ANCA-induced Neutrophil Activation Research Articles

FTY720 ameliorates experimental MPO-ANCA-associated vasculitis by regulating fatty acid oxidation via the neutrophil PPARα-CPT1a pathway.

Increasing studies demonstrated the importance of C5a and anti-neutrophil cytoplasmic antibody (ANCA)-induced neutrophil activation in the pathogenesis of ANCA-associated vasculitis (AAV). Sphingosine-1-phosphate (S1P) acts as a downstream effector molecule of C5a and enhances neutrophil activation induced by C5a and ANCA. The current study investigated the role of a S1P receptor modulator, FTY720, in experimental autoimmune vasculitis (EAV) and explored the immunometabolism-related mechanisms of FTY720 in modulating ANCA-induced neutrophil activation. The effects of FTY720 in EAV were evaluated by quantifying haematuria, proteinuria, crescent formation, tubulointerstitial injury and pulmonary haemorrhage. RNA sequencing of renal cortex and gene enrichment analysis were performed. The proteins of key identified pathways were analysed in neutrophils isolated from peripheral blood of patients with active AAV and normal controls. We assessed the effects of FTY720 on ANCA-induced neutrophil respiratory burst and neutrophil extracellular traps formation (NETosis). FTY720 treatment significantly attenuated renal injury and pulmonary haemorrhage in EAV. RNA sequencing analyses of renal cortex demonstrated enhanced fatty acid oxidation (FAO) and peroxisome proliferator-activated receptor (PPAR) signalling in FTY720-treated rats. Compared with normal controls, patients with active AAV showed decreased FAO in neutrophils. FTY720-treated differentiated HL-60 cells showed increased expression of carnitine palmitoyltransferase 1a (CPT1a) and PPARα. Blocking or knockdown of CPT1a or PPARα in isolated human neutrophils and HL-60 cells reversed the inhibitory effects of FTY720 on ANCA-induced neutrophil respiratory burst and NETosis. FTY720 attenuated renal injury in EAV through upregulating FAO via the PPARα-CPT1a pathway in neutrophils, offering potential immunometabolic targets in AAV treatment.

Regulation of NETosis and Inflammation by Cyclophilin D in Myeloperoxidase-Positive Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is pathologically characterized by focal fibrinoid necrosis, in which ANCA-mediated neutrophil extracellular trap (NET) formation and subsequent endothelial cell necrosis occur. Cyclophilin D (CypD) plays an important role in mediation of cell necrosis and inflammation via the opening of mitochondrial permeability transition pores. This study was undertaken to examine the role of CypD in AAV pathogenesis. We assessed the role and mechanism of CypD in ANCA-stimulated neutrophils in vitro by immunostaining and electron microscopy observation. We performed a comprehensive RNA-sequencing analysis on ANCA-treated murine neutrophils. To investigate the role of CypD in vivo, we assessed disease features in CypD-knockout mice and wild-type mice using 2 different murine AAV models: anti-myeloperoxidase IgG transfer-induced AAV and spontaneous AAV. In vitro experiments showed that pharmacologic and genetic inhibition of CypD suppressed ANCA-induced NET formation via the suppression of reactive oxygen species and cytochrome c release from the mitochondria. RNA-sequencing analyses in ANCA-treated murine neutrophils revealed the involvement of inflammatory responses, with CypD deficiency reducing ANCA-induced alterations in gene expression. Furthermore, analyses of upstream regulators revealed the relevance of intracellular calcium (CypD activator) and cyclosporin (CypD inhibitor) in ANCA stimulation, indicating that the CypD-dependent opening of mitochondrial permeability transition pores is associated with ANCA-induced neutrophil activation and NETosis. In both AAV mouse models, the genetic deletion of CypD ameliorated crescentic glomerulonephritis via the inhibition of CypD-dependent neutrophil and endothelial necrosis. CypD targeting is a novel and specific therapeutic strategy for AAV via the resolution of necrotizing vasculitis.

MO241: Nets and Terminal Complement Pathway as Potential Biomarkers for Complement Overactivation Assessment in Anca-Associated Vasculitis

Abstract BACKGROUND AND AIMS Clinical, in vitro, and animal model-derived evidence has demonstrated a critical involvement of the alternative complement pathway (aCP) in the pathogenesis of ANCA-associated vasculitis (AAV). In this regard, neutrophil extracellular traps (NETs) have been suggested to be a key element between ANCA-induced neutrophil activation and aCP [1]. However, the role of the terminal complement pathway (tCP) is less well studied [2]. METHOD A prospective, observational, multicenter study analyzing first episodes and relapses of patients with AAV, with a minimum follow-up of 6 months, was performed. Blood samples were collected at diagnosis (AAV-t1) and at remission (AAV-t2). Control population consisted of age and sex-matched individuals. Complement activation was assessed by analyzing the complement membrane attack complex (C5b-9) deposition on cultured endothelial cells (HMEC-1), by immunofluorescence, after exposing them to activated plasma (a-plasma: obtained by mixing patient's citrated plasma with healthy subjects’ sera pool, 1:1). C5b-9 deposits induced by patients’ a-plasma were calculated as percentage of labeled area with respect to the total area analyzed. Results from patient and control samples were expressed as fold increase (mean ± SEM) versus those obtained with the pool of a-plasma from healthy subjects. Plasma levels of tCP and aCP soluble factors, such as sC5b-9 and sFBb (respectively), were also measured (mean ± SEM). Circulating NETs were indirectly measured by quantifying circulant dsDNA plasmatic concentration (mean ± SEM) as a NET surrogate. RESULTS The present results were obtained with samples from 13 AAV-MPO patients who achieved complete remission (38% men, age 63 ± 14 years) and 10 controls (45% men, age 66 ± 6 years). At AAV-t1, there was a statistically significant increase (P < .05) of C5b-9 deposition on HMEC-1 in response to patients’ a-plasma (fold increase of 5.3 ± 1.3) compared with control samples (fold increase of 1.2 ± 0.2). Samples obtained at AAV-t2 induced less C5b-9 deposition than at AAV-t1 (fold increase of 0.9 ± 0.2; P < .05), with values similar to controls. Regarding soluble factors, levels of both sC5b-9 and sFBb were significantly increased in AAV-t1 (1882 ± 418 ng/mL and 3.2 ± 0.4 µg/mL, respectively; P < .05) versus AAV-t2 (852 ± 104 ng/mL and 1.9 ± 0.2 µg/mL, respectively; P < .05). At AAV-t2, levels were similar to controls (708 ± 42 ng/mL for sC5b-9 and 2.4 ± 0.2 µg/mL for sFBb). Circulating NETs were also increased in AAV-t1 (22.2 ± 3.5 µg/mL) compared with both AAV-t2 and controls (13.6 ± 1.1 µg/mL and 13.7 ± 0.3 µg/mL, respectively; P < .05). Moreover, NETs in AAV-t1 presented a significant correlation with sFBb levels, both in AAV-t1 (r = 0.709; P < .05) and in AAV-t2 (r = 0.585; P < .05). CONCLUSION There is a relationship between NETs, AAV activity and sFBb, which supports the role of NETs and cAP in AAV pathogenesis. Moreover, differences in C5b-9 deposition between the two stages of the disease suggest that tCP may be dysregulated in AAV. Further characterization of this dysregulation may lead to new diagnostic or disease activity biomarkers, as well as new therapeutic options for the management of patients with AAV.

Spleen tyrosine kinase inhibition is an effective treatment for established vasculitis in a pre-clinical model

The anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitides (AAV) are a group of life-threatening multi-system diseases characterized by necrotising inflammation of small blood vessels and crescentic glomerulonephritis. ANCA are thought to play a direct pathogenic role. Previous studies have shown that spleen tyrosine kinase (SYK) is phosphorylated during ANCA-induced neutrophil activation in vitro. However, the role of SYK in vivo is unknown. Here, we studied its role in the pathogenesis of experimental autoimmune vasculitis, a pre-clinical model of myeloperoxidase-ANCA-induced pauci-immune systemic vasculitis in the Wistar Kyoto rat. Up-regulation of SYK expression in inflamed renal and pulmonary tissue during early autoimmune vasculitis was confirmed by immunohistochemical and transcript analysis. R406, the active metabolite of fostamatinib, a small molecule kinase inhibitor with high selectivity for SYK, inhibited ANCA-induced pro-inflammatory responses in rat leucocytes in vitro. In an in vivo study, treatment with fostamatinib for 14 days after disease onset resulted in rapid resolution of urinary abnormalities, significantly improved renal and pulmonary pathology, and preserved renal function. Short-term exposure to fostamatinib did not significantly affect circulating myeloperoxidase-ANCA levels, suggesting inhibition of ANCA-induced inflammatory mechanisms in vivo. Finally, SYK expression was demonstrated within inflammatory glomerular lesions in ANCA-associated glomerulonephritis in patients, particularly within CD68+ve monocytes/macrophages. Thus, our data indicate that SYK inhibition warrants clinical investigation in the treatment of AAV.

Complement Factor H Inhibits Anti-Neutrophil Cytoplasmic Autoantibody-Induced Neutrophil Activation by Interacting With Neutrophils.

Our previous study demonstrated that plasma levels of complement factor H (FH) were inversely associated with the disease activity of patients with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV). In addition to serving as an inhibitor of the alternative complement pathway, there is increasing evidence demonstrating direct regulatory roles of FH on several cell types. Here, we investigated the role of FH in the process of ANCA-mediated activation of neutrophils and neutrophil–endothelium interaction. We demonstrated that FH bound to neutrophils by immunostaining and flow cytometry. Interestingly, ANCA-induced activation of neutrophils, including respiratory burst and degranulation, was inhibited by FH. Although FH enhanced neutrophils adhesion and migration toward human glomerular endothelial cells (hGEnCs), it inhibited ANCA-induced activation of neutrophils in the coculture system of hGEnCs and neutrophils. Moreover, the activation and injury of hGEnCs, reflected by the level of endothelin-1 in the supernatant of cocultures, was markedly reduced by FH. However, we found that FH from patients with active AAV exhibited a deficient ability in binding neutrophils and inhibiting ANCA-induced neutrophil activation in fluid phase and on endothelial cells, as compared with that from healthy controls. Therefore, our findings indicate a novel role of FH in inhibiting ANCA-induced neutrophil activation and protecting against glomerular endothelial injury. However, FH from patients with active AAV are deficient in their ability to bind neutrophils and inhibit neutrophil activation by ANCA. It further extends the current understanding of the pathogenesis of AAV, thus providing potential clues for intervention strategies.

Necroptosis controls NET generation and mediates complement activation, endothelial damage, and autoimmune vasculitis

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) constitutes life-threatening autoimmune diseases affecting every organ, including the kidneys, where they cause necrotizing crescentic glomerulonephritis. ANCA activates neutrophils and activated neutrophils damage the endothelium, leading to vascular inflammation and necrosis. Better understanding of neutrophil-mediated AAV disease mechanisms may reveal novel treatment strategies. Here we report that ANCA induces neutrophil extracellular traps (NETs) via receptor-interacting protein kinase (RIPK) 1/3- and mixed-lineage kinase domain-like (MLKL)-dependent necroptosis. NETs from ANCA-stimulated neutrophils caused endothelial cell (EC) damage in vitro. This effect was prevented by (i) pharmacologic inhibition of RIPK1 or (ii) enzymatic NET degradation. The alternative complement pathway (AP) was recently implicated in AAV, and C5a inhibition is currently being tested in clinical studies. We observed that NETs provided a scaffold for AP activation that in turn contributed to EC damage. We further established the in vivo relevance of NETs and the requirement of RIPK1/3/MLKL-dependent necroptosis, specifically in the bone marrow-derived compartment, for disease induction using murine AAV models and in human kidney biopsies. In summary, we identified a mechanistic link between ANCA-induced neutrophil activation, necroptosis, NETs, the AP, and endothelial damage. RIPK1 inhibitors are currently being evaluated in clinical trials and exhibit a novel therapeutic strategy in AAV.

High mobility group box-1 contributes to anti-myeloperoxidase antibody-induced glomerular endothelial cell injury through a moesin-dependent route

BackgroundOur previous study found that circulating and urinary levels of high mobility group box-1 (HMGB1) were closely associated with disease activity in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Moreover, HMGB1 participates in ANCA-induced neutrophil activation. Cross-reactivity between moesin and anti-myeloperoxidase (MPO) antibody has been reported in both human and mouse. The current study investigated whether HMGB1 participated in MPO-ANCA-induced glomerular endothelial cell (GEnC) injury, which is one of the most important aspects in the pathogenesis of AAV.MethodsThe effects of HMGB1 on expression of moesin on GEnCs and anti-MPO antibody binding to GEnCs were measured. MPO expression on GEnCs was explored. The effects of HMGB1 in MPO-ANCA induced GEnC injury were measured, during which the role of moesin was explored. Antagonists for various relevant receptors were employed.ResultsSera from AAV patients at the active stage could mediate GEnC injury, while this effect could be attenuated by preblocking HMGB1. HMGB1 could increase the expression of moesin on GEnCs and the binding of anti-MPO antibody to moesin. The colocalization of moesin expression and anti-MPO antibody binding can be detected. Little, if any, MPO was expressed in GEnCs. HMGB1 increased GEnC activation and injury in the presence of patient-derived MPO-ANCA-positive IgGs through moesin. The effects of HMGB1 on expression of moesin on GEnCs, anti-MPO antibody binding to GEnCs, GEnC activation and injury were mainly toll like receptor 4 (TLR4) dependent.ConclusionsHMGB1 can increase the expression of moesin but not MPO on GEnCs, and can further participate in MPO-ANCA-induced GEnC activation and injury by cross-reactivity between moesin and anti-MPO antibody.

Involvement of high mobility group box 1 in the activation of C5a-primed neutrophils induced by ANCA

C5a plays a central role in antineutrophil cytoplasmic antibody (ANCA)-mediated neutrophil recruitment and activation. A previous study showed that C5a played a crucial role in the regulation of high mobility group box 1 (HMGB1) release from human neutrophils. The current study further investigated the interaction between C5a and HMGB1 in ANCA-induced neutrophil activation. The effects of HMGB1 inhibitors on the translocation of ANCA antigens, ANCA-induced respiratory burst and degranulation of C5a-primed neutrophils were tested. We found that blocking HMGB1 decreased C5a-mediated translocation of ANCA antigens, as well as ANCA-induced respiratory burst and degranulation of C5a-primed neutrophils. Further study showed that supernatant of C5a-primed neutrophils, which contained HMGB1, also caused translocation of ANCA antigens of primary neutrophils, whereas blocking HMGB1 decreased the translocation. In conclusion, blocking HMGB1 may attenuate ANCA-induced activation of C5a-primed neutrophils. The interaction between HMGB1 and C5a might play an important role in ANCA-induced neutrophil activation.

High mobility group box 1 contributes to anti-neutrophil cytoplasmic antibody-induced neutrophils activation through receptor for advanced glycation end products (RAGE) and Toll-like receptor 4.

IntroductionHigh mobility group box-1 (HMGB1), a typical damage-associated molecular pattern (DAMP) protein, is associated with inflammatory conditions and tissue damage. Our recent study found that circulating HMGB1 levels could reflect the disease activity of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The current study aimed to investigate whether HMGB1 participated in ANCA-induced neutrophil activation, which is one of the most important pathogenic aspects in the development of AAV.MethodsThe various effects of HMGB1 in ANCA-induced neutrophil activation were measured. Antagonists for relevant receptors and signaling molecules were employed.ResultsANCA antigens translocation on neutrophils primed with HMGB1 was significantly higher than non-primed neutrophils. The levels of respiratory burst and degranulation increased significantly in HMGB1-primed neutrophils activated with ANCA-positive IgG, as compared with non-primed neutrophils. Furthermore, blocking Toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE), rather than TLR2, resulted in a significant decrease in HMGB1-induced ANCA antigens translocation, respiratory burst and degranulation. Similar effects were also found when blocking MyD88 and NF-κB.ConclusionsHMGB1 could prime neutrophils by increasing ANCA antigens translocation, and the primed neutrophils could be further induced by ANCA, resulting in the respiratory burst and degranulation. This process is TLR4- and RAGE-dependent through the MyD88/NF-κB pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0587-4) contains supplementary material, which is available to authorized users.

SYK inhibition in experimental autoimmune vasculitis and its glomerular expression in ANCA-associated vasculitis

BackgroundThe anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitides (AAV) are a group of life-threatening multisystemic diseases characterised by necrotising inflammation of small blood vessels and crescentic glomerulonephritis. Existing treatments are not always effective and are often complicated by substantial toxicity. Previous studies have shown that spleen tyrosine kinase (SYK) is phosphorylated during ANCA-induced neutrophil activation, and thus it represents a potential therapeutic target. We aimed to investigate the role of SYK in the pathogenesis of an in-vivo model of AAV, and to establish whether SYK is expressed in human disease. MethodsWe studied the effect of fostamatinib, a small molecule inhibitor selective for SYK, in experimental autoimmune vasculitis (EAV), which is a rodent model of AAV that is induced by immunisation of Wistar Kyoto rats with the ANCA target antigen, myeloperoxidase. Animals (eight per group) with established disease, as confirmed by onset of haematuria and proteinuria 4 weeks after immunisation, were treated with vehicle, fostamatinib 20 mg/kg, or fostamatinib 30 mg/kg by twice daily oral gavage from week 4 to 6 and then assessed for disease severity at the end of week 6. We also used immunohistochemical techniques to analyse SYK expression and activation in human renal biopsy specimens from patients with ANCA-associated glomerulonephritis. FindingsIn EAV, at the end of week 6, there was a dose-dependent reduction in proteinuria (median 2·88 mg/day [IQR 1·80–6·37] vs 0·13 [0·00–0·78] vs 0·23 [0·00–0·59] in vehicle, fostamatinib 20 mg/kg, and fostamatinib 30 mg/kg groups, respectively; p=0·0004), haematuria (3 arbitrary units [AU] [2–3] vs 0 [0–0·38] vs 0 [0–0·038], p=0·0018), glomerular histological abnormalities (12% [3–29] vs 5 [2–9] vs 0 [0–6], p=0·0262), glomerular macrophage infiltration (1·77 [1·28–2·58] AU vs 0·25 [0·20–0·41] vs 0·20 [0·06-0·41], p=0·0004), lung haemorrhage severity (3 AU [2·0–3·8] vs 1 [0·3–1·8] vs 0 [0·1–0·4], p<0·0001), and haemosiderin deposition in lung tissue (3·06 AU [0·93–4·14] vs 0·48 [0·17–0·70] vs 0·19 [0·06–0·42], p=0·0087). Immunohistochemical analysis confirmed SYK expression in human ANCA-associated glomerulonephritis. Staining for phospho-SYK demonstrated SYK activation that localised to diseased lesions (segmental necrosis, areas of extracapillary proliferation). Glomerular SYK expression correlated with histological class of disease (crescentic 3·0% [2·1–3·8], mixed 1·4% [0·92–2·4], focal 1·1% [0·7–1·8], sclerotic 0·3% [0·1–0·5]; p=0·01). InterpretationOur results suggest that, in a preclinical model, SYK inhibition with fostamatinib is an effective treatment for crescentic glomerulonephritis and lung haemorrhage, the life-threatening manifestations of AAV, even after onset of disease. SYK is activated in human ANCA-associated glomerulonephritis, and expression correlates with disease severity, suggesting that SYK contributes to the pathogenesis of human disease. These observations support the potential investigation of targeting SYK in clinical studies of AAV. FundingUK Medical Research Council.

Complement in ANCA-Associated Vasculitis

Antineutrophil cytoplasmic autoantibodies (ANCA) are the likely cause for necrotizing small-vessel vasculitis and crescentic glomerulonephritis. Unlike other forms of crescentic glomerulonephritis induced by immune complexes or anti-glomerular basement membrane antibodies that have conspicuous vessel wall immunoglobulin and complement, there is a paucity, although usually not an absence, of vessel wall immunoglobulin and complement in ANCA-associated glomerulonephritis. Despite this comparatively lower level and more localized distribution of vessel wall complement, experimental and clinical observations strongly incriminate alternative complement pathway activation as critically important in the pathogenesis of ANCA disease. Experimental data in animal models and in vitro experiments has shown that primed neutrophils are activated by ANCA, which generates C5a, which engages C5a receptors on neutrophils. This attracts and in turn primes more neutrophils for activation by ANCA. In patients with ANCA disease, plasma levels of C3a, C5a, soluble C5b-9, and Bb have been reported to be higher in active disease than in remission, whereas no difference was reported in plasma C4d in active versus ANCA disease remission. Thus, experimental and clinical data support the hypothesis that ANCA-induced neutrophil activation activates the alternative complement pathway and generates C5a. C5a not only recruits additional neutrophils through chemotaxis but also primes neutrophils for activation by ANCA. This creates a self-fueling inflammatory amplification loop that results in the extremely destructive necrotizing vascular injury.

P38MAPK, ERK and PI3K Signaling Pathways Are Involved in C5a-Primed Neutrophils for ANCA-Mediated Activation

BackgroundThe complement system is one of the important contributing factors in the development of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). C5a and the neutrophil C5a receptor play a central role in antineutrophil cytoplasmic antibody (ANCA)-mediated neutrophil recruitment and activation. The current study further investigated the signaling pathways of C5a-mediated priming of human neutrophils for ANCA-induced neutrophil activation.Methodology/Principal FindingsThe effects of the p38 mitogen-activated protein kinase (p38MAPK) inhibitor (SB202190), extracellular signal-regulated kinase (ERK) inhibitor (PD98059), c-Jun N-terminal kinase (JNK) inhibitor (6o) and phosphoinositol 3-kinase (PI3K) inhibitor (LY294002) were tested on respiratory burst and degranulation of C5a-primed neutrophils activated with ANCA, as well as on C5a-induced increase in expression of membrane-bound PR3 (mPR3) on neutrophils. For C5a-primed neutrophils for MPO-ANCA-induced respiratory burst, the mean fluorescence intensity (MFI) value was 254.8±67.1, which decreased to 203.6±60.3, 204.4±36.7, 202.4±49.9 and 188±47.9 upon pre-incubation with SB202190, PD98059, LY294002 and the mixture of above-mentioned three inhibitors (compared with that without inhibitors, P<0.01, P<0.05, P<0.01 and P<0.05), respectively. For PR3-ANCA-positive IgG, the MFI value increased in C5a-primed neutrophils, which decreased upon pre-incubation with above-mentioned inhibitors. The lactoferrin concentration increased in C5a-primed neutrophils induced by MPO or PR3-ANCA-positive IgG supernatant and decreased upon pre-incubation with above-mentioned three inhibitors. mPR3 expression increased from 923.3±182.4 in untreated cells to 1278.3±299.3 after C5a treatment and decreased to 1069.9±188.9, 1100±238.2, 1092.3±231.8 and 1053.9±200.3 by SB202190, PD98059, LY294002 and the mixture of above-mentioned three inhibitors (compared with that without inhibitors, P<0.01, P<0.05, P<0.01 and P<0.01), respectively.Conclusions/SignificanceActivation of p38MAPK, ERK and PI3K are important steps in the translocation of ANCA antigens and C5a-induced activation of neutrophils by ANCA.

Effects of p38 mitogen-activated protein kinase inhibition on anti-neutrophil cytoplasmic autoantibody pathogenicity in vitro and in vivo

ObjectiveTo determine whether inhibition of p38 mitogen-activated protein kinase (p38MAPK) reduces the pathogenicity of anti-neutrophil cytoplasmic autoantibodies (ANCAs) in vitro and in vivo.MethodsThe effects of the p38MAPK-specific inhibitor AR-447 were...

IgG Glycan Hydrolysis Attenuates ANCA-Mediated Glomerulonephritis

Anti-neutrophil cytoplasmic autoantibodies (ANCA) directed against myeloperoxidase (MPO) and proteinase 3 (Pr3) are considered pathogenic in ANCA-associated necrotizing and crescentic glomerulonephritis (NCGN) and vasculitis. Modulation of ANCA IgG glycosylation may potentially reduce its pathogenicity by abolishing Fc receptor-mediated activation of leukocytes and complement. Here, we investigated whether IgG hydrolysis by the bacterial enzyme endoglycosidase S (EndoS) attenuates ANCA-mediated NCGN. In vitro, treatment of ANCA IgG with EndoS significantly attenuated ANCA-mediated neutrophil activation without affecting antigen-binding capacity. In a mouse model of anti-MPO IgG/LPS-induced NCGN, we induced disease with either unmodified or EndoS-treated (deglycosylated) anti-MPO IgG. In separate experiments, we administered EndoS systemically after disease induction with unmodified anti-MPO IgG. Pretreatment of anti-MPO IgG with EndoS reduced hematuria, leukocyturia, and albuminuria and attenuated both neutrophil influx and formation of glomerular crescents. After inducing disease with unmodified anti-MPO IgG, systemic treatment with EndoS reduced albuminuria and glomerular crescent formation when initiated after 3 but not 24 hours. In conclusion, IgG glycan hydrolysis by EndoS attenuates ANCA-induced neutrophil activation in vitro and prevents induction of anti-MPO IgG/LPS-mediated NCGN in vivo. Systemic treatment with EndoS early after disease induction attenuates the development of disease. Thus, modulation of IgG glycosylation is a promising strategy to interfere with ANCA-mediated inflammatory processes.

Circulating neutrophil gelatinase-associated lipocalin: a useful biomarker for assessing disease activity of ANCA-associated vasculitis

Biomarkers for assessing disease activity of patients with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) are important in identifying relapse and guiding treatment. ANCA-induced neutrophil activation and degranulation played an important role in the pathogenesis of AAV and neutrophil gelatinase-associated lipocalin (NGAL) is a biomarker of neutrophil degranulation. The purpose of the current study is to investigate whether NGAL is a useful biomarker for assessing disease activity of patients with AAV. Sequential sera from 19 patients with AAV at initial onset, remission and relapse were collected. Serum NGAL was detected using commercial ELISA kits. The association between serum NGAL and the Birmingham Vasculitis Activity Score (BVAS), ESR, CRP as well as serum ANCA was further investigated. The 19 patients had 19 relapses. The levels of serum NGAL in patients at initial onset and relapse were both significantly higher than that at remission (285.5 +/- 65.5 ng/ml vs 96.7 +/- 22.2 ng/ml, P < 0.05; 430.3 +/- 98.7 ng/ml vs 96.7 +/- 22.2 ng/ml, P < 0.05, respectively). It was still the case after adjusting for renal function. The levels of serum NGAL closely correlated with BVAS as well as the level of ESR, CRP and ANCA. Moreover, serum NGAL level had a closer correlation with BVAS than ESR, CRP and ANCA did. Circulating NGAL could be used as a useful biomarker for assessing disease activity of patients with AAV.

Febrile temperatures control antineutrophil cytoplasmic autoantibody–induced neutrophil activation via inhibition of phosphatidylinositol 3‐kinase/Akt

Neutrophil activation by antineutrophil cytoplasmic autoantibodies (ANCAs) is central to the pathogenesis of the ANCA-associated vasculitides. Febrile infections occur frequently during these diseases, often in the context of immunosuppressive treatment. Heat exposure may affect the underlying pathophysiologic processes of the vasculitis. In this study we tested the hypothesis that short-term exposure to heat inhibits ANCA-induced neutrophil activation. After exposure to temperatures from 37 degrees C to 42 degrees C, human neutrophils were primed with either tumor necrosis factor alpha (TNFalpha) or granulocyte-macrophage colony-stimulating factor (GM-CSF) and stimulated with monoclonal antibodies to myeloperoxidase or to proteinase 3. Respiratory burst activity was assayed using rhodamine and a nitroblue tetrazolium reduction assay. Specific inhibition experiments against p38 MAPK, ERK, and phosphatidylinositol 3-kinase (PI 3-kinase)/Akt, and Western blotting with phospho-specific antibodies were used to identify key components in the antibody-induced respiratory burst. A temperature-dependent reduction in ANCA-induced respiratory burst was observed over a range of heat exposures from 37 degrees C to 42 degrees C. Inhibition of human ANCA-induced neutrophil stimulation was significant at 40 degrees C (after priming with 2 ng/ml TNFalpha, mean [+/- SEM] fluorescence intensity [MFI] 114 +/- 12 at 37 degrees C versus 53 +/- 6 at 40 degrees C; after priming with 20 ng/ml GM-CSF, MFI 92 +/- 16 at 37 degrees C versus 35 +/- 6 at 40 degrees C; both P < 0.01). In the priming phase, the transient activation of the p38 MAPK, ERK, and PI 3-kinase/Akt pathways by TNFalpha was blocked by prior exposure of the neutrophils to heat, but GM-CSF-induced activation was unaltered by heat. However, in the second, antibody-induced wave of kinase activation, exposure to heat inhibited only the PI 3-kinase/Akt pathway, and these effects were independent of the priming agent used. Short-term spikes of modest heat abrogate ANCA-induced activation of neutrophils via inhibition of PI 3-kinase/Akt signaling. Febrile responses in ANCA-mediated diseases may therefore have a physiologic purpose.

Injection of ANCA-No Neutrophils, No Glomerular Damage: The Role of Neutrophils in the Induction of Glomerulonephritis by Anti-Myeloperoxidase Antibodies. Am J Pathol 167: 39-45, 2005.

Since the seminal observations of Davies (1) and later of van der Woude (2), it is well known that antineutrophil cytoplasmic antibodies (ANCA) are found in approximately 80% of patients with pauci-immune necrotizing/crescentic glomerulonephritis and with systemic small vessel vasculitis. The most common epitopes recognized by ANCA are myeloperoxidase (MPO) and proteinase 3 (PR3) (3), the former more frequently, but not uniquely, in Wegener’s granulomatosis, the latter mostly in microscopic polyangiitis (4). ANCA stimulate cytokine-primed neutrophils and monocytes, causing for instance respiratory burst, release of toxic granule constituents such as oxygen metabolites and proteinases, as well as endothelial cell injury (5). However, it had long remained controversial whether ANCA are just a marker of disease or have a causal role in the genesis of necrotizing and crescentic glomerulonephritis. The elegant animal model of Xiao et al. (6) had provided definite proof that ANCA were both necessary and sufficient to cause necrotizing and crescentic glomerulonephritis, as well as systemic necrotizing arteritis and hemorrhagic pulmonary capillaritis. For this purpose the authors used MPO knockout mice for which mouse MPO is a “foreign” substance. They immunized these mice with mouse MPO to obtain mouse MPO antibodies. In a second step, mice which were unable to launch an immune response against such mouse MPO antibodies, i.e., knockout mice with a deletion of the recombinase activating gene 2 and therefore devoid of functional B and T cells, received purified mouse anti-MPO IgG or control IgG by intravenous injection. This maneuver reproduced the features of microscopic polyangiitis, i.e., focal necrotizing and crescentic glomerulonephritis with fibrinoid necrosis, crescent formation, and absence or paucity of glomerular IgG deposits. The same outcome was seen when mouse anti-MPO splenocytes were injected. These experiments with injection of mouse anti-MPO IgG or adoptive transfer of mouse anti-MPO splenocytes left no doubt that MPO antibodies alone were able to cause the disease in the absence of B or T cells. What had remained unclear, however, was the cellular target through which MPO antibodies mediated glomerular and vascular injury. To chase down the culprit and to provide evidence that indeed neutrophils—and not for instance endothelial cells, as postulated by others—were the target of the MPO antibodies and the key effector cells, the authors now recently carried out an additional experiment. They used the model of the neutropenic mouse. After injection of NIMP-R14, a rat monoclonal antibody, the mice were selectively depleted of circulating neutrophils, the number decreasing from 14% to 1%. Neutropenic mice or control mice received a low (50 μg/g body weight) or high (50 μg/g body weight on day 0 and day 3) dose of anti-MPO IgG by intravenous injection. Control mice received an injection of bovine serum albumin. Five days after injection of anti-MPO IgG, mice without neutrophil depletion developed hematuria, proteinuria, and leucocyturia. Renal histology documented focal glomerular necrosis and glomerular crescents. Glomerular neutrophil phenotyping revealed glomerular infiltration by neutrophils in foci of inflammation and necrosis as well as in a few afferent arterioles, while monocytes/macrophages were mainly observed in glomerular crescents. With the higher dose, blood urea nitrogen was increased as well, but interestingly necroses were seen on average only in 17.8 ± 7.8% of glomeruli. All these lesions were attenuated or even absent in neutropenic mice receiving the anti-MPO IgG. The observation of no glomerular injury in the absence of neutrophils points to the neutrophil as the key effector cell in the induction of the acute glomerular injury of MPO-induced glomerulonephritis, although an ancillary role of monocytes is not completely excluded. The smoking gun points to the neutrophil, and this fits in nicely with past observations documenting the ability of human MPO ANCA and PR3 ANCA to activate and degranulate neutrophils causing the release of toxic oxygen metabolites (7), proteases, nitric oxide (8) and inflammatory cytokines (9), facilitating attachment and killing of endothelial cells (10,11). Why is the observation of Xiao et al. important? It points to maneuvers interfering with ANCA-induced neutrophil recruitment and activation as potential therapeutic targets, if this can be achieved safely and without inducing harm.

Membrane proteinase 3 expression and ANCA-induced neutrophil activation

Proteinase 3 is the major autoantigen in Wegener's granulomatosis (WG). Membrane PR3 expression is bimodal; low expressing cells (mPR3(low)) can be distinguished from cells with high expression (mPR3(high)) within a given individual. High mPR3 expression is a WG risk factor and is associated with relapse. However, no mechanisms for this important clinical observation have been provided. We tested the hypothesis that mPR3 expression, rather than the expression of other membrane molecules implicated in anti-neutrophil cytoplasmic autoantibodies (ANCA) activation, determines the robustness of the PR3-ANCA-mediated response. mPR3(low) and mPR3(high) neutrophils from a given individual were separated by magnetic cell sorting. Superoxide was measured by the ferricytochrome assay, and Akt phosphorylation by Western blotting. Double staining and flow cytometry were used to assay Fc gamma-receptor and beta 2-integrin expression with respect to the mPR3 phenotype. Degranulation was measured via beta-glucuronidase activity, migration with fibronectin-coated transwells, and cell quantification by the myeloperoxidase (MPO) assay. PR3-ANCA-treated mPR3(high) versus mPR3(low) neutrophils showed more superoxide generation (33.7 +/- 15.2 nmol O(2) (-) to 14.6 +/- 8.4, P < 0.01), more degranulation (29%+/- 5 to 22%+/- 3, P < 0.05), and more PI3-K/Akt activation. In contrast, all responses in both mPR3 subsets were similar after other stimuli. We observed no differences in the beta 2-integrin, Fc gamma R IIa, and III expression with respect to the mPR3 subtype. Furthermore, we found no differences in the mobilization of PR3-containing granules and no differences in migration through fibronectin. The degree of neutrophil mPR3 expression has definitive functional consequences.

Are circulating neutrophils intravascularly activated in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides?

Vascular injury in vasculitis may be due to activation of circulating neutrophils resulting in their increased adhesiveness to locally activated endothelium (Shwartzman phenomenon). Previously, we demonstrated up-regulation of endothelial intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in biopsies from patients with ANCA-associated vasculitis. In the present study, we investigated the expression of adhesion molecules (CD11b, ICAM-1, VLA-4, L-selectin) and activation markers (CD66b, CD64, CD63) on circulating neutrophils from patients with ANCA-associated vasculitis in comparison with their expression on cells from healthy volunteers and patients with sepsis. We related these findings to parameters of disease activity. Surface marker expression was determined by using a non-activating whole blood flow cytometric assay. The expression of activation markers, but not the expression of adhesion molecules, was increased on neutrophils from patients with active vasculitis. The expression of CD63 and CD66b on neutrophils correlated with disease activity as determined by the Birmingham Vasculitis Activity Score (BVAS). In contrast to patients with active vasculitis, patients with sepsis showed up-regulation of all markers, including adhesion molecules, suggesting that circulating neutrophils are fully activated in sepsis. We conclude that in ANCA-associated vasculitis, circulating neutrophils are not fully activated, since they do not express increased levels of adhesion molecules as sepsis or in the Shwartzman reaction. These findings are compatible with the concept that in vivo vascular damage in ANCA-associated vasculitides does not occur due to a Shwarzman-like reaction but only after ANCA-induced neutrophil activation at the endothelial cell surface.

Antineutrophil cytoplasm autoantibodies from patients with systemic vasculitis activate neutrophils through distinct signaling cascades: comparison with conventional Fcγ receptor ligation

In systemic vasculitis, interactions between antineutrophil cytoplasm autoantibodies (ANCAs) and neutrophils initiate endothelial and vascular injury. ANCAs directed against either myeloperoxidase (MPO) or proteinase 3 (PR3) can activate cytokine-primed neutrophils by binding cell surface-expressed MPO or PR3, with the concurrent engagement of Fcgamma receptors (FcgammaR). Because roles for phospholipase D (PLD) and phosphatidylinositol 3 kinase (PI3K) have been demonstrated in FcgammaR activation of neutrophils, this study investigated the hypothesis that ANCA stimulation of neutrophils involved a similar engagement of FcgammaR and activation of PLD and PI3K. Pretreatment of tumor necrosis factor (TNF) alpha-primed neutrophils with antibodies against FcgammaRII and FcgammaRIII inhibited MPO-ANCA and PR3-ANCA induced superoxide generation, confirming that FcgammaR ligation is involved in ANCA-mediated neutrophil activation. However, although stimulation of TNF-alpha-primed neutrophils by conventional FcgammaR ligation, either using antibody-mediated cross-linking of FcgammaR or aggregated IgG, induced PLD activation, ANCA stimulation did not. Moreover, although ANCA-induced neutrophil activation results in significant PI3K activation-as assessed by phosphatidylinositol 3,4,5-triphosphate generation-conventional FcgammaR ligation, but not ANCA, activates the p85/p110 PI3K subtype. Inhibition of ANCA-induced superoxide generation with pertussis toxin suggests that ANCAs activate the p101/p110gamma PI3K isoform. In addition, the kinetics of activation of protein kinase B differs between conventional FcgammaR ligation and ANCA stimulation of neutrophils. These results demonstrate that though ligation of FcgammaRIIa and FcgammaRIIIb may be necessary, it is likely that ANCAs require other membrane cofactors for neutrophil activation.