KO mice demonstrate elevated levels of serum IgE and an exacerbated response to histamine-mediated passive systemic anaphylaxis. We previously found that Fyn-mediated Stat5 tyrosine phosphorylation is critical for Stat5-dependent effects. The current work demonstrates that Stat5b is also phosphorylated on serine residues during IgE signaling in mast cells, via a Src-independent pathway requiring PI3-kinase function. Preliminary data show that STAT5b tyrosine and serine phosphorylation either independently or in tandem regulate IgE-induced cytokine production. Elucidating the role of STAT5b in the inflammatory response as well as the key players in STAT5b serine phosphorylation will yield new targets for mast cell-associated diseases.

Anti-allergic activity of 2,4,6-trihydroxy-3-geranylacetophenone (tHGA) via attenuation of IgE-mediated mast cell activation and inhibition of passive systemic anaphylaxis

tHGA, a geranyl acetophenone compound originally isolated from a local shrub called Melicope ptelefolia, has been previously reported to prevent ovalbumin-induced allergic airway inflammation in a murine model of allergic asthma by targeting cysteinyl leukotriene synthesis. Mast cells are immune effector cells involved in the pathogenesis of allergic diseases including asthma by releasing cysteinyl leukotrienes. The anti-asthmatic properties of tHGA could be attributed to its inhibitory effect on mast cell degranulation. As mast cell degranulation is an important event in allergic responses, this study aimed to investigate the anti-allergic effects of tHGA in cellular and animal models of IgE-mediated mast cell degranulation. For in vitro model of IgE-mediated mast cell degranulation, DNP-IgE-sensitized RBL-2H3 cells were pre-treated with tHGA before challenged with DNP-BSA to induce degranulation. For IgE-mediated passive systemic anaphylaxis, Sprague Dawley rats were sensitized by intraperitoneal injection of DNP-IgE before challenged with DNP-BSA. Both in vitro and in vivo models showed that tHGA significantly inhibited the release of preformed mediators (β-hexosaminidase and histamine) as well as de novo mediators (interleukin-4, tumour necrosis factor-α, prostaglandin D2 and leukotriene C4). Pre-treatment of tHGA also prevented IgE-challenged RBL-2H3 cells and peritoneal mast cells from undergoing morphological changes associated with mast cell degranulation. These findings indicate that tHGA possesses potent anti-allergic activity via attenuation of IgE-mediated mast cell degranulation and inhibition of IgE-mediated passive systemic anaphylaxis. Thus, tHGA may have the potential to be developed as a mast cell stabilizer for the treatment of allergic diseases in the future.

Sexual dimorphism in the mast cell transcriptome and the pathophysiological responses to immunological and psychological stress.

BackgroundBiological sex plays a prominent role in the prevalence and severity of a number of important stress-related gastrointestinal and immune-related diseases including IBS and allergy/anaphylaxis. Despite the establishment of sex differences in these diseases, the underlying mechanisms contributing to sex differences remain poorly understood. The objective of this study was to define the role of biological sex on mast cells (MCs), an innate immune cell central to the pathophysiology of many GI and allergic disorders.MethodsTwelve-week-old C57BL/6 male and female mice were exposed to immunological stress (2 h of IgE-mediated passive systemic anaphylaxis (PSA)) or psychological stress (1 h of restraint stress (RS)) and temperature, clinical scores, serum histamine, and intestinal permeability (for RS) were measured. Primary bone marrow-derived MCs (BMMCs) were harvested from male and female mice and analyzed for MC degranulation, signaling pathways, mediator content, and RNA transcriptome analysis.ResultsSexually dimorphic responses were observed in both models of PSA and RS and in primary MCs. Compared with male mice, female mice exhibited increased clinical scores, hypothermia, and serum histamine levels in response to PSA and had greater intestinal permeability and serum histamine responses to RS. Primary BMMCs from female mice exhibited increased release of β-hexosaminidase, histamine, tryptase, and TNF-α upon stimulation with IgE/DNP and A23187. Increased mediator release in female BMMCs was not associated with increased upstream phospho-tyrosine signaling pathways or downstream Ca2+ mobilization. Instead, increased mediator release in female MCs was associated with markedly increased capacity for synthesis and storage of MC granule-associated immune mediators as determined by MC mediator content and RNA transcriptome analysis.ConclusionsThese results provide a new understanding of sexual dimorphic responses in MCs and have direct implications for stress-related diseases associated with a female predominance and MC hyperactivity including irritable bowel syndrome, allergy, and anaphylaxis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13293-016-0113-7) contains supplementary material, which is available to authorized users.

What makes children outgrow food allergy?

What makes children outgrow food allergy?

STAT5 tetramer formation is critical for mast cell function (HYP4P.312)

Abstract Signal transducers and activators of transcription (STATs) are latent transcription factors that are vital for cell survival, proliferation and function. Activated STAT proteins dimerize and translocate to the nucleus, where they bind to high-affinity motifs to modulate gene expression. These dimers can also form tetramers through an N-terminal domain-mediated oligomerization process, and bind to low-affinity tandem motifs. We previously generated Stat5a-Stat5b double knock-in (DKI) mice in which STAT5 proteins are phosphorylated and can form dimers but not tetramers. We have now found that bone marrow-derived mast cells from Stat5 DKI mice express FceRI and c-Kit at levels comparable to those from wild-type mice. However, these mast cells are defective in IgE-induced cytokine and chemokine production, and exhibit less SCF-mediated migration in vitro. Similarly, IgE-mediated passive systemic anaphylaxis is also altered in Stat5 DKI mice. Unraveling the role of STAT5 tetramers may reveal novel insights into mast cell function in allergic and inflammatory diseases.

Regulatory CD4+Foxp3+ T Cells Control the Severity of Anaphylaxis

ObjectiveAnaphylaxis is a life-threatening outcome of immediate-type hypersensitivity to allergen, consecutive to mast cell degranulation by allergen-specific IgE. Regulatory T cells (Treg) can control allergic sensitization and mast cell degranulation, yet their clinical benefit on anaphylactic symptoms is poorly documented. Here we investigated whether Treg action during the effector arm of the allergic response alleviates anaphylaxis.MethodsWe used a validated model of IgE-mediated passive systemic anaphylaxis, induced by intravenous challenge with DNP-HSA in mice passively sensitized with DNP-specific IgE. Anaphylaxis was monitored by the drop in body temperature as well as plasma histamine and serum mMCP1 levels. The role of Treg was analyzed using MHC class II-deficient (Aβ°/°) mice, treatment with anti-CD25 or anti-CD4 mAbs and conditional ablation of Foxp3+ Treg in DEREG mice. Therapeutic efficacy of Treg was also evaluated by transfer experiments using FoxP3-eGFP knock-in mice.ResultsAnaphylaxis did not occur in mast cell-deficient W/Wv mutant mice and was only moderate and transient in mice deficient for histamine receptor-1. Defects in constitutive Treg, either genetic or induced by antibody or toxin treatment resulted in a more severe and/or sustained hypothermia, associated with a rise in serum mMCP1, but not histamine. Adoptive transfer of Foxp3+ Treg from either naïve or DNP-sensitized donors similarly alleviated body temperature loss in Treg-deficient DEREG mice.ConclusionConstitutive Foxp3+ Treg can control the symptomatic phase of mast cell and IgE-dependent anaphylaxis in mice. This might open up new therapeutic avenues using constitutive rather than Ag-specific Treg for inducing tolerance in allergic patients.

Role of Mast Cells and Basophils in IgE Responses and in Allergic Airway Hyperresponsiveness

We established a diphtheria toxin (DT)-based conditional deletion system using Il4 enhancer elements previously shown to be specific for IL-4 production in mast cells (MCs) or basophils (Mas-TRECK and Bas-TRECK mice). DT treatment of Bas-TRECK mice resulted in specific deletion of basophils, whereas both MCs and basophils were deleted in Mas-TRECK mice. DT-treated Mas-TRECK mice had impaired passive cutaneous anaphylaxis, IgE-mediated passive systemic anaphylaxis, and IgE-mediated chronic allergic inflammation, whereas DT-treated Bas-TRECK mice had impaired IgE-mediated chronic allergic inflammation. Using these mice, we also sought to tease out the role of MCs and basophils in airway hyperresponsiveness (AHR). Although MC deletion resulted in a slight increase in basal Ag-specific IgE levels and significant increases in basal IgE levels, we found that this deletion markedly impaired the AHR effector phase and was accompanied by decreased histamine levels. By contrast, basophil deletion had no effect on the AHR effector phase or on IgE production induced by systemic OVA immunization. Our results, using these newly established Mas-TRECK and Bas-TRECK models, demonstrated an indispensable role for MCs as effector cells in AHR.

LAT Is Essential for FcεRI-Mediated Mast Cell Activation

The linker molecule LAT is a substrate of the tyrosine kinases activated following TCR engagement of T cells. LAT is also expressed in platelets, NK, and mast cells. Although LAT-deficient mice contain normal numbers of mast cells, we found that LAT-deficient mice were resistant to IgE-mediated passive systemic anaphylaxis. LAT-deficient bone marrow–derived mast cells (BMMC) showed normal growth and development. Whereas tyrosine phosphorylation of FcεRI, Syk, and Vav was intact in LAT-deficient BMMCs following FcεRI engagement, tyrosine phosphorylation of SLP-76, PLC-γ1, and PLC-γ2 and calcium mobilization were dramatically reduced. LAT-deficient BMMCs also exhibited profound defects in activation of MAPK, degranulation, and cytokine production after FcεRI cross-linking. These results show that LAT plays a critical role in FcεRI-mediated signaling in mast cells.

Modulation of immunoglobulin (Ig)E-mediated systemic anaphylaxis by low-affinity Fc receptors for IgG.

It is widely accepted that immunoglobulin (Ig)E triggers immediate hypersensitivity responses by activating a cognate high-affinity receptor, FcepsilonRI, leading to mast cell degranulation with release of vasoactive and proinflammatory mediators. This apparent specificity, however, is complicated by the ability of IgE to bind with low affinity to Fc receptors for IgG, FcgammaRII and III. We have addressed the in vivo significance of this interaction by studying IgE-mediated passive systemic anaphylaxis in FcgammaR-deficient mice. Mice deficient in the inhibitory receptor for IgG, FcgammaRIIB, display enhanced IgE-mediated anaphylactic responses, whereas mice deficient in an IgG activation receptor, FcgammaRIII, display a corresponding attenuation of IgE-mediated responses. Thus, in addition to modulating IgG-triggered hypersensitivity responses, FcgammaRII and III on mast cells are potent regulators of IgE-mediated responses and reveal the existence of a regulatory pathway for IgE triggering of effector cells through IgG Fc receptors that could contribute to the etiology of the atopic response.