Excessive neutrophil extracellular trap formation in ANCA-associated vasculitis is independent of ANCA

Abstract

Neutrophil extracellular traps (NETs) are auto-antigenic strands of extracellular DNA covered with myeloperoxidase (MPO) and proteinase3 (PR3) that can be a source for the formation of anti-neutrophil cytoplasmic autoantibodies (ANCAs). The presence of NETs was recently demonstrated in renal tissue of patients with ANCA-associated vasculitis (AAV). NET formation was enhanced in AAV, suggesting that MPO-ANCA could trigger NET formation, supporting a vicious circle placing NETs in the center of AAV pathogenesis. Here we investigated NET formation in 99 patients with AAV by a novel highly sensitive and automated assay. There was a significant excess of ex vivo NET formation in both MPO-ANCA- and PR3-ANCA-positive patients with AAV compared to healthy individuals. Excessive NET formation did not correlate with serum ANCA levels. Likewise, immunoglobulin G depletion had no effect on excessive NET formation in patients with AAV, indicating an ANCA-independent process. Next, we explored the relation of excessive NET formation to clinical disease in ten patients with AAV and showed that excessive NET formation was predominantly found during active disease, more so than during remission. Excessive NET formation was found in patients with AAV hospitalized for disease relapse but not during severe infection. Thus, excessive NET formation in AAV is independent of ANCA, and an excess of ex vivo NET formation was related to active clinical disease in patients with AAV and a marker of autoimmunity rather than infection.

Intrinsically Distinct Role of Neutrophil Extracellular Trap Formation in Antineutrophil Cytoplasmic Antibody–Associated Vasculitis Compared to Systemic Lupus Erythematosus

“…Recently, our group demonstrated that AAV‐induced NET formation occurred in a manner independent of IgG ANCAs , whereas SLE‐specific autoantibodies were reported to induce NET formation . We therefore further corroborated the involvement of autoantibodies in a direct comparison of AAV‐induced and SLE‐induced NET formation.…”

“…Even though we demonstrated that AAV‐induced NET formation was independent of IgG ANCAs, the exact factors in AAV serum that trigger NET formation remain unknown. In a previous study, our group showed that neither IgA depletion nor C5 blockade affected AAV‐induced NET formation, and there was no correlation with the serum levels of tumor necrosis factor (TNF), interleukin‐8, or C‐reactive protein . Interestingly, the necroptosis pathway was recently demonstrated to be involved in ANCA‐mediated NET formation .…”

“…We have previously demonstrated that serum from both AAV patients and SLE patients can induce excessive NET formation in healthy neutrophils . We therefore performed a side‐by‐side quantitative comparison of 2 large cohorts of patients with ANCA‐positive AAV (n = 80) and patients with ANA‐positive SLE (n = 59).…”

“…In clinical studies, we and others have demonstrated that excessive NET formation or impaired NET degradation is present both in patients with active AAV and in patients with severe SLE , and this is correlated with the severity of disease activity. Thus, preclinical and clinical studies have demonstrated an important role for NETs in the pathogenesis of both AAV and SLE.…”

In vivo evidence for extracellular DNA trap formation

“…Similarly, druginduced formation of NETs enriched in NE serving as auto-antigen, together with the release of B-cell activating factor (BAFF) resulting in B-cell activation, and the production of ANCA directed against NE that further enhance NET formation, have been assumed to be pathogenic mechanisms in cocaine and levamisoleassociated autoimmunity 84 . A role for NETs in ANCAassociated vasculitis is also suggested by the observation that excessive NET formation correlated with disease exacerbation 85 .…”