Arthritogenicity of annexin VII revealed by phosphoproteomics of rheumatoid synoviocytes.

OBJECTIVE: To identify novel proteins involved in the pathogenesis of rheumatoid arthritis (RA) and to characterise the identified proteins based on pathogenic and therapeutic aspects. METHODS: The authors applied differential phosphoproteomic analysis to articular synoviocytes between RA and osteoarthritis (OA) to identify proteins differently phosphorylated between RA and OA. Focusing on annexin VII (Anx7), one of the highly phosphorylated proteins in RA, the authors prepared Anx7-transgenic C57BL/6 (Anx7-Tg-B6) mice to evaluate their susceptibility to collagen-induced arthritis (CIA). In addition, the authors examined the effect of anti-Anx7 antibodies (Abs) on CIA and serum levels of cytokines in wild-type DBA/1J mice, which are known to be susceptible to CIA, and in Anx7-Tg-B6 mice. In vitro, the authors examined the effect of the Anx7 knockdown by small interfering RNA on the secretion of cytokines in rheumatoid synoviocytes and the human synovial sarcoma cell line SW982. RESULTS: The Anx7 transgene altered the CIA-resistant B6 mice to CIA-susceptible ones. The Abs treatment suppressed CIA even in the wild-type DBA/1J mice. The serum levels of cytokines including interleukin 6 (IL-6) and TNFα were not altered by the Abs treatment in vivo. On the other hand, the knockdown of Anx7 by small interfering RNA caused downregulation of IL-8 secretion in vitro. CONCLUSIONS: These results indicate that Anx7 participates in the pathogenesis of RA partly through the secretion of IL-8. The study data have demonstrated the pathogenic roles and therapeutic significance of Anx7 in RA for the first time.

Regulatory role of heme oxygenase 1 in inflammation of rheumatoid arthritis.

OBJECTIVE: To examine the expression and pathogenetic roles of heme oxygenase 1 (HO-1), an inducible heme-degrading enzyme with antiinflammatory properties, in rheumatoid arthritis (RA). METHODS: HO-1 expression in synovial tissue from patients with RA, patients with osteoarthritis, and patients with noninflammatory joint diseases was determined by immunoblotting and immunohistochemistry. Effects of various agents, such as hemin (a chemical inducer of HO-1), small interfering RNA (siRNA) specific for HO-1, HO-1 expression vector, and antirheumatic agents, on HO-1 expression in RA synovial cell lines were analyzed by real-time reverse transcription-polymerase chain reaction (PCR) and immunoblotting. Cytokine synthesis was evaluated by real-time PCR and enzyme-linked immunosorbent assay. RESULTS: HO-1 was expressed more abundantly in the lesions of synovial tissue from patients with RA than in those from the other patient groups. Hemin, auranofin, and HO-1 expression vector induced HO-1 and reduced expression of tumor necrosis factor alpha (TNFalpha) messenger RNA, lipopolysaccharide (LPS)-induced secretion of interleukin-6 (IL-6) and IL-8, and expression of cyclooxygenase 2 in the synovial cell lines. Treatment with HO-1-specific siRNA augmented the synthesis of TNFalpha, IL-6, and IL-8 and canceled the suppressive effects of auranofin on TNFalpha secretion. When hemoglobin, as a scavenger of carbon monoxide, was added to auranofin-treated synovial cell lines, LPS-dependent production of IL-6 and IL-8 was increased. CONCLUSION: Our data demonstrate that HO-1 is expressed in RA synovial tissues and plays a regulatory role in the development of inflammation. The pharmacologic effects of auranofin depend, in part, on the levels of HO-1, suggesting that HO-1 induction is a novel therapeutic strategy for RA.

Identification of novel citrullinated autoantigens of synovium in rheumatoid arthritis using a proteomic approach.

Recently, autoantibodies to some citrullinated autoantigens have been reported to be specific for rheumatoid arthritis (RA). However, an entire profile of and autoimmunity of the citrullinated proteins have been poorly understood. To understand the profile, we examined citrullinated autoantigens by a proteomic approach and further investigated the significance of citrullination in antigenicity of one of the autoantigens. Specifically, we detected citrullinated autoantigens in synovial tissue of a patient with RA by two-dimensional electrophoresis and Western blotting by using pooled sera from five patients with RA and anti-citrulline antibodies. After identifying the detected autoantigens by mass spectrometry, we investigated the contribution of citrullination to autoantigenicity by using a recombinant protein with or without citrullination on one of the identified novel citrullinated autoantigens. As a result, we found 51 citrullinated protein spots. Thirty (58.8%) of these spots were autoantigenic. We identified 13 out of the 30 detected citrullinated autoantigenic proteins. They contained three fibrinogen derivatives and several novel citrullinated autoantigens (for example, asporin and F-actin capping protein alpha-1 subunit [CapZalpha-1]). We further analyzed the contribution of citrullination to autoantigenicity in one of the detected citrullinated autoantigens, CapZalpha-1. As a result, frequencies of autoantibodies to non-citrullinated CapZalpha-1 were 36.7% in the RA group tested, 10.7% in the osteoarthritis (OA) group, and 6.5% in healthy donors. On the other hand, those to citrullinated CapZalpha-1 were 53.3% in the RA group, 7.1% in the OA group, and 6.5% in the healthy donors. This shows that autoantigenicity of citrullinated or non-citrullinated CapZalpha-1 is relevant to RA. The antibody titers to the citrullinated CapZalpha-1 were significantly higher than those to the non-citrullinated CapZalpha-1 in 36.7% of patients; however, the other patients showed almost equal antibody titers to both citrullinated and non-citrullinated CapZalpha-1. Therefore, the autoantibodies would target citrulline-related and/or citrulline-unrelated epitope(s) of CapZalpha-1. In conclusion, we report a profile of citrullinated autoantigens for the first time. Even though citrullination is closely related to autoantigenicity, citrullination would not always produce autoantigenicity in RA. Citrullinated and non-citrullinated autoantigens/autoepitopes would have different pathological roles in RA.