Antigen-stimulated Degranulation Research Articles

Antiallergic Activity of 3-O-Dodecyl-l-ascorbic Acid.

2-O-Alkyl-l-ascorbic acids and 3-O-alkyl-l-ascorbic acids were synthesized, and their degranulation inhibitory activities were evaluated. Among ascorbic acid derivatives with butyl, octyl, dodecyl, hexadecyl, and octadecyl groups introduced at the C-2 or C-3 positions, an AA derivative with a dodecyl group introduced at the C-3 position, 3-O-dodecyl-l-ascorbic acid (compound 8), showed the strongest inhibitory activity against antigen-stimulated degranulation. Compound 8 also inhibited calcium ionophore-stimulated degranulation. Compound 11, in which the hydroxyl group at the C-6 position of compound 8 was substituted with an amino group, and compound 12, in which the dodecyloxy group at the C-3 position of compound 8 was exchanged with a dodecylamino group, were synthesized, and these derivatives showed weaker inhibitory activity against antigen-stimulated degranulation than that of compound 8. In addition, orally administered compound 8 inhibited passive cutaneous anaphylaxis reactions in mice with a potency equal to that of oxatomide, an antiallergic agent. These results suggest that compound 8 may be a candidate for antiallergic treatment.

Antiallergic activities of Japanese leaf burdock extract in a rat basophilic leukemia cell line.

Although primarily cultivated for its edible root, the leaves and stems of "Japanese leaf burdock (Arctium lappa L)" are also edible. This study determined the antiallergic activity of root, leaf, and stem extracts of Japanese leaf burdock using the rat basophilic leukemia cell line. We demonstrate that the hot water extract of Japanese leaf burdock stems has a strong inhibitory effect on antigen-stimulated degranulation. This extract was further fractionated into five fractions using a glass column. The fraction obtained with 99% ethanol extracts (99% EtEx) elicits a concentration-dependent increase in degranulation inhibition. One of antiallergic compounds from the separation of components of the 99% EtEx fraction using high-performance liquid chromatography and mass spectrometry analyses had a value of m/z 393.16. From these results and those of previous studies, one of the antiallergic components was hypothesized to be onopordopicrin. Our findings demonstrate that the extract of Japanese leaf burdock exerts a strong antiallergic activity. PRACTICAL APPLICATIONS: "Japanese leaf burdock (Arctium lappa L)" is a traditional Japanese vegetable with edible leaves and stems. The antiallergic activity of the hot water extracts has been shown in vitro. One of the antiallergic components was hypothesized to be onopordopicrin. Therefore, "Japanese leaf burdock" could serve as a functional food for patients with allergic disorders, such as allergic rhinitis.

Antioxidant compounds of Petasites japonicus and their preventive effects in chronic diseases: a review.

Petasites japonicus (P. japonicus) is a plant of the Asteraceae family. Its roots and stems have been used for the treatment or the prophylaxis of migraine and tension headache as a traditional Chinese medicine in Japan and Korea. Sesquiterpenoids, lignans, and flavonoids are components of P. japonicus. Regarding the biological activity of P. japonicus, its anti-allergic effect has been researched extensively using IgE antigen-stimulated degranulation of RBL-2H3 cells or passive cutaneous anaphylaxis reaction in experimental animal models. The study of the antioxidant activity of P. japonicus was initiated approximately 15 years ago using in vitro assays. In addition, its in vivo effect has also been examined in animal models with induced oxidative injury. Moreover, recently, many types of antioxidant compounds have been rapidly and simultaneously identified using the liquid chromatography–mass spectrometry technique. The number of reports on the other functions of this plant, such as its neuroprotective and anti-inflammatory effects, has been increasing. In this review, I summarized the studies of functional foods derived from P. japonicus, which may provide a basis for the development of potential functional foods. Finally, I discuss the future research avenues in this field.

WZ3146 inhibits mast cell Lyn and Fyn to reduce IgE-mediated allergic responses in vitro and in vivo

Mast cells (MCs) play an important role as effector cells that cause allergic responses in allergic diseases. For these reasons, MC is considered an attractive therapeutic target for allergic disease treatment. In this study, we investigated the inhibitory effect of WZ3146, N-[3-[5-chloro-2-[4-(4-methylpiperazin-1-yl)anilino]pyrimidin-4-yl]oxyphenyl]prop-2-enamide, and the mechanisms of its actions on the MC activation and IgE-mediated allergic response by using three types of MCs such as rat basophilic leukemia (RBL)-2H3 cells, mouse bone marrow mast cells (BMMCs), and human Laboratory of Allergic Diseases 2 (LAD2) cells. WZ3146 inhibited antigen-stimulated degranulation in a dose-dependent manner (IC50, ~ 0.35 μM for RBL-2H3 cells; ~ 0.39 μM for BMMCs; ~ 0.41 for LAD2 cells). WZ3146 also suppressed the production of histamine, tumor necrosis factor (TNF)-α and interleukin (IL)-6, which mediate various allergic responses, in a dose-dependent manner. As the mechanism of WZ3146 to inhibit MCs, it inhibited the activation of spleen tyrosine kinase (Syk) and the downstream signaling proteins of Syk such as linker for activation of T cell (LAT) and phospholipase (PL) Cγ1 in the signaling pathway of FcεRI. In addition, WZ3146 inhibited the activation of Akt, extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK). However, WZ3146 did not inhibit degranulation of MCs by thapsigargin or ionomycin, which increase calcium concentration in cytosol. Notably, WZ3146 inhibited the activity of Lyn and Fyn, but not Syk. In an following animal experiment, WZ3146 inhibited IgE-dependent passive cutaneous anaphylaxis (PCA) in a dose-dependent manner (ED50, ~ 20 mg/kg). Taken together, in this study we show that the pyrimidine derivative, WZ3146, inhibits the IgE-mediated allergic response by inhibiting Lyn and Fyn Src-family kinases, which are initially activated by antigen stimulation in MCs. Therefore, we propose that WZ3146 could be used as a new therapeutic agent for the treatment of allergic diseases.

Inhibitory Effects of Phenylpropanoid Derivatives from Oenanthe javanica on Antigen-Stimulated Degranulation in RBL-2H3 Cells.

Two diacyldaucic acids (1 and 2), an α,β-unsaturated γ-lactone-type lignan (3) and its derivatives (4-6), and 12 known compounds were isolated from a traditional East Asian vegetable, Oenanthe javanica. The absolute configuration of 1 was validated by obtaining (+)-osbeckic acid through acid hydrolysis. The absolute configurations of 3-5 were determined by comparing their experimental and computed ECD data. The conclusion was supported by applying the phenylglycine methyl ester method to 3. Compound 6 was obtained as an interconverting mixture of isomers in a 3:1 trans- cis ratio. Several water-soluble components (1, 3, and 6) showed concentration-dependent inhibitory effects on antigen-stimulated degranulation in RBL-2H3 cells without producing any direct cytotoxicity against RBL-2H3 or HeLa cells.

Repositioning of anti-cancer drug candidate, AZD7762, to an anti-allergic drug suppressing IgE-mediated mast cells and allergic responses via the inhibition of Lyn and Fyn

Mast cells are critical effector cells in IgE-mediated allergic responses. The aim of this study was to investigate the anti-allergic effects of 3-[(aminocarbonyl)amino]-5-(3-fluorophenyl)-N-(3S)-3-piperidinyl-2-thiophenecarboxamide (AZD7762) in vitro and in vivo. AZD7762 inhibited the antigen-stimulated degranulation from RBL-2H3 (IC50, ∼27.9 nM) and BMMCs (IC50, ∼99.3 nM) in a dose-dependent manner. AZD7762 also inhibited the production of TNF-α and IL-4. As the mechanism of its action, AZD7762 inhibited the activation of Syk and its downstream signaling proteins, such as Linker of activated T cells (LAT), phospholipase (PL) Cγ1, Akt, and mitogen-activated protein (MAP) kinases (Erk1/2, p38, and JNK) in mast cells. In in vitro protein kinase assay, AZD7762 inhibited the activity of Lyn and Fyn kinases, which are important for the activation of Syk in mast cells. Furthermore, AZD7762 also suppressed the degranulation of LAD2 human mast cells (IC50, ∼49.9 nM) and activation of Syk in a dose-dependent manner. As observed in experiments with mast cells in vitro, AZD7762 inhibited antigen-mediated passive cutaneous anaphylaxis in mice (ED50, ∼35.8 mg/kg). Altogether, these results suggest that AZD7762 could be used as a new therapeutic agent for mast cell-mediated allergic diseases.

Facile Synthesis of Chalcone Glycosides Isolated from Aerial Parts of Brassica rapa l. ‘hidabeni'.

Background: The Cruciferous family of vegetables which includes Brassica Turnips showed antioxidant and hepatoprotective effects. The phytochemical investigations of the aerial parts of the traditional Japanese turnip vegetable (B. rapa L. 'hidabeni') revealed the presence of three chalcone glycosides, along with other glycoside components. As many natural products inhibited Ag-stimulated degranulation in cellular system, those chalcone glycosides have biological significance of suppressing antigen-stimulated degranulation in rat basophilic leukemia RBL-2H3 cells. Aim and Objective: Further investigation on the biological importance of those chalcone glycosides demands ample quantities of well-defined compounds. Therefore, we report herein a convenient and concise synthetic approach for the preparation of those chalcone glycosides. Materials and Methods: 4'-O-β-D-Glucopyransoyl-4-hydroxy-3'-methoxychalcone and 4'-O-(β-D-Glucopyransoyl)- 3',4-dimethoxychalcone were synthesized using a three-step strategy includes: i) O-glucosylation of 4-OH of 4'-hydroxy-3'-methoxy acetophenone (Acetovanillone); ii) introduction of the cinnamoyl residue by aldol condensation with p-benzyloxy benzaldehyde and p-methoxy benzaldehyde respectively; iii) full debenzylation of all the sugar hydroxyl groups. Meanwhile, 4,4'-Di-O-β-D-glucopyransoyl-3-methoxychalcone was synthesized by an alternative way where a double armed aglycon acceptor was utilized in a one pot double glycosylation reaction. Results: Constructing the target chalcone glycosides: 4'-O-β-D-Glucopyransoyl-4-hydroxy-3'-methoxychalcone, 4'-O-(β-D-Glucopyransoyl)-3',4-dimethoxychalcone and 4,4'-Di-O-β-D-glucopyransoyl-3-methoxychalcone were achieved in 13%, 14%, and 90% yields. Conclusion: A simple and practical synthetic procedure by which the target chalcone glycosides were synthesized could be a promising and viable method. Furthermore, this strategy could be utilized in the synthesis of various O-diglycosyl chalcones having more complicated structures.

The inhibition effects of Camellia japonica leaf extract on antigen-stimulated degranulation and inflammatory cytokines in mast cells

This study investigated the effects of leaf extract of Camellia japonica (LECJ) on antigen-stimulated degranulation in mast cells and its mechanism of action in cell lines. It was identified that the inhibitor activation in the examination of β-hexosaminidase, inflammatory cytokines and Syk kinase activation upon which LECJ were collected in the prime of antioxidant activity were 1/10 concentration lower than the patent published in 2007 and the paper published 2008. In the case of preparing LECJ, the inhibition of the concentration was 10 times greater than the start as reported in the existing paper published in 2007 and the patent published 2008. The effects of LECJ verified it as an effective material for cosmetic use. In the study, the antigen-stimulated activation of mast cells, the inhibition of degranulation on LECJ and its mechanism of action, LECJ exhibited an inhibitory effect on degranulation in mast cells. Also, the activation of signaling pathways in antigen-stimulated mast cells depends initially on the interaction of FceRI on the downstream activation of Syk and other tyrosine kinases. This study showed that degranulation in antigen-stimulated mast cells and the secretion of inflammatory cytokines decreased in a concentration dependent manner. These results showed that the LECJ in cell line is effective for its anti-allergic activity. In the case of LECJ, the study showed that the effects of degranulation, the effects of the expression and secretion of inflammatory cytokines, and the directly inhibiting activation of signaling molecules such as adaptors.

Inhibitory Effects of Echinochrome A, Isolated from Shell of the Sea Urchin Anthocidaris crassispina, on Antigen-Stimulated Degranulation in Rat Basophilic Leukemia RBL-2H3 Cells through Suppression of Lyn Activation

Echinochrome A (Echi-A) was isolated from the sea urchin Anthocidaris crassispina and its structure determined using ID and 2D-NMR. In the present study, we examined the inhibitory effect of Echi-A on antigen-stimulated degranulation in rat basophilic leukemia RBL-2H3 cells, which were suppressed in a dose dependent manner.. The antigens bind to the high affinity immunoglobulin E receptor, which is expressed on the surface of mast cells and basophils and activate intracellular signal transduction, resulting in the release of biologically active mediators such as histamine. In order to disclose the inhibitory mechanisms of degranulation by Echi-A, we examined the elevation in intracellular C²⁺ concentration ([Ca²⁺]i), production levels of intracellular reactive oxygen species (ROS) and early intracellular signaling events. Both elevation of [Ca²⁺] and intracellular ROS production were markedly suppressed in cells treated with Echi-A. Echi-A also suppressed the activation of Lyn, Syk, and PLCyl/2 in antigen-stimulated cells. These results indicated that inhibition of antigen-stimulated degranulation in RBL-2H3 cells by Echi-A is mainly due to the inactivation of Lyn/Syk/PLCy signaling pathways. Our findings suggest that Echi-A could be a beneficial agent for alleviating the symptoms of type I allergy.

OM-X®, Fermented Vegetables Extract Suppresses Antigen-Stimulated Degranulation in Rat Basophilic Leukemia RBL-2H3 Cells and Passive Cutaneous Anaphylaxis Reaction in Mice

OM-X® is a hand-made and naturally manufactured probiotic supplement. This fermented food product is made from vegetables, fruits, seaweeds and mushrooms, using 12 strains of lactic acid bacteria and bifidobacteria. OM-X® is also known to have beneficial health properties, and some of its components show effects on antigen (Ag)-stimulated degranulation activity, indicating that OM-X® may be useful in the treatment of allergy responses and symptoms. In this study, we evaluated the inhibitory effects of OM-X® on Ag-stimulated degranulation in rat basophilic leukemia RBL-2H3 cells, clarified the underlying mechanisms, and determined the active compounds in OM-X® for suppression of degranulation. Treatment with OM-X® gradually suppressed Ag-stimulated degranulation throughout the maturation period. OM-X® also gradually produced melanoidins by lactic acid bacterial fermentation during the maturation process. There was a high correlation between the suppression levels of Ag-stimulated degranulation and the browning of OM-X®. Furthermore, the inhibition of Ag-stimulated degranulation by OM-X® was found to be partially due to the direct inactivation of NADPH oxidase. To elucidate the in vivo effects of OM- X®, type I allergy model mice were orally administered with OM-X®, and the passive cutaneous anaphylaxis (PCA) reaction was measured. OM-X® intake remarkably suppressed the PCA reaction. Taken together, our findings suggest that OMX® could be a beneficial food to ameliorate allergic reactions.

An indoxyl compound 5-bromo-4-chloro-3-indolyl 1,3-diacetate, CAC-0982, suppresses activation of Fyn kinase in mast cells and IgE-mediated allergic responses in mice

Mast cells, constituents of virtually all organs and tissues, are critical cells in IgE-mediated allergic responses. The aim of this study was to investigate the effect and mechanism of an indoxyl chromogenic compound, 5-bromo-4-chloro-3-indolyl 1,3-diacetate, CAC-0982, on IgE-mediated mast cell activation and allergic responses in mice. CAC-0982 reversibly suppressed antigen-stimulated degranulation in murine mast cells (IC50, ~3.8μM) and human mast cells (IC50, ~3.0μM). CAC-0982 also inhibited the expression and secretion of IL-4 and TNF-α in mast cells. Furthermore, CAC-0982 suppressed the mast cell-mediated allergic responses in mice in a dose-dependent manner (ED50 27.9mg/kg). As for the mechanism, CAC-0982 largely suppressed the phosphorylation of Syk and its downstream signaling molecules, including LAT, Akt, Erk1/2, p38, and JNK. Notably, the tyrosine kinase assay of antigen-stimulated mast cells showed that CAC-0982 inhibited Fyn kinase, one of the upstream tyrosine kinases for Syk activation in mast cells. Taken together, these results suggest that CAC-0982 may be used as a new treatment for regulating IgE-mediated allergic diseases through the inhibition of the Fyn/Syk pathway in mast cells.

Rhamnus davurica leaf extract inhibits Fyn activation by antigen in mast cells for anti-allergic activity.

BackgroundComplementary and alternative herbal medicines are recently considered as a promising approach for treating various diseases. We screened approximately 100 plant extracts for anti-allergic activity. Rhamnus davurica leaf extract showed the most potent inhibitory effect on the activation of RBL-2H3 mast cells. Although Rhamnus davurica extract has been used to treat pruritus, dysuresia, and constipation as a traditional herbal medicine in some Asian countries, an anti-allergic effect of Rhamnus davurica has not yet been demonstrated. We aimed to investigate the effect and mechanism of the leaf extract of Rhamnus davurica (LERD) on mast cells in vitro and allergic responses in vivo.MethodsThe effects of LERD on the activation of mast cells and mast cell-mediated passive cutaneous anaphylaxis (PCA) were measured in mice and two types of mast cells, mouse bone marrow-derived mast cells (BMMCs) and RBL-2H3 cells in vitro. A mechanistic study of its inhibitory effect was performed by using degranulation assay, reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting analysis.ResultsLERD reversibly suppressed antigen-stimulated degranulation in BMMCs and RBL-2H3 cells, and also inhibited mRNA expression and secretion of TNF-α and IL-4 in a dose-dependent manner. In a PCA animal model, LERD significantly inhibited antigen-induced allergic response and degranulation of ear tissue mast cells. As for the mechanism of action, LERD inhibited the activation of Syk, which is the pivotal signaling protein for mast cell activation by antigen. Furthermore, LERD also impeded the activations of well-known downstream proteins such as LAT, Akt and three MAP kinases (Erk, p38 and JNK). In an in vitro kinase assay, LERD suppressed the activation of Fyn in antigen-stimulated mast cells.ConclusionThis study demonstrated for the first time that LERD has anti-allergic effects through inhibiting the Fyn/Syk pathway in mast cells. Therefore, this study provides scientific evidence for LERD to be used as an herbal medicine or health food for patients with allergic diseases.

Flavonoid glucuronides isolated from spinach inhibit IgE-mediated degranulation in basophilic leukemia RBL-2H3 cells and passive cutaneous anaphylaxis reaction in mice

We isolated three active constituents from an aqueous extract of spinach leaves by evaluating the inhibitory activity of IgE-mediated degranulation in rat basophilic leukemia RBL-2H3 cells, and determined their chemical structures. These compounds, referred to as SO-1, SO-2 and SO-3, were assessed to be flavonol glucuronides with the same fundamental structure. Among them, SO-1 exhibited the most potent granulation-inhibitory activity at the concentrations of 0.5~10 µg/ml. This inhibitory effect was completely exhibited by its aglycon, which was prepared by the enzymatic treatment of β-glucuronidase and the chromatographic isolation. These compounds hardly showed cytotoxicity at the same concentration range in RBL-2H3 cell culture. To reveal its inhibitory mechanism, we examined the effects of SO-1 on the signal transduction of antigen-stimulated degranulation in the cells. Both SO-1 and its aglycon reduced the elevation of intracellular Ca 2+ concentration in a dose-dependent manner and also effectively inhibited the activation of tyrosine kinases such as Syk and Lyn, which underlay in the upstream of intracellular signal transduction followed by antigen stimulation. Furthermore, both SO-1 and its aglycon remarkably suppressed the passive cutaneous anaphylaxis (PCA) reaction induced in ICR mouse ear, as compared with the authentic anti-allergic drugs. In this PCA reaction, the aglycon exerted more potent inhibitory activity than SO-1, indicating that SO-1 also functions in vivo through the hydrolysis to the aglycon in the gastrointestinal tract. Together with these results, we conclude that spinach contains the promising anti-allergic flavonoid glucuronide, suggesting that this compound and its original vegetable may be beneficial to prevent or ameliorate allergic constitutions of humans.

좀보리사초의 IgE 매개성 알레르기 반응 억제 효과 및 기전

Allergic diseases have increased rapidly over the past decades, affecting an estimated 20~30% of the population in developed countries. In this study, we investigated whether or not a typical costal sand dune plant Carex pumila (CPE) suppresses the activation of mast cells and IgE-mediated allergic response in vitro and in vivo. As the results, the extract of Carex pumila inhibited antigen-stimulated degranulation in RBL-2H3 cells and Bone marrow-derived mast cells (BMMCs), and IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. CPE also sup- pressed the production of pro-inflammatory cytokines, TNF-α and IL-4, in antigen-stimulated mast cells. As its mech- anism of action, CPE inhibited the activation of Syk in FceRI-mediated signalling pathway, and that of LAT, a downstream adaptor molecule of Syk, in a dose-dependent manner. CPE also suppressed the activation of mitogen- activated protein (MAP) kinases, p38, ERK1/2, JNK, and Akt. Altogether, CPE inhibited mast cell activation and IgE- mediated allergic response by antigen through suppressing the activation of Syk. These results suggest that CPE may be useful for the treatment of allergic diseases.

Mycoepoxydiene inhibits antigen-stimulated activation of mast cells and suppresses IgE-mediated anaphylaxis in mice

Mycoepoxydiene (MED) is a polyketide isolated from a marine fungus associated with mangrove forest. It has been shown that MED has many kinds of effects such as anti-cancer and anti-inflammatory activities. However, its effects on anaphylaxis are still unknown. Mast cells play a pivotal role in IgE-mediated allergic response. Aggregation of the high affinity 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. Our study showed that MED could significantly suppress antigen-stimulated degranulation and cytokine production in mast cells and IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. Furthermore, we found that MED suppressed antigen-induced activation of Syk, and subsequently inhibited the phosphorylation of PLCγ1, Akt, and MAPKs such as extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 in mast cells. Collectively, our study demonstrates that MED can inhibit the activation of mast cells and protect mice from mast cell-mediated allergic response through inhibiting the activation of Syk. These results suggest that MED is a potential compound for developing a promising anti-anaphylaxis drug.

TRPM2 contributes to antigen-stimulated Ca2+ influx in mucosal mast cells

Food allergy (FA) is a common allergic disease without any currently available effective drug therapies. Mucosal mast cells (MMCs) play a particularly important role in FA, and the increase in their cytosolic Ca(2+) concentration ([Ca(2+)]cyt) is considered to be a principal component of the degranulation process. However, the mechanisms governing Ca(2+) influx remain poorly understood in MMCs. Recent reports have highlighted the functions of the transient receptor potential melastatin 2 (TRPM2) channel in immunocytes, including its role in monocyte chemokine production and macrophage phagocytic activity. Although TRPM2 gene expression has been demonstrated in mast cells, the significance of such expression remains virtually unknown. In this study, we found that antigen-stimulated degranulation was significantly reduced in mucosal-type bone marrow-derived mast cells (mBMMCs) prepared from TRPM2-knockout (TRPM2-KO) mice (TRPM2-KO mBMMCs) and was suppressed following the administration of three TRPM2 inhibitors with different chemical structures, including econazole, flufenamic acid (FFA), and 2-aminoethoxydiphenyl borate. Furthermore, the antigen-stimulated increase in [Ca(2+)]cyt was significantly decreased in TRPM2-KO mBMMCs and was also suppressed by the TRPM2 inhibitors econazole and FFA. In addition, thapsigargin-induced increase in [Ca(2+)]cyt was significantly decreased in TRPM2-KO mBMMCs. These results suggest that TRPM2 may participate in antigen-induced extracellular Ca(2+) influx and subsequent degranulation. In addition, TRPM2 inhibitors were shown to improve food allergic reactions in a mouse model. Together, these results suggest that TRPM2 inhibitors suppress MMC degranulation via regulation of the increase in [Ca(2+)]cyt. Thus, TRPM2 may play a key role in degranulation by modulating intracellular Ca(2+) in MMCs.

Toxoplasma gondii inhibits mast cell degranulation by suppressing phospholipase Cγ-mediated Ca2+ mobilization

Toxoplasma gondii is well-known to subvert normal immune responses, however, mechanisms are incompletely understood. In particular, its capacity to alter receptor-activated Ca2+-mediated signaling processes has not been well-characterized. In initial experiments, we found evidence that T. gondii infection inhibits Ca2+ responses to fMetLeuPhe in murine macrophages. To further characterize the mechanism of inhibition of Ca2+ mobilization by T. gondii, we used the well-studied RBL mast cell model to probe the capacity of T. gondii to modulate IgE receptor-activated signaling within the first hour of infection. Ca2+ mobilization that occurs via IgE/FcεRI signaling leads to granule exocytosis in mast cells. We found that T. gondii inhibits antigen-stimulated degranulation in infected cells in a strain-independent manner. Under these conditions, we found that cytoplasmic Ca2+ mobilization, particularly antigen-mediated Ca2+ release from intracellular stores, is significantly reduced. Furthermore, stimulation-dependent activation of Syk kinase leading to tyrosine phosphorylation and activation of phospholipase Cγ is inhibited by infection. Therefore, we conclude that inhibitory effects of infection are likely due to parasite-mediated inhibition of the tyrosine kinase signaling cascade that results in reduced hydrolysis of phosphatidylinositol 4,5-bisphosphate. Interestingly, inhibition of IgE/FcεRI signaling persists when tachyzoite invasion is arrested via cytochalasin D treatment, suggesting inhibition is mediated by a parasite-derived factor secreted into the cells during the invasion process. Our study provides direct evidence that immune subversion by T. gondii is initiated concurrently with invasion.

Anti-inflammatory effects of low-molecular weight chitosan oligosaccharides in IgE–antigen complex-stimulated RBL-2H3 cells and asthma model mice

The anti-inflammatory effects of low-molecular weight chitosan oligosaccharides (LM-COS) prepared from high-molecular weight chitosan by enzymatic digestion were investigated against allergic reaction and allergic asthma in vivo and in vitro. Allergic asthma is an inflammatory disease of the airways associated with enhanced degranulation and cytokine generation. The LM-COS (<1kDa), consisting of glucosamine (GlcN)n, n=3–5, were capable of inhibiting both antigen-stimulated degranulation and cytokine generation in rat basophilic leukemia RBL-2H3 cells. The protective effect of LM-COS against ovalbumin (OVA)-induced lung inflammation in asthma model mice was also examined. Oral administration of LM-COS (16mg/kg body weight/day) resulted in a significant reduction in both mRNA and protein levels of interleukin (IL)-4, IL-5, IL-13, tumor necrosis factor (TNF)-α in the lung tissue and bronchoalveolar lavage fluid (BALF); The protein levels of IL-4, IL-13 and TNF-α in BALF were decreased by 5.8-fold, 3.0-fold and 9.9-fold, respectively, compared to those in the OVA-sensitized/challenged asthma control group. These results suggest that the oral administration of LM-COS is effective in alleviating the allergic inflammation in vivo and thus can be a good source material for the development of a potent therapeutic agent against mast cell-mediated allergic inflammatory responses and airway inflammation in allergic inflammatory diseases, including asthma.

검정콩 껍질 유래 안토시아닌의 RBL-2H3 세포에서 탈과립화와 사이토카인 생성 저해 효과

Anthocyanins belong to a group of flavonoid compounds and are well known for their various health beneficial effects, which include antioxidative activities. Among them, the major anthocyanins isolated from seed coat of black soybean (Glycine max L.) were previously characterized as glycosides containing glucopyranose. Asthma is an allergic disease that is strongly associated with various immune cells, including basophils and mast cells. Eosinophils, basophils, and mast cells play important roles in allergic asthma through the release of inflammatory mediators such as asthma-specific T-helper 2 (Th2) cytokines and subsequent amplification of asthma symptoms via degranulation. Rat basophilic leukemia RBL-2H3 cells are the most common in vitro models for evaluating allergic reactions. In this study, we examined the effects of anthocyanin from seed coat of black soybean on antigen-stimulated degranulation and Th2 cytokine production in RBL-2H3 cells. Cell degranulation was evaluated by measuring the release of β-hexosaminidase. β-Hexosaminidase release and Th2 cytokine production in RBL-2H3 cells was much higher upon stimulation with IgE-antigen complex than those in untreated control cells. Anthocyanins significantly suppressed IgE-antigen complex-induced degranulation of RBL-2H3 cells and inhibited IgE-antigen complex-mediated interleukin (IL)-4, IL-13, and tumor necrosis factor α (TNF-α) production in RBL-2H3 cells. These findings suggest that anthocyanins from seed coat of black soybean effectively inhibit allergic reactions and may have beneficial effects against allergic asthma.

A Novel Imidazo[1,5-b]isoquinolinone Derivative, U63A05, Inhibits Syk Activation in Mast Cells to Suppress IgE-Mediated Anaphylaxis in Mice

Mast cells play a pivotal role in IgE-mediated allergic responses. Development of specific inhibitors against FcεRI-associated proximal signaling molecules in mast cells may represent a promising therapeutic strategy for allergic diseases. We examined whether a novel synthetic compound, 3-butyl-1-chloro-8-(2-methoxycarbonyl)phenyl-5H-imidazo[1,5-b]isoquinolin-10-one (U63A05), could suppress antigen-stimulated degranulation and cytokine secretion in mast cells and IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. U63A05 reversibly and dose-dependently inhibited degranulation of rat basophilic leukemia (RBL)-2H3 mast cells and bone marrow–derived mast cells (BMMCs) stimulated by antigen (IC50 values for RBL-2H3 and BMMCs were 4.1 and 4.8 μM, respectively). The secretion of inflammatory cytokines was also suppressed in antigen-stimulated mast cells. However, degranulation by thapsigargin, a typical calcium inducer, was not inhibited by U63A05. U63A05 exerts its inhibitory effect, to the same extent as in degranulation, on the activating phosphorylation of Syk and downstream signaling molecules, including LAT and SLP-76. Further downstream, the activating phosphorylations of Akt, Erk1/2, p38, and JNK were also inhibited. Finally, antigen-stimulated PCA was dose-dependently suppressed in mice (ED50, 26.3 mg/kg). Taken together, the results suggest that U63A05 suppresses the activation of mast cells and the mast cell–mediated allergic response through the inhibition of Syk activation in mast cells.