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

Abstract

Background. Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitidies (AAV) are life-threatening systemic autoimmune conditions. ANCA directed against proteinase 3 (PR3) or myeloperoxidase (MPO) bind their cell surfacepresented antigen, activate neutrophils and cause vasculitis. An imbalance between PR3 and its major inhibitor 1-antitrypsin (AAT) was proposed to underlie PR3-but not MPO-AAV. We measured AAT and PR3 in healthies and AAV patients and studied protective AAT effects pertaining to PR3-and MPO-ANCA. Methods.Plasma and blood neutrophils were assessed for PR3 and AAT. Wild-type, mutant, and oxidation-resistant AAT species were produced to characterize AAT-PR3 interactions by flow cytometry, immunoblotting, FRET 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 both PR3-and MPO-AAV patients, however, only in PR3-AAV did the PR3 pool correlate with 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 PR3-AAV patients 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 two critical methionines were substituted by valine and leucine. Conclusion.Pathogenic differences between PR3-and MPO-AAV are related to AAT regulation of membrane-PR3, attenuating neutrophil activation by PR3-rather than MPO-ANCA. Oxidation-resistant AAT could serve as adjunctive therapy in PR3-AAV.

The neutrophil: A key resourceful agent in immune‐mediated vasculitis

“…145 Interestingly, recent studies showed that a competing peptide and alpha-1 antitrypsin could disrupt the interaction between PR3 and CD177 and that this could inhibit anti-PR3-induced activation, thus highlighting the importance of the PR3-CD177 complex. 99,146 Importantly, we have shown that PR3 can be expressed in the membrane of microvesicles and that these PR3-expressing vesicles could enhance the activity of NADPH oxidase in bystander neutrophils at the site of inflammation to further promote inflammation. 147…”

“…The association of anti-PR3 ANCA positivity with genes coding for HLA-DP, PR3 itself, and its inhibitor, SERPINA1 (also called alpha-1-antitrypsin) may relate to the levels of target antigen on the neutrophils and the ability of alpha-1 antitrypsin to regulate these levels. 99 In contrast, no direct association with MPO-related genes was found in anti-MPO-associated vasculitis. 100 Recently, the association between risk allele HLA-DPB1*04:01 and PR3-ANCA was further confirmed by functional studies showing that binding between HLA-DPB1*04:01 and PR3 225-239 could initiate an immune response.…”

“…Both GPA and MPA show specific HLA dependence‐implicating autoreactive T cells as being critical. The association of anti‐PR3 ANCA positivity with genes coding for HLA‐DP, PR3 itself, and its inhibitor, SERPINA1 (also called alpha‐1‐antitrypsin) may relate to the levels of target antigen on the neutrophils and the ability of alpha‐1 antitrypsin to regulate these levels 99 . In contrast, no direct association with MPO‐related genes was found in anti‐MPO‐associated vasculitis 100 .…”