4C3 Human Monoclonal Antibody: A Proof of Concept for Non-pathogenic Proteinase 3 Anti-neutrophil Cytoplasmic Antibodies in Granulomatosis With Polyangiitis.

Granulomatosis with polyangiitis (GPA) is a severe autoimmune vasculitis associated with the presence of anti-neutrophil cytoplasmic antibodies (ANCA) mainly targeting proteinase 3 (PR3), a neutrophilic serine proteinase. PR3-ANCA binding to membrane-bound PR3 on neutrophils induce their auto-immune activation responsible for vascular lesions. However, the correlation between PR3-ANCA level and disease activity remains inconsistent, suggesting the existence of non-pathogenic PR3-ANCA. In order to prove their existence, we immortalized B lymphocytes from blood samples of GPA patients in remission having persistent PR3-ANCA to isolate non-activating PR3-ANCA. We obtained for the first time a non-activating human IgG1κ anti-PR3 monoclonal antibody (mAb) named 4C3. This new mAb binds soluble PR3 with a high affinity and membrane-bound PR3 on an epitope close to the PR3 hydrophobic patch and in the vicinity of the active site. 4C3 is able to bind FcγRIIA and FcγRIIIB and has a G2F glycosylation profile on asparagine 297. 4C3 did not induce activation of neutrophils and could inhibit human polyclonal PR3-ANCA-induced activation suggesting that 4C3 is non-pathogenic. This characteristic relies on the recognized epitope on PR3 rather than to the Fc portion properties. The existence of non-pathogenic PR3-ANCA, which do not activate neutrophils, could explain the persistence of high PR3-ANCA levels in some GPA patients in remission and why PR3-ANCA would not predict relapse. Finally, these results offer promising perspectives particularly regarding the understanding of PR3-ANCA pathogenicity and the development of new diagnostic and therapeutic strategies in GPA.

Evidence for enhanced Bruton's tyrosine kinase activity in transitional and naïve B cells of patients with granulomatosis with polyangiitis.

OBJECTIVES: To determine Bruton's tyrosine kinase (BTK) protein and phosphorylation levels in B cell subsets of granulomatosis with polyangiitis (GPA) patients and to investigate the effect of BTK blockade on in vitro B cell cytokine production, subset distribution and (auto)antibody production. METHODS: BTK protein and phosphorylation levels were determined by flow cytometry in peripheral blood B cells of 29 untreated GPA patients [9 active and 20 remission GPA patients (10 ANCA- and 10 ANCA+)], 9 age- and sex-matched healthy controls (HCs) and 9 untreated active RA patients. The effect of BTK blockade on in vitro B cell cytokine production, subset distribution and (auto)antibody production was determined in the same donors in peripheral blood mononuclear cell cultures. RESULTS: BTK protein levels were significantly increased in transitional and naïve B cells of active GPA and RA patients compared with remission GPA patients and HCs. Both B cell subsets of active patients were more sensitive to B cell receptor stimulation, as BTK and phospholipase Cγ2 phosphorylation were increased in these patients. In vitro BTK blockade had profound effects on B cell cytokine production, plasma cell formation and (auto)antibody production in both GPA patients and HCs. Interestingly, the effect of BTK blockade was less pronounced in active GPA patients, possibly due to increased activation of B cells. CONCLUSION: We show that BTK protein and phosphorylation levels are most profoundly increased in newly emerging B cells of active GPA patients compared with remission patients. BTK blockade greatly inhibits in vitro B cell effector functions in GPA patients and HCs. These promising data identify BTK as an interesting novel therapeutic target in the treatment of GPA.

Proteinase 3 Is a Phosphatidylserine-binding Protein That Affects the Production and Function of Microvesicles.

Proteinase 3 (PR3), the autoantigen in granulomatosis with polyangiitis, is expressed at the plasma membrane of resting neutrophils, and this membrane expression increases during both activation and apoptosis. Using surface plasmon resonance and protein-lipid overlay assays, this study demonstrates that PR3 is a phosphatidylserine-binding protein and this interaction is dependent on the hydrophobic patch responsible for membrane anchorage. Molecular simulations suggest that PR3 interacts with phosphatidylserine via a small number of amino acids, which engage in long lasting interactions with the lipid heads. As phosphatidylserine is a major component of microvesicles (MVs), this study also examined the consequences of this interaction on MV production and function. PR3-expressing cells produced significantly fewer MVs during both activation and apoptosis, and this reduction was dependent on the ability of PR3 to associate with the membrane as mutating the hydrophobic patch restored MV production. Functionally, activation-evoked MVs from PR3-expressing cells induced a significantly larger respiratory burst in human neutrophils compared with control MVs. Conversely, MVs generated during apoptosis inhibited the basal respiratory burst in human neutrophils, and those generated from PR3-expressing cells hampered this inhibition. Given that membrane expression of PR3 is increased in patients with granulomatosis with polyangiitis, MVs generated from neutrophils expressing membrane PR3 may potentiate oxidative damage of endothelial cells and promote the systemic inflammation observed in this disease.

Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis.

Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4+ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology.

Allosteric modulation of proteinase 3 activity by anti-neutrophil cytoplasmic antibodies in granulomatosis with polyangiitis.

Anti-neutrophil cytoplasmic antibodies (ANCA) with proteinase 3 (PR3) specificity are a useful laboratory biomarker for the diagnosis of Granulomatosis with Polyangiitis (GPA) and are believed to be implicated in the pathogenesis. It has been repeatedly suggested that disease activity of GPA is more closely related to the appearance and rise of PR3-inhibiting ANCA than to an increase of total ANCA. Previous studies on a limited number of patient samples, however, have yielded inconclusive results. To overcome the previous methodological limitations, we established a new ultrasensitive method to quantify the inhibitory capacity of PR3-ANCA using small volumes of plasma from patients with GPA. A large collection of longitudinally-collected samples from the Wegener Granulomatosis Etanercept Trial (WGET) became available to us to determine the functional effects of ANCA on PR3 in comparison to clinical disease manifestations. In these patient samples we not only detected PR3-ANCA with inhibitory capacity, but also PR3-ANCA with enhancing effects on PR3 activity. However no correlation of these activity-modulating PR3-ANCA with disease activity at either the time of enrollment or over the course of disease was found. Only patients with pulmonary involvement, especially patients with nodule formation in the respiratory tract, showed a slight, but not significant, decrease of inhibitory capacity. Epitope mapping of the activity-modulating PR3-ANCA revealed a binding on the active site surface of PR3. Yet these ANCA were able to bind to PR3 with an occupied active site cleft, indicating an allosteric mechanism of inhibition. The recently described signal ratio between the MCPR3-3 and MCPR3-2 capture ELISA was consistent with the binding of activity-modulating ANCA to the active site surface. Evidence for a shared epitope between activity-modulating PR3-ANCA and MCPR3-7, however, was very limited, suggesting that a majority of PR3-ANCA species do not inhibit PR3 by the same mechanism as previously reported for MCPR3-7.

Anti-carbonic anhydrase III autoantibodies in vasculitis syndrome.

AIM: To identify autoantibodies useful in the diagnosis of primary vasculitides. METHODS: The presence of antibodies against proteins in the lysate of mouse blood vessels was examined by two-dimensional electrophoresis followed by Western blotting for the pooled serum sample from patients with various forms of vasculitis: polyarteritis nodosa (PAN), microscopic polyangiitis (MPA), Wegener's granulomatosis (WG) and Takayasu's arteritis (TA). Autoantigenicity in patients with vasculitides was examined by Western blotting and enzyme-linked immunosorbent assay (ELISA). Clinicopathological correlations between the positivity of the autoantibodies and clinical status of patients with the vasculitis were examined. RESULTS: The autoantigen detected in the lysate of pooled sera from patients with vasculitides was identified by mass spectrometry as carbonic anhydrase III (CAIII). ELISA showed significantly higher prevalence of anti-CAIII antibodies in MPA patients (MPA, 11/23 [47.8%]; healthy controls, 2/32 [6.3%]; P < 0.001). Further, anti-CAIII antibody-positive MPA patients had higher vasculitis activity scores compared to anti-CAIII antibody-negative patients, and a weak and not significant negative correlation was observed between anti-CAIII antibody levels and myeloperoxidase - anti-nuclear cytoplasmic antibody (MPO-ANCA) levels. No significant differences were found in anti-CAIII autoantibody levels between MPA and the other primary vasculitides. CONCLUSION: We found significantly high prevalence of anti-CAIII antibody levels in sera from MPA patients. Although the number of samples available in this study is small and anti-CAIII autoantibodies display weak specificity for MPA, anti-CAIII antibodies may be useful for diagnosing MPA in patients who have no ANCA, as well as for assessing disease activity.

Neutrophil proteinase 3 and dipeptidyl peptidase I (cathepsin C) as pharmacological targets in granulomatosis with polyangiitis (Wegener granulomatosis).

Neutrophils are among the first cells implicated in acute inflammation. Leaving the blood circulation, they quickly migrate through the interstitial space of tissues and liberate oxidants and other antimicrobial proteins together with serine proteinases. Neutrophil elastase, cathepsin G, proteinase 3 (PR3), and neutrophil serine protease 4 are four hematopoietic serine proteases activated by dipeptidyl peptidase I during neutrophil maturation and are mainly stored in cytoplasmic azurophilic granules. They regulate inflammatory and immune responses after their release from activated neutrophils at inflammatory sites. Membrane-bound PR3 (mbPR3) at the neutrophil surface is the prime antigenic target of antineutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis (GPA), a vasculitis of small blood vessels and granulomatous inflammation of the upper and/or lower respiratory tracts. The interaction of ANCA with mbPR3 results in excessive activation of neutrophils to produce reactive oxygen species and liberation of granular proteinases to the pericellular environment. In this review, we focus on PR3 and dipeptidyl peptidase I as attractive pharmacological targets whose inhibition is expected to attenuate autoimmune activation of neutrophils in GPA.

Increased generation of TRAP expressing multinucleated giant cells in patients with granulomatosis with polyangiitis.

BACKGROUND: Tissue-infiltrating multinucleated giant cells (MNGs) within geographic necrosis are pathologic hallmarks of granulomatosis with polyangiitis (GPA). However, the origin, phenotype, and function of these cells in GPA remain undefined. METHODOLOGY/PRINCIPAL FINDINGS: MNG phenotype in GPA lung tissue was examined by immunohistochemistry using antibody directed against cathepsin K and calcitonin-receptor. Tartrate-resistant-acid-phosphatase (TRAP) expression was assessed using enzymatic color reaction. Peripheral blood mononuclear cells (PBMCs) from 13 GPA patients (5 with localized and 8 with systemic disease) and 11 healthy controls were cultured in the presence of RANKL and M-CSF for 9 days, and TRAP+ MNGs containing 3 or more nuclei were identified. GPA lung granulomata contained numerous MNGs that expressed osteoclastic TRAP and cathepsin K but not calcitonin receptors. In the presence of RANKL and M-CSF, PBMCs of GPA patients formed significantly more MNGs than healthy controls (114 ± 29 MNG/well vs. 22 ± 9 MNG/well, P = 0.02). In a subgroup analysis, patients with systemic disease generated significantly more MNGs than patients with localized disease (161 ± 35 MNG/well vs. 39 ± 27 MNG/well, P<0.01) or healthy controls (P<0.01). MNG production did not differ between localized GPA and control subjects (P = 0.96). CONCLUSIONS/SIGNIFICANCE: MNGs in granulomata in the GPA lung express osteoclastic enzymes TRAP and cathepsin K. GPA patients have a higher propensity to form TRAP+ MNGs from peripheral blood than healthy controls. These data suggest that (i) the tendency to form MNGs is a component of the GPA phenotype itself, and (ii) that lesional MNGs might participate in the destructive process through their proteolytic enzymes.

Proteinase 3, the autoantigen in granulomatosis with polyangiitis, associates with calreticulin on apoptotic neutrophils, impairs macrophage phagocytosis, and promotes inflammation.

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.

Distinct proteinase 3-induced cytokine patterns in Wegener´s granulomatosis, Churg-Strauss syndrome, and healthy controls.

OBJECTIVES: To analyse whether a specific cytokine pattern is elicited in response to the autoantigen proteinase 3 (PR3) in active Wegener's granulomatosis (WG). METHODS: Six-colour flow cytometry was used to analyse cytokine production and surface markers of the total CD4+ T-cell population ex vivo and in PR3-stimulated T-cell lines of patients with active PR3-ANCA-positive WG, PR3-ANCA-negative Churg-Strauss syndrome (CSS), and healthy controls (HC). RESULTS: The cytokine response of the total PB CD4+ T cell population was skewed towards distinct pro-inflammatory cytokine patterns in WG (Th1-type) and CSS (Th17, Th1-/Th2-type). Th2-type as well as Th17 cell populations including Th17/Th1, Th17/Th2 and Th22 cells were elicited in response to PR3 stimulation in WG. In contrast, CSS patients displayed a Th2-type dominated response following PR3 stimulation. CONCLUSIONS: These data suggest that the cytokine response of the total CD4+ T-cell population and PR3-specific cells is influenced by the underlying disorder.

Mapping of conformational epitopes on human proteinase 3, the autoantigen of Wegener's granulomatosis.

Anti-neutrophil cytoplasmic Abs (cANCAs) against conformational epitopes of proteinase 3 (PR3) are regarded as an important pathogenic marker in Wegener's granulomatosis (WG). Although the three-dimensional structure of PR3 is known, binding sites of mAbs and cANCAs have not been mapped to date. Competitive binding and biosensor experiments suggested the existence of four nonoverlapping areas on the PR3 surface. In this paper, we present an approach to identify these discontinuous surface regions that cannot be mimicked by linear peptides. The very few surface substitutions found in closely related PR3 homologs from primates, which were further varied by the construction of functional human-gibbon hybrids, resulted in the differential loss of three Ab binding sites, two of which were mapped to the N-terminal beta-barrel and one to the linker segment connecting the N- and C-terminal barrels of PR3. The sera from WG patients differed in their binding to gibbon PR3 and the gibbon-human PR3 hybrid, and could be divided into two groups with similar or significantly reduced binding to gibbon PR3. Binding of almost all sera to PR3-alpha1-protease inhibitor (alpha1-PI) complexes was even more reduced and often absent, indicating that major antigenic determinants overlap with the active site surface on PR3 that associates with alpha1-PI. Similarly, the mouse mAbs CLB12.8 and 6A6 also did not react with gibbon PR3 and PR3-alpha1-PI complexes. Our data strongly suggest that cANCAs from WG patients at least in part recognize similar surface structures as do mouse mAbs and compete with the binding of alpha1-PI to PR3.

Serum chitotriosidase activity and Wegener's granulomatosis.

OBJECTIVE: We evaluated serum chitotriosidase activity in patients with Wegener's granulomatosis (WG) and compared the values to controls. DESIGN AND METHODS: We used a standard fluorometric assay to measure chitotriosidase enzyme activity. RESULTS: Serum chitotriosidase enzyme activity levels were higher in WG patients. We found no association between clinical disease activity and chitotriosidase enzyme activity. CONCLUSIONS: Serum chitotriosidase enzyme activity has limited utility as a biomarker in WG patients.

Caspase-4 may play a role in loss of proximal tubules and renal injury in nephropathic cystinosis.

Nephropathic cystinosis is characterized clinically by generalized proximal renal tubular dysfunction, renal Fanconi Syndrome and progressive renal failure. Glomerular-proximal tubule disconnection has been noted in renal biopsies from patients with nephropathic cystinosis. In vitro studies performed in cystinotic fibroblasts and renal proximal tubular cells support a role for apoptosis of the glomerulotubular junction, and we have further extended these studies to human native cystinotic kidney specimens. We performed semi-quantitative analysis of tubular density in kidney biopsies from patients with nephropathic cystinosis and demonstrated a significant reduction (p=0.0003) in the number of proximal tubules in the kidney tissue of patients with cystinosis compared to normal kidneys and kidneys with other causes of renal injury; this reduction appears to be associated with the over-expression of caspase-4. This study provides the first quantitative evidence of a loss of proximal tubules in nephropathic cystinosis and suggests a possible role of caspase-4 in the apoptotic loss of proximal tubular cells. Further work is needed to elucidate if this injury mechanism may be causative for the progression of renal functional decline in nephropathic cystinosis.

Interaction of proteinase 3 with its associated partners: implications in the pathogenesis of Wegener's granulomatosis.

PURPOSE OF REVIEW: This review focuses on proteinase 3 (PR3), the preferred target of antineutrophil cytoplasmic antibodies (ANCAs) in Wegener's granulomatosis. Deciphering the molecular associations that PR3 can make with its cognate partners might help to understand its pathophysiological significance in Wegener's granulomatosis and the potential role of ANCA as modulator of PR3 functions. RECENT FINDINGS: In neutrophils, PR3 is mainly localized within azurophilic granules but is also detected at the plasma membrane. Among PR3 partners (CD16, CD11b/CD18), CD177, a glycosylphosphatidylinositol (GPI)-anchored membrane protein is a potential receptor for PR3. In addition, PR3 can be externalized at the plasma membrane at a very early stage of neutrophil apoptosis, in the absence of degranulation. In these conditions, PR3 is associated with specific partners including phospholipidscramblase-1 and calreticulin. Interestingly, apoptosis-induced PR3 membrane expression significantly impaired macrophage phagocytosis. This new role of PR3 acting as a 'don't eat me signal' that delays neutrophil clearance might potentiate inflammation and autoimmunity. SUMMARY: Since PR3 membrane expression seems to represent a key element in the inflammatory and autoimmunity process, elucidation of the molecular basis of PR3 interaction with the plasma membrane or with receptor proteins led to the possibility of targeted therapy.

Catalytic activity and inhibition of wegener antigen proteinase 3 on the cell surface of human polymorphonuclear neutrophils.

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.

[Role of matrix metalloproteinases in primary systemic vasculitis]. / Rola metaloproteinaz macierzy w pierwotnych ukladowych zapaleniach naczyn.

Primary systemic vasculitis comprise a group of diseases such as Wegener's granulomatosis, Kawasaki disease, Takayasu arteritis, giant cell arteritis with various clinical manifestations, and an etiology not fully understood. The pathogenesis involves an inflammatory infiltration of the vessel wall, which results in its damage. Matrix metalloproteinases seem to participate not only in the degradation of structural components of a vessel wall which leads to bleeding and/or aneurysmal dilatation. In addition, they play a significant role in the in inflammatory cells migration and development of inflammatory infiltration. This process, and the proliferation and migration of smooth muscle cells may result in the narrowing of the affected vessels. This article outlines the role of matrix metalloproteinases in primary systemic vasculitis.

Skewed distribution of Th17 lymphocytes in patients with Wegener's granulomatosis in remission.

OBJECTIVE: The recently characterized interleukin-17 (IL-17)-producing T helper cell lineage (Th17), rather than the Th1 lineage, is involved in several autoimmune diseases. The possible role of Th17 cells in Wegener's granulomatosis (WG) has not yet been elucidated. We undertook this study to assess the distribution of Th1/Th2/Th17 cells and to investigate the presence of Th17 cells specific for the WG autoantigen proteinase 3 (PR3) in WG. METHODS: Peripheral blood from patients with WG in remission (n = 26) and healthy controls (n = 10) was stimulated in vitro with PR3 or with the control stimuli staphylococcal enterotoxin B (SEB), tetanus toxoid (TT), or phorbol myristate acetate/calcium ionophore, together with anti-CD28 and anti-CD49d. The frequencies of the various CD4+ T cell phenotypes responsive to stimuli were determined by 7-color flow cytometric detection of CD3, CD8, an early activation marker (CD69), and intracellular cytokines (IL-2, interferon-gamma [IFNgamma], IL-17, and IL-4). RESULTS: The percentage of CD69+,CD4+ T cells in patients with WG in remission was significantly decreased in response to PR3 and tended to be lower in response to other stimuli compared with the percentage in healthy controls. The percentages of Th17 cells (IL-4-,IL-17+,IFNgamma-) and Th2 cells (IL-4+,IL-17-,IFNgamma-) within the activated CD69+,CD4+ T cell population were significantly increased in patients with WG in remission, while no difference was found in Th1 cells (IL-4-,IL-17-,IFNgamma+) compared with the percentage in healthy controls. Increased percentages of Th17 cells in response to TT and SEB were found both in antineutrophil cytoplasmic antibody (ANCA)-positive and in ANCA-negative patients, while an increased frequency of PR3-specific Th17 cells was restricted to ANCA-positive patients. CONCLUSION: A skewed Th17 response found in ANCA-positive WG patients following stimulation with the autoantigen PR3 suggests that IL-17 is involved in disease pathogenesis and could constitute a new therapeutic target.

Proteinase 3, the Wegener autoantigen, is externalized during neutrophil apoptosis: evidence for a functional association with phospholipid scramblase 1 and interference with macrophage phagocytosis.

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.

ANCA-associated vasculitis: diagnostic and therapeutic strategy.

Among small-vessel vasculitides, microscopic polyangiitis (MPA), Wegener's granulomatosis (WG), and allergic granulomatous angiitis (AGA) are known collectively as ANCA-associated vasculitis (AAV) because of the involvement of anti-neutrophil cytoplasmic antibodies (ANCA) as the common pathogenesis. Major target antigens of ANCA associated with vasculitis are myeloperoxidase (MPO) and proteinase 3 (PR3). MPO-ANCA is related to MPA and AGA, and PR3-ANCA is the marker antibody in WG. MPO-ANCA-associated vasculitis is more frequent in Japan, whereas PR3-ANCA-associated vasculitis is more common in Europe and USA. ANCA appears to induce vasculitis by directly activating neutrophils. Therefore, no immunoglobulins or complement components are detected in the vasculitis lesions; hence, AAV is called pauci-immune vasculitis (pauci = few/little). Untreated patients with severe AAV with multi-organ involvement have a poor prognosis, which is improved by combination therapy with cyclophosphamide and high-dose corticosteroid. Randomized controlled trials (RCT) regarding induction and maintenance of remission of AAV indicated that the rate of remission induction by the standard regimen is approximately 90% in 6 months, that maintenance of remission can be achieved with oral azathioprine as well as cyclophosphamide, and that methotrexate can be used only for non-renal mild AAV. As these data were obtained mostly in patients positive for PR3-ANCA, caution must be taken in applying these findings to Japanese patients, most of whom are positive for MPO-ANCA. A prospective study is now underway to clarify the effectiveness of the standard regimen in Japanese patients with MPO-ANCA-associated vasculitis. This article describes the diagnostic criteria and the recent evidence-based therapeutic strategy of AAV.

Inhibition of oxidation activity of myeloperoxidase (MPO) by propylthiouracil (PTU) and anti-MPO antibodies from patients with PTU-induced vasculitis.

Propylthiouracil (PTU) could induce antineutrophil cytoplasmic antibody (ANCA) associated vasculitis. This study aimed to investigate the inhibitory effects on MPO oxidation activity by PTU and MPO-ANCA from patients with primary microscopic polyangiitis (MPA) and PTU-induced vasculitis. IgG preparations were purified from MPO-ANCA-positive sera from seven patients with PTU-induced vasculitis and ten patients with primary MPA. The oxidation activity of MPO was measured in the presence of PTU and MPO-ANCA-positive IgG preparations from patients with PTU-induced vasculitis and primary MPA respectively. PTU could competitively inhibit the oxidation activity of MPO dose dependently. MPO-ANCA-positive IgG preparations from 6/7 patients with PTU-induced vasculitis and only 3/10 from patients with primary MPA could inhibit the MPO activity in a dose-dependent manner. In conclusions, the oxidation activity of MPO could be inhibited by PTU and PTU-induced MPO-ANCA in a dose-dependent manner, which might be involved in the pathogenesis PTU-induced vasculitis.