anti-DNP IgE Research Articles

Degranulation of basophilic leukemia cells on branched-chain peptide array with an OVA–DNP double epitope

Simple method for identification of heterovalent allergen epitopes was constructed to reflect the allergic reaction initiated by the cross-linking of the IgE receptors. The peptide array-based degranulation assay that enables interaction between solid support-bound peptide pairs and IgE sensitized basophilic leukemia cells was developed. Using lysine side chains, a chicken egg white ovalbumin (OVA) epitope and a dinitrophenol (DNP) modified amino acid was synthesized in the branched-chain peptide array. The basophilic leukemia cells sensitized with anti-OVA IgE and anti-DNP IgE induced degranulation directly on the branched-chain peptide array. The OVA–DNP double peptide disk caused the release of 37.5% of β-hexosaminidase whereas the DNP-only peptide disk caused the release by 20.9%. These results show that the double epitope branched-chain peptide array can induce degranulation, and would represent a suitable model for screening the heterovalent epitope pairs of allergens.

IgE Receptor-Mediated Mast-Cell Renin Release

Renin is a newly discovered constituent of mast cells. Given that mast cells play a major role in IgE-mediated allergic hypersensitivity, we investigated whether activation of the high-affinity IgE receptor FcεRI elicits release of mast-cell renin. Cross-linking of FcεRI on the surface of mature bone marrow-derived mast cells elicited release of enzymatically active renin protein. The angiotensin I-forming activity of the renin protein was completely blocked by the selective renin inhibitor BILA 2157, which excludes formation of angiotensin I by proteases other than renin. FcεRI-mediated mast-cell renin release was inhibited by dexamethasone and potentiated by the proinflammatory mediator PGE2. Furthermore, cross-linking of mast-cell FcεRI in exvivo murine hearts passively sensitized with monoclonal anti-DNP IgE also resulted in mast-cell degranulation and overflow of renin. Our findings indicate that IgE-mediated allergic hypersensitivity provokes release of renin from both cultured and resident cardiac mast cells, a process likely to be exacerbated in a chronic inflammatory background. Given the widespread distribution of mast cells, and the presence of angiotensinogen and angiotensin-converting enzyme in many tissues, renin release in immediate hypersensitivity reactions could result in local angiotensin II generation and multiorgan dysfunctions.

Influences of "Antiphlogistic No.1" on the degranultion of Activated RBL-2H3 cells

Objective To investigate the direct effect ofAntiphlogistic No.1 on the degranultion of RBL-2H3 mast cells stimulated by anti-DNP IgE and DNP-BSA complex.Methods The effect of Antiphlogistic No.1 on stabilization of RBL-2H3 cell membrane was assessed by degranultion inhibition rate.β-hexosaminidase and IL-4 released from RBL-2H3 cells were detected by PNAG colorimetric assay and ELISA.Results β-hexosaminidase and cytokine IL-4 production releases from RBL-2H3 cells was reduced after they had been treated with Antiphlogistic No.1.The inhibition rate of β-hexosaminidase release was 42.47％ at the concentration of 8 μg/ml (P＜0.01) and dose dependently increased to 52.40％,68.26％ and 72.15％ respectively when drug concentration up to 80 μg/ml,800 μg/ml,8000 μg/ml,while inhibition rate of IL-4 generation were 13.87％,23.27％,31.95％,39.99％ (P＜0.01).Antiphlogistic No.1 maximum inhibition rate of β-hexosaminidase release is 84.48％ of dexamethason maximum inhibition rate while Antiphlogistic No.l maximum inhibition rate of IL-4 is close to dexamethason maximum inhibition rate.Conclusion Antiphlogistic No.1 could stabilize the cellular membrane of RBL-2H3 and provide protection against Ⅰ type allergic response. Key words: Antiphlogistic No.1; RBL-2H3; Degranultion; β-hexosaminidase; IL-4; Dexamethasone

Highly cytokinergic SPE-7 anti-dinitrophenyl-immunoglobulin E induces antigen-dependent thermal hyperalgesia and inflammatory cytokine production in the mouse hindpaw (P6000)

Abstract SPE-7 anti-DNP-IgE initiates inflammatory cytokine production without antigen stimulation in vitro. In contrast, ϵ2624 anti-DNP IgE does not induce inflammatory cytokines independent of antigen-stimulation. However, their effects in vivo have not been described. We tested two well-characterized anti-mouse IgE antibody clones in our model of murine thermal hyperalgesia. We found pronounced hindpaw hyperalgesic responses lasting to at least 6 hours following antigen challenge after sensitization with SPE-7. These responses were accompanied by histamine release and neutrophil influx as we have previously shown in c48/80-mediated thermal hyperalgesia. In contrast, we found that antigen challenge following sensitization with clone ϵ2624 produced slight hyperalgesia at 1 hr that was resolved by 3 hours. We demonstrate a novel functional, behavioral difference between reactions mediated by these IgE antibody clones in vivo in mice. Also, SPE-7 does not lead to antigen-independent cytokine production in the mouse hind paw. After antigen challenge, both IgEs produce equivalent levels of TNF-α, IL-1, and IL-6, and cause equivalent histamine release and neutrophil influx. Thus tissue-resident sentinel mast cells may be critical initiators of allergen-evoked hyperalgesia in mice and differences in the nature and repertoire of IgE antibodies produced during an allergic response likely have pathophysiological implications for various health outcomes including pain.

6]-Shogaol inhibits the production of proinflammatory cytokines via regulation of NF-κB and phosphorylation of JNK in HMC-1 cells

[6]-Shogaol is a major bioactive component of Zingiber officinale. Although [6]-shogaol has a number of pharmacological activities including antipyretic, analgesic, antitussive and anti-inflammatory effects, the specific mechanisms of its anti-allergic effects have not been studied. In this study, we present the effects of [6]-shogaol on mast cell-mediated allergic reactions in vivo and in vitro. Sprague–Dawley rats received intradermal injections of anti-DNP IgE was injected into dorsal skin sites. After 48 h, [6]-shogaol was administered orally 1 h prior to challenge with DNP-HSA in saline containing 4% Evans blue through the dorsal vein of the penis. In addition, rat peritoneal mast cells (RPMCs) were cultured and purified to investigate histamine release. In vitro, we evaluated the regulatory effects of [6]-shogaol on the level of inflammatory mediators in phorbol 12-myristate 13-acetate plus calcium ionomycin A23187-stimulated human mast cells (HMC-1). [6]-Shogaol reduced the passive cutaneous anaphylaxis reaction compared to the control group, and histamine release decreased significantly following the treatment of RPMCs with [6]-shogaol. In HMC-1 cells, [6]-shogaol inhibited the production of TNF-α, IL-6 and IL-8, as well as the activation of nuclear factor-κB (NF-κB) and phosphorylation of JNK in compound 48/80-induced HMC-1 cells. [6]-shogaol inhibited mast cell-mediated allergic reactions by inhibiting the release of histamine and the production of proinflammatory cytokines with the involvement of regulation of NF-κB and phosphorylation of JNK.

교맥의 RBL-2H3 비만세포 탈과립과 cytokine 생산 억제 효과

Objectives : Fagopyrum esculentum(FE) has been used for removal of inflammation of internal organs and treatment of sore and ulcer by heat toxin in Korean herbal medicines. In this study, To investigated the protective effect of FE on allergic response, we determined whether FE inhibits allergic response. Methods : The effect of FE was analyzed by ELISA, RT-PCR and Western blot in RBL-2H3 cells. We investigated cell viability, <TEX>${\beta}$</TEX>-hexosaminidase, as a marker of degranulation, cytokne, and intracellular ROS and MAPK and NF-<TEX>${\kappa}B$</TEX> signaling. Results : We found that FE suppressed <TEX>${\beta}$</TEX>-hexosaminidase release, the production of IL-4 and TNF-<TEX>${\alpha}$</TEX> and intracellular ROS level in RBL-2H3 by the anti-DNP IgE plus DNP-HSA stimulation. FE also significantly inhibited cytokine mRNA expressions, such as IL-<TEX>$1{\beta}$</TEX>, IL-2, IL-3, IL-4, IL-5, IL-6, IL-13, TNF-<TEX>${\alpha}$</TEX> and GM-CSF in RBL-2H3. In addition, PF suppressed the phospholyation of ERK1/2, JNK1/2, p38 and <TEX>$I{\kappa}B{\alpha}$</TEX> and NF-<TEX>${\kappa}B$</TEX> signal transduction pathway. Conclusions : Our results indicate that FE protects against allergic response and exerts an anti-inflammatory effect through the inhibition of degranulation and production of cytokines and ROS via the suppression MAPK and NF-<TEX>${\kappa}B$</TEX> of signal transduction. Abbrevations : FE, Fagopyrum esculentum; RBL-2H3, rat basophilic leukemia cell line; ROS, reactive oxygen species; MAPK, Mitogen-activated protein kinase; <TEX>$NF{\kappa}B$</TEX>, nuclear factor <TEX>${\kappa}B$</TEX>; <TEX>$TNF{\alpha}$</TEX>, Tumor necrosis factor alpha; GM-CSF, Granulocyte macrophage colony-stimulating factor; ERK, extracellular-signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; p38, p38 MAP kinase; <TEX>$I{\kappa}B{\alpha}$</TEX>, inhibitory-kappa B alpha.

Archetypical Conductive Polymer Structure for Specific Interaction with Proteins

A series of 2,4-dinitrophenyl (DNP) functionalized polypyrrole terpolymers, capable of specific binding to IgE antibodies (proteins), have been synthesized using oxidizing initiator, ammonium persulfate and were characterized by 1H-NMR, IR, DSC, Light Scattering etc. The terpolymers were composed of monomers and macromonomers: monomer (pyrrole), macromonomer A (pyrrole with pendant ethylene glycol) and macromonomer B (pyrrole with pendant DNP) with specific functionality of conductance, processiblity and binding, respectively. The terpolymers are found to be semiconductive, (5 × 10−6 S cm−1) by itself without the addition of doping agents. Molecular dynamics simulation of terpolymer shows that the DNP-(2,4-dinitrophenyl) functional group extends out from the polymer backbone and thus, is available for binding. The DNP functional groups on the terpolymer achieve steady state binding with anti-DNP IgE proteins at nanomolar concentrations in solution. The terpolymer was blended with sulfonated polystyrene and processed in fibers which exhibited effective specific binding to fluorescently tagged IgE proteins and therefore, possessed the potential to be an active component in biosensor.

Antiallergic effects of Scutellaria baicalensis on inflammation in vivo and in vitro

Ethnopharmacological relevanceScutellaria baicalensis (SB) is one of the most widely used medicinal herbs for the treatment of inflammation. In this study, we investigated the antiallergic effect of SB in vivo and in vitro. Materials and methodsSprague–Dawley (SD) rats received intradermal injections of anti-DNP IgE at each of three dorsal skin sites. Forty-eight hours later, each rat received an injection of DNP-HSA in saline containing 4% Evans blue through the dorsal vein of the penis. One hour before injection, SB extract was administered orally. The dorsal skin of the rats was removed and the pigment area measured. In addition, rat peritoneal mast cells (RPMCs) were cultured and purified to investigate histamine release. In vitro, human mast cells (HMC-1) were pretreated with SB extract for 30min before stimulation with phorbol 12-myristate 13-acetate (PMA) plus A23187. The effects on pro-inflammatory cytokine expression and mitogen activated protein (MAP) kinase expression were investigated using TNF-α and IL-8 assays, and Western blotting analysis of HMC-1 cells. Results and conclusionsSB treatment inhibited the passive cutaneous anaphylaxis reaction compared to the control group, and histamine release decreased significantly following treatment of RPMCs with SB. In HMC-1 cells, SB restored IL-8 and TNF-α expression and inhibited MAP kinase expression in compound 48/80-induced HMC-1 cells. These data suggest that SB may prove to be a useful anti-inflammatory agent through its downregulation of the expression of various inflammatory mediators.

Determination of Mast Cell/Basophil Inhibitory Activities of Traditional Chinese Herbal Medicines

IgE-stimulated mast cells degranulate and release several potent mediators in response to specific antigen exposure, and are key effector cells in food allergy. Traditional Chinese medicines have showed potential for treating allergies. The aim of this study was to identify TCM herbal medicines which inhibit mast cells degranulation. Dried water extracts of 296 Chinese herbal medicines (CHM), previously reported to have anti-inflammation and allergy effects, were labeled with code numbers. These CHM were dissolved into PBS before in vitro experiment. Effects of 100 μg/mL and 500 μg/mL of each extract on RBL-2H3 cells were determined. The cells were sensitized with anti-DNP IgE and challenged with DNP-BSA. β-hexosaminidase released into the culture media was measured. Potential toxicity was analyzed by MTT assays. The 3 most effective herbs on β-exosaminidase release were also tested on human basophile activation using peripheral blood samples from patients with food allergy by flow cytometry. Among 296 herbal extracts tested 43 inhibited the release of β-hexosaminidase by more than 50% and 16 inhibited more than 80% at 500 μg/mL. 18 CHMs inhibited more than 50% at 100 μg/mL, 3 of which by more than 80%. These 3 CMHs (code 0173, 0070 and 0249) also inhibited human basophil activation by 50-100% in vitro to peanut stimulation. This study found several CMHs that significantly inhibited mast cell β-hexosaminidase release in vitro and peripheral blood basophil activation in a non-toxic manner. Further investigation of these herbal medicines might lead to the development of novel mast cell/basophil inhibitors for allergy treatment.

SHIP-1 Is Critical in Regulation of Severe Anaphylaxis

RationaleSHIP-1 regulates signaling pathways triggered by antigens and cytokines. SHIP-1 deficient mice develop a spontaneous allergic pulmonary inflammation but have impaired adaptive immune response to allergen stimulation. To determine the roles of SHIP-1 in regulation of anaphylaxis, we examined IgE-dependent passive (PSA) and OVA-induced active systemic anaphylactic (ASA) responses in mice lacking SHIP-1.MethodsSHIP-1-/- mice were compared with BALB/c wild type (WT) mice. For PSA, 24 h after passive immunization with 10 μg anti-DNP IgE, mice were challenged i.v. with 1 mg of DNP-HSA. For ASA, mice were immunized i.p. with 50 μg of OVA/Alum and challenged i.v. with 1 mg of OVA 1 week (ASA-1W) or 2 weeks (ASA-2W) later. The core body temperature and clinical symptoms were monitored and blood was collected for serum immunoglobulins.ResultsCompared to WT mice, SHIP-1-/- mice developed significantly enhanced IgE-dependent PSA as expected. In ASA-1W, however, SHIP-1-/- mice showed diminished ASA, possibly due to lower serum levels of OVA-specific IgE and IgG1. Strikingly, SHIP-1-/- mice in ASA-2W showed a high mortality rate (8 out of 9) compared to WT mice (0 out of 9). In SHIP-1-/- mice serum OVA-specific IgE was significantly lower, IgG1 was comparable, but IgG2a was significantly higher.ConclusionsThese studies demonstrate that SHIP-1 deficiency leads to enhanced PSA but diminished ASA due to decreased production of IgE and IgG1. However, SHIP-1 deficiency leads to fatal anaphylaxis even with low IgE and comparable IgG1, suggesting that SHIP-1 plays a critical role in regulating fatal anaphylaxis in IgE-dependent and IgE-independent pathways. RationaleSHIP-1 regulates signaling pathways triggered by antigens and cytokines. SHIP-1 deficient mice develop a spontaneous allergic pulmonary inflammation but have impaired adaptive immune response to allergen stimulation. To determine the roles of SHIP-1 in regulation of anaphylaxis, we examined IgE-dependent passive (PSA) and OVA-induced active systemic anaphylactic (ASA) responses in mice lacking SHIP-1. SHIP-1 regulates signaling pathways triggered by antigens and cytokines. SHIP-1 deficient mice develop a spontaneous allergic pulmonary inflammation but have impaired adaptive immune response to allergen stimulation. To determine the roles of SHIP-1 in regulation of anaphylaxis, we examined IgE-dependent passive (PSA) and OVA-induced active systemic anaphylactic (ASA) responses in mice lacking SHIP-1. MethodsSHIP-1-/- mice were compared with BALB/c wild type (WT) mice. For PSA, 24 h after passive immunization with 10 μg anti-DNP IgE, mice were challenged i.v. with 1 mg of DNP-HSA. For ASA, mice were immunized i.p. with 50 μg of OVA/Alum and challenged i.v. with 1 mg of OVA 1 week (ASA-1W) or 2 weeks (ASA-2W) later. The core body temperature and clinical symptoms were monitored and blood was collected for serum immunoglobulins. SHIP-1-/- mice were compared with BALB/c wild type (WT) mice. For PSA, 24 h after passive immunization with 10 μg anti-DNP IgE, mice were challenged i.v. with 1 mg of DNP-HSA. For ASA, mice were immunized i.p. with 50 μg of OVA/Alum and challenged i.v. with 1 mg of OVA 1 week (ASA-1W) or 2 weeks (ASA-2W) later. The core body temperature and clinical symptoms were monitored and blood was collected for serum immunoglobulins. ResultsCompared to WT mice, SHIP-1-/- mice developed significantly enhanced IgE-dependent PSA as expected. In ASA-1W, however, SHIP-1-/- mice showed diminished ASA, possibly due to lower serum levels of OVA-specific IgE and IgG1. Strikingly, SHIP-1-/- mice in ASA-2W showed a high mortality rate (8 out of 9) compared to WT mice (0 out of 9). In SHIP-1-/- mice serum OVA-specific IgE was significantly lower, IgG1 was comparable, but IgG2a was significantly higher. Compared to WT mice, SHIP-1-/- mice developed significantly enhanced IgE-dependent PSA as expected. In ASA-1W, however, SHIP-1-/- mice showed diminished ASA, possibly due to lower serum levels of OVA-specific IgE and IgG1. Strikingly, SHIP-1-/- mice in ASA-2W showed a high mortality rate (8 out of 9) compared to WT mice (0 out of 9). In SHIP-1-/- mice serum OVA-specific IgE was significantly lower, IgG1 was comparable, but IgG2a was significantly higher. ConclusionsThese studies demonstrate that SHIP-1 deficiency leads to enhanced PSA but diminished ASA due to decreased production of IgE and IgG1. However, SHIP-1 deficiency leads to fatal anaphylaxis even with low IgE and comparable IgG1, suggesting that SHIP-1 plays a critical role in regulating fatal anaphylaxis in IgE-dependent and IgE-independent pathways. These studies demonstrate that SHIP-1 deficiency leads to enhanced PSA but diminished ASA due to decreased production of IgE and IgG1. However, SHIP-1 deficiency leads to fatal anaphylaxis even with low IgE and comparable IgG1, suggesting that SHIP-1 plays a critical role in regulating fatal anaphylaxis in IgE-dependent and IgE-independent pathways.

Effects of Angelica acutiloba on mast cell-mediated allergic reactions in vitro and in vivo

The root of Angelica acutiloba is a widely used herbal medicine which has been used as a typical therapeutic for allergic diseases in traditional medicine. This study was aimed to investigate the effects of A. acutiloba on allergic reactions in in vitro and in vivo models and its mechanism of action. A. acutiloba was extracted by maceration with 80% ethanol (AAE) and standardized by high-performance liquid chromatography. We investigated the effect of AAE on phorbol-12-myristate-13-acetate plus calcium ionophore A23187 (PMACI)-induced cytokine release; phosphorylation of JNK, ERK, and p38 in human mast cell-1 (HMC-1); and compound 48/80-induced release of histamine in rat peritoneal mast cells (RPMCs). We also investigated the effects on Evans blue (EB) extravasation induced by anti-DNP IgE in rats. Treatment with 1, 10 and 100 μg/ml AAE concentration-dependently inhibited the release of cytokines (tumor necrosis factor-α, interleukin (IL) -6, and IL-8) and phosphorylation of ERK and JNK induced by PMACI in HMC-1 cells, but it did not inhibit the phosphorylation of p38. It also inhibited compound 48/80-induced histamine release in RPMCs. Oral administration of 271 mg/kg AAE inhibited EB extravasation in a passive cutaneous anaphylaxis rat model. In conclusion, AAE inhibited mast cell-derived allergic reactions by inhibiting the release of histamine, the production of pro-inflammatory cytokines, and the phosphorylation of ERK and JNK.

Engineered Nanostructures of Antigen Provide an Effective Means for Regulating Mast Cell Activation

Nanostructures containing 2,4-dinitrophenyl (DNP) as antigen were designed and produced to investigate antibody-mediated activation of mast cells. The design consists of nanogrids of DNP termini inlaid in alkanethiol self-assembled monolayers (SAMs). Using scanning probe-based nanografting, nanometer precision was attained for designed geometry, size, and periodicity. Rat basophilic leukemia (RBL) cells exhibited high sensitivity to the geometry and local environment of DNP presented on these nanostructures. The impact included cellular adherence, spreading, membrane morphology, cytoskeleton structure, and activation. The highest level of spreading and activation was induced by nanogrids of 17 nm line width and 40 nm periodicity, with DNP haptens 1.4 nm above the surroundings. The high efficacy is attributed to two main factors. First, DNP sites in the nanostructure are highly accessible by anti-DNP IgE during recognition. Second, the arrangement or geometry of DNP termini in nanostructures promotes clustering of FcεRI receptors that are prelinked to IgE. The clustering effectively initiates Lyn-mediated signaling cascades, ultimately leading to the degranulation of RBL cells. This work demonstrates an important concept: that nanostructures of ligands provide new and effective cues for directing cellular signaling processes.

Mast Cells Increase Vascular Permeability by Heparin-Initiated Bradykinin Formation In Vivo

Activated mast cells trigger edema in allergic and inflammatory disease. We report a paracrine mechanism by which mast cell-released heparin increases vascular permeability invivo. Heparin activated the protease factor XII, which initiates bradykinin formation in plasma. Targeting factor XII or kinin B2 receptors abolished heparin-triggered leukocyte-endothelium adhesion and interfered with a mast cell-driven drop in blood pressure in rodents. Intravital laser scanning microscopy and tracer measurements showed heparin-driven fluid extravasation in mouse skin microvessels. Ablation of factor XII or kinin B2 receptors abolished heparin-induced skin edema and protected mice from allergen-activated mast cell-driven leakage. In contrast, heparin and activated mast cells induced excessive edema in mice deficient in the major inhibitor of factor XII, C1 esterase inhibitor. Allergen exposure triggered edema attacks in hereditary angioedema patients, lacking C1 esterase inhibitor. The data indicate that heparin-initiated bradykinin formation plays a fundamental role in mast cell-mediated diseases.

Living cell-based allergen sensing using a high resolution two-dimensional surface plasmon resonance imager

Recently, several papers indicated that the surface plasmon resonance (SPR) technique was available to monitor stimulation responses of mammalian cells adhered on sensor chips. On the other hand, the newly developed two-dimensional SPR (2D-SPR) imager system can obtain 2D-images of local refractive index change on the surface of a gold thin film. From these backgrounds, we expected that the 2D-SPR imager can visualize the individual response of many mammalian cells, simultaneously. Here, we report the observation of an allergenic response of a model mast cell, rat basophilic leukaemia cell (RBL-2H3), by using the high magnification 2D-SPR imaging system after pre-sensitization with 0.1 μg mL(-1) anti-dinitrophenyl immunoglobulin E (anti-DNP IgE). The response of the cells was successfully observed as the increment of the SPR signal (reflection intensity) upon stimulation with 0.1-1000 ng mL(-1) DNP-modified bovine serum albumin (DNP-BSA).

Zebrafish mast cells possess an FcɛRI-like receptor and participate in innate and adaptive immune responses

We previously identified a zebrafish mast cell (MC) lineage and now aim to determine if these cells function analogously in innate and adaptive immunity like their mammalian counterparts. Intraperitoneal (IP) injection of compound 48/80 or live Aeromonas salmonicida resulted in significant MC degranulation evident histologically and by increased plasma tryptase compared with saline-injected controls ( p = 0.0006, 0.005, respectively). Pre-treatment with ketotifen abrogated these responses ( p = 0.0004, 0.005, respectively). Cross-reactivity was observed in zebrafish to anti-human high-affinity IgE receptor gamma (FcɛRIγ) and IgE heavy chain-directed antibodies. Whole mount in situ hybridization on 7-day embryos demonstrated co-localization of cpa5, a MC-specific marker, with myd88, a toll-like receptor adaptor, and zebrafish FcɛRI subunit homologs. Zebrafish injected IP with matched dinitrophenyl-sensitized mouse (anti-DNP) IgE and DNP-BSA or trinitrophenyl-sensitized mouse (anti-TNP) IgE and TNP-BSA demonstrated increased plasma tryptase compared with mismatched controls ( p = 0.03, 0.010, respectively). These results confirm functional conservation and validate the zebrafish model as an in vivo screening tool for novel MC modulating agents.

Effect of Chlorogenic acid on mast cell-dependent anaphylactic reaction

Chlorogenic acid (CGA), a naturally occurring polyphenol compound, has a number of biological activities. However, roles of CGA in the mast cell-dependent anaphylactic reaction have not been fully examined. In the present study, the effect and mechanism of CGA on mast cell-dependent anaphylactic reaction were investigated using in vivo and in vitro models. CGA inhibited compound 48/80-induced systemic anaphylactic shock in mice and skin vascular permeability in rats. CGA also inhibited anti-dinitrophenyl (DNP) IgE-mediated passive cutaneous anaphylaxis (PCA). Moreover, CGA dose-dependently reduced histamine and TNF-α release from RBL-2H3 cells activated by anti-DNP IgE. Pretreatment with CGA suppressed IgE–antigen complex induced calcium uptake into RBL-2H3 cells. When CGA was added, the level of intracellular cyclic adenosine monophosphate (cAMP) in RBL-2H3 cells was significantly elevated compared with the untreated cells. Decreased calcium uptake and increased cAMP level might be involved in the inhibitory effect of CGA on mast cell activation. These results suggest a possible therapeutic application of CGA in allergic diseases.

Dual Roles of Inositol Phosphatase SHIP-1 in Active and Passive Systemic Anaphylactic Responses (91.16)

Abstract SHIP-1 is a negative regulator of signaling pathways triggered by antigens and cytokines. We reported that SHIP-1 knockout (KO) mice develop a spontaneous allergic pulmonary inflammation. To determine the roles of SHIP-1 in innate and adaptive immune responses, we examined IgE-dependent passive (PSA) and OVA-induced active systemic anaphylactic (ASA) responses in mice deficient in SHIP-1. Heterozygous SHIP-1 (hSHIP) mice were compared with C57BL/6 wild type (WT) mice. For PSA, 24 h after passive immunization with 5 µg anti-DNP IgE, mice were challenged i.v. with 1 mg of DNP-HSA. For ASA, mice were immunized i.p. once or twice with 50 µg of OVA/Alum on day 1 and 7 and then challenged with 1 mg of OVA on day 14 or 21. The core body temperature was monitored and blood was collected for serum immunoglobulins. Compared to WT controls, hSHIP mice developed significantly enhanced IgE-dependent PSA, but surprisingly, with one-time sensitization, hSHIP mice developed diminished ASA responses. Serum OVA-specific IgE and IgG1 were significantly lower but OVA-specific IgG2a was significantly higher in hSHIP mice. These studies demonstrate that SHIP-1 deficiency leads to enhanced PSA by increased mast cell response but diminished ASA, possibly due to impaired Th2 response to allergen and decreased production of IgE and IgG1. Thus, in contrast to its role as a negative regulator in innate immunity, SHIP-1 may play a critical role in the Th2 adaptive immunity as a positive regulator.

Scutellaria baicalensis Inhibits Mast Cell-Mediated Anaphylactic Reactions

Mast cells play a critical role in the effector phase of immediate hypersensitivity and allergic diseases. Scutellaria baicalensis is a widely used herb in traditional oriental medicine with anticancer, antiviral, antibacterial and anti-inflammatory properties. However, the roles of Scutellaria baicalensis in mast cell-mediated anaphylactic reactions have not fully been investigated. In this study, we examined the influences of the methanol extract of Scutellaria baicalensis (MESB) on compound 48/80- or anti-dinitrophenyl (DNP) IgE-induced anaphylaxis-like response in vivo. To further prove these in vivo results, the inhibitory effect of MESB on mast cell activation was evaluated, focusing on the histamine release from rat peritoneal mast cells (RPMC). MESB inhibited compound 48/80-induced systemic anaphylaxis-like reaction, plasma histamine release and ear swelling response in mice. MESB also attenuated passive systemic and cutaneous anaphylaxis evoked by anti-DNP IgE. In in vitro experiments, MESB dose-dependently reduced histamine release from RPMC activated by compound 48/80 or anti-DNP IgE. Moreover, compound 48/80-elicited calcium uptake was suppressed in a concentration-dependent manner of MESB. Furthermore, MESB transiently increased the level of intracellular cAMP. From these results, it is suggested that MESB possesses effective anti-anaphylactic activity.

Formation of a mast cell synapse: Fc epsilon RI membrane dynamics upon binding mobile or immobilized ligands on surfaces.

Fc epsilonRI on mast cells form a synapse when presented with mobile, bilayer-incorporated Ag. In this study, we show that receptor reorganization within the contacting mast cell membrane is markedly different upon binding of mobile and immobilized ligands. Rat basophilic leukemia mast cells primed with fluorescent anti-DNP IgE were engaged by surfaces presenting either bilayer-incorporated, monovalent DNP-lipid (mobile ligand), or chemically cross-linked, multivalent DNP (immobilized ligand). Total internal reflection fluorescence imaging and electron microscopy methods were used to visualize receptor reorganization at the contact site. The spatial relationships of Fc epsilonRI to other cellular components at the synapse, such as actin, cholesterol, and linker for activation of T cells, were also analyzed. Stimulation of mast cells with immobilized polyvalent ligand resulted in typical levels of degranulation. Remarkably, degranulation also followed interaction of mast cells, with bilayers presenting mobile, monovalent ligand. Receptors engaged with mobile ligand coalesce into large, cholesterol-rich clusters that occupy the central portion of the contacting membrane. These data indicate that Fc epsilonRI cross-linking is not an obligatory step in triggering mast cell signaling and suggest that dense populations of mobile receptors are capable of initiating low-level degranulation upon ligand recognition.

FcepsilonRI-alpha siRNA inhibits the antigen-induced activation of mast cells.

FcepsilonRI, The high affinity receptor for IgE plays a critical role in triggering the allergic reactions. It is responsible for inducing mast cell degranulation and deliberation of allergy mediators when it is aggregated by allergen and IgE complexes. FcepsilonRI on the mast cells consists of three subunits; alpha chain directly binds IgE, beta chain and dimmer of gamma chains together mediate intracellular signaling. Cross-linking of IgE-bound FcepsilonRI on the surface of mast cells and basophils by the multivalent antigen induces release of chemical mediators. The present in vitro study was designed to investigate the effect of synthetic FcepsilonRI-alpha siRNA on the antigen-induced activation of MC/9 cells. MC/9 cells which are murine mast cells were transfected by FcepsilonRI-alpha siRNA and negative control siRNA. After 6 h, anti-DNP (Dinitrophenyl) IgE was used for the cells sensitization. Then the cells were challenged with Dinitrophenyl-Human Serum Albumin (DNP-HSA) for mast cell degranulation induction before collection of supernatants. The amount of mRNA and protein expression was measured by Real Time PCR and western blot analysis, respectively. Determination of the expression rate of FcepsilonRI-alpha on cell surface was achieved by flow cytometry. ELISA and spectrophotometry methods were used subsequently for measuring the effects of FcepsilonRI-alpha siRNA on antigen-induced histamine and beta-hexosaminidase release. FcepsilonRI-alpha siRNA treated cells showed significant decrease in FcepsilonRI-alpha mRNA and protein expression in comparison to control cells. FcepsilonRI-mediated mast cell release of beta-hexosaminidase and histamine were also inhibited. In this study it was shown that FcepsilonRI-alpha siRNA could suppress FcepsilonRI-alpha expression and inhibited degranulation and histamine release in antigen-stimulated MC/9 cells. In conclusion, knock-down of FcepsilonRI-alpha by siRNA could be a promising method for inhibition of the mast cell-mediated allergic reactions.