ABSTRACT
BACKGROUND: Despite their central role in peanut allergy, human monoclonal IgE antibodies have eluded characterization. OBJECTIVE: We sought to define the sequences, affinities, clonality, and functional properties of human monoclonal IgE antibodies in peanut allergy. METHODS: We applied our single-cell RNA sequencing-based SEQ SIFTER discovery platform to samples from allergic individuals who varied by age, sex, ethnicity, and geographic location in order to understand commonalities in the human IgE response to peanut allergens. Select antibodies were then recombinantly expressed and characterized for their allergen and epitope specificity, affinity, and functional properties. RESULTS: We found striking convergent evolution of IgE monoclonal antibodies (mAbs) from several clonal families comprising both memory B cells and plasmablasts. These antibodies bound with subnanomolar affinity to the immunodominant peanut allergen Ara h 2, specifically a linear, repetitive motif. Further characterization of these mAbs revealed their ability to single-handedly cause affinity-dependent degranulation of human mast cells and systemic anaphylaxis on peanut allergen challenge in humanized mice. Finally, we demonstrated that these mAbs, reengineered as IgGs, inhibit significant, but variable, amounts of Ara h 2- and peanut-mediated degranulation of mast cells sensitized with allergic plasma. CONCLUSIONS: Convergent evolution of IgE mAbs in peanut allergy is a common phenomenon that can reveal immunodominant epitopes on major allergenic proteins. Understanding the functional properties of these molecules is key to developing therapeutics, such as competitive IgG inhibitors, that are able to stoichiometrically outcompete endogenous IgE for allergen and thereby prevent allergic cascade in cases of accidental allergen exposure.
Subject(s)
Peanut Hypersensitivity , Humans , Animals , Mice , Immunodominant Epitopes , Antigens, Plant , Glycoproteins , Immunoglobulin E , Epitopes , Antibodies, Monoclonal , Allergens , Arachis , 2S Albumins, PlantABSTRACT
Neobavaisoflavone (NBIF), a monomolecular compound extracted from Psoralea corylifolia (Leguminosae), is commonly used in traditional Chinese medicine for multiple purposes. NBIF is known to exert anti-fungal and anti-tumor effects, and promote bone formation. Whether NBIF exhibits anti-allergic effects by regulating mast cell activation remains unclear. Therefore, we designed this study to investigate the anti-allergic effects of NBIF on IgE/Ag-induced mouse bone marrow-derived mast cells and ovalbumin-induced asthma, and the passive systemic anaphylaxis (PSA) reaction in mice. Our results showed that NBIF suppresses the production of leukotriene C4, prostaglandin D2 and inflammatory cytokines, and decreases the degranulation of BMMCs stimulated by IgE/Ag. A thorough investigation ascertained that NBIF suppresses the phosphorylation of mitogen-activated protein kinases, and represses the nuclear factor-κB-related signaling pathway. In addition, the oral administration of NBIF in mice inhibited the IgE-induced PSA reaction in a dose-dependent manner. Overall, we provide new insights into how NBIF regulates the IgE/Ag-mediated signaling pathways. Moreover, our investigation promotes the potential use of NBIF in treating allergy and asthma.
Subject(s)
Anaphylaxis , Anti-Allergic Agents , Asthma , Hypersensitivity , Anaphylaxis/drug therapy , Animals , Anti-Allergic Agents/pharmacology , Anti-Allergic Agents/therapeutic use , Asthma/drug therapy , Asthma/metabolism , Cell Degranulation , Hypersensitivity/drug therapy , Immunoglobulin E/metabolism , Isoflavones , Mast Cells , Mice , Mice, Inbred BALB CABSTRACT
Introduction: Asthma is a chronic and recurring airway disease, which related to mast cell activation. Many compounds derived from Chinese herbal medicine has promising effects on stabilizing mast cells and decreasing inflammatory mediator production. Safranal, one of the active compounds from Crocus sativus, shows many anti-inflammatory properties. In this study, we evaluated the effect of safranal in ovalbumin (OVA)-induced asthma model. Furthermore, we investigate the effectiveness of safranal on stabilizing mast cell and inhibiting the production of inflammatory mediators in passive systemic anaphylaxis (PSA) model. Methods: OVA-induced asthma and PSA model were used to evaluate the effect of safranal in vivo. Lung tissues were collected for H&E, TB, IHC, and PAS staining. ELISA were used to determine level of IgE and chemokines (IL-4, IL-5, TNF-α, and IFN-γ). RNA sequencing was used to uncovers genes that safranal regulate. Bone marrow-derived mast cells (BMMCs) were used to investigate the inhibitory effect and mechanism of safranal. Cytokine production (IL-6, TNF-α, and LTC4) and NF-κB and MAPKs signaling pathway were assessed. Results: Safranal reduced the level of serum IgE, the number of mast cells in lung tissue were decreased and Th1/Th2 cytokine levels were normalized in OVA-induced asthma model. Furthermore, safranal inhibited BMMCs degranulation and inhibited the production of LTC4, IL-6, and TNF-α. Safranal inhibits NF-κB and MAPKs pathway protein phosphorylation and decreases NF-κB p65, AP-1 nuclear translocation. In the PSA model, safranal reduced the levels of histamine and LTC4 in serum. Conclusions: Safranal alleviates OVA-induced asthma, inhibits mast cell activation and PSA reaction. The possible mechanism occurs through the inhibition of the MAPKs and NF-κB pathways.
Subject(s)
Allergens/immunology , Asthma/etiology , Cyclohexenes/pharmacology , Mast Cells/drug effects , Mast Cells/immunology , Ovalbumin/adverse effects , Terpenes/pharmacology , Animals , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/pharmacology , Asthma/drug therapy , Asthma/metabolism , Asthma/pathology , Cell Degranulation/drug effects , Cell Degranulation/immunology , Cyclohexenes/administration & dosage , Cytokines/metabolism , Disease Models, Animal , Disease Susceptibility , Female , Immunoglobulin E/immunology , Inflammation Mediators/metabolism , Mast Cells/metabolism , Mice , NF-kappa B/metabolism , Ovalbumin/immunology , Signal Transduction/drug effects , Terpenes/administration & dosageABSTRACT
Mast cells are involved in many physiological reactions in which their functions can be very diverse. Models of allergic skin inflammation and systemic anaphylactic reactions in mice are validated methods in which the role of mast cells is well established. In this chapter, we will therefore present protocols for passive cutaneous anaphylaxis and contact hypersensitivity, i.e., models which can be used to identify and characterize the role of mast cells as well as mast cell mediators and receptors in allergic IgE-dependent and IgE-independent skin inflammation, and for passive systemic anaphylaxis, a model ideally suited to characterize the systemic effects of mast cell-derived mediators and mast cell receptors.
Subject(s)
Anaphylaxis/immunology , Dermatitis, Contact/immunology , Disease Models, Animal , Immunization/methods , Mast Cells/immunology , Mast Cells/metabolism , Skin Tests/methods , Animals , Cell Degranulation/immunology , Dermatitis, Contact/metabolism , Ear, External/immunology , Ear, External/pathology , Edema/chemically induced , Edema/immunology , Haptens/administration & dosage , Mast Cells/chemistry , Mice , Skin/immunology , Skin/pathologyABSTRACT
Mercaptopyruvate sulfurtransferase (Mpst) and its homolog thiosulfate sulfurtransferase (Tst = rhodanese) detoxify cyanide to thiocyanate. Mpst is attracting attention as one of the four endogenous hydrogen sulfide (H2S)/reactive sulfur species (RSS)-producing enzymes, along with cystathionine ß-synthase (Cbs), cystathionine γ-lyase (Cth), and cysteinyl-tRNA synthetase 2 (Cars2). MPST deficiency was found in 1960s among rare hereditary mercaptolactate-cysteine disulfiduria patients. Mpst-knockout (KO) mice with enhanced liver Tst expression were recently generated as its model; however, the physiological roles/significances of Mpst remain largely unknown. Here we generated three independent germ lines of Mpst-KO mice by CRISPR/Cas9 technology, all of which maintained normal hepatic Tst expression/activity. Mpst/Cth-double knockout (DKO) mice were generated via crossbreeding with our previously generated Cth-KO mice. Mpst-KO mice were born at the expected frequency and developed normally like Cth-KO mice, but displayed increased urinary 3-mercaptolactate excretion and enhanced passive systemic anaphylactic responses when compared to wild-type or Cth-KO mice. Mpst/Cth-DKO mice were also born at the expected frequency and developed normally, but excreted slightly more 3-mercaptolactate in urine compared to Mpst-KO or Cth-KO mice. Our Mpst-KO, Cth-KO, and Mpst/Cth-DKO mice, unlike semi-lethal Cbs-KO mice and lethal Cars2-KO mice, are useful tools for analyzing the unknown physiological roles of endogenous H2S/RSS production.
Subject(s)
Amino Acid Metabolism, Inborn Errors/etiology , Amino Acid Metabolism, Inborn Errors/metabolism , Sulfhydryl Compounds/urine , Sulfurtransferases/deficiency , Alleles , Amino Acid Metabolism, Inborn Errors/urine , Animals , Biomarkers , Disease Models, Animal , Gene Targeting , Genotype , Liver/metabolism , Mice , Mice, Knockout , MutationABSTRACT
The systemic anaphylactic reaction is a life-threatening allergic response initiated by activated mast cells. Sphingolipids are an essential player in the development and attenuation of this response. De novo synthesis of sphingolipids in mammalian cells is inhibited by the family of three ORMDL proteins (ORMDL1, 2, and 3). However, the cell and tissue-specific functions of ORMDL proteins in mast cell signaling are poorly understood. This study aimed to determine cross-talk of ORMDL2 and ORMDL3 proteins in IgE-mediated responses. To this end, we prepared mice with whole-body knockout (KO) of Ormdl2 and/or Ormdl3 genes and studied their role in mast cell-dependent activation events in vitro and in vivo. We found that the absence of ORMDL3 in bone marrow-derived mast cells (BMMCs) increased the levels of cellular sphingolipids. Such an increase was further raised by simultaneous ORMDL2 deficiency, which alone had no effect on sphingolipid levels. Cells with double ORMDL2 and ORMDL3 KO exhibited increased intracellular levels of sphingosine-1-phosphate (S1P). Furthermore, we found that concurrent ORMDL2 and ORMDL3 deficiency increased IκB-α phosphorylation, degranulation, and production of IL-4, IL-6, and TNF-α cytokines in antigen-activated mast cells. Interestingly, the chemotaxis towards antigen was increased in all mutant cell types analyzed. Experiments in vivo showed that passive cutaneous anaphylaxis (PCA), which is initiated by mast cell activation, was increased only in ORMDL2,3 double KO mice, supporting our in vitro observations with mast cells. On the other hand, ORMDL3 KO and ORMDL2,3 double KO mice showed faster recovery from passive systemic anaphylaxis, which could be mediated by increased levels of blood S1P presented in such mice. Our findings demonstrate that Ormdl2 deficiency potentiates the ORMDL3-dependent changes in mast cell signaling.
Subject(s)
Mast Cells/immunology , Mast Cells/metabolism , Membrane Proteins/deficiency , Signal Transduction , Amino Acid Sequence , Anaphylaxis/etiology , Anaphylaxis/metabolism , Animals , Biomarkers , Calcium/metabolism , Calcium Signaling , Chemotaxis/immunology , Cytokines/metabolism , Disease Susceptibility , Gene Expression , Lysophospholipids/blood , Lysophospholipids/metabolism , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Knockout , Multigene Family , Passive Cutaneous Anaphylaxis/genetics , Passive Cutaneous Anaphylaxis/immunology , Sphingolipids/blood , Sphingolipids/metabolism , Sphingosine/analogs & derivatives , Sphingosine/blood , Sphingosine/metabolismABSTRACT
Basophils, which are the rarest granulocytes, play crucial roles in protective immunity against parasites and development of allergic disorders. Although immunoglobulin (Ig)E-dependent responses via receptor for IgE (FcεRI) in basophils have been extensively studied, little is known about cell surface molecules that are selectively expressed on this cell subset to utilize the elimination in vivo through treatment with monoclonal antibody (mAb). Since CD200 receptor 3 (CD200R3) was exclusively expressed on basophils and mast cells (MCs) using a microarray screening, we have generated anti-CD200R3 mAb recognizing CD200R3A. In this study we examined the expression pattern of CD200R3A on leukocytes, and the influence of the elimination of basophils by anti-CD200R3A mAb on allergic responses. Flow cytometric analysis showed that CD200R3A was primarily expressed on basophils and MCs, but not on other leukocytes. Administration with anti-CD200R3A mAb led to the prominent specific depletion of tissue-resident and circulating basophils, but not MCs. Furthermore, in vivo depletion of basophils ameliorated IgE-mediated systemic and local anaphylaxis. Taken together, these findings suggest that CD200R3A is reliable cell surface marker for basophils in vivo, and targeting this unique molecule with mAb for the elimination of basophils may serve as a novel therapeutic strategy in ameliorating the allergic diseases.
ABSTRACT
Objective:To ascertain whether the immune complexes (ICs) formed by Dengue virus 1 non-structure protein 1 (DENV1 NS1)and its IgG antibodies could mediate passive systemic anaphylaxis (PSA) and to explain the pathogenesis of Dengue hemorrhagic fever or Dengue shock syndrome (DHF/DSS).Methods:The monoclonal antibodies (mAbs) or mAb cocktails from 20 IgG mAbs of DENV1 NS1 prepared in this lab were screened to initiate PSA and passive cutaneous anaphylaxis (PCA) in mice.Meanwhile, the effects of GdCl3 and platelet activating factor ( PAF) antagonist CV-3988 on PSA induced by the NS1-IgG ICs were observed.Results:Two groups of monoclonal antibody cocktails with purified NS 1 were proved to be capable of provoking PCA and PSA in mice,whereas the other mAbs or mAb cocktails could be not .The murine PSA initiated by NS1-IgG(5D25+3B1) ICs could be sig-nificantly inhibited by in vivo treatment with GdCl3 or PAF antagonist CV-3988.Conclusion: The NS1-IgG ICs formed with DENV1 NS1 and IgG mAb cocktails can mediate PSA and PCA ,but not all of ICs formed by DENV 1 NS1 mAbs or mAb cocktails with DENV 1 NS1 can induce PSA ,indicating that it may be related to the special epitopes of DENV 1 NS1.The monocyte/macrophages and PAF may be as major effector cells and the major mediator for PSA induced by NS 1-IgG ICs,respectively.
ABSTRACT
BACKGROUND: Mast cells are a critical component of allergic responses in humans, and animal models that allow the in vivo investigation of their contribution to allergy and evaluation of new human-specific therapeutics are urgently needed. OBJECTIVE: To develop a new humanized mouse model that supports human mast cell engraftment and human IgE-dependent allergic responses. METHODS: This model is based on the NOD-scid IL2rg(null)SCF/GM-CSF/IL3 (NSG-SGM3) strain of mice engrafted with human thymus, liver, and hematopoietic stem cells (termed Bone marrow, Liver, Thymus [BLT]). RESULTS: Large numbers of human mast cells develop in NSG-SGM3 BLT mice and populate the immune system, peritoneal cavity, and peripheral tissues. The human mast cells in NSG-SGM3 BLT mice are phenotypically similar to primary human mast cells and express CD117, tryptase, and FcεRI. These mast cells undergo degranulation in an IgE-dependent and -independent manner, and can be readily cultured in vitro for additional studies. Intradermal priming of engrafted NSG-SGM3 mice with a chimeric IgE containing human constant regions resulted in the development of a robust passive cutaneous anaphylaxis response. Moreover, we describe the first report of a human mast cell antigen-dependent passive systemic anaphylaxis response in primed mice. CONCLUSIONS: NSG-SGM3 BLT mice provide a readily available source of human mast cells for investigation of mast cell biology and a preclinical model of passive cutaneous anaphylaxis and passive systemic anaphylaxis that can be used to investigate the pathogenesis of human allergic responses and to test new therapeutics before their advancement to the clinic.
Subject(s)
Anaphylaxis/immunology , Disease Models, Animal , Mast Cells/immunology , Passive Cutaneous Anaphylaxis/immunology , Animals , Hematopoietic Stem Cell Transplantation , Humans , Immunoglobulin E/immunology , Liver Transplantation , Mice , Thymus Gland/transplantationABSTRACT
Antigen-induced mast cell (MC) activation via cross-linking of IgE-bound high-affinity receptors for IgE (FcεRI) underlies type I allergy and anaphylactic shock. Comprehensive knowledge of FcεRI regulation is thus required. We have identified a functional interaction between FcεRI and CD13 in murine MCs. Antigen-triggered activation of IgE-loaded FcεRI results in cocapping and cointernalization of CD13 and equivalent internalization rates of up to 40%. Cointernalization is not unspecific, because ligand-driven KIT internalization is not accompanied by CD13 internalization. Moreover, antibody-mediated cross-linking of CD13 causes IL-6 production in an FcεRI-dependent manner. These data are indicative of a functional interaction between FcεRI and CD13 on MCs. To determine the role of this interaction, CD13-deficient bone marrow-derived MCs (BMMCs) were analyzed. Intriguingly, antigen stimulation of CD13-deficient BMMCs results in significantly increased degranulation and proinflammatory cytokine production compared to wild-type cells. Furthermore, in a low-dose model of passive systemic anaphylaxis, antigen-dependent decrease in body temperature, reflecting the anaphylactic reaction, is substantially enhanced by the CD13 inhibitor bestatin (-5.9 ± 0.6°C) and by CD13 deficiency (-8.8 ± 0.6°C) in contrast to controls (-1.2 ± 1.97°C). Importantly, bestatin does not aggravate anaphylaxis in CD13-deficient mice. Thus, we have identified CD13 as a novel negative regulator of MC activation in vitro and in vivo-Zotz, J. S., Wölbing, F., Lassnig, C., Kauffmann, M., Schulte, U., Kolb, A., Whitelaw, B., Müller, M., Biedermann, T., Huber, M. CD13/aminopeptidase N is a negative regulator of mast cell activation.
Subject(s)
CD13 Antigens/metabolism , Mast Cells/physiology , Anaphylaxis , Animals , CD13 Antigens/antagonists & inhibitors , CD13 Antigens/genetics , Cell Proliferation , Dinitrophenols/immunology , Gene Expression Regulation/physiology , Leucine/analogs & derivatives , Leucine/pharmacology , Macrophages , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, IgE/genetics , Receptors, IgE/metabolism , Serum Albumin/immunologyABSTRACT
BACKGROUND: Steroid receptor coactivator 3 (SRC-3) is a multifunctional protein that plays an important role in malignancy of several cancers and in regulation of bacterial LPS-induced inflammation. However, the involvement of SRC-3 in allergic response remains unclear. Herein we used passive systemic anaphylaxis (PSA) and passive cutaneous anaphylaxis (PCA) mouse models to assess the role of SRC-3 in allergic response. RESULTS: SRC-3-deficient mice exhibited more severe allergic response as demonstrated by a significant drop in body temperature and a delayed recovery period compared to wild-type mice in PSA mouse model, whereas no significant difference was observed between two kinds of mice in PCA mouse models. Mast cells play a pivotal role in IgE-mediated allergic response. Antigen-induced aggregation of IgE receptor (FcϵRI) on the surface of mast cell activates a cascade of signaling events leading to the degranulation and cytokine production in mast cells. SRC-3-deficient bone marrow derived mast cells (BMMCs) developed normally but secreted more proinflammatory cytokines such as TNF-α and IL-6 than wild-type cells after antigen stimulation, whereas there was no significant difference in degranulation between two kinds of mast cells. Further studies showed that SRC-3 inhibited the activation of nuclear factor NF-κB pathway and MAPKs including extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 in antigen-stimulated mast cells. CONCLUSIONS: Our data demonstrate that SRC-3 suppresses cytokine production in antigen-stimulated mast cells as well as PSA in mice at least in part through inhibiting NF-κB and MAPK signaling pathways. Therefore, SRC-3 plays a protective role in PSA and it may become a drug target for anaphylactic diseases.
ABSTRACT
IgG-induced passive systemic anaphylaxis (PSA), a serious adverse effect of passive immune therapy using therapeutic monoclonal antibodies, has been greatly emphasized. However, controversy exists regarding the type of effector cells involved in IgG-induced anaphylaxis as a result of the induction of PSA by different IgG subtypes or the use of mice with varying genetic backgrounds. To clarify the effector cells for PSA, the PSA model with serious hypothermia was established by IgG monoclonal antibody (mAb) against natural protein or complete antigen, not hapten conjugate, in BALB/c and C57BL/6 mice. The results indicated that PSA could be remarkably inhibited by the depletion of macrophages but not by the depletion of whole leukocytes, basophils, neutrophils or monocytes. We further confirmed that macrophages are indispensable for the PSA induced by all six IgG-natural antigen complexes in both strains of mice. Additionally, platelet-activating factor (PAF) was found to be the major effector mediator for IgG-induced anaphylaxis. Moreover, gene knock-out of the third component of complement (C3) did not affect PSA-related hypothermia in C57BL/6 mice. These results indicate that macrophages and PAF act as dominant effector cells and mediator molecules, respectively, and are indispensable components in the induction of IgG-mediated PSA induced by IgG mAb and natural protein antigen. Based on the above results, we hypothesize that inconsistencies in effector cells for PSA may be associated with different features of the mAb-antigen system that might affect the magnitude of FcγRs cross-linking on effector cells.
Subject(s)
Anaphylaxis/immunology , Immunoglobulin G/immunology , Macrophages/immunology , Platelet Activating Factor/immunology , Animals , Antibodies, Monoclonal/immunology , Basophils/immunology , Complement C3/genetics , Female , Hypothermia/immunology , Immunization, Passive/adverse effects , Leukocytes, Mononuclear/immunology , Mast Cells/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Neutrophils/immunology , Platelet-Derived Growth Factor/immunology , Receptors, IgG/immunologyABSTRACT
Curcumin is naturally occurring polyphenolic compound found in turmeric and has many pharmacological activities. The present study was undertaken to evaluate anti-allergic inflammatory activity of curcumin, and to investigate its inhibitory mechanisms in immunoglobulin E (IgE)/Ag-induced mouse bone marrow-derived mast cells (BMMCs) and in a mouse model of IgE/Ag-mediated passive systemic anaphylaxis (PSA). Curcumin inhibited cyclooxygenase-2 (COX-2) dependent prostaglandin D2 (PGD2) and 5-lipoxygenase (5-LO) dependent leukotriene C4 (LTC4) generation dose-dependently in BMMCs. To probe the mechanism involved, we assessed the effects of curcumin on the phosphorylation of Syk and its downstream signal molecules. Curcumin inhibited intracellular Ca(2+) influx via phospholipase Cγ1 (PLCγ1) activation and the phosphorylation of mitogen-activated protein kinases (MAPKs) and the nuclear factor-κB (NF-κB) pathway. Furthermore, the oral administration of curcumin significantly attenuated IgE/Ag-induced PSA, as determined by serum LTC4, PGD2, and histamine levels. Taken together, this study shows that curcumin offers a basis for drug development for the treatment of allergic inflammatory diseases.
ABSTRACT
To clarify the role of Janus kinase (JAK) in and the efficacy of JAK inhibitors on food allergy, we investigated the effect of the clinically available JAK inhibitor ruxolitinib on mouse food allergy and the functions of cultured mast cells in vitro. Anaphylactic symptoms including diarrhea and decreases in body temperature pursuant to oral ovalbumin (OVA) challenges in food allergy mice were attenuated by the daily oral administration of ruxolitinib. This drug inhibited increases in mouse mast cell protease-1 concentrations in the serum and mast cell numbers in the intestines of these mice as well as degranulation, IL-13 production, and the spontaneous and IL-9-dependent survival of mouse bone marrow-derived mast cells in spite of the absence of an effect of ruxolitinib on passive systemic anaphylaxis. Anti-OVA IgG2a, IgE, and IgG1 serum levels and the release of IFN-γ, IL-4, IL-9, and IL-10 from the OVA-restimulated splenocytes of food allergy mice were also decreased by the treatment. Moreover, ruxolitinib administration to mice that had already exhibited anaphylactic responses to previous challenges reduced anaphylactic responses to further oral OVA challenges, which suggested that ruxolitinib has a therapeutic potential on food allergy. Our results showed that ruxolitinib remitted food allergy in mice mainly through immunosuppression and the prevention of mast cell hyperplasia, and partially through the inhibition of mast cell activation. We consider JAK inhibition to be a promising strategy for the prevention of food allergy, and ruxolitinib along with its derivatives inhibiting JAK as good candidates for therapeutic drugs to treat food allergy.
Subject(s)
Food Hypersensitivity/drug therapy , Janus Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/therapeutic use , Pyrazoles/therapeutic use , Allergens/immunology , Animals , Bone Marrow Cells/cytology , Cell Line , Chymases/blood , Cytokines/immunology , Food Hypersensitivity/blood , Food Hypersensitivity/immunology , Goblet Cells/pathology , Immunoglobulin E/blood , Immunoglobulin G/blood , Intestines/cytology , Male , Mast Cells/immunology , Mast Cells/pathology , Mice , Mice, Inbred BALB C , Nitriles , Ovalbumin/immunology , Pyrimidines , Spleen/cytologyABSTRACT
Curcumin is naturally occurring polyphenolic compound found in turmeric and has many pharmacological activities. The present study was undertaken to evaluate anti-allergic inflammatory activity of curcumin, and to investigate its inhibitory mechanisms in immunoglobulin E (IgE)/Ag-induced mouse bone marrow-derived mast cells (BMMCs) and in a mouse model of IgE/Ag-mediated passive systemic anaphylaxis (PSA). Curcumin inhibited cyclooxygenase-2 (COX-2) dependent prostaglandin D2 (PGD2) and 5-lipoxygenase (5-LO) dependent leukotriene C4 (LTC4) generation dose-dependently in BMMCs. To probe the mechanism involved, we assessed the effects of curcumin on the phosphorylation of Syk and its downstream signal molecules. Curcumin inhibited intracellular Ca2+ influx via phospholipase Cgamma1 (PLCgamma1) activation and the phosphorylation of mitogen-activated protein kinases (MAPKs) and the nuclear factor-kappaB (NF-kappaB) pathway. Furthermore, the oral administration of curcumin significantly attenuated IgE/Ag-induced PSA, as determined by serum LTC4, PGD2, and histamine levels. Taken together, this study shows that curcumin offers a basis for drug development for the treatment of allergic inflammatory diseases.
Subject(s)
Animals , Mice , Administration, Oral , Anaphylaxis , Arachidonate 5-Lipoxygenase , Curcuma , Curcumin , Cyclooxygenase 2 , Histamine , Immunoglobulin E , Immunoglobulins , Leukotriene C4 , Mast Cells , Mitogen-Activated Protein Kinases , Phospholipases , Phosphorylation , Prostaglandin D2ABSTRACT
Curcumin is naturally occurring polyphenolic compound found in turmeric and has many pharmacological activities. The present study was undertaken to evaluate anti-allergic inflammatory activity of curcumin, and to investigate its inhibitory mechanisms in immunoglobulin E (IgE)/Ag-induced mouse bone marrow-derived mast cells (BMMCs) and in a mouse model of IgE/Ag-mediated passive systemic anaphylaxis (PSA). Curcumin inhibited cyclooxygenase-2 (COX-2) dependent prostaglandin D2 (PGD2) and 5-lipoxygenase (5-LO) dependent leukotriene C4 (LTC4) generation dose-dependently in BMMCs. To probe the mechanism involved, we assessed the effects of curcumin on the phosphorylation of Syk and its downstream signal molecules. Curcumin inhibited intracellular Ca2+ influx via phospholipase Cgamma1 (PLCgamma1) activation and the phosphorylation of mitogen-activated protein kinases (MAPKs) and the nuclear factor-kappaB (NF-kappaB) pathway. Furthermore, the oral administration of curcumin significantly attenuated IgE/Ag-induced PSA, as determined by serum LTC4, PGD2, and histamine levels. Taken together, this study shows that curcumin offers a basis for drug development for the treatment of allergic inflammatory diseases.
Subject(s)
Animals , Mice , Administration, Oral , Anaphylaxis , Arachidonate 5-Lipoxygenase , Curcuma , Curcumin , Cyclooxygenase 2 , Histamine , Immunoglobulin E , Immunoglobulins , Leukotriene C4 , Mast Cells , Mitogen-Activated Protein Kinases , Phospholipases , Phosphorylation , Prostaglandin D2ABSTRACT
BACKGROUND: Studies with c-kit mutant mast cell (MC)-deficient mice and antibody-mediated depletion of basophils suggest that both MCs and basophils can contribute to peanut-induced anaphylaxis (PIA). However, interpretation of data obtained by using such approaches is complicated because c-kit mutant mice have several phenotypic abnormalities in addition to MC deficiency and because basophil-depleting antibodies can also react with MCs. OBJECTIVE: We analyzed (1) the changes in the features of PIA in mice after the selective and inducible ablation of MCs or basophils and (2) the possible importance of effector cells other than MCs and basophils in the PIA response. METHODS: Wild-type and various mutant mice were orally sensitized with peanut extract and cholera toxin weekly for 4 weeks and challenged intraperitoneally with peanut extract 2 weeks later. RESULTS: Peanut-challenged, MC-deficient Kit(W-sh/W-sh) mice had reduced immediate hypothermia, as well as a late-phase decrease in body temperature that was abrogated by antibody-mediated depletion of neutrophils. Diphtheria toxin-mediated selective depletion of MCs or basophils in Mcpt5-Cre;iDTR and Mcpt8(DTR) mice, respectively, and treatment of wild-type mice with the basophil-depleting antibody Ba103 significantly reduced peanut-induced hypothermia. Non-c-kit mutant MC- and basophil-deficient Cpa3-Cre;Mcl-1(fl/fl) mice had reduced but still significant responses to peanut. CONCLUSION: Inducible and selective ablation of MCs or basophils in non-c-kit mutant mice can significantly reduce PIA, but partial responses to peanut can still be observed in the virtual absence of both cell types. The neutrophilia in Kit(W-sh/W-sh) mice might influence the responses of these mice in this PIA model.
Subject(s)
Anaphylaxis/immunology , Arachis/immunology , Basophils/immunology , Diphtheria Toxin/immunology , Mast Cells/immunology , Peanut Hypersensitivity/complications , Peanut Hypersensitivity/immunology , Anaphylaxis/etiology , Anaphylaxis/metabolism , Animals , Antibodies/immunology , Antibodies/metabolism , Basophils/metabolism , Mast Cells/metabolism , Mice , Mice, Inbred C57BL , Mutation , Peanut Hypersensitivity/metabolism , Proto-Oncogene Proteins c-kit/immunology , Proto-Oncogene Proteins c-kit/metabolismABSTRACT
BACKGROUND: Rapid desensitization transiently prevents severe allergic reactions, allowing administration of life-saving therapies in previously sensitized patients. However, the mechanisms underlying successful rapid desensitization are not fully understood. OBJECTIVES: We sought to investigate whether the mast cell (MC) is an important target of rapid desensitization in mice sensitized to exhibit IgE-dependent passive systemic anaphylaxis in vivo and to investigate the antigen specificity and underlying mechanisms of rapid desensitization in our mouse model. METHODS: C57BL/6 mice (in vivo) or primary isolated C57BL/6 mouse peritoneal mast cells (PMCs; in vitro) were passively sensitized with antigen-specific anti-2,4-dinitrophenyl IgE, anti-ovalbumin IgE, or both. MCs were exposed over a short period of time to increasing amounts of antigen (2,4-dinitrophenyl-human serum albumin or ovalbumin) in the presence of extracellular calcium in vitro or by means of intravenous administration to sensitized mice in vivo before challenging the mice with or exposing the PMCs to optimal amounts of specific or irrelevant antigen. RESULTS: Rapidly exposing mice or PMCs to progressively increasing amounts of specific antigen inhibited the development of antigen-induced hypothermia in sensitized mice in vivo and inhibited antigen-induced PMC degranulation and prostaglandin D2 synthesis in vitro. Such MC hyporesponsiveness was induced antigen-specifically and was associated with a significant reduction in antigen-specific IgE levels on MC surfaces. CONCLUSIONS: Rapidly exposing MCs to progressively increasing amounts of antigen can both enhance the internalization of antigen-specific IgE on the MC surface and also desensitize these cells in an antigen-specific manner in vivo and in vitro.
Subject(s)
Anaphylaxis/therapy , Antigens/immunology , Desensitization, Immunologic/methods , Immunoglobulin E/immunology , Mast Cells/immunology , 2,4-Dinitrophenol/immunology , Animals , Antibodies, Anti-Idiotypic/immunology , Antibody Specificity , Antigens/administration & dosage , Antigens/metabolism , Humans , Hypersensitivity , Mice , Mice, Inbred C57BL , Ovalbumin/immunology , Time FactorsABSTRACT
Allergy is an acquired hypersensitivity reaction of the immune system mediated by cross-linking of the allergen-specific IgE-bound high-affinity IgE receptors, leading to immediate mast cell degranulation. Rottlerin is an active molecule isolated from Mallotus philippinensis, a medicinal plant used in Ayurvedic Medicine System for anti-allergic and anti-helminthic treatments. The present study investigated potential anti-allergic effects of rottlerin in animal models of IgE-dependent anaphylaxis and the anti-allergic mechanisms of action of rottlerin in mast cells. Anti-allergic actions of rottlerin were evaluated in passive cutaneous anaphylaxis and passive systemic anaphylaxis mouse models, and in anaphylactic contraction of bronchial rings isolated from sensitized guinea pigs. Direct mast cell-stabilizing effect of rottlerin was examined in RBL-2H3 mast cell line. Anti-allergic signaling mechanisms of action of rottlerin in mast cells were also examined. Rottlerin prevented IgE-mediated cutaneous vascular extravasation, hypothermia, elevation in plasma histamine level and tracheal tissue mast cell degranulation in mice in a dose-dependent manner. In addition, rottlerin suppressed ovalbumin-induced guinea pig bronchial smooth muscle contraction. Furthermore, rottlerin concentration-dependently blocked IgE-mediated immediate release of ß-hexosaminidase from RBL-2H3 mast cells. Rottlerin was found to inhibit IgE-induced PLCγ1 and Akt phosphorylation, production of IP3 and rise in cytosolic Ca²âº level in mast cells. We report here for the first time that rottlerin possesses anti-allergic activity by blocking IgE-induced mast cell degranulation, providing a foundation for developing rottlerin for the treatment of allergic asthma and other mast cell-mediated allergic disorders.
Subject(s)
Acetophenones/therapeutic use , Anaphylaxis/prevention & control , Anti-Allergic Agents/therapeutic use , Benzopyrans/therapeutic use , Mallotus Plant , Mast Cells/pathology , Passive Cutaneous Anaphylaxis/drug effects , Acetophenones/pharmacology , Animals , Benzopyrans/pharmacology , Cell Degranulation/drug effects , Cell Line, Tumor , Female , Guinea Pigs , Mallotus Plant/chemistry , Mast Cells/drug effects , Mice , Mice, Inbred BALB C , Plant Extracts/therapeutic use , RatsABSTRACT
BACKGROUND: Aggregation of FcεRI activates a cascade of signaling events leading to mast cell activation, followed by inhibitory signals that turn off the activating signals. However, the overall view of negative signals in mast cells is still incomplete. Although AMP-activated protein kinase (AMPK), which is generally known as a regulator of energy metabolism, is also associated with anti-inflammation, little is known about the role of AMPK in mast cells. OBJECTIVES: We investigated the role of AMPK and its regulatory mechanism in mast cells. METHOD: The roles of AMPK in FcεRI-dependent activation of bone marrow-derived mast cells (BMMCs) were evaluated by using chemical agents, small interfering RNAs (siRNAs), or adenovirus that modulated the activity or expression of AMPK signaling components. In addition, AMPKα2(-/-) mice were used to verify the role of AMPK in anaphylactic models. RESULTS: FcεRI signaling and associated effector functions in BMMCs were suppressed by the AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-4-ribofuranoside (AICAR) and were conversely augmented by siRNA knockdown of AMPKα2 or liver kinase B1 (LKB1), an upstream kinase of AMPK. Furthermore, AMPKα2 deficiency led to increased FcεRI-mediated BMMC activation and anaphylaxis that were insensitive to AICAR, whereas enforced expression of AMPKα2 in AMPKα2(-/-) BMMCs reversed the hypersensitive FcεRI signaling to normal levels. Pharmacologic inhibition or siRNA knockdown of Fyn mimicked AMPK activation, suggesting that Fyn counterregulates the LKB1-AMPK axis. Mechanistically, Fyn controlled AMPK activity by regulating LKB1 localization. CONCLUSIONS: The Fyn-regulated LKB1-AMPK axis acts as a novel inhibitory module for mast cell activation, which points to AMPK activators as therapeutic drugs for allergic diseases.