Anti-myeloperoxidase antibodies: immunological characteristics and clinical associations.

Antibodies directed against myeloperoxidase (anti-MPO) were detected, using a solid-phase ELISA and purified sputum myeloperoxidase as the substrate, in 54 sera from 22 patients. Anti-MPO were present in 17 patients with crescentic glomerulonephritis (CGN), Wegener's granulomatosis (WG) and microscopic polyarteritis (MPA), and thus are associated with different forms of vasculitis. Anti-MPO were also present in five out of 20 patients with systemic lupus erythematosus (SLE). Anti-MPO activity in SLE sera was low, in contrast to the high titers observed in patients with vasculitis. All positive sera had IgG anti-MPO (except two SLE sera) and most of them also contained low-titered IgM anti-MPO. Only three patients had high IgM anti-MPO activities, the significance of which remains to be determined. In patients with CGN, WG or MPA, the anti-MPO titer decreased following therapy and paralleled the disease activity. Thus, anti-MPO constitute a useful diagnostic tool and a sensitive marker of disease activity in this group of patients with vasculitis.     

Factors involved in the pathogenesis of neutrophilic vasculitis in MRL/Mp-lpr/lpr mice: a model for human microscopic angiitis

Anti-neutrophil cytoplasm antibodies (ANCA) directed against myeloperoxidase (MPO) are detected in patients with microscopic angiitis. Human MPO autoantibodies stimulate neutrophil degranulation in vitro and are thought to be pathogenic. We have previously shown that MRL-lpr mice with MPO autoantibodies have a higher incidence of vasculitis than their seronegative littermates. The aim of the present study is to determine the relationship between MPO autoantibodies and microscopic angiitis. The neutrophil binding properties of anti-MPO monoclonal antibodies (mAbs) from MRL-lpr mice were tested using murine heterophils (neutrophils) present in blood and induced peritoneal exudates. MRL anti-MPO mAbs selectively bind activated neutrophils which express MPO in vitro. The pathogenicity of an IgG2b anti-MPO mAb, C6, was investigated in vivo. Anti-MPO mAb, C6 was administered to young MRL mice which had been primed with exogenous TNF alpha to induce neutrophil activation and expression of MPO. Neutrophilic vasculitis similar to microscopic angiitis occurred in 33% of MRL mice which had been treated with anti-MPO mAb. The lesions were mainly restricted to sites of previous endothelial insult which suggests an active role for injured endothelium in this pathology.     

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.     

Anti-myeloperoxidase antibodies in sera from patients with propylthiouracil-induced vasculitis might recognize restricted epitopes on myeloperoxidase molecule

Myeloperoxidase (MPO) is one of the major target antigens of antineutrophil cytoplasmic antibodies (ANCA) in primary systemic vasculitis. It is known that propylthiouracil (PTU) could induce MPO-ANCA-positive vasculitis. The production of anti-MPO antibodies in patients with PTU-induced vasculitis may be different from that in patients with primary microscopic polyangiitis (MPA). One possible reason for this may be differences in epitope recognition. The aim of this study is to compare the epitopes of antibodies to MPO in sera from patients with PTU-induced vasculitis (n = 10) and MPA (n = 10). The sera were collected and used to inhibit monoclonal antibodies against human MPO (3D8 and 6B9) and affinity purified, horseradish peroxidase conjugated human anti-MPO antibodies (Pab1-HRP, Pab2-HRP) in a competitive inhibition enzyme-linked immunosorbent assay (ELISA) system using soluble human MPO as solid phase ligand. The Pab1-HRP and Pab2-HRP were affinity purified from plasma exchanges of a patient with PTU-induced vasculitis and a patient with MPA, respectively. The inhibition rates were evaluated and compared between the PTU and primary MPA groups. In the PTU group all 10 sera could inhibit 3D8: the average inhibition rate was 44.7% +/- 5.0%; 9/10 sera could inhibit 6B9: the average inhibition rate was 35.6% +/- 6.0%. However, in the MPA group all 10 sera could inhibit 3D8 and 6B9; the average inhibition rates were 68.4% +/- 16.1% (P < 0.01) and 62.2% +/- 17.2% (P < 0.01), respectively. Sera in both the PTU and MPA groups could inhibit Pab1-HRP and the inhibition rates were 81.4% +/- 9.4%versus 86.6% +/- 17.2% (P > 0.05). However, the average inhibition rate for Pab2-HRP in the MPA group was significantly higher than that in the PTU group (76.3% +/- 7.8%versus 58.9% +/- 15.5%, P < 0.01). We conclude that anti-MPO antibodies from patients with PTU-induced vasculitis and from patients with primary MPA could recognize more than one epitope on the native MPO molecule. Although the epitopes overlapped between the two groups, the epitopes of anti-MPO antibodies from patients with PTU-induced vasculitis might be more restricted.     

Titre and affinity of propylthiouracil-induced anti-myeloperoxidase antibodies are closely associated with the development of clinical vasculitis

Substantial evidences suggested that propylthiouracil (PTU) could induced anti-myeloperoxidase (MPO) antibodies in sera from patients with hyperthyroidism, however, only a subgroup of the PTU-induced anti-MPO antibody positive patients developed clinical evident vasculitis. The aim of this study is to compare the titres and affinities of PTU induced anti-MPO antibodies in sera from patients with hyperthyroidism with and without clinical vasculitis. Anti-MPO antibody positive sera from patients diagnosed hyperthyroidism with (n = 13) and without (n = 14) clinical evident vasculitis were collected. The titre was determined by MPO-ELISA and expressed as logarithm value (lgT). The affinity constant (aK) of anti-MPO IgG was measured by antigen inhibition assay. The titre and aK values were compared between patients with and without vasculitis. In patients with vasculitis, the mean lgT of anti-MPO antibodies was 3.62 +/- 0.66; the median aK was 4.47 x 10(7)M(-1). In patients without vasculitis, the mean lgT was 2.54 +/- 0.29; the median aK was 0.14 x 10(7)M(-1), and both were significant lower than those in patients with vasculitis (t = 5.464; P = 0.000 & z = -4.373; P = 0.000, respectively). We concluded that the titre and affinity of anti-MPO antibodies might be associated with the development of clinical vasculitis in patients with PTU-induced ANCA.     

Role of myeloperoxidase in the host defense against fungal infection]

Neutrophils are believed to be the first line of defense against invading microorganisms, but in vivo roles of reactive oxygens produced by neutrophils are not well known. Myeloperoxidase (MPO) catalyzes reaction of hydrogen peroxide with chloride ion to produce hypochlorous acid that is used for microbial killing by phagocytic cells. To define the in vivo role of MPO, we generated mice having no peroxidase activity in their neutrophils or monocytes. MPO-deficient (MPO-KO) mice showed severely reduced cytotoxicity to Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, and other microorganisms, demonstrating that an MPO-dependent oxidative system is important for host defense against fungi. However, the significance of MPO compared to the NADPH-oxidase is still unclear because individuals with MPO deficiency are usually healthy in contrast to patients with chronic granulomatous disease (CGD) who present clinical symptoms early in life. To better understand the contributions of MPO and NADPH-oxidase to antifungal defense mechanisms, we compared the susceptibility of MPO-KO mice and CGD mice to infections by C. albicans. Interestingly, at the highest dose, the mortality of MPO-KO mice was comparable to CGD mice, but was the same as normal mice at the lowest dose. These results suggest that MPO and NADPH-oxidase are equally important for early host defense against a large inocula of Candida. Our present results suggest that MPO-deficient individuals could exhibit similar problems as CGD patients if exposed to a large number of microorganisms.     

Arthritogenicity of collagen type II is increased by chlorination

During inflammation, activated neutrophils, monocytes and macrophages produce and release myeloperoxidase (MPO). MPO converts hydrogen peroxide to hypochlorous acid, a highly reactive and oxidizing agent. Proteins subjected to hypochlorous acid become chlorinated. We analysed how chlorination of the cartilage antigen collagen type II (CII) affects its immunogenic and arthritogenic properties by studying immune responses to chlorinated CII in comparison to immune responses to CII and by studying the development of arthritis in rats immunized with CII-Cl. CII-Cl immunization of LEW.1AV1 rats caused a 100% incidence of arthritis with a mean maximum score of 9.2 (maximal score possible 16). The same dose of non-chlorinated CII did not induce arthritis at all. Rats immunized with CII-Cl developed high anti-CII-Cl IgG titres and also developed IgG antibodies recognizing the non-chlorinated form of CII. Analysis of cytokine mRNA expression in lymph nodes 10 days after immunzation revealed an increased expression of interferon (IFN)-gamma mRNA and interleukin (IL)-1beta mRNA in CII-Cl-immunized rats compared to CII-immunized rats. Thus, chlorination of CII increased its immunogenicity as well as its arthritogenicity. As neutrophils, monocytes and macrophages are abundant cells in arthritic joints of patients with rheumatoid arthritis, chlorination might be a mechanism by which immunoreactivity to CII is induced and by which chronic joint inflammation is supported.     

Analysis of immunoglobulin variable region genes of a human IgM anti-myeloperoxidase antibody derived from a patient with vasculitis.

Circulating antibodies to myeloperoxidase (MPO) are associated primarily with pauci-immune glomerulonephritis and systemic vasculitis. Anti-MPO antibodies belong to a group of autoantibodies, anti-neutrophil cytoplasmic antibodies, that may play a pathogenic role in vasculitis. We have generated a human monoclonal anti-MPO antibody (E3-MPO) using peripheral blood lymphocytes from a patient with microscopic polyarteritis. Variable region gene analysis of E3-MPO showed that the VH region had 90% homology with the germ line gene VH4-21. E3-MPO was also shown to carry the 9G4 idiotope, which so far has been associated only with human antibodies that utilize the VH4-21 gene. The 9G4 idiotope was also expressed on anti-MPO antibodies in sera from the donor patient and from 4/7 additional patients with active, untreated vasculitis. The nucleotide sequences of both the variable heavy and light chains of E3-MPO showed evidence of an antigen-driven response.     

An IgM class anti-neutrophil cytoplasm antibody inhibits neutrophil adhesion and apoptosis via a Syk dependent signaling cascade

Anti-neutrophil cytoplasm antibodies (ANCA) with specificity for myeloperoxidase (MPO) are implicated as pathogenic agents in pauci-immune systemic vasculitis. In agreement with previously published observations we show that human neutrophils incubated with an MPO-specific IgG class monoclonal antibody are pro-adhesive and undergo apoptosis more readily in vitro. If apoptotic neutrophils are incubated with this antibody they are readily phagocytosed by macrophages and we show that 'blocking' antibodies to FcgammaRIIa (CD32) on the macrophage inhibit this process. We also examined the effect of E3MPO, a monoclonal anti-MPO antibody derived from a patient with severe systemic vasculitis. E3MPO is closely related to the cold-agglutinins and bears an epitope recognized by the monoclonal antibody 9G4 which is expressed on antibodies derived from the V4-34 germ-line immunoglobulin gene. In previous studies, we have shown that anti-MPO antibodies present in sera from patients with vasculitis often bear this epitope. In contrast to the IgG-class antibody, incubation of neutrophils with E3MPO inhibited neutrophil adhesion and apoptosis. Apoptotic neutrophils however were phagocytosed more readily by macrophages in the presence of E3MPO. The effects of E3MPO on neutrophil adhesion and apoptosis were inhibited by piceatannol, an inhibitor of Syk-family kinases; activation of which is associated with cross-linking of the beta(2)-integrins. We show that surface-expressed MPO co-localizes with these beta(2)-integrins and suggest that cross-linking of beta(2)-integrin-bound MPO by polyvalent antibodies could result in signaling through these receptors. We have demonstrated that there are different consequences of Fcgamma-receptor-dependent and -independent signaling mediated by ANCA.     

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.     

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.     

Severe impairment in early host defense against Candida albicans in mice deficient in myeloperoxidase

Myeloperoxidase (MPO) catalyzes the reaction of hydrogen peroxide with chloride ion to produce hypochlorous acid (HOCl), which is used for microbial killing by phagocytic cells. Despite the important role of MPO in host defense, however, MPO deficiency is relatively common in humans, and most of these individuals are in good health. To define the in vivo role of MPO, we have generated by gene targeting mice having no MPO activity in their neutrophils and monocytes. The mice without MPO developed normally, were fertile, and showed normal clearance of intraperitoneal Staphylococcus aureus. However, they showed increased susceptibility to pneumonia and death following intratracheal infection with Candida albicans. Furthermore, the lack of MPO significantly enhanced the dissemination of intraperitoneally injected C. albicans into various organs during the first 7 days. Thus, MPO is important for early host defense against fungal infection, and the inability to generate HOCl cannot be compensated for by other oxygen-dependent systems in vivo in mice. The mutant mice serve as a model for studying pulmonary and systemic candidiasis.     

Anti-idiotypic activity against anti-myeloperoxidase antibodies in pooled human immunoglobulin.

We investigated the ability of six different pooled human immunoglobulin (PHIG) preparations to inhibit the binding of anti-myeloperoxidase (MPO) antibodies to MPO. All six PHIG preparations inhibited the binding of anti-MPO antibodies from six sera to MPO in a concentration-dependent manner in the concentration range 0.016-10 mg/ml. There was considerable variation in the ability of each PHIG preparation to inhibit the binding of anti-MPO antibody in a given serum. Further differences were seen in the ability of a given PHIG to inhibit anti-MPO binding in different sera. F(ab')2 fragments from two PHIG preparations also inhibited in a concentration-dependent manner anti-MPO binding to MPO in all six sera in the concentration range 0.002-2.65 mg/ml, with a maximum inhibition of 42%. Little inhibition was seen with F(ab')2 of normal human IgG from individual donors (1.8-12.2% at the maximum concentration of 2 mg/ml). F(ab')2 fragments from three anti-MPO containing sera and two affinity-purified anti-MPO antibodies were eluted by affinity chromatography from Sepharose-bound PHIG F(ab')2 and showed anti-MPO antibody activity. We have shown that PHIG and F(ab')2 fragments of PHIG inhibit anti-MPO binding to MPO, and further that F(ab')2 fragments of PHIG bind to F(ab')2 fragments of anti-MPO antibodies. These observations indicate binding between the variable regions of PHIG and the antigen binding site of anti-MPO antibodies, and are consistent with an anti-idiotypic reaction. The variability seen in the inhibitory effect of the different PHIG preparations in anti-MPO-positive sera implies differences in their anti-idiotype content, while the variability of the inhibitory effect of a particular PHIG preparation between different sera suggests heterogeneity in the idiotypic repertoire of anti-MPO antibodies. Such variations in the inhibitory effect of different PHIG preparations on antibody binding may be an important determinant of their therapeutic effect.     

Expression of myeloperoxidase (MPO) by neutrophils is necessary for their activation by anti-neutrophil cytoplasm autoantibodies (ANCA) against MPO

Anti-neutrophil cytoplasm autoantibodies (ANCA) directed against proteinase-3 and myeloperoxidase (MPO) activate tumor necrosis factor-alpha-primed neutrophils in vitro. We used neutrophils from one completely and one partially MPO-deficient donor to assess the requirement of MPO expression for neutrophil activation by anti-MPO antibodies. The MPO deficiencies were defined enzymatically, by immunocytochemistry and by immunoblotting. The mutations in the MPO genes of these donors were identified as a combination of a novel splice-site mutation at the 3' end of intron 11 (A-2-->C), a deletion of 14 nucleotides in exon 9 (A1555-C1568), and a novel C1907 --> T (636Thr-->Met) substitution in exon 11 in the completely MPO-deficient donor and as the same splice-site mutation and a novel C995 --> T (332Ala-->Val) substitution in exon 7 in the partially MPO-deficient donor. Monoclonal antibody 4.15 against MPO and MPO-ANCA-immunoglobulin G induced no superoxide anion production in these MPO-deficient neutrophils despite a normal production induced by other stimuli. Thus, the presence of MPO is a conditio sine qua non for neutrophil activation by anti-MPO antibodies. Moreover, we demonstrated that by means of these MPO-deficient cells, hydrogen peroxide may diffuse from neutrophils to surrounding cells, which may contribute to the damage induced by oxygen radicals in the pathology of systemic vasculitides.     

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.     

Myeloperoxidase autoantibodies distinguish vasculitis mediated by anti-neutrophil cytoplasm antibodies from immune complex disease in MRL/Mp- lpr/lprmice: a spontaneous model for human microscopic angiitis

Anti-neutrophil cytoplasm antibodies (ANCA) with specificity for myeloperoxidase (MPO) occur in the sera of patients with microscopic angiitis, an autoimmune disease characterized by necrotizing vasculitis and crescentic glomerulonephritis. These autoantibodies have been shown to stimulate neutrophil degranulation and are believed to participate in pathogenesis. A neutrophilic vasculitis has been reported in MRL- lpr mice which has histological appearances similar to microscopic angiitis. In the present study we show that 22 % of female MRL- lpr mice develop MPO autoantibodies. These animals develop a clinical syndrome of vasculitis and glomerulonephritis that is distinct from immune complex disease. Anti-MPO monoclonal antibodies derived from these mice are polyreactive and react with double-stranded DNA. They bind a conformational epitope on human MPO which is also expressed by activated human neutrophils. The results suggest that a subset of MRL- lpr mice develop ANCA-related vasculitis rather than systemic lupus erythematosus and may be used as a model for human microscopic angiitis.     

IgM anti-myeloperoxidase antibody-secreting lymphocytes are present in the peripheral repertoire of lupus mice but rarely differentiate into IgG-producing cells.

Two IgM, kappa anti-myeloperoxidase (MPO) monoclonal antibodies, 6D6 and 9B5, bound to MPO in a solid-phase enzyme-linked immunosorbent assay were derived from the splenocytes of (NZB x NZW) F1 and MRL/lpr-lpr mice, respectively. 6D6 gave a characteristic perinuclear immunofluorescence staining pattern on ethanol-fixed human neutrophils, bound to the native form of MPO by immunoblotting and had a high constant affinity for MPO as demonstrated by real-time specific interaction. 9B5 produced a cytoplasmic immunofluorescence staining pattern, reacted with the heavy chain of MPO and had a low constant affinity for MPO. The heavy-and light-chain variable region genes of monoclonal antibodies (mAb) 6D6 and 9B5 were sequenced and found to be highly homologous to germline genes and to contain negatively charged amino acids in the complementarity determining regions. IgM MPO-binding activity was observed in most BW and MRL/lpr-lpr mouse sera, which may correspond to polyclonal activation of B cells, whereas IgG anti-MPO antibodies could be rarely detected. Thus, this study indicates that (i) BW and MRL/lpr-lpr mice do not delete IgM anti-MPO secreting B cells, do not maintain these B cells in a state of anergy, but most individuals are not able to spontaneously induce the class-switching of this autoantibody population; (ii) IgM anti-MPO antibodies can recognize different epitopes on MPO and produce different immunofluorescence staining pattern on ethanol-fixed human neutrophils, as demonstrated by the immunochemical properties of the two lupus-mouse derived mAb.     

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.     

Hypogalactosylation of serum IgG in patients with ANCA-associated systemic vasculitis

The triad of small vessel vasculitides (SVV) comprise Wegener's granulomatosis (WG), microscopic polyangiitis (MPA) and Churg-Strauss syndrome (CS). All three are associated with presence of circulating IgG antineutrophil cytoplasm antibodies (ANCA) which target autoantigens contained, primarily, within neutrophil azurophilic granules. The widely accepted model of pathogenesis suggests that ANCA activate cytokine-primed neutrophils within the microvasculature, leading to by-stander damage to endothelial cells, and rapid escalation of inflammation with recruitment of mononuclear cells. Activation may be initiated, in vitro, by the coligation of the PR3 or MPO antigen, translocated to the cell surface, and FcgammaRIIa/FcgammaRIIIb receptors. This suggests that the IgG subclass profile of ANCA and, possibly, its glycosylation status could influence the inflammatory mechanisms activated. The glycosylation status of total IgG isolated from the sera of patients with WG (13), MPA (6) and CSS (1) was determined by analysis of the released oligosaccharides. A deficit in IgG galactosylation is demonstrated for all patient samples, compared to controls. The mean percentage values for the agalactosylated (G0) oligosaccharides were 57% (SD +/- 9.71), 47% (SD +/- 4.25) and 28% (SD +/- 4.09) for WG, MPO and control samples, respectively. The G0 levels for polyclonal IgG isolated from the sera of both WG and MPA patients were significantly increased compared to controls (P < 0.0001). The major glycoform present therefore is agalactosylated (G0) IgG. In previous studies the G0 glycoform of IgG has been shown to bind and activate mannan binding lectin, and hence to activate the complement cascade, and to facilitate mannose receptor binding and the uptake of IgG complexes by macrophages and dendritic cells. Both of these activities could impact on the processing and presentation of self-antigens in autoimmune disease.