High redox thioredoxin but low thioredoxin reductase activities in the serum of patients with rheumatoid arthritis

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Abstract

Background

Thioredoxin (Trx)/thioredoxin reductase (TrxR) is a redox-active system induced by oxidative stress. We investigated its status as a function of RA disease activity.

Methods

64 consecutive RA patients and 27 healthy subjects were enrolled in the study. Serum Trx protein levels were evaluated using an immunoassay and immunoblot, while redox Trx and TrxR activities and oxidative stress markers (carbonyl groups, thiols), were determined using spectrophotometric methods.

Results

Redox Trx activity and Trx protein concentrations were significantly higher in RA patients than in controls (redox Trx activity: 37.7 ± 22.6 versus 21.1 ± 7.9 ng/mL, p < 0.01; Trx protein: 25.5 ± 12.0 versus 12.3 ± 5.1 ng/mL, p < 0.0001). Redox Trx activity correlated with the DAS score (r = 0.45, p = 0.004) and with the tender joint count (r = 0.49, p = 0.002) whereas there was no correlation with Trx protein concentrations. Immunoblot analysis showed that circulating Trx was partially aggregated. TrxR activity was lower in the serum of RA patients than in healthy subjects (197 ± 70 versus 263 ± 56 U/L, p = 0.002). TrxR activity was correlated with the DAS score (r = 0.53, p < 0.001) and with the tender joint count (r = 0.36, p < 0.01). There were no correlations between oxidative stress marker levels and redox Trx activity, Trx protein concentrations or TrxR activity.

Conclusion

Redox Trx and TrxR activities correlated with the disease activity of RA patients consistent with the hypothesis that Trx/TrxR activities may contribute to disease activity in RA.

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory disease, characterized by synovial proliferation and excessive mononuclear infiltration, leading to the development of cartilage and subchondral bone erosions [1]. A variety of factors are implicated in the development of oxidative stress in RA patients: chronic inflammatory conditions, and vascular changes including post-ischemic reperfusion injury leading to the destruction of cartilage and bone [2], [3]. Oxidative stress has been described as an important mechanism that underlies destructive proliferative synovitis [4] and may also modulate T cellular activities [5]. Oxidative damage to cells, such as the rupture of membranes due to raised levels of lipid peroxidation or alterations to the structure or function of proteins have been reported in studies relating to RA [6], [7]. Lowered serum levels of antioxidants have also been found to predispose patients to the development of RA, and the dietary intake of antioxidants has decreased oxidant levels in several animal models of arthritis [8], [9], [10].

An important regulator of the redox state of proteins is the thioredoxin reductase/thioredoxin system. Thioredoxin reductase (TrxR) is a redox-active selenoprotein that efficiently regenerates oxidized Trx to its reduced form, requiring selenium for its activity [11]. It is over-expressed and released in the event of oxidative stress and has recently been detected in plasma [12]. Thioredoxin (Trx), a 12-kDa ubiquitous protein with a highly conserved active site (–Cys–Gly–Pro–Cys–) plays a variety of redox-related roles in living organisms [13]. This protein has been shown to catalyze protein disulfide reduction in combination with thioredoxin reductase and nicotinamide–adenine dinucleotide phosphate as part of the thioredoxin–thioredoxin reductase system. Trx is currently known to possess both intracellular and extracellular activities. Several of the biological activities of Trx have been classified as antioxidant, anti-apoptotic growth-promoting, or inflammation modulating, and seem to be dependent on signal transduction pathways and gene expressions [14], [15], [16]. Trx appears to be beneficial in reducing inflammation, because circulating Trx may be also involved in the suppression of cytokine and chemokine production, resulting in the attenuation of leukocyte infiltration [17]. A link between elevated serum Trx levels and the inhibition of chemotaxis at the local site of inflammation has been demonstrated [18]. Trx appears to play a crucial role in controlling oxidative stress during many human diseases. It was recently shown that Trx levels are elevated in the synovial fluid and serum of RA patients [19], [20], [21]. This increase also correlated with levels of urinary 8-OHdG, a biomarker of oxidative DNA damage [21]. Trx mainly circulates in the serum in an oxidized form that easily aggregates, and the immunoassays used during almost all these studies may have underestimated these aggregated forms with a high molecular weight [22].

In order to improve our knowledge of the Trx/TrxR system in RA, we first of all investigated serum Trx redox activity using spectrophotometry and then compared it with TrxR activity and Trx protein determinations performed using a commercial immunoassay kit. Finally, we determined the potential links between the Trx/TrxR system and disease activity and with imbalanced redox status, evaluated by determining protein carbonyl and thiol groups.

Section snippets

Patients

Sixty-four consecutive, hospitalized patients responding to the American College of Rheumatology RA criteria [23] were studied. Five patients were taking non-steroidal anti-inflammatory drugs (NSAID), thirty-eight patients were receiving methotrexate (MTX), twelve MTX combined with salazopyrine, and nine MTX plus hydrocloroquine. Patients taking methothrexate were also treated with folic acid substitution. All patients were receiving a stable prednisone dose (less than 10 mg per day). We

Results

Sixty-four consecutive rheumatoid arthritis patients (54 of them women [85%]; mean age [± SD]: 54 ± 14 years and a disease duration of 12 ± 9 years) were included in the study. Their clinical and biological characteristics are shown in Table 1. Twenty-seven healthy control subjects (21 of them women [78%]; mean age 46 ± 17 years) displayed CRP levels ≤ 2.5 mg/L. There were no statistically significant differences in terms of age and sex distribution between the control and RA groups.

Discussion

It has previously been reported that an increase in Trx protein levels is associated with various diseases, including viral infections, primary tumours, heart failure and chronic inflammatory diseases, suggesting that Trx could constitute a useful clinical parameter [27], [28], [29]. During this study, we investigated the implications of serum Trx in RA disease. We showed that serum redox thioredoxin activity and protein were significantly increased in patients with RA when compared to healthy

References (36)

  • P.I. Mapp et al.

    Hypoxia, oxidative stress and rheumatoid arthritis

    Br Med Bull

    (1995)
  • C.A. Hitchon et al.

    Oxidation in rheumatoid arthritis

    Arthritis Res Ther

    (2004)
  • P.H.J. Remans et al.

    Intracellular free radical production in synovial T lymphocytes from patients with rheumatoid arthritis

    Arthritis Rheum

    (2005)
  • S. Bashir et al.

    Oxidative DNA damage and cellular sensitivity to oxidative stress in human autoimmune diseases

    Ann Rheum Dis

    (1993)
  • B. Halliwell

    Oxygen radicals, nitric oxide and human inflammatory joint disease

    Ann Rheum Dis

    (1995)
  • M. Heliovaara et al.

    Serum antioxidants and risk of rheumatoid arthritis

    Ann Rheum Dis

    (1994)
  • L. Hagfors et al.

    Antioxidant intake, plasma antioxidants and oxidative stress in a randomized, controlled, parallel, Mediterranean dietary intervention study on patients with rheumatoid arthritis

    Nutr J

    (2003)
  • S. Gromer et al.

    Active sites of thioredoxin reductases: why selenoproteins?

    Proc Natl Acad Sci U S A

    (2003)
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