The phenoxazine derivative Phx-1 suppresses IgE-mediated degranulation in rat basophilic leukemia RBL-2H3 cells

Antigen-induced aggregation of the high affinity IgE receptor (FcepsilonRI) on mast cells induces degranulation to release chemical mediators, leading to acute allergic inflammation. We have demonstrated that the treatment of rat mast cells, RBL-2H3, with a phenoxazine derivative Phx-1 (2-amino-4,4alpha-dihydro-4alpha,7-dimethyl-3H-phenoxazine-3-one) suppresses the antigen-induced degranulation. Biochemical analysis reveals that the complementary signaling pathway through Gab2 and Akt is inhibited by this compound in mast cells. These findings suggest that phenoxazine derivatives may have a therapeutic potential for allergic diseases by inhibiting mast cell degranulation.     

Application of human Fc epsilon RI alpha-chain-transfected RBL-2H3 cells for estimation of active serum IgE

We transfected the alpha-chain of human FcepsilonRI into rat basophilic leukemia cell line RBL-2H3, established several stable transfected cells, and screened them by beta-hexosaminidase release induced by sensitization with human IgE and stimulation with anti-human IgE antibody. A cloned cell line RBL-hEIa-2B12 was the strongest responder among the transfected cell clones. The concentrations of cytosolic free Ca2+ concentration in the human IgE-sensitized cells increased after stimulation with anti-human IgE antibody. Thus, it is suggested that the alpha-chain of human FcepsilonRI is associated with the beta-chain and/or gamma-chain of rat FcepsilonRI, and that they form functional high affinity IgE receptor complexes. The total IgE concentrations of the sera from allergic patients were determined by using the beta-hexosaminidase release assay, where the transfected cells were sensitized with diluted and heat-inactivated (at 56 degrees C for 30 min) serum and stimulated with anti-human IgE antibody. The IgE concentration obtained correlated with those measured by an enzyme immunoassay method. beta-Hexosaminidase release induced by stimulation with 5 times diluted serum was sometimes less than the release induced by the same serum; diluted 25 times or 125 times, suggesting that these serum contained factors that blocked IgE binding to FcepsilonRI or cross-linking by anti-human IgE antibody. The results suggested that our system will be useful for detecting FcepsilonRIalpha-bindable IgE in human serum.     

Evaluation of an in vitro method for the measurement of specific IgE antibody responses: the rat basophilic leukemia (RBL) cell assay

The evaluation of allergenic potential is a key parameter in the safety assessment of novel proteins, including those expressed in genetically modified crops and foodstuffs. The majority of allergic reactions to food proteins are immediate type hypersensitivity reactions in which the principal biological effector is IgE antibody; the accurate measurement of specific IgE antibody is therefore a critical factor in experimental systems designed to characterize protein allergenic potential. Due to the presence of much higher concentrations of other immunoglobulin isotypes, the assessment of specific serum IgE antibody poses substantial technical challenges. We have examined the utility of the rat basophilic leukemia (RBL) cell line for the measurement of murine IgE responses. RBL cells were sensitized with mouse monoclonal anti-dinitrophenyl (DNP) IgE antibody and challenged with DNP-albumin conjugates with various hapten substitution ratios (SR). Polyclonal anti-OVA IgE antisera were also assessed for activity in the RBL assay. Results were compared with titers measured in homologous passive cutaneous anaphylaxis (PCA) assay. Marked degranulation of RBL cells was induced by conjugates with SRs of between 16 and 32, whereas conjugates with lower SRs (of 10 or 3) failed to elicit significant serotonin release. All conjugates were able to induce mast cell degranulation in vivo in a PCA assay. Anti-OVA antisera with PCA titers of 1/32 to 1/64 failed to stimulate RBL cell degranulation, whereas high titer antibody (1/2048 to 1/4096 by PCA) induced a positive RBL cell response. Successful stimulation of RBL cell degranulation requires not only appropriate epitope densities but also high affinity antibody. These data indicate that this assay is inappropriate for the routine analysis of specific polyclonal IgE antibody responses such as those that are induced by exposure to complex protein allergens.     

Inhibitory effects of parthenolide on antigen-induced microtubule formation and degranulation in mast cells

Pharmacological modulation of IgE-mediated mast cell activation is important to the development of anti-allergic reagents. In this study, we investigated the effects of parthenolide (PTL) on high-affinity IgE receptor (FcepsilonRI)-induced degranulation in mast cells. PTL dose-dependently inhibited degranulation induced by IgE.antigen stimulation in RBL-2H3 cells and BMMCs. Although PTL is a potent NF-kappaB inhibitor by targeting IkappaB kinase complex, NF-kappaB inhibition by other IkappaB kinase inhibitors did not inhibit degranulation in mast cells. IgE.antigen-induced microtubule formation is well known to be critical for degranulation in mast cells. Immunocytochemical study with anti-alpha-tubulin antibody revealed that PTL significantly inhibited IgE.antigen-induced microtubule formation. However, PTL, as well as nocodazol, had no significant effects on degranulation in the fyn-deficient BMMCs, suggesting that inhibitory effects of PTL in the microtubule formation are fyn dependent. We further demonstrated that in vivo administration of PTL in mice strongly inhibited passive cutaneous anaphylaxis reaction. The present study provides a possibility to develop potent reagents against mast cell activation based on an inhibition of microtubule formation.     

Upregulation of heme oxygenase-1 by degranulation in rat basophilic leukemia cells

Heme oxygenase (HO)-1, which is a rate-limiting enzyme involved in the catabolism of heme, is upregulated by a variety of stresses including oxidative stresses and inflammatory cytokines, in many cell types. Recent studies have suggested that upregulation of HO-1 might provide cytoprotection and immunomodulatory functions in addition to its obvious role in heme metabolism. In this study, we examined whether HO-1 was upregulated following degranulation in mast cells that initiate vigorous immunity reactions. To trigger degranulation, rat basophilic leukemia (RBL)-2H3 cells were passively sensitized using an antiserum collected from ovalbumin (OA) immunized-Brown Norway rats, and the cells were stimulated by treatment with OA. Degranulation was confirmed by measuring the release of beta-hexosaminidase. HO-1 mRNA and presence of HO-1 protein were detected using Northern blot and Western blot analyses, respectively. The effect of the antioxidant N-acetyl-L-cysteine (NAC) on HO-1 expression was also tested. HO-1 mRNA transiently increased at 1--2 h after RBL-2H3 cells were stimulated to degranulate. Its mRNA increases were dependent on the extent of degranulation. Following the upregulation of HO-1 mRNA, HO-1 protein was also increased. We also detected intracellular production of reactive oxygen species following degranulation in RBL-2H3 cells. NAC attenuated the HO-1 expression in a dose-dependent manner. This is the first report to reveal induction of both HO-1 mRNA and protein by degranulation in RBL-2H3 cells. We showed that NAC inhibited HO-1 upregulation. These results suggest that oxidative stress in activated RBL-2H3 cells results in the upregulation of HO-1.     

Gab2 antisense oligonucleotide blocks rat basophilic leukemic cell functions

Adapter molecule Grb2-associated binder-like protein 2 (Gab2) plays a critical role in FcepsilonRI-induced mast cell degranulation and activation. The present study aimed to investigate the pharmacological effects of an antisense oligonucleotide (ASO) targeted at Gab2 on the immune responses of rat basophilic leukemic (RBL)-2H3 cells. Gab2 ASOs were rationally designed and transfected into RBL-2H3 cells. Gab2 mRNA and protein knockdown was confirmed by real-time RT-PCR and immunoblotting, respectively. Effects of Gab2 ASO on FcepsilonRI-induced release of histamine and beta-hexosaminidase was measured by EIA and an enzymatic assay, respectively; signaling events by immunoblotting; and cytokine mRNA expression by RT-PCR. Effects of Gab2 ASO on cell adhesion and migration were performed on fibronectin-coated 96-well plate and transwells cell culture chambers, respectively. We have characterized a phosphorothioate-modified ASO targeted at Gab2 mRNA that was able to knockdown Gab2 mRNA and protein in RBL-2H3 cells. Gab2 ASO significantly blocked IgE-mediated mast cell release of beta-hexosaminidase and histamine; phosphorylation of Akt, p38 mitogen-activated protein kinase and PKCdelta; and up-regulation of cytokine mRNA levels (e.g. IL-4, -6, -9 and -13, and TNF-alpha). In addition, Gab2 ASO markedly prevented mast cell adhesion to fibronectin-coated plates and restrained random migration of RBL-2H3 cells in cell culture chambers. Our findings show that Gab2 knockdown in RBL-2H3 cells by ASO strategy can suppress many aspects of the mast cell functions and, therefore, a selective Gab2 ASO may have therapeutic potential for mast cell-dependent allergic disorders.     

Investigation into the effects of amyloid (1-42) beta-peptide upon basal and antigen-stimulated hexosaminidase and serotonin release from rat RBL-2H3 basophilic leukemia cells

There is evidence that beta-amyloid (A beta) peptide infusion in vivo produces a degranulation of vascular mast cells. It would be useful to investigate the interaction between A beta and mast cells in a simple in vitro model system in order to determine the cellular mechanism by which exposure to A beta peptides results in mast cell degranulation. In the present study, the effect of A beta(1-42) upon the release of granular hexosaminidase and serotonin has been investigated using the cognate rat mast cell line, RBL-2H3. Sensitization of these cells for 1 h with anti-DNP IgE (monoclonal anti-dinitrophenyl) results in a large release of hexosaminidase and serotonin due to degranulation when the cells are exposed to DNP-HSA (albumin, human dinitrophenyl). Pretreatment overnight with A beta(1-42) (10 and 30 microM) did not affect either the basal or antigen-stimulated release of hexosaminidase or serotonin. A similar lack of effect of A beta(25-35) and the lipid peroxidation product HNE upon antigen-stimulated release of hexosaminidase or serotonin was also found. It is concluded that RBL-2H3 cells are not a useful model for mechanistic studies into the effects of beta-amyloid peptides upon vascular mast cells.     

Inhibition of the antigen-induced activation of RBL-2H3 cells by sinomenine

The present study was designed to investigate the effect of sinomenine (SIN), an alkaloid extracted from sinomenium acutum, on the antigen-induced activation of RBL-2H3. For this investigation, the RBL-2H3 cells were sensitized with dinitrophenyl (DNP)-specific IgE overnight in 1.0 ml of Eagle's MEM (EMEM), and varying doses of SIN were added to the culture medium for 30 min and challenged with dinitrophenyl-human serum albumin (DNP-HSA) to induce mast cell degranulation before supernatants were collected. The effects of SIN on antigen-induced release of beta-hexosaminidase were measured by enzymatic assay, calcium influx by FACS, cytokines by ELISA, and signaling events by immunoblotting. The results showed that treatment with SIN was followed by a decrease in FcepsilonRI-mediated mast cell release of beta-hexosaminidase, production of IL-4 and TNF-alpha, phosphorylation of Gab2 (Scaffolding adapter Grb2-associated binder 2), Akt and p38 mitogen-activated protein kinase (MAPK). In addition, SIN had no effect on the phosphorylation of LAT and no significant difference on calcium mobilization was observed between control and SIN treated group. These results suggested that SIN might suppress the antigen-induced activation of RBL-2H3 cells via a Ca2+ independent pathway.     

3-O-(2,3-dimethylbutanoyl)-13-O-decanoylingenol from Euphorbia kansui suppresses IgE-mediated mast cell activation

Aggregation of the high affinity receptor for IgE (FcepsilonRI) on mast cells by antigen and IgE complex induces release of chemical mediators, leading to acute allergic inflammation. We recently found that 3-O-(2,3-dimethylbutanoyl)-13-O-decanoylingenol (DBDI), purified from the Euphorbia kansui L., inhibits degranulation in rat basophilic leukemia 2H3 cells upon aggregation of the FcepsilonRI. In the present study, we demonstrated that the DBDI significantly inhibits release of beta-hexosaminidase, synthesis of eicosanoids, and mobilization of intracellular Ca(2+) in the bone marrow-derived mouse mast cells stimulated with IgE and antigen. Furthermore, we revealed that phosphorylation of Syk, phospholipase C-gamma(2), and extracellular signal-related kinase 1/2 is significantly suppressed in the DBDI-treated mast cells. These findings suggest that the DBDI may have a therapeutic potential for allergic diseases by inhibiting intracellular signaling pathways for activation and chemical mediator release in mast cells.     

FcepsilonRI cross-linking-induced actin assembly mediates calcium signalling in RBL-2H3 mast cells

1. To determine the role of actin assembly in the Ca(2+) signalling of mast cells activated by cross-linking of FcepsilonRI, we examined the effects of cytochalasin D, an inhibitor of actin polymerization. 2. In the RBL-2H3 cells, F-actin content was increased by sensitization with anti-dinitrophenol (DNP) IgE. In these cells, cytochalasin D induced oscillatory increases in cytosolic Ca(2+) ([Ca(2+)](i)); these increase were inhibited by jasplakinolide, a stabilizer of actin filaments. 3. In the IgE-sensitized RBL-2H3 cells, DNP-human serum albumin (DNP-HSA) augmented actin assembly. DNP-HSA also increased the production of IP(3), [Ca(2+)](i) and degranulation. Cytochalasin D enhanced all of these DNP-HSA-induced effects. 4. In a Ca(2+)-free solution, DNP-HSA induced a transient increase in [Ca(2+)](i), and this increase was accelerated by cytochalasin D. After cessation of the DNP-HSA-induced Ca(2+) release, the re-addition of Ca(2+) induced a sustained increase in [Ca(2+)](i) through capacitative Ca(2+) entry (CCE), and this increase was enhanced by cytochalasin D. 5 The effect of cytochalasin D in enhancing the CCE activity was prevented by xestospongin C. 6. In contrast, neither the Ca(2+) release nor the CCE activation that was induced by thapsigargin was affected by cytochalasin D. 7. These results suggest that actin de-polymerization stimulates the FcepsilonRI-mediated signalling to augment the release of Ca(2+) from the endoplasmic reticulum in RBL-2H3 cells.     

Peroxisome proliferator-activated receptor alpha-independent effects of peroxisome proliferators on cysteinyl leukotriene production in mast cells

The effects of peroxisome proliferators, the ligands of a nuclear receptor peroxisome proliferator-activated receptor (PPAR) alpha, on cysteinyl leukotriene production were investigated in rodent mast cells. Peroxisome proliferators Wy-14,643 (30 microM) and fenofibrate (100 microM) significantly inhibited the cysteinyl leukotriene production that was induced by antigen (Ag) treatment after overnight sensitization to Ag specific immunoglobulin E (IgE) in a rat basophilic leukemia (RBL)-2H3 mast cell line. Similar inhibition by these drugs was observed in IgE and Ag-treated mouse bone marrow-derived mast cells, A23187-treated RBL-2H3 and A23187-treated mouse peritoneal macrophages. Wy-14,643 (30 microM) and fenofibrate (100 microM) did not affect the release of radioactivity from RBL-2H3 pre-incubated with [(3)H]-arachidonic acid, which is considered an index of phospholipase A(2) activity. Wy-14,643 (30 microM) and fenofibrate (100 microM) did not directly inhibit 5-lipoxygenase activity. Troglitazone was found to directly inhibit the activity of 5-lipoxygenase. The PPARalpha mRNA level was at less than the limit of detection for the realtime polymerase chain reaction both in RBL-2H3 and bone marrow-derived mast cells. Wy-14,643 (30 microM) and fenofibrate (100 microM) did not induce acyl-CoA oxidase mRNA in RBL-2H3, which was reported to be induced by peroxisome proliferators via PPARalpha in hepatocytes. Wy-14,643 (30 microM) and fenofibrate (100 microM) inhibited the cysteinyl leukotriene production in bone marrow-derived mast cells from PPARalpha-null mice. It was concluded that the inhibitory effects of these peroxisome proliferators on cysteinyl leukotriene production are independent of PPARalpha in mast cells.     

中药注射剂所含吐温-80与过敏反应关系的研究

目的：观察不同浓度吐温-80溶液、吐温-80含量不同的中药注射剂对RBL-2H3细胞脱颗粒的影响,探讨中药注射剂所含吐温-80与过敏反应的关系。方法：体外培养RBL-2H3细胞,加入不同浓度（40、20、10、2、1、0.2、0.1、0.05mg/mL）的吐温-80溶液,之后加中性红染液,计数不同浓度吐温-80溶液各组及对照组的脱颗粒细胞,并计算其百分率,同时检测细胞上清液中β-氨基己糖苷酶及组胺的释放量;测定穿琥宁注射液和香丹注射液中吐温-80的含量,2种中药注射剂对RBL-2H3细胞的半数抑制浓度（IC50）以及加入2种注射液各组细胞释放组胺的量。结果：中性红染色实验显示吐温-80可导致RBL-2H3细胞脱颗粒,表现为肥大细胞体积变大,内有空泡产生;浓度为40、20、10、2、1、0.2、0.1mg/mL的吐温-80溶液各组和RPMI1640对照组导致细胞的脱颗粒百分率分别为（57.38±0.47）、（32.54±2.33）、（21.74±0.72）、（16.96±0.26）、（11.40±1.70）、（9.71±0.26）、（7.22±0.15）和（1.51±1.39）%,2组相比差异有统计学意义（P〈0.05,P〈0.01）;浓度为40、20、2、1、0.2mg/mL的吐温-80溶液各组和RPMI1640对照组致细胞β-氨基己糖苷酶的释放率分别为（52.44±1.53）、（18.91±0.77）、（7.50±1.82）、（6.65±0.20）、（6.15±0.27）和（0.35±0.06）%,2组相比差异有统计学意义（P〈0.05,P〈0.01）;不同浓度吐温-80溶液引起RBL-2H3细胞释放组胺的量也不同;当吐温-80溶液浓度为20～0.1mg/mL时,RBL-2H3细胞脱颗粒百分率、β-氨基己糖苷酶的释放率及其释放组胺的量均与吐温-80溶液的浓度呈线性关系（r=0.9862,r=0.9849,r=0.9740）。穿琥宁注射液和香丹注射液中吐温-80的含量分别为（0.086±0.004）和（0.070±0.008）mg/mL,2种注射液对RBL-2H3细胞的IC50分别为（57.4±1.2）、（1.0±0.2）μL/mL,穿琥宁注射液和香丹注射液组组胺释放量分别为（2.39±0.01）和（1.87±0.00）ng/mL。结论：吐温-80可引起RBL-2H3细胞脱颗粒释放炎症介质;RBL-2H3细胞组胺的释放量与中药注射剂中吐温-80的含量有关;中药注射剂中所含的吐温-80可能与过敏反应的发生有关。     

Inhibitory mechanism of anti-allergic peptides in RBL2H3 cells

Here we report the identification and functional characterization of several anti-allergic peptides identified through biopanning of pooled sera of patients with various allergies. Several peptides, including LSYLLWRSRLP (LSY), LVAHVGAGGVL (LVA), RVSSCRGRNHIV (RVS), ETIGARWVRIE (ETI), TDGVTYTNDCL (TDG), RVVRYDADFWI (RVV), GFWCRRSGLVGV (GFW), were further characterized. These peptides inhibited the release of histamine from antigen-stimulated mast cells isolated from lung tissues of guinea pigs. Furthermore, the peptides inhibited calcium influx in ovalbumin-stimulated mast cells isolated from lung tissues of guinea pigs. Likewise, the peptides inhibited the release of beta-hexosaminidase from antigen-stimulated rat basophilic leukemia (RBL2H3) cells and decreased calcium influx and intracellular reactive oxygen species production as well. We found that the peptides significantly decreased phosphorylation of extracellular regulated kinase (ERK) and that this was responsible for the decreased calcium influx and beta-hexosaminidase in antigen-stimulated RBL2H3 cells, suggesting that ERK plays an important role in allergic reactions. The peptides identified in this study also affected upstream signaling of allergic inflammation. In other words, these peptides decreased phosphorylation of Lyn, PKCalpha, and -delta. Lyn and PKC are known to be responsible for the phosphorylation of FcepsilonRI, in response to receptor aggregation. The peptides inhibited interaction between IgE and FcepsilonRI, suggesting that these peptides exert anti-allergic effects by inhibiting receptor cross-linking. These peptides also inhibited interaction between FcepsilonRI and PKCdelta. Taken together, these data suggest that peptides exert anti-allergic effect through the inhibition of upstream signaling, involving receptor cross-linking, and downstream multiple signaling.     

Enhancement of FcɛRI-mediated degranulation response in the rat basophilic leukemia cell line RBL2H3 by the fluorosurfactants perfluorooctanoic acid and perfluorooctane sulfonate

The effect of two fluorosurfactants, perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), on degranulation of rat basophilic leukemia RBL2H3 cells was investigated. PFOA and PFOS promoted IgE-mediated release of granule components of RBL2H3 cells at 10-300μM. At low concentrations (<30μM), the fluorosurfactants failed to induce degranulation, but promoted IgE-mediated degranulation without affecting cell viability. The absence of extracellular Ca(2+) removed the promoting effect of the fluorosurfactants on IgE-mediated degranulation. On the other hand, the fluorosurfactants at high concentrations (>100μM) induced release of granule components without IgE-mediated activation in parallel with cell death. Pretreatment of tetradecanoyl-phorbol-acetate, a protein kinase C activator, inhibited both the promoting effect of the fluorosurfactants at low concentration on IgE-mediated degranulation and cell death-associated granule component release by high concentration of the fluorosurfactants. These findings indicate that PFOA and PFOS affect granule component release of mast cells by two different mechanisms, namely enhancement of active degranulation machinery at low concentrations and cell lysis at high concentrations.     

中药制剂苓槐丸提取物对小鼠过敏性皮炎的治疗作用及机制研究

目的研究中药制剂苓槐丸对过敏性皮炎的治疗作用及机制。方法制备苓槐丸甲醇提取物（MELH）,观察其对过敏性皮炎模型鼠耳肿胀程度及炎症因子释放的影响;而后,检测MELH 对肥大细胞RBL鄄2H3 脱粒作用及细胞信号通路的影响。结果MELH 可有效抑制过敏性皮炎模型鼠耳炎症性肿大（P 〈0. 05）,及抑制鼠耳组织中IFN-γ和TNF-α水平升高（P 〈0. 05）。MELH 可剂量依赖性抑制佛波酯（PMA）和钙离子载体（A23187）联合引起的RBL鄄2H3 细胞的组胺释放（P〈0. 05）和p38 MAPK 磷酸化。结论MELH 通过抑制T 细胞炎症因子的释放、肥大细胞脱粒作用及p38 MAPK 通路的活化,阻止T细胞向Th1免疫偏离,总体说苓槐丸可起到明显的抗过敏性皮炎作用。     

Anti-allergic activity of sesquiterpenes from the rhizomes of <Emphasis Type="Italic">Cyperus rotundus</Emphasis>

From the 70% ethanol extract of the rhizomes of Cyperus rotundus (CRE), several major constituents including the sesquiterpene derivatives (valencene, nootkatone, and caryophyllene α-oxide), monoterpenes (β-pinene, 1,8-cineole, and limonene) and 4-cymene were isolated and examined for their anti-allergic activity in vitro and in vivo. In rat basophilic leukemia (RBL)-1 cells, the sesquiterpenes strongly inhibited 5-lipoxygenase-catalyzed leukotrienes production. In addition, they inhibited β-hexosaminidase release by antigen-stimulated RBL-2H3 cells, with valencene having the highest inhibitory effect. CRE inhibited leukotrienes production and β-hexosaminidase release at 300 μg/mL. It was also found that the most active sesquiterpene (valencene) and CRE inhibited β-hexosaminidase degranulation by inhibiting the initial activation reaction, Lyn phosphorylation, in IgE-stimulated RBL-2H3 cells. Moreover, CRE, valencene and nootkatone significantly inhibited the delayed-type hypersensitivity reaction in mice when administered orally at 50–300 mg/kg. In conclusion, C. rotundus and its constituents, valencene, nootkatone, and caryophyllene α-oxide, exert anti-allergic activity in vitro and in vivo. These sesquiterpenes, but not monoterpenes, certainly contribute to the anti-allergic activity of the rhizomes of C. rotundus.     

Selinidin suppresses IgE-mediated mast cell activation by inhibiting multiple steps of Fc epsilonRI signaling

IgE-mediated mast cell activation is critical for development of allergic inflammation. We have recently found that selinidin, one of the coumarin derivatives isolated from Angelica keiskei, attenuates mast cell degranulation following engagement of the high-affinity receptor for IgE (Fc epsilonRI) with IgE and antigen. In the present study, we investigated the effects of selinidin on intracellular signaling and mast cell activation employing bone marrow-derived mast cells. Here, we report that selinidin attenuates the release of beta-hexosaminidase, synthesis of leukotriene C4, and production of tumor necrosis factor-alpha without affecting IgE-Fc epsilonRI binding. Furthermore, biochemical analyses of the Fc epsilonRI-mediated signaling pathway demonstrated that selinidin decreases phosphorylation of phospholipase C-gamma1, p38 mitogen-activated protein kinase, and IkappaB-alpha upon FcepsilonRI stimulation. These results suggest that this compound suppresses IgE-mediated mast cell activation by inhibiting multiple steps of FcepsilonRI-dependent signaling pathways and would be beneficial for the prevention of allergic inflammation.     

Enhancement of allergic responses in vivo and in vitro by butylated hydroxytoluene

The effect of butylated hydroxytoluene (BHT), which is used widely as an antioxidant, on IgE-dependent allergic responses in vivo and in vitro was investigated. For in vivo study, passive cutaneous anaphylaxis (PCA) was elicited in rats by i.d. injection of anti-DNP IgE and 48 h later by i.v. injection of DNP-HSA. BHT was i.p. given immediately after anti-DNP IgE injection. For in vitro studies, the rat mast cell line RBL2H3 sensitized with monoclonal anti-dinitrophenol (DNP) IgE was challenged with the multivalent antigen DNP-human serum albumin (DNP-HSA) in the presence or absence of BHT. beta-Hexosaminidase and histamine released from RBL2H3 cells, as indicators of degranulation of the cells, the concentration of intracellular Ca2+, the level of phosphorylated-Akt, and global tyrosine phosphorylation as indicators of mast cell activation, were measured. The results showed that BHT given to anti-DNP IgE-sensitized rats augmented DNP-specific PCA in a dose-dependent manner. In the presence of BHT, IgE-induced releases of beta-hexosaminidase and histamine from RBL2H3 cells were increased. BHT also further elevated IgE-mediated increased concentrations of intracellular Ca2+ and the levels of phosphorylated-Akt, but did not affect global tyrosine phosphorylation, in RBL2H3 cells. Moreover, the PI3K inhibitor LY294002 inhibited IgE-dependent degranulation and its enhancement by BHT. These findings indicate that BHT may upregulate PCA by enhancing mast cell degranulation associated with enhancements of intracellular Ca2+ concentration and PI3K activation, suggesting that BHT might affect allergic diseases such as allergic rhinitis and asthma.     

Inhibition of IgE-mediated phosphorylation of FcepsilonRIgamma protein by antiallergic drugs in rat basophilic leukemia (RBL-2H3) cells: a novel action of antiallergic drugs

We examined the effect of antiallergic drugs, azelastine and epinastine, on the expression of FcepsilonRIalpha, beta, and gamma chains and phosphorylation of the gamma chains in rat basophilic leukemia (RBL-2H3) cells. The cells were cultured for 24 h with IgE treatment in the presence of azelastine or epinastine at the concentration of 10(-5) M. The FcepsilonRIalpha mRNA expression was determined by northern blot analysis. The protein level of FcepsilonRI expressed on the plasma membrane was examined following IgE treatment by immunoprecipitation with anti-IgE light chain, followed by western blot analysis with anti-gamma chain of FcR. Azelastine and epinastine had no effect on the FcepsilonRIalpha, beta and gamma mRNA levels. Although the amount of gamma chain assembled into IgE-bound FcepsilonRI was not changed by treatment with azelastine nor epinastine, phosphorylation levels of gamma chains of IgE-bound FcepsilonRI were inhibited by azelastine. The inhibitory effect of azelastine on the IgE-mediated expression of FcepsilonRIgamma protein is not due to their inhibition of mRNA and protein expression, but due to abrogating phosphorylation of the gamma chains, which is important for initiation of FcepsilonRI signaling cascade elicited by IgE interaction.