Enhanced formation and disordered regulation of NETs in myeloperoxidase-ANCA-associated microscopic polyangiitis.

Microscopic polyangiitis (MPA) is an ANCA-associated vasculitis that affects small vessels, especially renal glomeruli. We recently demonstrated that the abnormal formation and impaired degradation of neutrophil extracellular traps (NETs) may be crucially involved in the generation of myeloperoxidase (MPO)-ANCA and subsequent development of MPA. This study assessed the formation and regulation of NETs in patients with MPO-ANCA-associated MPA. Peripheral blood samples were obtained from 38 patients with MPO-ANCA-associated MPA, 23 patients with systemic lupus erythematosus (SLE), and 8 healthy controls. IgG eluted from MPO-ANCA-associated MPA sera demonstrated the highest ability to induce NETs, and this ability correlated with disease activity and paralleled ANCA affinity for MPO. Moreover, addition of recombinant human MPO to these IgG samples reduced NET induction. Additionally, MPO-ANCA-associated MPA sera exhibited lower rates of NET degradation that recovered partially upon depletion of IgG. The activity of DNase I, an important regulator of NETs, was also lower in MPO-ANCA-associated MPA and SLE sera. IgG depletion from MPO-ANCA-associated MPA sera partially restored the rate of NET degradation, and addition of DNase I synergistically enhanced this restoration. Addition of anti-MPO antibodies did not inhibit DNase I activity, and some MPO-ANCA-associated MPA sera contained anti-NET antibodies at levels not correlated with MPO-ANCA titers, suggesting the involvement of unidentified autoantibodies as well. The collective evidence suggests a vicious cycle involving MPO-ANCA and the regulation of NETs could be critically involved in the pathogenesis of MPO-ANCA-associated MPA.

Anti-carbonic anhydrase III autoantibodies in vasculitis syndrome.

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

The immunodominant myeloperoxidase T-cell epitope induces local cell-mediated injury in antimyeloperoxidase glomerulonephritis.

Microscopic polyangiitis is an autoimmune small-vessel vasculitis that often manifests as focal and necrotizing glomerulonephritis and renal failure. Antineutrophil cytoplasmic Abs (ANCAs) specific for myeloperoxidase (MPO) play a role in this disease, but the role of autoreactive MPO-specific CD4(+) T cells is uncertain. By screening overlapping peptides of 20 amino acids spanning the MPO molecule, we identified an immunodominant MPO CD4(+) T-cell epitope (MPO(409-428)). Immunizing C57BL/6 mice with MPO(409-428) induced focal necrotizing glomerulonephritis similar to that seen after whole MPO immunization, when MPO was deposited in glomeruli. Transfer of an MPO(409-428)-specific CD4(+) T-cell clone to Rag1(-/-) mice induced focal necrotizing glomerulonephritis when glomerular MPO deposition was induced either by passive transfer of MPO-ANCA and LPS or by planting MPO(409-428) conjugated to a murine antiglomerular basement membrane mAb. MPO(409-428) also induced biologically active anti-MPO Abs in mice. The MPO(409-428) epitope has a minimum immunogenic core region of 11 amino acids, MPO(415-426), with several critical residues. ANCA-activated neutrophils not only induce injury but lodged the autoantigen MPO in glomeruli, allowing autoreactive anti-MPO CD4(+) cells to induce delayed type hypersensitivity-like necrotizing glomerular lesions. These studies identify an immunodominant MPO T-cell epitope and redefine how effector responses can induce injury in MPO-ANCA-associated microscopic polyangiitis.

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

Proteinase 3 (PR3) is the target of anti-neutrophil cytoplasm Abs in granulomatosis with polyangiitis, a form of systemic vasculitis. Upon neutrophil apoptosis, PR3 is coexternalized with phosphatidylserine and impaired macrophage phagocytosis. Calreticulin (CRT), a protein involved in apoptotic cell recognition, was found to be a new PR3 partner coexpressed with PR3 on the neutrophil plasma membrane during apoptosis, but not after degranulation. The association between PR3 and CRT was demonstrated in neutrophils by confocal microscopy and coimmunoprecipitation. Evidence for a direct interaction between PR3 and the globular domain of CRT, but not with its P domain, was provided by surface plasmon resonance spectroscopy. Phagocytosis of apoptotic neutrophils from healthy donors was decreased after blocking lipoprotein receptor-related protein (LRP), a CRT receptor on macrophages. In contrast, neutrophils from patients with granulomatosis with polyangiitis expressing high membrane PR3 levels showed a lower rate of phagocytosis than those from healthy controls not affected by anti-LRP, suggesting that the LRP-CRT pathway was disturbed by PR3-CRT association. Moreover, phagocytosis of apoptotic PR3-expressing cells potentiated proinflammatory cytokine in vitro by human monocyte-derived macrophages and in vivo by resident murine peritoneal macrophages, and diverted the anti-inflammatory response triggered by the phagocytosis of apoptotic cells after LPS challenge in thioglycolate-elicited murine macrophages. Therefore, membrane PR3 expressed on apoptotic neutrophils might amplify inflammation and promote autoimmunity by affecting the anti-inflammatory "reprogramming" of macrophages.

The oxidation induced by antimyeloperoxidase antibodies triggers fibrosis in microscopic polyangiitis.

Lung fibrosis is considered a severe manifestation of microscopic polyangiitis (MPA). Antimyeloperoxidase (anti-MPO) antibodies in MPA patients' sera can activate MPO and lead to the production of reactive oxygen species (ROS). While high levels of ROS are cytotoxic, low levels can induce fibroblast proliferation. Therefore, we hypothesised that the oxidative stress induced by anti-MPO antibodies could contribute to lung fibrosis. 24 MPA patients (45 sera) were enrolled in the study, including nine patients (22 sera) with lung fibrosis. Serum advanced oxidation protein products (AOPP), MPO-induced hypochlorous acid (HOCl) and serum-induced fibroblast proliferation were assayed. AOPP levels, MPO-induced HOCl production and serum-induced fibroblast proliferation were higher in patients than in healthy controls (p<0.0001, p=0.0001 and p=0.0005, respectively). Increased HOCl production was associated with active disease (p=0.002). Serum AOPP levels and serum-induced fibroblast proliferation were higher in patients with active MPA and lung fibrosis (p<0.0001). A significant linear relationship between fibroblast proliferation, AOPP levels and HOCl production was observed only in patients with lung fibrosis. Oxidative stress, in particular the production of HOCl through the interaction of MPO with anti-MPO antibodies, could trigger the fibrotic process observed in MPA.

[Inhibition of oxidative activity of myeloperoxidase by anti-myeloperoxidase antibodies from patients with microscopic polyangiitis].

OBJECTIVE: To investigate the inhibitory effects on myeloperoxidase (MPO) oxidation activity by affinity-purified anti-MPO antibodies from patients with microscopic polyangiitis (MPA) and to further investigate the interaction between MPO, ceruloplasmin and anti-MPO antibodies. METHODS: Human IgG fractions were purified from plasma of 11 patients with anti-MPO antibody positive MPA and sera of 12 normal controls. Anti-MPO antibodies were further purified from anti-MPO antibody containing IgG fractions using MPO affinity chromatography. The enzyme activity of MPO was measured, in the presence of anti-MPO antibodies and normal IgG preparations, using a classical MPO oxidation assay. Interaction between ceruloplasmin, MPO and anti-MPO antibodies was further investigated using ELISA. RESULTS: Anti-MPO antibodies from 7/11 patients with MPA could inhibit the MPO activity as non-competitive inhibitors in a dose-dependent manner. Ceruloplasmin could competitively inhibit the oxidation activity of MPO in a dose-dependent and time-dependent manner. Anti-MPO antibodies could inhibit the binding between MPO and ceruloplasmin to a maximum of (75.4+/-11.6)%. CONCLUSION: Anti-MPO antibodies, from the majority of patients with MPA, could inhibit the oxidation activity of MPO and interfere with the binding between MPO and ceruloplasmin.