Tang S, Zhang Y, Yin S-W, Gao X-J, Shi W-W, Wang Y, Huang X, Wang L, Zou L-Y, Zhao J-H, Huang Y-J, Shan L-Y, Gounni A S, Wu Y-Z, Zhang J-B
Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, China.
Institute of Immunology of PLA, Third Military Medical University, Chongqing, 400038, China.
Clin Exp Immunol. 2015 Jun;180(3):408-18. doi: 10.1111/cei.12589. Epub 2015 Apr 29.
Increasing evidence indicates that aberrant neutrophil extracellular trap (NET) formation could contribute to the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Recent research has provided evidence that a novel type of ANCA autoantibody, anti-lysosomal membrane protein-2 (LAMP-2) antibody, may have a pathogenic role in AAV. We have shown previously that anti-LAMP-2 antibody-stimulated NET formation contains autoantigens and anti-microbial peptides. The current study sought to determine whether LAMP-2, as a novel antigen of ANCA, was present on NETs in AAV patients, the influence of the anti-LAMP-2 antibody on the neutrophil apoptosis rate and the role of autophagy in anti-LAMP-2 antibody-induced NET formation. NET formation was assessed using immunofluorescence microscopy, scanning electron microscopy or live cell imaging. The neutrophil apoptosis rate was analysed using fluorescence activated cell sorting (FACS). Autophagy was detected using LC3B accumulation and transmission electron microscopy. The results showed that enhanced NET formation, which contains LAMP-2, was observed in kidney biopsies and neutrophils from AAV patients. The apoptosis rate decreased significantly in human neutrophils stimulated with anti-LAMP-2 antibody, and this effect was attenuated by the inhibitors of autophagy 3-methyladenine (3MA) and 2-morpholin-4-yl-8-phenylchromen-4-one (LY294002). The anti-LAMP-2 antibody-stimulated NET formation was unaffected by benzyloxycarbonyl-Val- Ala-Asp (OMe)-fluoromethylketone (zVAD-fmk) and necrostatin-1 (Nec-1), which are inhibitors of apoptosis and necrosis, respectively, but was inhibited by 3MA and LY294002. Moreover, the proportion of LC3BI that was converted to LC3BII increased significantly (P=0.0057), and massive vacuolizations that exhibited characteristics typical of autophagy were detected in neutrophils stimulated with anti-LAMP-2 antibody. Our results provide further evidence that autophagy is involved in ANCA-induced NET formation in human neutrophils.
越来越多的证据表明，异常的中性粒细胞胞外陷阱（NET）形成可能参与抗中性粒细胞胞浆抗体（ANCA）相关血管炎（AAV）的发病机制。最近的研究提供了证据，表明一种新型的ANCA自身抗体，即抗溶酶体膜蛋白2（LAMP-2）抗体，可能在AAV中具有致病作用。我们之前已经表明，抗LAMP-2抗体刺激的NET形成包含自身抗原和抗菌肽。当前的研究旨在确定作为ANCA新抗原的LAMP-2是否存在于AAV患者的NETs上，抗LAMP-2抗体对中性粒细胞凋亡率的影响以及自噬在抗LAMP-2抗体诱导的NET形成中的作用。使用免疫荧光显微镜、扫描电子显微镜或活细胞成像评估NET形成。使用荧光激活细胞分选（FACS）分析中性粒细胞凋亡率。使用LC3B积累和透射电子显微镜检测自噬。结果显示，在AAV患者的肾活检组织和中性粒细胞中观察到包含LAMP-2的NET形成增强。用抗LAMP-2抗体刺激的人中性粒细胞凋亡率显著降低，并且自噬抑制剂3-甲基腺嘌呤（3MA）和2-吗啉-4-基-8-苯基色原酮-4-酮（LY294002）减弱了这种作用。抗LAMP-2抗体刺激的NET形成不受分别为凋亡和坏死抑制剂的苄氧羰基-Val-Ala-Asp（OMe）-氟甲基酮（zVAD-fmk）和坏死抑制因子-1（Nec-1）的影响，但受到3MA和LY294002的抑制。此外，转化为LC3BII的LC3BI比例显著增加（P = 0.0057），并且在用抗LAMP-2抗体刺激的中性粒细胞中检测到大量具有自噬典型特征的空泡化。我们的结果提供了进一步的证据，表明自噬参与了人中性粒细胞中ANCA诱导的NET形成。
