Protective α1-antitrypsin effects in autoimmune vasculitis are compromised by methionine oxidation

BACKGROUND. Antineutrophil cytoplasmic autoantibody-associated (ANCA-associated) vasculitidies (AAV) are life -threatening systemic autoimmune conditions. ANCAs directed against proteinase 3 (PR3) or myeloperoxidase (MPO) bind their cell surface-presented antigen, activate neutrophils, and cause vasculitis. An imbalance between PR3 and its major inhibitor alpha 1-antitrypsin (AAT) was proposed to underlie PR3-but not MPO-AAV. We measured AAT and PR3 in healthy individuals and patients with AAV and studied protective AAT effects pertaining to PR3-and MPO-ANCA. METHODS. Plasma and blood neutrophils were assessed for PR3 and AAT. WT, mutant, and oxidation-resistant AAT species were produced to characterize AAT-PR3 interactions by flow cytometry, immunoblotting, fluorescence resonance energy transfer assays, and surface plasmon resonance measurements. Neutrophil activation was measured using the ferricytochrome C assay and AAT methionine-oxidation by Parallel Reaction Monitoring. RESULTS. We found significantly increased PR3 and AAT pools in patients with both PR3-and MPO-AAV; however, only in PR3-AAV did the PR3 pool correlate with the ANCA titer, inflammatory response, and disease severity. Mechanistically, AAT prevented PR3 from binding to CD177, thereby reducing neutrophil surface antigen for ligation by PR3-ANCA. Active patients with PR3-AAV showed critical methionine-oxidation in plasma AAT that was recapitulated by ANCA-activated neutrophils. The protective PR3-related AAT effects were compromised by methionine-oxidation in the AAT reactive center loop but preserved when 2 critical methionines were substituted with valine and leucine. CONCLUSION. Pathogenic differences between PR3-and MPO-AAV are related to AAT regulation of membrane-PR3, attenuating neutrophil activation by PR3-ANCA rather than MPO-ANCA. Oxidation-resistant AAT could serve as adjunctive therapy in PR3-AAV.

Automated summary

The levels of PR3 and AAT were significantly increased in patients with both PR3-AAV and MPO-AAV, but only in PR3-AAV did the level of PR3 correlate with the ANCA titer, inflammatory response, and disease severity. Mechanistically, AAT prevented PR3 from binding to CD177, reducing the binding of PR3-ANCA to neutrophil surface antigens. Active patients with PR3-AAV showed methionine oxidation in plasma AAT, which was replicated by ANCA-activated neutrophils. The protective effects of PR3-related AAT were compromised by oxidation in the AAT reactive center loop, but preserved with valine and leucine substitution for critical methionines.