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Objectives: To determine the spectrum of anti-neutrophil cytoplasmic antibody (ANCA) antigen-specificities in eosinophilic granulomatosis with polyangiitis (EGPA), an ANCA-associated vasculitis (AAV) entity., Methods: We conducted a retrospective analysis of 73 EGPA patients from three German tertiary referral centres for vasculitis. In addition to in-house ANCA testing, pentraxin 3 (PTX3)- and olfactomedin 4 (OLM4)-ANCA were determined using a prototype cell-based assay for research (EUROIMMUN, Lübeck, Germany). Patient characteristics and clinical manifestations were evaluated and compared based on ANCA status., Results: Myeloperoxidase (MPO)-ANCA positive patients (n=8; 11%) significantly more frequently displayed peripheral nervous system (PNS) and pulmonary involvement and less frequently heart involvement compared to MPO-ANCA negative patients. PTX3-ANCA positive patients (n=5; 6.8%) had a significantly higher prevalence of ear, nose and throat, pulmonary, gastrointestinal and PNS involvement, and a lower prevalence of renal and central nervous system involvement compared to PTX3-ANCA negative patients. Proteinase 3 (PR3)-ANCA and OLM4-ANCA were detected in 2 patients (2.7%) each with multiorgan involvement. One PR3-ANCA positive patient was also positive for bactericidal permeability increasing protein (BPI)-ANCA., Conclusions: In addition to MPO, the spectrum of ANCA antigen specificities includes various other target antigens such as PR3, BPI, PTX3, and OLM4, potentially segregating further EGPA subgroups. A lower prevalence of MPO-ANCA was detected in this study compared with other studies. OLM4 is reported as novel ANCA antigen-specificity in EGPA, and thus AAV.

Objectives: To evaluate the autoantibody profile in eosinophilic granulomatosis and polyangiitis (EGPA) patients., Methods: 33 EGPA patients were tested for anti-neutrophil cytoplasmic antibodies (ANCA), antinuclear antibodies (ANA), rheumatoid factor (RF), anti-alpha-enolase antibodies, and anti-eosinophil peroxidase (EPO) antibodies. Granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), hypereosinophilic syndrome (HES), rheumatoid arthritis (RA), primary biliary cirrhosis (PBC) patients and healthy subjects were tested as a control group., Results: Anti-alpha-enolase antibodies were positive in 82% of EGPA patients at high titers. Although a high sensitivity was shown for an anti-alpha-enolase antibody titer above 1/100 (82%), the specificity for EGPA remained low (44%) (AUC=0.653, p=0.008). Anti-alpha-enolase antibodies predominated in males with EGPA (p=0.048) and were associated with skin involvement (p=0.040). Most of the EGPA patients positive for anti-alpha enolase antibodies (20 out of 27) had a negative indirect immunofluorescence test (IFT) for ANCA. ANCA were positive in 8 EGPA patients (24%) with a perinuclear pattern in all but one patient. The ANCA-target antigen was myeloperoxidase (MPO) and/or alpha-enolase. A usually fine-speckled ANA pattern was observed in 42% of the EGPA patients. RF was positive in 1 (6%) of the 18 EGPA patients tested. There was no association between the presence and levels of autoantibodies and EGPA disease activity. None of the patients and controls was positive for anti-EPO antibodies., Conclusions: Alpha-enolase may be a target of autoimmunity in EGPA patients and shows usually negative ANCA IFT results.

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: To assess the diagnostic performance of 11 commercial PR3- and MPO-ANCA ELISA systems (direct, capture and high sensitive [hs] ELISA)., Methods: Sera from 90 patients with AAV (GPA, MPA and CSS) and 20 disease controls (SLE; RA) and healthy individuals were tested for the presence of ANCA by IFT and by different ELISAs for the presence of PR3-and MPO-ANCA, respectively. Furthermore, the binding capacity of the IUIS-CDC reference sera for PR3-/MPO-ANCA in different commercial assays was analysed., Results: Commercial ELISA kits for PR3-ANCA differed moderately in their sensitivity (from 45% to 62.5%). The highest sensitivity for PR3-ANCA was obtained with hs ELISA (kit A) and capture ELISA (kit N). Testing for MPO-ANCA the highest sensitivity (85%) was obtained with direct ELISA (kit D and I). Specificity was high in all kits. Only three PR3-ANCA commercial kits and three MPO-ANCA kits produced binding at the expected value for the IUIS-CDC reference sera (100 U/ml). In all of the kits, serial dilutions of the reference sera did not yield linearity., Conclusions: Second (capture) and third (high sensitivity) generation PR3-ANCA ELISA kits are superior to conventional ELISAs. Direct and capture MPO-ANCA ELISAs showed a good overall performance in all kits. Most of the kits have not been standardised to allow their results to be compared.

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.

Background: Interferon-alpha has been successfully used for induction of remission in patients with Churg-Strauss syndrome, but data on its ability to prevent relapses and its safety during long-term use are lacking., Objectives: To examine the safety and efficacy of interferon-alpha for mainten-ance of remission in Churg-Strauss syndrome., Patients and Methods: In a prospective open-label long-term observational study, 13 patients with CSS in stable remission received interferon-alpha (3 x 3 Mio. I.U/week s.c.) for maintenance of remission. Primary end point was the incidence of relapses. Secondary end points were the doses of concomitant prednisolone and the frequency adverse events., Results: After a median follow up of 64 month three patients were still on treatment with interferon-alpha all with a dose of 9 million units/week. In nine patients, interferon-alpha was discontinued for lack of efficacy (n=5), due to adverse events (n=2), or both (n=2) after median of 63 months (15-153) of therapy. A total of 3 major and 18 minor relapses occurred in 10 of the 13 patients with a median time to first relapse of 17 months (range 5-46). Sera of relapsing patients did not contain antibodies against interferon-alpha. In 6 relapsing patients treatment was switched to cyclophosphamide (n=4) or methotrexate (n=2). Four episodes of worsened asthmatic symptoms associated with a mild rise of blood eosinophils occurred in 3 patients and resolved following a transient increase of the oral prednisolone dosage. After 49 months one patient died probably due to a relapse. IFN-alpha was ceased prematurely, because of autoimmune-thyreoiditis in one, depression in another and cerebral leukoencephalopathy in two patients. Overall, 18 infectious episodes with need of antimicrobial treatment were observed.CONCLUSIONS. Recombinant interferon-alpha appears to be partially effective in the prevention of major relapses in patients with Churg-Strauss syndrome. Due to numerous side effects and infections during long-term administration its use should be restricted to patients with contraindications against conventional immunosuppressive therapies.

Defects in regulatory T (Treg) cells have been implicated in the pathogenesis of chronic inflammatory and autoimmune diseases, such as Wegener's granulomatosis (WG). This study aimed at evaluating numbers, phenotype and suppressive capacity of Treg cells in WG. Peripheral blood (PB) mononuclear cells from 22 WG-patients (17 active, 5 remission) and 22 sex- and age-matched healthy controls (HC) were examined for Treg cells by flow cytometry measuring CD4, CD25, transcription factor forkhead box P3 (FoxP3), chemokine receptor CCR4 and interferon receptor I (IFNRI). Suppressive function of CD4+CD25high Treg cells from 3 WG-patients and 3 HC was analysed using a carboxyfluoresceindiacetate-succinimidylester-based in vitro proliferation assay. Endonasal biopsies of 10 WG- and 5 sinusitis-patients were investigated for CD3+FoxP3+ cells, employing double immunohistochemistry. WG-patients displayed elevated numbers of CD4+CD25med T cells and of CD4+CD25high Treg cells. CD4+ T cells of WG-patients contained higher numbers of CCR4+ cells. However, CD4+CD25high Treg cells of WG-patients exhibited decreased numbers of cells co-expressing FoxP3 and CCR4. A low but significant increase of CD4+CD25highIFNRI+ Treg cells was detected in WG-patients. 9 days following stimulation with interferon (IFN)alpha + proteinase 3 (PR3), a reduced suppression of proliferation of responder T cells was observed for WG and proliferated CD4+CD25high Treg cells still showed downregulated co-expressions of FoxP3 and CCR4. Wegener's granuloma exhibited increased numbers of CD3+FoxP3+ cells. The results indicate upregulated numbers of Treg cells in PB and nasal mucosa as well as phenotypical and functional alterations of PB Treg cells in WG, some presumably mediated through PR3 and IFN-alpha.

Objectives: The aim of this study was to analyse mPR3 expression on neutrophils in two Anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitides (AAV), namely WG (localised vs. generalised) and Churg-Strauss syndrome (CSS) and other inflammatory disorders, in order to evaluate (i) whether the pattern of mPR3 expression is specific for AAV and (ii) to assess whether the mPR3high status is associated with clinically distinct disease stages of WG (localised vs. generalised)., Methods: Localised WG (n=15), generalised WG (n=55), Churg-Strauss Syndrome (CSS) (n=20), systemic lupus erythematosus (SLE) (n=15), Rheumatoid Arthritis (RA) (n=22) and healthy controls (n=30) were analysed. mPR3 and CD63 expression on surface of neutrophils were assessed by flow cytometric analysis on isolated neutrophils and whole blood., Results: In patients with genWG and SLE, an increased percentage of mPR3+ neutrophils and an elevated level of mPR3 expression compared to healthy controls were found (percentage: p=0.001, p=0.000; MFI ratio: p=0.038, p=0.019, respectively). There was no increased frequency of mPR3+ neutrophils in CSS. Within the group of WG, an elevated level of mPR3 expression was significantly associated with disease stage (genWG and not locWG), and in genWG with disease activity and the presence of ANCA., Conclusions: The mPR3high status is associated with generalised WG and correlates with disease activity and ANCA status in generalised WG. An increased proportion of mPR3-positive neutrophils is not specific for AAV.

Objectives: The aim of the study was to assess PAR-2 expression on dendritic cell (DC) subsets and other immune cells of Wegener's granulomatosis (WG) patients and healthy controls (HC) and to investigate whether Proteinase 3 (PR3, a serine protease which can activate PAR2) induces maturation of human DC-like monocytes and murine Flt-3 ligand- and GM-CSF-generated DC., Methods: Human peripheral blood cells including DC subsets and Flt-3l- and GM-CSF-generated mouse DC were analysed for expression of PAR-2 and DC maturation markers by flow cytometry before and after stimulation with PR3, trypsin, PAR-2 agonist or LPS for 24 h., Results: There was no difference of PAR-2 expression on PMNs, monocytes, lymphocytes and DC between all WG samples and HC. However, in inactive WG, expression of PAR-2 was downregulated on the cell surface of PMNs, monocytes, lymphocytes, and CD11c+DC compared to active WG and HC. PR3 and PAR2-agonists did not induce upregulation of PAR-2 or maturation markers of human DC-like monocytes in WG and HC. Likewise, murine PR3 did not induce upregulation of PAR-2 or maturation markers in murine DC., Conclusions: PAR-2 expression is downregulated on human peripheral blood cells including CD11c+ DC in inactive WG compared to active WG and HC, possibly reflecting a non-activated status of these cells in inactive disease. PR3 and PAR-2- agonists did not induce maturation of human ex vivo DC-like monocytes in WG and HC and of murine DC, suggesting this pathway is not singularly involved in the maturation of these cell subsets.

Objectives: Nasal colonisation with Staphylococcus aureus (S. aureus) has been implicated in Wegener's granulomatosis (WG) disease activity. In this study, the frequency of nasal colonisation with S. aureus in WG was compared to healthy and disease control groups for the first time. Moreover, endonasal activity was correlated to colonisation., Patients and Methods: Nasal carriage of S. aureus of a well-defined group of 89 patients with WG was compared to 40 patients with chronic rhinosinusitis with nasal polyps (CRS), 35 patients with rheumatoid arthritis (RA), 50 hospital staff members and 25 subjects without regular hospital contact and correlation analysis of nasal carriage and endonasal activity of WG was performed., Results: WG patients showed significantly higher rates (72%) of nasal colonisation with S. aureus compared to CRS patients (28%) and healthy subjects without regular hospital contact (25%, 95%-CI), but not to RA patients (46%) and hospital staff members (58%). WG patients with nasal carriage of S. aureus had significantly higher endoscopically proven endonasal activity (p=0.01), significantly more often first manifestation of WG in the upper respiratory tract (p=0.02) and higher relapse-rates (p=0.052) than WG patients without such carriage., Conclusions: Endonasal activity in WG is associated with higher nasal S. aureus colonisation rates and subsequent higher relapse rates. The higher frequency of S. aureus colonisation could be a consequence of a recently shown mucosal barrier defect in WG and facilitate chronic inflammation and granuloma formation in the upper respiratory tract.

Proteinase 3 (PR3) is a multifunctional neutrophil-derived serine protease influencing cell cycle, differentiation, and cell death. This molecule is the main target antigen of autoantibodies in Wegener's granulomatosis (WG) known as antineutrophil cytoplasmic antibodies (PR3-ANCA). WG usually starts as granulomatous inflammation of the upper respiratory tract (localized phase) and progress to systemic disease with PR3-ANCA-associated vasculitis (generalized phase). PR3-ANCA is thought to play a critical role in the pathogenesis of vascular damage in WG. In contrast, it is not clear how the granulomatous inflammation, the hallmark of WG, is driven, and what is the relationship between granuloma and autoimmunity. Recent findings provide evidence that PR3 might function as endogenous "danger/alarm" signal that communicates the presence of tissue injury to dendritic cells (DC) via protease-activated receptor-2 (PAR-2), triggers their maturation and instructs DC to induce Th1-type cell responses in WG. Furthermore, PR3 has the capacity to bind and activate IL-32, a recently discovered proinflammatory cytokine that has emerged as an important player in innate and adaptive immune response.Collectively, these results delineate new pathogenic pathways at the molecular level and provide insights into the mechanisms by which PR3 may contribute to the early pathogenesis of WG supporting the pivotal role of the interaction of Wegener's autoantigen with the "gateway" receptor PAR-2 in mediating both innate and adaptive immune response in WG.

Objective: Conventional direct enzyme-linked immunosorbent assays (ELISA) for the detection of anti-neutrophil cytoplasm antibodies (ANCA) often lack sensitivity because epitopes of the target antigen are hidden by binding to the ELISA plate. This study was designed to evaluate a novel ELISA method for detection of ANCA against proteinase-3 (PR3) for the diagnosis of Wegener's granulomatosis (WG) using PR3 presented in its native form., Methods: Sera from four subgroups of patients with a diagnosis of WG (n=86), 80 healthy controls and 450 disease controls were tested for the presence of C-ANCA/PR3-ANCA by anchor ELISA, direct ELISA, capture ELISA, indirect immunofluorescence (IFT) and immunoblotting., Results: In prospectively analysed consecutive patients, anchor ELISA showed the highest sensitivity for a diagnosis of WG of 96.0% (95% CI: 79.6-99.3), followed by IFT 92.0% (73.9-98.8), capture ELISA 72.0 (50.6-87.9) and direct ELISA 60.0 (38.7-78.8). Specificity was high for all methods and ranged from 98.5 (97.0-99.4) to 95.5% (97.9-99.8). Receiver operating characteristics curve analysis revealed that the overall diagnostic performance of the anchor ELISA was significantly superior compared to the direct ELISA and the capture ELISA in patients with generalized WG, and also compared to IFT and immunoblotting in patients with localised WG., Conclusion: Anchor ELISA is a novel highly sensitive and specific method for the detection of PR3-ANCA in patients with WG, which may replace the need for a combined analysis with IFT and ELISA in the future.

Objective: To determine the prevalence, target antigens and clinical associations of antineutrophil cytoplasmic antibodies (ANCA) in chronic hepatitis C without extrahepatic manifestations and in chronic hepatitis C virus (HCV)-associated mixed cryoglobulinemia (MC) in two European centers., Methods: 50 sera from patients with chronic hepatitis C and 116 sera from HCV-associated MC were tested for cytoplasmic or perinuclear pattern (C-ANCA/P-ANCA) by indirect immunofluorescence test (IFT). ANCA target antigens were determined by enzyme-linked immunosorbent assay (ELISA)., Results: Clinical characteristics of the patients were not different between the two centers. Cryoglobulinemic vasculitis (CV) was biopsy-proven in about 90% of the MC patients. Two patients with HCV-associated MC and 1 patient with chronic hepatitis C had a P-ANCA. A C-ANCA was detected in 1 patient with HCV-associated MC. Eight patients with a HCV-associated MC and 5 patients with chronic hepatitis C had an ANCA either directed against bactericidal/permeability increasing protein (BPI) or cathepsin G (CG). BPI- or CG-ANCA positivity was not associated with a more severe disease course. The C-ANCA titer followed disease activity in one C-ANCA positive HCV-associated MC patient. The subspecificity of the C-ANCA was not determinable in that patient., Conclusion: Two new target antigens of ANCA have been identified in HCV-associated MC and chronic hepatitis C in this study. BPI-ANCA and GC-ANCA were present in about 10% of patients with HCV-associated MC or chronic hepatitis C. ELISA proved to be more sensitive in the detection of ANCA than IFT. The present study on chronic HCV infection adds to various reports on the induction of CG- and BPI-ANCA in chronic infections.

Objective: To determine the prevalence and clinical associations of ANCA against the antibiotic proteins and peptides: Bactericidal/permeability-increasing protein (BPI), Azurocidin (AZ), Calprotectin (CP) and beta-Defensin-1 and -2 (DF)., Methods: Patients with ANCA-associated vasculitides (n = 99), other vasculitides and rheumatic connective tissue diseases (n = 303), HIV-infection (n = 66), other infectious diseases (n = 134) Crohn's disease (n = 12) and ulcerative colitis (n = 12) were tested for BPI-, AZ-, CP-, DF-, PR3-, and MPO-ANCA in indirect immunofluorescence technique (IFT) and ELISA., Results: In ANCA associated vasculitides BPI-ANCA were detected in 6% of patients. In HIV infection, BPI was the main target antigen of ANCA-IFT positive sera (74%). BPI-ANCA was associated with higher inflammatory activity. In Crohn's disease and ulcerative colitis BPI-ANCA was prominent (34% of patients). AZ-ANCA were found in 5% of patients. No ANCA were detected against defensin and calprotectin., Conclusion: BPI-ANCA is the main autoantibody in HIV and is associated with higher inflammatory activity. In inflammatory bowel diseases BPI-ANCA is predominant, AZ-ANCA are also present to a lesser extend. Both were not useful characterize clinical subgroups. No ANCA were detected against calprotectin or defensins.

Churg-Strauss syndrome (CSS) is a rare form of systemic vasculitis occurring in patients with asthma. The cause of CSS is unknown, and yet little data are available regarding its pathogenesis. The presence of a marked tissue- and blood-eosinophilia, as well as secretory products of eosinophils in blood and tissues, implicates a pathogenetic role of eosinophil granulocytes. Prolonged survival of eosinophils due to inhibition of CD95-mediated apoptosis by soluble CD95 seems to contribute to eosinophilia in CSS. Although the mechanisms involved in eosinophil-activation in CSS have not been elucidated, recent data suggest a possible role of T lymphocytes secreting eosinophil-activating cytokines. This review describes the current insights into the pathogenesis of CSS in the light of its putative nature as a type 2 granulomatous disease. Recent clinical, experimental and epidemiologic data regarding the possible role of inflammatory cells and their secretory products, anti neutrophil cytoplasm antibodies (ANCA), epidemiologic factors and anti-asthma treatments are summarized.

Objective: To assess circulating immunoregulatory cytokines and soluble surface markers of T and B cell activation in the plasma of patients with Wegener's granulomatosis (WG), Churg-Strauss syndrome (CSS) and microscopic polyangiitis (MPA) during active and inactive disease, in order to establish their value in discriminating between disease entities and as markers of disease activity., Methods: Plasma levels of IL-4, IL-5, IL-10, IL-12, IL-13, IFN-gamma and soluble CD23, CD26 and CD30 were determined by enzyme-linked immunosorbent assay in patients with WG (n = 21), CSS (n = 19) and MPA (n = 14) during active disease and remission., Results: Concerning cytokines, no differences were observed for IFN-gamma, IL-4, IL-5 and IL-13. Plasma levels of IL-12 were decreased in all subgroups of patients. On the contrary, IL-10 levels were significantly elevated only in patients with CSS. Levels of sCD30 were significantly increased in patients with active generalized WG and CSS, but not in those with MPA and localized WG, correlating with the disease extent and activity. sCD26 levels were markedly decreased in patients with generalized WG, CSS and MPA and increased towards remission. sCD23 levels were slightly, but not significantly increased in CSS and generalized WG., Conclusion: Regarding the investigated immunoregulatory cytokines (Th1/Th2 type), only the measurement of plasma levels of IL-10 discriminated CSS from WG and MPA. The reported data could indicate a similar status of T cell activation in generalized WG and CSS, and possibly a shift in peripheral immunity towards a more humoral dominated immune response. The differences observed between patients with the localized and generalized forms of WG seem to reflect the clinically known biphasic course of this disease.