RBL-2H3细胞不适合用于类过敏模型的建立

目的考察RBL-2H3细胞是否适用于建立类过敏反应模型。方法用荧光定量聚合酶链反应考察RBL-2H3细胞上Mas相关G蛋白偶联受体(Mas-related G protein cou-pled receptor,Mrgpr)B2的表达情况;用显微镜观察和MTS法考察Compound 48/80对RBL-2H3细胞活力的影响;测定不同浓度Compound 48/80刺激RBL-2H3细胞脱颗粒释放的β-己糖胺酶含量,对比RBL-2H3细胞、人肥大细胞系LAD2和大鼠腹腔肥大细胞(rat peritoneal mast cells,RPMCs)对Compound 48/80响应性的差异。结果RBL-2H3细胞可表达MrgprB2受体。Compound 48/80能剂量依赖性地诱导RBL-2H3细胞释放β-己糖胺酶,但在高剂量(≥20 mg·L^-1)时对RBL-2H3的细胞活力产生明显影响,此时释放的β-己糖胺酶应当是由于其细胞毒作用引起细胞破裂所致。同在无毒剂量(10 mg·L^-1)的Compound 48/80刺激下,LAD2和RPMCs的响应性良好,β-己糖胺酶释放量分别为空白对照的15.02倍和16.05倍,而RBL-2H3细胞仅为空白对照的2.35倍。结论RBL-2H3细胞对Compound 48/80的响应性差,表明其并不适用于建立类过敏反应模型。     

牛膝多糖对抗原诱导的肥大细胞活化的影响

目的观察牛膝多糖(achyranthes bidentata polysaccha-rides,ABPS)对肥大细胞活化脱颗粒的影响。方法运用大鼠被动皮肤过敏反应(PCA)实验,用比色测定法检测ABPS在体内对肥大细胞(MC)影响;体外实验将ABPS分为高、中、低3个剂量组,然后分别将其加入抗原致敏的RBL-2H3细胞中(大鼠嗜碱性粒细胞白血病细胞,国际公认的MC研究模型细胞),观察ABPS对RBL-2H3细胞脱颗粒的影响。结果ABPS能明显抑制大鼠PCA、RBL-2H3细胞脱颗粒,并能抑制RBL-2H3细胞释放组胺、肿瘤坏死因子α及白细胞介素4;抑制RBL-2H3细胞中Akt和p38的磷酸化。结论ABPS的抗过敏作用与抑制肥大细胞的脱颗粒及炎性物质释放有关;ABPS抑制肥大细胞的活化与抑制Akt和p38的活性相关。     

参麦注射液致RBL-2H3细胞脱颗粒的研究

目的 探讨参麦注射液(SMI)对RBL-2H3细胞脱颗粒的影响及其原因。方法 以C48/80为工具药建立RBL-2H3细胞脱颗粒模型, 检测不同浓度C48/80与RBL-2H3细胞作用不同时间后细胞β-己糖苷酶、类胰蛋白酶和组胺的释放率以及细胞活力, 在细胞活力大于80%情况下, 选择释放程度较高的指标和条件为优选考察指标和条件。将SMI和其溶剂(Tween-80)原液等比稀释成不同浓度后与RBL-2H3细胞共同培养, 通过中性红染色法观察细胞脱颗粒的形态学变化, 分别用显色法和间接荧光法检测细胞上清的β-己糖苷酶和组胺释放率, 采用细胞计数法(CCK-8)检测细胞的活力。结果 RBL-2H3细胞脱颗粒模型的最佳作用时间为30 min, 最佳指标为β-己糖苷酶和组胺释放率。与空白组相比, SMI质量浓度低于13.3 g生药/L(3倍临床浓度)时, 细胞中性红染色未见脱颗粒现象, 组胺和β-己糖苷酶释放率亦无差异;而在Tween-80质量浓度为1.00 g/L时, SMI 40 g生药/L(9倍临床浓度)组和溶剂1.00 g/L组细胞中性红染色均可见脱颗粒现象, 细胞上清组胺和β-己糖苷酶释放率亦明显增加。此外, CCK-8结果显示, 与空白组相比, 各浓度的SMI对细胞活力均无影响。结论 SMI低于3倍临床浓度无明显RBL-2H3细胞脱颗粒作用;而在9倍临床浓度能刺激细胞脱颗粒, 这种脱颗粒作用可能与所含溶剂(Tween-80)有关, 与其对RBL-2H3的细胞毒性作用无关。提示SMI在低于3倍临床浓度相对安全, 在9倍临床浓度时有致类过敏反应的风险。     

聚山梨酯-80致RBL-2H3肥大细胞脱颗粒的研究

目的:研究聚山梨酯-80对RBL-2H3肥大细胞脱颗粒释放组胺的影响.方法:培养大鼠来源的RBL-2H3肥大细胞,取不同厂家来源的聚山梨酯-80与RBL-2H3细胞共培养60 min,用荧光分光光度法定量检测RBL-2H3细胞释放的组胺量,计算组胺释放率.结果:不同厂家来源的聚山梨酯-80与RBL-2H3细胞作用60 min后,细胞的组胺释放率与空白对照组相比均显著增加,在一定浓度范围内,组胺的释放随聚山梨酯-80浓度的增加而增加.结论:聚山梨酯-80可导致RBL-2H3肥大细胞脱颗粒,并存在着明显的量效关系,为研究聚山梨酯80致过敏反应机制提供了一定的依据.     

绿原酸致RBL-2H3细胞脱颗粒反应及机制研究

目的 研究绿原酸对大鼠嗜碱性白血病细胞 RBL-2H3 脱颗粒作用及可能机制。方法 将不同质量浓度(3.125、6.250、12.500、25.000、50.000、100.000、200.000 μg·mL-1)的绿原酸作用于 RBL-2H3 细胞,通过实时细胞分析(RTCA)系统检测绿原酸干预后引起的细胞指数(CI)值变化,以评价 RBL-2H3 细胞脱颗粒情况;利用甲苯胺蓝染色观察细胞形态变化;化学荧光法测定组胺释放率;底物显色法测定β-氨基己糖苷酶释放率、类胰蛋白酶释放量;网络药理学预测绿原酸诱导RBL-2H3 细胞脱颗粒的潜在机制,并应用实时荧光定量PCR(qRT-PCR)对预测通路的主要靶点细胞外调节蛋白激酶(ERK1)、c-Jun氨基末端激酶(JNK)、p38丝裂原活化蛋白激酶(MAPK)mRNA水平进行检测。结果 绿原酸质量浓度 ≥ 6.25 μg·mL^-1时,可使RBL-2H3细胞的CI值在给药20min后呈现先快速上升后下降的趋势;绿原酸质量浓度 ≥ 12.5 μg·mL^-1时,RBL-2H3 细胞逐渐变圆甚至呈现多边形;与对照组比较,β-氨基己糖苷酶、组胺和类胰蛋白酶的释放量显著增加(P<0.05、0.01、0.001)。网络药理学预测发现,绿原酸致细胞脱颗粒作用可能与MAPK、磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)等信号通路相关;qRT-PCR 结果证实,与对照组比较,绿原酸可显著增加 ERK1、p38MAPK mRNA表达(P<0.05、0.01),但对JNK mRNA表达影响不显著。结论 绿原酸可通过激活ERK1、p38 MAPK通路诱导 RBL-2H3 细胞发生脱颗粒,具有潜在的诱发类过敏反应作用。     

基于实时细胞分析技术评价注射用益气复脉(冻干)类过敏反应

目的 建立中药注射剂体外类过敏反应评价方法,快速评价不同批次注射用益气复脉（冻干）类过敏反应表现。方法 体外培养嗜碱性白血病细胞株RBL-2H3细胞,选择Compound 48/80为阳性药,采用实时细胞分析（real-time cell analysis,RTCA）系统检测药物干预后引起的细胞指数（CI）值变化,并利用甲苯胺蓝、鬼笔环肽染色观察细胞形态、骨架变化,以及检测组胺和β-已糖苷酶释放量验证RBL-2H3细胞的脱颗粒情况。选择20个批次的注射用益气复脉（冻干,100 μg/mL）作用于RBL-2H3细胞,进行基于RTCA技术的类过敏反应评价。结果 阳性药Compound 48/80（20 μg/mL）能够使RBL-2H3细胞的CI值在加药后30 min内呈先快速上升后下降趋势;形态学研究发现,Compound 48/80使细胞形态和细胞骨架均发生明显改变,发生明显的脱颗粒现象;组胺和β-己糖苷酶释放实验进一步证实Compound 48/80导致炎症介质的释放,引起了明显的脱颗粒现象;提示RTCA系统可以用于快速敏感的评价RBL-2H3细胞脱颗粒。不同批次的注射用益气复脉（冻干）对RBL-2H3细胞CI值无明显影响,提示所选批次为合格批次,无类过敏反应现象的发生。结论 建立了一套基于RTCA系统的类过敏反应体外快速评价技术,可用于注射用益气复脉（冻干）等中药注射剂类过敏反应的体外快速评价。     

非免疫性过敏反应细胞模型的建立

目的:通过非免疫性刺激物C48/80诱导大鼠嗜碱性粒细胞白血病细胞株RBL-2H3细胞脱颗粒反应正交试验,优化非免疫性过敏反应细胞模型建立条件.方法:在不同的孵育时间下,C48/80与不同浓度的RBL-2H3细胞共孵育,通过测定β-氨基己糖苷酶的释放率确定建立非免疫型过敏反应细胞模型的最优实验条件,并且分析不同检测方法间的差异.结果:不同浓度、不同孵育时间下的RBL-2H3细胞均可受C48/80诱导发生典型的脱颗粒反应,但不同条件下的脱颗粒程度和β-氨基己糖苷酶释放率都有显著差异.结论:当RBL-2H3细胞浓度为2×105 mL、孵育时间60 min时,细胞的感受性较好,其脱颗粒程度与药物浓度呈正相关.     

聚山梨酯80对多源肥大细胞脱颗粒的影响研究

目的 通过比较不同浓度聚山梨酯80溶液对RBL-2H3、P815、Ku812细胞脱颗粒的影响,探究聚山梨酯80的量与过敏反应的关系,进而筛选出一套灵敏、稳定的体外肥大细胞脱颗粒模型。方法 体外培养RBL-2H3、P815和Ku812细胞,测定3株细胞的生长曲线,在细胞生长对数期,以不同浓度（0.04、0.20、1.00、5.00、10.00、20.00、40.00、80.00 mg/mL）聚山梨酯80分别刺激3株细胞,采用中性红染色法显微镜下观察不同浓度聚山梨酯80溶液对3株细胞脱颗粒的影响,并计算脱颗粒百分率,同时以化学发光法分别检测组胺和β-氨基己糖苷酶释放率,以酶联免疫吸附法测定类胰蛋白酶的释放量;并进一步检测聚山梨酯80对人的肥大细胞IgE释放的影响。结果 3株肥大细胞脱颗粒模型中,各细胞的脱颗粒指标均随聚山梨酯80浓度的升高而升高。相同浓度聚山梨酯80对人源、大鼠、小鼠肥大细胞的类胰蛋白酶释放量无较大统计学差异;与RBL-2H3细胞系比较,P815细胞和组胺释放率显著降低（P<0.05、0.01）,Ku812细胞组胺释放率和β-氨基己糖苷酶释放率显著升高（P<0.05、0.01）,Ku812细胞脱颗粒最灵敏。但在实验过程中发现Ku812细胞模型稳定性、可重复性较差,而RBL-2H3细胞稳定性、可重复性均优于Ku812和P815细胞。0.04~80.00 mg/mL的聚山梨酯80溶液作用于Ku812细胞产生的IgE均低于检测限。结论 聚山梨酯80诱导的过敏反应可能是不经过IgE介导的类过敏反应,随着聚山梨酯80浓度的增大,3个细胞模型脱颗粒现象显著,相比于Ku812和P815细胞,RBL-2H3细胞更适合作为体外肥大细胞脱颗粒检测模型。     

肥大细胞脱颗粒肽调控肥大细胞脱颗粒的实验研究

目的 研究肥大细胞脱颗粒肽(Mast cell degranulating peptide,MCDP)对小鼠肥大细胞Pik3r、Akt2mRNA表达的影响,探讨MCDP调控肥大细胞脱颗粒的机制.方法 采用小鼠肥大细胞瘤细胞(P815),随机分为正常组、MCDP(高、中、低)剂量组.CCK8法检测MCDP对细胞的毒性作用,ELISA法检测MCDP刺激细胞脱颗粒释放组胺的变化,RT-PCR法检测MCDP干预前后PI3K/Akt信号通路上相关基因Pik3r3、Akt2的表达量变化.结果 1)MCDP对P815细胞的毒性作用随着干预时间的延长而增加,在0.25～2.00 h与时间呈正相关性,(P＜0.05);2)MCDP高剂量组(1 μM)可使P815细胞组胺释放率达56.73％,与对照组相比组胺释放率显著增加(P＜0.05);3)MCDP高、低剂量组Pik3r3的mRNA平均吸光度值比分别为(5.16±0.25)和(2.06±0.54);Akt2的mRNA平均吸光度值比分别为(3.87±0.21)和(1.57±0.35),与对照组相比表达量均有增加(P＜0.05);MCDP高剂量组的Pik3r3与Akt2 mRNA表达量较低剂量组均明显升高(P＜0.05).结论 MCDP调控肥大细胞脱颗粒可能与激活PI3K/Akt信号转导通路有关.     

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。     

Inhibition of the antigen-induced activation of rodent mast cells by putative Janus kinase 3 inhibitors WHI-P131 and WHI-P154 in a Janus kinase 3-independent manner

We analyzed the effects of the Janus kinase 3 (Jak3)-specific inhibitor WHI-P131 (4-(4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline) and the Jak3/Syk inhibitor WHI-P154 (4-(3'-bromo-4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline) on the antigen-induced activation of mast cells. In the rat mast cell line RBL-2H3, both WHI-P131 and WHI-P154 inhibited the antigen-induced degranulation and phosphorylation of p44/42 mitogen-activated protein kinase (MAPK), p38 MAPK and c-Jun N-terminal kinase (JNK). The phosphorylation of Gab2, Akt and Vav was also inhibited by WHI-P131 and WHI-P154, indicating that these inhibitors suppress the activation of phosphatidylinositol 3-kinase (PI3K). In bone marrow-derived mast cells (BMMCs) from Jak3-deficient (Jak3-/-) mice, degranulation and activation of MAPKs were induced by the antigen in almost the same extent as in BMMCs from wild-type mice. In addition, the antigen-induced degranulation and activation of MAPKs were inhibited by WHI-P131 and WHI-P154 in both groups of BMMCs, indicating that these compounds inhibit a certain step except for Jak3. The antigen-induced increase in the activity of Fyn, a probable tyrosine kinase of Gab2, was also inhibited by WHI-P131 and WHI-P154 in RBL-2H3 cells. In BMMCs from Jak3-/- mice, the antigen stimulation induced tyrosine phosphorylation of Fyn, which was inhibited by WHI-P131, as well as in BMMCs from wild-type mice and in RBL-2H3 cells. These findings suggest that Jak3 does not play a significant role in the antigen-induced degranulation and phosphorylation of MAPKs, and that WHI-P131 and WHI-P154 inhibit the PI3K pathway by preventing the antigen-induced activation of Fyn, thus inhibiting the antigen-induced degranulation and phosphorylation of MAPKs in mast cells.     

Vancomycin-induced release of histamine from rat peritoneal mast cells and a rat basophil cell line (RBL-1).

Rapid intravenous administration of the glycopeptide antibiotic, vancomycin, may cause a hypotensive reaction which can usually be prevented by infusing vancomycin in dilute solutions. The release of histamine from circulating cells such as basophils and tissue mast cells has been implicated in hypotensive reactions since the effects can be prevented by antihistamine pretreatment. The direct effects of vancomycin on histamine release were therefore investigated in rat peritoneal mast cells and rat leukemic basophils (RBL-1 cells). Suspension cultures of mast cells or RBL-1 cells were exposed to vancomycin for 30-60 minutes at concentrations comparable to those infused clinically (2.28 or 4.56 mg/ml). Vancomycin induced a time- and dose-dependent release of histamine into the culture media from both cell types. The reference degranulating agent, Compound 48/80 (CP 48/80), was also shown to induce histamine release from mast cells and RBL-1 cells. Mast cells were significantly more sensitive to vancomycin and CP 48/80 than RBL-1 cells and, unlike RBL-1 cells, were responsive to the inhibitory effects of cromolyn sodium on histamine release. Cromolyn sodium did not inhibit vancomycin-induced histamine release in RBL-1 or mast cells. Morphologically, mast cells exposed to either vancomycin or CP 48/80 exhibited dose-related degranulation. On the other hand, treatment-related degranulation effects of either vancomycin or CP 48/80 on RBL-1 cells could not be reliably distinguished from controls by qualitative evaluation. Based upon these findings it is concluded that mast cells may represent a more useful model to evaluate the potential of investigational agents to release histamine and to study mechanisms of histamine release than RBL-1 cells.     

Houttuynia cordata water extract suppresses anaphylactic reaction and IgE-mediated allergic response by inhibiting multiple steps of FcεRI signaling in mast cells

Houttuynia cordata has been used as a traditional medicine in Korea and is known to have antioxidant, anti-cancer and anti-allergic activities. The precise effect of H. cordata, however, remains unknown. In this study, we investigated the effects of H. cordata water extract (HCWE) on passive cutaneous anaphylaxis (PCA) in mice and on IgE-mediated allergic response in rat mast RBL-2H3 cells. Oral administration of HCWE inhibited IgE-mediated systemic PCA in mice. HCWE also reduced antigen (DNP-BSA)-induced release of β-hexosaminidase, histamine, and reactive oxygen species in IgE-sensitized RBL-2H3 cells. In addition, HCWE inhibited antigen-induced IL-4 and TNF-α production and expression in IgE-sensitized RBL-2H3 cells. HCWE inhibited antigen-induced activation of NF-κB and degradation of IκB-α. To investigate the inhibitory mechanism of HCWE on degranulation and cytokine production, we examined the activation of intracellular FcεRI signaling molecules. HCWE suppressed antigen-induced phosphorylation of Syk, Lyn, LAT, Gab2, and PLC γ2. Further downstream, antigen-induced phosphorylation of Akt and MAP kinases (ERK1/2 and JNK1/2 but not p38 MAP kinase) were inhibited by HCWE. Taken together, the in vivo/in vitro anti-allergic effect of HCWE suggests possible therapeutic applications of this agent in inflammatory allergic diseases through inhibition of cytokines and multiple events of FcεRI-dependent signaling cascades in mast cells.     

G protein coupled receptor specificity for C3a and compound 48/80-induced degranulation in human mast cells: roles of Mas-related genes MrgX1 and MrgX2

Although human mast cells express G protein coupled receptors for the anaphylatoxin C3a, previous studies indicated that C3a causes mast cell degranulation, at least in part, via a C3a receptor-independent mechanism similar to that proposed for polycationic molecules such as compound 48/80. The purpose of the present study was to delineate the receptor specificity of C3a-induced degranulation in human mast cells. We found that C3a, a C3a receptor "superagonist" (E7) and compound 48/80 induced Ca(2+) mobilization and degranulation in a differentiated human mast cell line, LAD2. However, C3a and E7 caused Ca(2+) mobilization in an immature mast cell line, HMC-1 but compound 48/80 did not. We have previously shown that LAD2 cells express MrgX1 and MrgX2 but HMC-1 cells do not. To delineate the receptor specificity for C3a and compound 48/80 further, we generated stable transfectants expressing MrgX1 and MrgX2 in a rodent mast cell line, RBL-2H3 cells. We found that compound 48/80 caused degranulation in RBL-2H3 cells expressing MrgX1 and MrgX2 but C3a did not. By contrast, E7 activated RBL-2H3 cells expressing MrgX2 but not MrgX1. These findings demonstrate that in contrast to previous reports, C3a and compound 48/80 do not use a shared mechanism for mast cell degranulation. It shows that while compound 48/80 utilizes MrgX1 and MrgX2 for mast cell degranulation C3a does not. It further reveals the novel finding that the previously characterized synthetic peptide, C3a receptor "superagonist" E7 activates human mast cells via two mechanisms; one involving the C3a receptor and the other MrgX2.     

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.     

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.     

Inhibitory mechanism of saponins derived from roots of Platycodon grandiflorum on anaphylactic reaction and IgE-mediated allergic response in mast cells

The purpose of this study was to investigate the protective effects of saponins isolated from the root of Platycodi Radix (Changkil saponins: CKS) anti-allergic effects in mice and mast cells. Oral administration of CKS inhibited the dinitrophenyl (DNP)–IgE antibody-induced systemic PCA reaction in mice. CKS reduced the β-hexosaminidase and histamine release from anti-DNP–IgE-sensitized RBL–2H3 cells. In addition, CKS inhibited the IgE antibody-induced increases in IL-4 and TNF-α production and expression in RBL-2H3 cells. In order to explore the inhibitory mechanism of CKS in PCA and mast cell degranulation, we examined the activation of intracellular signaling molecules. CKS suppressed DNP–IgE antibody-induced Syk phosphorylation. Further downstream, CKS also inhibited the phosphorylation of Akt and MAP kinases. Taken together, the in vivo/in vitro anti-allergic effects of CKS suggest possible therapeutic applications for this agent in allergic diseases through the inhibition of inflammatory cytokines and Syk-dependent signaling cascades.     

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 (IC(50) 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 (ED(50), 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.     

Effects of Clostridium botulinum C2 toxin-induced depolymerisation of actin on degranulation of suspended and attached mast cells

We studied the effects of C. botulinum C2 toxin, which ADP-ribosylates G-actin, on mast cell degranulation. C2 toxin inhibited degranulation of suspended rat peritoneal mast cells induced by compound 48/80 and dinitrophenyl-conjugated bovine serum albumin (DNP-BSA) maximally by about 50 and 90%, respectively. Inhibition by C2 toxin occurred in a time- and concentration-dependent manner. Half-maximal inhibition of DNP-BSA-induced degranulation by C2 toxin occurred at about 0.015 ng/ml, whereas stimulation of mast cells induced by compound 48/80 was half-maximally inhibited at 0.15 ng/ml C2 toxin. C2 toxin also inhibited stimulated [³H]serotonin release from suspended mast cells. Phorbol 12-myristate 13-acetate (PMA)-induced histamine release of suspended mast cells was inhibited by C2 toxin by about 80–90%. C2 toxin had no effect on calcium ionophore A23187-induced histamine release. Toxin treatment of mast cells caused ADP-ribosylation of actin and depolymerisation of F-actin. Attachment of mast cells, which largely increased the diameter of the subcortical actin network, reduced degranulation stimulated by compound 48/80, antigen and calcium ionophore but not by PMA. Opposite to its effect on suspended cells, in adherent mast cells C2 toxin stimulated degranulation by compound 48/80, antigen, and calcium ionophore but not by PMA. The data indicate that mast cell degranulation and responsiveness towards the actin-depolymerising C2 toxin depend largely on mast cell attachment. Received: 7 October 1996 / Accepted: 22 November 1996     

Ethyl acetate extract of Psidium guajava inhibits IgE-mediated allergic responses by blocking FcεRI signaling

Psidium guajava (P. guajava) is an important food crop and medicinal plant with antioxidant, anti-inflammatory, and anti-allergic activities, supporting its traditional uses. However, its precise effects remain unknown. We investigated the effects of P. guajava ethyl acetate extract (PGEA) on IgE-mediated allergic responses in rat mast RBL-2H3 cells. PGEA reduced antigen (DNP-BSA)-induced release of β-hexosaminidase and histamine in IgE-sensitized RBL-2H3 cells. In addition, it inhibited antigen-induced IL-4 and TNF-α mRNA expression and protein production in IgE-sensitized RBL-2H3 cells. PGEA also suppressed antigen-induced COX-2 mRNA and protein expression in these cells, as well as antigen-induced activation of NFAT and reactive oxygen species. Moreover, it inhibited antigen-induced activation of NF-κB and degradation of IκB-α. To identify the mechanisms underpinning the inhibition of degranulation and cytokine production by PGEA, we examined the activation of intracellular FcεRI signaling molecules. PGEA suppressed antigen-induced phosphorylation of Syk, LAT, Gab2, and PLCγ2 but not Lyn, and inhibited antigen-induced phosphorylation of downstream signaling intermediates including MAP kinases and Akt. Collectively, the anti-allergic effects of PGEA in vitro suggest its possible therapeutic application to inflammatory allergic diseases, in which its inhibition of inflammatory cytokine production and FcεRI-dependent signaling events in mast cells may be hugely beneficial.