溶酶体膜蛋白-2抗体诱导中性粒细胞胞外捕网形成的实验研究

目的研究溶酶体膜蛋白-2抗体(LAMP-2-antibody)诱导中性粒细胞胞外捕网形成(NETosis)的作用,为进一步探讨这种新型细胞死亡模式在系统性小血管炎中的作用及机制奠定基础。方法采用PolymorphprepTM进行密度梯度离心法分离健康志愿者外周静脉血中性粒细胞,抗LAMP-2抗体刺激中性粒细胞,以及通过NADPH氧化酶抑制剂二甲苯基碘(DPI)阻断活性氧簇的产生,电镜、免疫荧光观测胞外捕网的形成,激光共聚焦检测自身抗原LAMP-2、PR3、MPO的表达。结果 LAMP-2抗体促进中性粒细胞胞外捕网NETs形成,自身抗原MPO、PR3及LAMP-2包含在NETs内,抑制NADPH氧化酶的活性可以阻断NETs形成。结论 LAMP-2抗体可能通过诱导中性粒细胞异常死亡,释放内源性危险信号,激活自身免疫反应等来维持血管炎的恶性持续状态。

Effects of anti-LAMP-2 antibody on neutrophil extracellular trap formation

The aim of this study is to investigate the role of anti-LAMP-2 antibody on neutrophil extracellular trap formation(NETosis).Human neutrophils were isolated from peripheral vein blood of health individuals by density gradient centrifugation using PolymorphprepTM,and then stimulated by anti-LAMP-2 antibody.NETosis was detected by immunofluorescence staining,laser confocal microscopy,and scanning electron microscopy.To further examine the role of anti-LAMP-2 antibody-induced reactive oxygen species(ROS) in NETosis,the NADPH oxidase inhibitor diphenylene iodonium(DPI) which inhibits production of ROS were used.We found that NETosis could be induced by anti-LAMP-2 antibody.Immunofluorescence analysis revealed that PR3,MPO,and LAMP-2 were all localized in extracellular chromatin fibers.Activation of NETosis has been shown to involve with NADPH oxidase activity.In conclusion,anti-LAMP-2 antibody may perpetuate a vicious circle of NET production that maintains the delivery of endogenous danger signals to the immune system.Further studies are required to address whether suppression of NETosis,for instance with reactive oxygen species scavengers,can ameliorate vasculitis and abrogate chronic autoimmunity in individuals suffering from systemic small-vessel vasculitis.