大鼠慢性肺部感染刺激抗中性粒细胞胞质抗体产生及其机制

Investigation on the production of anti-neutrophil cytoplasmic antibodies in chronic bronchitis rats

目的探讨大鼠慢性支气管炎(chronic bronchitis,CB)诱导产生抗中性粒细胞胞质抗体(antineutrophil cytoplasmic antibodies,ANCA)的机制.方法用烟熏法联合气管内注入脂多糖(LPS)诱导建立CB大鼠模型,LPS和佛波酯(PMA)反复刺激CB发作.分为正常对照组(n=5)、对照+PMA组(n=5)、CB组(n=5)、CB+PMA组(n=6).检测4组大鼠肾功能,HE染色观察大鼠肺、肾组织病理变化,ELISA法检测血清中髓过氧化物酶(MPO)-ANCA、蛋白酶3(PR3)-ANCA和中性粒细胞胞外诱捕网(NETs)标志物瓜氨酸化组蛋白H3(CitH3)的水平.采用Spearman秩相关分析CitH3与MPO-ANCA的相关性.应用免疫荧光染色和激光共聚焦显微镜观察MPO、CitH3在肺脏、肾脏组织的表达和定位.结果(1)CB+PMA组大鼠血清CitH3、MPO-ANCA水平随时间变化均呈上升趋势,第6周末CitH3、MPO-ANCA水平均高于正常对照组、CB组(均P<0.05).大鼠血清CitH3水平与血清MPO-ANCA水平呈正相关(rs=0.490,P=0.024).(2)CB组、CB+PMA组大鼠肺组织存在CB的病理表现,正常对照组、对照+PMA组大鼠肺组织未见明显异常.CB+PMA组大鼠肾脏组织中肾小球内及肾小管周围大量淋巴细胞浸润,炎性细胞黏附于血管壁,但尚未发现肾小球坏死.正常对照组、对照+PMA组和CB组大鼠肾脏组织未见明显异常.(3)CB+PMA组大鼠肺脏、肾脏冰冻组织免疫荧光双染激光共聚焦显微镜可观察到NETs沉积.结论CB大鼠在反复感染过程中,NETs在体内发生蓄积,其所含有的MPO抗原组分反复暴露,最终可导致MPO-ANCA的产生.

Objective To investigate the pathogenesis of the production of anti-neutrophil cytoplasmic antibodies(ANCA)in the rat models of chronic bronchitis(CB)with recurrent infections.Methods The CB models were made by double element of smoking and lipopolysaccharide(LPS)stimulation.The rats were divided into four groups,including normal control group(n=5),phorbol-12-myristate-13-acetate(PMA)-treated healthy rats control group(n=5),CB rats group(n=5)and PMA-treated CB rats group(n=6).Renal function of rats was detected.The histopathological lung and kidney tissues were observed by HE staining of paraffin section.Immunological markers,including myeloperoxidase anti-neutrophil cytoplasmic antibodies(MPO-ANCA),proteinase 3 anti-neutrophil cytoplasmic antibodies(PR3-ANCA)and citrullinated histone H3(CitH3),were measured by enzyme-linked immune-sorbent assay(ELISA)at different time points.Correlation between CitH3 and MPO-ANCA was analyzed by the Spearman rank correlation.NETs components were further detected in lung and kidney tissue by confocal immunofluorescence and colocalization analysis.Results(1)The serum levels of CitH3 and MPO-ANCA in CB+PMA group showed an increased trend.Compared with those in the normal control group and CB rats group,the serum levels of CitH3 and MPO-ANCA in CB+PMA group increased significantly at the sixth week(both P<0.05).Serum CitH3 levels in rats were positively correlated with serum MPO-ANCA levels(rs=0.490,P=0.024).(2)There were pathological manifestations of CB in the lung tissues of rats in CB group and CB+PMA group,and no obvious abnormalities in the lung tissues of rats in the normal control group and control group.In the rat kidney tissue of CB+PMA group,there were inflammatory cells infiltrated in the glomerular and around the renal tubules,but glomerular necrosis was not found.No obvious abnormalities were observed in the kidney tissues of rats in the normal control group,PMA-treated healthy rats control group and CB group.(3)In the lung and kidney tissues of CB+PMA group NETs could be detected by confocal immunofluorescence analysis.Conclusion CB rats with the recurrent infections can release large amounts of NETs,in which the exposure of MPO antigen will break the immune tolerance and result in the production of MPO-ANCA.