Monoclonal anti-double-stranded DNA antibodies cross-react with phosphoglycerate kinase 1 and inhibit the expression and production of IL-2 in activated Jurkat T cell line
Introduction
Autoantibodies to double-stranded DNA are highly specific for SLE. The titers of these antibodies fluctuate with the disease activity and they appear to participate in the development of organ damage, especially in the kidney [1]. However, their crucial role in lupus nephritis and tissue damage remains unclear. In addition to non-specifically deposit in the kidney by binding to DNA, anti-double strand DNA antibodies (anti-dsDNA) may also directly cross-react with protein antigens to trigger cell dysfunction and tissue damage [2], [3], [4], [5], [6], [7]. Moreover, a 30-amino acid peptide corresponding to the combined sequences of CDR 2 and 3 of heavy chain variable region of anti-DNA has been demonstrated to penetrate into the cells [8]. These findings suggest the role of protein antigens in lupus pathogenesis.
The modulation of cytokines has been considered to be important in the pathogenesis of SLE, especially the involvement of Th1 cytokine IL-12, Th2 cytokine IL-10, proinflammatory cytokine TNF-α, and cytokine BLyS [9]. Proinflammatory cytokines produced by activated macrophages and monocytes may have potent stimulatory effects on T cells, B cells, and neutrophils in increasing the biosynthesis of prostaglandins and acute-phase proteins [10]. IL-12 may induce the differentiation of Th1 to produce IL-2 and IFN-γ and modulate cellular immune responses. IL-4 may induce the differentiation of Th2 to produce IL-4 and IL-10 and modulate humoral immune reactions [11]. Anti-dsDNA may stimulate the release of IL-1 and IL-6 from endothelial cells and cause inflammatory injury of vascular endothelium in SLE [12]. Moreover, anti-dsDNA monoclonal antibodies (anti-dsDNA mAb) has been demonstrated to penetrate Jurkat T cells and stimulate IL-1β, IL-8, TNF-α, and IL-10 production in human mononuclear cells [7].
To identify new cross-reactive antigens bound by anti-dsDNA, we established the anti-dsDNA mAb 9D7 and found two possible cognate protein antigens with molecular weights of 35 kDa and 45 kDa. The 35-kDa antigen was identified to be heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) [13]. In this study, the 45-kDa antigen was determined to be phosphoglycerate kinase-1 (PGK-1). In Jurkat T cells, the 9D7 mAb was also found to affect PGK-1 mRNA expression and decrease IL-2 expression and production. These findings indicate that anti-dsDNA may involve in the pathogenesis of SLE by interacting with PGK-1 and decreasing IL-2 production.
Section snippets
Cell lines
Human acute T cell leukemia (Jurkat) and RAW 264.7 mouse macrophage cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA). RAW 264.7 were cultured in DMEM and Jurkat T cells were cultured in RPMI-1640. These culture media were supplemented with 10% FBS and the cell cultures were maintained in 5% CO2 at 37°C.
Sera
Serum samples were collected from SLE patients and normal subjects according to the IRB protocol in the Section of Arthritis, Immunology and Rheumatology,
The 45-kDa cognate protein of anti-dsDNA mAb 9D7 is human PGK-1
By immunoblotting, we identified a 45-kDa protein to be a cognate antigen of anti-dsDNA. However, this protein was not detectable in a small amount (1 × 106) of cell lysates by 2D gel electrophoresis and Coomassie brilliant blue staining [13]. In this study, 2D gel electrophoresis (Fig. 1A) was performed on a larger amount (9 × 106) of Jurkat lysates and then immunoblotted with the 9D7 mAb. Two cognate protein antigens of anti-dsDNA mAb with molecular weights of 35 kDa and 45 kDa were visualized (
Discussion
SLE is a systemic autoimmune disease that affects many organs and tissues of the body, causing glomerulonephritis, arthritis, anemia, and cerebritis. This disease is characterized by B cell hyperactivity and the presence of autoantibodies. Among the autoantibodies, anti-dsDNA are most commonly found in SLE specimens [16]. Because polyreactivity is a common property of natural and disease-associated human autoantibodies [17], anti-DNA may cross-react with DNase I [2], myosin 1 [3], calreticulin
Acknowledgments
This work was supported in part by grants from the National Science Council (NSC 93-2314-B-010-016), Mackay Memorial Hospital-Yang-Ming University (MMHYM 93-P-010-006), Veteran General Hospital-University System of Taiwan (VGHUST93-P7-34), and a grand from Ministry of Education, Aim for the Top University Plan, ROC.
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