anti-DNP IgE Research Articles

Nematode Galectin Inhibits Basophilic Leukaemia RBL-2H3 Cells Apoptosis in IgE-Mediated Activation.

Mast cells are essential immune cells involved in the host's defence against gastrointestinal nematodes. To evade the immune response, parasitic nematodes produce a variety of molecules. Galectin 1, produced by Teladorsagia circumcincta (Tci-gal-1), reduces mast cell degranulation and selectively regulates mediator production and release in an IgE-dependent manner. To uncover the activity of Tci-gal-1, we have examined the effect of the protein on gene expression, protein production, and apoptosis in activated basophilic leukaemia RBL-2H3 cells. Rat RBL-2H3 cells were activated with anti-DNP IgE and DNP-HSA, and then treated with Tci-gal-1. Microarray analysis was used to examine gene expression. The levels of several apoptosis-related molecules and cytokines were determined using antibody arrays and ELISA. Early and late apoptosis was evaluated cytometrically. Degranulation of cells was determined by a β-hexosaminidase release assay. Treatment of activated RBL-2H3 cells with Tci-gal-1 resulted in inhibited apoptosis and decreased degranulation, although we did not detect significant changes in gene expression. The production of pro-apoptotic molecules, receptor for advanced glycation end products (RAGE) and Fas ligand (FasL), and the cytokines IL-9, IL-10, IL-13, TNF-α, and IL-2 was strongly inhibited. Tci-gal-1 modulates apoptosis, degranulation, and production of cytokines by activated RBL-2H3 cells without detectable influence on gene transcription. This parasite protein is crucial for modulation of the protective immune response and the inhibition of chronic inflammation driven by mast cell activity.

Comparative analyses of various IgE-mediated and non-IgE-mediated inducers of mast cell degranulation for in vitro study.

In vitro investigations of mast cell (MC) degranulation are essential for studying many diseases, particularly allergy and urticaria. Many MC-degranulation inducers are currently available. However, there is no previous systematic comparative analysis of these available inducers in term of their efficacies to induce MC degranulation. Herein, we performed systematic comparisons of efficacies of five well-known and commonly used MC-degranulation inducers. RBL-2H3 cells were sensitized with 50ng/ml anti-DNP IgE or biotinylated IgE followed by stimulation with 100ng/ml DNP-BSA or streptavidin, respectively. For non-IgE-mediated inducers, the cells were treated with 5µg/ml substance P, compound 48/80, or A23187. At 15-, 30-, 45- and 60-min post-induction, several common MC-degranulation markers (including intracellular [Ca2+], β-hexosaminidase release, tryptase expression by immunofluorescence staining, cellular tryptase level by immunoblotting, secretory tryptase level by immunoblotting, CD63 expression by immunofluorescence staining, and CD63 expression by flow cytometry) were evaluated. The data showed that all these markers significantly increased after activation by all inducers. Among them, A23187 provided the greatest degrees of increases in intracellular [Ca2+] and β-hexosaminidase release at all time-points and upregulation of CD63 at one time-point. These data indicate that all these IgE-mediated (anti-DNP IgE/DNP-BSA and biotinylated IgE/streptavidin) and non-IgE-mediated (substance P, compound 48/80, and A23187) inducers effectively induce MC degranulation, while A23187 seems to be the most effective inducer for MC degranulation.

Human placental extract suppresses mast cell activation and induces mast cell apoptosis

BackgroundHuman placental extract (HPE) has been documented to facilitate the healing of certain disorders including allergy. However, the effects of HPE on the functionality of mast cells, a critical cell type in allergic diseases, have not been reported.MethodsTo investigate the effects of HPE on the regulation of allergy with respect to the biological functions of mast cells, the mast cell line C57 or HMC-1 cells were treated with HPE followed by the assessment of cell proliferation, apoptosis, activation, chemotaxis and phagocytosis. Mouse peritoneal mast cells were also investigated for their responses to induction of apoptosis by HPE in vivo. Furthermore, the effect of HPE on mast cell degranulation was confirmed using the passive cutaneous anaphylaxis (PCA) assay, an acute allergy model.ResultsHPE was capable of suppressing mast cell proliferation and inducing mast cell apoptosis. Mast cell degranulation in response to compound 48/80- or anti-DNP IgE and DNP-mediated activation was suppressed. In addition, treatment with HPE compromised the production of cytokines by mast cells and cell chemotaxis. These observations were consistent with the dampened passive cutaneous anaphylaxis (PCA) assay following treatment with HPE.ConclusionThis study revealed a suppressive effect of HPE on overall mast cell activities, suggesting a potential regulatory role of HPE on the alleviation of allergic diseases through mast cells.

Systematic comparisons of various markers for mast cell activation in RBL-2H3 cells.

Mast cell activation plays a key role in various allergic diseases and anaphylaxis. Several methods/techniques can be used for detection of mast cell activation. However, there was no previous systematic evaluation to compare the efficacy of each method/technique. The present study thus systematically compared various markers for mast cell activation induced by IgE cross-linking. The widely used RBL-2H3 mast cells were sensitized with anti-DNP (dinitrophenyl) IgE overnight and activated with DNP-BSA (bovine serum albumin) for up to 4h. The untreated cells and those with anti-DNP IgE sensitization but without DNP-BSA activation served as the controls. Intracellular calcium level gradually increased to ~2-fold at 1h, reached its peak (~5-fold) at 2h, and returned to the basal level at 3-h post-activation. The increases in cellular tryptase level (by Western blotting) (~0.3- to 0.4-fold) and average cell size (~2.5-fold) and decrease of nucleus/cytoplasm ratio (~0.4- to 0.5-fold) were marginal at all time-points. By contrast, β-hexosaminidase release and CD63 expression (by both flow cytometry and immunofluorescence detection/localization), secreted tryptase level (by Western blotting), and tryptase expression (by immunofluorescence detection/localization) stably and obviously increased (~10-fold as compared with the untreated control and sensitized-only cells or detectable only after activation). Based on these data, the stably obvious increases (by ≥ 10-fold) in β-hexosaminidase release, CD63 expression (by both flow cytometry and immunofluorescence staining), secreted tryptase level (by Western blotting), and tryptase expression (by immunofluorescence staining) are recommended as the markers of choice for the in vitro study of mast cell activation using RBL-2H3 cells.

Multifunctional regulation of VAMP3 in exocytic and endocytic pathways of RBL-2H3 cells.

Mast cells (MCs) are inflammatory cells involved in allergic reactions. Crosslinking of the high-affinity receptor for IgE (FcϵRI) with multivalent antigens (Ags) induces secretory responses to release various inflammatory mediators. These responses are largely mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Vesicle-associated membrane protein 3 (VAMP3) is a vesicular-SNARE that interacts with targeted SNARE counterparts, driving the fusion of MC secretory granules with the membrane and affecting subsequent assembly of the plasma membrane. However, the role of VAMP3 in FcϵRI-mediated MC function remains unclear. In this study, we comprehensively examined the role of VAMP3 and the molecular mechanisms underlying VAMP3-mediated MC function upon FcϵRI activation. VAMP3 shRNA transduction considerably decreased VAMP3 expression compared with non-target shRNA-transduced (NT) cells. VAMP3 knockdown (KD) cells were sensitized with an anti-DNP IgE antibody and subsequently stimulated with Ag. The VAMP3 KD cells showed decreased degranulation response upon Ag stimulation. Next, we observed intracellular granule formation using CD63-GFP fluorescence. The VAMP3 KD cells were considerably impaired in their capacity to increase the size of granules when compared to NT cells, suggesting that VAMP3 mediates granule fusion and therefore promotes granule exocytosis in MCs. Analysis of FcϵRI-mediated activation of signaling events (FcϵRI, Lyn, Syk, and intracellular Ca2+ response) revealed that signaling molecule activation was enhanced in VAMP3 KD cells. We also found that FcϵRI expression on the cell surface decreased considerably in VAMP3 KD cells, although the amount of total protein did not vary. VAMP3 KD cells also showed dysregulation of plasma membrane homeostasis, such as endocytosis and lipid raft formation. The difference in the plasma membrane environment in VAMP3 KD cells might affect FcϵRI membrane dynamics and the subsequent signalosome formation. Furthermore, IgE/Ag-mediated secretion of TNF-α and IL-6 is oppositely regulated in the absence of VAMP3, which appears to be attributed to both the activation of FcϵRI and defects in VAMP3-mediated membrane fusion. Taken together, these results suggest that enhanced FcϵRI-mediated signal transduction in VAMP3 KD cells occurs due to the disruption of plasma membrane homeostasis. Hence, a multifunctional regulation of VAMP3 is involved in complex secretory responses in MCs.

Inhibitory Effect of Rhododendron Mucronulatum Root Extract on Allergic Inflammation

Objective: In this study, we investigated the protective effect of rhododendron mucronulatum extract (RME) on allergic reactions and inflammation.Methods: The effect of RME was determined using ELISA and RT-PCR in RBL-2H3 mast cells and RAW 264.7 macrophage cells. We determined cell viability, β-hexosaminidase release, and the synthesis of IL-4 and TNF-α in RBL-2H3 cells. In addition, we determined NO from RAW 264.7 and the gene expression of IL-1β, iNOS, IL-6, TNF-α, and IL-10.Results: RME inhibited β-hexosaminidase release and synthesis of IL-4 and TNF-α in RBL-2H3 by the anti-DNP IgE plus DNP-HSA stimulation. In addition, RME inhibited the production of NO and the gene expression of IL-1β, iNOS, IL-6, and TNF-α in LPS-stimulated RAW 264.7 cells.Conclusion: From these results, we concluded that RME possesses anti-allergic activity and anti-inflammatory activity due to the inhibition of mast cells and macrophage function.

꼬막 함유 혼합분말이 RBL-2H3 세포에서 IgE 매개 알레르기 반응에 미치는 영향

The purpose of this study was to investigate the anti-allergic effects of a powder mixture containing ark shells (MP) on rat basophilic leukemia (RBL)-2H3 mast cells. When RBL-2H3 cells were sensitized with anti-dinitrophenyl (anti-DNP) IgE and subsequently stimulated with DNP-human serum albumin (HSA), β-hexosaminidase was released from the cells following degranulation of the activated mast cells. However, pretreatment with MP extract (MPCAS) before stimulation with DNP-HSA attenuated the release of β-hexosaminidase in IgE–antigen complex-stimulated RBL-2H3 cells. The levels of interleukin-4 (IL-4), IL-13, and TNF-α in the IgE-antigen complex-stimulated RBL-2H3 cells were measured using ELISA. Activated RBL-2H3 cells induced the expression of the abovementioned inflammatory cytokines that are critical for the pathogenesis of allergic diseases. MPCAS inhibited IL-4, IL-13, and TNF-α secretion from activated RBL-2H3 cells. In addition, the present study investigated the effects of MPCAS as an antigen on IgE-mediated activation of RBL-2H3 cells. When RBL-2H3 cells were sensitized with IgE and subsequently stimulated with MPCAS, β-hexosaminidase, IL-4, IL-13, and TNF-α levels did not change compared with those in the control. Thus, MPCAS does not act as an allergen in IgE-stimulated RBL-2H3 cells. These results indicate that MPCAS can be developed as an allergy-reducing food material by suppressing degranulation and inflammatory cytokine expression.

Ephedra Herb, Mao, Inhibits Antigen-Induced Mast Cell Degranulation by the Induction of the Affinity Receptor for IgE Internalization

Ephedra herb (Mao in Japanese) is traditionally used in Japanese herbal medicine (Kampo) for treatment of allergic diseases. Mao exerts potent anti-allergic effects, however the underlying mechanisms remain elusive. In this study, we investigated the mechanisms of Mao on allergic inflammation using in vitro cultured mast cell (MC) and in vivo mouse model of allergy. Mao water extracts were applied on bone marrow-derived mast cells (BMMCs). The BMMCs were pre-sensitized with anti-DNP IgE and challenged by antigen (DNP-HSA). Degranulation responses and localization of IgE receptor (FcεRI) in BMMCs were observed with/without Mao treatment. Passive systemic anaphylaxis (PSA)-treated mice were subjected to Mao administration and the pathophysiological responses were evaluated. Mao inhibited the antigen-induced BMMCs degranulation. Mao-treated BMMCs exhibited significant reduction for the levels of IgE and its receptor FcεRI expressions on cell surface. Analysis of their subcellular localization of FcεRI by confocal laser scanning microscopy revealed that Mao treatment induced IgE-FcεRI internalization in BMMCs. Additionally, FcεRI-induced tyrosine phosphorylation and calcium mobilization in the antigen-stimulated BMMCs were attenuated by Mao treatment, indicating that Mao-treated BMMCs failed to respond to the antigen. Further, Mao administration to the PSA models resulted in the prevention of antigen-induced hypothermia. Peritoneal mast cells (PMCs) in the PSA mice showed significant reduction of surface FcεRI by Mao administration, indicating Mao-induced FcεRI internalization in the PMCs. Mao induced IgE-FcεRI internalization in MCs, and may thereby inhibit the antigen-induced IgE-dependent degranulation. These findings provide novel mechanism of Mao on MC functions and allergic responses.

Phospholipid arrays on porous polymer coatings generated by micro-contact spotting.

Nanoporous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (HEMA-EDMA) is used as a 3D mesh for spotting lipid arrays. Its porous structure is an ideal matrix for lipid ink to infiltrate, resulting in higher fluorescent signal intensity as compared to similar arrays on strictly 2D substrates like glass. The embedded lipid arrays show high stability against washing steps, while still being accessible for protein and antibody binding. To characterize binding to polymer-embedded lipids we have applied Streptavidin as well as biologically important biotinylated androgen receptor binding onto 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (Biotinyl Cap PE) and anti-DNP IgE recognition of 2,4-dinitrophenyl[1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[6-[(2,4-dinitrophenyl)amino]hexanoyl] (DNP)] antigen. This approach adds lipid arrays to the range of HEMA polymer applications and makes this solid substrate a very attractive platform for a variety of bio-applications.

A novel mast cell co-culture microfluidic chip for the electrochemical evaluation of food allergen

In this study a novel cell-to-cell electrochemical microfluidic chip was developed for qualitative and quantitative analysis of food allergen. Microfluidic cell culture, food allergen-induced cell morphological changes, and cell metabolism measurements were performed simultaneously using the aforementioned device. RBL-2H3 mast cells and ANA-1 macrophages have been used within a cell co-culture model to observe their allergic response when they are introduced to the antigen stimulus. Two cell cultivation microfluidic channels are located in the microfluidic chip, which is fabricated with four groups of gold electrodes, with an additional “capillary”. In order to detect the allergic response, the cells were stimulated with dinitrophenylated bovine serum albumin (DNP-BSA) without anti-DNP IgE incubation. When exocytosis occurs, the cell-secreted inflammatory cytokines were measured by enzyme-linked immuno sorbent assay (ELISA) and cell impedance changes were detected using cell-based electrochemical assay. Results indicate that the real-time cell allergic response are accurately monitored by this electrochemical microfluidic chip, which provides a general example of rapidly prototyped low-cost biosensor technology for applications in both food allergen detection and investigation.

Inhibitory Effects of Angelica Polysaccharide on Activation of Mast Cells.

This study was designed to investigate the inhibitory effects of Angelica polysaccharide (AP) on activation of mast cells and its possible molecular mechanism. In our study, we determined the proinflammatory cytokines and allergic mediators in anti-DNP IgE stimulated RBL-2H3 cells and found that AP (50, 100, and 200 μg/mL) significantly decreased the release of histamine, β-hexosaminidase, leukotrienes C4 (LTC4), IL-1, IL-4, TNF-α, IL-6, and human monocyte chemotactic protein-1 (MCP-1/CCL2) (p < 0.05). In addition, Ca2+ entry was inhibited by treatment with AP. AP also downregulated the protein expressions of p-Fyn, p-Akt, p-P38, IL-4, TNF-α, and NF-κB p65 in both Fyn gene upregulated and normal RBL-2H3 cells (p < 0.05). Collectively, our results showed that AP could inhibit the activation of mast cells via suppressing the releases of proinflammatory cytokines allergic mediators, Gab2/PI3-K/Akt and Fyn/Syk pathways.

Nano-Snowflower of Gold Nanoparticles-Ruthenium Metallopolymer-Carbon Nanotubes Binding Anti-DNP IgE Antibody.

Gold nanoparticles (AuNPs) have been chemically functionalized onto multiwalled carbon nanotubes (MWCNT) through a metallopolymer linker-bis (2,2':6'2"-terpyridine) ruthenium(II)-connected diblock poly(N-isopropyacryamide). A "nano-snowflower" pattern was formed by self-assembly MWCNT-AuNP nanocomposite with anti-DNP IgE antibody. MWCNT-AuNP nanohybrid has unique biocompatibility and electronic current-voltage properties. This nanohybrid shows the potential application for IgE biosensor to diagnose cancer cells. We represent a step towards building complex electronic circuits response by providing molecular recognition properties.

Acidic environment augments FcεRI-mediated production of IL-6 and IL-13 in mast cells

Although blood pH is maintained in a narrow range of around pH 7.4 in living organisms, inflammatory loci are characterized by acidic conditions. Mast cells tend to reside close to the surface of the body in areas such as the mucosa and skin where they may be exposed to exogenous acids, and they play an important role in immune responses. However, little is known about the effects of extracellular acidification on the functions of mast cell. Here, we found that extracellular acidification increased the dinitrophenyl-conjugated human serum albumin (DNP-HSA)-induced production of interleukin (IL)-6 and IL-13 in MC/9 cells or bone marrow-derived mouse mast cells sensitized with anti-DNP IgE. Extracellular acidification also inhibited migration of MC/9 cells toward DNP-HSA. In addition, acidic pH stimulated antigen-induced activation of p38 mitogen-activated protein kinase (MAPK) and protein kinase B (Akt). These findings suggest that extracellular acidification augmented antigen/IgE-induced and FcεRI-mediated production of IL-6 and IL-13 in mast cells, and that this was associated with the enhancement of p38 MAPK and Akt activation.

Establishment of a mouse model for immunological contact urticaria

Objective To establish an animal model for immunological contact urticaria in mice. Methods A total of 60 BALB/c mice were randomly and equally divided into 5 groups: anti-dinitrophenol IgE monoclonal antibody(anti-DNP IgE) + 2, 4-dinitrofluorobenzene (DNFB) group and anti-DNP IgE + trimellitic anhydride (TMA) group both injected with anti-DNP IgE via tail veins firstly, followed by topical treatment with DNFB and TMA respectively on the ears at 24 hours after the injection, DNFB group, TMA group and normal saline (NS) group all injected with NS via the tail vein firstly, followed by topical treatment with DNFB, TMA and NS on the ears 24 hours after the injection. In the following 14 days, mice were observed daily for the appearance of wheals and for scratching behavior. All the mice were sacrificed at the end of the study followed by determination of the percentage of degranulated mast cells and spleen index as well as observation of pathological changes. Results Wheals were observed in all the mice (12/12) in the anti-DNP IgE + DNFB group, some mice (8/12) in the anti-DNP IgE + TMA group, but not observed in any mice in the other 3 groups. Compared with the NS group, both the anti-DNP IgE + DNFB group and anti-DNP IgE + TMA group showed a significant increase in the percentage of degranulated mast cells (70.21%±26.01% and 54.25%±39.57% vs. 14.45%±6.79%, F= 14.41, P= 0.000), spleen index (7.54±1.56 and 7.87±1.18 vs. 5.37±1.16, F= 4.29, P= 0.004) and scratching frequency ((31.58±3.58)/h and (22.17±3.81)/h vs. (2.00±0.85)/h at 30 minutes, F = 437.86, P< 0.01). Conclusion A stable mouse model for immunological contact urticaria can be established quickly by sensitization with anti-DNP IgE and challenge with DNFB. Key words: Urticaria; Disease models, animal; Mice; Dermatitis, allergic contact; Dinitrobenzenes; Dinitrochlorobenzene

Desensitization induces hyporesponsiveness and cell-surface phenotype changes on wild-type and humanized mouse mast cells

Background Rapid drug desensitization (DST) protocols have been developed based on clinical evidence, but in vitro studies are lacking. Understanding the mechanisms involved in the early stages of DST will allow improvements in patients’ treatment. The aim of this study is to demonstrate and characterize the induction of hyporesponsiveness in murine mast cells by desensitization. Methods We assessed the effect of rapid desensitization on murine bone marrow derived mast cells (BMMC). Experiments with three diferent types of murine BMMC were done: wild-type, LILRB4-/-, and transgenic expressing the human receptor FceRI. Wild-type and LILRB4-/- BMMC were sensitized with anti-DNP IgE and stimulated with DNP, while transgenic BMMC were sensitized with concentrated human serum of allergic subjects and stimulated with the respective allergen. BMMC were stimulated through desensitization or single activation, with optimal or suboptimal doses of antigen. Comparisions were made according to the percentage of b-Hexosaminidase (b-Hex) release by BMMC and the expression of LAMP-1 on the surface of these cells. Furthermore, expression of FceRI, PDL1 and LILRB4 on the surface of BMMC were also assessed. Results

A novel model of IgE-mediated passive pulmonary anaphylaxis in rats.

Mast cells are central effector cells in allergic asthma and are augmented in the airways of asthma patients. Attenuating mast cell degranulation and with it the early asthmatic response is an important intervention point to inhibit bronchoconstriction, plasma exudation and tissue oedema formation. To validate the efficacy of novel pharmacological interventions, appropriate and practicable in vivo models reflecting mast cell-dependent mechanisms in the lung, are missing. Thus, we developed a novel model of passive pulmonary anaphylaxis in rats. Rats were passively sensitized by concurrent intratracheal and intradermal (ear) application of an anti-DNP IgE antibody. Intravenous application of the antigen, DNP-BSA in combination with Evans blue dye, led to mast cell degranulation in both tissues. Quantification of mast cell degranulation in the lung was determined by (1) mediator release into bronchoalveolar lavage, (2) extravasation of Evans blue dye into tracheal and bronchial lung tissue and (3) invasive measurement of antigen-induced bronchoconstriction. Quantification of mast cell degranulation in the ear was determined by extravasation of Evans blue dye into ear tissue. We pharmacologically validated our model using the SYK inhibitor Fostamatinib, the H1-receptor antagonist Desloratadine, the mast cell stabilizer disodium cromoglycate (DSCG) and the β2-adrenergic receptor agonist Formoterol. Fostamatinib was equally efficacious in lung and ear. Desloratadine effectively inhibited bronchoconstriction and ear vascular leakage, but was less effective against pulmonary vascular leakage, perhaps reflecting the differing roles for histamine receptor sub-types. DSCG attenuated both vascular leakage in the lung and bronchoconstriction, but with a very short duration of action. As an inhaled approach, Formoterol was more effective in the lung than in the ear. This model of passive pulmonary anaphylaxis provides a tissue relevant readout of early mast cell activity and pharmacological benchmarking broadly reflects responses observed in patients with asthma.

Clonal differences in IgE antibodies affect cutaneous anaphylaxis-associated thermal sensitivity in mice

Cellular and molecular mediators of immune responses are increasingly implicated in acute and chronic pain pathophysiologies. Here we demonstrate that passive cutaneous IgE/Ag anaphylaxis provokes increased thermal sensitivity in the hind paw tissue of mice. The murine anti-DNP IgE antibodies SPE-7 and ɛ26 are known to induce differential cytokine production in bone marrow cultured mast cells in vitro without antigen challenge. We found a novel, antigen-dependent heterogeneity in the thermal pain responses elicited in the hind paws between SPE-7 and ɛ26 sensitized DNP-challenged mice. Mice experienced pronounced hind paw thermal sensitivity lasting 6h after DNP challenge when sensitized with SPE-7 but not ɛ26 IgE. The two IgE clones induced equivalent hind paw edema, neutrophil influx, cytokine production, and reduction in tissue histamine content in vivo, and bound to the same or overlapping epitopes on the DNP antigen in vitro. Therefore IgE antibodies against the same antigen can induce comparable inflammation, yet contribute to markedly different anaphylaxis-associated pain within an allergic response, suggesting that non-canonical IgE binding partners such as sensory neurons may play a role in allergy-related pain responses.

Studies on anti-allergic activity of Sargassum horneri extract

Chlorophyll c2 in a Sargassum horneri extract suppressed the degranulation of rat basophilic leukaemia RBL-2H3 cells. In this contribution, the mode of action of S. horneri extract was revealed. The extract inhibited the elevation of intracellular Ca2+ concentration induced by the antigen. Immunoblot analysis showed that S. horneri extract down-regulated the phosphorylation of both Syk and PI3K in the signalling pathways involved in degranulation triggered by antigen–antibody interaction. In addition, oral administration of S. horneri extract inhibited mice nasal rubbing behaviour sensitized with anti-DNP IgE. These findings suggest that S. horneri extract has anti-allergic activity via the suppression of degranulation of mast cells and basophils.

SPE-7 but not Epsilon-26 anti-dinitrophenyl-Immunoglobulin E induces antigen-dependent thermal hyperalgesia accompanying equivalent inflammatory responses during passive cutaneous anaphylaxis reactions in mice (HYP3P.405)

Abstract Sensitization of mast cells with SPE-7 anti-DNP-IgE alone initiates inflammatory cytokine production in vitro. In contrast, ε26 anti-DNP IgE does not induce inflammatory cytokines independent of antigen-stimulation. We tested these two anti-DNP IgE clones in our model of murine thermal hyperalgesia. We found pronounced hind paw hyperalgesic responses lasting to at least 6 hours following antigen challenge 24 hours after sensitization with SPE-7 but not ε26. SPE-7/DNP did not induce greater levels of mast cell degranulation in the hind paw. Differential hyperalgesic responses were accompanied by otherwise equivalent inflammatory changes measured including histamine release, neutrophil influx, expression of TNF-α, IL-1β, and IL-6 genes and changes in levels of TNF-α, IL-1, and IL-6 proteins. Inflammatory responses and hyperalgesia were dependent on antigen challenge and were not detected in sham-challenged mice. Hyperalgesia responses or other inflammatory changes were not detected in the hind paw tissue post-sensitization in the 24 hours prior to antigen challenge. We demonstrate a novel behavioral, antigen-dependent difference between reactions mediated by these IgE antibody clones in vivo in mice. We are currently further investigating the inflammatory cytokine milieu and measuring changes in mRNA and protein levels of nociceptive signaling molecules provoked by IgE/Ag-induced cutaneous anaphylaxis to elucidate the mechanisms underlying this differential pain outcome.

Signal-Transducing Adaptor Protein-2 Controls the IgE-Mediated, Mast Cell–Mediated Anaphylactic Responses

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that regulates immune and inflammatory responses through interactions with a variety of signaling and transcriptional molecules. In the current study, we clarified the physiological role of STAP-2 in mast cell function, a key mediator of IgE-associated allergic responses. STAP-2 is constitutively expressed in mast cells. STAP-2 deficiency in mast cells greatly enhances FcεRI-mediated signals, resulting in the increased tyrosine phosphorylation of the phospholipase C-γ isoform, calcium mobilization, and degranulation. Of importance, STAP-2-deficient mice challenged with DNP-BSA after passive sensitization with anti-DNP IgE show more severe rectal temperature decrease than do wild-type mice. STAP-2-deficient mice also show increased vascular permeability and more severe cutaneous anaphylaxis after DNP-BSA injection. These regulatory functions performed by STAP-2 indicate that there is an interaction between STAP-2 and FcεRI. In addition, our previous data indicate that STAP-2 binds to the phospholipase C-γ isoform and IκB kinase-β. Therefore, our data described in this article strongly suggest that manipulation of STAP-2 expression in mast cells may control the pathogenesis of allergic diseases and have the potential for treating patients with allergy.