Background/Purpose: Anti-endothelial cell antibodies (AECA) are autoantibodies that are detected frequently in vasculitis caused by, for example, Kawasaki disease (KD). However, AECA target molecules have been poorly identified, which hampers our understanding of the roles of AECA. We aimed to detect target proteins of AECA comprehensively by proteomics and investigate their clinical importance in patients with KD.

Methods: We separated proteins extracted from human umbilical vein endothelial cells (HUVECs) and HeLa cells by 2-dimensional electrophoresis and transferred them onto membranes. Antigens that were positive only in the HUVEC samples but not in the HeLa cell samples were detected by western blot, using serum samples from patients with vasculitis. Next, we identified the detected proteins by peptide mass finger-printing and characterized their antigenicity by preparing recombinant antigens and antibodies to them. Furthermore, we investigated the frequency, clinical significance, and pathological roles of AECA against individual identified target autoantigens.

Results: We identified 63 proteins, one of which was peroxiredoxin 2 (Prx2), an anti-oxidative enzyme. Using immunocytochemistry, antibodies against Prx2 were found to bind to the cell surface of live HUVECs. IgG antibodies to Prx2 were detected in 60% (18/30) of the patients with KD, but not in healthy controls. Furthermore, IgG antibodies to Prx2 were detected in all tested KD patients with coronary artery lesions. Stimulating endothelial cells (ECs) with anti-Prx2 antibodies resulted in increased H₂O₂levels in the cell lysates. The anti-Prx2 antibodies also increased various inflammatory cytokine secretions significantly; in particular, IL-6 in HUVECs and G-CSF in human coronary artery ECs. In addition, anti-Prx2 antibodies induced the increased expression of adhesion molecules, such as E-selectin and ICAM-1, on ECs. EC ELISA indicated that Prx2 proteins were released from ECs stimulated by inflammatory cytokines. By themselves, Prx2 proteins induced various inflammatory cytokine secretions, such as IL-6, IL-8, TNF-α and GM-CSF, from ECs, and the expression of adhesion molecules such as E-selectin and ICAM-1 on ECs. Interestingly, these effects of Prx2 proteins were enhanced significantly by the appearance of anti-Prx2 antibodies and were inhibited significantly by blocking Toll-like receptor 4 (TLR4) signaling with a TLR4-specific inhibitory peptide. Clinically, the duration of fever (>37.5°C) in KD patients was significantly longer in the anti-Prx2–positive group than in the anti-Prx2–negative group. There was no significant difference in inflammatory markers such as white blood cell counts and C-reactive protein between both groups.

Conclusion: IgG antibodies to Prx2 are expected to be useful markers for KD. Expression of endothelial adhesion molecules and inflammatory cytokine production are induced by the binding of anti-Prx2 antibodies to Prx2 proteins on ECs, and by the activation of TLR4 by extracellular Prx2 proteins, which could result in endothelial injury. Furthermore, anti-Prx2 antibodies may cause endothelial injury by inhibiting the anti-oxidative activity of intracellular Prx2 proteins.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/pathological-roles-of-the-anti-oxidative-enzyme-peroxiredoxin-2-in-patients-with-kawasaki-disease/