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Native myeloperoxidase is required to make the experimental vasculitis model
Arthritis Research & Therapy volume 21, Article number: 296 (2019)
Myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA) is a pathogenic autoantibody . Wistar-Kyoto (WKY) rats immunized with human native MPO produce anti-human MPO antibody cross-reactive with rat MPO, resulting in the development of MPO-ANCA-associated vasculitis (MPO-AAV) . MPO is a heterotetramer composed of two light chains (14 kDa) and two heavy chains (59 kDa) . In this study, we examined if immunization of WKY rats with the recombinant light chain of human MPO could induce MPO-AAV.
WKY rats (4–5 weeks old) were immunized with the recombinant light chain of human MPO (1600 μg/kg; Cloud-Clone, Katy, TX, USA; group 1) or human native MPO (1600 μg/kg; RayBiotech, Peachtree Corners, GA, USA; group 2) on day 0. These rats were given an intraperitoneal injection of pertussis toxin (800 ng; Sigma-Aldrich, St. Louis, MO, USA) on days 0 and 2. A subgroup of group 1 was given an intraperitoneal injection of lipopolysaccharide (LPS; 100 μM/week; Sigma-Aldrich) through days 7 to 35. Urine samples were collected using a metabolic cage on day 40. All rats were euthanized on day 42.
Flow cytometry (FCM) using human neutrophils demonstrated the presence of ANCA in sera of group 2 but not group 1 (Fig. 1a). Correspondingly, sera of group 2 but not group 1 induced neutrophil extracellular traps (NETs) from tumor necrosis factor (TNF)-primed neutrophils (Fig. 1b). Immunoblot of neutrophil lysates demonstrated that antibody reactive with the MPO light chain (14 kDa) was produced in group 1, whereas antibodies reactive with the MPO heavy chain (59 kDa) and light chain (14 kDa) were produced in group 2 (Fig. 1c). The collective findings indicated that the anti-MPO light chain antibody produced in group 1 did not bind to native MPO. Renal tissue damage represented by hematuria and erythrocyte casts in renal tubules was evident in group 2 but not group 1 regardless of the disease boost by LPS (Fig. 1d, e). The degree of pulmonary hemorrhage that represents capillaritis in the lungs tended to be severe in group 2 compared to group 1 (Fig. 1f).
The majority of MPO-AAV patients produced MPO-ANCA that recognizes an epitope in the heavy chain of MPO, whereas a few number of patients produced MPO-ANCA against an epitope in the light chain of MPO [4, 5]. The collective findings suggested that the recombinant light chain of human MPO has a low potential to induce MPO-AAV in rats compared to native human MPO.
Availability of data and materials
The data sheets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
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This work was supported by a grant from Ono Pharmaceutical (Osaka, Japan).
Ethics approval and consent to participate
Experiments using rats were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals in Hokkaido University (Permission No. 15-0034). Experiments using human materials were permitted by the Ethics Committee of the Faculty of Health Sciences, Hokkaido University (Permission No. 18-34).
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The authors declare that they have no competing interests.
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Nonokawa, M., Suzuki, K., Hayashi, H. et al. Native myeloperoxidase is required to make the experimental vasculitis model. Arthritis Res Ther 21, 296 (2019) doi:10.1186/s13075-019-2084-7