持续吸入变应原引起支气管哮喘小鼠免疫耐受

Continuous inhalation of allergen induces immunotolerance in a mouse asthma model

目的探讨吸入变应原引起支气管哮喘(简称哮喘)过敏性气道炎症免疫耐受形成的机制。方法BALB/c小鼠60只,按随机数字表法分为实验组(50只)和空白对照组(10只),实验组小鼠先给予腹腔注射卵清白蛋白(OVA)1mg,每周1次,共3周。雾化吸入OVA每天1h(含OVA80μg),连续10d。依据吸入OVA时间分为A、B、C、D、E5组,每组10只。A组雾化吸入10d后处死。B、D组继续每天1次,每次1h,每周5次,分别吸入OVA4周及8周,然后每天1h,连续10d吸入OVA后处死。C组停止吸入OVA4周后再次吸入OVA,每天1h,连续10d后处死。E组每天1次,每次1h,每周5次,吸入OVA4周,停止雾化吸入OVA4周,然后每天1h,连续10d吸入OVA后处死。测定各组小鼠支气管肺泡灌洗液(BALF)中细胞总数,嗜酸粒细胞、淋巴细胞、CD4+、CD8+、CD4+IL-10+分类及BALF中白细胞介素4(IL-4)、γ干扰素(IFN-γ)、IL-10的含量。测定血清中IL-4、IFN-γ、IL-10、OVA、IgE、IgG1、IgG2a水平,并对各组小鼠肺组织病理学进行分析。结果空白对照组BALF中嗜酸粒细胞、B淋巴细胞、CD4+IL-10+细胞分别为0.010±0.000、2.1±1.9、4.9±1.5,A组分别为0.480±0.110、5.1±2.6、5.1±2.3,B组分别为0.120±0.020、8.9±3.6、10.4±3.6,C组分别为0.560±0.050、4.7±1.7、6.3±3.1,D组分别为0.070±0.030、10.1±2.9、12.7±4.5,E组分别为0.680±0.030、5.6±3.2、6.1±3.4,各组间比较差异有统计学意义(F值分别为36.46、31.89、167.89,P均<0.01)。B、D组BALF中CD4+IL-10+细胞数与A组比较差异有统计学意义(q=5.8、6.4,P均<0.05);空白对照组BALF中IL-4、IL-10水平分别为(21±3)pg/ml、(44±12)pg/ml,A组分别为(128±23)pg/ml、(68±18)pg/ml,B组分别为(54±12)pg/ml、(127±27)pg/ml,C组分别为(133±21)pg/ml、(78±17)pg/ml,D组分别为(8±18)pg/ml、(135±34)pg/ml,E组分别为(143±26)pg/ml、(76±15)pg/ml,组间比较差异有统计学意义(F分别为37.20、143.78,P均<0.01)。B、D两组BALF中IL-10水平与A组比较差异有统计学意义(q分别为7.8、9.6,P均<0.05)。结论持续吸入变应原可使小鼠气道炎症减轻,产生免疫耐受,调节T淋巴细胞产生的IL-10参与了耐受形成。

Objective To investigate the mechanism of immunotolerance caused by allergen immunotherapy in allergen-induced asthmatic airway inflammation. Methods Sixty ovalbumin （OVA）-sensitized BALB/c mice were randomly divided into two groups, 50 in the experimental group and 10 in the control group. The mice in the experimental group were treated with 3 injections of ovalbumin intraperitoneally （ 1 mg for each separated for two weeks） and challenged by ovalbumin inhalation 1 h/day for 10 successive days. Then the mice were divided further into group A, group B, group C, group D and E with 10 mice in each group. The mice in group A were sacrificed after 10 day challenge. The mice in group B and D were continuously exposed to inhaled OVA for 4 and 8 weeks （ 1 h/day, 5 days a week）, respectively, then to inhaled OVA 1 h/day for 10 successive days. The inhalation was interrupted for 4 weeks in group C after initial challenge and restarted for another 10 days （ 1 h/day） afterwards. The mice in group E were exposed continuously to inhaled OVA for 4 weeks （ 1 h/day, 5 days a week） after initialchallenge, which was interrupted for 4 weeks, and restarted for 10 days （ 1 h/day）. Bronchoalveolar lavage （BAL） was performed, and total cells, EOS inophils, lymphocytes were assessed; CD4^＋, CD8^＋, CD4^＋ IL-10 ^＋ cells were determined using flow cytometry, and IL-4, IFN-γ, and IL-10 in the BAL fluid were measured by ELISA. Serum ovalbumin-specific IgE, IL-4, IFN-γ/ and IL-10 were also determined. Pathologic manifestation of the lung was analyzed. Results The percentage of EOS, B lymphocytes, CD4^＋ IL-10^＋cells in BAL were 0. 010 ±0. 000, 2. 1 ± 1.9 and 4.9± 1.5, respectively in the control group; 0. 480 ±0. 110, 5. 1 ± 2.6 and 5. 1 ± 2.3, respectively, in group A; 0. 120 ± 0.020, 8.9 ±3.6, and 10.4 ± 3.6, respective, in group B ; 0. 560 ± 0. 050, 4. 7 ± 1.7 and 6. 3 ± 3.1, respectively, in group C ; 0. 070 ± 0. 030, 10. 1 ± 2. 9 and 12. 7 ±4. 5, respectively, in group D ; 0. 680 ± 0. 030, 5.6 ± 3.2 and 6. 1 ± 3.4, respectively, in group E. The difference was significant among different groups （ F = 36. 46, 31.89, 167. 89 respectively ; all P 〈0. 01 ）. The percentage of CD4^＋ IL-10^＋ ceils in BAL was increased in group B and group D, which were significantly higher than those in group A （q = 5.8,6.4, P 〈 0. 05 ）. The levels of IL-4 and IL-10 in BAL fluid were （21 ±3） pg/ml and （44 ± 12） pg/ml, respectively, in the control group; （ 128 ± 23） pg/ml and （68 ± 18） pg/ml,respectively, in group A; （54 ±12） pg/ml and （127 ±27） pg/ml, respectively, in group B ; （ 133 ± 21 ） pg/ml and （78±17） pg/ml, respectively, in group C ; （ 8 ± 18 ） pg/ ml and （135 ±34） pg/ml, respectively, in group D; （143 ± 26） pg/ml and （76 ± 15） pg/ml, respectively, in group E. The difference was statistically significant among different groups （F = 37.20, 143.78 respectively; all P 〈0. 01 ）. The levels of IL-10 in BAL fluid were increased in group B and group D, which were significantly higher than that in group A （ q = 7.8, 9. 6, all P 〈 0. 05 ）. Conclusion The results show that continuous allergen inhalation suppresses allergen-induced airway inflammation and produces immunotolerance, in which IL-10 may play an important role.