anti-MPO Antibodies Research Articles

Risk factors of peritonitis during earlyperitoneal dialysis in patients with ANCA-associated systemic vasculitis .

This study investigated the risk factors of peritonitis during early peritoneal dialysis for renal failure in patients with antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis (AASV). This study enrolled hospitalized AASV patients with renal failure who underwent peritoneal dialysis at the Department of Nephrology of our hospital between 2008 and 2015. The primary aim of this study was to perform a retrospective analysis to characterize peritonitis during the early period (≤8weeks) of dialysis, and to compare the differences in initial clinical manifestations, vasculitis activity, immunosuppressive therapies prior to the renal replacement therapy, and prognosis between patients with or without peritonitis. The secondary aim of this study was to conduct a literature review of peritoneal dialysis in patients with vasculitis. All 14 enrolled AASV patients had myeloperoxidase (MPO)-positive microscopic polyangiitis (MPA). Six patients (of whom 2 died) withdrew from peritoneal dialysis due to peritonitis caused by enteric bacteria during the early period of dialysis. Their mean Birmingham vasculitis activity score (BVAS) during the early AASV onset was 31.67±3.98, with significant symptoms of gastrointestinal involvement. Also, their BVAS score, anti-MPO antibody titer, erythrocyte sedimentation rate, C-reactive protein level, serum ANCA level, and glucocorticoid dose were higher than the AASV patients without peritonitis (all p<0.05). AASV patients with a higher level of vasculitis (BVAS score >30) during onset as well as gastrointestinal symptoms had a higher risk for intestinal peritonitis. Therefore, peritoneal dialysis is not recommended as a first-choice renal replacement therapy for such patients. .

The C3aR promotes macrophage infiltration and regulates ANCA production but does not affect glomerular injury in experimental anti-myeloperoxidase glomerulonephritis.

The anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitides are autoimmune diseases associated with significant morbidity and mortality. They often affect the kidney causing rapidly progressive glomerulonephritis. While signalling by complement anaphylatoxin C5a though the C5a receptor is important in this disease, the role of the anaphylatoxin C3a signalling via the C3a receptor (C3aR) is not known. Using two different murine models of anti-myeloperoxidase (MPO) glomerulonephritis, one mediated by passive transfer of anti-MPO antibodies, the other by cell-mediated immunity, we found that the C3aR did not alter histological disease severity. However, it promoted macrophage recruitment to the inflamed glomerulus and inhibited the generation of MPO-ANCA whilst not influencing T cell autoimmunity. Thus, whilst the C3aR modulates some elements of disease pathogenesis, overall it is not critical in effector responses and glomerular injury caused by autoimmunity to MPO.

Clinical Characteristics and Outcomes of Propylthiouracil-Induced Antineutrophil Cytoplasmic Antibody-Associated Vasculitis in Patients with Graves' Disease: A Median 38-Month Retrospective Cohort Study from a Single Institution in China.

This study sought to investigate the clinical characteristics and outcomes of propylthiouracil (PTU)-induced antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis in patients with Graves' disease. Sixteen patients diagnosed with PTU-induced ANCA-associated vasculitis at the authors' hospital from January 2010 to June 2017 were analyzed retrospectively. All 16 patients with PTU-induced ANCA-associated vasculitis were female. The mean age ± standard deviation of the patients was 39.4 ± 15.3 years (range 19-69 years), and the median time of onset was 36 months (range 1-193 months) post-PTU initiation. The median dose at the onset of PTU-induced ANCA-associated vasculitis was 150 mg/day (range 50-300 mg/day). All patients had a positive serum perinuclear staining pattern (p-ANCA) and antibodies directed against myeloperoxidase (anti-MPO). Six patients tested positive for both anti-MPO antibodies and antibodies directed against proteinase-3. Seven (43.8%) patients presented with involvement of a single organ. The kidney was the organ most commonly affected, as 12 (75%) patients were found to have disease involving this organ. PTU was stopped in all patients, corticosteroids were administered to two patients, and immunosuppressive agents and corticosteroids were administered to five patients. Three patients were lost to follow-up. However, the remaining patients achieved remission after a median follow-up period of 38 months (range 6-76 months). Patients who were positive for pANCA and displayed cytoplasmic staining showed negative findings at rates of approximately 53.8% (7/13) and 100% (6/6), respectively, following treatment. PTU-induced ANCA-positive vasculitis occurs at varying times and after exposure to various doses of PTU. The condition has a milder course and has a better prognosis after PTU cessation.

Identification and characterization of myeloperoxidase in orange-spotted grouper (Epinephelus coioides)

Cryptocaryon irritans is an important protozoan ciliate, which has led to heavy economic losses in marine aquaculture. Previous studies have indicated that C. irritans infection could induce the migration of neutrophils to infection sites. Myeloperoxidase (MPO) mainly exists in the cytoplasmic granules of the neutrophil and performs its function by a unique enzymatic capacity to produce hypohalous acid and other toxic oxidants. To determine the involvement of MPO and neutrophils against C. irritans infection in the host, we amplified MPO cDNA (EcMPO) from orange-spotted grouper (Epinephelus coioides). The open reading frame (ORF) of EcMPO encodes a putative polypeptide of 770 amino acids and has typical structural characteristics of mammalian MPO, including a signal peptide, a propeptide, a light chain, a heavy chain, and a peroxidase domain. Bioinformatics analysis has demonstrated that the most important functional sites in mammalian MPO were also conserved in grouper and other piscine MPO, implying the functional conservation of this protein during evolution. A rabbit anti-MPO recombinant protein polyclonal antibody was produced, which could recognize the native MPO protein. The expression of EcMPO was higher in the lympho-hematopoietic organs, such as head kidney, trunk kidney, spleen, but lower in muscle, heart, and brain. After infection with C. irritans, the EcMPO transcript was significantly up-regulated at specific time points in the infection sites (skin and gill) and systemic immune organs (head kidney and spleen); The number of EcMPO positive cells first increased and then decreased in the gill, but was still higher than the control after 7 days. These results demonstrated that EcMPO and its positive cells may be involved in anti-C. irritans infection in the grouper, which is attributed to the innate immune mechanisms of the host against parasite infection.

Monocytes Promote Crescent Formation in Anti-Myeloperoxidase Antibody–Induced Glomerulonephritis

Neutrophils and monocytes express anti-neutrophil cytoplasmic antibody (ANCA) antigens, and activation of these cells by ANCA is central to ANCA-associated vasculitis and necrotizing crescentic glomerulonephritis (NCGN). The importance of neutrophils is established; however, any role of monocytes is less clear. We tested the hypothesis that depletion of CCR2+ inflammatory monocytes and their derivatives would abrogate anti-myeloperoxidase (MPO) antibody-induced NCGN in a mouse model. We used passive anti-MPO antibody transfer for NCGN induction in wild-type mice or mice expressing the CCR2 promoter-controlled diphtheria toxin receptor. Both mouse strains showed similar circulating Ly6Chi and Ly6Clo monocytes and neutrophils at baseline. Diphtheria toxin robustly depleted circulating monocytes only in CCR2 promoter-controlled diphtheria toxin receptor mice, whereas neutrophil numbers were similar. Anti-MPO antibody transfer resulted in nephritic urine by dipstick and albuminuria by enzyme-linked immunosorbent assay, and monocyte depletion had no effect. However, monocyte depletion significantly reduced glomerular necrosis and crescent formation and abrogated monocyte, macrophage, and dendritic cell increase in the affected kidneys, whereas renal neutrophil numbers were not affected. Soluble CD163 increased in serum, but not in urine, with anti-MPO antibody treatment and was completely abolished with monocyte depletion. Our findings establish an important role of monocytes/macrophages for glomerular necrosis and crescent formation in a renal ANCA-associated vasculitis model.

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.

Effect of Continuous B Cell Depletion With Rituximab on Pathogenic Autoantibodies and Total IgG Levels in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

To evaluate the effect of rituximab on pathogenic autoantibodies and total Ig levels, and to identify serious adverse events in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) treated with continuous B cell depletion. We conducted a retrospective analysis of 239 patients with AAV treated with rituximab-induced continuous B cell depletion. Two treatment cohorts were analyzed: an induction group (n = 52) and a maintenance group (n = 237). Changes in ANCA titers and total Ig levels over time were evaluated using mixed-effects models. Risk factors for serious infections during maintenance treatment were evaluated using Poisson regression. During induction, IgG levels fell at a mean rate of 6% per month (95% confidence interval [95% CI] 4, 8%), while ANCA levels declined at a mean rate of 47% per month (95% CI 42, 52%) and 48% per month (95% CI 42, 54%) for patients with antimyeloperoxidase (anti-MPO) antibodies and those with anti-proteinase 3 (anti-PR3) antibodies, respectively. During maintenance treatment, with a median duration of 2.4 years (interquartile range 1.5, 4.0 years), IgG levels declined a mean of 0.6% per year (95% CI -0.2, 1.4%). New significant hypogammaglobulinemia (IgG level of <400 mg/dl) during maintenance treatment occurred in 4.6% of the patients, all of whom were in the lowest baseline IgG quartile. Serious infections during maintenance therapy occurred at a rate of 0.85 per 10 patient-years (95% CI 0.66, 1.1) and were independently associated with an IgG level of <400 mg/dl. B cell-targeted therapy causes a preferential decline in ANCA titers relative to total IgG levels. Despite prolonged maintenance therapy with rituximab, IgG levels remain essentially constant. Serious infections were rare.

WS6_1 Anti-MPO antibodies cause granulomatosis in mice: an animal model of GPA

WS6_1 Anti-MPO antibodies cause granulomatosis in mice: an animal model of GPA

Association of serum levels of anti-myeloperoxidase antibody with retinal photoreceptor ellipsoid zone disruption in diabetic retinopathy

AimTo study the association of serum levels of anti-myeloperoxidase (MPO) antibody with retinal photoreceptor ellipsoid zone (EZ) disruption in diabetic retinopathy. MethodsConsecutive patients with type 2 DM [diabetes mellitus with no retinopathy (NODR; n=20); non-proliferative diabetic retinopathy (NPDR; n=18); proliferative diabetic retinopathy (PDR; n=16)] and healthy controls (n=20) between the ages of 40 and 65years were included. Disruption of EZ was graded by spectral domain optical coherence tomography as no disruption of EZ and disrupted EZ. The serum levels of anti-MPO antibody was analyzed using standard protocol. Association between the variables was evaluated using multiple regression analysis. ResultsA significant difference was found between the serum levels of anti-MPO antibody in various study groups (p<0.001). A positive association was found between EZ disruption and levels of anti-MPO antibody [adjusted odd's ratio (AOR)=1.079, CI 1.010–1.124, p=0.04]. A significant positive correlation was found between logMAR visual acuity and grade of disruption (AOR=1.008, CI 1.006–5.688, p=0.04). ConclusionsAn increased serum anti-MPO antibody levels is associated with retinal photoreceptor EZ disruption and decreased visual acuity in diabetic retinopathy.

Systemic Lupus Erythematosus and Antineutrophil Cytoplasmic Antibody-Associated Vasculitis Overlap Syndrome in Patients With Biopsy-Proven Glomerulonephritis

The aim of the study was to report the clinical, biological, and pathological characteristics of patients with glomerulonephritis (GN) secondary to systemic lupus erythematosus (SLE)/antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) overlap syndrome.A nationwide survey was conducted to identify cases of SLE/AAV overlap syndrome. Data were collected from SLE and AAV French research groups. Inclusion criteria were diagnosis of both SLE and AAV according to international classification criteria and biopsy-proven GN between 1995 and 2014. Additional cases were identified through a systematic literature review. A cohort of consecutive biopsy-proven GN was used to study the prevalence of overlapping antibodies and/or overlap syndrome.The national survey identified 8 cases of SLE/AAV overlap syndrome. All patients were female; median age was 40 years. AAV occurred before SLE (n = 3), after (n = 3), or concomitantly (n = 2). Six patients had rapidly progressive GN and 3/8 had alveolar hemorrhage. All patients had antinuclear antibodies (ANA); 7/8 had p-ANCA antimyeloperoxidase (MPO) antibodies. Renal biopsies showed lupus nephritis (LN) or pauci-immune GN. Remission was obtained in 4/8 patients. A literature review identified 31 additional cases with a similarly severe presentation. In the GN cohort, ANCA positivity was found in 30% of LN, ANA positivity in 52% of pauci-immune GN, with no correlation with pathological findings. The estimated prevalence for SLE/AAV overlap syndrome was 2/101 (2%).In patients with GN, SLE/AAV overlap syndrome may occur but with a low prevalence. Most patients have an aggressive renal presentation, with usually both ANA and anti-MPO antibodies. Further studies are needed to assess shared pathogenesis and therapeutic options.

Antimyeloperoxidase antibody is a biomarker for progression of diabetic retinopathy

AimTo study the correlation between serum antimyeloperoxidase (MPO) antibody levels with severity of diabetic retinopathy (DR). MethodsStudy subjects included 60 consecutive cases of type 2 diabetes mellitus (DM): no diabetic retinopathy (NODR, n=20); nonproliferative DR (NPDR, n=20); proliferative DR (PDR, n=20) and 20 healthy controls. Best corrected visual acuity (BCVA) was measured on logMAR scale. Serum anti-MPO antibody levels were evaluated using ELISA IgG kit. Serum urea and creatinine was measured using standard protocol. Data were analysed statistically. ResultsMean serum anti-MPO antibody (RU/ml) was 16.94±4.85 in controls, 17.66±4.78 in NODR, 21.51±5.27 in NPDR and 37.27±11.92 in PDR groups. On ANOVA, significant difference in visual acuity was found among the study groups (F=73.46, p<0.001). Serum anti-MPO antibody was correlated significantly with decrease in visual acuity (F=48.40, p<0.001), increase in serum urea (F=128.13, p<0.001) and creatinine (F=77.10, p<0.001). ConclusionIncrease in serum anti-MPO antibody levels correlate with increased severity of DR. Serum anti-MPO antibody may be a noteworthy biochemical marker for progression of retinopathy from nonproliferative to proliferative stage.

Intermediate monocytes in ANCA vasculitis: increased surface expression of ANCA autoantigens and IL-1β secretion in response to anti-MPO antibodies.

ANCA vasculitis encompasses several autoimmune conditions characterised by destruction of small vessels, inflammation of the respiratory tract and glomerulonephritis. Most patients harbour autoantibodies to myeloperoxidase (MPO) or proteinase 3 (PR3). Clinical and experimental data suggest that pathogenesis is driven by ANCA-mediated activation of neutrophils and monocytes. We investigated a potential role for distinct monocyte subsets. We found that the relative proportion of intermediate monocytes is increased in patients versus control individuals, and both MPO and PR3 are preferentially expressed on these cells. We demonstrate that MPO and PR3 are expressed independently of each other on monocytes and that PR3 is not associated with CD177. MPO expression correlates with that of Fc receptor CD16 on intermediate monocytes. Monocyte subsets respond differently to antibodies directed against MPO and PR3, with anti-MPO but not anti-PR3 leading to increased IL-1β, IL-6 and IL-8 production. In concordance with the observed higher surface expression of MPO on intermediate monocytes, this subset produces the highest quantity of IL-1β in response to anti-MPO stimulation. These data suggest that monocytes, specifically, the intermediate subset, may play a role in ANCA vasculitis, and also indicate that substantial differences exist between the effect of anti-MPO and anti-PR3 antibodies on these cells.

An immunological method to combine the measurement of active and total myeloperoxidase on the same biological fluid, and its application in finding inhibitors which interact directly with the enzyme

Myeloperoxidase (MPO) is a pro-oxidant enzyme involved in inflammation, and the measurement of its activity in biological samples has emerged essential for laboratory and clinical investigations. We will describe a new method which combines the SIEFED (specific immunological extraction followed by enzymatic detection) and ELISA (ELISAcb) techniques to measure the active and total amounts of MPO on the same human sample and with the same calibration curve, as well as to define an accurate ratio between both the active and total forms of the enzyme.The SIEFED/ELISAcb method consists of the MPO extraction from aqueous or biological samples by immobilized anti-MPO antibodies coated onto microplate wells. After a washing step to eliminate unbound material, the activity of MPO is measured in situ by adding a reaction solution (SIEFED). Following aspiration of the reaction solution, a secondary anti-MPO antibody is added into the wells and the ELISAcb test is carried out in order to measure the total MPO content.To validate the combined method, a comparison was made with SIEFED and ELISA experiments performed separately on plasma samples isolated from human whole blood, after a neutrophil stimulation.The SIEFED/ELISAcb provides a suitable tool for the measurement of specific MPO activity in biological fluids and for the estimation of the inhibitory potential of a fluid. The method can also be used as a pharmacological tool to make the distinction between a catalytic inhibitor, which binds to MPO and inhibits its activity, and a steric inhibitor, which hinders the enzyme and prevents its immunodetection.

Abstract 69: Possible Involvement Of Proline-rich Tyrosine Kinase 2 (pyk2) In The Pathogenesis Of Kawasaki Disease

Introduction: Although etiology of Kawasaki disease (KD) remains elusive, a line of recent experimental studies implies that some kinds of infectious stimuli are implicate in the vasculitis through uncontrolled innate immune systems such as pattern recognition receptor (PPR)-mediated inflammatory signaling. It has already known that Candida albicans water-soluble fraction (CAWS) inducing KD-like vasculitis in mice function through PRP. Furthermore, it is reported that proline-rich tyrosine kinase (Pyk2), which is molecule involved in the PRPs-dependent signaling pathways, plays an important role in activation of NF-κB. Therefore, we investigated a possible relevance of Pyk2 in the pathogenesis of KD. Methods: Pyk2-knock out (Pyk2-KO) and wild-type C57BL/6 mice (WT) were administered CAWS to induce KD-like vasculitis. Extension of the experimental vasculitis was immunohistochemically determined with anti-MPO antibody. CAWS-stimulated NF-κB activation was evaluated by quantifying nuclear translocation of NF-κB p65 subunit in peritoneal macrophages isolated from PYK2-KO and wild-type mice in vitro. Cytokines and chemokines across each mice were compared by cytokine array. Results: Pyk2-KO mice didn’t show any apparent defective phenotype. While marked inflammation was observed in the aortic root of CAWS-treated WT mice, such vasculitis was barely detected in CAWS-treated Pyk2-KO mice. CAWS-induced NF-κB activation was also less observed in macrophages from Pyk2-KO mice. There were differences in some cytokines and chemokines production between mice. Conclusion: We speculate that Pyk2 is involved in the pathogenesis of KD. Pyk2 might be a potential therapeutic target for KD.

Autoantibodies in systemic vasculitis.

Autoantibodies in systemic vasculitis.

Microscopic polyangiitis associated with antineutrophil cytoplasmic antibodies: clinical features

To study the clinical features of early- and extended-stage microscopic polyangiitis (MPA) and its outcomes on the basis of a long-term follow-up in a rheumatologist's practice. The clinical features of early- and extended-stage MPA were studied in detail and the premorbid background and possible precipitating factors were analyzed in 70 patients with MPA and the proven hyperproduction of antineutrophil cytoplasmic antibodies (anti-proteinase-3 (anti-PR3) antibodies in 55% and anti-myeloperoxidase (anti-MPO) antibodies in 45%) who had been followed up for more than a year. There is evidence for the nosological unity of the two immunological types of MPA associated with anti-PR-3 or anti-MPO antibodies. MPA has been demonstrated to be an aggressive, polysyndromic disease prone to recurrences (52%), the typical manifestation of which is glomerulonephritis (94%) that is rapidly progressive in every four cases and accompanied by hemorrhagic alveolitis (69%) and involvement of other organs. ENT organs and lungs have been noted to be commonly involved in early-stage MPA, which was observed in 61% of the patients in the premorbid period, and to become the first manifestation of MPA (63%) concurrent with body temperature rises (64%), arthralgia or arthritis (41%). Respiratory tract involvement in MPA may be asymptomatic. Anti-PR-3-associated MPA may manifest itself more aggressively and in the first 2 years it is characterized by a poorer prognosis than of anti-MPO-associated MPA (survival rates, 82 and 94%, respectively; p = 0.04). With time, the differences were levelled off; recurrences in the patients with anti-PR-3 and anti-MPO develop equally frequently and proceed showing the similar clinical picture; the survival curves converge by age 3. Anti-MPO-associated MPA is characterized by the highest rate of lung involvement in the clinical phase of the disease (61%) and by a propensity to develop hemorrhagic alveolitis, diffuse interstitial (22%) or circumscribed pulmonary fibrosis in the outcome. CONCLUSION. The findings emphasize how important to diagnose MPA early and to prescribe long-term active treatment using the entire current arsenal of medications as soon as possible until severe injury to organs and systems develops. To specify regularities in the development of MPA may be of value for the better diagnosis of the disease and the further elaboration of optimal treatment policy.

FcγRIIB regulates T-cell autoreactivity, ANCA production, and neutrophil activation to suppress anti-myeloperoxidase glomerulonephritis.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis involves innate and adaptive immune cells in the induction of autoimmunity and in autoimmune effector responses. Most Fcγ receptors (FcγRs) activate immune cells, but FcγRIIB, found in humans and mice on B cells and innate cells, is an inhibitory receptor. Here we tested whether endogenous FcγRIIB negatively regulates autoreactivity and effector responses in experimental anti-myeloperoxidase (MPO) glomerulonephritis, using wild-type and FcγRIIB(-/-) mice. After MPO immunization, FcγRIIB(-/-) mice developed higher MPO-ANCA titers and increased anti-MPO T-cell responses. Transfer of FcγRIIB-deficient dendritic cells loaded with a nephritogenic MPO peptide (MPO409-428) into wild-type mice induced stronger autoimmunity than dendritic cells derived from wild-type mice. Transferring anti-MPO antibodies into lipopolysaccharide-primed mice resulted in increased glomerular neutrophil accumulation and injury in FcγRIIB(-/-) mice, showing a role for FcγRIIB in suppressing neutrophil activation. Inducing active autoimmunity to MPO followed by triggering T cell-mediated glomerular injury by transfer of sub-nephritogenic doses of lipopolysaccharide and anti-MPO antibodies resulted in more disease in FcγRIIB(-/-) mice. Thus, endogenous FcγRIIB negatively regulates anti-MPO autoimmunity and glomerulonephritis by dendritic cells, B cells, and neutrophils to limit MPO-ANCA production, T-cell responses, and neutrophil activation.

A dual-fixed neutrophil substrate improves interpretation of antineutrophil cytoplasmic antibodies by indirect immunofluorescence.

To determine whether the addition of a formalin-fixed neutrophil substrate could improve interpretation and prediction of autoantigenic specificity in antineutrophil cytoplasmic antibody (ANCA) testing. Routine diagnostic samples sent for ANCA testing were analyzed prospectively on a dual substrate of both ethanol- and formalin-fixed neutrophils. Positive samples on ethanol-fixed neutrophils were deemed "typical" if formalin-fixed neutrophils also stained, and "atypical" if not. Indirect immunofluorescence (IIF) results were correlated with antimyeloperoxidase (MPO) and anti-proteinase 3 (PR3) results with an enzyme-linked immunosorbent assay (ELISA). Of 1,426 samples, 201 from unique patients were ANCA-positive (200 on IIF, 1 on ELISA alone). Thirty-two (45%) of 71 typical ANCA staining patterns were positive for either an anti-MPO or anti-PR3 antibodies, whereas only one (0.8%) of 129 atypical patterns was ELISA-positive, in a patient without systemic vasculitis. Only one (3%) of 34 ELISA-positive samples had a negative IIF-ANCA (1/1,426 patients, 0.07%), and this patient did not have vasculitis. Concomitant staining on formalin fixation of IIF-positive ethanol-fixed ANCA samples improves the interpretation of ANCA testing and is predictive of vasculitis autoantigens MPO and PR3.

Phagocyte NADPH oxidase restrains the inflammasome in ANCA-induced GN.

ANCA-activated phagocytes cause vasculitis and necrotizing crescentic GN (NCGN). ANCA-induced phagocyte NADPH oxidase (Phox) may contribute by generating tissue-damaging reactive oxygen species. We tested an alternative hypothesis, in which Phox restrains inflammation by downregulating caspase-1, thereby reducing IL-1β generation and limiting NCGN. In an antimyeloperoxidase (anti-MPO) antibody-mediated disease model, mice transplanted with either gp91(phox)-deficient or p47(phox)-deficient bone marrow showed accelerated disease with increased crescents, necrosis, glomerular monocytes, and renal IL-1β levels compared with mice transplanted with wild-type bone marrow. IL-1β receptor blockade abrogated aggravated NCGN in gp91(phox)-deficient mice. In vitro, challenge with anti-MPO antibody strongly enhanced caspase-1 activity and IL-1β generation in gp91(phox)-deficient and p47(phox)-deficient monocytes compared with wild-type monocytes. This enhanced IL-1β generation was abrogated when caspase-1 was blocked. ANCA-induced superoxide and IL-1β generation were inversely related in human monocytes. Furthermore, transplantation of gp91(phox)/caspase-1 double-deficient bone marrow rescued the accelerated NCGN phenotype in gp91(phox) bone marrow-deficient mice. These results suggest that Phox-generated reactive oxygen species downregulate caspase-1, thereby keeping the inflammasome in check and limiting ANCA-induced inflammation. IL-1 receptor blockade may provide a promising strategy in NCGN, whereas our data question the benefit of antioxidants.

FcγRIIB regulates T cell autoreactivity, ANCA production and neutrophil activation to suppress anti-myeloperoxidase glomerulonephritis (THER5P.834)

Abstract Anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis involves innate and adaptive immune cells in the induction of autoimmunity and in autoimmune effector responses. Most Fcγ receptors (FcγRs) activate immune cells, but FcγRIIB, found in humans and mice on B cells and innate cells, is an inhibitory receptor. These studies tested the hypothesis that endogenous FcγRIIB negatively regulates autoreactivity and effector responses in experimental anti-myeloperoxidase (MPO) glomerulonephritis, using wild type and FcγRIIB-/- mice. After MPO immunization, FcγRIIB-/- mice developed higher MPO-ANCA titers and increased anti-MPO T cell responses. Transfer of FcγRIIB deficient dendritic cells loaded with a nephritogenic MPO peptide (MPO409-428) into wild type mice induced stronger autoimmunity than dendritic cells derived from wild type mice. Transferring anti-MPO antibodies into LPS primed mice resulted in increased glomerular neutrophil accumulation and injury in FcγRIIB-/- mice, showing a role for FcγRIIB in suppressing neutrophil activation. Inducing active autoimmunity to MPO followed by triggering T cell mediated glomerular injury by transfer of subnephritogenic doses of LPS and anti-MPO antibodies resulted in more disease in FcγRIIB-/- mice. Therefore endogenous FcγRIIB negatively regulates anti-MPO autoimmunity and glomerulonephritis via dendritic cells, B cells and neutrophils to limit MPO-ANCA production, T cell responses and neutrophil activation.