Abnormal conformation and impaired degradation of propylthiouracil-induced neutrophil extracellular traps: implications of disordered neutrophil extracellular traps in a rat model of myeloperoxidase antineutrophil cytoplasmic antibody-associated vasculiti

Neutrophil extracellular traps (NETs) are composed of DNA and antimicrobial proteins, including myeloperoxidase (MPO). Recent studies have demonstrated that impaired regulation of NETs could trigger an autoimmune response. Propylthiouracil (PTU), an antithyroid drug, is associated with a risk of MPO antineutrophil cytoplasmic antibody (ANCA) production and MPO ANCA-associated vasculitis (MPO AAV). This study was undertaken to clarify the mechanism of MPO ANCA production, using the PTU-induced model of MPO AAV. NETs were induced by treating human neutrophils with phorbol myristate acetate (PMA) in vitro. We examined whether the addition of PTU influenced the NET formation induced by PMA and the degradation of NETs by DNase I, which is regarded as a regulator of NETs. Furthermore, we examined whether NETs generated by the combination of PMA and PTU induced MPO ANCA and MPO AAV in vivo in rats. When NETs were induced by PMA with PTU using human neutrophils in vitro, abnormal conformation of NETs was observed. Interestingly, the abnormal NETs were hardly digested by DNase I. Moreover, rats immunized with the abnormal NETs, which had been induced by PMA with PTU using rat neutrophils, produced MPO ANCA and developed pulmonary capillaritis. When rats were given oral PTU with intraperitoneal injection of PMA, pauci-immune glomerulonephritis and pulmonary capillaritis occurred with MPO ANCA production in the serum. Our findings indicate that abnormal conformation and impaired degradation of NETs induced by PTU are involved in the pathogenesis of PTU-induced MPO ANCA production and MPO AAV. These findings suggest that disordered NETs can be critically implicated in the pathogenesis of MPO AAV.

Nakazawa D ，Tomaru U ，Suzuki A ，Masuda S ，Hasegawa R ，Kobayashi T ，Nishio S ，Kasahara M ，Ishizu A ... -  《-》

Possible implication of disordered neutrophil extracellular traps in the pathogenesis of MPO-ANCA-associated vasculitis.

Nakazawa D ，Tomaru U ，Ishizu A 《-》

Peptidylarginine Deiminase Inhibitor Suppresses Neutrophil Extracellular Trap Formation and MPO-ANCA Production.

Kusunoki Y ，Nakazawa D ，Shida H ，Hattanda F ，Miyoshi A ，Masuda S ，Nishio S ，Tomaru U ，Atsumi T ，Ishizu A ... -  《-》

Impact of anti-glomerular basement membrane antibodies and glomerular neutrophil activation on glomerulonephritis in experimental myeloperoxidase-antineutrophil cytoplasmic antibody vasculitis.

Antineutrophil cytoplasmic antibody (ANCA) and neutrophil interactions play important roles in ANCA-associated vasculitis (AAV) pathogenesis. However, mechanisms underlying the pathogenesis of crescent formation in ANCA-associated vasculitis have not been completely elucidated. To ascertain the involvement of these interactions in necrotizing crescentic glomerulonephritis (NCGN), we used an AAV rat model and investigated the effects of the anti-myeloperoxidase (MPO) antibody (Ab) titer, tumor necrosis factor α (TNF-α), granulocyte colony-stimulating factor (G-CSF) and subnephritogenic anti-glomerular basement membrane (GBM) Abs, as proinflammatory stimuli. NCGN was induced in Wistar Kyoto rats by human MPO (hMPO) immunization. Renal function, pathology, and glomerular cytokine and chemokine expression were evaluated in hMPO-immunized rats with/without several co-treatments (TNF-α, G-CSF or subnephritogenic anti-GBM Abs). Rat neutrophils activation by IgG purified from rat serum in each group was examined in vitro. The hMPO-immunized rats had significantly higher level of anti-hMPO Ab production. The induced anti-hMPO Abs cross-reacted with TNF-α- or G-CSF-primed rat neutrophils secreting TNF-α and interleukin-1β in vitro. The reactivity of anti-MPO Abs against rat MPO, crescent formation with neutrophil extracellular traps and glomerular-activated neutrophil infiltration in the rat model were significantly enhanced by subnephritogenic anti-GBM Ab but not by TNF-α or G-CSF administration. The model rats injected with the subnephritogenic anti-GBM Abs showed increased urinary albumin excretion and serum TNF-α, chemokine (C-X-C) ligand 1 (CXCL1) and CXCL2 levels. TNF-α, CXCL1, CXCL2 and CXCL8 increased in the glomeruli with significant amounts of crescent formation. In addition, in vitro activated neutrophils decreased CXC chemokine receptor 1 (CXCR1) and CXCR2 expressions. The coexistence of subnephritogenic anti-GBM Abs leads to the inflammatory environment in glomeruli that is amplified by the interaction of ANCA and neutrophils. Development of NCGN in MPO-AAV may be necessary for not only the accumulation of neutrophils in glomeruli, but also the aberrant neutrophil activation on glomerulonephritis.

Kanzaki G ，Nagasaka S ，Higo S ，Kajimoto Y ，Kanemitsu T ，Aoki M ，Nagahama K ，Natori Y ，Tsuboi N ，Yokoo T ，Shimizu A ... -  《-》

Myeloperoxidase anti-neutrophil cytoplasmic antibody affinity is associated with the formation of neutrophil extracellular traps in the kidney and vasculitis activity in myeloperoxidase anti-neutrophil cytoplasmic antibody-associated microscopic polyangii

Anti-neutrophil cytoplasmic antibody (ANCA) is associated with small-vessel vasculitis particularly in the kidneys and can induce the formation of neutrophil extracellular traps (NETs) from primed neutrophils. Recently we have reported that the induction of NETs correlates with ANCA affinity for myeloperoxidase (MPO) and disease activity in patients with MPO-ANCA-associated microscopic polyangiitis. To investigate whether MPO-ANCA affinity is associated with the formation of NETs in vivo, we examined the occurrence of NETs in the renal tissues of patients with MPO-ANCA-associated microscopic polyangiitis and ANCA affinity by double immunofluorescence staining for NET components of citrullinated histone, MPO and PAD4 and by ELISA competition with MPO, respectively. We divided 30 MPO-ANCA-associated microscopic polyangiitis patients into 2 groups based on their ANCA affinity levels (IC50 for the high: 0.11 ± 0.04 µg/mL (Group1) and IC50 for the low: 0.66 ± 0.24 µg/mL (Group2)). Group1 showed a higher Birmingham vasculitis activity score (15.6 ± 5.7) and 73% of the patients presented clinically with rapidly progressive glomerulonephritis and histologically with focal/crescentic glomerulonephritis (GN). Group 2 showed a lower Birmingham vasculitis activity score (9.2 ± 4.9) and 73% of the patients presented clinically with chronic renal failure and histologically with mixed/sclerotic GN. Group 1 showed a much higher occurrence of NETs than Group 2. Our findings indicate that ANCA affinity was associated with the in vivo formation of NETs, which might be involved in the pathophysiology of patients with MPO-ANCA-associated microscopic polyangiitis.

Yoshida M ，Yamada M ，Sudo Y ，Kojima T ，Tomiyasu T ，Yoshikawa N ，Oda T ，Yamada M ... -  《-》