anti-PR3 Antibodies Research Articles

Sialylation levels of anti-proteinase 3 antibodies are associated with the activity of granulomatosis with polyangiitis (Wegener's)

To investigate whether the glycosylation and sialylation levels of anti-proteinase 3 (anti-PR3) antibodies could affect their pathogenicity, and whether these levels could be correlated with the activity of granulomatosis with polyangiitis (Wegener's) (GPA). Forty-two serum samples positive for anti-PR3 antibodies from 42 patients with active or weakly active/inactive GPA were included. Anti-PR3 antibodies were assayed by enzyme-linked immunosorbent assay, and their levels of glycosylation and sialylation were assessed by enzyme-linked lectin assay. The glycosylation and sialylation levels of IgG purified from the serum of healthy donors and patients with active, remitted, or weakly active disease were assessed by permethylation and mass spectrometry analysis of glycans, following neuraminidase digestion. The neutrophil oxidative burst induced by purified IgG was assayed by spectrofluorimetry. The mean sialylation ratio of anti-PR3 antibodies was significantly lower in patients with active disease than in patients with weakly active or inactive disease, and this was inversely correlated with the Birmingham Vasculitis Activity Score (BVAS) (P < 0.0001). Similar results were obtained using the BVAS/GPA. The area under the receiver operating characteristic curve for the sialylation ratio of anti-PR3 antibodies, as a test to determine the activity of GPA, was 0.82 (P = 0.0006). The characterization of N-glycans showed a decrease in 2,6-linked sialylated N-glycans and an increase in dHex₁ Hex₃ HexNAc₄ (mass/charge 1,836) agalactosylated structures in purified IgG from patients with active disease compared with controls. The anti-PR3 antibody-induced oxidative burst of neutrophils was inversely correlated with the sialylation levels of anti-PR3 IgG. The sialylation level of anti-PR3 antibodies contributes to the clinical activity of GPA, by modulating the oxidative burst of neutrophils induced by these autoantibodies.

Adult-onset Henoch–Schonlein purpura with positive c-ANCA (anti-proteinase 3): case report and review of literature

Anti-neutrophil cytoplasmic antibodies (ANCA) have two common patterns detected by indirect immunoXuorescence test (IIF test). The cytoplasmic pattern (c-ANCA) that is strongly associated with antibodies against proteinase-3 (PR3) and the perinuclear pattern (p-ANCA) that is mostly directed against myeloperoxidase (MPO). Anti-proteinase 3 and anti-MPO are characteristic for pauci-immune small vessel vasculitis like Wegener’s granulomatosis, microscopic polyangiitis, Churg–Strauss syndrome and idiopathic crescentic glomerulonephritis [1]. In Wegener’s granulomatosis (WG), the combination of c-ANCA with anti-PR3 antibodies has a sensitivity of 58% and a speciWcity of 99% [2]. On the other hand, the presence of c-ANCA (anti-PR3) in immune-complex-mediated vasculitis such as Henoch–Schonlein purpura (HSP) is very unusual. We report a case of biopsy proven HSP associated with IgG c-ANCA (anti-PR3).

Anti-neutrophil cytoplasmic antibodies target sequential functional proteinase 3 epitopes in the sera of patients with Wegener's granulomatosis

Many patients with Wegener’s granulomatosis (WG) have anti-neutrophil cytoplasmic antibodies (c-ANCA). Aside from being a diagnostic marker, these autoantibodies may play roles in disease pathogenesis. Proteinase 3 (PR3) is the primary target of c-ANCA in WG patient sera. Of 60 c-ANCA-positive patients, 10 patients were selected for detailed humoral epitope analysis, contingent upon serum availability, using samples with positive levels of anti-PR3 by enzyme-linked immunosorbent assay (ELISA). Sequential epitope specificities of anti-PR3 antibodies detected by screening the maximally overlapping solid-phase octapeptides of PR3 showed seven major common antigenic targets bound by WG patient sera. These include novel and previously identified sequential PR3 epitopes bound by c-ANCA. B cell epitope prediction algorithms identified all or part of the seven defined epitopes. Several epitopes share sequence and structural proximity with functional sites, including the catalytic triad and proposed binding sites of other potential proteins [PR3 complementary peptide and soluble endothelial protein C receptor (sEPCR)]. Epitope 4 (VVLGAHNVRTQ) had the highest binding prevalence (90%) and epitope 2 (AQPHSRPYMAS) has the highest average reactivity of the antigenic regions. Epitope 4 includes the interaction site between sEPCR and PR3 which may serve as an important interaction to down-regulate inflammation. Epitopes 3, 5 and 7 are in direct proximity to amino acids that form the catalytic triad of the protein. c-ANCA targets both unique and previously known sequential PR3 peptides. This information may prove useful in understanding anti-PR3-mediated disease pathogenesis in systemic vasculitides.

Longitudinal studies of patients with ANCA vasculitis demonstrate concurrent reactivity to complementary PR3 protein segments cPR3m and cPR3C and with no reactivity to cPR3N

Antibodies recognizing the complement of the middle of PR3 (cPR3m) occur in ∼30% of PR3-anti-neutrophil cytoplasmic autoantibodies (ANCA)-vasculitis patients and immunization of animals with a peptide complementary to the middle of PR3 (cPR3m) induces not only anti-complementary PR3 antibodies, but also anti-PR3 antibodies derived through an anti-idiotypic response. PR3 epitopes recognized by patient ANCA, however, are not restricted to the middle of PR3. This prompted us to test for antibodies that react with proteins complementary to the terminal regions of PR3 (cPR3C and cPR3N) in PR3-ANCA patients. Anti-cPR3C reactivity was detected in 28% of patients but anti-cPR3N reactivity in only 15%. Ranked anti-cPR3C and anti-cPR3m reactivity correlated in the cohort, whereas there was no significant relationship between cPR3C and cPR3N reactivity. Serial samples from 3 patients' revealed that anti-cPR3C and anti-cPR3m reactivity followed a similar pattern over time. Serial samples from a fourth patient demonstrated an anti-cPR3N response without concurrent cPR3m or cPR3C reactivity. Epitope determination by mass spectrometry identified a 13-amino acid sequence on cPR3C that contained a common binding site recognized by antibodies from three patients. This peptide sequence contains a “PHQ” motif which was reported to be the basis for cross-reactivity of anti-cPR3m antibodies with plasminogen. Why these antibodies are detected in only ∼30% of the patients remains unclear. The data reveal that it is not due to lack of inclusion of flanking regions of complementary PR3 during screening. Instead, quite unexpectedly, the data demonstrate that patients' antibodies react with a restricted epitope that exists in both cPR3m and cPR3C.

Generation of Anti-Proteinase 3 Monoclonal Antibodies and Development of Immunological Methods to Detect Endogenous Proteinase 3

Proteinase 3 (PR3), a neutrophil granule serine protease, is the major autoantigen for autoantibodies in the systemic vasculitic disease, Wegener's granulomatosis. It is also found to be involved in various inflammatory diseases including Crohn's disease, rheumatoid arthritis, cystic fibrosis, and gingivitis. However, there is no high quality antibody available to detect endogenous PR3 in biological samples such as plasma and tissue. Several commercial anti-PR3 monoclonal antibodies (MAbs) were obtained by using HMC-1/PR3 cell granule extracts as the antigen, but the resulting antibodies could not be applied for immunoblotting or other immunological methods. Therefore, we produced human recombinant PR3 in Escherichia coli and developed several MAbs that are highly sensitive and can be used for immunoblotting, FACS analysis, and immunofluorescent staining. The PR3 MAbs recognized both rhPR3 and human plasma-derived neutrophil PR3 in reducing and non-reducing conditions at low nanogram levels. In addition, new MAbs detect endogenous PR3 from normal human plasma and urine with high specificity. The new anti-PR3 MAbs will be an essential tool for investigating the role of PR3 in inflammatory and autoimmune diseases.

A composite elisa for the rapid diagnosis of ANCA-associated vasculitis and ANTI-GBM mediated disease

Background Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis and Goodpasture’s disease are conditions requiring rapid institution of treatment for optimal outcomes. Consequently a rapid diagnosis is important. We undertook an audit of the clinical and laboratory features of patients from Christchurch Hospital on whom a rapid vasculitis and anti-glomerular basement membrane (GBM) antibody screen was performed in the period 1 August 2003 to 30 July 2008. Methods 109 patient samples were tested on the rapid screen using a modified in-house composite ELISA for anti-proteinase 3 (PR3), anti-myeloperoxidase (MPO) and anti-GBM antibodies. Fifty-three of these samples were also tested on routine ELISAs for those antibodies. Results The rapid screen for anti-GBM antibodies had a clinical sensitivity of 75% (95%CI 24.23–98.75) and specificity of 97.14% (92.42–99.27) for anti-GBM mediated disease. The MPO and PR3 screening tests had a sensitivity of 100% (79.42–100) and specificity of 93.83% (86.85–97.7) for ANCA-associated vasculitis. Conclusions The composite ELISA provides a rapid result with high specificity for anti-GBM, anti-MPO and anti-PR3 antibodies.

C-ANCA-induced neutrophil-mediated lung injury: a model of acute 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.

Use of Likelihood Ratios Can Improve the Clinical Usefulness of Enzyme Immunoassays for the Diagnosis of Small-Vessel Vasculitis

Antineutrophil cytoplasmic antibodies (ANCAs)1 are associated with small-vessel vasculitis (SVV), such as Wegener granulomatosis, microscopic polyangiitis, and Churg–Strauss syndrome (1). Indirect immunofluorescence (IIF) analyses can distinguish 2 major patterns: cytoplasmic and perinuclear. The cytoplasmic ANCA pattern is typically associated with antibodies to proteinase 3 (PR3), whereas the perinuclear ANCA pattern is typically associated with antibodies to myeloperoxidase (MPO). PR3 ANCAs are predominantly seen in patients with Wegener granulomatosis, whereas MPO ANCAs are predominantly seen in patients with microscopic polyangiitis(1)(2)(3). Studies that have addressed the clinical usefulness of specific enzyme immunoassays (EIAs) for PR3 and MPO for the diagnosis of SVV have used a single cutoff value. In this letter, we illustrate how the likelihood ratio (LR) for SVV depends on the titer of anti-PR3 or anti-MPO antibodies. Our calculations are based on a clinically well-defined group of 37 consecutive patients with newly diagnosed SVV (data collected over a 10-year period) and 285 consecutive control individuals with disease (data collected over 21 months) that a physician suspected to be a …

Manifestations cardiaques au cours la granulomatose de Wegener : à propos de quatre observations et revue de la littérature

Introduction Discordance exists between the results of post-mortem studies and the low number of clinical reported cases of cardiac involvements in Wegener's granulomatosis. Case reports Data from four patients were studied retrospectively. Three patients had associated airway localization and three had kidney involvement. All patients had positive test for anti-PR3 antineutrophil antibodies. Two patients presented with dilated cardiomyopathy (one with terminal cardiac failure), another patient with complete atrioventricular block and pericarditis, and the remaining one with myopericarditis. One patient was asymptomatic. For three of these patients, the cardiac manifestations were contemporary of the diagnosis of Wegener's granulomatosis and had a severe disease course. Conclusion Cardiac events in Wegener's granulomatosis are probably underestimated, given the various type of heart damage and the clinical presentation. Cardiac involvement seems to be associated with a poor prognosis. Thus, we recommend systematic and regular cardiac assessment in the follow-up of patients with Wegener's granulomatosis.

High‐sensitivity Detection of Autoantibodies Against Proteinase‐3 by a Novel Third‐generation Enzyme‐linked Immunosorbent Assay

The aim of this study was to evaluate a novel third-generation enzyme-linked immunosorbent assay (ELISA) for the high-sensitivity detection of autoantibodies to proteinase-3 (PR3) in patients with Wegener's granulomatosis (WG). First- and second-generation ELISA for the detection of antineutrophil cytoplasmic antibodies (ANCA) frequently demonstrate insufficient sensitivity due to inadequate presentation of autoantigenic epitopes. Human PR3 was immobilized on the solid phase of ELISA plates by anchoring technique. Anti-PR3 reactivity was measured in 34 C-ANCA positive patients with WG, 11 MPO-ANCA-positive patients with other autoimmune vasculitides, 65 patients with systemic lupus erythematosus (SLE), and 137 healthy blood donors. Thirty-three of 34 patients with WG (97.1%) showed positive anti-PR3 IgG antibody reactivity. None of 11 MPO-ANCA positive vasculitis patients, none of 137 blood donors, and 3 of 65 SLE patients expressed elevated IgG reactivity to PR3 (specificity: 98.4%). Comparison with another third-generation ELISA did not reveal different qualitative results. However, there was no significant correlation between quantitative results of both assays. Receiver operating characteristic (ROC) curve analysis revealed a significantly better assay performance compared with first (direct)- and second (capture)-generation assays (P = 0.011 and P = 0.001, respectively). Third-generation (anchor) anti-PR3 ELISA exhibit significantly higher sensitivity than previous generation assays. Anchoring of PR3 renders the granulocyte protein more autoantigenic compared with direct or capture immobilization.

Coexpression of CD177 and membrane proteinase 3 on neutrophils in antineutrophil cytoplasmic autoantibody–associated systemic vasculitis: Anti–proteinase 3–mediated neutrophil activation is independent of the role of CD177‐expressing neutrophils

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. 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. 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. 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.

Anti-idiotype antibodies in immune regulation of ANCA associated vasculitis

Background:Anti-idiotype antibodies (anti-ids) have a potential role in the immunomodulation of various autoimmune disorders. The immunoregulatory role of anti-idiotypic antibodies in ANCA-associated vasculitis needs to be studied. This study was conducted in clinically and histopathologically diagnosed ANCA-associated vasculitis (AAV) patients.Methods:Anti-ids were tested in 100 AAV patients of which 80 had anti-MPO and 20 had anti-PR3 antibodies at various stages of disease over a period of 2-3 years. The disease activity was estimated by the Birmingham vasculitis activity score (BVAS). The affinity-purified ANCA F(ab')2 fragments were prepared using three each of anti-MPO and anti-PR3 high titer sera and were used as idiotype coats for anti-idiotype antibody detection by ELISA. Positivity was confirmed by fluid phase inhibition ELISA.Results:Patients who went into remission showed 53.8% anti-ids to anti-MPO and 52.9% to anti-PR3 with low BVAS values (0-8), whereas in patients with active disease, only 12.5% had anti-ids to anti-MPO and 10% had anti-ids to anti-PR3 with comparatively high BVAS (18-32), while five cases who had relapse (BVAS 18-20) did not have anti-ids to anti-MPO or anti-PR3. An inverse correlation was noted between ANCA and anti-ids (r = −0.901).Conclusions:High prevalence of anti-ids in remission cases and low prevalence in active cases with absence of anti-ids in relapse cases as well as an inverse correlation of ANCA and anti-ids indicate its beneficial effect on the disease process, thus suggesting the dynamic role of anti-idiotype networks in the immunoregulation of AAV.

Anti-PR3 and Anti-MPO IgG ANCA in Autistic Children with Chronic GI Disease

Aim To assess both ant-PR3 and anti-MPO IgG anti-neutrophil cytoplasmic antibodies (ANCA), in autistic children with gastrointestinal symptoms and controls, and to test the hypothesis that there is an association between the presence of these antibodies and inflammatory GI disease seen in many autistic children. Subjects and Methods ELISA's were used to measure anti-PR3 and anti-MPO IgG in 40 autistic children with chronic digestive disease (many with ileo-colonic lymphoid nodular hyperplasia (LNH) and inflammation of the colorectum, small bowel and/or stomach), and 41 controls (21 age matched autistic children with no GI disease and 20 age matched children without autism or GI disease). Results Six of 40 autistic children with chronic digestive disease had anti-PR3 antibodies compared to none of the 41 controls (p &lt;0.01). Thirteen of the 40 autistic children with chronic digestive disease had anti-MPO IgG compared to only 3 (1 autistic control with no GI disease and 2 non-autistic controls without GI disease) of 41 controls (p &lt;0.001). All six of those with anti-PR3 IgG also had anti-MPO antibodies. These results suggest a relationship between individuals with ANCA and severity of intestinal disease in a subset. Discussion These results suggest an association between anti-PR3 and anti-MPO antibodies and a subset of ASD children with GI inflammation.

Pauci-Immune Lupus Nephritis: A Case Report

A 26-year-old female with systemic lupus erythematosus was admitted because of dyspnea and progressive lower extremity edema. Laboratory testing showed blood urea nitrogen levels of 147 mg/dL, creatinine of 6.7 mg/dL, serum albumin of 1.7 g/dL and the daily protein loss was 12.7 g. Her C3 level was 60.4mg/dL and C4 level was 10.2 mg/dL. The antinuclear antibody titer was 1:320, with a homogeneous pattern, but she was negative for anti-dsDNA. ELISA testing for anti-PR3 antibodies and anti-MPO antibodies were all negative. She was also negative for circulating lupus anticoagulant. Renal biopsy revealed diffuse proliferation of glomerular cells, but immunofluorescent microscopy showed no immune deposits and electron microscopy revealed only scanty electron-dense deposits. She received 1 g/day of methylprednisolone intravenously for 3 days, followed by 60 mg/day of prednisolone. She was discharged with serum creatinine decreased to 4.7 mg/dL, and a great improvement in dyspnea. Diffuse proliferative lupus nephritis that contains little or no subendothelial deposits is rare. The differential diagnosis, possible mechanisms and treatment are discussed.

A rare cause of reversible unilateral third nerve palsy

Case reportA 59-year-old Chinese male presented in January 2007 with acute left retro-orbital headache, diplopia and left partial ptosis. Isolated left third nerve palsy was diagnosed. Imaging studies and cerebral angiography excluded a posterior communicating artery aneurysm. Anti-nuclear (titre 1/800, speckled pattern), anti-PR3, anti-Ro and anti-La antibodies were present. Sjogren's syndrome (SS) was considered in view of positive anti-Ro and La antibodies, and was confirmed with focal lymphocytic infiltrates on labial salivary gland biopsy and a positive Schirmer's test (6 mm of tear flow over 5 min). Immunosuppressive therapy was started 2 months after his initial presentation and within 2 weeks, the patient experienced an almost complete recovery of both ptosis and diplopia. He has been on tapering doses of prednisolone since and his condition remains stable. ConclusionThis patient has cranial neuropathy secondary to Sjogren's syndrome. The rapid reversibility of the oculomotor nerve palsy with immunosuppression suggests lymphocytic infiltration or autoantibodies as the cause rather than a vasculitic process, which would have led to irreversible or slowly, partially reversible ischaemic damage.

Alpha 1-antitrypsin deficiency and anti-proteinase 3 antibodies in anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis.

alpha 1-antitrypsin (alpha 1-AT) is a naturally occurring inhibitor of proteinase 3 (PR3) and elastase, two of the target antigens of anti-neutrophil cytoplasmic antibodies (ANCA). An increased incidence of alpha 1-AT phenotypes associated with dysfunctional alpha 1-AT or low serum levels has been reported in patients with anti-PR3 antibodies. We have studied the relationship between ANCA, and phenotypes and serum levels of alpha 1-AT. Phenotypes usually associated with a moderate or severe reduction in alpha 1-AT serum levels or in dysfunctional activity were found more often in individuals with anti-PR3 antibodies than in the general population: four of the 31 patients (13%) with anti-PR3 antibodies had phenotypes MZ (n = 2), S (n = 1) or Z (n = 1) (P < 0.05). However, the corresponding alpha 1-AT serum levels were normal (n = 3) or elevated (n = 1). None of the 31 sera with anti-PR3 antibodies had low levels of alpha 1-AT. No abnormal alpha 1-AT phenotype was demonstrated in seven patients with anti-elastase antibodies, despite a low level of alpha 1-AT in one serum. Anti-myeloperoxidase antibodies are common in patients with ANCA, but no abnormal phenotype or low serum alpha 1-AT level was demonstrated in any of 29 sera containing these antibodies. Finally anti-glomerular basement membrane (GBM) antibodies occur occasionally in patients with ANCA-associated diseases, but again none of 10 sera had an abnormal alpha 1-AT phenotype or low serum level. ANCA were not demonstrated by indirect immunofluorescence in any serum from 73 patients with abnormal alpha 1-AT phenotypes. These results confirm that patients with anti-PR3 antibodies often have alpha 1-AT phenotypes that are usually associated with low serum levels of alpha 1-AT or with dysfunctional protein. Nevertheless, the incidence of anti-PR3 antibodies in patients with abnormal alpha 1-AT phenotypes is very low. This probably reflects the rarity of Wegener's granulomatosis, the major disease associated with anti-PR3 antibodies, and the relative frequency of abnormal alpha 1-AT phenotypes. The mechanism for the development of anti-PR3 antibodies in patients with abnormal alpha 1-AT phenotypes is not clear, but may relate to the increased propensity of unbound and uninhibited PR3 to stimulate autoantibody production.

Epitope mapping of anti-proteinase 3 and anti-myeloperoxidase antibodies.

Anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO) autoantibodies are present in many patients with Wegener's granulomatosis (WG) and microscopic polyarteritis. The aim of this study was to determine whether these antibodies bound to linear peptide sequences on their target antigens. If common linear epitopes were demonstrated, then these could be manufactured and used in diagnostic ELISAs for anti-PR3 and anti-MPO antibodies. In addition, any homology between these epitopes and bacterial or viral sequences might implicate those microorganisms in the development of these antibodies and the pathogenesis of the associated diseases. The presence of linear epitopes on PR3 and MPO was suggested by the binding of the corresponding autoantibodies to these proteins after they had been reduced with beta-mercaptoethanol (beta-ME) and denatured with SDS or boiling, and digested with proteases. Four of the 22 sera with anti-PR3 antibodies bound to PR3 in Western blots after treatment with SDS, beta-ME and boiling for 5 min. Thermal denaturation reduced the amount of binding more than other forms of denaturation. One serum with anti-PR3 antibodies bound to Lys-C and Glu-C-digested PR3 in dot blots. Linear epitopes could not be further defined by their binding in an ELISA using overlapping peptides corresponding to the PR3 molecule because of non-specific binding. Three of the five sera with anti-MPO antibodies bound to MPO in Western blots after treatment with SDS, beta-ME and boiling for 5 min. One serum with anti-MPO antibodies bound to Lys-C and Glu-C-digested MPO in dot blots. Again, linear epitopes could not be further defined using an ELISA with overlapping peptides because of non-specific binding. Some anti-PR3 and anti-MPO antibodies are likely to recognize linear epitopes, but these cannot be defined by use of a PIN ELISA system.

Relevance of classic anti-neutrophil cytoplasmic autoantibody (C-ANCA)-mediated inhibition of proteinase 3-alpha 1-antitrypsin complexation to disease activity in Wegener's granulomatosis.

In the sera of patients with Wegener's granulomatosis (WG), C-ANCA can be detected that are directed against proteinase 3 (PR3). We have previously observed that C-ANCA interfere with PR3 proteolytic activity and with complexation of PR3 with its major physiologic inhibitor, alpha 1-antitrypsin (alpha 1AT). In the present study we investigated whether this inhibitory effect of C-ANCA on PR3-alpha 1AT complexation correlates with clinical activity of WG. Serial serum samples of eight consecutive patients with histologically proven relapses of WG were tested. At the moment of relapse all sera revealed inhibitory activity towards PR3-alpha 1AT complexation (median 22%, range 10-59%). Disease activity score (r = 0.87, P < 0.02) and C-reactive protein (CRP) levels (r = 0.66, P < 0.1) correlated with C-ANCA inhibition of PR3-alpha 1AT complexation, while they did not correlate with the C-ANCA titre detected by indirect immunofluorescence (IIF) nor with IgG anti-PR3 antibody level measured by ELISA. The inhibitory effect of C-ANCA on PR3-alpha 1AT complexation had risen significantly at the moment of relapse compared with values 3 months (P < 0.05) and 6 months (P < 0.01) before relapse. Eight patients with established WG and positive for C-ANCA but without clinical evidence of relapse served as controls. In this group no inhibitory effect of C-ANCA on PR3-alpha 1AT complexation was observed in 7/8 patients sera. Sera of one control patient contained moderate C-ANCA inhibitory activity towards PR3-alpha 1AT complexation, which remained at a constant level during the 6 months period of observation. Thus, disease activity in WG appears to be more closely related to C-ANCA inhibitory activity towards PR3-alpha 1AT complexation.

Antibodies to Proteinase 3 Prime Human Oral, Lung, and Kidney Epithelial Cells To Secrete Proinflammatory Cytokines upon Stimulation with Agonists to Various Toll-Like Receptors, NOD1, and NOD2

Antineutrophil cytoplasmic antibodies (ANCA) are autoantibodies, the detection of which in serum can be used in the diagnosis of Wegener's granulomatosis (WG). Proteinase 3 (PR3) is a major target antigen of ANCA in WG patients, and the interaction of PR3 ANCA with leukocytes causes a debilitating autoimmune disease. The first signs and symptoms in WG patients are observed in the oral cavity, lungs, and kidneys. Human epithelial cells generally do not secrete proinflammatory cytokines upon stimulation with pathogen-associated molecular patterns (PAMPs). In this study, anti-PR3 antibodies (Abs) and PR3 ANCA-containing sera from WG patients endowed human oral, lung, and kidney epithelial cells with responsiveness to PAMPs in terms of the production of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-8, monocyte chemoattractant protein-1, and tumor necrosis factor alpha. Protease-activated receptor-2 (PAR-2) agonist peptides mimicked the priming effects of PR3 ANCA against PAMPs. Furthermore, the anti-PR3 Ab-mediated cell activation was significantly abolished by RNA interference targeting PAR-2 and NF-kappaB. This is the first report of priming effects of anti-PR3 Abs (PR3 ANCA) on epithelial cells. The results suggest that anti-PR3 Abs (PR3 ANCA) prime human epithelial cells to produce cytokines upon stimulation with various PAMPs, and these mechanisms may be involved in severe chronic inflammation in WG.

Neutrophil surface presentation of the anti-neutrophil cytoplasmic antibody-antigen proteinase 3 depends on N-terminal processing

The neutrophil serine protease proteinase 3 (PR3) is a main autoantigen in anti-neutrophil cytoplasmic antibody-associated vasculitis. PR3 surface presentation on neutrophilic granulocytes, the main effector cells, is pathogenically important. PR3 is presented by the NB1 (CD177) glycoprotein, but how the presentation develops during neutrophil differentiation is not known. An N-terminally unprocessed PR3 (proPR3) is produced early during neutrophil development and promotes myeloid cell differentiation. We therefore investigated if PR3 presentation depended on NB1 during neutrophil differentiation and if PR3 and proPR3 could both be presented by NB1. In contrast to mature neutrophils, differentiating neutrophils showed an early NB1-independent PR3 surface display that was recognized by only two of four monoclonal anti-PR3 antibodies and occurred in parallel with proPR3, but not PR3 secretion, suggesting that the NB1-independent surface PR3 was proPR3. PR3 gene expression preceeded NB1. When the NB1 receptor was detected on the surface, a mode of PR3 surface display similar to mature neutrophils developed together with the degranulation system. Ectopic expression studies showed that NB1 was a sufficient receptor for PR3 but not proPR3. ProPR3 display on the plasma membrane may influence the bone marrow microenvironment. NB1-mediated PR3 presentation depended on PR3 N-terminal processing implicating the PR3-N-terminus as NB1-binding site.