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. 2015 Apr 29;180(3):408–418. doi: 10.1111/cei.12589

Neutrophil extracellular trap formation is associated with autophagy-related signalling in ANCA-associated vasculitis

S Tang *,1, Y Zhang *,1, S-W Yin *, X-J Gao *, W-W Shi *, Y Wang *, X Huang *, L Wang †, L-Y Zou †, J-H Zhao *, Y-J Huang *, L-Y Shan ‡, A S Gounni ‡, Y-Z Wu †, J-B Zhang *,✉
PMCID: PMC4449769  PMID: 25644394

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.

Keywords: anti-neutrophil cytoplasmic antibody, apoptosis, autophagy, neutrophil extracellular traps, vasculitis

Introduction

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of autoimmune diseases characterized by the abnormal infiltration of neutrophils, accumulation of unscavenged leucocytoclasis in perivascular tissues and fibrinoid necrosis of the vessel walls 1. Patients with AAV frequently exhibit rapidly progressive renal failure caused by crescentic glomerulonephritis (GN). Myeloperoxidase (MPO) and proteinase 3 (PR3) have been shown to be two major ANCA antigens 2. The lysosomal membrane protein-2 (LAMP-2) autoantibody represents an additional ANCA subtype 3,4.

ANCAs, which activate neutrophils through Fab and Fc fragments that adhere to endothelial cells and produce damage, is believed to play a direct role in AAV pathogenesis 5. Kain and colleagues recently provided evidence that another type of autoantibody, anti-LAMP-2 (directed against lysosomal membrane protein-2), may have a pathogenic role in AAV and molecular mimicry between LAMP-2 and the bacterial adhesion protein FimH 4. However, the prevalence of autoantibodies to LAMP-2 in AAV remains controversial 6,7. Our previous data showed that an anti-LAMP-2 antibody could activate neutrophils in vitro to release neutrophil extracellular traps (NETs) that contained autoantigens and anti-microbial peptides 8.

Neutrophils are the most abundant group of leucocytes in the circulation, and they have a very short lifespan before undergoing apoptosis. Neutrophils form the first line of defence against invading pathogens with several effector mechanisms, including phagocytosis, release of bactericidal products and reactive oxygen species (ROS) production 9. NET formation, also termed NETosis, represents a unique, recently discovered, innate immune defence mechanism characterized by the active release of chromatin fibres decorated with a variety of granular proteins to the extracellular space in response to bacteria 10. Although NETs are bactericidal and contribute to the innate host defence, excessive NET formation has been linked to autoimmune disease pathogenesis 11. Our and others' data have demonstrated that NETs trigger vasculitis and perpetuate the autoimmune response against neutrophil components, such as MPO, PR3 and cathelicidin LL37, in AAV patients with 8,12–14 and in animal models 15.

NETosis is a complex process that differs depending on the stimulus. Although the regulation of subcellular events during NETosis remains unclear, increasing evidence indicates that superoxide production, Raf/mitogen extracellular signal-regulated kinase (MEK)/extracellular signal-related kinase (ERK) activation, autophagy and histone citrullination could be engaged in NET formation 16. Considering that LAMP-2 is critical for autophagy 17, we hypothesized that autophagy-related signalling may be involved in anti-LAMP-2 antibody-induced NET formation.

Autophagy is a critical homeostatic mechanism involved in the clearance of damaged organelles or proteins and in cellular survival during periods of nutrient depletion, and provides an essential nutrient supply by recycling cytosolic macromolecules and organelles 18. PI3K signalling is crucial for the initiation of autophagy, whereas the fusion of early autophagosomes with lysosomes and the acidification of autophagosomes constitute a terminal event in autophagy. In contrast, PI3K inhibition with 3-methyladenine (3MA) or 2-morpholin-4-yl-8-phenylchromen-4-one (LY294002) has been suggested to inhibit autophagy 19.

Based on these findings, we sought to determine whether LAMP-2, as a novel ANCA-targeted autoantigen, was present on NETs in AAV patients and the influence of the anti-LAMP-2 antibody on neutrophil apoptosis while activating NET release. Furthermore, we investigated the role of autophagy-related signalling in anti-LAMP-2 antibody-stimulated NET formation.

Materials and methods

Subjects

Fifteen AAV patients diagnosed according to the Chapel Hill definition 20 were recruited from the Xinqiao Hospital Department of Nephrology of the Third Military Medical University. AAV patient demographic characteristics are presented in Table1. Peripheral blood collected from these patients and healthy controls (HC) as well as paraffin sections of renal biopsy specimens obtained from six patients with crescentic GN were included in the present study. All biopsies were reviewed and classified by an experienced nephropathologist based on the revised criteria for crescentic GN. PR3-ANCA and MPO-ANCA were evaluated via indirect immunofluorescence (IIF) (FA-1200-2010; Euroimmun, Singapore) using EUROBlot kits (DL-1200-6421-3G; Euroimmun), according to the manufacturer's instructions. The serum levels of creatinine (Cr) were determined using routine techniques. This study was approved by the ethics committee of Xinqiao Hospital, and all subjects provided written informed consent to participate in this study.

Table 1.

Demographic characteristics of the study subjects.

All AAV patients Crescentic+ AAV patients Healthy controls
Total number 15 6 10
Male/female, no. 8/7 3/3 4/6
Age, mean (range) years 48 (7–73) 50 (8–73) 41 (25–57)
MPO+, number 11 5 n.a.
PR3+, number 4 1 n.a.
Serum Cr, median (range), μmol/l 462·28 (52·0–947·3) 610·70 (165·7–947·3) n.a.
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AAV = anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis; MPO = myeloperoxidase; PR3 = proteinase 3; n.a. = not applicable.

Immunofluorescence analysis of kidney biopsies

Kidney needle biopsies from AAV patients with crescentic GN (n = 6) were fixed and embedded in paraffin. Subsequently, 5-μm sections were prepared and mounted on glass slides. After deparaffination using xylene, rehydration using an ethanol gradient and antigen retrieval using citrate buffer, we treated the specimens with blocking buffer and, subsequently, fluorescently labelled antibodies against LAMP-2, MPO or PR3 (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). Histone was stained using primary antibodies against histone H3 (Abcam, Hong Kong, China) followed by fluorescently labelled specific secondary antibodies. DNA was stained using 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China). These specimens were analysed using a Leica SP5 confocal microscope.

Human neutrophil isolation

Human neutrophils were isolated by density centrifugation using Polymorphprep™ (Axis-Shield, Oslo, Norway). Briefly, 5 ml of blood containing ethylenediamine tetraacetic acid (EDTA) was layered on 5 ml of Polymorphprep™. After 35 min of centrifugation at 500 rcf, the neutrophils were separated from the polymorphonuclear neutrophils (PMN)-rich pellet. The residual erythrocytes were then eliminated via red blood cell lysis. Neutrophil purity was routinely ∼95%, as assessed by CD16 (BioLegend, San Diego, CA, USA) labelling via cytometric analysis. Unless stated otherwise, the cells were resuspended in RPMI medium (phenol red-free) (neither calf nor human serum was used) supplemented with 1% penicillin/streptomycin; 5 × 105−106 neutrophils/ml were seeded on culture plates or glass coverslips for immunofluorescence analysis. The incubations were performed at 37°C in the presence of 5% CO2.

Immunofluorescence and quantification of NET formation

Neutrophils were seeded on lysine-coated glass slides in 24-well cell culture plates and incubated for 1 h to adhere to glass coverslips. In the stimulation experiments, cells were stimulated with phorbol myristate acetate (PMA) (100 nM) (Sigma-Aldrich, St Louis, MO, USA), an anti-LAMP-2 antibody (20 μg/ml, azide free) (Abcam), or an isotype-specific control antibody (20 μg/ml, azide-free mouse IgG1) (Abcam) for up to 180 min. For inhibition experiments, the neutrophils were pretreated with benzyloxycarbonyl-Val- Ala-Asp (OMe)-fluoromethylketone (zVAD-fmk) (10 μM), necrostatin-1(Nec-1) (10 μM), LY294002 (50 μM) (all from Sigma-Aldrich) or 3MA (5 mM) (InvivoGen, San Diego, CA, USA) for 15 min before anti-LAMP-2 antibody application. Subsequently, the cells were fixed and permeabilized. After rehydration with phosphate-bufferd saline (PBS) at room temperature, the cells were incubated in blocking buffer overnight at 4°C. Then, the specimens were incubated in fluorescein isothiocyanate (FITC)-labelled antibodies against LAMP-2, MPO or PR3. Chromatin was stained using a rabbit anti-histone H3 antibody and then stained with fluorescently labelled goat anti-rabbit immunoglobulin (Ig)G secondary antibodies. DNA was stained using DAPI. The method of NET formation quantification as reported previously 12,21. In brief, using a ×20 objective, the percentage of neutrophil-releasing NETs was quantified in blinded samples by evaluating neutrophils displaying expanded nuclei [nuclei without lobulation and exceeding the normal average diameter of DNA staining (10 μm)] and releasing DNA fibres in at least five random microscope fields. The image files were analysed with the Image pro-Plus version 6·0 software. The NET percentage was calculated as follows: NET-rate (%) = 100 × number of neutrophils displaying expanded nuclei and releasing DNA fibres/total number of neutrophils.

Scanning electron microscopy (SEM)

Freshly purified neutrophils were stimulated with 20 µg/ml anti-LAMP-2 antibody or isotype control antibody for up to 3 h, and then cells were fixed in 2·5% glutaraldehyde for 2 h at 4°C. After washing three times with physiological saline, the samples were dehydrated using a graded ethanol followed by a tert-butyl alcohol series, and then dried in a critical point dryer. The specimen surfaces were coated with a 5 nm platin/carbon layer using a thin layer evaporator.

Live cell imaging

Neutrophils were diluted to 2 × 106 cells/ml in a glass-bottomed culture dish for live cell observation (NEST, Shanghai, China) and incubated for 30 min at 37°C in the presence of 10 μg/ml Hoechst 33342 (Beyotime, Shanghai, China) and 1 μM Sytox green (Invitrogen, Grand Island, NJ, USA). Then, the cells were treated with anti-LAMP-2 antibody or isotype-specific control antibody for 180 min. The fluorescent signals were detected and imaged using a Leica SP5 workstation. Cell morphology was observed using differential interference contrast (DIC). The cells were monitored at various time-points, and multiple image stacks were captured every 3 min.

Fluorescence activated cell sorting (FACS) analysis

The neutrophil apoptosis rate was measured with flow cytometry (BD FACSCantoII) using an Annexin-V/PI apoptosis detection kit (4Abio, Beijing, China), according to the manufacturer's instructions.

Western blot analysis

Western blot analysis was performed on cells treated for 30 min. At this time-point, the most prominent conversion of LC3BI to LC3BII was observed after treatment with anti-LAMP-2 antibody. Neutrophils were pelleted and resuspended in lysis buffer supplemented with 1 mM phenylmethylsulphonyl fluoride (PMSF), and cell debris was removed via centrifugation after incubating the lysate for 30 min on ice. Then, 30 µg of protein was separated on 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Subsequently, overnight incubation of polyvinylidene difluoride (PVDF) membranes at 4°C in an anti-LC3B antibody (1 : 3000) (Novus, Littleton, CO, USA) was performed followed by probing with a horseradish peroxidase ( HRP)-conjugated secondary antibody (1 : 1000) for 1 h at room temperature.

Transmission electron microscopy (TEM)

After stimulation, the neutrophils were collected via centrifugation (1500 rpm for 10 min). Then, the cell pellet was fixed using 2·5% glutaraldehyde, post-fixed using 1% osmium tetroxide, contrast-stained with uranyl acetate and tannic acid, dehydrated, embedded on moulds containing pure Spurr's resin and allowed to solidify at 70°C. After polymerization, the specimens were sliced into 60-nm sections and contrast-stained with lead citrate.

Statistical analysis

The data are expressed as the means ± standard error of the mean (s.e.m.). Statistical analysis included unpaired Student's t-test and was performed using GraphPad Prism version 5 software. Probability values of P < 0·05 were considered to be statistically significant.

Results

NETs deposit in the kidneys of AAV patients with crescentic GN

To assess the role of NETs in renal inflammation, we examined a panel of paraffin-embedded tissue sections from kidney needle biopsies from AAV patients with crescentic GN and acute deterioration of kidney function (n = 6). As expected, we found typical components of NETs, which appeared as ‘cloud-like' or ‘string-like' stained DNA structures and co-localization of histone and granular proteins, such as MPO (Fig. 1a,f) and PR3 (Fig. 1b,g), as reported previously 12. We also detected LAMP-2, a novel targeted ANCA autoantigen, observed on NETs located to affected glomeruli (Fig. 1c) and in the interstitium (Fig. 1e) of kidney biopsies from AAV patients with crescentic GN. However, only mild immunofluorescence signals positive for histone and MPO were observed in the renal tissues from AAV patients with non-crescentic GN (Fig. 1d,h). Our results suggest that NET deposition may be contributes to the crescentic formation in rapid progressive AAV subjects.

Fig 1.

Fig 1

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Neutrophil extracellular traps (NETs) deposit in kidney biopsies from anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) patients. Immunofluorescence analysis of paraffin-embedded kidney biopsy sections from AAV patients with or without crescentic glomerulonephritis (GN). NETs were identified by co-localization with extracellular DNA (blue), histones (red) and the neutrophil granule proteins myeloperoxidase (MPO), proteinase 3 (PR3) or lysosomal membrane protein-2 (LAMP-2) (green). (a–c) Co-localization of autoantigens (green), histone (red) and DNA (blue) in the glomerulus (defined by white line). (e–g) Co-localization of autoantigens (green), histone (red) and DNA (blue) in the renal tubule interstitium (defined by white line). (d,h) Co-localization of MPO (green), histone (red) and DNA (blue) in kidney biopsy sections from AAV patients without crescentic GN.

Enhanced NET formation in peripheral neutrophils from AAV patients

Kessenbrock et al. 12 suggested that ANCA-induced NET formation was involved in vasculitis and chronic autoimmunity in AAV patients. To evaluate NET formation and determine whether LAMP-2 co-localized to NETs in AAV patients, peripheral blood neutrophils were isolated from AAV patients (n = 15) and healthy controls (HC) (n = 10), and were then incubated in a CO2 incubator at 37°C for 1 h prior to NET detection with fluorescence imaging. We detected enhanced NET formation in AAV patient neutrophils, as several nuclei had altered their typical lobulated structure and released DNA fibres (Fig. 2a) compared with HC neutrophils (Fig. 2b). We found that 19·57 ± 1·051% of neutrophils from AAV patients produced NETs compared with 12·86 ± 1·528% of HC neutrophils (Fig. 2c, **P < 0·01). Next, we sought to determine whether the ANCA-targeted autoantigens PR3, MPO and LAMP-2 were present on AAV patient NETs. Immunofluorescence analysis revealed that LAMP-2, MPO and PR3 co-localized to the extracellular chromatin fibres from AAV patients (Fig. 2d). As reported recently, the peculiar structure of NETs favours neutrophil cytoplasmic antigen up-loading into myeloid dendritic cells (mDC) and induces ANCA production and autoimmunity 22. These data suggest that the intracellular autoantigens released from NETosis may cause neutrophil constituents, as endogenous danger signals expose the immune system and break immune tolerance.

Fig 2.

Fig 2

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Release of neutrophil extracellular traps (NETs) in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) patients peripheral blood neutrophils. (a,b) Fluorescence imaging of NET formation by neutrophils isolated from AAV patients (a) and healthy controls (HC) (b) after incubation for 1 h in vitro and staining for DNA using 4′,6-diamidino-2-phenylindole (DAPI). (c) Quantification of NET-forming neutrophils from HC or AAV patients via fluorescence microscopic analysis (**P < 0·01). (d) Immunofluorescence analysis of AAV-NETs identified by co-localization with extracellular DNA (blue), histones (red) and the neutrophil granule proteins myeloperoxidase (MPO), proteinase 3 (PR3) or lysosomal membrane protein-2 (LAMP-2) (green).

Anti-LAMP-2 antibody promotes NET formation in neutrophils

The human LAMP-2 antibody represents a novel ANCA subtype, although its role in AAV remains controversial 23. In contrast to MPO and PR3, human LAMP-2 is expressed abundantly on both neutrophil and endothelial surfaces and shuttles between lysosomes and the cell membrane; thus LAMP-2 is directly accessible to circulating antibodies 24. Kessenbrock et al. 12 reported that ANCAs, which are specific for MPO and PR3, induce primed human neutrophils to form NETs in vitro; therefore, we examined whether antibodies raised against human LAMP-2 have the same effect on unprimed neutrophils. Some neutrophils isolated from HC stimulated with H4B4 (20 μ/ml), a monoclonal anti-human LAMP-2 IgG, for 180 min, displayed typical NET characteristics, including ‘morphologically changed nuclei' and ‘web-like structures', compared with the isotype-specific control antibody treated neutrophils, which were visualized by SEM (Fig. 3a) and fluorescence microscopy (Fig. 3b). H4B4 (20 μ/ml) caused 25·5 ± 1·88% of these neutrophils to form NETs compared with 11·7 ± 0/94% for isotype-specific antibody (Fig. 3c, ***P < 0·001). To examine further the dynamic process of NET formation, H4B4-stimulated neutrophils were monitored via live cell imaging using the cell-impermeable DNA dye Sytox green together with the cell-permeable DNA dye Hoechst33342. Over time, NETs characterized by intracellular chromatin decondensation followed by plasma membrane disintegration were detected in neutrophils treated with H4B4 (Fig. 3d).

Fig 3.

Fig 3

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Neutrophils treated with anti-lysosomal membrane protein-2 (LAMP-2) antibody release neutrophil extracellular traps (NETs). (a,b) Healthy control (HC) neutrophils were incubated with H4B4 or an isotype-specific control antibody for 180 min. (a) Scanning electron microscopy (SEM) analysis of NET formation based on the appearance of flattened cells producing many membrane protrusions. (b) Fluorescence microscopic analysis of NET formation based on the appearance enlarged nuclei releasing extracellular DNA fibres stained with 4′,6-diamidino-2-phenylindole (DAPI). (c) Quantification of NET-forming neutrophils (***P < 0·001). (d) Neutrophils stimulated with H4B4 were monitored every 3 min for up to 180 min via live cell imaging for three parameters: morphology using differential interface contrast (DIC), chromatin using the cell-permeable DNA marker Hoechst 33342 (blue) and extracellular DNA using the cell-impermeable DNA dye Sytox Green (green). Representative time-points are shown.

Anti-LAMP-2 antibody inhibits spontaneous neutrophil apoptosis

To maintain homeostatic cell counts, aged neutrophils die constitutively via apoptosis 25. It has been reported that neutrophil apoptosis in AAV is dysregulated, thereby contributing to the accumulation of neutrophils in AAV tissues 26. Therefore, we evaluated the influence of the anti-LAMP-2 antibody on the spontaneous in-vitro apoptosis rate of neutrophils. The neutrophil apoptosis rate of healthy individuals treated with H4B4 or isotype-specific control antibody was quantified with FACS after 24 h in culture. The results revealed a significantly lower rate of apoptosis in H4B4-treated neutrophils compared with the isotype controls (Fig. 4a,c, ***P < 0·001). This result was consistent with the finding that anti-LAMP-2 antibody activate neutrophils to form NETs rather than undergo apoptosis. Interestingly, we found that pretreatment of neutrophils with 3MA or LY294002, which were employed as specific inhibitors of autophagy, attenuated this effect of H4B4 by elevating the apoptosis rate of the H4B4-treated neutrophils (Fig. 4b,c, *P < 0·05, **P < 0·01).

Fig 4.

Fig 4

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Anti-lysosomal membrane protein-2 (LAMP-2) antibody inhibits spontaneous neutrophil apoptosis. Apoptotic cells were detected via flow cytometry using annexin V-Alexa Fluor 488/PI. (a) The neutrophils were incubated in the isotype-specific control antibody or H4B4 for 24 h. (b) The neutrophils were pretreated with 3-methyladenine (3MA) or 2-morpholin-4-yl-8-phenylchromen-4-one (LY294002) (LY294002) for 15 min and then stimulated with H4B4 for 24 h. (c) The percentages of apoptotic neutrophils are shown (*P < 0·05; **P < 0·01; ***P < 0·001 compared with H4B4-treated cells).

Anti-LAMP-2 antibody-induced NET formation involves autophagy

To examine further the possible role of the autophagic machinery in NET release induced by anti-LAMP-2 antibody, 3MA and LY294002 were employed as PI3K inhibitors. Pretreatment (15 min) of HC neutrophils with these agents prior to H4B4 addition reduced significantly the percentage of cells releasing NETs (Fig. 5a,b). However, these H4B4-induced NETs were insensitive to zVAD-fmk, which prevents apoptosis, and Nec-1, an inhibitor of necrosis via the RPI1 kinase pathway (Fig. 5b). Together, these findings suggest that the autophagic machinery is involved in anti-LAMP-2 antibody-induced NET formation.

Fig 5.

Fig 5

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Phosphatidylinositol 3-kinase (PI3K) signalling is required for anti-lysosomal membrane protein-2 (LAMP-2) antibody-induced neutrophil extracellular trap (NET) formation. (a) Pretreatment with 3-methyladenine (3MA) or 2-morpholin-4-yl-8-phenylchromen-4-one (LY294002) for 15 min prevents the release of NETs from neutrophils treated withH4B4 for 180 min. NETs were detected by co-staining with DNA (blue) and myeloperoxidase (MPO) (green) and were visualized via immunofluorescence microscopy. (b) The effect of 3MA, LY294002, benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone (zVAD-fmk) or necrostatin-1 (Nec-1) pretreatment on the percentage of NET-releasing neutrophils stimulated with -H4B4 (*P < 0·05; n.s. = non-significant compared with H4B4-treated cells).

Moreover, the activation of autophagy in neutrophils treated with H4B4 was monitored based on the localization of LC3B immunofluorescence. During the early period of autophagy induction, LC3B translocates from the cytoplasm to newly formed autophagosomes 27. Within 30 min of H4B4 stimulation, we observed punctate LC3B structures, which are typical of autophagy, and this effect was diminished by 3MA (Fig. 6a). The ability of H4B4 to promote autophagy was demonstrated further via immunoblotting for LC3B (Fig. 6b, **P < 0·01). It has been reported that many cell types exhibit massive vacuolization during autophagy-associated cell death 28. TEM analysis of neutrophils revealed the presence of massive vacuolization typical of autophagy when neutrophils were treated with H4B4 for 30 min (Fig. 6c). These vacuoles were not observed in isotype-specific antibody-treated neutrophils, which are characterized by multi-lobulated nuclei and various types of granules (Fig. 6d).

Fig 6.

Fig 6

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Anti-lysosomal membrane protein-2 (LAMP-2) antibody promotes autophagy in neutrophils. (a) Immunofluorescence staining for LC3B in neutrophils treated with H4B4 or the isotype-specific control antibody for 30 min. Autophagy was assessed based on LC3B-positive autophagosome formation. DNA was labelled using 4′,6-diamidino-2-phenylindole (DAPI) (blue), and LC3B was detected using a polyclonal anti-LC3B antibody (red). (b) Analysis and quantification of the conversion from LC3BI to LC3BII in neutrophils treated the isotype control antibody (lane I) or H4B4 (lane II) for 30 min and the effect of 3-methyladenine (3MA) (lane III) and 2-morpholin-4-yl-8-phenylchromen-4-one (LY294002) (lane IV) on H4B4-treated cells, as assessed by immunoblotting for LC3B (**P < 0·01 compared with H4B4-treated cells). (c,d) Transmission electron microscopy (TEM) shows massive vacuolization in the neutrophils treated with H4B4 for 30 min (c) but not in the isotype control antibody-treated cells (d).

Discussion

NETosis plays an important role in autoantigen exposure to the immune system and producs destructive necrotizing vasculitis in various conditions, such as systemic lupus erythematosus (SLE) 29 and AAV 12. LAMP-2 is a novel target of ANCA along with the more commonly known targets PR3 and MPO. LAMP-2 is a major constituent of the lysosomal membrane that is found not only lining neutrophil granules but also on the cell surface, and plays a role in autophagy 23.

This study is the first to demonstrate that LAMP-2 ANCA-induced NET formation involves the initiation of autophagic machinery. Our experimental data indicate that anti-LAMP-2 antibody inhibited spontaneous apoptosis and activated neutrophil autophagy while activating in vitro NETs release in neutrophils, and that PI3K signalling inhibition reduced autophagy and hindered NET release. We also provide evidence that enhanced NETs were deposited in kidney biopsies and released by peripheral neutrophils in AAV patients, and that LAMP-2 co-localized to NETs in AAV patients.

Neutrophils can release ROS and proteases into the tissue microenvironment leading to tissue inflammation and injury, which are thought to serve as the critical effector cells responsible for vascular necrotizing inflammation in AAV. When cultured in vitro, these cells undergo spontaneous apoptosis in the absence of sufficient concentrations of neutrophil survival factors 30. Neutrophils are removed from tissues via necrosis or apoptosis followed by marophagy phagocytosis 31. Defects in apoptotic pathways could lead to the persistence of autoreactive T or B cells and the development of autoimmune diseases, including AAV 32. In the past 20 years, apoptosis and secondary necrosis have been considered the major sources of autoantigens in systemic autoimmune diseases pathogenesis. Because NETosis represents an effective illustration of the mechanism by which intracellular autoantigens are exposed to immune responses, NETosis has recently gained increasing interest regarding AAV 33,34. NETs composed largely of intracellular and intranuclear materials could be potential autoantigens to the immune system 35 and directly injure vascular endothelial cells 36. Enhanced NET formation has been observed in SLE 37, rheumatoid arthritis (RA) 35, dermatomyositis (DM) 38 and AAV 12, among others. Our study shows that enhanced NETs were deposited in AAV patient kidney biopsies and released by peripheral neutrophils, which is a phenomenon that exposes normally intracellular autoantigens to the immune system, suggesting their pathogenic role in inflammation induction in AAV. Moreover, these fibrous DNA deposits, which contain the targeted autoantigens LAMP-2, MPO and PR3 that render the NET structures highly immunogenic, are primed to activate plasmacytoid dendritic cells (pDCs) and autoreactive B cells in a Toll-like receptor 9-dependent manner, as reported previously 39,40.

Apoptosis and necrosis are two major forms of neutrophil death. In addition, autophagy has been implicated recently in neutrophil cell death 41. Autophagy is a well-conserved, essential, intracellular degradation process that is known to regulate protein and organelle turnover and that constitutes a crucial mechanism of innate immunity in many cell types 42,43. Autophagy is also involved adaptive immune responses, playing a key role in antigen processing for major histocompatibility complex (MHC) presentation and in lymphocyte development, survival and proliferation, which appears to be regulated abnormally in autoimmune diseases 44. In this study, zVAD-fmk and Nec-1 were used as apoptosis and necrosis inhibitors, respectively, whereas 3MA and LY294002 were utilized as PI3K inhibitors. We found that neither inhibition of caspases by zVAD-fmk nor RIP1 kinases inhibition by Nec-1 affected anti-LAMP-2 antibody-induced NET formation. These results were consistent with NETosis differing between apoptosis and necrosis not only morphologically but also biochemically. However, both 3MA and LY294002 significantly inhibited anti-LAMP-2 antibody-induced NET formation. Therefore, we propose that autophagic machinery is required for the release of these extracellular structures. This suggestion is consistent with a recent report demonstrating that the inhibition of autophagy in neutrophils treated with PMA prevented NETosis and led to apoptotic cell death 45. However, PI3K signalling inhibition did not completely inhibit anti-LAMP-2 antibody-induced NET formation to control levels. Other regulatory mechanisms must underlie NETosis. At least three different types of autophagy exist, including macroautophagy, chaperone-mediated autophagy (CMA) and microautophagy. Macroautophagy, usually referred to simply as autophagy, is the subject of our study. CMA and microautophagy did not complicated in this study and should be investigated in further studies. Genetic data on the mechanisms of enhanced NET formation in AAV patients are lacking. Therefore, great efforts will be required in this area.

In conclusion, our data indicate that NETs are formed during autoimmune AAV attacks and suggest an association between anti-LAMP-2-antibody-induced NET release and autophagy-related signalling. Further studies are required to evaluate the in-vivo autophagic mechanism in NET-forming neutrophils of AAV patients and to investigate the precise signalling pathways leading to ANCA-stimulated NETosis.

Acknowledgments

The authors thank all patients and healthy volunteers who participated in this study. We thank our associate Ms Jin Peng for providing assistance in the confocal microscopic and live cell imaging analyses. We also thank Ms Xiaolan Fu for her assistance in the FACS analyses. This study was supported by the National Science Foundation of China (30971366), the International Cooperation Projects of Chongqing Science & Technology Committee (CSTC201110004) and the Clinical Research Project of the Third Military Medical University (2011XLC37).

Author contributions

J. B. Z. was involved in all aspects of the study conception, design and direction and provided the final approval of the submitted manuscript. J. B. Z., A. S. G. and Y. Z. W. designed the study. J. B. Z., S. T., L. Y. S., L. W. and S. W. Y. were involved in data acquisition, data analysis and interpretation of the results. S. T., Y. Z., X. J. G., W. W. S. and L. Y. Z. performed the laboratory tests. Y. W., X. H., J. H. Z. and Y. J. H. were involved in the clinical analysis. All authors read and approved the manuscript.

Disclosure

The authors declare that they have no conflicts of interest.

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