Antineutrophil Cytoplasmic Antibody Antigens Research Articles

Eosinophilic granulomatosis with polyangiitis: etiopathogenesis, classification and clinical phenotypes

Eosinophilic granulomatosis with polyangiitis (EGPA) is a rare systemic disease that can be classified as both a hypereosinophilic condition and an antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis and is characterized by granulomatous inflammation. The pathogenesis of EGPA is not completely understood. It is likely that this disease is Th2-mediated, and blood and tissue eosinophilia serves as the main diagnostic criterion. The hallmarks and main effectors of organ damage in EGPA include asthma-associated necrotizing vasculitis of small-to-medium vessels and eosinophilic proliferation. Endothelial injury and vascular inflammation in EGPA is caused by ANCA via activation of circulating neutrophils. Two clinical phenotypes of the disease have been described based on the detection of ANCA: ANCA-negative with manifestations of hypereosinophilia (for example, pulmonary infiltrates and cardiomyopathy) and ANCA-positive with clinical signs of vasculitis (for example, glomerulonephritis, purpura, and mononeuritis multiplex). Both phenotypes were confirmed by histological and genomic research. However, these two coexisting mechanisms cannot be separated in clinical practice.The aim of the article is to present current knowledge of eosinophilic and ANCA-mediated aspects of the pathogenesis, classification and clinical phenotypes of EGPA, and consider prospects for future research.Conclusion. The development of EGPA is based on eosinophilic dysfunction. This dysfunction means that patients with a genetically determined predisposition to recognize the ANCA antigen and with HLA-DQ (human leukocyte antigen DQ) alleles produce anti-myeloperoxidase autoantibodies and later develop an aberrant autoimmune process. Further comprehensive post-genomic studies are needed to identify the pathogenetic mechanisms and characterize molecular features of EGPA clinical phenotypes. The elaboration of molecular endotypes will lead to the identification of new activity biomarkers and therapeutic targets that can improve the diagnosis of EGPA and the treatment outcomes.

Beyond Systemic Lupus Erythematosus and Anti-Phospholipid Syndrome: The Relevance of Complement From Pathogenesis to Pregnancy Outcome in Other Systemic Rheumatologic Diseases.

Evidence about the relevance of the complement system, a highly conserved constituent of the innate immunity response that orchestrates the elimination of pathogens and the inflammatory processes, has been recently accumulated in many different rheumatologic conditions. In rheumatoid arthritis, complement, mainly the classical pathway, contributes to tissue damage especially in seropositive subjects, with complement activation occurring in the joint. Data about complement pathways in psoriatic arthritis are dated and poorly consistent; among patients with Sjögren syndrome, hypocomplementemia exerts a prognostic role, identifying patients at risk of extra-glandular manifestations. Hints about complement involvement in systemic sclerosis have been recently raised, following the evidence of complement deposition in affected skin and in renal samples from patients with scleroderma renal crisis. In vasculitides, complement plays a dual role: on one hand, stimulation of neutrophils with anti-neutrophil cytoplasmic antibodies (ANCA) results in the activation of the alternative pathway, on the other, C5a induces translocation of ANCA antigens, favouring the detrimental role of antibodies. Complement deposition in the kidneys identifies patients with more aggressive renal disease; patients with active disease display low serum levels of C3 and C4. Even though in dermatomyositis sC5b-9 deposits are invariably present in affected muscles, data on C3 and C4 fluctuation during disease course are scarce. C3 and C1q serum levels have been explored as potential markers of disease activity in Takayasu arteritis, whereas data in Behçet disease are limited to in vitro observations. Pregnancies in women with rheumatologic conditions are still burdened by a higher rate of pregnancy complications, thus the early identification of women at risk would be invaluable. A fine-tuning of complement activation is required from a physiological progression of pregnancy, from pre-implantation stages, through placentation to labour. Complement deregulation has been implicated in several pregnancy complications, such as recurrent abortion, eclampsia and premature birth; low complement levels have been shown to reliably identify women at risk of complications. Given its physiologic role in orchestrating pregnancy progression and its involvement as pathogenic effector in several rheumatologic conditions, complement system is an attractive candidate biomarker to stratify the obstetric risk among women with rheumatologic conditions.

Neutrophil fixation protocols suitable for substrates to detect anti-neutrophil cytoplasmic antibodies by indirect immunofluorescence

Anti-neutrophil cytoplasmic antibodies (ANCAs) are autoantibodies that recognize neutrophil cytoplasmic antigens. The major ANCA antigens are myeloperoxidase and proteinase 3. Necrotizing small vessel vasculitis accompanied by ANCA production is called ANCA-associated vasculitis (AAV). In addition to AAV, ANCA is sometimes produced in patients with connective tissue diseases, such as systemic lupus erythematosus, and inflammatory bowel diseases. Indirect immunofluorescence (IIF) and enzyme immunoassay (EIA) have been used to detect ANCAs. Recently, the accuracy of EIA has improved and it has become the gold standard for ANCA detection. However, IIF does not lose its role in ANCA detection because EIA cannot detect ANCAs that recognize antigens other than those coated on the plate. For IIF, neutrophil substrates prepared with two different fixations, namely, ethanol fixation and formalin fixation, are used. There is a recommended protocol for ethanol fixation but not for formalin fixation. This study prepared neutrophil substrates according to the recommended protocol for ethanol fixation and protocols in the literature and original protocols for formalin fixation and then examined ANCA specificity and how storage period would influence the number of cells, antigen distribution, and antigenicity of the substrates. As a result, the number of cells and antigen distribution did not change after storage for up to 2 months regardless of fixation protocols, whereas a time-dependent decline in ANCA antigenicity and a fixation protocol-dependent difference in ANCA specificity were observed. How neutrophils are fixed on the glass slide needs to be checked upon evaluation of ANCAs by IIF.

Fluorine (19F) MRI for Assessing Inflammatory Cells in the Kidney: Experimental Protocol.

Inflammation is one underlying contributing factor in the pathology of acute and chronic kidney disorders. Phagocytes such as monocytes, neutrophils and dendritic cells are considered to play a deleterious role in the progression of kidney disease but may also contribute to organ homeostasis. The kidney is a target of life-threatening autoimmune disorders such as the antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV). Neutrophils and monocytes express ANCA antigens and play an important role in the pathogenesis of AAV. Noninvasive in vivo methods that can quantify the distribution of inflammatory cells in the kidney as well as other organs in vivo would be vital to identify the causality and significance of inflammation during disease progression. Here we describe an noninvasive technique to study renal inflammation in rodents in vivo using fluorine (19F) MRI. In this protocol we chose a murine ANCA-AAV model of renal inflammation and made use of nanoparticles prepared from perfluoro-5-crown-15-ether (PFCE) for renal 19F MRI.This chapter is based upon work from the COST Action PARENCHIMA, a community-driven network funded by the European Cooperation in Science and Technology (COST) program of the European Union, which aims to improve the reproducibility and standardization of renal MRI biomarkers. This experimental protocol chapter is complemented by two separate chapters describing the basic concept and data analysis.

Circulating B Cells, Plasma Cells, and Treg Associate with ANCA Levels in ANCA-associated Vasculitis

Circulating B Cells, Plasma Cells, and Treg Associate with ANCA Levels in ANCA-associated Vasculitis

Anti-neutrophil cytoplasmic antibodies (ANCA): Antigen interactions and downstream effects.

Neutrophils are the most abundant leukocytes in circulation and are key "first responders" in the immune response to infectious and non-infectious stimuli. Unlike other immune cells, neutrophils can mount a robust response (including a change in surface markers and the production of extracellular traps and reactive oxygen species) just minutes after sensing a disturbance. It has been speculated that, in some individuals, the activation of neutrophils inadvertently leads to the generation of anti-neutrophil cytoplasmic autoantibodies (ANCA) against particular neutrophil proteins (antigens) such as myeloperoxidase (MPO) and proteinase 3 (PR3). In these individuals, continuous ANCA-antigen interactions are thought to drive persistent activation of neutrophils, chronic immune activation, and disease, most notably, small vessel vasculitis. There are significant gaps however in our understanding of the underlying mechanisms and even the pathogenicity of ANCA given that vasculitis can develop in the absence of ANCA, and that ANCA have been found in circulation in other conditions with no apparent contribution to disease. These gaps are particularly evident in the context of human studies. Herein, we review knowledge on neutrophil-derived ANCA antigens PR3 and MPO, ANCA generation, and ANCA-antigen interaction(s) that may promote immune activation and disease.

Detection by flow cytometry of anti-neutrophil cytoplasmic antibodies in a novel approach based on neutrophil extracellular traps

Background: Anti-neutrophil-cytoplasmic antibodies (ANCA) are auto-antibodies directed against components of neutrophil granulocytes and may be found in various inflammatory conditions, like small-vessel vasculitis or ulcerative colitis (UC). Routine ANCA screening is performed on ethanol-fixed neutrophils using indirect immunofluorescence technique. Yet, how neutrophil granule proteins become available to immunologic presentation is a matter of debate. In recent years, various studies have shown that neutrophils are able to extrude their chromatin decorated with granular proteins as neutrophil extracelullar traps (NETs).Aim: We hypothesized that (I) ANCA immunoreactivity may be found on NETs and (II) NETs may serve as a useful tool in a novel approach for ANCA detection.Methods: Sera from patients suffering from either ANCA-associated vasculitis (n = 10), UC (n = 30) or sera from patients without diagnosed ANCA-associated diseases (n = 20), respectively, were subjected to indirect immunofluorescence and a newly developed method to detect ANCA by flow cytometry employing microbead technology.Results: ANCA-related immunofluorescence was readily detectable on ethanol-fixed NETs, establishing NETs as a structure carrying ANCA target antigens. Moreover, we observed that neutrophils form NETs in response to microbeads and stick to the surface of these beads. Using these NET-coated microbeads in flow cytometry, we were capable of reliably detecting p-ANCA, c-ANCA, and a-ANCA in tested patient sera. UC-related complex DNase-1-sensitive ANCA (NET-ANCA) antigens were also detected on NET-coated microbeads.Conclusion: NET-coated microbeads may be commercially developed as a novel tool for automated ANCA screening assays using flow cytometry.

New concepts in ANCA detection and disease classification in small vessel vasculitis: the role of ANCA antigen specificity.

Anti-neutrophil cytoplasmic antibodies (ANCA) play a central role in the diagnosis and pathogenesis of patients with ANCA-associated vasculitis. ANCA-associated vasculitis is a rare disease characterized by necrotizing inflammation of small/medium-sized blood vessels with and without granuloma in different organs. The main syndromes are granulomatosis with polyangiitis, microscopic polyangiitis, and eosinophilic GPA. ANCA in these diseases are almost always directed against proteinase 3 and myeloperoxidase. Most laboratories worldwide use as standard the indirect immunofluorescence technique to screen for ANCA and then confirm positive IFT results with antigen specific immunoassyas for PR3- and MPO-ANCA. New guidelines for ANCA testing have been developed based on a recent European multicentre study, and according to the revised 2017 international consensus recommendations, testing for ANCA in small vessel vasculitis can be done by PR3- and MPO-ANCA immunoassays, without the categorical need for IIF. The new testing strategy for ANCA in vasculitis directly identifies the ANCA target antigen and has a particular value for the AAV sub-classification. Recent studies have shown that AAV can be classified based on ANCA serotype. ANCA presence and the antigen specificity also may have important value as a prognostic factor and may serve as a guide for immunosuppressive therapy. The clinical utility of ANCA depends on the type of assay performed and the appropiate ordering of testing the right clinical setting. Accurate identification of all patients with AAV and the avoidance of misdiagnosis can be achieved using a “gating policy” based on clinical information given to the laboratory at the time of request.

Macrophage migration inhibitory factor contributes to anti-neutrophil cytoplasmic antibody-induced neutrophils activation

BackgroundMacrophage migration inhibitory factor (MIF) is an inflammatory mediator released by macrophages that is central to the innate immune system, with an upstream role in the inflammatory cascade. MIF is one of the most important pathogenic factors in the development of the autoimmune diseases. In the current study, we investigated the role of MIF in anti-neutrophil cytoplasmic antibody (ANCA)-induced neutrophil activation. MethodsPlasma levels of MIF from 31 patients with active ANCA-associated vasculitis (AAV) were analyzed by ELISA. The various effects of MIF in ANCA-induced neutrophil respiratory burst and degranulation were measured. ResultsPlasma levels of circulating MIF were significantly higher in AAV patients with active disease compared with those in remission and healthy controls. Compared with MIF-primed neutrophils, the MFI value increased significantly in MIF-primed neutrophils further activated with MPO-ANCA-positive IgG or PR3-ANCA-positive IgG (270.8±9.7 vs. 421.5±9.7, P<0.001; 270.8±9.7 vs. 414.1±15.6, P<0.001, respectively). Compared with MIF-primed neutrophils, the lactoferrin concentration increased significantly in the supernatant of MIF-primed neutrophils further activated by MPO-ANCA-positive IgG (567.8±61.2ng/ml vs. 1677.0±42.5ng/ml, P<0.001) or PR3-ANCA-positive IgG (567.8±61.2ng/ml vs. 1546.0±116.2ng/ml, P<0.001), respectively. Interleukin-8 (IL-8), IL-6 and IL-23 were involved in ANCA-induced activation of MIF-primed neutrophils. ConclusionsMIF primes neutrophils by increasing ANCA antigen translocation. The primed neutrophils can be further induced by ANCA, resulting in respiratory burst and degranulation.

Detection of Anti-Pentraxin-3 Autoantibodies in ANCA-Associated Vasculitis.

ObjectivesPentraxin 3 (PTX3), in common with myeloperoxidase and proteinase 3, is stored in human neutrophil granules and is expressed on apoptotic neutrophil surface. We therefore investigated the presence of anti-PTX3 autoantibodies (aAbs) in the sera of antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) patients.MethodsPresence of anti-PTX3 autoantibodies was analysed by a specific enzyme-linked immunosorbent assay in sera from 150 patients with microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA), and eosinophilic granulomatosis with polyangiitis (EGPA), and in sera of 227 healthy subjects (HS), 40 systemic sclerosis (SSc) patients, and 25 giant cell arteritis patients (GCA). Using indirect immunofluorescence on fixed human neutrophils, we also analyzed the staining pattern associated with the presence of anti-PTX3 aAbs.ResultsAnti-PTX3 aAbs were detected in 56 of 150 (37.3%) of the AAV patients (versus 12 of 227 (5.3%) of HS, p<0.001) and, interestingly, in 7 of 14 MPO and PR3 ANCA negative AAV patients. Moreover, by indirect immunofluorescence on fixed neutrophils, anti-PTX3 aAbs gave rise to a specific cytoplasmic fluorescence pattern distinct from the classical cytoplasmic (c-ANCA), perinuclear (p-ANCA), and atypical (a-ANCA) pattern. Anti-PTX3 aAbs levels were higher in patients with active AAV as compared to patients with inactive disease.ConclusionOur work suggests that PTX3 is as a novel ANCA antigen. Anti-PTX3 aAbs appear thus as a promising novel biomarker in the diagnosis of AAV, including in patients without detectable MPO and PR3 ANCA.

High-mobility group box 1 potentiates antineutrophil cytoplasmic antibody-inducing neutrophil extracellular traps formation.

BackgroundRecent studies found that the circulating high-mobility group box 1 (HMGB1) levels could reflect the disease activity of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). HMGB1 could prime neutrophils by increasing ANCA antigens translocation for ANCA-mediated respiratory burst and degranulation. The current study aimed to investigate whether HMGB1 participates in ANCA-induced neutrophil extracellular traps (NETs) formation, which is one of the most important pathogenic aspects in the development of AAV.MethodsNETs were induced by treating neutrophils with HMGB1 and ANCA-positive IgG in vitro. NETs formation was assessed using immunofluorescence microscopy and fluorescence probe. Antagonist for relevant receptors Toll-like receptor (TLR)2, TLR4 and the receptor for advanced glycation end products (RAGE), as well as NADPH oxidase molecules were employed.ResultsThe percentage of NETs formation was significantly higher in neutrophils stimulated with HMGB1 plus ANCA-positive IgG than that in neutrophils incubated with HMGB1 or ANCA-positive IgG alone. Consistently, compared with the nonstimulated neutrophils, the cell-free DNA (cfDNA) concentration of NETs was significantly increased from 334.09 ± 46.89 ng/ml to 563.32 ± 122.07 ng/ml in the neutrophils incubated with HMGB1 plus MPO-ANCA-positive IgG (P < 0.001), and from 303.44 ± 37.14 ng/ml to 563.79 ± 145.94 ng/ml in the neutrophils incubated with HMGB1 plus PR3-ANCA-positive IgG (P < 0.001). The aforementioned effect was significantly attenuated by antagonist for relevant receptors TLR2, TLR4 and RAGE, as well as blocking NADPH oxidase.ConclusionsHMGB1 can potentiate ANCA-inducing NETs formation and may be involved in the pathogenesis of AAV. HMGB1 exerts effects on NETs formation through interaction with TLR2, TLR4 and RAGE, and the process is NADPH oxidase dependent.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0903-z) contains supplementary material, which is available to authorized users.

Can antineutrophil cytoplasmic antibody levels be used to inform treatment of pauci-immune vasculitis?

The role of serial antineutrophil cytoplasmic antibody (ANCA) measurements to guide immunosuppressive therapy in pauci-immune vasculitis is an area of ongoing controversy. Evidence elucidating the relationship between ANCA levels and disease activity continues to grow. The finding that proteinase 3-ANCA and myeloperoxidase-ANCA-associated diseases differ genetically has challenged the traditional classification and study of pauci-immune vasculitis. Multiple studies have also found that the clinical features and course of disease differ by ANCA antigen specificity more than clinical diagnosis. Advances in diagnostic assays and new techniques for the evaluation of ANCA levels over time have yielded improved assay performance in specific subsets of patients. In addition, increasing use of rituximab has added to our understanding of the relationship of ANCA levels to B-cell counts and disease relapse, which may differ by treatment regimen. The relationship between ANCA levels and disease activity is impacted by multiple factors, including antigen specificity, disease manifestations, clinical assay, and therapeutic regimen. Each of these must be taken into account when determining the significance of ANCA levels during long-term follow-up of pauci-immune vasculitis.

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.

The role of neutrophils in causing antineutrophil cytoplasmic autoantibody-associated vasculitis.

Antineutrophil cytoplasmic antibody (ANCA)-activated phagocytes cause vasculitis and necrotizing crescentic glomerulonephritis. Experimental data support the notion that activation of neutrophils and monocytes by ANCA immunoglobulin G with generation of reactive oxygen species, degranulation of proteases, and formation of neutrophil extracellular traps play a role in tissue injury. We discuss novel findings regarding the expression of ANCA antigens and the mechanisms involved in myeloid cell activation by ANCA immunoglobulin G. The contribution of neutrophil serine proteases and their specific role in the generation of interleukin-1beta (IL-1β) is highlighted. ANCA-induced reactive oxygen species generation plays an important role in downregulating inflammation by inhibition of the inflammasome-dependent caspase-1 activation and subsequent IL-1β generation. Neutrophil extracellular trap generation by ANCA-activated neutrophils and their potential role in the pathogenesis of the disease will be discussed. Lastly, the pathogenic role of the complement system will be discussed. ANCA-induced activation of both neutrophils and monocytes is one of the main pathogenic mechanisms involved in disease induction. Therefore, a better understanding of the fundamental processes involved here are necessary. Specifically, the mechanisms involved in IL-1β generation have been recently identified and could lead to better targeted novel therapies.

Involvement of protein kinase C in C5a-primed neutrophils for ANCA-mediated activation

C5a and the neutrophil C5a receptor play a central role in antineutrophil cytoplasmic antibody (ANCA)-mediated neutrophil recruitment and activation. Our recent study found that activation of p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase (ERK) and phosphoinositol 3-kinase (PI3K) are all important steps in the translocation of ANCA antigens by C5a-mediated priming and activation of neutrophils. The current study further investigated the protein kinase C (PKC) pathway of C5a-mediated neutrophils for ANCA-induced activation.The effect of the PKC inhibitor (bisindolylmaleimide I, BIS) was tested on respiratory burst and degranulation of C5a-primed neutrophils activated with ANCA, as well as on C5a-induced increase in expression of PR3 and MPO.For C5a-primed neutrophils for MPO-ANCA-induced respiratory burst, the mean fluorescence intensity (MFI) value was 369.8±18.8, which decreased to 308.3±14.2 upon pre-incubation with BIS (P<0.001). For PR3-ANCA-positive IgG, the MFI value increased in C5a-primed neutrophils, which decreased upon pre-incubation with BIS. The lactoferrin concentration increased from 414.8±26.9ng/ml in the non-primed neutrophils supernatant to 1099.8±80.7ng/ml in C5a-primed neutrophils induced by MPO-ANCA-positive IgG supernatant (P<0.001), and decreased to 814.5±45.3ng/ml upon pre-incubation with BIS (P<0.01). The lactoferrin concentration also increased in C5a-primed neutrophils induced by PR3-ANCA-positive IgG supernatant and decreased upon pre-incubation with BIS. Membrane expression of PR3 (mPR3) expression increased from 788.0±87.5 in untreated cells to 1071.3±81.3 after C5a treatment and decreased to 827.3±48.1 by BIS (P<0.05).Activation of PKC is an important step in the translocation of ANCA antigens and C5a-induced activation of neutrophils by ANCA.

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.

Antineutrophil cytoplasmic antibodies against myeloperoxidase, proteinase 3, elastase, cathepsin G and lactoferrin in Japanese patients with rheumatoid arthritis

Antineutrophil cytoplasmic antibodies (ANCAs) against myeloperoxidase (MPO), proteinase 3 (PR-3), lactoferrin (LF), cathepsin G (CG) and elastase (EL) were determined to investigate whether the presence of ANCAs is closely related to extra-articular manifestations in Japanese patients with rheumatoid arthritis (RA). Antibodies against MPO, PR-3, LF, CG and EL were determined in sera from 125 patients with RA and 83 sera from patients with other rheumatic diseases by enzyme-linked immunosorbent assay. Clinical manifestations and laboratory parameters of the patients were studied from medical records. Thirty of the 125 (24.0%) RA patients were positive for ANCAs for at least one of these 5 ANCA antigens. Among the 5 ANCAs, anti-LF antibody (anti-LF) (16.8%) was most commonly observed in patients with RA. A higher joint score (JS) and an elevated ESR were demonstrated in ANCA-positive RA patients compared to those of ANCA-negative patients (40.8±43.3, 24.3±26.2, p<0.05, 44.4±22.4, 28.9±23.6, p<0.05, respectively). No statistical differences in the presence of interstitial pneumonia, cutaneous vasculitis, rheumatoid nodules and mononeuropathy multiplex were observed between ANCA-positive and ANCA-negative patients. The presence of anti-LF is expected to be of pathological relevance, as the action of anti-LF towards LF results in the inhibition of the anti-inflammatory activity of LF.

LAMPs and NETs in the Pathogenesis of ANCA Vasculitis

Strong evidence from in vitro studies, animal models, and clinical observations suggests that anti-neutrophil cytoplasmic antibodies (ANCA) play a critical role in the vascular damage of ANCA-associated vasculitis (AAV).1 Understanding the key pathogenic mechanisms of AAV may provide a safer and more rational therapeutic approach. Although the pathogenic role of ANCA in AAV has been studied extensively, it is still not clear why ANCAs appear. Two recent papers in Nature Medicine 2,3 provide a previously undescribed molecular explanation of the origin, development, and perpetuation of injury in AAV that may have important clinical implications. In the first article, Kain et al. 2 reported that infection by fimbriated bacteria triggers cross-reactive autoimmunity to a previously characterized ANCA antigen, lysosomal membrane protein 2 (LAMP-2), resulting in the production of autoantibodies that activate neutrophils and damage human microvascular endothelium in vitro and cause pauci-immune focal necrotizing glomerulonephritis (FNGN) in rats.2 In the second article, Kessenbrock et al. 3 demonstrated that the formation of neutrophil extracellular traps (NETs), which are involved in neutrophil cell death during infection, trigger vasculitis, and perpetuate the autoimmune response against neutrophil components in patients with AAV. It has long been suspected that infection plays a role in the development of ANCAs. They were discovered in 1982 while Davies et al. 4 were studying antinuclear antibodies in serum from patients with FNGN, many of whom were infected with the Ross River virus. Several ANCA autoantigens are leukocyte proteins implicated in host defense against infectious diseases.5,6 Wegener's granulomatosis may be an atypical cell-mediated immune response to an exogenous or endogenous antigen in the respiratory tract that results in granuloma formation and the development of humoral autoimmunity to proteinase 3 (PR3).6 One theory of PR3-directed autoimmunity involves the complementary peptide of PR3, which is …

Activation of granulocytes by anti-neutrophil cytoplasmic antibodies (ANCA): a Fc gamma RII-dependent process.

ANCA have been demonstrated to induce the respiratory burst in primed neutrophils. In this study we have extended the investigations on neutrophil activation by ANCA directed against proteinase 3 (PR3), myeloperoxidase (MPO) and lactoferrin (LF), and we have analysed the underlying mechanisms. All three ANCA antigens were expressed on the cell surface of primed neutrophils. Superoxide production assayed by both cytochrome c reduction and oxidation of dihydrorhodamine 123, was induced by heterologous polyclonal anti-MPO and anti-LF antibodies, and ANCA-positive plasma samples. Induction of superoxide production was dose-dependent. F(ab')2 fragments did not induce the respiratory burst. Blockade of Fc receptors by specific MoAbs showed that anti-Fc gamma RII antibodies were able to turn off the ANCA-induced respiratory burst, whereas anti-Fc gamma RIII antibodies did not. Plasma samples that induced the respiratory burst did not differ from samples that did not induce superoxide production with respect to ANCA titre, but had higher levels of the IgG3 subclass of ANCA. Levels of the other subclasses of ANCA were comparable between those samples. We conclude that ANCA-induced activation of primed neutrophils is Fc gamma RII-dependent, and appears to be facilitated by antibodies of the IgG3 subclass.