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- MATERIALS AND METHODS
Antibodies to the anti-oxidative peroxiredoxin (Prx) enzymes occur in both systemic autoimmune disease and vasculitis in adulthood. Because increased oxidative stress induces vasculitis in Kawasaki disease (KD), autoimmunity to Prxs in patients with KD was investigated. The presence of antibodies to Prx 1, 2 and 4 was analyzed by ELISA and Western blot. Of 30 patients with KD, 13 (43.3%) possessed antibodies to Prx 2, whereas these antibodies were present in only 1 of 10 patients (10.0%) with sepsis (4 with purulent meningitis and 6 with septicemia). In contrast, antibodies to Prx 1 and 4 were not detected in either group. There was no significant correlation among the titers of the three antibodies. Clinical parameters were compared between anti-Prx 2-positive and -negative patients. The presence of anti-Prx 2 antibodies correlated with a longer period of fever and poor response to high-dose γ-globulin therapy in patients with KD. Anti-Prx 2-positive patients had significantly greater excretion of urinary 8-isoprostaglandin than did anti-Prx 2-negative patients. These results provide the first evidence for an antibody to Prx 2 in patients with KD. They also suggest that this antibody might serve as a marker of disease severity and be involved in the pathophysiology of vasculitis in some patients with KD.
Kawasaki disease is a type of acute vasculitis of unknown etiology that occurs in childhood. We and others have reported that AECA is both found in KD and cytotoxic to endothelial cells in patients with KD (1–3). AECA induces complement-dependent cytotoxicity (1, 2), enhances expression of adhesion molecules, and causes monocyte adhesion to HUVECS (3). In addition, immune dysregulation in KD patients leads to abnormal apoptosis of neutrophils or mononuclear cells (4, 5), which may result in oxidative stress and autoimmunity. Prxs have peroxidase activity (ROOH + 2e−→ ROH + H2O), which results in reduction of hydrogen peroxide, peroxynitrite and ROOH in various organs (6–10). Recently, we detected autoantibodies to Prxs 1 and 4 in sera from autoimmune individuals with systemic vasculitis, such as occurs in SLE or RA (6). Anti-Prx 2 antibody is also found more frequently in systemic vasculitis (7). Increased oxidative stress induces vasculitis in KD (8); therefore we speculated that antibodies to antioxidative enzymes might be involved in the pathogenesis of this disease. The present study was undertaken to determine whether antibodies to Prxs are present in sera from patients with KD, and whether these antibodies are predictive of the severity of vasculitis. We focused on Prx 2, which is located in both the cytosol and cell membrane and has high antioxidative activity in many types of cells, including the vascular endothelium (9).
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- MATERIALS AND METHODS
Peroxiredoxin are a recently discovered and characterized family of thiol-specific antioxidant proteins (9). Six Prxs have been reported in humans. Prxs are found in various subcellular locations, including Prx 1 in the cytosol and nucleus; Prx 2 in the cytosol and cell membrane; and Prx 4 in the cytosol, Golgi and secretory fluid. Reversible modifications of Prxs from HUVECs based on changes in isoelectric points after exposure to peroxide have been described (10, 11). Prx 2 is a non-secreted cytosolic protein that has the most powerful peroxidase activity of the six Prxs (12–14). Prx oxidation status has been shown to reflect oxidative stress in the vasculature and to correlate with the extent of atherosclerotic lesions in an animal model (15). Since oxidative stress is known to cause inflammation, including vasculitis (16) and KD (8, 17), impaired function of Prx 2 triggered by anti-Prx antibodies may initiate vasculitis and KD.
We detected anti-Prx 2 antibodies in 43.3% of patients with KD, this rate being significantly higher than that for patients with sepsis or for controls. Thus, anti-Prx 2 antibody may be involved in the pathophysiology of vasculitis in some patients with KD. A specific role of anti-Prx 2 antibody in KD was further suggested by correlations between the presence of autoantibody and both disease severity and poorer response to high-dose γ-Glb therapy. We examined two γ-Glb preparations, both with final concentrations corresponding to 500 mg/dL (IgG), and obtained OD values of 0.303 and 0.289. We evaluated these values as negative for anti-Prx 2 antibody. In this study, mean IgG concentrations in diluted serum samples (× 200) from eight patients with KD were very low (3.5 mg/dL for pre-γ-Glb therapy and 9.0 mg/dL for post-γ-Glb therapy). We found that titers of anti-Prx2 antibody decreased 2 to 4 weeks after administration of high-dose γ-Glb therapy. In addition, the ratio of anti-Prx 2 antibody titers to serum immunoglobulin G concentrations (g/L) decreased significantly between pre-γ-Glb and post-γ-Glb therapy in eight patients with KD. Therefore, it is unlikely that the γ-Glb preparation has a major influence on anti-Prx 2 antibody titer. Although anti-Prx 2 antibody activity and function decrease with treatment, it is also possible that this antibody is spontaneously eliminated slowly from sera of patients with KD. Further studies are required to examine serial long-term changes in anti-Prx 2 antibody titer, especially in patients where γ-Glb ceases to be effective or the KD flares up.
Karasawa et al. examined the subcellular localization of Prx 2, the binding affinity of anti-Prx 2 antibodies to live endothelial cells and the effects of anti-Prx antibodies on secretion of cytokines and chemokines from endothelial cells (7). Prx 2 has been detected in the membrane and organelle fractions of endothelial cells lines, as well as in whole cell lysates. Anti-Prx 2 antibody bound to the surface of live HUVECs and stimulation of these cells with rabbit anti-Prx 2 antibody upregulates secretion of interleukin-6, interleukin-8 and granulocyte colony-stimulating factor by more than 2-fold compared to stimulation with rabbit IgG (7). Suzuki et al. showed that KD patients with CALs had significantly higher concentrations of serum granulocyte colony-stimulating factor than did patients without CALs (18). However, we could not confirm whether the function and binding sites of human anti-Prx 2 antibody are the same as for rabbit anti-Prx 2 antibody. In addition, the concentrations of anti-Prx 2 antibodies in patients with CALs were not extremely high, compared to the other KD patients with anti-Prx 2 antibodies. Therefore, anti-Prx 2 antibody might be a predictive marker for refractoriness to high dose γ-Glb therapy.
A homolog of Prx 2 is conserved in many microorganisms and is expressed in BCG at a higher level than in Mycobacterium tuberculosis (19). Thus, immune responses triggered by a microorganism with cross-reactive antigens might be associated with the development of KD. In a mouse model of KD, Nakamura et al. showed that intravenous injection with anti-Prx 2 antibodies resulted in coronary arteritis, but only after prior inoculation with BCG (20). In the present study, the titers of anti-Prx 2 antibody in the three patients with CALs were not higher than those in the other ten patients in the anti-Prx 2 antibody-positive group who did not have CALs. However, the three patients with CALs had a significantly longer duration of fever (10.3 ± 2.7 vs. 9.2 ± 4.0 days, P= 0.045), higher CRP concentrations and increased WBC counts compared to the ten patients without CALs. The rates of BCG vaccination and local swelling were the same in the antibody-positive and -negative subgroups (data not shown). These findings suggest that coronary arteritis in KD is not induced by anti-Prx 2 antibodies alone, and that other microorganisms besides BCG, as well as autoantibodies against other endothelial antigens (especially human coronary artery endothelial antigens), might also be required to induce coronary arteritis.
The reason for the higher prevalence of the anti-Prx 2 antibody compared to the anti-Prx 1 and 4 antibodies is unclear. The amino acid sequence identities are 78% between Prx 2 and Prx 1, and 70% between Prx 2 and Prx 4 (6, 21). However, we found no significant correlations among the occurrence and titers for anti-Prx 1, 2, and 4 antibodies. Thus, generation of these three antibodies appears to be independent; therefore we cannot assume cross-reactivity among them.
On the other hand, this study revealed that oxidative stress evaluated by urinary excretion of 8-iso PG contributes to the acute phase of KD, a finding which is supported by previous report (8). In addition, the present study showed a positive correlation between anti-Prx 2 antibody titers and urinary 8-iso-PG concentrations. Therefore, the presence of anti-Prx 2 antibody might reflect the production of oxidative stress.
In conclusion, we identified a novel antigen, Prx 2, as an AECA target. Anti-Prx 2 antibody may be a predictor of disease severity and may also be involved in the pathophysiology of vasculitis in some patients with KD. However, further studies are needed to examine the diagnostic value of the autoantibody and to elucidate the mechanism of its possible association with development of CALs on a large scale, since the patients number in the present study was small and only three patients with KD developed CALs.