Sequential carbonyl derivatives and hydrazone adduct formation on myeloperoxidase contribute to development of ANCA vasculitis
Gang Xi, Elizabeth A. Mclnnis, Olivier Lardinois, Peiqi Hu, John S. Poulton, Meghan E. Free, Dhruti P. Chen, Evan M. Zeitler, Eveline Y. Wu, Nicole M. Orzechowski, Vimal K. Derebail, J. Charles Jennette, Ronald J. Falk
Gang Xi, Elizabeth A. Mclnnis, Olivier Lardinois, Peiqi Hu, John S. Poulton, Meghan E. Free, Dhruti P. Chen, Evan M. Zeitler, Eveline Y. Wu, Nicole M. Orzechowski, Vimal K. Derebail, J. Charles Jennette, Ronald J. Falk
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Research Article

Sequential carbonyl derivatives and hydrazone adduct formation on myeloperoxidase contribute to development of ANCA vasculitis

Abstract

Drug-induced autoimmune diseases are increasingly recognized, although mechanistic insight into disease causation is lacking. Hydralazine exposure has been linked to autoimmune diseases, including antineutrophil cytoplasmic autoantibody (ANCA) vasculitis. Our hypothesis posits that hydralazine covalently binds to myeloperoxidase (MPO), triggering the autoimmune response in ANCA vasculitis. In vitro, we observed formation of carbonyl derivatives on amine groups in the presence of acrolein. This facilitated the subsequent binding of hydralazine to heme-containing proteins, including MPO, via a Michael addition. Our studies demonstrated that carbonyl derivatives and hydrazone adducts induced conformational changes in the MPO heavy chain, potentially changing its immunogenicity. We identified hydrazone adducts on circulating MPO in patients with hydralazine-associated ANCA vasculitis. These patients exhibited elevated anti-MPO IgM levels, while anti-MPO IgG levels were comparable between hydralazine-associated and nonhydralazine-associated vasculitis patients. IgM isolated from patients with hydralazine-associated MPO ANCA demonstrated a heightened affinity to hydralazine-modified MPO and activated neutrophil-like HL-60 cells. Hydralazine-modified MPO was pathogenic, as demonstrated by splenocyte transfer in a mouse model of ANCA vasculitis. Our findings unveil a mechanism of drug-induced autoimmunity wherein stepwise chemical modifications of MPO lead to conformational changes and hydrazone adduct formation, producing a neoantigen that generates pathogenic autoantibodies.

Authors

Gang Xi, Elizabeth A. Mclnnis, Olivier Lardinois, Peiqi Hu, John S. Poulton, Meghan E. Free, Dhruti P. Chen, Evan M. Zeitler, Eveline Y. Wu, Nicole M. Orzechowski, Vimal K. Derebail, J. Charles Jennette, Ronald J. Falk

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Figure 4

IgM is a primary subtype of immunoglobins that is generated against hydralazine-modified MPO ex vivo.

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IgM is a primary subtype of immunoglobins that is generated against hydr...
(A) Plasma anti-myeloperoxidase IgG and IgM from patients with hydralazine-associated ANCA (n = 10), patients with nonhydralazine-associated ANCA (n = 25), and participants in a healthy control group (n = 19) were measured following the protocol described in the Methods section. The multiple comparisons of IgM or IgG for any 2 groups of patients’ P values (*P < 0.05; ***P < 0.001; ****P < 0.0001) were calculated by Dunn’s multiple comparisons test. (B and C) IgG and IgM were purified from plasma obtained from healthy participants (HC, n = 5), patients with nonhydralazine-associated ANCA (ANCA, n = 4), and patients with hydralazine-associated ANCA (HA-ANCA, n = 6). The same amount of native MPO and hydralazine-modified MPO were separated using SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were immunoblotted with IgM (B) and IgG (C). Densitometry values were obtained using Image J (NIH, 1.53K version) and graph was drawn using GraphPad Prism (GraphPad Software, Version 9.5.1). *P < 0.05 assessed by Mann-Whitney test for comparison between 2 groups.