title: Neutrophil extracellular trap formation is associated with autophagy-related signalling in ANCA-associated vasculitis.
abstract: 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.
authors: Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, China.
authors: S
authors: S
authors: Tang
authors: Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, China.
authors: Y
authors: Y
authors: Zhang
authors: Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, China.
authors: S-W
authors: SW
authors: Yin
authors: Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, China.
authors: X-J
authors: XJ
authors: Gao
authors: Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, China.
authors: W-W
authors: WW
authors: Shi
authors: Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, China.
authors: Y
authors: Y
authors: Wang
authors: Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, China.
authors: X
authors: X
authors: Huang
authors: Institute of Immunology of PLA, Third Military Medical University, Chongqing, 400038, China.
authors: L
authors: L
authors: Wang
authors: Institute of Immunology of PLA, Third Military Medical University, Chongqing, 400038, China.
authors: L-Y
authors: LY
authors: Zou
authors: Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, China.
authors: J-H
authors: JH
authors: Zhao
authors: Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, China.
authors: Y-J
authors: YJ
authors: Huang
authors: Department of Immunology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, R3E 0T5, Canada.
authors: L-Y
authors: LY
authors: Shan
authors: Department of Immunology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, R3E 0T5, Canada.
authors: A S
authors: AS
authors: Gounni
authors: Institute of Immunology of PLA, Third Military Medical University, Chongqing, 400038, China.
authors: Y-Z
authors: YZ
authors: Wu
authors: Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, China.
authors: J-B
authors: JB
authors: Zhang
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D056648:Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis / Q000276:immunology* / Q000378:metabolism* / Q000473:pathology; D017209:Apoptosis / Q000276:immunology; D001323:Autoantibodies / Q000276:immunology; D001343:Autophagy / Q000276:immunology; D002648:Child; D065206:Extracellular Traps / Q000276:immunology* / Q000378:metabolism*; D005260:Female; D005921:Glomerulonephritis / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007668:Kidney / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D052119:Lysosomal-Associated Membrane Protein 2 / Q000276:immunology; D008297:Male; D008875:Middle Aged; D009504:Neutrophils / Q000276:immunology* / Q000378:metabolism* / Q000648:ultrastructure; D015398:Signal Transduction; D055815:Young Adult
chemical_list: D001323:Autoantibodies; D052119:Lysosomal-Associated Membrane Protein 2
keywords: anti-neutrophil cytoplasmic antibody; apoptosis; autophagy; neutrophil extracellular traps; vasculitis
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