白细胞介素-33在过敏性哮喘小鼠体内的变化及作用

The change and role of interleukin-33 in mouse allergic asthma

目的探讨白细胞介素-33(IL-33)在粉尘螨1类变应原(Der f 1)诱导的过敏性哮喘小鼠中的变化及作用。方法将40只雌性BALB/c小鼠用随机数字表法分为哮喘组、免疫治疗组、抑制剂组和阴性对照组(PBS组),每组10只。哮喘组、免疫治疗组和抑制剂组分别在第0、7和14天腹腔注射100μl粉尘螨变应原提取液(含100μg/ml Der f 1),抑制剂组小鼠同时注射抑制剂(可溶性ST2 100μl,用于抑制IL-33),PBS组注射等量PBS。第21天起,各组小鼠雾化吸入粉尘螨变应原提取液(0.5μg/ml),30 min/次,1次/d,连续7 d。免疫治疗组小鼠第25天起于雾化前0.5 h,分别用200μl的Der f 1蛋白溶液(100μg/ml)腹腔注射进行免疫治疗,PBS组用PBS代替粉尘螨变应原。第27天雾化吸入后24 h处死小鼠,收集小鼠支气管肺泡灌洗液(BALF)和眼球血,并对BALF中嗜酸粒细胞进行计数,制作肺组织病理切片。ELISA法检测BALF中IL-5、IL-13和γ干扰素(IFN-γ)的含量及血清中特异性Ig E和Ig G2a水平。结果除PBS组小鼠外,其他组小鼠自第21天开始出现不同程度的哮喘症状。自第25天开始,免疫治疗组小鼠的哮喘症状开始缓解。哮喘组和PBS组小鼠BALF中嗜酸粒细胞总数分别为(4.41±0.36)×10~5/ml和(0.37±0.08)×10~5/ml,两组差异有统计学意义(t=24.50,P<0.01);免疫治疗组和抑制剂组小鼠的嗜酸粒细胞总数分别为(1.43±0.14)×10~5/ml和(2.73±0.33)×10~5/ml,均明显低于哮喘组(F=129.72,P<0.01)。ELISA检测结果显示,哮喘组、免疫治疗组、抑制剂组和PBS组BALF中IFN-γ的水平分别为(83.06±11.38)、(277.97±22.46)、(175.13±13.41)和(224.77±19.97)pg/ml,免疫治疗组和抑制剂组均明显高于哮喘组(t=17.31,t=11.71,P<0.01)。哮喘组、免疫治疗组、抑制剂组和PBS组小鼠BALF中IL-5水平分别为(208.64±11.55)、(106.87±11.39)、(140.71±14.58)和(90.15±9.49)pg/ml,哮喘组的水平明显高于其他各组(F=97.19,P<0.01)。哮喘组和免疫治疗组BALF中IL-13水平分别为(308.37±13.67)pg/ml和(175.66±11.79)pg/ml,两者差异有统计学意义(P<0.01);抑制剂组小鼠的为(221.12±21.08)pg/ml,明显低于哮喘组(t=16.44,P<0.01);PBS组为(97.57±18.38)pg/ml。免疫治疗组与哮喘组相比,小鼠肺部炎性症状均减轻,几乎无炎症细胞。抑制剂组小鼠的支气管周围也有嗜酸粒细胞增多症、上皮细胞脱落和支气管上皮细胞肥大,但较哮喘组要减轻许多。血清Ig E抗体水平的检测结果显示,哮喘组、免疫治疗组、抑制剂组和PBS组分别为(31.97±3.48)、(12.86±2.22)、(18.43±2.30)和(9.68±1.27)IU/ml,免疫治疗组和抑制剂组均低于哮喘组(t=-7.77,P<0.01)。Ig G2a抗体水平的变化和Ig E相反,哮喘组、免疫治疗组和PBS组分别为(26.94±2.96)、(35.06±2.57)和(10.31±1.48)μg/ml,免疫治疗组高于哮喘组(t=6.55,P<0.01)。结论 ELISA检测结果表明IL-33/ST2信号通路在小鼠过敏性哮喘中发挥着重要的作用。

Objective To investigate the change and the role of interleukin-33（IL-33） in mice with allergic asthma induced by Dermatophagoides farinae group 1 allergen（Der f 1）. Methods Forty female BALB/c mice were randomized into four groups using a random number table method： asthma group（sensitized by Der f 1 allergen）,SIT group（specific immunotherapy with Der f 1）, inhibitor administration group and negative control group（PBS group）（n = 10 for each）. All mice except the PBS group were intraperitoneally injected with Dermatophagoides farinae allergen extract containing 100 μg/ml Der f 1 on days 0, 7 and 14, and those in the inhibition group also received an intraperitoneal injection of inhibitor（soluble ST2） at the same time. From day 21, mice in the three groups were sensitized through aerosol inhalation of the extract（0.5 μg/ml for 30 min, once per day for consecutive days）. Mice in the immunotherapy group began to receive an immunotherapy through intraperitoneal injection of 200 μl of Der f 1 protein solution（100 μg/ml） 0.5 h prior to aerosol inhalation, from day 25 on, while in the PBS group,PBS was used instead of Der f 1. Twenty-four hours after the final aerosol inhalation on day 27, all the mice were sacrificed to collect the bronchoalveolar lavage fluid（BALF） and eyeball blood. The total eosinophils in BALF were counted and pulmonary histopathological sections were prepared. ELISA was performed to measure the levels of cytokines IL-5, IL-13, IFN-γ and IL-33 in the BALF, as well as serum levels of Ig E and Ig G2 a. Results All groups except for the PBS group displayed different degrees of asthma symptoms since day 21. From day 25, the asthma symptoms in the immunotherapy group began to show remission. The total numbers of BALF eosinophils in the immunotherapy group and the inhibitor group were（1.43 ± 0.14） × 10~5/ml and（2.73 ± 0.33） × 10~5/ml, respectively（t = 24.50, P〈0.01 between the two groups）. The numbers of eosinophils in SIT group（1.43 ± 0.14） × 10~5/ml and the inhibitor administration group（2.73 ± 0.33） × 10~5/ml were both significantly lower than that in the asthma group（ F = 129.72, P〈0.01）. ELISA results showed that the levels of IFN-γ in BALF in the asthma group, the SIT group, the inhibitor administration group and the negative control group were（ 83.06 ± 11.38）,（ 277.97 ± 22.46）,（ 175.13 ± 13.41） and（ 224.77 ± 19.97） pg/ml, respectively. The BALF IFN-γ levels in the SIT group and t he inhibitor administration group were both significantly higher than that in the asthma group（t = 17.31, t = 11.71, P〈0.01）. The levels of IL-5 in BALF in the asthma group, the SIT group, the inhibitor administration group and the negative control group were（ 208.64 ± 11.55）,（ 106.87 ± 11.39）,（ 140.71 ± 14.58） and（ 90.15 ± 9.49） pg/ml,respectively. The BALF IL-5 level in the asthma group was significantly higher than that in the other groups（ F =97.19, P〈0.01）. There was a significant difference in the BALF IL-13 level between the asthma group [（308.37 ±13.67） pg/ml] and the SIT group [（175.66 ± 11.79） pg/ml]（P〈0.01）, and that in the inhibition group was significantly lower than that in the asthma group（t = 16.44, P〈0.01）. The BALF IL-13 level in the PBS group was（97.57 ± 18.38） pg/ml. Compared with the asthma group, mice in the SIT group showed improved inflammation in the lungs. Although the inhibition group also showed eosinophilia, epithelial cell shedding and bronchial epithelial cells around the bronchus, the symptoms were much attenuated compared with the asthma group. Serum Ig E levels in the asthma group, the SIT group, the inhibitor administration group and the PBS group were（31.97 ± 3.48）,（1 2.86 ±2.22）,（1 8.43 ± 2.30） and（9.68 ± 1.27） IU/ml, respectively. The serum Ig E levels in the SIT group and the inhibitor administration group were both significantly lower than that in the asthma group（ t =-7.77, P〈0.01）. In contrast,the Ig G2 a level in the SIT group [（35.06 ± 2.57） μg/ml] was significantly higher than that in the asthma group[（26.94 ± 2.96） μg/ml]（t = 6.55, P〈0.01）. The Ig G2 a level in the PBS group was（10.31 ± 1.48） μg/ml.Conclusion The IL-33/ST2 signaling pathway may play a critical role in allergic asthma by ELISA.