IgE-dependent Mast Cell Degranulation Research Articles

Deficiency of BMAL1 promotes ROS generation and enhances IgE-dependent degranulation in mast cells

BackgroundBmal1 (Brain and muscle arnt-like, or Arntl) is a bHLH/PAS domain transcription factor central to the transcription/translation feedback loop of the circadian clock. Mast cells are crucial for effector functions in allergic reaction and their activity follows a circadian rhythm. However, the functional roles of Bmal1 in mast cells remain to be determined. PurposeThis study aimed to elucidate the specific roles of Bmal1 in IgE-dependent mast cell degranulation. ResultsIgE-dependent degranulation was enhanced in bone marrow–derived mast cells (BMMCs) derived from Bmal1-deficient mice (Bmal1-KO mice) compared to that in BMMCs derived from wild-type mice (WT mice) in the absence of 2-Mercaptoethanol (2-ME) in culture. Mast cell–deficient KitW-sh mice reconstituted with Bmal1-KO BMMCs showed more robust passive cutaneous anaphylactic (PCA) reactions, an in vivo model of IgE-dependent mast cell degranulation, than KitW-sh mice reconstituted with WT BMMCs. In the absence of 2-ME in culture, the mRNA expression of the anti-oxidative genes NF-E2-related factor 2 (Nrf2), superoxide dismutase 2 (SOD2), and heme oxygenase-1 (HO-1) was lower and reactive oxygen species (ROS) generation was higher in Bmal1-KO BMMCs than in WT BMMCs at steady state. The IgE-dependent ROS generation and degranulation were enhanced in Bmal1-KO BMMCs compared to WT BMMCs in the absence of 2-ME in culture. The addition of 2-ME into the culture abrogated or weakened the differences in anti-oxidative gene expression, ROS generation, and IgE-dependent degranulation between WT and Bmal1-KO BMMCs. ConclusionThe current findings suggest that Bmal1 controls the expression of anti-oxidative genes in mast cells and Bmal1 deficiency enhanced IgE-dependent degranulation associated with promotion of ROS generation. Thus, Bmal1 may function as a key molecule that integrates redox homeostasis and effector functions in mast cells.

MITF Downregulation Induces Death in Human Mast Cell Leukemia Cells and Impairs IgE-Dependent Degranulation.

Activating mutations in KIT (CD117) have been associated with several diseases, including gastrointestinal stromal tumors and mastocytosis. Rapidly progressing pathologies or drug resistance highlight the need for alternative treatment strategies. Previously, we reported that the adaptor molecule SH3 binding protein 2 (SH3BP2 or 3BP2) regulates KIT expression at the transcriptional level and microphthalmia-associated transcription factor (MITF) expression at the post-transcriptional level in human mast cells and gastrointestinal stromal tumor (GIST) cell lines. Lately, we have found that the SH3BP2 pathway regulates MITF through miR-1246 and miR-5100 in GIST. In this study, miR-1246 and miR-5100 were validated by qPCR in the SH3BP2-silenced human mast cell leukemia cell line (HMC-1). MiRNA overexpression reduces MITF and MITF-dependent target expression in HMC-1. The same pattern was observed after MITF silencing. In addition, MITF inhibitor ML329 treatment reduces MITF expression and affects the viability and cell cycle progression in HMC-1. We also examine whether MITF downregulation affected IgE-dependent mast cell degranulation. MiRNA overexpression, MITF silencing, and ML329 treatment reduced IgE-dependent degranulation in LAD2- and CD34+-derived mast cells. These findings suggest MITF may be a potential therapeutic target for allergic reactions and deregulated KIT mast-cell-mediated disorders.

Nanoparticles Displaying Allergen and Siglec-8 Ligands Suppress IgE-FcεRI-Mediated Anaphylaxis and Desensitize Mast Cells to Subsequent Antigen Challenge.

Siglec-8 is an inhibitory receptor expressed on eosinophils and mast cells. In this study, we took advantage of a novel Siglec-8 transgenic mouse model to assess the impact of modulating IgE-dependent mast cell degranulation and anaphylaxis using a liposomal platform to display an allergen with or without a synthetic glycan ligand for Siglec-8 (Sig8L). The hypothesis is that recruitment of Siglec-8 to the IgE-FcεRI receptor complex will inhibit allergen-induced mast cell degranulation. Codisplay of both allergen and Sig8L on liposomes profoundly suppresses IgE-mediated degranulation of mouse bone marrow-derived mast cells or rat basophilic leukemia cells expressing Siglec-8. In contrast, liposomes displaying only Sig8L have no significant suppression of antigenic liposome-induced degranulation, demonstrating that the inhibitory activity by Siglec-8 occurs only when Ag and Sig8L are on the same particle. In mouse models of anaphylaxis, display of Sig8L on antigenic liposomes completely suppresses IgE-mediated anaphylaxis in transgenic mice with mast cells expressing Siglec-8 but has no protection in mice that do not express Siglec-8. Furthermore, mice protected from anaphylaxis remain desensitized to subsequent allergen challenge because of loss of Ag-specific IgE from the cell surface and accelerated clearance of IgE from the blood. Thus, although expression of human Siglec-8 on murine mast cells does not by itself modulate IgE-FcεRI-mediated cell activation, the enforced recruitment of Siglec-8 to the FcεRI receptor by Sig8L-decorated antigenic liposomes results in inhibition of degranulation and desensitization to subsequent Ag exposure.

Gender differences in anaphylaxis.

Is sexual dimorphism also true in anaphylaxis as described in other allergic diseases? Possible gender differences in the epidemiology, triggers, severity, outcomes of anaphylaxis as well as in the pathogenesis of the disease are discussed. Hormonal status and the X-chromosome-coded factors deeply involved in the regulation of T-cell and B-cell responses may influence the gender difference noticed in allergic diseases, such as asthma and rhinitis. Little is known if sex is also relevant for anaphylaxis, although the description of catamenial anaphylaxis is intriguing. However, epidemiologic bias, lack of reliable animal models for the human disease, differences into diagnostic codes and not harmonized clinical grading unfortunately represent hurdles to obtain meaningful information on this topic. The female sex predisposes to a dysregulation of the immune response as suggested by the increased prevalence of autoimmunity and atopy. In anaphylaxis, pathomechanisms are not fully disclosed, triggers are numerous and IgE-dependent mast cell degranulation only represents a part of the story. Improvement into the definition of the disease including a more careful coding system and better investigations about triggers seem the only way to allow a more precise assessment of the possible different risk for women to develop anaphylaxis.

Mas-Related G Protein-Coupled Receptor-X2 (MRGPRX2) in Drug Hypersensitivity Reactions.

The human ortholog MRGPRX2 and the mice ortholog, Mrgprb2 are activated by basic secretagogues and neurokinins. A number of commonly used small-molecule drugs (e.g., neuromuscular blocking agents, fluoroquinolones, vancomycin) have been recently shown to activate these receptors under in vitro experimental conditions, what results in mast cell degranulation. The above drugs are also known to cause IgE-mediated anaphylactic reactions in allergic patients. The new findings on mechanisms of drug-induced mast cell degranulation may modify the current management of drug hypersensitivity reactions. Clinical interpretation of mild drug-provoked hypersensitivity reactions, interpretation of skin test with a drug of interest or further recommendations for patients suspected of drug allergy are likely to be reconsidered. In the paper we discussed future directions in research on identification and differentiation of MRGPRX2-mediated and IgE-dependent mast cell degranulation in patients presenting clinical features of drug-induced hypersensitivity reactions.

Innate function of house dust mite allergens: robust enzymatic degradation of extracellular matrix at elevated pH

Background Exposure to the house dust mite Dermatophagoides pteronyssinus (D.p.) increases the risk for developing allergic diseases in humans and their best friends, the dogs. Here, we explored whether this allergenic mite via its enzymes may impact the cutaneous extracellular matrix (ECM), which critically determines epithelial barrier integrity both structurally and functionally.Methods Two extracts obtained from either dust-purified or cultured D.p. bodies were used in the present study. To assess the potential impact of D.p. on protein components of the ECM, proteolytic activity of the D.p. extracts were determined by casein and gelatin gel zymography, and their N-acetyl-β-hexosaminidase activity determined colorimetrically. In addition, IgE-dependent and innate degranulation potential of D.p. was examined in canine MPT-1 mast cells and neurite outgrowth assay using rat pheochromocytoma PC-12 cells.Results In gel zymography, both extracts digested the substrates casein and gelatin in a dose-dependent manner, especially at alkaline pH, and effective in a wide range of temperatures (30 °C−42 °C). In particular, a 25-kDa band corresponding to Der p 1, the major D.p. allergen for humans, was found enzymatically active in both casein and gelatin gels regardless of the presence of metal ions and of alkaline conditions. Besides protease activity, N-acetyl-β-hexosaminidase activity was detected in both extracts, suggesting that D.p. affects the cutaneous ECM through deteriorating both proteins and glycosaminoglycans. While both D.p. extracts induced IgE-dependent mast cell degranulation, much less innate effects on mast- and neuronal cells were observed.Conclusions Our data highlight that D.p. is a robust source of several distinct enzymes with protease- and N-acetyl-β-hexosaminidase activities. In alkaline milieu they can degrade components of the ECM. Therefore, D.p. may contribute to epithelial barrier disruption especially when the skin surface pH is elevated.

Bacteria and viruses modulate FcεRI-dependent mast cell activity

Undoubtedly, mast cells play a central role in allergic processes. Specific allergen cross-linking of IgE bound to the high affinity receptors (FcεRI) on the mast cell surface leads to the release of preformed mediators and newly synthesized mediators, i.e. metabolites of arachidonic acid and cytokines. More and more data indicate that bacteria and viruses can influence FcεRI-dependent mast cell activation. Some bacterial and viral components can reduce the surface expression of FcεRI. There are also findings that ligation of Toll-like receptors (TLRs) by bacterial or viral antigens can affect IgE-dependent mast cell degranulation and preformed mediator release as well as eicosanoid production. The synergistic interaction of TLR ligands and allergen can also modify cytokine synthesis by mast cells stimulated via FcεRI. Moreover, data suggest that specific IgE for bacterial or viral antigens can influence mast cell activity. What is more, some bacterial and viral components or some endogenous proteins produced during viral infection can act as superantigens by interacting with the VH3 domain of IgE. All these observations indicate that bacterial and viral infections modify the course of allergic diseases by affecting FcεRI-dependent mast cell activation.

Critical role of P1-Runx1 in mouse basophil development

AbstractRunx1P1N/P1N mice are deficient in the transcription factor distal promoter-derived Runt-related transcription factor 1 (P1-Runx1) and have a > 90% reduction in the numbers of basophils in the BM, spleen, and blood. In contrast, Runx1P1N/P1N mice have normal numbers of the other granulocytes (neutrophils and eosinophils). Although basophils and mast cells share some common features, Runx1P1N/P1N mice have normal numbers of mast cells in multiple tissues. Runx1P1N/P1N mice fail to develop a basophil-dependent reaction, IgE-mediated chronic allergic inflammation of the skin, but respond normally when tested for IgE- and mast cell–dependent passive cutaneous anaphylaxis in vivo or IgE-dependent mast cell degranulation in vitro. These results demonstrate that Runx1P1N/P1N mice exhibit markedly impaired function of basophils, but not mast cells. Infection with the parasite Strongyloides venezuelensis and injections of IL-3, each of which induces marked basophilia in wild-type mice, also induce modest expansions of the very small populations of basophils in Runx1P1N/P1N mice. Finally, Runx1P1N/P1N mice have normal numbers of the granulocyte progenitor cells, SN-Flk2+/−, which can give rise to all granulocytes, but exhibit a > 95% reduction in basophil progenitors. The results of the present study suggest that P1-Runx1 is critical for a stage of basophil development between SN-Flk2+/− cells and basophil progenitors.

Enhancement of IgE- and mast cell-dependent cutaneous inflammation by mast cell expression of C3aR and C5aR (177.5)

Abstract Mast cells express receptors for complement anaphylatoxins C3a and C5a (i.e., C3aR and C5aR) and can be activated by C3a or C5a in vitro, and C3a and C5a are generated during IgE-dependent immediate hypersensitivity reactions in vivo. However, it is not clear to what extent expression of C3aR or C5aR by mast cells influences either C3a- or C5a-induced cutaneous responses, or IgE-dependent passive cutaneous anaphylaxis (PCA), in vivo. In this study we assessed whether the expression of C3aR or C5aR by mouse skin mast cells influences: 1) the cells’ responsiveness to i.d. injections of exogenous C3a or C5a, or 2) the extent of IgE-dependent mast cell degranulation and IgE-dependent PCA, in vivo. We measured the magnitude of cutaneous responses to i.d. injections of C3a or C5a, and the extent of IgE-dependent mast cell degranulation and IgE-dependent PCA responses, in mice containing mast cells that did or did not express C3aR or C5aR. We found that the majority of the skin swelling induced by C3a or C5a required that mast cells expressed C3aR or C5aR, respectively, and the extent of IgE-dependent degranulation of skin mast cells and of IgE-dependent PCA were significantly reduced when mast cells lacked either C3aR or C5aR. We conclude that the expression of C3aR and C5aR by skin mast cells contributes to the ability of C3a and C5a to induce skin swelling and can enhance the extent of mast cell degranulation and inflammation during IgE-dependent PCA in vivo.

The significance of chloride in the inhibitory action of disodium cromoglycate on immunologically-stimulated rat peritoneal mast cells

BackgroundThe microelectrode array (MEA) was used to investigate the pharmacological relevance of chloride (Cl−) ions in antigen-dependent mast cell activation and the inhibitory effect of disodium cromoglycate (DSCG) on mast cell activation. MethodsThe movements of ions across the cellular membrane and the potential relationship between Cl− channels and DSCG during immunological activation were investigated using the MEA. The results were then subsequently compared with the amount of histamine released from anti-IgE activated peritoneal mast cells. ResultsThe inclusion of charybdotoxin (ChTX) in Cl−-free buffer showed that the measured field potentials during antigen-stimulated peritoneal mast cell were a combination of Cl− influx and K+ efflux. The delayed onset time of Cl− influx indicated the presence of a delayed outwardly-rectifying Cl− current in the antigen-stimulated peritoneal mast cells. The use of 5-nitro-2-(3-phenylpropylamino) benzoic acid demonstrated that the activated mast cell membrane potential can be stabilised, thereby reducing the amount of histamine released from the anti-IgE activated mast cells. The correlation between the results of the histamine release assay and the electrophysiological measurements demonstrated the importance of Cl− to anti-IgE dependent mast cell activation. The inhibitory effect of DSCG on anti-IgE activated cells, however, did not correlate with the presumed influx of Cl−. ConclusionsThe MEA data suggest that Cl− influx is crucial to IgE-dependent mast cell degranulation. General significanceWhile the MEA cannot yield information about single channel properties, it is convenient to use and can provide information on the global changes in electrophysiological responses of non-excitable cells.

Pharmacological Manipulation of Immune-Induced Food Aversion in Rats

Background: Mice allergic to ovalbumin (OVA) avoid drinking a solution containing this antigen. This was interpreted as related to IgE-dependent mast cell degranulation and sensory C fiber activation. Methods: We employed pharmacological manipulation to further investigate the mediators involved in immune-induced food aversion. Results: While nonimmunized rats preferred a sweetened OVA solution, immunized rats avoided it. We also employed a paradigm in which rats are conditioned to drink water for two 10-min sessions a day. Tolerant rats presented lower IgE titers, and this manipulation abrogated food aversion. Dexamethasone (1.0 mg/kg) prevented the aversion of OVA-immunized rats to the antigen-containing solution. Combined blockade of H₁ and 5-hydroxytryptamine (5-HT)₂ receptors by promethazine (3.0 mg/kg) plus methysergide (5.0 mg/kg) was unable to alter food aversion. Blockade of 5-HT₃ receptors by ondansetron (1.0 mg/kg) caused a twofold increase in the ingestion of the sweetened OVA solution by immunized rats, suggesting the involvement of 5-HT₃ receptors in food aversion. Finally, we showed that dexamethasone or promethazine plus methysergide, but not ondansetron, effectively prevented the IgE-dependent mast-cell-degranulation-induced increase in vascular permeability in rats. Conclusion: We suggest that regardless of whether or not they cause edema, IgE-mediated mast cell degranulation and consequent 5-HT₃ signaling are involved in the process that triggers avoidance to the source of the allergen in allergic rats.

Surfactant protein D decreases pollen-induced IgE-dependent mast cell degranulation

Mast cells play a key role in allergy and asthma. They reside at the host-environment interface and are among the first cells to make contact with inhaled microorganisms and particulate antigens. Pulmonary surfactant proteins A and D (SP-A and SP-D) function in lung host defense by enhancing microbe phagocytosis and mediating other immune cell functions, but little is known about their effects on mast cells. We hypothesized that SP-A and/or SP-D modulate IgE-dependent mast cell functions. Pollen starch granules (PSG) extracted from Dactylis glomerata and coated with trinitrophenol (TNP) were used as a model of an inhaled organic particulate allergen. Our data revealed that SP-D inhibited by 50% the release of beta-hexosaminidase by peritoneal mast cells sensitized with IgE anti-TNP and stimulated with TNP-PSG. In contrast, SP-A had no effect. Furthermore, SP-D aggregated PSG in a dose-dependent manner, and this aggregation was mediated by SP-D's carbohydrate recognition domain. A single arm SP-D mutant (RrSP-Dser15,20) neither aggregated PSG nor inhibited degranulation, suggesting that multimerization of SP-D is required for maximal PSG aggregation and inhibition of PSG-induced mast cell degranulation. This study is the first to demonstrate that SP-D modulates IgE-mediated mast cell functions, which are important in asthma and allergic inflammation.

Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation.

Fc epsilon RI activation of mast cells is thought to involve Lyn and Syk kinases proximal to the receptor and the signaling complex organized by the linker for activation of T cells (LAT). We report here that Fc epsilon RI also uses a Fyn kinase-dependent pathway that does not require Lyn kinase or the adapter LAT for its initiation, but is necessary for mast cell degranulation. Lyn-deficiency enhanced Fyn-dependent signals and degranulation, but inhibited the calcium response. Fyn-deficiency impaired degranulation, whereas Lyn-mediated signaling and calcium was normal. Thus, Fc epsilon RI-dependent mast cell degranulation involves cross-talk between Fyn and Lyn kinases.

Mast cell alpha-chymase reduces IgE recognition of birch pollen profilin by cleaving antibody-binding epitopes.

In sensitized individuals birch pollen induces an allergic response characterized by IgE-dependent mast cell degranulation of mediators, such as alpha-chymase and other serine proteases. In birch and other plant pollens, a major allergen is profilin. In mammals, profilin homologues are found in an intracellular form bound to cytoskeletal or cytosolic proteins or in a secreted form that may initiate signal transduction. IgE specific to birch profilin also binds human profilin I. This cross-reactivity between airborne and endogenous proteins may help to sustain allergy symptoms. The current work demonstrates that cultured mast cells constitutively secrete profilin I, which is susceptible to degranulation-dependent proteolysis. Coincubation of chymase-rich BR mastocytoma cells with Ala-Ala-Pro-Phe-chloromethylketone (a chymase inhibitor) blocks profilin cleavage, which does not occur in degranulated HMC-1 mast cells, which are rich in tryptase, but chymase deficient. These data implicate chymase as the serine protease cleaving secreted mast cell profilin. Sequencing of chymase-cleaved profilins reveals hydrolysis at Tyr(6)-Val(7) and Trp(35)-Ala(36) in birch profilin and at Trp(32)-Ala(33) in human profilin, with all sites lying within IgE-reactive epitopes. IgE immunoblotting studies with sera from birch pollen-allergic individuals demonstrate that cleavage by chymase attenuates binding of birch profilin to IgE. Thus, destruction of IgE-binding epitopes by exocytosed chymase may limit further mast cell activation by this class of common plant allergens, thereby limiting the allergic responses in sensitized individuals.

Mast-cell chymase: a new target for anti-inflammatory drugs

Human mast cells have complex roles in allergic and inflammatory disorders and might contribute to atherosclerosis by releasing cytokines and vasoactive and proinflammatory mediators. Modulating mast-cell pro-inflammatory activities is, therefore, a plausible target for developing new anti-inflammatory drugs. Chymase is a chymotrypsin-type serine protease mainly localized in mast cells. Following IgE-dependent mast-cell degranulation, chymase appears to participate in inflammatory processes, including extracellular-matrix degradation, collagenase activation, target-cell apoptosis, processing of inflammatory cytokines and induction of cytokine secretion by bystander leukocytes.

Recruitment of neutrophils during IgE-dependent cutaneous late phase reactions in the mouse is mast cell-dependent. Partial inhibition of the reaction with antiserum against tumor necrosis factor-alpha.

Much of the clinically important pathology associated with IgE-dependent disorders is thought to reflect the actions of the blood-borne leukocytes recruited during these responses. To evaluate the extent to which mast cells are responsible for the leukocyte infiltration associated with IgE-dependent cutaneous reactions, we attempted to elicit these responses in normal mice, genetically mast cell-deficient W/Wv mice, and in W/Wv mice selectively repaired of their mast cell deficiency by the intradermal injection of cultured mast cells derived from the congenic normal (+/+) mice. We found that the tissue swelling associated with IgE-dependent passive cutaneous anaphylaxis reactions developed rapidly and diminished markedly from 2 to 4 h after antigen challenge, but remained detectable for at least 24 h after elicitation of the responses. Infiltration of leukocytes (predominantly neutrophils) also occurred at these sites, but reached maximal levels 6-12 h after antigen challenge, persisted at high levels for 24 h, and largely waned by 48 h. Virtually all of the tissue swelling and leukocyte infiltration associated with IgE-dependent cutaneous reactions was mast cell dependent. Intradermal injection of 40 U of recombinant murine TNF-alpha (rmTNF-alpha) elicited neutrophil infiltration similar in magnitude and kinetics to that observed after IgE-dependent mast cell degranulation. A rabbit anti-rmTNF-alpha (R anti-rmTNF-alpha) antiserum, which was able to inhibit 84% of the neutrophil infiltration observed after i.d. injection of rmTNF-alpha, inhibited IgE-, and mast cell-dependent leukocyte infiltration by 47 +/- 7% in three separate experiments. These findings indicate that TNF-alpha contributes to mast cell-dependent recruitment of leukocytes during IgE-dependent cutaneous late phase reactions, but suggest that other mast cell-associated mediators probably also contribute to this response.

IGE-MEDIATED LATE PHASE REACTIONS - THE PATHO-PHYSIOLOGIC BACKGROUND OF CHRONIC BRONCHIAL ASTHMA?

Late phase reactions (LPR) have been considered generally as type III allergy according to the classification of Coombs and Gell. IgE-antibodies, however, are able to initiate not only immediate, but also late cutaneous reactions (LCR). Using the skin blister technique we could show LCR to be mediated by inflammatory mediators generated during the preceeding immediate reaction. Various mediators as histamine, active kallikrein, platelet activating properties, leukotrienes and thromboxanes were demonstrable in skin blister fluids their concentration being in part related to the time course of the skin reactions. LCRs were diminished by eikosatetraynoic acid (not by indomethacin), by dazoxiben as well as by ethanolic onion extract. Betamimetics as salbutamol and anticholinergics as ipratropiumbromide could reverse late bronchial reactions (LBR), ethanolic onion extract could prevent LBR. Other authors found prostaglandin D2 and chemotactic substances during LPR as well as protective effects of corticosteroids, disodium cromoglicicum, tranexamic acid and combined histamine H1/H2 receptor antagonists. Two types of LPR have to be distinguished: type I and type III. The further is initiated by a IgE-dependent mast cell degranulation and maintained by a complex inter action of mediators and cells. It represents the patho-physiolggic correlate of long lasting bronchial asthma.