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Objectives: Neutrophils play a key role in ANCA-associated vasculitis, both as targets of autoimmunity and as facilitators of vascular damage. In granulomatosis with polyangiitis (GPA), the data regarding the production of reactive oxygen species (ROS) in neutrophils are unclear. Further, recent data suggests that ROS production could have an anti-inflammatory effect through the regulation of inflammasomes and IL-1-related cytokines. We aimed to analyse ROS production in neutrophils from patients with GPA and investigate its association with IL-1-related cytokines and the autoantigen PR3., Methods: Seventy-two GPA patients with disease flare were included in the NEUTROVASC prospective cohort study. ROS production in whole blood of patients with active GPA was evaluated and compared with that in the same patients in remission or healthy controls. Associations between ROS production, PR3 membrane expression on neutrophils, serum levels of IL-1-related cytokines as well as inflammasome-related proteins were analysed., Results: We observed a robust defect in ROS production by neutrophils from patients with active GPA compared with healthy controls, independent of glucocorticoid treatment. Serum levels of IL-1-related cytokines were significantly increased in GPA patients, particularly in patients with kidney involvement, and levels of these cytokines returned to normal after patients achieved remission. Further, inflammasome-related proteins were significantly dysregulated in the cytosol of neutrophils as well as the serum from GPA patients., Conclusion: Our data suggests that ROS production and regulation of inflammasomes in neutrophils from patients with GPA are disturbed and may be a potential therapeutic target., Trial Registration: ClinicalTrials.gov, https://www.clinicaltrials.gov, NCT01862068., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

CD177 is a neutrophil-specific receptor presenting the proteinase 3 (PR3) autoantigen on the neutrophil surface. CD177 expression is restricted to a neutrophil subset, resulting in CD177 pos /mPR3 high and CD177 neg /mPR3 low populations. The CD177 pos /mPR3 high subset has implications for antineutrophil cytoplasmic autoantibody (ANCA)-associated autoimmune vasculitis, wherein patients harbor PR3-specific ANCAs that activate neutrophils for degranulation. Here, we generated high-affinity anti-CD177 monoclonal antibodies, some of which interfered with PR3 binding to CD177 (PR3 "blockers") as determined by surface plasmon resonance spectroscopy and used them to test the effect of competing PR3 from the surface of CD177 pos neutrophils. Because intact anti-CD177 antibodies also caused neutrophil activation, we prepared nonactivating Fab fragments of a PR3 blocker and nonblocker that bound specifically to CD177 pos neutrophils. We observed that Fab blocker clone 40, but not nonblocker clone 80, dose-dependently reduced anti-PR3 antibody binding to CD177 pos neutrophils. Importantly, preincubation with clone 40 significantly reduced respiratory burst in primed neutrophils challenged with either monoclonal antibodies to PR3 or PR3-ANCA immunoglobulin G from ANCA-associated autoimmune vasculitis patients. After separating the two CD177/mPR3 neutrophil subsets from individual donors by magnetic sorting, we found that PR3-ANCAs provoked significantly more superoxide production in CD177 pos /mPR3 high than in CD177 neg /mPR3 low neutrophils, and that anti-CD177 Fab clone 40 reduced the superoxide production of CD177 pos cells to the level of the CD177 neg cells. Our data demonstrate the importance of the CD177:PR3 membrane complex in maintaining a high ANCA epitope density and thereby underscore the contribution of CD177 to the severity of PR3-ANCA diseases., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Granulomatosis with polyangiitis (GPA) is a rare but serious necrotizing auto-immune vasculitis. GPA is mostly associated with the presence of Anti-Neutrophil Cytoplasmic Antibody (ANCA) targeting proteinase 3 (PR3-ANCA), a serine protease contained in neutrophil granules but also exposed at the membrane. PR3-ANCAs have a proven fundamental role in GPA: they bind neutrophils allowing their auto-immune activation responsible for vasculitis lesions. PR3-ANCAs bind neutrophil surface on the one hand by their Fab binding PR3 and on the other by their Fc binding Fc gamma receptors. Despite current therapies, GPA is still a serious disease with an important mortality and a high risk of relapse. Furthermore, although PR3-ANCAs are a consistent biomarker for GPA diagnosis, relapse management currently based on their level is inconsistent. Indeed, PR3-ANCA level is not correlated with disease activity in 25% of patients suggesting that not all PR3-ANCAs are pathogenic. Therefore, the development of new biomarkers to evaluate disease activity and predict relapse and new therapies is necessary. Understanding factors influencing PR3-ANCA pathogenicity, i.e. their potential to induce auto-immune activation of neutrophils, offers interesting perspectives in order to improve GPA management. Most relevant factors influencing PR3-ANCA pathogenicity are involved in their interaction with neutrophils: level of PR3 autoantigen at neutrophil surface, epitope of PR3 recognized by PR3-ANCA, isotype and glycosylation of PR3-ANCA. We detailed in this review the advances in understanding these factors influencing PR3-ANCA pathogenicity in order to use them as biomarkers and develop new therapies in GPA as part of a personalized approach., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Granel, Korkmaz, Nouar, Weiss, Jenne, Lemoine and Hoarau.)

Objective: To determine to neutrophil-to-lymphocyte ratio (NLR) in granulomatosis with polyangiitis (GPA) patients and to study its relation to disease manifestations and activity., Methods: The study included 44 GPA patients and 44 matched age and sex controls. Full history taking, thorough clinical examination with more attention to ocular examination, laboratory and radiological investigations were considered. Disease activity was assessed using the Birmingham Vasculitis Activity Score (BVAS)., Results: The patients (21 males and 23 females) had a mean age of 45.66±7.24 years, disease duration 6.8±3.6 years and BVAS 50.1±14.3. All patients had a positive cytoplasmic anti-neutrophil cytoplasmic antibody (c-ANCA) while only 5 had a positive p-ANCA. The NLR was significantly increased in the GPA patients (5.1±2.4) compared to the control (1.5±0.8) (P<.0001). Ten patients with uveitis had a significantly higher NLR (6.5±1.9) compared to those without (4.7±2.4) (0.03) while those with proptosis (n=10), cutaneous manifestations (n=17) or ischemic heart disease (n=9) had a significantly lower NLR than those without (P=.0001, P=.017 and P=.046 respectively). The NLR did not significantly correlate with any of the patients' characteristics. The NLR inversely yet insignificantly correlated with the disease activity (r=-0.02, P=.93)., Conclusion: The NLR may have a significant role in the pathogenesis of GPA, the development of uveitis or proptosis, cutaneous manifestations and ischemic heart disease. NLR may serve as a future potential companion to c-ANCA positivity in diagnosing and evaluating GPA and may play a role in the tissue-specific and clinical characteristics., (Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Reumatología y Colegio Mexicano de Reumatología. All rights reserved.)

Granulomatosis with polyangiitis (GPA) is an autoimmune vasculitis associated with anti-neutrophil-cytoplasmic antibodies (ANCA) against proteinase 3 leading to kidney damage. Neutrophils from those patients have increased expression of membrane proteinase 3 during apoptosis. Here we examined whether neutrophils from patients with GPA have dysregulated protein expressions associated with apoptosis. A global proteomic analysis was performed comparing neutrophils from patients with GPA, with healthy individuals under basal conditions and during apoptosis. At disease onset, the cytosolic proteome of neutrophils of patients with GPA before treatment was significantly different from healthy controls, and this dysregulation was more pronounced following ex vivo apoptosis. Proteins involved in cell death/survival were altered in neutrophils of patients with GPA. Several proteins identified were PR3-binding partners involved in the clearance of apoptotic cells, namely calreticulin, annexin-A1 and phospholipid scramblase 1. These proteins form a platform at the membrane of apoptotic neutrophils in patients with GPA but not healthy individuals and this was associated with the clinical presentation of GPA. Thus, our study shows that neutrophils from patients with GPA have an intrinsic dysregulation in proteins involved in apoptotic cell clearance, which could contribute to the unabated inflammation and autoimmunity in GPA. Hence, harnessing these dysregulated pathways could lead to novel biomarkers and targeted therapeutic opportunities to treat kidney disease., (Copyright © 2019 International Society of Nephrology. All rights reserved.)

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV), including granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA), are autoimmune conditions associated with small vessel inflammation. Earlier studies indicate that complement activation via the alternative pathway plays a major role in the pathogenesis. In this study we have investigated if ANCA-activation of neutrophils from AAV patients leads to activation of the alternative complement pathway. C5a-primed neutrophils (PMN) from 10 AAV patients and 10 healthy controls (HC) were stimulated with PMA or IgG purified from PR3-ANCA positive patients (ANCA IgG). The supernatants were analyzed for release of complement proteins and markers of different granules by ELISA, and release of microparticles (MP) by flow cytometry. The ability of the supernatants to activate the alternative complement pathway was determined by incubation with normal serum and C3bBbP and C5a were measured by ELISA. MP were analyzed by flow cytometry and removed by centrifugation. The supernatants from the AAV patients' neutrophils produced significantly more C3bBbP compared with HCs (p = 0.0001). C3bBbP levels correlated with the number of MP. After removal of MP from the supernatants, alternative pathway activation was significantly lower. This study shows that primed and ANCA-stimulated neutrophils from AAV patients have a greater ability to activate the alternative complement pathway compared to primed neutrophils from healthy controls. This finding emphasizes the role of complement in the pathogenesis of AAV - underlining the therapeutic potential of C5a and other complement blockade., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Delta neutrophil index (DNI) represents the immature granulocytes count associated with neutrophil-consumption. We investigated whether DNI might be associated with Birmingham vasculitis activity score (BVAS) at diagnosis and could predict relapse during the follow-up in patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV)., Materials and Methods: We reviewed the medical records of 97 patients having DNI results. Twenty patients had granulomatosis with polyangiitis (GPA), 58 had microscopic polyangiitis (MPA), and 19 had eosinophilic GPA (EGPA). We collected clinical and laboratory data including BVAS, five factor score (FFS), and DNI. The correlation coefficient and cumulative relapse free survival rate were obtained. The optimal cut-off of DNI was extrapolated by calculating the area under the receiver operator characteristic curve., Results: DNI was significantly related to cross-sectional BVAS. Furthermore, among continuous variables, only DNI could reflect BVAS of GPA and MPA, but not EGPA. Severe AAV was defined as BVAS ≥20 (the highest quartile). At diagnosis, patients having DNI ≥0.65% had a significantly higher risk of severe GPA and MPA than those having not (relative risk 4.255) at diagnosis. During the follow-up, DNI ≥0.65% could predict the higher relapse rate., Conclusion: DNI could reflect BVAS at diagnosis and furthermore, DNI ≥0.65% could not only identify severe AAV at diagnosis, but also predict relapse during the follow-up in patients with GPA and MPA., Competing Interests: The authors have no financial conflicts of interest., (© Copyright: Yonsei University College of Medicine 2018.)

Neutrophils are critical in the defense against bacterial and fungal pathogens, and they also modulate the inflammatory process. The areas where neutrophils are studied have expanded from the restricted field of antibacterial defense to the modulation of inflammation and finally, to fine-tuning immune responses. As a result, recent studies have shown that neutrophils are implicated in several systemic autoimmune diseases, although exactly how neutrophils contribute to these diseases and the molecular mechanisms responsible are still under investigation. In a group of autoimmune vasculitides associated with anti-neutrophil cytoplasmic antibodies (AAVs), granulomatosis with polyangiitis (GPA) illustrates the concept that autoimmunity can develop against one specific neutrophil protein, namely, proteinase 3 (PR3), one of the four serine protease homologs contained within azurophilic granules. In this review, we will focus on recent molecular analyses combined with functional studies that provide clear evidence that the pathogenic properties of PR3 are not only a result of its enzymatic activity but also mediated by a particular structural element-the hydrophobic patch-which facilitates associations with various proteins and lipids and permits anchorage into the plasma membrane. Furthermore, these unique structural and functional characteristics of PR3 might be key contributors to the systemic inflammation and to the immune dysregulation observed in GPA., (© Society for Leukocyte Biology.)

Objectives: The aim of this study was to analyse the role of netting neutrophils in the pathogenesis of granulomatosis with polyangiitis (GPA), especially their interplay with peripheral blood mononuclear cells (PBMCs)., Methods: The amount of cell-free DNA (cfDNA) was determined in sera from GPA patients (pairs active/inactive state of disease, n=18) and from healthy controls (HCs, n=10). Furthermore, we performed in vitro incubation experiments using PBMCs and NETs from patients and HCs for accessing the effect of NETs on PBMC behaviour. We determined proliferation of T- and B-cells (CSFE assay), B-cell maturation (CD38 staining and flow cytometry), production of IgG (ELISpot, ELISA), and secretion of the cytokines IFN-γ, IL-4, IL-10, IL-17A (ELISA)., Results: We detected a significant increase in serum cfDNA levels of GPA patients compared to HCs. The concentration of cfDNA was associated with disease activity. NETs of patients and HCs induced proliferation of CD4+ T- cells and CD19+ B-cells and maturation of B-cells. Furthermore, we detected an increase in IL-17A secretion after stimulating PBMCs with NETs. A significant difference between PBMCs from GPA patients and HCs was not detectable., Conclusions: NETs activate PBMCs of HCs and GPA patients. Our findings give supportive evidence that NETosis plays a role in the pathogenesis of GPA.

Objectives: Eosinophilic granulomatosis with polyangiitis (EGPA) is associated with an inflammation and the presence of antineutrophil cytoplasmic antibodies (ANCA). Thus, we investigated the impact of ANCAs and eosinophilic inflammation on neutrophil activation and extracellular traps (NETs) formation., Methods: We recruited 29 patients in the remission of EGPA (17 ANCA-negative and 12 ANCA-positive, including 7 p-ANCA-positive and 5 c-ANCA-positive patients). Healthy donors' neutrophils were stimulated with EGPA patients' serum. NETs formation was assessed by immunofluorescence and scanning electron microscopy., Results: EGPA patients presented enhanced ability to generate NETs compared to healthy subjects (20.3±8.2% vs. 2.7±1.5%, p=0.0036). However, there were no differences in NETs formation between ANCA-positive and ANCA-negative patients (23±11.2% vs. 17±6.1%, p=0.15). There was also no correlation between NETs generation and the amount of circulating DNA in EGPA patients. Among ANCA-positive patients, p-ANCA-positives showed the highest percentage of NETs as compared to cANCA-positive and ANCA-negative patients (27.3±10.3% vs. 17.8±10.5% and vs. 17±6.1%, both p<0.01, respectively). Eosinophils number correlated with the percentage of NETs in the whole EGPA group (r=0.53, p=0.039), but we failed to observe the correlation with an eosinophil cationic protein (r=0.49, p=0.058)., Conclusions: EGPA patients' serum has the ability to induce NETosis with no regard to the ANCA status in contrast to other vasculitides, where p-ANCA were considered as the main factor. Interestingly, NETs formation in EGPA patients connected with the number of eosinophils might be of major relevance. Further studies are required to assess which eosinophil-derived factors might be responsible for the neutrophils activation in EGPA patients.

We studied 91 patients with granulomatosis with polyangiitis (GPA; Wegener's granulomatosis) who were dealt with by otolaryngologists in Japan. The upper respiratory tract (URT) alone was involved in 56%. Regarding findings of PR3-ANCA, histology and initial diagnosis, of the 40 patients in whom sites other than the URT were involved, 64 and 73% were positive for PR3-ANCA and histology, respectively. On the other hand, of 51 patients in whom only the URT was involved, only 49 and 31% were positive for PR3-ANCA and histology, respectively. With regard to diagnosis, definitive and probable diagnoses were made in 78 and 20%, respectively, of patients involving sites other than the URT. On the other hand, definitive and probable diagnoses were made in only 22 and 37%, respectively, of patients involving the URT alone; the Japanese diagnostic criteria were inapplicable to 41%. Of the 21 patients to whom the diagnostic criteria were inapplicable, 13 (62%) developed additional symptoms and signs during the observation period, and they then fulfilled the Japanese diagnostic criteria. Among these patients, 8 were MPO-ANCA-positive and 3 patients had 2 or more sub-lesions in the URT. If the diagnostic criteria included cases with MPO-ANCA (±) and 2 lesions in the URT, the diagnostic rates increased from 59 to 86% even though they were of limited form. Thus, about 60% of the patients with URT symptoms alone did not satisfy the Japanese diagnostic criteria at the initial visit.

Proteinase 3 (PR3) is the target of anti-neutrophil cytoplasm Abs in granulomatosis with polyangiitis, a form of systemic vasculitis. Upon neutrophil apoptosis, PR3 is coexternalized with phosphatidylserine and impaired macrophage phagocytosis. Calreticulin (CRT), a protein involved in apoptotic cell recognition, was found to be a new PR3 partner coexpressed with PR3 on the neutrophil plasma membrane during apoptosis, but not after degranulation. The association between PR3 and CRT was demonstrated in neutrophils by confocal microscopy and coimmunoprecipitation. Evidence for a direct interaction between PR3 and the globular domain of CRT, but not with its P domain, was provided by surface plasmon resonance spectroscopy. Phagocytosis of apoptotic neutrophils from healthy donors was decreased after blocking lipoprotein receptor-related protein (LRP), a CRT receptor on macrophages. In contrast, neutrophils from patients with granulomatosis with polyangiitis expressing high membrane PR3 levels showed a lower rate of phagocytosis than those from healthy controls not affected by anti-LRP, suggesting that the LRP-CRT pathway was disturbed by PR3-CRT association. Moreover, phagocytosis of apoptotic PR3-expressing cells potentiated proinflammatory cytokine in vitro by human monocyte-derived macrophages and in vivo by resident murine peritoneal macrophages, and diverted the anti-inflammatory response triggered by the phagocytosis of apoptotic cells after LPS challenge in thioglycolate-elicited murine macrophages. Therefore, membrane PR3 expressed on apoptotic neutrophils might amplify inflammation and promote autoimmunity by affecting the anti-inflammatory "reprogramming" of macrophages.

Granulomatosis with polyangiitis (Wegener's ) is a rare autoimmune disease associated with the presence of antibodies directed against neutrophil antigen, proteinase-3 (PR3). The mechanisms by which anti-neutrophil cytoplasmic antibodies (ANCA) may activate neutrophils are still not well understood. In the present study we analyzed neutrophil gene expression profile after anti-PR3 antibodies stimulation. Briefly neutrophils isolated from 12 healthy volunteers, who tested negative for anti-PR3 autoantibodies, were stimulated with anti-PR3 IgG and activation of 147 genes was analyzed with the use of TaqMan low-density arrays. In stimulated neutrophils we observed up-regulation of 13 genes (CCL2, CXCL2, VCAM1, MMP9, PLCB4, PDE4C, PLA2G4C, RAC1, RHOA, IRAK1, CACNA1D, CACNB2, PTGDR), further 11 genes were up-regulated only in some donors (IL13, PF4, IL2RG, ITGB1, CD83, PLA2G7, ALOX12, AXNA1, AXNA5, LTA4H, MCR2) yet two others (HRH3 and PLA2G2D) were up-regulated in a few samples and undetectable in others. The obtained results demonstrate that c-ANCA mediated activation of neutrophils involve several pathways mediated via FcγRs like calcium signaling, phosphatidylinositol 3-kinase AKT pathway or MAPK signaling systems, but also inducts others, like G-protein signaling. Neutrophil is a very sensitive cell, responding to many environment changes. As our results showed, some anti-PR3 responses are highly variable across donors. Perhaps, this variablity also contribute to the susceptibility for granulocyte vasculitis and requires future studies.

The physiological and pathological functions of proteinase 3 (PR3) are not well understood due to its close similarity to human neutrophil elastase (HNE) and the lack of a specific inhibitor. Based on structural analysis of the active sites of PR3 and HNE, we generated mutants derived from the polyvalent inhibitor SerpinB1 (monocyte/neutrophil elastase inhibitor) that specifically inhibit PR3 and that differ from wt-SerpinB1 by only 3 or 4 residues in the reactive center loop. The rate constant of association between the best SerpinB1 mutant and PR3 is 1.4 × 10⁷ M⁻¹ · s⁻¹, which is ∼100-fold higher than that observed with wt-SerpinB1 and compares with that of α1-protease inhibitor (α1-PI) toward HNE. SerpinB1(S/DAR) is cleaved by HNE, but due to differences in rate, inhibition of PR3 by SerpinB1(S/DAR) is only minimally affected by the presence of HNE even when the latter is in excess. SerpinB1(S/DAR) inhibits soluble PR3 and also membrane-bound PR3 at the surface of activated neutrophils. Moreover, SerpinB1(S/DAR) clears induced PR3 from the surface of activated neutrophils. Overall, these specific inhibitors of PR3 will be valuable for defining biological functions of the protease and may prove useful as therapeutics for PR3-related inflammatory diseases, such as Wegener's granulomatosis.

Anti-neutrophil cytoplasmic antibodies (c-ANCA) targeting proteinase 3 (PR3) are implicated in the pathogenesis of Wegener's granulomatosis (WG). Fulminant disease can present as acute lung injury (ALI). In this study, a model of ALI in WG was developed using isolated rat lungs. Isolated human polymorphonuclear leukocytes (PMNs) were primed with tumour necrosis factor (TNF) to induce surface expression of PR3. Co-perfusion of TNF-primed neutrophils and monoclonal anti-PR3 antibodies induced a massive weight gain in isolated lungs. This effect was not observed when control immunoglobulin G was co-perfused with TNF-primed PMNs. The c-ANCA-induced oedema formation was paralleled by an increase in the capillary filtration coefficient as a marker of increased pulmonary endothelial permeability. In contrast, pulmonary artery pressure was not affected. In the presence of the oxygen radical scavenger superoxide dismutase and a NADPH oxidase inhibitor, c-ANCA-induced lung oedema could be prevented. Inhibition of neutrophil elastase was equally effective in preventing c-ANCA-induced lung injury. In conclusion, anti-PR3 antibodies induced neutrophil mediated, elastase- and oxygen radical-dependent ALI in the isolated lung. This experimental model supports the hypothesis of a pathogenic role for c-ANCA in WG and offers the possibility of the development of therapeutic strategies for the treatment of lung injury in fulminant WG.

Proteinase 3 (Pr3), the main target of anti-neutrophil cytoplasmic antibodies, is a neutrophil serine protease that may be constitutively expressed at the surface of quiescent circulating neutrophils. This raises the question of the simultaneous presence in the circulation of constitutive membrane-bound Pr3 (mPr3) and its plasma inhibitor alpha1-protease inhibitor (alpha1-Pi). We have looked at the fate of constitutive mPr3 at the surface of circulating blood neutrophils and of induced mPr3 on triggered neutrophils. We found significant Pr3 activity at the surface of activated neutrophils but not at the surface of quiescent neutrophils whatever the constitutive expression. This suggests that constitutive mPr3 is enzymatically inactive or its active site is not accessible to the substrate. Supporting this conclusion, we have not been able to demonstrate any interaction between constitutive mPr3 and alpha1-Pi, whereas induced mPr3 is cleared from the cell surface when activated cells are incubated with this inhibitor. But, unlike membrane-bound elastase that is also cleared from the surface of activated cells, mPr3 remained bound to the membrane when inhibited by elafin or by a low molecular weight chloromethyl ketone inhibitor, which shows that it binds more tightly to the neutrophil membrane. mPr3 may thus be present at the surface of circulating neutrophils in an environment replete with alpha1-Pi. The permanent presence of inactive Pr3 at the surface of quiescent neutrophils may explain why Pr3 is a major target of anti-neutrophil cytoplasmic antibodies, whose binding activates neutrophils and triggers inflammation, as in Wegener granulomatosis.

Objective: Wegener's granulomatosis (WG) is strongly associated with antineutrophil cytoplasmic autoantibodies (ANCAs) directed against proteinase 3 (PR3). Recent studies have shown that membrane-bound PR3 (mPR3) is differentially expressed and colocalizes with CD177/NB1 on circulating neutrophils. We undertook this study to assess the differential expression of CD177 on neutrophils from patients with ANCA-associated systemic vasculitis (ASV) in comparison with patients with systemic lupus erythematosus (SLE), patients with rheumatoid arthritis (RA), and healthy individuals, and to investigate whether colocalization of mPR3 and CD177 affects anti-PR3-mediated neutrophil activation., Methods: Expression of CD177 and mPR3 was analyzed by flow cytometry on isolated neutrophils from patients with ASV (n=53), those with SLE (n=30), those with RA (n=26), and healthy controls (n=31). Neutrophil activation mediated by anti-PR3 antibodies was assessed by measuring the oxidative burst with a dihydrorhodamine assay., Results: Percentages of CD177-expressing neutrophils were significantly higher in patients with ASV and those with SLE than in healthy controls. In 3 healthy donors, CD177 expression was not detected. After priming with tumor necrosis factor alpha, neutrophils remained negative for CD177 while mPR3 expression was induced. Neutrophils from CD177-negative donors or CD177- neutrophils sorted from donors with bimodal expression were susceptible to anti-PR3-mediated oxidative burst. Variation in the extent of anti-PR3-mediated neutrophil activation among different donors occurred independent of the percentage of CD177-expressing neutrophils., Conclusion: Membrane expression of CD177 on circulating neutrophils is increased in patients with ASV and in those with SLE, but not in RA patients. However, primed neutrophils from CD177-negative individuals also express mPR3 and are susceptible to anti-PR3-mediated oxidative burst, suggesting that recruitment of CD177-independent mPR3 is involved in anti-PR3-induced neutrophil activation.

Wegener's Granulomatosis (WG) is a life-threatening autoimmune disease. A pathogenic role for anti-neutrophil cytoplasmic autoantibodies (ANCAs) by inducing necrotizing damage to the vessel wall has been strongly suggested by in vitro and in vivo experimental data. Proteinase 3 (PR3), a serine protease mainly stored in the azurophilic granules of neutrophils, has been identified as a major ANCA-antigen in WG. Elevated expression levels of membrane-bound PR3 (mPR3) has been observed in WG and some other chronic inflammatory diseases, suggesting a pathogenic role of mPR3 by allowing interaction with PR3-ANCA. Recent studies revealed CD177 as a receptor for mPR3 on the neutrophil membrane. However, we recently showed that CD177 negative neutrophils also express mPR3 and are susceptible to PR3-ANCA induced neutrophil activation. Therefore, it is of interest to further investigate the functional consequences of binding of mPR3 to CD177, to explore other binding partners for mPR3 on the neutrophil membrane, and to study the relevance of colocalization of these molecules for disease pathogenesis. This review gives updated information on the mechanism of mPR3 expression and the relevance of colocalization of mPR3 with other molecules on the neutrophil membrane for the pathophysiological events occurring in WG.

Objectives: To gain insight into the immune pathogenesis of primary ANCA-associated vasculitides, the prevalence of circulating T lymphocytes expressing CD11b as a marker for activation was analysed in patients with WG or microscopic polyangiitis. METHODS; Receptor expression and IFNgamma synthesis were measured in T cells of patients with active disease by cytofluorometry and compared with expression in patients in remission and in healthy donors., Results: During active disease, a small but conspicuous population of CD8+CD28+CD11b+ was found which produced IFNgamma. In healthy donors and in patients in remission or undergoing immunosuppressive therapy, CD11b was exclusively associated with CD8+CD28- cells, the latter being more frequent in patients with long-lasting or severe disease. In vitro experiments confirmed that CD11b is up-regulated when T cells are activated. After multiple rounds of restimulation, the CD11b expression persists whereas CD28 expression is lost, compatible with the notion that CD8+CD28+CD11b+ represents a transient phenotype in the course of T-cell activation. The IFNgamma-producing T cells activated polymorphonuclear neutrophils (PMN) to express MHC class II, thus generating the same PMN phenotype as in patients with active ANCA-associated vasculitis. A similar PMN phenotype could be generated by cultivation with supernatants of activated T cells or by IFNgamma alone, but not by antibodies to proteinase 3., Conclusions: In active primary vasculitis, a small population of CD8+ T cells, identified by the expression of CD11b, expands, producing IFNgamma. These T cells could activate PMN, thus generating a long-living and potentially destructive PMN phenotype.

The expression and production of cytokines by cells of the innate immune system, including monocytes/macrophages, dendritic and NK cells, play a critical role not only in defensive and inflammatory but also in immunoregulatory and anti-/pro-tumoral processes. Studies performed in the last years have well ascertained that polymorphonuclear neutrophils can also be induced to express and produce chemokines, proinflammatory, anti-inflammatory, immunoregulatory, angiogenic and fibrogenic cytokines, as well as ligands belonging to the TNF superfamily. Among the latter group of molecules, B-cell-activating factor (BAFF)/B lymphocyte stimulator (BLyS), known to be essential for B lymphocyte homeostasis and related pathologies, has recently been identified as one of the factors potentially expressed by human neutrophils. The addition of this novel TNF superfamily member, and more recently also of the closely related "A Proliferation-Inducing Ligand" (APRIL), to the list of cytokines produced by neutrophils not only testifies to the continuous growth of this area of investigation, but also implies the involvement of neutrophils in B-cell-dependent autoimmune diseases and tumors.

Wegener's Granulomatosis (WG) is closely associated with antineutrophil cytoplasmic autoantibodies (ANCA), particularly those directed to proteinase 3 (PR3). ANCA directed to myeloperoxidase (MPO) are associated with microscopic polyangiitis (MPA) and the Churg Strauss syndrome. PR3-ANCA associated vasculitis differs from MPO-ANCA associated vasculitis particularly in the occurrence of granulomatous inflammation and more widespread disease in the former condition. Studies in experimental animals strongly suggest a pathogenic role for MPO-ANCA, but this is less clear for PR3-ANCA. Here, we review pathogenic pathways in PR3-ANCA associated vasculitis focussing on unique patterns of PR3 expression on the neutrophil membrane in WG, as surface expression of PR3 is a prerequisite for stimulation by PR3-ANCA; (PR3-specific) cellular immunity in WG; and the role of Staphylococcus aureus carriage in this disease. These factors, together, may explain, at least in part, differences in clinical expression between MPO-ANCA and PR3-ANCA associated vasculitis.

Proteinase 3 (PR3), a serine proteinase contained in neutrophil azurophilic granules, is considered a risk factor for vasculitides and rheumatoid arthritis when expressed on the outer leaflet of neutrophil plasma membrane and is the preferred target of antineutrophil cytoplasm autoantibodies (ANCA) in Wegener granulomatosis. ANCA binding to PR3 expressed at the surface of neutrophils activates them. Evidence is provided that neutrophil apoptosis induced significantly more membrane PR3 expression without degranulation (but no enhanced membrane CD35, CD66b, CD63, myeloperoxidase, or elastase expression). This observation was confirmed on cytoplasts, a model of granule-free neutrophils. We hypothesized that PR3 could interact with proteins involved in membrane flip-flop (eg, phospholipid scramblase 1 [PLSCR1]). PR3-PLSCR1 interaction in neutrophils was demonstrated by confocal microscopy and coimmunoprecipitation. In the RBL-2H3 rat mast-cell line stably transfected with PR3 or its inactive mutant (PR3S203A), PR3 externalization depended on PLSCR1, as shown by less PR3 externalization in the presence of rPLSCR1 siRNA, but independently of its serine-proteinase activity. Finally, apoptosis-externalized PR3 decreased the human macrophage-phagocytosis rate of apoptotic PR3 transfectants. Therefore, in addition to ANCA binding in vasculitis, the proinflammatory role of membrane PR3 expression may involve interference with macrophage clearance of apoptotic neutrophils.

Purpose of Review: Recent progress in the identification of leukemia antigens has stimulated the development of vaccines to treat hematological malignancies. Here we review the identification and characterization of the myeloid leukemia-specific antigens proteinase 3 and neutrophil elastase found in the primary (azurophil) granule proteins of granulocytes and their precursors. A peptide 'PR1' derived from these proteins induces powerful HLA-A0201-restricted CD8 T-cell proliferation. PR1-specific T cells are cytotoxic to leukemia and myelodysplastic syndrome progenitors, and occur at low frequencies in normal individuals. Frequencies are higher in patients with myeloid leukemias, and highest in patients with chronic myeloid leukemia entering molecular remission after allogeneic stem cell transplantation., Recent Findings: These observations, together with the known association of autoimmunity to proteinase 3 and neutrophil elastase in Wegener's granulomatosis, support the concept that there is a natural immunity to primary granule proteins which can be boosted to enhance immunity to leukemia. Preliminary reports indicate that PR1 peptide vaccination induces significant increases in PR1-specific cytotoxic T cells with rapid and durable remissions in some patients with advanced myeloid leukemias., Summary: These promising developments in antileukemia vaccines have stimulated research to optimize vaccine delivery and modify regulation of natural T-cell immunity to primary granule proteins to improve treatment of otherwise refractory myeloid leukemias and myelodysplastic syndrome.

Proteinase 3 (PR3) is found in neutrophil and monocyte lysosomal granules. Anti-neutrophil cytoplasmatic antibodies (ANCA) with specificity for PR3 are characteristic for patients with Wegener's granulomatosis. The interaction of ANCA with neutrophilic ANCA antigens is necessary for the development of ANCA-associated diseases. ANCA bind to membrane-expressed PR3 and induce full-blown activation in primed neutrophils. We discuss two different aspects of membrane PR3 (mPR3). The first aspect is the amount of PR3 and mechanisms controlling this issue. The second aspect is the presence of two neutrophil subsets that differ in the mPR3 expression phenotype.

Background: Recently, the in vivo pathogenic role of anti-neutrophil cytoplasm autoantibodies (ANCA) in ANCA-associated vasculitis has been challenged by Abdel-Salam et al. In their report, they observed that ANCA directed against proteinase 3 (PR3) cannot bind to their target autoantigen PR3 on circulating neutrophils (PMN). Here we present evidence that human PR3-ANCA do specifically bind to PMN that express PR3 on their membrane., Methods: PMN were isolated from donors showing bimodal membrane PR3 expression on their PMN (N= 3). TNFalpha-primed PMN or PMA-stimulated PMN were incubated with serum or plasma from PR3-ANCA-positive patients with Wegener's granulomatosis (WG) (N= 8) or healthy controls (N= 8). Binding of IgG in serum or plasma samples to PMN was assessed by indirect immunofluorescence., Results: Binding of IgG in undiluted plasma or serum from PR3-ANCA-positive WG-patients to PMN was significantly increased compared to plasma or serum from healthy controls. Dilution of plasma and serum showed concentration-dependent binding of IgG. Double staining for PR3 and IgG demonstrated that IgG in plasma or serum from PR3-ANCA-positive patients only bound to those PMN that expressed PR3, and not to PMN that lacked PR3 expression on their membrane., Conclusion: PR3-ANCA in undiluted serum or plasma from PR3-ANCA-positive WG patients bind to TNFalpha- primed and PMA-stimulated PMN that express PR3 on their membrane. Therefore, the hypothesis that PR3-ANCA can bind and activate primed PMN is still the most attractive explanation for the contribution of PR3-ANCA to the pathogenesis of Wegener's granulomatosis.

A large membrane proteinase 3 (mPR3)-positive neutrophil subset (mPR3high) is a risk for Wegener's granulomatosis (WG). The relationship between mPR3 expression and clinical manifestations was investigated in 81 WG patients and mPR3 expression was studied in CD34+ stem cell-derived human neutrophils. The mPR3high neutrophil percentage correlated with renal function, anemia, and albumin at the time of presentation. The mPR3high neutrophil percentage and renal failure severity correlated directly after 5 yr. For elucidating mechanisms that govern mPR3 expression, studies were conducted to determine whether the genetic information that governs mPR3 expression resides within the neutrophils, even without stimuli possibly related to disease. CD34+ hematopoietic stem cells were differentiated to neutrophils, and their mPR3 expression was determined. A two-step amplification/differentiation protocol was used to differentiate human CD34+ hematopoietic stem cells into neutrophils with G-CSF. The cells progressively expressed the neutrophil surface markers CD66b, CD35, and CD11b. The ferricytochrome C assay demonstrated a strong respiratory burst at day 14 in response to PMA but none at day 0. Intracellular PR3 was detectable from day 4 by Western blotting. An increasing percentage of a mPR3-positive neutrophil subset became detectable by flow cytometry, whereas a second subset remained negative, consistent with a bimodal expression. Finally, human PR3-anti-neutrophil cytoplasmic autoantibodies induced a stronger respiratory burst, compared with human control IgG in stem cell-derived neutrophils. Taken together, these studies underscore the clinical importance of the WG mPR3 phenotype. The surface mPR3 on resting cells is probably genetically determined rather than being dictated by external factors.

Wegener's granulomatosis (WG) is an etiologically obscure entity with multiple systemic manifestations. Recently, cytoplasmic anti-neutrophil cytoplasmic antibody (C-ANCA) has become recognized as a valuable adjunct in the diagnosis of this disorder. WG typically involves the upper airway, lungs, and kidneys, but any other organ can be involved, including the skin. We encountered a unique case in which a 27-year-old Japanese man with WG presented with various typical cutaneous manifestations. Purpuric skin lesions and erythematous rash on the lower extremities progressively involved and changed into a necrotizing ulceration on his toe. Additionally, several nodules developed on the extensor surfaces of his elbows. His serum C-ANCA level increased remarkably. Leukocytoclastic vasculitis, the most common histopathological finding in WG patients, was detected in a purpuric lesion on his hand. A biopsy of a nodule on his elbow revealed palisading epithelioid histiocyte granulomas with features of leukocytoclastic vasculitis. The distinctive pattern of papules has been referred to as "palisading neutrophilic granulomatous dermatitis". An open lung biopsy confirmed WG with focal necrotizing granuloma. A renal biopsy demonstrated necrotizing vasculitis and crescentic glomerulonephritis. He showed a good response to oral corticosteroids and cyclophospamide with total remission of symptoms. We believe that a careful balance between the clinical manifestations and the histopathological evidence allows for timely treatment of WG, which may prevent serious morbidity or death. Although uncommon, WG can present with various types of accompanying cutaneous lesions. Therefore, clinicians should keep this diagnosis in mind when presented with these manifestations.

Wegener's granulomatosis (WG) is strongly associated with the presence of antineutrophil cytoplasmic autoantibodies (ANCAs). Within WG these ANCAs are usually (80-90%) directed against the azurophilic enzyme proteinase 3, the so called PR3-ANCA. A pathophysiological role for these autoantibodies, supported by numerous in vitro and in vivo studies, is specifically based on their capacity to bind and activate neutrophils and potentially may damage vessels. In this review, the pathogenic potential of different developmental stages of the neutrophil in the pathogenesis of WG is discussed. After release from the bone marrow into the circulation, neutrophils can be primed by TNFalpha and become attached to locally activated endothelium. Once attached to the endothelium, ANCAs can fully activate these primed neutrophils. In this activation process, the degree of activation after stimulation with PR3-ANCAs associates with the level of PR3 expression on the membrane of the neutrophil. Following activation, infiltrated neutrophils become apoptotic with further membrane expression of PR3. In WG patients, clearance of apoptotic neutrophils can be disturbed due to the opsonization of PR3-expressing apoptotic neutrophils with PR3-ANCAs, thereby perpetuating inflammation by the release of proinflammatory cytokines during clearance; or it may favor autoimmunity by PR3 presentation in an inflammatory environment. Furthermore, the presence of ANCAs and the release of the vessel-related pentraxin PTX3 may lead to the persistence of late apoptotic neutrophils in tissues, thereby inducing leukocytoclastic lesions that are characteristic in patients with WG. All together, alive neutrophils as well as apoptotic neutrophils play a key role in different inflammatory phenomena seen in patients suffering from WG.

Antineutrophil cytoplasm autoantibodies with specificity for proteinase 3 (PR3) are thought to play a major role in the pathogenesis of Wegener's granulomatosis (WG), presumably by their potential to activate neutrophils. In patients with WG, high expression of PR3 on the surface of nonprimed neutrophils is associated with an increased incidence and rate of relapse. In this study, we analyzed the functional significance of constitutive PR3 expression for neutrophil activation as induced by anti-PR3 antibody. Therefore, primed and nonprimed neutrophils were stimulated with the monoclonal anti-PR3 antibody PR3G-3. Activation was measured as actin polymerization by the phalloidin assay as an early, detectable activation event and oxidative burst by the dihydrorhodamine assay, as a late, detectable activation event. In contrast to the oxidative burst, we found that anti-PR3 antibody-induced actin polymerization could be triggered in neutrophils without priming with tumor necrosis factor alpha (TNF-alpha). In addition, a correlation was found between the level of PR3 expression on the surface of these nonprimed neutrophils and the degree of actin polymerization. However, after priming with TNF-alpha, no correlation was found between membrane expression of PR3 and the level of actin polymerization or respiratory burst as induced by anti-PR3 antibody. These data suggest that the presence of PR3 on the surface of nonprimed neutrophils has consequences for their susceptibility to the initial activation step by anti-PR3 antibodies. These data may be relevant in view of the observed relation between membrane expression of PR3 on nonprimed neutrophils of patients with WG and their susceptibility for relapses.

Background: Autoantibodies to neutrophil cytoplasmic antigens (ANCA), particularly to proteinase 3 (PR3), are found in the majority of patients with systemic Wegener's granulomatosis. The autoantibodies are widely used as diagnostic markers. Their role in the development and progression of the disease, however, is still under investigation. The primary target of ANCA, PR3, is located in the cytoplasm of polymorphonuclear neutrophils (PMN) or monocytes and is translocated to the cell surface upon stimulation. In patients with Wegener's granulomatosis PR3 is up-regulated most prominently during active disease. Despite the fact that both autoantibodies to PR3 and PMN expressing PR3 are present in patients with Wegener's granulomatosis, there is no evidence for binding of the autoantibodies to PMN. The present study was designed to analyze binding characteristics of autoantibodies to PR3 on PMN., Methods and Results: PMN of patients with active Wegener's granulomatosis (N= 10) were tested for autoantibody binding. Despite high autoantibody titer and PR3 expression on the PMN, no surface-bound IgG was found on PMN ex vivo. When ANCA-containing plasma from patients was incubated with isolated PMN, stimulated to express PR3, again no specific binding of the autoantibody could be detected. Also keeping the samples on ice did not allow surface detection of IgG, ruling out degradation or internalization of the autoantibodies. Only when purified IgG fractions were used, binding to PMN was seen in 14 of 25 patients. Already 1% of plasma, however, was sufficient to greatly reduce the IgG binding. Reduced binding of the IgG fraction was also seen when a larger reaction volume was used., Conclusion: Our data indicate that autoantibodies to PR3 have a rather low affinity for surface-associated PR3 on PMN. This, in turn, argues against the hypothesis that ANCA contributes to the pathogenesis of the disease by stimulating viable PMN in whole blood.

Wegener's granulomatosis is a systemic disease characterized by the presence of antineutrophil cytoplasm autoantibodies specific for proteinase 3 (PR3-ANCA). The functional characteristics of PR3-ANCA differ between quiescent and active disease, suggesting changes in the properties of the autoantibodies in time. Using biosensor technology, we found that PR3-ANCA of different patients (n = 8) recognize a limited number of overlapping regions on PR3 at the time of diagnosis of Wegener's granulomatosis. This area might cover an immunodominant epitope, common for PR3-ANCA from all patients, irrespective of the size of the total area recognized by an individual autoantibody. Experiments with sera (n = 4) collected at the moment of diagnosis and at the time of relapse showed that the individual epitope specificities of PR3-ANCA change during the course of the disease. These changes in epitope specificity of PR3-ANCA may be responsible for the differences in functional properties of these autoantibodies between various stages of the disease.

Upon cultivation with interferon-gamma (IFN-gamma ) and granulocyte/macrophage-colony stimulating factor (GM-CSF) polymorphonuclear neutrophils (PMN) acquire characteristics of dendritic cells, including expression of major histocompatibility complex (MHC) class II antigens, of the co-stimulatory antigens CD80, CD86 and of CD83, the latter considered to be specific for dendritic cells. Dendritic-like PMN were also able to present to T cells antigens in a MHC class II-restricted manner. To assess whether dendritic-like PMN are also generated in vivo, cells of patients with acute bacterial infections and of patients with chronic inflammatory diseases (primary vasculitis) were tested. During acute infection up to 80% of PMN acquired CD83, but remained negative for MHC class II, CD80 or CD86. PMN of patients with primary vasculitis expressed MHC class II antigens, CD80 and CD86, but not CD83, indicating that up-regulation of MHC class II and of CD83 are not necessarily linked to each other. Indeed, parallel studies with PMN of healthy donors showed that while IFN-gamma and granulocyte/macrophage colony stimulating factor (GM-CSF) induced both, MHC class II and CD83, tumour necrosis factor (TNF)-alpha selectively induced de novo synthesis of CD83. The function of CD83 on PMN is still elusive. A participation in the MHC class II-restricted antigen presentation could be ruled out, consistent with the segregation of MHC class II and CD83 expression. Regardless, however, of its function, CD83 expression could serve as a marker to differentiate between acute and chronic inflammation.

Background: Anti-neutrophil cytoplasmic autoantibodies (ANCA) induce neutrophil activation in vitro with release of injurious products that can mediate necrotizing vasculitis in vivo. The importance of ANCA IgG F(ab')2-antigen binding versus Fcgamma receptor engagement in this process is controversial. We propose that ANCA-antigen binding affects cell signaling pathways that can result in changes of gene expression., Methods: Microarray GeneChip analysis and real-time, quantitative PCR (TaqMan(R)) was used to probe for transcripts in leukocytes from patients (in vivo gene expression study) and in leukocytes treated with ANCA IgG or ANCA-F(ab')2 (in vitro gene expression study)., Results: Microarray gene chip analysis showed that ANCA IgG and ANCA-F(ab')2 stimulate transcription of a distinct subset of genes, some unique to whole IgG, some unique to F(ab')2 fragments, and some common to both. DIF-2, COX-2, and IL-8 were identified as genes responsive to ANCA signaling and were selected for in depth evaluation. In vitro DIF-2 and IL-8 were increased by both ANCA IgG and F(ab')2, but COX-2 only by MPO-ANCA F(ab')2. In vivo DIF-2 levels were increased in leukocytes of ANCA patients, which correlated strongly with disease activity and ANCA titer. DIF-2 was not increased in patients in remission or in disease control patients (systemic lupus erythematosus and IgA nephropathy). COX-2 gene expression was significantly increased in patients with active disease, while IL-8 was increased in remission., Conclusions: The data indicate that leukocyte genes are activated in vitro by both ANCA Fc and ANCA F(ab')2 pathways and that in vitro activation mimics changes in circulating leukocytes of patients with ANCA disease. Increased levels of DIF-2 in patient leukocytes strongly correlate with severity of disease in kidney tissue. The observations indicate a previously unrecognized role for DIF-2 in ANCA-mediated inflammation, which raises the possibility that DIF-2 has an important role in other types of inflammation.

Wegener granulomatosis (WG) is strongly associated with the presence of antineutrophil cytoplasm autoantibodies (ANCA) with specificity for proteinase 3 (PR3). Relapses of WG are frequently preceded by a rise of autoantibody titer and PR3-ANCA are able to activate primed neutrophils in vitro. Except being stored intracellularly and translocated to the cell surface upon neutrophil stimulation, PR3 can also be detected on the surface of non-stimulated neutrophils (membrane PR3 or mPR3), with an interindividual variability in percentages of mPR3(-)-positive cells and level of mPR3 expression. This study began with the hypothesis that the presence of PR3 on the surface of non-stimulated neutrophils enables interaction with PR3-ANCA and influences clinical manifestations of the disease. It analyzed mPR3 expression on neutrophils of 89 WG patients in complete remission and 72 healthy controls to evaluate whether the presence of PR3 on the surface of resting neutrophils is related to clinical manifestations of WG and/or to the susceptibility to develop relapses. The number of patients with a bimodal mPR3 expression on resting neutrophils did not differ between patients and controls. However, in WG patients, an increased percentage of mPR3(+) neutrophils and an elevated level of mPR3 expression compared with healthy individuals (P = 0.037) were found. Within the group of WG patients, an elevated level of mPR3 expression was significantly associated with an increased risk for relapse (P = 0.021) and with an increased relapse rate (P = 0.011), but not with the disease extent or particular manifestations at diagnosis or at relapse. These data support the hypothesis that PR3 expression on the membrane of neutrophils plays a role in the pathophysiology of PR3-ANCA associated vasculitis.

Background: Constitutive expression of major histocompatibility complex (MHC) class II antigens and of the co-stimulatory receptors CD80 and CD86 is restricted to professional antigen presenting cells. Polymorphonuclear neutrophils (PMN) of healthy donors are negative for those antigens. Our recent study, however, found that PMN of patients with active Wegener's granulomatosis acquired MHC class II antigens., Methods: To continue and extend the previous study results, PMN and monocytes of 60 patients with Wegener's granulomatosis, 24 patients with microscopic polyangiitis (MPA), 20 patients with acute bacterial infection, and 53 healthy donors were analyzed for the expression of MHC class II antigens as well as of CD80 and CD86. Moreover, induction on PMN of MHC class II expression was studied, as was antigen presentation as a possible functional consequence., Results: PMN of patients with acute, active Wegener's granulomatosis expressed MHC class II antigens, CD80 and CD86; on monocytes up-regulation of MHC class II was seen. In contrast, PMN of patients with inactive disease, or with relapse, patients with microscopic polyangiitis or with bacterial infections expressed neither MHC class II, nor CD80 or CD86. PMN of healthy donors acquired these antigens when cultured in the presence of T cells or T cell-derived cytokines. The PMN were then able to present to T cell antigens in a MHC-class II restricted manner., Conclusion: During active disease, the PMN of patients with Wegener's granulomatosis acquire characteristics of antigen presenting cells, whereas the PMN of patients with MPA or bacterial infection do not. The finding reflects differences in the pattern of the respective inflammatory response and suggests new effector functions of PMN. Moreover, MHC class II expression on PMN could serve as a novel marker for active Wegener's granulomatosis.

We report a case of sarcoidosis with mediastinal and pulmonary localizations associated with diffuse antineutrophil cytoplasmic antibodies (c-ANCA). This led to discuss the differential diagnosis with vascularitis and the specificity of c-ANCA in Wegener's granulocytosis.

Background: Interactions between anti-neutrophil cytoplasmic autoantibody (ANCA) and primed neutrophils (PMNs) may be central to the pathogenesis of primary small vessel vasculitis. PMNs from patients are primed, expressing proteinase 3 (PR3) on the cell surface, which permits interaction with ANCA. In vitro ANCA activates primed PMN to degranulate and generate a respiratory burst. Resultant reactive oxygen species are important in triggering apoptosis, but the fate of PMN in ANCA-associated vasculitis is unknown. Failure to remove apoptotic PMN in a nonphlogistic manner may sustain the inflammatory response., Methods: PMNs from patients or controls were isolated, and the basal production of superoxide was measured by the superoxide dismutase-inhibitable reduction of ferricytochrome C. ANCA antigen expression on apoptotic PMN was assessed at 0, 12, and 18 hours by flow cytometry using dual staining with FITC-conjugated annexin V and PE-conjugated anti-murine IgG against monoclonal ANCA. Apoptosis was also assessed by morphology. In further studies, apoptotic PMNs were opsonized with monoclonal anti-myeloperoxidase (MPO) or anti-proteinase-3 (PR3) or irrelevant isotype-matched IgG (N IgG) and phagocytosis by macrophages was measured using interaction assays. Cytokines interleukin-8 (IL-8) and interleukin-1 were measured by enzyme-linked immunosorbent assay (ELISA)., Results: Proteinase-3 expression (active 63.04 +/- 5.6% of total number of cells, remission 51.47 +/- 7.9% of total number of cells, control 17.7 +/- 4.7% of total number of cells, P < 0.05) and basal superoxide production (active 6.9 +/- 0.8 nmol/L x 10(6) cells, remission 5.15 +/- 0.4 nmol/L/10(6) cells, control 3.63 +/- 0.3 nmol/L/10(6) cells, P < 0.001) were significantly greater with freshly isolated PMN from patients than controls. PR3 expression and superoxide generation were positively correlated. PMN from patients with active disease became apoptotic at a greater rate than those of controls (at 18 hours, patients 72.3 +/- 3.9% apoptosis, controls 53.2 +/- 2.7% apoptosis, P < 0.05). PR3 and MPO expression were significantly greater on PMN isolated from patients at 12 and 18 hours. Opsonization of apoptotic PMN with ANCA significantly enhanced recognition and phagocytosis by scavenger macrophages (anti-MPO 88.95 +/- 6.27, anti-PR3 93.98 +/- 4.90, N IgG 44.89 +/- 3.44, P < 0.01) with increased secretion of IL-1 (anti-PR3 34.73 +/- 6.8 pg/mL, anti-MPO 42.01 +/- 12.3 pg/mL, N IgG 8.04 +/- 6.3 pg/mL, P < 0.05) and IL-8 (anti-PR3 8.97 +/- 0.93 ng/mL, anti-MPO 8.45 +/- 1.46 ng/mL, N IgG 0.96 +/- 0.15 ng/mL, P < 0.01)., Conclusion: In vivo circulating PMNs are primed as assessed by PR3 expression and basal superoxide production, thereby enhancing their inflammatory potential. These PMNs undergo apoptosis more readily, at which times they express PR3 and MPO on their surface. These antigens may then provide targets for ANCA. Opsonization of apoptotic PMN will enhance clearance by macrophages but will also trigger the release of pro-inflammatory cytokines that may contribute to chronic inflammation.

Objective: Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA) have been reported to be pathologically and clinically different. The aim of this study was to assess whether these differences could be explained by differing abilities of proteinase 3-antineutrophil cytoplasmic antibody (PR3-ANCA)-positive IgG preparations or myeloperoxidase-ANCA (MPO-ANCA)-positive IgG preparations to activate neutrophils (polymorphonuclear cells [PMN]) in vitro., Methods: Using Percoll density gradients, PMN were isolated (concentration 2 x 10(6)/ml) and primed with cytochalasin B (1 ng/ml) and tumor necrosis factor alpha (TNFalpha; 2 ng/ml). The PMN were activated with 200 microg/ml of normal IgG or ANCA. Activation was determined by 1) superoxide anion generation as determined by the superoxide dismutase-inhibitable reduction of ferricytochrome c, 2) monitoring fluxes in Ca2+ concentration using Fura 2-AM-loaded PMN, and 3) degranulation using an MPO assay. Surface expression of PR3 and MPO was determined by fluorescence-activated cell sorter analysis. ANCA isotypes were investigated by enzyme-linked immunosorbent assay., Results: Activation of PMN by MPO-ANCA-positive IgG preparations compared with PR3-ANCA-positive IgG preparations resulted in greater generation of superoxide anions (MPO-ANCA-positive IgG preparations 9.13 +/- 0.39 nmoles [mean +/- SEM], PR3-ANCA-positive IgG preparations 6.32 +/- 0.35 nmoles; P < 0.001), Ca2+ fluxes (MPO-ANCA-positive IgG preparations 0.735 +/- 0.10, PR3-ANCA-positive IgG preparations 0.33 +/- 0.098; P < 0.01), and MPO degranulation (MPO-ANCA-positive IgG preparations 251.98 +/- 26.7 ng, PR3-ANCA-positive IgG preparations 145.19 +/- 19.4 ng; P < 0.001). The increased activation seen with MPO-ANCA-positive IgG preparations was not due to increased expression of MPO on the cell surface, because following TNFalpha priming PR3 was expressed on significantly more cells than was MPO (PR3 expression 54.2 +/- 5.18%, MPO 31.6 +/- 3.55%; P < 0.001). IgG1 and IgG4 were the predominant isotypes in both MPO-ANCA-positive IgG preparations and PR3-ANCA. MPO-ANCA contained significantly more IgG1 than did PR3-ANCA, and PR3-ANCA-positive IgG preparations contained significantly more IgG3., Conclusion: In vitro MPO-ANCA-positive IgG preparations are more activating than PR3-ANCA-positive IgG preparations. The increased activation cannot be explained by increased MPO expression on the cell surface or greater IgG3 present in MPO-ANCA-positive IgG preparations. Differences in activation of PMN by these antibodies may determine some differences between WG and MPA.

Since proteinase 3 (PR3)-ANCA interact with PR3 on the surface of apoptotic polymorphonuclear neutrophils (PMN) and ingestion of apoptotic PMN is known to modulate macrophage inflammatory reactions, we raised the question whether PR3-ANCA-opsonized apoptotic PMN influence the uptake by macrophages and their state of activation. We therefore analysed the effects of PR3-ANCA-opsonized apoptotic PMN on the uptake process by enzymatic assay. We further investigated the production of TNF-alpha, IL-10, IL-12 and the secretion of lipid inflammatory mediators (TxB2, leukotriene B4 (LTB4) and prostaglandin E2 (PGE2)) by human monocyte-derived macrophages using FACS and ELISA methods. We show that PMN-opsonization by PR3-ANCA substantially enhances phagocytosis by macrophages and thereby triggers the production of TNF-alpha and TxB2. These in vitro findings indicate that PR3-ANCA opsonization of apoptotic PMN might be an important mechanism in the pathogenesis of Wegener's granulomatosis (WG), prompting macrophages to produce proinflammatory mediators. These mediators, mainly TNF-alpha, might prime further PMN leading to perpetuation of the known priming-dependent mechanisms of ANCA action.

Background: Wegener's granulomatosis is a systemic inflammatory disease of unknown etiology. Many studies suggest that autoimmune reactions are involved, and there is good evidence for the participation of immunocompetent cells. In that context, we examined the activation of polymorphonuclear neutrophils (PMNs) of patients with Wegener's granulomatosis., Methods: In a prospective study, the expression on the surface of PMNs of CD64 and of the major histocompatibility class II (MHC II) antigen was measured by cytofluorometry in whole blood. The expression of those antigens was correlated to disease activity., Results: Up to 15% of the peripheral PMNs of patients with active disease expressed MHC II. Follow-up studies showed that expression correlated closely with disease activity and that it decreased rapidly under immunosuppressive therapy. Expression of CD64 was seen in approximately 50% of the patients, regardless of disease activity., Conclusion: MHC II expression on PMNs might serve as a novel diagnostic marker for active disease and appears to be suitable for monitoring immunotherapy. Moreover, our data provide evidence that PMNs, which are normally MHC II negative, acquire MHC II antigens in the course of disease and may be an unrecognized function within the afferent limb of the immune response.

Wegener's granulomatosis is characterized by crescentic necrotizing glomerulonephritis and systemic vasculitis. Both proteinase 3 (PR3) and anti-neutrophil cytoplasmic antibodies (ANCA), directed against this enzyme, are thought to play a pathogenic role. PR3 has been shown to cause detachment and cytolysis of human umbilical vein endothelial cells (HUVEC) in vitro and to induce apoptosis of bovine pulmonary artery endothelial cells. In the present study we investigated the effect of PR3 and ANCA on the induction of apoptosis of human endothelial cells in vitro. HUVEC were cultured in the absence or presence of varying concentrations of PR3 for different time periods and apoptosis was assessed by three different methods. Staining of the cells with Hoechst 33258 and assessment of nuclear morphology by ultraviolet (UV) light microscopy revealed a dose-dependent induction of apoptosis, as determined by cell counts. A concentration of 8 microg/ml PR3 was found to induce 16% apoptosis after 16 h incubation. Analysis of apoptosis by flow cytometry using the terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labelling (TUNEL) method also demonstrated a dose-dependent induction of apoptosis by PR3. DNA fragmentation was confirmed by agarose gel electrophoresis. To investigate the effect of ANCA on PR3-mediated apoptosis, HUVEC were exposed to immunoglobulin G (IgG) from patients with Wegener's granulomatosis or systemic vasculitis, and from normal controls, in the presence or absence of PR3. Enhancement of PR3-mediated apoptosis was found in two of 10 IgG samples with anti-PR3 activity, whereas a reduction in apoptosis was observed in two others. Anti-MPO (myeloperoxidase)-positive IgG, six additional anti-PR3 positive IgG samples and control IgG samples did not have any detectable effect on apoptosis. These studies suggest that ANCA may modulate the relative degree of injury in some cases of Wegener's granulomatosis.

Fifty-six sera from patients with autoimmune thyroiditis and 33 sera from patients with MPO-ANCA were examined in order to ascertain whether a cross reactivity between MPO-ANCA and anti-thyroperoxidase (aTPO) was present. Sera from 20 healthy donors aTPO and aMPO negative were used as control. About 95% of heat inactivated sera from patients with autoimmune thyroiditis and from controls gave positive results (atypical pANCA pattern) on ethanol-fixed neutrophils. The prevalence of positive results was significantly lower when unheated aTPO positive sera were used (17.8%). On the other hand, only 9% of sera with MPO-ANCA were positive on cryostatic sections of human thyroid. Indirect immunofluorescence tests (IF) on human neutrophils with MPO defect were negative with sera from patients with MPO-ANCA, but uninactivated sera with aTPO and positive for pANCA on normal neutrophils showed a very high prevalence of positive results (90%). According to our data only few sera positive for aTPO recognize "normal" MPO, but the majority of sera from patients with autoimmune thyroiditis and positive for pANCA on normal neutrophils recognize also an "abnormal" MPO. On the other hand MPO-ANCA usually recognize epitopes presently only on the normal enzyme, a small proportion of these autoantibodies can react with TPO. Heat inactivated sera give false positive results for pANCA on ethanol fixed human neutrophils.

To test the hypothesis that anti-neutrophil cytoplasm autoantibodies (ANCAs) interfere with the functions of proteinase 3 (PR3) (the Wegener autoantigen) and alpha 1-antitrypsin (alpha 1AT), complexes of PR3/alpha 1 AT and PR3/PR3-ANCA-IgG were assayed Plasma samples were obtained from 44 patients with Wegener's granulomatosis (WG): 34 had active disease (88% ANCA positive) whereas 10 patients were in remission (20% ANCA positive). Plasma samples from 14 of the patients with active disease were also available at the time of remission. The complexes of PR3/alpha 1 AT and PR3/PR3-ANCA-IgG were detected by capture enzyme-linked immunoassays (ELISAs) alpha 1 AT deficiency was evaluated by determining PiZ alleles by ELISA. Eight (18%) of the patients were PiZ positive. The frequency of this alpha 1-antitrypsin phenotype in the Scandinavian population is 4.7% (P < 0.001). The median PR3/alpha 1AT complex level in the PiZ-positive group with active disease (n = 5) was similar to the level in the PiZ-negative group with active disease. During remission the median level for the PR3/alpha 1AT complex was significantly higher than in the acute group (P < 0.001) including both PiZ-positive and PiZ-negative patients. No difference between PiZ positivity and PiZ negativity could be found in the remission group. PR3/PR3-ANCA-IgG complexes were found in patients with acute disease as well as in patients in remission, in almost equal frequency. This complex was also present in 13/18 ANCA-negative samples from patients in remission. Finally, purified IgG fractions from WG patients were examined for their capacity to inhibit binding between PR3 and alpha 1AT. An effect on the binding between PR3 and alpha 1AT by PR3-ANCA could not be demonstrated. Thus, our results do not support the hypothesis that PR3-ANCA interferes with the binding between PR3 and alpha 1AT. However, the high prevalence of the PiZ alleles among WG patients suggests that an imbalance between proteinases and alpha 1AT may be of importance in this disease. The clinical usefulness of both the PR3/alpha 1AT and the PR3/PR3-ANCA-IgG complexes and the possible influence on ANCA detection need to be examined in prospective longitudinal studies.

The crystal structure of PR3, a serine proteinase from the azurophilic granules of human polymorphonuclear neutrophils, has been solved by molecular replacement using the human leukocyte elastase structure. The PR3 structure has been refined to an R-factor (= sigma parallel Fo magnitude of-Fc parallel/sigma magnitude of Fo) of 0.201 for all data in the range of 10.0 to 2.2 A resolution. The enzyme was crystallized in space group P21 with four molecules in the asymmetric unit (Vm approximately equal to 2.6 A/Da). The overall fold consists of two domains of beta-barrel structures typical of the chymotrypsin family of serine proteinases. In general, the substrate binding sites, S4 to S3', are more polar than comparable sites in the related proteinase, human leukocyte elastase. The experimentally observed preference of PR3 for small aliphatic residues at the P1 position of a substrate is explained by the Val to Ile substitution at position 190 when compared to the elastase structure. The substitution of Ala by Asp at position 213 at the back of S1 should not affect its specificity greatly, as the Asp side-chain points back into the interior of the protein. The PR3 structure includes a disaccharide unit (N-linked 2-acetamido-2-deoxy-beta-D-glucopyranose and 1,6-linked alpha-L-fucopyranose) covalently attached to Asn 159. The linear antigenic sites of PR3 reported to react with Wegener's granulomatosis autoantibodies occur in regions of the three-dimensional structure that may implicate the inactive pro-form of the enzyme in the pathogenesis of the disease.

Proteinase 3 is the major target antigen of antineutrophil cytoplasmic autoantibodies (ANCA) in Wegener's granulomatosis and is contained in the azurophilic granules of polymorphonuclear neutrophils, the dominant cell type in vascular lesions during the early stages of systemic vasculitis. This study questioned whether neutrophil lysosomal enzymes, once released at the site of inflammation, are able to potentiate the influx of additional neutrophils by enhancing the production of the chemotactic cytokine interleukin-8 (IL-8) by endothelial cells. Therefore, human umbilical vein endothelial cells in culture were incubated with varying concentrations of highly purified proteinase 3, human neutrophil elastase, and cathepsin G for different time periods. The supernatants were subsequently assessed for IL-8 antigen by using a sandwich ELISA. The presence of both proteinase 3 and elastase resulted in an increased production of IL-8, up to 15.6- and 4.2-fold, respectively, in a dose- and time-dependent fashion. Cathepsin G did not influence IL-8 production. Although the addition of an alpha 1-proteinase inhibitor completely abrogated elastase-mediated IL-8 production, it did not significantly influence the effect of proteinase 3. Both proteinase 3-and elastase-mediated production of IL-8 was inhibited by cycloheximide, indicating de novo synthesis. This was supported by the finding of increased IL-8 mRNA levels in proteinase 3-treated human umbilical vein endothelial cells by using Northern blot analysis. Taken together, the neutrophil lysosomal enzymes proteinase 3 and human neutrophil elastase may contribute to a self-perpetuating process of neutrophil recruitment in acute inflammation by increasing de novo synthesis of IL-8 by endothelial cells. The studies presented here also show that proteinase 3 mediates its effect independently of its enzymatic activity, indicating a hitherto unknown mode of action on endothelial cells.

Proteinase 3, which is known as an intracellular serine protease of neutrophils, was detected at the surface of a subpopulation of freshly isolated PMN. The proportion of PR3-positive and -negative PMN, observed by flow cytometry with anti-PR3 mAbs or ANCA autoantibodies, varies among individuals but is extremely stable for each individual over prolonged time periods. After PMN degranulation by FMLP with cyt. B, membrane PR3 expression increases but the proportion of low and high PR3-expressing cells remains stable. The existence of a subset of PMN which spontaneously expresses PR3 and varies among individuals, may be relevant to the pathogenesis of anti-PR3 ANCA autoantibody-related vasculitis.

Antineutrophil cytoplasmic autoantibodies (ANCA) have been described in sera of patients with several forms of systemic vasculitis, including Wegener's granulomatosis and microscopic polyarteritis. The two main targets of ANCA in vasculitis are proteinase 3 (PR3) and myeloperoxidase (MPO). ANCA are capable of activating neutrophils primed by tumor necrosis factor-alpha (TNF-alpha) in vitro, which may be relevant for the induction of the vascular inflammation observed in vivo. Recently, it has been suggested that engagement of Fc gamma receptor IIa (Fc gamma RIIa) on the neutrophils is involved in the activation by ANCA. In the present study, we show that activation of the neutrophil respiratory burst by anti-PR3 and anti-MPO is strongly enhanced after TNF priming and lost on removal of the Fc parts of the antibodies. Similar results were obtained when the neutrophils were activated with antibodies against known membrane antigens without major changes in the expression of the target antigens. The TNF-induced enhancement of the neutrophil activation was not observed when adherence of the cells was prevented by continuous stirring of the suspension or by the addition of CD18 antibodies before TNF exposure. Hence, our results indicate that engagement of both Fc gamma RIIa and beta 2 integrins is instrumental in neutrophil activation induced by ANCA.

Objective: To investigate the pathogenetic role of antineutrophil cytoplasmic antibodies (ANCA) in Wegener's granulomatosis (WG)., Methods: BALB/c mice were immunized with human IgG ANCA from a patient with WG. Control mice were immunized with normal human IgG. Levels of mouse ANCA and other autoantibodies were determined. Mouse ANCA were tested for their ability to induce adhesion and respiratory burst of neutrophils. The mouse lungs and kidneys were examined for the development of vasculitis., Results: Mice immunized with human ANCA developed anti-human ANCA and anti-anti-human ANCA (mouse ANCA), while the controls did not develop these antibodies. Mouse ANCA were capable of inducing adhesion of neutrophils to fibronectin and activating the respiratory burst in neutrophils. Moreover, the mice that were immunized with human ANCA developed perivascular mononuclear cell infiltrates in the lungs, suggesting vasculitis., Conclusion: The results suggest a pathogenic role of ANCA in WG, and may imply that activation of neutrophils is the initiating event in the development of vasculitis in WG.