阻断IgG-FcγR结合短肽在抗中性粒细胞胞浆抗体促进中性粒细胞凋亡中的作用

Inhibitory effect of an small peptide able to interfere with Fcγ-receptor recognition on ANCA-accelerated apoptosis of neutrophils

目的利用阻断IgG-FcγR结合的短肽,观察FcγR在抗中性粒细胞胞浆抗体(ANCA)促进中性粒细胞凋亡过程中的作用。方法人工合成短肽tg19320并鉴定其与人IgG的结合,玫瑰花环形成试验鉴定其阻断IgG-FcγR间的作用。提纯ANCA IgG并鉴定,体外分离健康人中性粒细胞。tg19320预处理经TNF-α预激的粒细胞1．5 h后,ANCA(200μg／ml)刺激粒细胞,分别在3、6、9、12和18 h收获细胞。流式细胞仪检测细胞线粒体膜电位及凋亡细胞之Sub- G1峰变化,结合DNA缺口末端标记技术观察tg19320对ANCA促进中性粒细胞凋亡的抑制作用。结果(1)tg19320剂量依赖性与人IgG结合(P<0．01),并显著阻断IgG与FcγR的相互作用(玫瑰花环形成率20．3％比53．2％,P<0．01)。(2)正常粒细胞凋亡率随孵育时间增加,TNF-α在6 h前促进粒细胞凋亡而之后抑制粒细胞的凋亡:ANCA进行性促进经TNF-α预处理的粒细胞凋亡(P<0．01)。tg19320处理组粒细胞凋亡率在各时间点均低于ANCA作用组(P<0．01),而与正常对照组比较无显著差异。结论分支短肽tg19320可干预ANCA对中性粒细胞凋亡的促进, FcγR在ANCA促进中性粒细胞凋亡过程中具有关键作用。

Objective To investigated the inhibitory effect of an IgG-Fc region specific inhibitory peptide on the ANCA-accelerated apoptosis of neutrophils. Methods The peptide was prepared by solid-phase peptide synthesis and its biological activity was identified by rosette formation assay. ANCA was prepared from the sera of active Wegener＇s granulomatosis （WG） and microscopic polyangiitis （MPA） patients. Neutrophils isolated from the blood of healthy volunteers were primed with TNF-α（2 ng/ml） then incubated with ANCA. At different intervals（3, 6, 12, 18 hours） the neutrophils were harvested to assess the apoptesis by flow cytometric analysis of JC-1 staining, Sub- G1 population and formation TUNEL technique. Results Tg19320 bound tightly to human IgG dosedependently and inhibited statistically the rosette formation between SRBC-IgG and U937 cens（20.3% vs 53.2% ,P 〈 0.01）.Furthermore,tg19320 blocked ANCA-accelerated apoptosis of neutrophils at different intervals and made the apoptesis have no significant difference with control groups. Conclusion The small peptide can intervene the apoptesis induced by ANCA, which supports the role of FcγR in primary systemic small vasculitis（PSV） pathogenesis and perhaps opens new avenues for the development of drugs to treat PSV.