支气管哮喘患者外周血炎性细胞凋亡的研究

A study on the heterogeneous apoptosis of lymphocytes, eosinophils,and neutrophils from peripheral blood of asthmatic patients

目的 观察支气管哮喘患者外周血嗜酸粒细胞 (EOS)、淋巴细胞和中性粒细胞对地塞米松及孟鲁司特凋亡诱导作用的反应性 ,并探讨差异的分子机制。方法 18例支气管哮喘患者外周静脉血分离淋巴细胞、EOS和中性粒细胞 ,体外培养时分别加入地塞米松和孟鲁司特 ,流式细胞仪检测细胞凋亡率和Fas受体表达率 ,用酶联免疫吸附测定 (ELISA)法检测细胞中半胱氨酸天冬氨酸蛋白酶 3 (caspase 3 )水平。结果 ( 1)凋亡率 :体外培养时淋巴细胞、EOS和中性粒细胞的自发凋亡率分别为 ( 6 9± 0 7) %、( 3 1± 11) %、( 3 2± 3 0 ) % ,地塞米松刺激后的凋亡率分别为 ( 17 1± 10 8) %、( 4 4±2 2 ) %、( 3 5± 2 4) % ,孟鲁司特刺激后的凋亡率分别为 ( 2 2 5± 17 6) %、( 50± 2 7) %和 ( 55± 2 2 ) % ,地塞米松和孟普司特组淋巴空白细胞和EOS凋亡率与空白对照组比较 ,差异有显著性 (P <0 0 1、0 0 5) ;而中性粒细胞与EOS比较 ,差异无显著性 (P >0 0 5)。 ( 2 )Fas表达率 :淋巴细胞、EOS和中性粒细胞的Fas基础表达率分别为 ( 1 50± 0 0 7) %、( 2 2 0± 0 10 ) %和 ( 1 2 1± 0 0 9) % ,地塞米松刺激后分别为( 6 58± 2 10 ) %、( 7 52± 3 2 0 ) %和 ( 3 2 4± 2 3 4 ) % ,孟鲁司特刺激后分别为 (

Objectives To observe different responsiveness of lymphocytes, eosinophils, and neutrophils from peripheral blood of asthmatic patients to dexamethasone and montelukast induced apoptosis and to explore the roles of Fas antigen and caspase 3 in the heterogeneity of cell apoptosis Methods Lymphocytes, eosinophils, and neutrophils were isolated from peripheral blood of 18 asthmatic patients Cells were incubated in vitro and treated with dexamethasone and leukotriene receptor antagonist montelukast respectively Cell apoptosis rates and Fas expression rates were examined by flowcytometry whereas caspase 3 levels in these cells were detected by enzyme linked immunosorbent assay (ELISA) Results (1) Apoptosis rates: in vitro lymphocytes, eosinophils and neutrophils were compromised of spontaneous apoptosis at lower rates [(6 9±0 7)%, (31±11)% and (32±30)%, respectively] With induction of dexamethasone, the apoptosis rates were (17 1±10 8)%, (44±22)% and (35±24)% Montelukast markedly elevated the apoptosis rates of these three cells [(22 5±17 6)%, (50±27)% and (55±22)%, respectively] (compared to control, P< 0 01, <0 05,>0 05, respectively) (2) Fas expression: lymphocytes, eosinophils and neutrophils expressed low levels of Fas antigen at baseline [(1 50± 0 07)%, (2 20±0 10)% and (1 21±0 09)%, respectively] Dexamethasone induced Fas antigen expression levels of these cells of (6 58±2 10)%, (7 52±3 20)% and (3 24±2 34)%, and montelukast induced the expression levels of (5 06±1 66, 7 45±2 63, 3 03±2 47, P< 0 01, <0 01, > 0 05, respectively). (3) caspase 3 levels: lymphocytes, eosinophils and neutrophils expressed constitutive caspase 3 levels of [(3 3±2 9)ng/L, (5±4)ng/L and (4 3±2 6)ng/L, respectively] The dexamethasone induced caspase 3 levels were (6 7±3 1) ng/L, (6±3) ng/L and (3 1±1 8) ng/L The montelukast induced levels were (5 2± 3 7) ng/L, (8±4) ng/L, and (3 1±2 0) ng/L (compared to control, P< 0 01, <0 01, >0 05, respectively) It was demonstrated that dexamethasone and montelukast significantly induced apoptosis of lymphocytes and eosinophils which were assocreased with increased expression of Fas antigen and caspase 3 Dexamethasone was incapable of inducing neutrophils to apoptosis and had no significant effects on Fas expression and caspase 3 activity Neurtophils underwent significant apoptosis after montelukast treatment, however, the induction was unlikely to be regulated by Fas and caspase 3 pathway Conclusions In asthmatic inflammatory modulating and effective cells, neutrophils is distinct from lymphocytes and eosinophils in profile of apoptosis induced by glucocorticoids and leukotriene receptor antagonist. The signal pathway contributing neutrophil apoptosis heterogeneity may involve deficient caspase cascade or Fas/FasL