Evaluation of a model for post-partum arthritis and the role of oestrogen in prevention of MRL-lpr associated rheumatic conditions.

Binding of both proteinase 3 (PR3) and myeloperoxidase (MPO) to endothelial cells (EC) has been suggested to be involved in the vascular damage seen in patients with Wegener's granulomatosis or microscopic polyangiitis. In the present study we investigated in detail the interaction of MPO and PR3 with cultured human umbilical vein endothelial cells (HUVEC) and its matrix products. In addition, we investigated whether interaction of PR3 or MPO with HUVEC monolayers also resulted in antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by anti-neutrophil cytoplasmic antibody (ANCA)-positive patient sera or rabbit IgG anti-PR3 or anti-MPO. Preincubation of HUVEC monolayers with PR3 or MPO resulted in a dose-dependent binding of both PR3 and MPO. However, HUVEC, preincubated with PR3 or MPO, followed by ANCA or by rabbit anti-PR3 or anti-MPO, were not susceptible to ADCC. Detailed analysis of the binding of PR3 to HUVEC monolayers showed that PR3 binds primarily to the extracellular matrix of endothelial cells, and to a very limited extent to the cells themselves. For MPO it was shown that it binds both to the extracellular matrix and to the endothelial cells themselves. However, after binding to HUVEC cultures, MPO was not detectable by polyclonal rabbit or human antibodies specific for MPO, probably because MPO is bound to sites not accessible for immunoglobulins. Binding of PR3 to HUVEC cultures (cells + matrix) was inhibited by fetal calf serum and by alpha 1-antitrypsin, but inactivation of enzymatic activity of PR3 by PMSF did not influence binding of PR3 to HUVEC cultures. Binding of MPO to HUVEC cultures was not influenced by fetal calf serum.     

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.     

Characterization of monoclonal antibodies to proteinase 3 (PR3) as candidate tools for epitope mapping of human anti-PR3 autoantibodies

Anti-neutrophil cytoplasmic antibodies directed against PR3 (PR3-ANCA) in patients with Wegener's granulomatosis are supposedly involved in the pathophysiology of this disease as different functional characteristics of the autoantibodies correlate with disease activity. However, little is known about the epitopes of PR3 that are recognized by PR3-ANCA and how epitope specificity may relate to functional characteristics of PR3-ANCA. As candidate tools for epitope mapping we studied 13 anti-PR3 MoAbs, including nine widely used and four newly raised MoAbs, for their mutual binding characteristics to PR3 using biosensor technology. Antigen specificity was confirmed by indirect immunofluorescence, immunoblotting, FACS analysis and antigen-specific ELISA. Competition between anti-PR3 MoAbs in binding to PR3 was investigated in a capture system set up in a BIAcore. In this system grouping of 12 of the 13 anti-PR3 MoAbs based on their mutual recognition patterns was achieved. Four MoAbs, from different research groups, namely 12.8, PR3G-2, 6A6 and Hz1F12, recognized comparable epitopes (group 1). Group 2 MoAbs including PR3G-4 and PR3G-6 bound to overlapping regions on PR3. The MoAbs PR3G-3, 4A5 and WGM2 recognized similar epitopes as they inhibited binding of each other (group 3). The fourth group of related MoAbs consisted of MC-PR3-2, 4A3 and WGM3. Because of its binding characteristics MoAb WGM1 could not be grouped. These results demonstrate that eight well-established anti-PR3 MoAbs produced by different research groups and four newly produced anti-PR3 MoAbs recognize four separate epitope areas on PR3, including one area detected with newly raised MoAbs only.     

Les nouveaux ANCA : leur mode d'emploi

Antineutrophil cytoplasmic autoantibodies (ANCA) are a useful diagnostic tool for ANCA-associated systemic small vessel vasculitis. For these patients, combining indirect immunofluorescence (IIF) with PR3 and MPO specific Elisa leads to the best diagnostic performance. However, ANCA targeting other autoantigens with positive IIF, anti-PR3 and/or anti-MPO positivity without IIF may be found and special measures must be taken to help and avoid clinical misinterpretation.     

Characterisation of anti-neutrophil cytoplasmic antibody target antigens using electrophoresis and western blotting techniques.

Anti-neutrophil cytoplasmic antibodies (ANCA) are a family of autoantibodies which react with components of phagocytic cells, and are associated with vasculitis and other idiopathic inflammatory disorders. However, the antigenic targets of many of these autoantibodies have not been defined yet. In this study, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) were evaluated for characterising the antigenic specificity of unidentified ANCA. The uncharacterised sera included those from patients with ulcerative colitis (n = 21), Crohn's disease (n = 5), cystic fibrosis (n = 16) and sarcoidosis (n = 2). In addition, sera from patients with antibodies to the phagocytic enzymes proteinase 3 (PR3) (n = 11) and myeloperoxidase (MPO) (n = 5) were also included. The sub-cellular localisation of antigens was determined by testing sera against crude neutrophil extract and sub-cellular fractions consisting of azurophilic granules, specific granules and cytosolic, fractions using enzyme-linked immunosorbent assays (ELISAs). All sera reacted with the crude and azurophilic granule extracts. The native system of IEF followed by capillary immunoblotting successfully detected anti-PR3 and anti-MPO in azurophilic granule extracts. In contrast, SDS-PAGE Western blotting failed to detect any reactivity, either to PR3 or MPO, in the crude extract or azurophilic granule extract. However, the antibody specificity of patient sera with uncharacterised autoantibodies could not be detected by IEF/capillary immunoblotting or SDS-PAGE. This study showed that the sub-cellular azurophilic granules are the antigenic target of a variety of uncharacterised ANCA. It also showed that IEF characterised both anti-PR3 and anti-MPO but failed to detect other forms of ANCA. In contrast, the majority of common ANCA were not detected by SDS-PAGE.     

Priming by tumor necrosis factor-alpha of human neutrophil NADPH-oxidase activity induced by anti-proteinase-3 or anti-myeloperoxidase antibodies

Anti-proteinase-3 (anti-PR3) or anti-myeloperoxidase (anti-MPO) antibodies are capable of activating human neutrophils primed by TNF-alpha in vitro. We described previously the involvement of FcgammaRIIa and beta(2) integrins in this neutrophil activation. In the literature, the requirement of TNF priming has been attributed to an effect of TNF-alpha on the expression of PR3 or MPO on the cell surface. Under our experimental conditions, TNF-alpha (2 ng/ml) increased the binding of the antibody against PR3, whereas binding of the antibody against MPO could hardly be detected, not even after TNF-alpha treatment. The aim of this study was to consider (an)other(s) role(s) for TNF-alpha in facilitating the NADPH-oxidase activation by these antibodies. We demonstrate the early mobilization of the secretory vesicles as a result of TNF-induced increase in intracellular-free calcium ions, the parallel colocalization of gp91(phox), the main component of the NADPH oxidase with beta(2) integrins and FcgammaRIIa on the neutrophil surface, and the FcgammaRIIa clustering upon TNF priming. TNF-alpha also induced redistribution of FcgammaRIIa to the cytoskeleton in a dose- and time-dependent manner. Moreover, blocking CD18 MHM23 antibody, cytochalasin B, and D609 (an inhibitor of phosphatidylcholine phospholipase C) inhibited this redistribution and the respiratory burst in TNF-treated neutrophils exposed to anti-PR3 or anti-MPO antibodies. Our results indicate direct effects of TNF-alpha in facilitating neutrophil activation by these antibodies and further support the importance of cytoskeletal rearrangements in this priming process.     

Anti-neutrophil cytoplasmic antibody (ANCA) in malaria is directed against cathepsin G

Autoantibodies of diverse specificities are detected in sera of patients with acute malaria. The clinical relevance of these autoantibodies is not clear, though there are reports associating some autoantibodies with specific disease manifestations. We have investigated the occurrence of ANCA in the sera of 93 patients during episodes of acute malaria. Sera were tested by indirect immunofluorescence (IIF) and by ELISA for antibodies to neutrophil cytoplasmic components proteinase 3 (PR3), myeloperoxidase (MPO), cathepsin G (CG), human leucocyte elastase (HLE), and lactoferrin (LF). Forty-seven sera samples (50.5%) were positive by IIF, all except one with the atypical ANCA pattern (a-ANCA). When screened by ELISA, anti-CG antibodies were detected in 52 samples (56%), while anti-PR3 and anti-MPO antibodies were detected in three and one samples, respectively. Antibody binding to HLE and LF was not significant. Anti-CG antibodies were detected in 93% of the IIF-positive sera. A combination of anti-CG and anti-PR3 antibodies was noted in three samples. Our study demonstrates the presence of ANCA in sera from patients with acute malaria, almost all with the a-ANCA pattern on IIF. The antibody specificity, noted for the first time in our study, appears to be predominantly directed against CG. The significance of CG and CG-ANCA in the pathogenesis and clinical manifestations of malaria has yet to be elucidated.     

Audit of the clinical usefulness of a rapid qualitative ELISA screen for antimyeloperoxidase and antiproteinase 3 antibodies in the assessment of patients with suspected vasculitis

BACKGROUND: The need for urgent antineutrophil cytoplasmic antibody (ANCA) results when assessing patients with acute renal failure, pulmonary renal syndrome, or mononeuritis multiplex has led to the development of a rapid qualitative ELISA screening assay for antibodies to myeloperoxidase (MPO) and proteinase 3 (PR3). AIMS: To report the use of a rapid qualitative ELISA screen for PR3-ANCA and MPO-ANCA in a regional immunology laboratory and its correlation with standard indirect immunofluorescence (IIF) and quantitative ELISA for PR3-ANCA and MPO-ANCA. METHODS: Over 12 months, 103 samples requiring urgent ANCA testing were screened by a rapid qualitative ELISA and the results compared with IIF and quantitative ELISA assays for PR3-ANCA and MPO-ANCA. RESULTS: There was an excellent correlation between the rapid qualitative ELISA and standard ANCA IIF and a routine ELISA for MPO/PR3-ANCA, with sensitivities ranging from 82% to 100%. There were two false negatives, which gave weak to moderately positive values as determined by routine ELISA. However, the clinical relevance of these two cases is doubtful. CONCLUSIONS: The rapid ELISA for anti-MPO and anti-PR3 correlates well with quantitative ELISA and IIF ANCA, and urgent management decisions in patients with suspected small vessel vasculitis can be based with confidence on this test.     

Anti-myeloperoxidase autoantibodies react with native but not denatured myeloperoxidase.

We wondered whether anti-myeloperoxidase (MPO) autoantibodies (MPO-ANCA) found in patients with systemic vasculitis react with a conformational epitope or epitopes on the MPO molecule. Sera from 15 human MPO-ANCA, and a polyclonal and a monoclonal anti-MPO antibodies were reacted with MPO in native and denatured states. Human MPO-ANCA and mouse monoclonal anti-MPO reacted with native MPO, and a 120-kD band representing the MPO hologenzyme, but not with denatured MPO fragments; however, MPO-ANCA and mouse anti-MPO did not demonstrate competitive inhibition of binding to MPO. Polyclonal rabbit anti-MPO reacted with both native and denatured MPO. All MPO-ANCA tested showed the same patterns of reactivity with native and denatured MPO in dot blot and Western blot analyses. Both polyclonal and monoclonal anti-MPO antibodies inhibited MPO's protein iodination by over 90%, whereas MPO-ANCA IgGs, normal IgGs and disease control IgGs did not. These data suggest that (i) MPO-ANCA interact with a conformational epitope on the MPO molecule; and (ii) MPO-ANCA from different patients have similar reactivity with native versus denatured MPO.     

B cell epitope specificity in ANCA‐associated vasculitis: does it matter?

Pauci‐immune idiopathic small‐vessel vasculitis is strongly associated with the presence of antineutrophil cytoplasm autoantibodies (ANCA). Antibodies to PR3 predominate in patients with Wegener's granulomatosis; antibodies to myeloperoxidase (MPO) are found more frequently in patients with microscopic polyangiitis. There is increasing in vivo and in vitro evidence for a pathogenic role of ANCA in systemic vasculitis based on associations of ANCA with disease activity. If ANCA are pathogenic, why is the course of disease different from one patient to another? Antibodies can recognize different binding sites (epitopes) on their corresponding antigens. Differences in binding specificity may influence the pathogenic potential of the antibodies. Differences between epitope specificity of ANCA between patients or changes in epitope specificity of ANCA in time in an individual patient may, accordingly, result in differences in disease expression. This review will focus on epitope specificity of autoantibodies in systemic autoimmune diseases and especially on the epitope specificity of PR3– and MPO–ANCA. We will discuss whether PR3–ANCA or MPO–ANCA recognize different epitopes on PR3 and MPO, respectively, and whether the epitopes recognized by ANCA change in parallel with the disease activity of ANCA‐associated vasculitis. Finally, we will speculate if the direct pathogenic role of ANCA can be ascribed to one relapse‐ or disease‐inducing epitope. Characterization of relapse‐ or disease‐inducing epitopes bound by PR3–ANCA and MPO–ANCA is significant for understanding initiation and reactivation of ANCA‐associated vasculitis. Elucidating a disease‐inducing epitope bound by ANCA may lead to the development of epitope‐specific therapeutic strategies.     

False-positive myeloperoxidase binding activity due to DNA/anti-DNA antibody complexes: a source for analytical error in serologic evaluation of anti-neutrophil cytoplasmic autoantibodies

Anti-myeloperoxidase antibodies (anti-MPO) are a major type of anti-neutrophil cytoplasmic antibody (ANCA). While evaluating anti-MPO monoclonal antibodies from SCG/Kj mice, we observed several hybridomas that appeared to react with both MPO and DNA. Sera from some patients with systemic lupus erythematosus (SLE) also react with MPO and DNA. We hypothesized that the MPO binding activity is a false-positive result due to the binding of DNA, contained within the antigen binding site of anti-DNA antibodies, to the cationic MPO. Antibodies from tissue culture supernatants from 'dual reactive' hybridomas were purified under high-salt conditions (3 M NaCl) to remove any antigen bound to antibody. The MPO and DNA binding activity were measured by ELISA. The MPO binding activity was completely abrogated while the DNA binding activity remained. The MPO binding activity was restored, in a dose-dependent manner, by the addition of increasing amount of calf-thymus DNA (CT-DNA) to the purified antibody. Sera from six patients with SLE that reacted with both MPO and DNA were treated with DNase and showed a decrease in MPO binding activity compared with untreated samples. MPO binding activity was observed when CT-DNA was added to sera from SLE patients that initially reacted with DNA but not with MPO. These results suggest that the DNA contained within the antigen binding site of anti-DNA antibodies could bind to the highly cationic MPO used as substrate antigen in immunoassays, resulting in a false-positive test.     

Antineutrophil cytoplasmic antibodies induce decreased CD62L expression and enhanced metabolic activity in monocytes

Monocyte in vitro activation by antimyeloperoxidase (anti-MPO)- and antiproteinase-3 (anti-PR3)-positive sera, corresponding immunoglobulin G (IgG) fractions and monoclonal antibodies against MPO and PR3 was evaluated. The expression of adhesion molecules, l-selectin (CD62L) and CR3 (CD11b), involved in leucocyte endothelial adhesion, and metabolic activity, measured as the production of hydrogen peroxide, were analysed. Decreased expression of CD62L was demonstrated in monocytes after incubation with antineutrophil cytoplasmic antibody (ANCA)-positive sera. This finding was not accompanied by changes in CD11b expression. Metabolic activity was increased in monocytes after incubation with ANCA-positive IgG fractions as well as after incubation with monoclonal anti-MPO and anti-PR3. These findings support the concept that the pathophysiological effect of ANCA is partly mediated through the action on crucial events in monocyte activation, such as CD62L downregulation and oxygen radical production.     

A novel capture-ELISA for detection of anti-neutrophil cytoplasmic antibodies (ANCA) based on c-myc peptide recognition in carboxy-terminally tagged recombinant neutrophil serine proteases

Testing for antineutrophil cytoplasmic antibodies (ANCA) reacting with proteinase 3 (PR3) is part of the routine diagnostic evaluation of patients with small vessel vasculitis. For PR3-ANCA detection, capture ELISAs are reported to be superior to direct ELISAs. Standard capture ELISAs, in which PR3 is anchored by anti-PR3 monoclonal antibodies (moAB), have two potential disadvantages. First, the capturing moAB may compete for epitopes recognized by some PR3-ANCA, causing occasional false-negative results. Second, the capture of recombinant PR3 mutant molecules becomes unpredictable as modifications of specific conformational epitopes may not only affect the binding of PR3-ANCA, but also the affinity of the capturing anti-PR3 moAB. Here, we describe a new capture ELISA, and its application for PR3-ANCA detection. This new assay is based on the standardized capture of a variety of different carboxy-terminally c-myc tagged recombinant ANCA target antigens using anti-c-myc coated ELISA plates. Antigen used include c-myc tagged human rPR3 variants (mature and pro-form conformations), mouse mature rPR3 and human recombinant neutrophil elastase. This new anti-c-myc-capture ELISA for PR3-ANCA detection has an intra- and inter-assay coefficient of variation of 3.6% to 7.7%, and 15.8% to 18.4%, respectively. The analytical sensitivity and specificity for PR3-ANCA positive serum samples were 93% and 100%, respectively when rPR3 with mature conformation was used as target antigen, and 83% and 100% when the pro-enzyme conformation was employed. In conclusion, this new anti-c-myc capture ELISA compares favorably to our standard capture ELISA for PR3-ANCA detection, enables the unified capture of different ANCA target antigens through binding to a c-myc tag, and allows capture of rPR3 mutants necessary for PR3-ANCA epitope mapping studies.     

Anti-native and recombinant myeloperoxidase monoclonals and human autoantibodies

Myeloperoxidase (MPO) is one of the main antigen targets of anti-neutrophil cytoplasmic antibodies (ANCA) in systemic vasculitides. It has been suggested that anti-MPO antibodies may recognize a single epitope on recombinant MPO. If confirmed on native MPO, this might allow specific therapeutic intervention with anti-idiotypic MoAbs to prevent antibody–antigen interaction which is thought to cause activation of neutrophils and vasculitis. We searched for restriction in the epitope recognition profile in 50 patients with anti-MPO autoantibodies, using both native and recombinant MPO. Mouse monoclonals were purified and tested in competition assays. At least four epitopes were identified on native MPO using these monoclonals and only two were conserved on recombinant MPO. We found that human MPO autoantibody response was not restricted to a single epitope on native MPO, as all sera tested did not show the same profile in competitive studies with monoclonals. Furthermure, 30% of human anti-native MPO sera failed to recognize rMPO.     

IgG subclass distribution and relative functional affinity of anti-myeloperoxidase antibodies in systemic vasculitis at presentation and during follow-up.

Circulating IgG autoantibodies to myeloperoxidase (MPO) are associated with renal vasculitis and have been implicated in its pathogenesis. However, raised levels of these autoantibodies may persist during clinical remission. We tested whether this paradox could be explained by immunoglobulin subclass switching during disease evolution, since different subclasses have different immunological and biochemical properties. Sera with anti-myeloperoxidase (anti-MPO) activity from 33 patients with active disease and 20 anti-MPO positive follow-up sera were studied by an ELISA using a panel of anti-human IgG subclass monoclonal reagents previously calibrated on human myeloma proteins. Anti-MPO subclass distribution in initial sera was: IgG1, 31 (94%); IgG2, 10 (30%); IgG3, 24 (73%); and IgG4, 22 (67%). IgG3 anti-MPO decreased during follow-up (P less than 0.02), with no change in IgG1 and IgG4. Relative functional affinity of anti-MPO antibodies in purified IgG subclasses was studied by the diethylamine method. IgG3 fractions consistently had a greater affinity for MPO than the other subclasses. Sequential studies in four patients demonstrated an affinity maturation for IgG1 and IgG4 anti-MPO as IgG3 anti-MPO disappeared. We conclude that dynamic changes of subclass distribution and affinity may explain discrepancies between anti-MPO antibody titre and disease expression.     

Monoclonal antibodies against different epitopes of nonstructural protein sigmaNS of avian reovirus S1133

Ten monoclonal antibodies (MAbs) were prepared against the nonstructural protein sigmaNS of avian reovirus S1133. Eight of them were selected for two-way competitive binding assay after coupling with horseradish peroxidase. The results allowed the definition of three epitopes, designated A, B, and C. Blocking assay of poly(A)-Sepharose binding activity of sigmaNS with MAbs indicated that MAb recognizing epitope B was able to block poly(A) oligomer binding, suggesting that epitope B is involved in ssRNA binding of sigmaNS. An immuno-dot binding assay was used to analyze the effect of denaturation on antibody recognition of the epitopes. All MAbs bound to protein sigmaNS in its native form. After denaturation by boiling in SDS and 2-mercaptoethanol, the binding of MAbs recognizing epitopes B and C was not affected. The reactivity of MAbs recognizing epitope A was fully abolished by denaturation. These results suggest that the binding of MAbs directed against epitope A is conformation-dependent; however, the recognition by MAbs of epitopes B and C is not conformation-dependent. In addition, the results from the cross-reactivity of MAbs to heterologous avian reovirus strains suggest that the three epitopes are highly conserved among these virus strains.     

Anti-lactoferrin antibodies and other types of anti-neutrophil cytoplasmic antibodies (ANCA) in reactive arthritis and ankylosing spondylitis.

Fifty-five serum samples from patients with reactive arthritis (ReA), 40 from patients with ankylosing spondylitis (AS) and three from patients with chronic sacroiliac joint arthritis were analysed for the presence of ANCA of IgG class by means of enzyme immunosorbent assay using lactoferrin (Lf), myeloperoxidase (MPO) and antigen extracted from azurophil granules ('alpha-antigen') containing proteinase 3 (PR3) as substrate. IgG-ANCA were found in 31 (56%) patients with ReA. Twenty-three (42%) had anti-Lf antibodies, nine (16%) had anti-MPO and eight (15%) had anti-alpha-antigen antibodies, none of which reacted with PR3. Only six (14%) AS or sacroiliac joint arthritis patients had ANCA (P < 0.001). Three (7%) had anti-Lf, two (5%) anti-MPO and two (5%) anti-alpha-antigen antibodies. Yersinia and Salmonella bacteria were separated by SDS-PAGE and blots were incubated with serum from rabbits immunized with human Lf. The hyperimmune serum recognized a band of 78 kD from both bacteria which was not seen when preimmune serum was used. The reaction to the 78-kD antigen could be completely inhibited when anti-Lf antibodies were absorbed on Lf coupled to cyanogen bromide-activated Sepharose, possibly indicating cross-reacting epitopes in Lf and enterobacterial antigen.     

Antibodies to selected minor target antigens in patients with anti-neutrophil cytoplasmic antibodies (ANCA)

In patients with anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, indirect immunofluorescence (IF) distinguishes between cytoplasmic (C-ANCA) and perinuclear (P-ANCA) neutrophil staining patterns. In patients with primary systemic vasculitis such as Wegener's granulomatosis, microscopic polyangiitis and Churg-Strauss syndrome, these IF staining patterns correspond broadly with antibodies to the two major antigens: the C-ANCA pattern is associated generally with antibodies to serine protease 3 (PR3) and the P-ANCA pattern with antibodies to myeloperoxidase (MPO). However, some sera positive for ANCA by IF are negative for anti-PR3 and anti-MPO antibodies, suggesting the presence of antibodies to minor antigens of PMN granules. We tested sera from a previously well-defined clinical cohort of patients for antibodies to four possible minor antigens: bactericidal permeability increasing protein, elastase, cathepsin G and lactoferrin. IF-positive (+) sera had significantly higher antibody frequencies to the minor antigens than did the IF-negative (-) sera (P < 0.01). Patients with IF(+) PR3(-)MPO(-) sera showed the most varied reactivity to the minor antigens. Among the IF(+) groups, the IF(+) PR3(+)/MPO(-) sera showed the lowest reactivity to the minor antigens. Patients with well-defined ANCA specificities, e.g. the PR3-ANCA response associated with Wegener's granulomatosis, are less likely than are other patient subsets to have antibodies to minor antigen targets. Autoantibodies to these minor antigens contribute to the overall pattern of ANCA identified by IF and help to explain why the correlation between IF and enzyme immunoassays show discrepancies. While the pathophysiological significance of antibodies to minor target antigens needs further evaluation, they may be markers of inflammation associated with disease processes.     

A comparison of the characteristics of circulating anti-myeloperoxidase autoantibodies in vasculitis with those in non-vasculitic conditions

Although circulating anti-neutrophil cytoplasmic antibodies (ANCA) specific for myeloperoxidase (MPO) are strongly associated with the presence of vasculitis, they have been described in sera from patients with other conditions. High levels of anti-MPO antibodies can also persist in sera from patients with vasculitis despite the achievement of clinical remission. One possible interpretation is that a potentially pathogenic subset of anti-MPO antibodies exists, which is only present in patients with active vasculitis. We therefore compared the characteristics of anti-MPO antibodies in sera from patients with active vasculitis (n = 18) with those present in remission (n = 9) and in a disease control group (n = 10) without clinical evidence of vasculitis. The class, subclass and ability of anti-MPO antibodies from the three groups of patients to recognize three different conformational epitopes were analysed using ELISA-based techniques. The expression of an idiotope, designated 9G4, was also examined. Epitope recognition by anti-MPO antibodies from all patients tested was found to be similar. Sera from patients with active vasculitis showed an over-representation of IgG4 subclass anti-MPO antibodies and a more frequent presence of IgM class anti-MPO antibodies. In disease controls, IgG1 anti-MPO antibodies were predominant. In vitro, neutrophil activation by ANCA has been shown to be dependent on engagement of neutrophil FcγRIIa receptors following binding of these autoantibodies to surface-expressed ANCA antigens. We found that active vasculitis may be associated with the presence of circulating anti-MPO antibodies which do not significantly bind this receptor, suggesting that mechanisms other than those dependent on FcγRIIa binding should be explored. In addition, the expression of the 9G4 idiotope on anti-MPO antibodies in 60% (12/18) of patients with active vasculitis and 20% (2/10) of disease control patients may indicate a common origin for anti-MPO antibodies in different individuals.