甲泼尼龙对慢性咳嗽患者诱导痰巨噬细胞中Toll样受体2表达的影响

Methylprednisolone enhances the expression of Toil-like receptor 2 on macrophages in induced sputum from patients with chronic cough

目的 探讨Toll样受体2（TLR2）在慢性咳嗽患者诱导痰巨噬细胞中的表达与气道炎症的关系以及甲泼尼龙对TLR2表达的影响.方法 收集2008年9月至2010年3月泸州医学院附属医院呼吸科门诊的慢性咳嗽患者86例,其中咳嗽变异性哮喘（CVA）组31例,上气道咳嗽综合征（UACS）组14例,嗜酸细胞性支气管炎（EB）组16例,胃食管反流性咳嗽（GERC）组25例.对照组为20名健康志愿者.各组均行痰液诱导检查,HE染色,免疫荧光染色测定TLR2表达,酶联免疫吸附法（ELISA）测定诱导痰上清液中细胞因子的浓度.结果 （1）CVA、UACS、EB和GERC各组患者诱导痰中细胞总数[分别为（3.4±1.1）×106/L、（2.6±0.6）×106/L、（2.6±0.6）×106/L、（2.7±0.5）×106/L]均明显高于对照组[（1.4±0.6）×106/L],差异均有统计学意义（均P＜0.01） 分类计数显示以上4组痰中巨噬细胞比例（分别为0.35±0.04、0.40±0.06、0.38±0.04、0.43±0.05）均明显低于对照组（0.63±0.08） 中性粒细胞比例（分别为0.52±0.06、0.58±0.06、0.52±0.04、0.54±0.06）明显高于对照组,差异均有统计学意义（均P＜0.01）.CVA和EB组患者嗜酸细胞比例（分别为0.115±0.054和0.099±0.034）明显高于其他各组,差异有统计学意义（均P＜0.01）,但两组间差异无统计学意义（t=1.18,P＞0.05）.（2）CVA、UACS、EB和GERC各组患者诱导痰上清液白细胞介素-8浓度[分别为（7.0±1.2）μg/L、（10.2±3.1）μg/L、（6.3±1.9）μg/L、（7.7±2.5）μg/L]和肿瘤坏死因子-α浓度[分别为（30±11）ng/L、（46±16）ng/L、（2l±5）ng/L、（33±15）ng/L]明显高于对照组[分别为（1.6±0.8）μg/L和（13±6）ng/L],差异均有统计学意义（均P＜0.01）.（3）CVA、UACS、EB和GERC各组患者诱导痰上清液中白细胞介素-8与痰中中性粒细胞数呈正相关（r值分别为0.628、0.816、0.769、0.733,均P＜0.05） CVA、UACS、EB和GERC各组患者诱导痰上清液中肿瘤坏死因子-α与痰中中性粒细胞数呈正相关（r值分别为0.579、0.865、0.730、0.755,均P＜0.05）.（4）CVA、UACS、EB和GERC各组患者诱导痰巨噬细胞TLR2表达量（56±15、38±7、65±17、27±4）明显低于对照组（135±14）,甲泼尼龙可上调以上各组患者诱导痰巨噬细胞中TLR2（75±17、53.29±16、94±30、41±5）的表达.结论 CVA、UACS、EB和GERC等慢性咳嗽患者诱导痰巨噬细胞中TLR2的表达下降,甲泼尼龙可上调巨噬细胞TLR2的表达.

Objective To explore the relationship between airway inflammation and Toll-like receptor 2 （ TLR2 ） expression on macrophages in induced sputum from chronic cough patients, and to observe the effect of methylprednisolone on this receptor. Methods Eighty-six outpatients with chronic cough were enrolled in this study from the respiratory department in the Affiliated Hospital of Luzhou Medical College from September 2008 to March 2010. These diseases were diagnosed according to the guideline of Chinese Society of Respiratory Diseases in 2005, excluding severe liver, heart and kidney pathology and pregnant women. Among 86 cases, 31 were cough variant asthma （ CVA）, 14 upper airway cough syndrome （UACS）, 16 eosinophilic bronchitis （EB） and 25 gastroesophageal reflux （GERC）. Twenty healthy volunteers were included as controls. Induced sputum was collected and cell counts were analyzed. TLR2 expression on macrophages was determined by immunofluorescence staining. Cytokine levels were measured with enzyme linked immunosorbent assay （ELISA）. Results （1） The total inflammatory cells [（3.4±1.1） ×106/L, （2.6 ±0.6）×106/L, （2.6±0.6）×106/L, （2.7±0.5） × 106/L] and neutrophils （0.52±0.06, 0.58 ±0.06, 0.52±0.04, 0.54 ±0.06） in induced sputum from CVA, UACS, EB and GERC were significantly increased（t value = 5.27, 4.09, 3. 16, 3.92 and 3.62, 3.49, 4.82, 6.17respectively, all P〈0.01 ）, with decreased macrophages （0.35±0.04, 0.40±0.06,0.38±0.04,0.43 ±0. 05） in comparison with the control group （t value =4. 76,5.24,4. 57,3. 18, all P 〈0. 01 ）. There was a significant increase in eosinophils（0.118±0.054,0.099±0.034） in CVA and EB group （t value =4.46,3.87,5. 19 and 3.51,4. 06,4. 38 respectively, all P〈0.01）. （2） The concentration of IL-8[（7.0±1.2）μg/L,（10.2±3.1）μg/L,（6.3±1.9）μg/L,（7.7±2.5）μg/L] and TNF-α[（30 ± 11）ng/L, （46±16）rng/L, （21 ±5）ng/L, （33 ± 15）ng/L] from induced sputum in CVA, UACS, EB and GERC groups were significantly increased, compared with the control group（ t value =4. 58,3.67,4. 26,5. 39 and 5.16,3.97,4. 59,3.34 respectively, all P 〈 0.01 ）. （3） The concentration of IL-8 was positively correlated with neutrophils in each group（ R value = 0.628,0. 816,0. 769 and 0. 733 respectively, all P〈0. 05 ）. The concentration of TNF-o was also positively correlated with neutrophils in each group （ R value = 0. 579,0. 865,0. 730 and 0. 755 respectively,all P 〈 0. 05 ）. （4） The expression of TLR2 （ 56 ± 15,38 ± 7,65 ±17,27 ±4） on macrophages in CVA, UACS,EB and GERC was significantly decreased. The expression of TLR2 （75±17, 53±16,94±30,41±5 ） on macrophages was enhanced by methylprednisolone.Conclusions The expression of TLR2 on macrophages from induced sputum in CVA, UACS, EB and GERC was decreased, but could be enhanced by methylprednisolone, suggesting that TLR2 might play a regulatory role in airway inflammatory response.