RBL-2H3 Degranulation Research Articles

Formononetin inhibits IgE by huPlasma/PBMCs and mast cells/basophil activation via JAK/STAT/PI3-Akt pathways.

Food allergy is a prevalent disease in the U.S., affecting nearly 30 million people. The primary management strategy for this condition is food avoidance, as limited treatment options are available. The elevation of pathologic IgE and over-reactive mast cells/basophils is a central factor in food allergy anaphylaxis. This study aims to comprehensively evaluate the potential therapeutic mechanisms of a small molecule compound called formononetin in regulating IgE and mast cell activation. In this study, we determined the inhibitory effect of formononetin on the production of human IgE from peripheral blood mononuclear cells of food-allergic patients using ELISA. We also measured formononetin's effect on preventing mast cell degranulation in RBL-2H3 and KU812 cells using beta-hexosaminidase assay. To identify potential targets of formononetin in IgE-mediated diseases, mast cell disorders, and food allergies, we utilized computational modeling to analyze mechanistic targets of formononetin from various databases, including SEA, Swiss Target Prediction, PubChem, Gene Cards, and Mala Cards. We generated a KEGG pathway, Gene Ontology, and Compound Target Pathway Disease Network using these targets. Finally, we used qRT-PCR to measure the gene expression of selected targets in KU812 and U266 cell lines. Formononetin significantly decreased IgE production in IgE-producing human myeloma cells and PBMCs from food-allergic patients in a dose-dependent manner without cytotoxicity. Formononetin decreased beta-hexosaminidase release in RBL-2H3 cells and KU812 cells. Formononetin regulates 25 targets in food allergy, 51 in IgE diseases, and 19 in mast cell diseases. KEGG pathway and gene ontology analysis of targets showed that formononetin regulated disease pathways, primary immunodeficiency, Epstein-Barr Virus, and pathways in cancer. The biological processes regulated by formononetin include B cell proliferation, differentiation, immune response, and activation processes. Compound target pathway disease network identified NFKB1, NFKBIA, STAT1, STAT3, CCND1, TP53, TYK2, and CASP8 as the top targets regulated at a high degree by formononetin. TP53, STAT3, PTPRC, IL2, and CD19 were identified as the proteins mostly targeted by formononetin. qPCR validated genes of Formononetin molecular targets of IgE regulation in U266 cells and KU812 cells. In U266 cells, formononetin was found to significantly increase the gene expression of NFKBIA, TP53, and BCL-2 while decreasing the gene expression of BTK TYK, CASP8, STAT3, CCND1, STAT1, NFKB1, IL7R. In basophils KU812 cells, formononetin significantly increased the gene expression of NFKBIA, TP53, and BCL-2 while decreasing the gene expression of BTK, TYK, CASP8, STAT3, CCND1, STAT1, NFKB1, IL7R. These findings comprehensively present formononetin's mechanisms in regulating IgE production in plasma cells and degranulation in mast cells.

Six sesquiterpenoids from the stems and leaves of Chrysanthemum morifolium Ramat and their anti-asthma activities

Six previously undescribed sesquiterpenoids, chrysanthterpenoids H-M (1–6), were isolated from the stem and leaves of Chrysanthemum morifolium Ramat. Structure elucidation of isolated compounds was unambiguously determined based on extensive 1D and 2D NMR spectroscopic analyses. Furthermore, computational prediction of ECD was used to propose the absolute configurations of the compounds. All compounds were evaluated for their anti-asthma effects on RBL-2H3 cells in vitro. The results showed that Compounds 2 and 3 significantly inhibited the release of β-aminohexosidase and improved RBL-2H3 degranulation by chromogenic substrate and high-content imaging. These results suggest that Compounds 2 and 3 may exhibit anti-asthma activities.

Discovery of a traditional Chinese herbal combination for the treatment of atopic dermatitis: saposhnikoviae radix, astragali radix and cnidium monnieri.

Atopic dermatitis (AD) is a skin disease characterized by pruritus. The present study aimed to discover a herbal combination with anti-allergic and anti-inflammatory activities to treat AD. First, the anti-allergic and anti-inflammatory activities of herbs were evaluated by RBL-2H3 degranulation and HaCaT inflammatory models. Subsequently, the optimal proportion of herbs was determined by uniform design-response surface methodology. The effectiveness and synergistic mechanism was further verified. Cnidium monnieri (CM) suppressed β-hexosaminidase (β-HEX) release, saposhnikoviae radix (SR), astragali radix (AR), and CM inhibited the release of IL-8 and MCP-1. The optimal proportion of herbs was SR∶AR∶CM = 1: 2: 1. The in vivo experiments results indicated that the topical application of combination at high (2 ×) and low (1 ×) doses improved dermatitis score and epidermal thickness, and attenuated mast cell infiltration. Network pharmacology and molecular biology further clarified that the combination resisted AD by regulating the MAPK, JAK signaling pathways, and the downstream cytokines such as IL-6, IL-1β, IL-8, IL-10, and MCP-1. Overall, the herbal combination could inhibit inflammation and allergy, improving AD-like symptoms. The present study discovers a promising herbal combination, worthy of further development as a therapeutic drug for AD.

Anti-allergic property of 4,8-sphingadienine stereoisomers in vivo and in vitro model

4,8-Sphingadienines (SD), metabolites of glucosylceramides (GlcCer), are sometimes determined as key mediators of the biological activity of dietary GlcCer, and cis/trans geometries of 4,8-SD have been reported to affect its activity. Since regulating excessive activation of mast cells seems an important way to ameliorate allergic diseases, this study was focused on cis/trans stereoisomeric-dependent inhibitory effects of 4,8-SD on mast cell activation. Degranulation of RBL-2H3 was inhibited by treatment of 4-cis-8-trans- and 4-cis-8-cis-SD, and their intradermal administrations ameliorated ear edema in passive cutaneous anaphylaxis reaction, but 4-trans-8-trans- and 4-trans-8-cis-SD did not. Although the activation of mast cells depends on the bound IgE contents, those stereoisomers did not affect IgE contents on RBL-2H3 cells after the sensitization of anti-TNP IgE. These results indicated that 4-cis-8-trans- and 4-cis-8-cis-SD directly inhibit the activation of mast cells. In conclusion, it was assumed that 4,8-SD stereoisomers with cis double bond at C4-position shows anti-allergic activity by inhibiting downstream pathway after activation by the binding of IgE to mast cells.

A TCM formula VYAC ameliorates DNCB-induced atopic dermatitis via blocking mast cell degranulation and suppressing NF-κB pathway

Ethnopharmacological relevanceA Traditional Chinese Medicine (TCM) formula (VYAC) consists of three herbs including Viola yedoensis Makino, herb (Violaceae, Viola), Sophora flavescens Aiton, root (Fabaceae, Sophora) and Dictamnus dasycarpus Turcz, root and rhizome (Rutaceae, Dictamnus), has been traditionally prescribed to treat various skin diseases in clinic. Aim of the studyThis study aims to investigate the therapeutic effects of VYAC on the 2,4-dinitrobenzene (DNCB) induced atopic dermatitis (AD)-like mice and to explore the underlying mechanisms. Materials and methodsVYAC was extracted with 70 % aqueous ethanol and lyophilized powder was used. AD-like mice were challenged by DNCB, VYAC (150 and 300 mg/kg) were oral administration daily from day 7 to day 28. At the end of experiment, the clinical scores were recorded, serum and skin in the dorsal were isolated to evaluate the therapeutic effects of VYAC. RBL-2H3 cells were stimulated with C48/80 for degranulation and plasmids expressing constitutively active form of Syk (Silence or overexpression) were transfected into RBL-2H3 cells to explore the underlying mechanisms in vitro. ResultsVYAC significantly ameliorated the cardinal symptoms in the DNCB-induced AD-like mice by repairing the skin barrier function, inhibiting mast cells infiltration, restraining the serum IgE and histamine release and decreasing TNF-α, IL-4 as well as Syk mRNA level in dorsal skin and alleviating inflammation. Besides, VYAC significantly blocked RBL-2H3 cells degranulation, reduced β-hexosaminidase and histamine release, and suppressed NF-κB pathway. What's more, the degranulation of RBL-2H3 was reduced after Syk silence and increased after Syk overexpression. ConclusionOur findings clearly suggested that VYAC treat AD through inhibiting the inflammatory mediator productions and blocking mast cell degranulation via suppressing Syk mediated NF-κB pathway.

Immunomodulatory Effect of Laccase/Caffeic Acid and Transglutaminase in Alleviating Shrimp Tropomyosin (Met e 1) Allergenicity.

This work aimed to investigate the effect of enzymatic cross-linking on the allergenic potential of shrimp tropomyosin (TM), Met e 1. The cross-linked TM with laccase (CL), laccase/caffeic acid (CLC and CLC+), and transglutaminase (CTG and CTG+) formed macromolecules and altered the allergen conformation. The IgG/IgE-binding potentials of the cross-linked TM were reduced as confirmed by Western blotting and ELISA. Enzymatic cross-linking improved the gastrointestinal digestibility and induced a lower level of degranulation in RBL-2H3 and KU812 cells. Moreover, cross-linked TM decreased anaphylactic symptoms, as well as reduced the serum levels of IgG1, IgE, histamine, tryptase, and mMCP-1. In spleen cells, CLC+ and CTG+ downregulated the Th2-related cytokines and upregulated IFN-γ and IL-10. These findings revealed that CTG+ has shown more potential than CLC+ in mitigating the allergenicity of TM by influencing the conformational structure, enhancing the digestibility, decreasing the cellular degranulation process, and positively modulating the Th1/Th2 immunobalance.

5-HMF induces anaphylactoid reactions in vivo and in vitro

AimExcessive exposure to 5-hydroxymethylfurfural (5-HMF), which is a common impurity in various sugar-containing products, induces serious side effects. Our previous study revealed that 5-HMF exerted immune sensitizing potential when injected into rodents. In this study, we explored 5-HMF mediated anaphylactoid reactions and its underlying molecular mechanisms. MethodsWe investigated anaphylactoid reactions in Brown Norway (BN) rats and Institute of Cancer Research (ICR) mice to identify 5-HMF mediated in vivo anaphylactoid reactions. RBL-2H3 and P815 cell degranulation models were also established, and degranulation, enzyme-linked immunosorbent, filamentous actin (F-actin) microfilament staining, and western blot assays were performed in these cells. ResultsWe showed that 5-HMF induced anaphylactoid reactions by increasing blood vessel permeability in mice, and significantly elevating histamine (His) and glutathione peroxidase-1 (Gpx-1) levels in rat serum. Moreover, after incubation with 5-HMF, β-hexosaminidase (β-Hex), His, IL-4 and IL-6 levels were all significantly increased, thereby inducing cellular degranulation in RBL-2H3 and P815 cells. Finally, 5-HMF also upregulated Lyn, Syk, p38 and JNK protein phosphorylation levels. ConclusionsOur findings suggest that 5-HMF induces anaphylactoid reactions both in vivo and in vitro, therefore 5-HMF limits in sugar-containing products should receive more regulatory attention.

Qi-Wei-Du-Qi-Wan and its major constituents exert an anti-asthmatic effect by inhibiting mast cell degranulation

Ethnopharmacological relevanceIn Asia, Qi-Wei-Du-Qi-Wan (QWDQW) is a traditional Chinese medicine that has been used to treat chest tightness, cough, shortness of breath, night sweats, frequent urination and asthma. QWDQW is recorded in Yi Zong Yi Ren Pian (Medical Physician's Compilation), which was written by Yang Cheng Liu during the Qing Dynasty. Aim of the studyThe traditional Chinese medicine QWDQW is composed of 7 ingredients and has been used in the treatment of asthma in Asia for hundreds of years. However, the mechanism through which QWDQW affects the immune system in the treatment of asthma is not known. Therefore, this study aimed to investigate whether QWDQW alleviates asthmatic symptoms in mice with chronic asthma induced by repeated stimulation with Dermatophagoides pteronyssinus (Der p) and to explore the underlying immune modulatory mechanism. Materials and methodsBALB/c mice were stimulated intratracheally (i.t.) with Der p (40 μl, 2.5 μg/μl) once weekly for 6 weeks. Thirty minutes prior to Der p stimulation, the mice were treated with QWDQW (0.5 g/kg and 0.17 g/kg) orally. Three days after the last stimulation, the mice were sacrificed, and infiltration of inflammatory cells, lung histological characteristics, gene expression of lung and serum total IgE were assessed. In other experiments, RBL-2H3 cells were stimulated with DNP-IgE/DNP-BSA and then treated with QWDQW, quercetin, β-carotene, luteolin or a mixture of the three chemicals (Mix13) for 30 min, and the effects of the drugs on RBL-2H3 cell degranulation after DNP stimulation were determined. ResultsQWDQW significantly reduced Der p-induced airway hyperreactivity (AHR) and decreased total serum IgE and Der p-specific IgE levels. Histopathological examination showed that QWDQW reduced inflammatory cell infiltration and sputum secretion from goblet cells in the lungs. Gene expression analysis indicated that QWDQW reduced overproduction of IL-12、IFN-γ、IL-13、IL-4、RNATES、Eotaxin and MCP-1in lung. Additionally, QWDQW and Mix13 suppressed DNP induced RBL-2H3 degranulation, and the effect was maximal when quercetin, β-carotene and luteolin were administered together. ConclusionThese results indicate that QWDQW plays a role in suppressing excessive airway reaction and in specific immune modulation in a mouse model of chronic asthma and that QWDQW suppresses mast cell degranulation at defined doses of quercetin, β-carotene and luteolin.

The novel H2 S donor 4-carboxy-phenyl isothiocyanate inhibits mast cell degranulation and renin release by decreasing intracellular calcium.

Hydrogen sulfide (H2 S) modulates many pathophysiological processes, including inflammation and allergic reactions, in which mast cells act as major effector cells. IgE receptor (FcεRI) cross linking leads to an increase in intracellular calcium ([Ca+2 ]i ), a critical step in mast cell degranulation. The aim of this study was to investigate the role of H2 S in [Ca+2 ]i -dependent mast cell activation. We investigated the effects of H2 S, either endogenously produced or released by the slow H2 S donor 4-carboxy-phenyl isothiocyanate (PhNCS-COOH), on antigenic- and non-antigenic degranulation of native murine mast cells, and human and rat (RBL-2H3) mast cell lines. We measured the release of specific mast cell degranulation markers (β-hexosaminidase and renin), as well as changes in [Ca+2 ]i and phosphorylation of proteins downstream of FcεRI activation. Endogenously produced H2 S inhibited antigen-induced degranulation in RBL-2H3. Similarly, H2 S released by PhNCS-COOH (10-300μM) reduced, in a concentration-dependent manner, antigenic and non-antigenic degranulation and renin release in all mast cell types. Notably, PhNCS-COOH also prevented in a concentration-dependent mode the increase in [Ca+2 ]i elicited by Ca+2 ionophore, thapsigargin and FcεRI activation. Moreover, PhNCS-COOH attenuated the phosphorylation of Syk, cPLA-2 and PLCγ1 in antigen-stimulated RBL-2H3 cells. Collectively, our results demonstrate that, by attenuating the phosphorylation of proteins downstream of FcεRI cross-linking on mast cells, H2 S diminishes [Ca+2 ]i availability and thus mast cell degranulation and renin release. These findings suggest that PhNCS-COOH could be a strategic therapeutic tool in mast cell-mediated allergic conditions.

Effects of a Moderately Lower Temperature on the Proliferation and Degranulation of Rat Mast Cells.

Mast cells are traditionally considered as key effector cells in IgE-mediated allergic diseases. However, the roles of mast cells have also been implicated in diverse physiological and pathological processes. Mast cells are distributed in various organs and tissues of various species. Some of the organs and tissues, such as testis, skin, and the upper part of the respiratory tract, have a temperature that is lower than the body's core temperature. The purpose of the present study was to investigate the effects of a lower temperature on the proliferation and degranulation of rat mast cells. Here, we demonstrate that cell growth was retarded at 35°C compared to 37°C for both rat peritoneal mast cells (RPMC) and RBL-2H3, a rat mast cell line. Furthermore, RPMC became more susceptible to degranulation at 35°C compared to 37°C. In contrast, degranulation of RBL-2H3 was not as sensitive to temperature change as RPMC. The functionality of mast cells in unique organs with a lower temperature warrants further analysis.

Rosette Nanotubes Alter IgE-Mediated Degranulation in the Rat Basophilic Leukemia (RBL)-2H3 Cell Line.

In this study, the effects of rosette nanotube (RNT) exposure on immune cell viability and function were investigated in vitro using the rat basophilic leukemia (RBL)-2H3 cell line. RBL-2H3 viability was decreased in a dose- and time-dependent manner after lysine-functionalized RNT (K-RNT) exposure. In addition, K-RNTs had a significant effect on RBL-2H3 degranulation. When K-RNT exposure was concurrent with IgE sensitization, 50 and 100 mg l(-1) K-RNTs elicited a heightened degranulatory response compared with IgE alone. Exposure to 50 and 100 mg l(-1) K-RNTs also caused degranulation in RBL-2H3 cells not sensitized with IgE (0 ng ml(-1) IgE). Furthermore, in cells preexposed to K-RNTs for 2 h and subsequently washed, sensitized, and stimulated with IgE, a potentiated degranulatory response was observed. Using confocal laser scanning microscopy and a fluorescein isothiocyanate (FITC)-functionalized RNT construct (termed FITC(1)/TBL(19)-RNT), we demonstrated a strong and direct affiliation between RNTs and RBL-2H3 cell membranes. We also demonstrated cellular internalization of RNTs after 2 h of exposure. Together, these data demonstrate that RNTs may affiliate with the cellular membrane of RBL-2H3 cells and can be internalized. These interactions can affect viability and alter the ability of these cells to elicit IgE-FcεR mediated degranulation.

Antimicrobial agent triclosan is a proton ionophore uncoupler of mitochondria in living rat and human mast cells and in primary human keratinocytes

Triclosan (TCS) is an antimicrobial used widely in hospitals and personal care products, at ~10 mm. Human skin efficiently absorbs TCS. Mast cells are ubiquitous key players both in physiological processes and in disease, including asthma, cancer and autism. We previously showed that non-cytotoxic levels of TCS inhibit degranulation, the release of histamine and other mediators, from rat basophilic leukemia mast cells (RBL-2H3), and in this study, we replicate this finding in human mast cells (HMC-1.2). Our investigation into the molecular mechanisms underlying this effect led to the discovery that TCS disrupts adenosine triphosphate (ATP) production in RBL-2H3 cells in glucose-free, galactose-containing media (95% confidence interval EC50 = 7.5-9.7 µm), without causing cytotoxicity. Using these same glucose-free conditions, 15 µm TCS dampens RBL-2H3 degranulation by 40%. The same ATP disruption was found with human HMC-1.2 cells (EC50 4.2-13.7 µm), NIH-3 T3 mouse fibroblasts (EC50 4.8-7.4 µm) and primary human keratinocytes (EC50 3.0-4.1 µm) all with no cytotoxicity. TCS increases oxygen consumption rate in RBL-2H3 cells. Known mitochondrial uncouplers (e.g., carbonyl cyanide 3-chlorophenylhydrazone) previously were found to inhibit mast cell function. TCS-methyl, which has a methyl group in place of the TCS ionizable proton, affects neither degranulation nor ATP production at non-cytotoxic doses. Thus, the effects of TCS on mast cell function are due to its proton ionophore structure. In addition, 5 µm TCS inhibits thapsigargin-stimulated degranulation of RBL-2H3 cells: further evidence that TCS disrupts mast cell signaling. Our data indicate that TCS is a mitochondrial uncoupler, and TCS may affect numerous cell types and functions via this mechanism. Copyright © 2015 John Wiley & Sons, Ltd.

Raft localization of Type I Fcε receptor and degranulation of RBL-2H3 cells exposed to decavanadate, a structural model for V2O5

Vanadium oxides (VOs) have been identified as low molecular weight sensitizing agents associated with occupational asthma and compromised pulmonary immunocompetence. Symptoms of adult onset asthma result, in part, from increased signal transduction by Type I Fcε receptors (FcεRI) leading to release of vasoactive compounds including histamine from mast cells. Exposure to (VOs) typically occurs in the form of particles which are insoluble. Upon contact with water or biological fluids, (VOs) form a series of soluble oxoanions, one of which is decavanadate, V10O28(6-) abbreviated V10, which is structurally related to a common vanadium oxide, that is vanadium pentoxide, V2O5. Here we investigate whether V10 may be initiating plasma membrane events associated with activation of FcεRI signal transduction. We show that exposure of RBL-2H3 cells to V10 causes a concentration-dependent increase in degranulation of RBL-2H3 and, in addition, an increase in plasma membrane lipid packing as measured by the fluorescent probe, di-4-ANEPPDHQ. V10 also increases FcεRI accumulation in low-density membrane fragments, i.e., lipid rafts, which may facilitate FcεRI signaling. To determine whether V10 effects on plasma membrane lipid packing were similarly observed in Langmuir monolayers formed from dipalmitoylphosphatidylcholine (DPPC), the extent of lipid packing in the presence and absence of V10 and vanadate was compared. V10 increased the surface area of DPPC Langmuir monolayers by 6% and vanadate decreased the surface area by 4%. These results are consistent with V10 interacting with this class of membrane lipids and altering DPPC packing.

Galectin‐9 induced by dietary synbiotics is involved in suppression of allergic symptoms in mice and humans

Prebiotic galacto- and fructo-oligosaccharides (scGOS/lcFOS) resembling non-digestible oligosaccharides in human milk reduce the development of atopic disorders. However, the underlying mechanisms are still unclear. Galectins are soluble-type lectins recognizing β-galactoside containing glycans. Galectin-9 has been shown to regulate mast cell degranulation and T-cell differentiation. In this study, the involvement of galectin-9 as a mechanism by which scGOS/lcFOS in combination with Bifidobacterium breve M-16V protects against acute allergic symptoms was investigated. Mice were sensitized orally to whey, while being fed with a diet containing scGOS/lcFOS and Bifidobacterium breve M-16V (GF/Bb) or a control diet. Galectin-9 expression was determined by immunohistochemistry in the intestine and measured in the serum by ELISA. T-cell differentiation was investigated in the mesenteric lymph nodes (MLN) as well as in galectin-9-exposed peripheral blood mononuclear cells (PBMC) cultures. Sera of the mice were evaluated for the capacity to suppress mast cell degranulation using a RBL-2H3 degranulation assay. In addition, in a double-blind, placebo-controlled multicenter trial, galectin-9 levels were measured in the sera of 90 infants with atopic dermatitis who received hydrolyzed formulae with or without GF/Bb. Galectin-9 expression by intestinal epithelial cells and serum galectin-9 levels were increased in mice and humans following dietary intervention with GF/Bb and correlated with reduced acute allergic skin reaction and mast cell degranulation. In addition, GF/Bb enhanced T(h)1- and T(reg)-cell differentiation in MLN and in PBMC cultures exposed to galectin-9. Dietary supplementation with GF/Bb enhances serum galectin-9 levels, which associates with the prevention of allergic symptoms.

Studies on the Degranulation of RBL-2H3 Cells Induced by Traditional Chinese Medicine Injections

Aims: To study RBL-2H3 cell degranulation phenomena induced by some TCMIs through cell morphological and ultra-structural observation, released enzyme activity and establish RBL-2H3 cell degranulation test indicated by β- hexosaminidase activity as a method to evaluate TCMIs at nonclinical stage. Methods: RBL-2H3 cells were used to study the degranulation by co-culture with positive control C48/80 and some TCMIs through morphological and ultra-structure observation, β-hexosaminidase activity detection. RBL-2H3 cell degranulation test was established to detect β-hexosaminidase activity caused by 17 kinds of TCMIs and their ingredients. The cytotoxicity effect of some TCMIs on both RBL 2H3 and BRL cells was measured by CCK-8 assay. Results: Toluidine blue staining and ultra-structure of electronic microscope observation of treated RBL-2H3 cells showed degranulation morphologically. Detection of β-hexosaminidase activity in the supernatant of treated cells showed some TCMIs had elevated enzyme release rates. Further analysis of the ingredients and compound in Tanreqing Injection and Shengmai Injection showed Scutellaria baicalensis Georgi in Tanreqing Injection, Red ginseng and Fructus Schisandrae Chinensis in Shengmai Injection were responsible to the degranulation of RBL-2H3 cells. Osmotic pressures and pH influenced RBL-2H3 degranulation. High Osmotic pressure of Tanreqing Injection and low pH of chlorogenic acid at 2.5 and 5.0 mmol/L congcentration might be responsible to high β-hexosaminidase activity. Most of the TCMIs inducing degranulation had cytotoxicity effect for both RBL-2H3 and BRL cells, but some TCMIs inducing degranulation had no cytotoxicity effect. Conclusion: Some TCMIs can induce degranulation of RBL-2H3 cells; RBL-2H3 cell degranulation test can be used in non-clinical stage to detect the risk causing anaphylactoid reactions. Osmotic pressures and pH influenced RBL-2H3 degranulation, and they should be measured before testing. The mechanism of degranulation caused by some TCMIs is cytotoxic, and some are non-cytotoxic and may be through exicytosis.

Aspergillus oryzae Lectin Induces Anaphylactoid Oedema and Mast Cell Activation Through its Interaction With Fucose of Mast Cell–Bound Non‐specific IgE

We investigated whether Aspergillus oryzae lectin (AOL), a fucose-specific lectin, induces anaphylactoid reactions and mast cell activation. The injection of AOL into footpads of mice produced a dose-related acute paw oedema. The AOL-induced oedema was attenuated by predose of histamine H1 receptor blocker or pretreatment of the lectin with fucose before injection and was not observed in SCID and mast cell-deficient WBB6F1-W/Wv mice. These results suggested that the AOL-induced anaphylactoid reaction was mediated by histamine released from mast cells. In addition, the activation of mast cells was seemed to be induced by the crosslinking of IgE on the cell surface following the binding of AOL to fucose residues in IgE. Consistent with the in vivo results, AOL induced the degranulation of the rat mast cell line RBL2H3 sensitized with monoclonal IgE. As AOL induced the increase in intracellular Ca(2+) concentration of IgE-sensitized RBL2H3 cells as well as antigen stimulation, AOL could input signals from FcεRI. The degranulation of IgE-sensitized RBL2H3 cells by AOL was diminished by pretreatment of AOL with fucose. Defucosylated IgE did not induce degranulation of RBL2H3 cells in response to AOL stimulation, in spite of its ability to induce degranulation by antigen stimulation as intact IgE. These results indicated that AOL bound to fucose residue of IgE causing antigen-independent IgE-mediated mast cell activation and anaphylactoid reactions in vitro and in vivo, respectively. AOL bound to human IgE as well as to mouse IgE, suggesting the possible implication of AOL in the allergic response to Aspergillus oryzae in humans.

Comparison of RBL-2H3 and P815 Cell Lines for Establishing in vitro Model of Mast Cell Degranulation

Aim To investigated the difference between C48/80-induced degranulation in RBL-2H3 and that in P815 cells, and to find an early, stable and sensitive in vitro mast cell degranulation model. All findings might provide reference for establishing in vitro models to screen allergens and testing anti-allergic agents. Methods Cells were treated with 10 μg·mL −1 C48/80 for fifteen to sixty minutes. Cell degranulation was evaluated by detecting the release of histamine, β-hexosaminidase and tryptase by colorimetric assays, observing the morphological changes by neutral red staining, and detecting the ratio of Annexin V positive cell by flow cytometry. Results After treatment with C48/80, the histamine release in RBL-2H3 and P815 cells was significantly increased ( P < 0.05), and the ratio of histamine release in both cells was almost equivalent. β-hexosaminidase release, tryptase release, the percentage of Annxin V positive cells and the degranulation ratio were markedly elevated in P815 cells treated with C48/80 for more than 15 min, but the same concentration of C48/80 induced similar changes in RBL-2H3 cells only when the cells were treated for more than 30 to 45 min. Upon the same stimulating conditions, allergic mediators release, the percentage of Annxin V positive cells and the degranulation ratio in P815 cells were much higher than those in RBL-2H3 cells. Conclusion Compared with the RBL-2H3 cells, cell degranulation of P815 cells occurred faster and more severe than that of RBL-2H3 under the s ame stimulating conditions. These data sustain our contention that P815 cells might be utilized as a potential early, fast and sensitive in vitro model for screening suspicious allergens and searching for anti-allergic agents.

Induction of Prostaglandin D2 through the p38 MAPK Pathway Is Responsible for the Antipruritic Activity of Sertaconazole Nitrate

Prostaglandin D(2) (PGD(2)) is known to have antipruritic activity by suppressing histamine release. However, agents that can topically induce PGD(2) for itch relief are not well established. The antimycotic sertaconazole nitrate (STZ) has been shown to exhibit anti-itch properties; however, the mechanism for this activity has not been elucidated. STZ mitigated degranulation of RBL-2H3 (rat basophilic leukemia) mast cells induced by compound 48/80, a pruritogenic agent known to promote the release of histamine, and augmented PGD(2) production in mast cells and macrophages. Addition of exogenous PGD(2) abrogated compound 48/80-induced degranulation by acting through the prostanoid D receptor 1 (DP1). STZ induced p38 mitogen-activated protein kinase (MAPK) phosphorylation in mast cells and a pharmacological inhibitor of p38 MAPK, SB203580, resulted in the attenuation of PGD(2) levels. Finally, in a murine model of pruritus, the scratching behavior induced by compound 48/80 was mitigated by topical application of STZ. This effect was reversed by the addition of the cyclooxygenase inhibitor, ibuprofen, or a DP1 receptor antagonist (MK0524). Collectively, these results suggest that STZ mediates its anti-itch effects by boosting the antipruritic agent, PGD(2), by the activation of the p38-MAPK pathway. This is the first report to demonstrate a promising approach to topically induce PGD(2) for improving pruritus.

ChemInform Abstract: Inhibition of Antigen‐Induced Degranulation by Aryl Compounds Isolated from the Bark of Betula platyphylla in RBL‐2H3 Cells.

Abstract The methanolic extract of the bark of Betula platyphylla was found to suppress antigen mediated degranulation of RBL-2H3 cells. Four arylbutanoids ( 1 – 4 ) and eight diarylhepatonoids ( 5 – 12 ) were isolated from the methanolic extract using bioassay-guided fractionation. Among them, compounds 4 and 12 were isolated and assigned for the first time. Compounds 1 , 2 , 3 , 5 , 10 , and 12 showed remarkable inhibitory activity against the degranulation of RBL-2H3 by antigen stimulation in a dose dependent manner at the concentrations ranging from 10 μM to 100 μM.

Syk and Lyn mediate distinct Syk phosphorylation events in FcɛRI-signal transduction: Implications for regulation of IgE-mediated degranulation

Spleen tyrosine kinase (Syk) is a key regulatory factor in the IgE-mediated allergic signal transduction pathway in mast cells and basophils. Syk is phosphorylated on a number of tyrosines following the binding of IgE/allergen complexes to FcɛRI receptors leading to initiation of inflammatory signaling via downstream enzymes and scaffolding proteins. We examined the kinases responsible for the phosphorylation of key Syk tyrosines in rat RBL-2H3 basophilic cells and bone marrow-derived mast cells (BMMCs). The phosphorylation of Syk tyrosine 346 was completely blocked by the novel Src family kinase inhibitor BIRA766, suggesting this tyrosine is a pure substrate for Src family kinases. This was supported by the findings that kinase-dead (KD) Syk was efficiently phosphorylated on this tyrosine and that a specific Syk inhibitor BAY61-3606 was without effect. The phosphorylation of other Syk tyrosines 317, 342, 519 and 520 was reduced by Syk and Src family inhibitors, suggesting a role for auto- and trans-phosphorylation. Lyn was the predominant Src family kinase expressed and activated in RBL-2H3 cells, meanwhile Lyn knockdown with a specific siRNA interfered with the phosphorylation of all Syk tyrosines and the Syk substrates SLP-76 and LAT. Pharmacological inhibition of Syk completely blocked the degranulation of RBL-2H3 and BMMCs. However, Lyn knockdown sensitized RBL-2H3 cells to FcɛRI-induced degranulation. We showed that whilst interference with Lyn expression disrupts FcɛRI proximal signaling via Syk and its direct substrates including SLP-76 and LAT, distal activation of downstream proteins including Erk is enhanced. This study identifies the responsible kinases for the phosphorylation of key Syk tyrosines and the propagation of FcɛRI receptor mediated signal transduction in allergic responses.