Skip Navigation


Rheumatology Advance Access originally published online on February 19, 2007
Rheumatology 2007 46(5):790-795; doi:10.1093/rheumatology/kem010
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
46/5/790    most recent
kem010v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (5)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Iwata, Y.
Right arrow Articles by Sato, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Iwata, Y.
Right arrow Articles by Sato, S.
Related Collections
Right arrow Systemic Sclerosis
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Autoantibody against peroxiredoxin I, an antioxidant enzyme, in patients with systemic sclerosis: possible association with oxidative stress

Y. Iwata1,3, F. Ogawa1, K. Komura1, E. Muroi1, T. Hara1, K. Shimizu1, M. Hasegawa2, M. Fujimoto2, Y. Tomita3 and S. Sato1

1Department of Dermatology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, 2Department of Dermatology, Kanazawa University Graduate School of Medical Science, Kanazawa and 3Department of Dermatology, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Correspondence to: S. Sato, Department of Dermatology, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan. E-mail: s-sato{at}nagasaki-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Objectives. To determine the prevalence and clinical correlation of autoantibody to peroxiredoxin (Prx) I, an antioxidant enzyme, in patients with systemic sclerosis (SSc).

Methods. Serum samples from SSc patients (n = 70) and healthy controls (n = 23) were examined by ELISA using human recombinant Prx I. The presence of anti-Prx I antibody was further evaluated by immunoblotting analysis. To determine the functional relevance of anti-Prx I antibody in vivo, we assessed whether anti-Prx I antibody was able to inhibit Prx I enzymatic activity using yeast thioredoxin reductase system.

Results. IgG anti-Prx I antibody levels in SSc patients were significantly higher than healthy controls and this autoantibody was detected in 33% of SSc patients. The presence of IgG anti-Prx I antibody was associated with longer disease duration, more frequent presence of pulmonary fibrosis, heart involvement, and anti-topoisomerase I antibody and increased levels of serum immunoglobulin and erythrocyte sedimentation rates. IgG anti-Prx I antibody levels also correlated positively with renal vascular damage and negatively with pulmonary function tests. Furthermore, anti-Prx I antibody levels correlated positively with serum levels of 8-isoprostane, a marker of oxidative stress. Immunoblotting analysis confirmed the presence of anti-Prx I antibody. Remarkably, Prx I enzymatic activity was inhibited by IgG isolated from SSc sera containing IgG anti-Prx I antibody.

Conclusions. These results suggest that elevated IgG anti-Prx I autoantibody is associated with the disease severity of SSc and that anti-PrxI antibody may enhance the oxidative stress by inhibiting Prx I enzymatic activity.

KEY WORDS: Systemic sclerosis, Oxidative stress, Peroxiredoxin, Pulmonary fibrosis, Autoantibody


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Systemic sclerosis (SSc) is a connective tissue disease that is characterized by fibrosis and vascular changes in the skin and other internal organs with autoimmune background [1]. Although the pathogenesis of SSc remains unknown, it has been proposed that oxidative stress may play an important role in the development of SSc [2]. Ischaemia and reperfusion injury following Raynaud's phenomenon can generate reactive oxygen species that may result in vascular endothelial damage [3, 4]. Reactive oxygen species are also released from skin fibroblasts of SSc patients in vitro [5] and have been shown to stimulate fibroblast proliferation, which may result in fibrosis [6, 7]. Furthermore, enhanced oxidative stress in SSc has been demonstrated, since 8-isoprostane, a reliable biomarker of oxidative stress, increases in urine [7, 8], bronchoalveolar lavage [9], and serum samples [10] in patients with SSc and serum 8-isoprostane levels correlate with the disease severity of SSc [10].

Exposure to reactive oxygen species from a variety of sources has led organisms to develop a series of defence mechanism [11]. One of the important defence mechanisms against oxidative stress is defence by antioxidant enzymes, including peroxiredoxin (Prx), which is a recently discovered and characterized family of thiol-specific antioxidant enzyme [12–14]. Prxs have peroxidase activity (ROOH + 2e– -> ROH + H2O) and thereby reduce hydrogen peroxide, peroxynitrate, and various organic hydroperoxides [15–17]. Prxs, which are present in a large variety of organisms, are produced at high levels in cells [15, 16, 18]. Six Prx isoforms (Prxs I–VI) have been identified [14] and Prx I is the most abundant and ubiquitously distributed member of the mammalian Prxs [16]. Recently, Karasawa et al. [19] have shown that 33% of patients with systemic autoimmune diseases, including systemic lupus erythematosus, primary vasculitis syndrome and rheumatoid arthritis, possess autoantibodies to Prx I. However, the prevalence and clinical correlation of anti-Prx I antibody (Ab) in patients with SSc and whether anti-Prx I Ab was able to inhibit the enzymatic activity of Prx I remained unknown in their study.

Autoantibody production is one of the central features in SSc, since more than 90% of patients have antinuclear Abs [20]. Although it remains controversial whether SSc-specific autoantibodies, such as anti-topoisomerase I, anticentromere and anti-RNA-polymerase Abs, directly contribute to the clinical manifestations of SSc, there may be possibilities that several autoantobodies play a pathogenetic role [1, 21]. Therefore, we hypothesized that anti-Prx I Ab could also be detected in patients with SSc and contribute to enhanced oxidative stress by inhibiting Prx I enzymatic activity, leading to tissue damage of SSc. To test this possibility, the presence or levels of anti-Prx I Ab, its clinical correlation and its functional significance were investigated in the current study.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Serum samples
Serum samples were obtained from 70 Japanese patients with SSc (61 women and 9 men). All patients fulfilled the criteria proposed by the American College of Rheumatology [22]. Patients were grouped according to the classification system proposed by LeRoy et al. [23]: 30 patients (28 women and 2 men) had limited cutaneous SSc (lSSc) and 40 patients (33 women and 7 men) had diffuse cutaneous SSc (dSSc). The age of patients (mean ± S.D.) was 50 ± 16 yrs. Patients with dSSc were aged 49 ± 18, while those with lSSc were 53 ± 14-yrs-old. The disease duration of patients with lSSc and dSSc was 8.3 ± 9.3 and 3.0 ± 2.9 yrs, respectively. None of SSc patients was treated with oral corticosteroid, D-penicillamine or other immunosuppressive therapy at the evaluation. Antinuclear antibody (Ab) was determined by indirect immunofluorescence using HEp-2 cells as the substrate, and specificities of autoantibody were further assessed by ELISA and immunoprecipitation. Anticentromere Ab was positive for 23 patients (2 dSSc and 21 lSSc), anti-topoisomerase I Ab for 32 (26 dSSc and 6 lSSc), anti-U1RNP Ab for 2 (all lSSc), anti-U3RNP Ab for 1 (dSSc), anti-RNA polymerases I and III Ab for 7 (all dSSc) and Th/To Ab for 1 (lSSc). The remaining 4 patients were negative for autoantibodies. Twenty-three age- and sex-matched healthy Japanese individuals were used as normal controls. Smokers were excluded in this study. Fresh venous blood samples were centrifuged shortly after clot formation. All samples were stored at –70°C prior to use.

Clinical assessment
Complete medical histories, physical examinations, and laboratory tests, including vital capacity (VC) and diffusion capacity for carbon monoxide (DLco), were conducted for all patients. When the DLco and VC were <75% and <80%, respectively, of the predicted normal values, they were considered to be abnormal. Skin score was measured by scoring technique of the modified Rodnan total skin thickness score (modified Rodnan TSS) as previously described [24]: The anatomical areas were rated as 0 (normal skin thickness), 1+ (mild but definite thickening), 2+ (moderate skin thickening) and 3+ (severe skin thickening) and the modified Rodnan TSS was derived by summation of the score from all 17 areas (range 0–51). Organ involvement was defined as described previously with some modifications [25]: pulmonary fibrosis = bibasilar fibrosis on chest radiography and high-resolution computed tomography; isolated pulmonary hypertension = clinical evidence of pulmonary hypertension and increased systolic pulmonary arterial pressure (>35 mmHg) by Doppler echocardiography, in the absence of severe pulmonary interstitial fibrosis; oesophagus = hypomotility shown by barium radiography; joints = inflammatory polyarthralgias or arthritis; heart = pericarditis, congestive heart failure, arrhythmias requiring treatment; kidney = malignant hypertension and rapidly progressive renal failure with no other explanation; muscle = proximal muscle weakness and elevated serum creatine kinase. Renal vascular resistance was determined as pulsatility index by colour-flow Doppler ultrasonography of the renal interlobar arteries of both kidneys [26]. The pulsatility index, which represents vascular impedance, was calculated as A-B/mean, where A is the peak systolic frequency, B is the end diastolic frequency and the mean is the time-averaged frequency. The pulsatility index was calculated as an average value obtained with eight waveforms on the renal interlobar arteries of both kidneys. The protocol was approved by Kanazawa University Graduate School of Medical Science and Kanazawa University Hospital and informed consent was obtained from all patients.

ELISA for anti-Prx I Ab
ELISA was performed as previously described [27]. Briefly, 96-well plates were coated with human recombinant Prx I (0.3 µg/µl in 50 mM Hepes buffer, pH 7.0, and 5% trehalose; Sigma-Aldrich Co., St. Louis, MO, USA) at 4°C overnight. The wells were blocked with 2% bovine serum albumin (BSA) and 1% gelatin in Tris-buffered saline (TBS) for 1 hour at 37°C. After washing twice with TBS, the serum samples (100 µl) diluted to 1:100 in TBS containing 1% BSA were added to triplicate wells and incubated for 90 minutes at 20°C. After washing four times with TBS containing 0.05% Tween-20, the plates were incubated with alkaline phosphatase-conjugated goat anti-human IgG or IgM Abs (Cappel, Durham, NC, USA) for 1 hour at 20°C. After washing 4 times with TBS containing 0.05% Tween-20, substrate solution containing 0.91 µg/µl p-nitrophenyl phosphate (Sigma-Aldrich) in diethanolamine buffer (1 M diethanolamine, 0.5 M MgCl2) was added and the optical density (OD) of the wells at 405 nm was subsequently determined. Absorbance values greater than the mean + 2 S.D. of normal controls were considered positive in this study.

Relative levels of autoantibodies were determined for each group of patients and normal controls using pooled serum samples as described previously [27]. Among each disease or control group, the same amounts of all the serum samples were pooled into a single tube. Then, the pooled sera were diluted at log intervals (1:10–1:105) and were subjected to ELISA assays to obtain OD vs dilution (log scale). The dilutions of pooled sera giving half-maximal OD values were determined by linear regression analysis, thus generating mean arbitrary unit per millilitre values for comparison between sets of sera. We could not generate arbitrary unit in each serum sample because of a large amount of the serum sample required for this purpose.

ELISA for 8-isoprostane
ELISA for serum 8-isoprostane levels was performed as previously described [10] using specific ELISA kits (Cayman, Ann Arbor, MI, USA), according to the manufacturer's protocol. Each sample was tested in duplicate. The detection limit of this assay was 5 pg/ml.

Immunoblotting
Human recombinant Prx I (3 µg/lane; Sigma-Aldrich) was subjected to electrophoresis and electrotransferred to nitrocellulose sheets. The nitrocellulose sheets were cut into strips and incubated overnight with serum samples diluted 1:100. Then, the strips were incubated for 1.5 hours with alkaline phosphatase-conjugated goat anti-human IgG Ab (Cappel). Colour was developed using 5-bromo-4-chloro-3-indrolyl phosphate and nitro blue tetrazolium (Sigma-Aldrich). Ten SSc patients positive for IgG anti-Prx I Ab by ELISA, 9 SSc patients positive for either anti-topoisomerase I or anticentromere Ab, but not for IgG anti-Prx I Ab by ELISA, and 5 healthy individuals were evaluated.

Prx I activity assay
IgG was purified from serum samples using magnetic beads coated with recombinant protein G covalently coupled to the surface (Dynal, Lake Success, NY, USA). Final IgG concentration was measured by spectrophotometer (Gene Quant II, Amersham Biosciences, Piscataway, NJ, USA). Prx I activity was determined using yeast thioredoxin reductase system as described with some modifications [28]: first, a pre-reaction cocktail, which contained 50 mM Hepes-NaOH buffer (pH 7.0), 1 mM EDTA, 200 µM NADPH (Sigma-Aldrich), 1.5 µM yeast thioredoxin (LabFrontier, Seoul, Korea), and 0.8 µM yeast thioredoxin reductase (LabFrontier) in 180 µl total volume, was prepared. Next, 2 µg of Prx I (6.7 µl) was incubated with 60 µg of purified IgG (60 µl) for 30 minutes at 20°C. Then, Prx I treated with IgG (66.7 µl in total volume) and 20 µl of 100 µM H2O2 were added to each well in the ELISA plate, and immediately 180 µl of pre-reaction cocktail was added to each well. Five minutes later, absorbance values at 340 nm were determined. Similarly, absorbance values for 2 µg and 0 µg of Prx I untreated with IgG were determined in triplicate wells, respectively. The mean values for 2 µg and 0 µg of Prx I untreated with IgG were 0.572 and 0.643, respectively. Thus, absorbance reduction per 5 minutes at 340 nm due to 2 µg of Prx I-mediated NADPH oxidation was calculated as 0.071. To clearly demonstrate the difference of Prx I activity for each group, absorbance value was converted into % Prx I activity by calculating the ratio of its absorbance value to controls. Ten SSc patients positive for IgG anti-Prx I Ab, 10 SSc patients positive for either anti-topoisomerase I Ab, anti-centromere Ab, or anti-U1RNP Ab but not for IgG anti-Prx I Ab, and 10 healthy individuals were assessed in the current assay.

Statistical analysis
Statistical analysis was performed using the Mann–Whitney U-test for determining the level of significance of differences between sample means and Fisher's exact probability test for comparison of frequencies, and Bonferroni's test for multiple comparisons. Spearman's rank correlation coefficient was used to examine the relationship between two continuous variables. A P-value < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Anti-Prx I autoantibody by ELISA
The levels and presence of anti-Prx I Ab in serum samples from SSc patients and normal controls were assessed by ELISA (Fig. 1). The dilution of pooled sera giving half-maximal OD values in ELISA was also determined to generate mean arbitrary units per millilitre that could be directly compared between patient group and control group. IgG anti-Prx I Ab levels in lSSc patients were significantly 3.5-fold higher than those found in normal controls (P < 0.01). Similarly, dSSc patients exhibited significantly elevated IgG anti-Prx I Ab levels by 2.8-fold compared with normal controls (P < 0.05). In contrast, IgM anti-Prx I Ab levels in patients with dSSc or lSSc were not significantly elevated relative to controls. IgG or IgM anti-Prx I Ab levels in dSSc patients were similar to those in lSSc patients. OD values greater than the mean + 2 S.D. (0.537 for IgG anti-Prx I Ab and 0.683 for IgM anti-Prx I Ab) of normal controls were considered positive in this study (Fig. 1). In total patients with SSc, IgG or IgM anti-Prx I Ab was detected in 39% (27/70). IgG or IgM anti-Prx I Ab was detected in 37% (11/30) of lSSc patients and similar positivity was observed in dSSc patients (40%, 16/40). In contrast, IgG or IgM anti-Prx I Ab was detected in only one healthy individual (4%, 1/23). Thus, IgG but not IgM anti-Prx I Ab levels were elevated in SSc.


Figure 1
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 1. IgG and IgM anti-Prx I Ab levels in serum samples from patients with lSSc, those with dSSc, and healthy controls (CTL). Anti-Prx I Ab levels were determined by ELISA. The short bar indicates the mean value in each group; broken line indicates the mean + 2 S.D. level of healthy controls. Values in parentheses represent the dilutions of pooled sera giving half-maximal optical density (OD) values in ELISAs, which were determined by linear regression analysis to generate mean arbitrary units per millilitre that could be directly compared between each group of SSc and healthy controls.

 
Clinical correlation
Then, we assessed clinical correlation of anti-Prx I Ab in SSc patients. SSc patients positive for IgG anti-Prx I Ab had significantly longer disease duration (P < 0.05), more frequent presence of pulmonary fibrosis (P < 0.05) and cardiac involvement (P < 0.01), and decreased %VC (P < 0.05) and %DLco (P < 0.01) than those negative (Table 1). Regarding correlation of IgG anti-Prx I Ab levels with clinical parameters, IgG anti-Prx I Ab levels also correlated inversely with %DLco (R = –0.37, P < 0.005; Fig. 2a) or %VC (R = –0.36, P < 0.005; Fig. 2b). Furthermore, anti-Prx I Ab levels correlated positively with disease duration (R = 0.284, P < 0.05), renal vascular resistance, which was determined as the pulsatility index value in the renal interlobular arteries by colour-flow Doppler scans (R = 0.33, P < 0.05; Fig. 2c) [26], and positively with serum levels of 8-isoprostane, a reliable marker of oxidative stress (R = 0.43, P < 0.001; Fig. 2d). However, IgG anti-Prx I Ab levels did not correlate with any other clinical parameters, including the modified Rodnan TSS. Frequency of IgM anti-Prx I Ab positivity did not correlate with the presence or absence of any organ involvement (data not shown). In addition, IgM anti-Prx I Ab levels did not correlate with any clinical parameters.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Clinical and laboratory features of SSc patients with IgG anti-Prx I Ab

 

Figure 2
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 2. The correlation of anti-Prx I Ab levels against %DLco (a), %VC (b), pulsatility index (c) and serum 8-isoprostane levels (d) in SSc patients. Anti-Prx I Ab levels and serum 8-isoprostane levels were determined by ELISA. The pulsatility index is a parameter for renal vascular resistance determined by colour-flow Doppler ultrasonography of the renal interlobar arteries of both kidneys.

 
SSc patients positive for IgG anti-Prx I Ab had significantly higher frequency of elevated levels of serum IgG, IgA, and IgM and erythrocyte sedimentation rates (ESR) compared with those negative (P < 0.01, P < 0.05, P < 0.05 and P < 0.05, respectively; Table 1). Anti-topoisomerase I Ab was more frequently detected in SSc patients with anti-Prx I Ab than in those negative (P < 0.05). Regarding correlation of IgG anti-Prx I Ab levels with immunological parameters, IgG anti-Prx I Ab levels correlated positively with levels of serum IgG (R = 0.422, P < 0.001), IgA (R = 0.275, P < 0.05) and IgM (R = 0.322, P < 0.01) and ESR (R = 0.323, P < 0.01). However, IgG anti-Prx I Ab levels did not correlate with any other immunological parameters. In addition, IgM anti-Prx I Ab levels did not correlate with any immunological parameters. Thus, the presence of IgG anti-Prx I Ab was associated with longer disease duration, the severity of pulmonary fibrosis, renal vascular damage, increased serum immunoglobulin, increased serum 8-isoprostane levels, elevated ESR and more frequent presence of anti-topoisomerase I Ab in SSc.

Immunoblotting analysis for anti-Prx I Ab
The presence of anti-Prx I Ab was evaluated by immunoblotting analysis using human recombinant Prx I. Serum samples from SSc patients positive for IgG anti-Prx I Ab by ELISA exhibited reactivity with Prx I (25 kDa) by immunoblotting (Fig. 3, lanes 2–5). By contrast, no reactivity with Prx I was observed using serum samples with either anti-topoisomerase I Ab, anticentromere Ab, or anti-U1RNP Ab, but without IgG anti-Prx I Ab by ELISA (lane 6 and data not shown). Furthermore, serum samples from healthy individuals did not react with Prx I (lane 7). Thus, the presence of anti-Prx I Ab in patients with SSc was confirmed by immunoblotting analysis.


Figure 3
View larger version (78K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 3. Immunoblotting analysis of IgG anti-Prx I Ab in serum samples from SSc patients. Lane 1: colloidal gold-stained Prx I. Lanes 2–5: serum samples from SSc patients positive for IgG anti-Prx I Ab by ELISA. Lane 6: a serum sample from an SSc patient positive for anti-topoisomerase I Ab, but not for IgG anti-Prx I Ab by ELISA. Lane 7: a serum sample from a healthy individual. Markers for molecular weights (kDa) are shown to the left.

 
Inhibition of Prx I activity by IgG isolated from serum samples of SSc patients which contained IgG anti-Prx I Ab
To determine the functional relevance of anti-Prx I Ab in vivo, we assessed whether anti-Prx I Ab was able to inhibit Prx I enzymatic activity. Prx I activity was determined using yeast thioredoxin reductase system. Absorbance reduction of Prx I-mediated NADPH oxidation was measured by ELISA. The Prx I activity was not inhibited by IgG isolated from healthy individuals (Fig. 4). In contrast, IgG isolated from serum samples of SSc patients positive for IgG anti-Prx I Ab by ELISA significantly inhibited the Prx I activity by 59% compared with healthy controls (P < 0.01). The Prx I activity was not inhibited by IgG isolated from serum samples that contained autoantibodies against topoisomerase I, centromere, or U1RNP, but not IgG anti-Prx I Ab. Thus, IgG isolated from serum samples of SSc patients, which contained anti-Prx I Abs, was able to inhibit the Prx I enzymatic activity.


Figure 4
View larger version (10K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 4. Inhibition of Prx I enzymatic activity by IgG isolated from serum samples that contained anti-Prx I Ab. IgG was purified from serum samples of SSc patients positive for IgG anti-Prx I Ab by ELISA [anti-Prx I (+)], those positive for either anti-topoisomerase I Ab, anti-centromere Ab, or anti-U1RNP Ab but not for IgG anti-Prx I Ab [anti-Prx I (–)], and healthy controls (CTL). Prx I enzymatic activity is shown as percentage of IgG-untreated Prx I that was defined as 100%. Each histogram shows the mean (+SEM) values obtained from 10 subjects of each group.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The present study is the first to reveal that IgG anti-Prx I Ab levels were significantly elevated in serum samples from SSc patients relative to normal controls by ELISA. The presence of anti-Prx I Ab was further confirmed by immunoblotting analysis. However, this autoantibody is not specific for SSc, since it is detected in patients with other autoimmune disorders [19]. Interestingly, IgG anti-Prx I Ab levels correlated with the severity of lung fibrosis, the extent of renal vascular damage, and the presence of heart involvement. Moreover, the presence of IgG anti-Prx I Ab was related to the immunological abnormalities, including hyperproduction of immunoglobulins and inflammation as shown by elevated ESR. Thus, the results of the present study indicate that elevated anti-Prx I Ab is generally associated with the disease severity in SSc.

There were no significant differences in anti-Prx I Ab levels between dSSc and lSSc patients. Furthermore, the presence and levels of anti-Prx I Ab did not correlate with skin involvements including modified Rodnan TSS, the presence of pitting scars, diffuse pigmentation, and contracture of phalanges. Similarly, when analysed in dSSc patients, there was no correlation between anti-Prx I Ab levels and modified Rodnan TSS (data not shown). Thus, although anti-Prx I Ab correlated with the severity of the pulmonary fibrosis, it did not correlate with the extent of skin sclerosis. The reasons for this discrepancy remained unknown. Several longitudinal studies have shown that in dSSc patients, skin scores rapidly increase over the first several years, and then gradually decrease [29, 30]. The finding that anti-Prx I Ab correlated with longer disease duration in SSc suggests that anti-Prx I Ab is not likely to reflect ongoing skin fibrosis but reflects the accumulation of oxidative stress in some internal organs. Collectively, these results suggest that anti-Prx I Ab is not a suitable marker for predicting the activity or stage of SSc but useful in determining the accumulation of oxidative stress and the severity of visceral involvement.

Remarkably, in the current study, IgG isolated from serum samples of SSc patients, which contained anti-Prx I Abs, was able to inhibit Prx I enzymatic activity. In contrast, the Prx I activity was not inhibited by IgG isolated from serum samples of healthy individuals and those of SSc patients that had autoantibodies against topoisomerase I, centromere, or U1RNP, but not IgG anti-Prx I Ab. These results suggest that IgG anti-Prx I Ab inhibits Prx enzymatic activity. However, as IgG anti-Prx I Ab was not purified from total IgG, the current study did not formally prove that IgG anti-Prx I Ab itself inhibits Prx I activity. Nevertheless, this is the first study suggesting a possible functional relevance of anti-Prx I Ab in vivo. Moreover, anti-Prx I Ab levels closely correlated with serum levels of 8-isoprostane, a marker of oxidative stress (Fig. 2d). These results suggest the possibility that anti-Prx I Ab could enhance the oxidative stress by inhibiting Prx I enzymatic activity in SSc. However, it remains still controversial whether autoantibodies found in SSc have a pathogenetic role or they are merely by-products of underlying disease process [31], because autoantigens in SSc are generally intracellular molecules, and there is little evidence that autoantibody can penetrate into viable cells. Prx I also exists in the cytosol and nucleus [16, 32], and is not secreted outside the cells under physiological condition. Therefore, it remains unknown whether the presence of anti-Prx I Ab is related to the development of SSc.

There are two possibilities that might explain the pathogenetic role of anti-Prx I Ab in SSc. One possibility is that Prx I could be secreted or leaked from cells in SSc. Overexpression and cellular release of Prx I are shown in patients with lung cancer and in cultured astrocyte cells [33, 34]. Prx I overexpression is also induced by cellular proliferation, differentiation, oxidative stress and pro-inflammatory cytokines [18]. Furthermore, it has been suggested that excessive Prx I leads to self-aggregation or aggregation with other intracellular proteins, resulting in cell death [18], because Prx I tends to form large insoluble aggregates [12, 35]. Taken together, it is possible that Prx I is also overexpressed/released or leaked from apoptotic cells in SSc patients. Another possibility is cross-reactivity of anti-Prx I Ab with membrane or secreted isoforms of Prxs II and IV [15]: the structure and sequence of the peroxidatic active site is highly conserved among Prxs I–IV [14, 15] and all Prxs I–IV can form both homodimers and heterodimers [18]. In fact, it has been found that Prx I and Prx IV form a heterodimer [36]. If released or leaked, Prx I could play an important role as an extracellular antioxidant enzyme like extracellular superoxide dismutase and glutathione peroxidases [37]. Alternatively, if anti-Prx I Ab cross-reacts with other extracellular Prxs, anti-Prx I Ab could contribute to enhance the oxidative stress by inhibiting enzymatic activity of extracellular Prxs. In the current study, we could not detect serum Prx I in SSc patients by immunoblotting analysis (data not shown) and did not assess whether Prx I was detected in sera from SSc patients and whether anti-Prx I Ab cross-reacted with Prxs I, II and IV. Therefore, further research will be required to address these issues.

In SSc, ischaemic-reperfusion injury due to Raynaud's phenomenon increases the production of reactive oxygen species [38]. Because endothelial cells lack catalase, an antioxidant enzyme [39], and are susceptible to reactive oxygen species, which results in endothelial cell damage, including intimal proliferation and fibrous thickening of the media. Furthermore, the exposure of nuclear contents of damaged endothelial cells may provoke an autoimmune response to various nuclear antigens, leading to autoantibody formation and further recruitment of inflammatory cells [40]. That Prx I is also abundantly expressed in endothelial cells and overexpressed in proliferating cells, which suggests that long-term injury of endothelial cells by oxidative stress increases the chance of Prx I overexpression and fragmentation, leading to anti-Prx I autoantibody production. Consistent with this, the presence of anti-Prx I Ab correlated with longer disease duration and inflammation in SSc. On the other hand, there was strong correlation between anti-Prx I Ab levels and the severity of lung fibrosis. Several studies have shown Prx I overexpression in lung diseases, such as sarcoidosis and cancer [33, 41]. Interestingly, in patients with non-small cell lung cancer, both anti-Prx I Ab and Prx I are detected in their serum samples [33]. Collectively, anti-Prx I Ab may be secondarily produced by the exposure of the cryptic epitope produced by the long-term exposure of oxidative stress, which in turn could enhance oxidative stress in SSc.

In conclusion, our study suggests that IgG anti-Prx I autoantibody is related to enhanced oxidative stress and is a useful serological marker for the disease severity of SSc. In addition, this autoantibody may contribute to the development of SSc by inhibiting Prx I enzymatic activity.

Formula

The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Sato S, Hayakawa I, Hasegawa M, Fujimoto M, Takehara K. (2003) Function blocking autoantibodies against matrix metalloproteinase-1 in patients with systemic sclerosis. J Invest Dermatol 120:542–7.[CrossRef][Web of Science][Medline]
  2. Murrell DF. (1993) A radical proposal for the pathogenesis of scleroderma. J Am Acad Dermatol 28:78–85.[Web of Science][Medline]
  3. Butler AR, Flitney FW, Williams DL. (1995) NO, nitrosonium ions, nitroxide ions, nitrosothiols and iron-nitrosyls in biology: a chemist's perspective. Trends Pharmacol Sci 16:18–22.[CrossRef][Medline]
  4. Suematsu M, Wakabayashi Y, Ishimura Y. (1996) Gaseous monoxides: a new class of microvascular regulator in the liver. Cardiovasc Res 32:679–86.[CrossRef][Web of Science][Medline]
  5. Sambo P, Baroni SS, Luchetti M, et al. (2001) Oxidative stress in scleroderma: maintenance of scleroderma fibroblast phenotype by the constitutive up-regulation of reactive oxygen species generation through the NADPH oxidase complex pathway. Arthritis Rheum 44:2653–64.[CrossRef][Web of Science][Medline]
  6. Murrell GA, Francis MJ, Bromley L. (1990) Modulation of fibroblast proliferation by oxygen free radicals. Biochem J 265:659–65.[Web of Science][Medline]
  7. Stein CM, Tanner SB, Awad JA, Roberts LJ 2nd, Morrow JD. (1996) Evidence of free radical-mediated injury (isoprostane overproduction) in scleroderma. Arthritis Rheum 39:1146–50.[Web of Science][Medline]
  8. Volpe A, Biasi D, Caramaschi P, et al. (2006) Levels of F2-isoprostanes in systemic sclerosis: correlation with clinical features. Rheumatology 45:314–20.[Abstract/Free Full Text]
  9. Montuschi P, Ciabattoni G, Paredi P, et al. (1998) 8-Isoprostane as a biomarker of oxidative stress in interstitial lung diseases. Am J Respir Crit Care Med 158:1524–7.[Abstract/Free Full Text]
  10. Ogawa F, Shimizu K, Muroi E, et al. (2006) Serum levels of 8-isoprostane, a marker of oxidative stress, are elevated in patients with systemic sclerosis. Rheumatology 45:815–8.[Abstract/Free Full Text]
  11. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. (2007) Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39:44–84.[CrossRef][Web of Science][Medline]
  12. Chae HZ, Chung SJ, Rhee SG. (1994) Thioredoxin-dependent peroxide reductase from yeast. J Biol Chem 269:27670–8.[Abstract/Free Full Text]
  13. Chae HZ, Robison K, Poole LB, Church G, Storz G, Rhee SG. (1994) Cloning and sequencing of thiol-specific antioxidant from mammalian brain: alkyl hydroperoxide reductase and thiol-specific antioxidant define a large family of antioxidant enzymes. Proc Natl Acad Sci USA 91:7017–21.[Abstract/Free Full Text]
  14. Rhee SG, Chae HZ, Kim K. (2005) Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radic Biol Med 38:1543–52.[CrossRef][Web of Science][Medline]
  15. Wood ZA, Schroder E, Robin Harris J, Poole LB. (2003) Structure, mechanism and regulation of peroxiredoxins. Trends Biochem Sci 28:32–40.[CrossRef][Web of Science][Medline]
  16. Immenschuh S and Baumgart-Vogt E. (2005) Peroxiredoxins, oxidative stress, and cell proliferation. Antioxid Redox Signal 7:768–77.[CrossRef][Web of Science][Medline]
  17. Hofmann B, Hecht HJ, Flohe L. (2002) Peroxiredoxins. Biol Chem 383:347–64.[CrossRef][Web of Science][Medline]
  18. Butterfield LH, Merino A, Golub SH, Shau H. (1999) From cytoprotection to tumor suppression: the multifactorial role of peroxiredoxins. Antioxid Redox Signal 1:385–402.[Medline]
  19. Karasawa R, Ozaki S, Nishioka K, Kato T. (2005) Autoantibodies to peroxiredoxin I and IV in patients with systemic autoimmune diseases. Microbiol Immunol 49:57–65.[Web of Science][Medline]
  20. Okano Y. (1996) Antinuclear antibody in systemic sclerosis (scleroderma). Rheum Dis Clin North Am 22:709–35.[CrossRef][Web of Science][Medline]
  21. Kodera M, Hayakawa I, Komura K, et al. (2005) Anti-lipoprotein lipase antibody in systemic sclerosis: association with elevated serum triglyceride concentrations. J Rheumatol 32:629–36.[Abstract/Free Full Text]
  22. Committee SfscotARADaTC. (1980) Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis Rheum 23:581–90.[Web of Science][Medline]
  23. LeRoy EC, Black C, Fleischmajer R, et al. (1988) Scleroderma (systemic sclerosis): classification, subsets and pathogenesis. J Rheumatol 15:202–5.[Web of Science][Medline]
  24. Clements PJ, Lachenbruch PA, Seibold JR, et al. (1993) Skin thickness score in systemic sclerosis: an assessment of interobserver variability in 3 independent studies. J Rheumatol 20:1892–6.[Web of Science][Medline]
  25. Sato S, Ihn H, Kikuchi K, Takehara K. (1994) Antihistone antibodies in systemic sclerosis. Association with pulmonary fibrosis. Arthritis Rheum 37:391–4.
  26. Nishijima C, Sato S, Hasegawa M, et al. (2001) Renal vascular damage in Japanese patients with systemic sclerosis. Rheumatology 40:406–9.[Abstract/Free Full Text]
  27. Sato S, Hasegawa M, Fujimoto M, Tedder TF, Takehara K. (2000) Quantitative genetic variation in CD19 expression correlates with autoimmunity. J Immunol 165:6635–43.[Abstract/Free Full Text]
  28. Kim JA, Park S, Kim K, Rhee SG, Kang SW. (2005) Activity assay of mammalian 2-cys peroxiredoxins using yeast thioredoxin reductase system. Anal Biochem 338:216–23.[CrossRef][Web of Science][Medline]
  29. Clements PJ. (2000) Systemic sclerosis (scleroderma) and related disorders: clinical aspects. Baillieres Best Pract Res Clin Rheumatol 14:1–16.[CrossRef][Medline]
  30. Mayes MD, O’Donnell D, Rothfield NF, Csuka ME. (2004) Minocycline is not effective in systemic sclerosis: results of an open-label multicenter trial. Arthritis Rheum 50:553–7.[CrossRef][Web of Science][Medline]
  31. Ho KT and Reveille JD. (2003) The clinical relevance of autoantibodies in scleroderma. Arthritis Res Ther 5:80–93.[Web of Science][Medline]
  32. Masutani H, Ueda S, Yodoi J. (2005) The thioredoxin system in retroviral infection and apoptosis. Cell Death Differ 12:Suppl 1, 991–8.[CrossRef][Web of Science][Medline]
  33. Chang JW, Lee SH, Jeong JY, et al. (2005) Peroxiredoxin-I is an autoimmunogenic tumor antigen in non-small cell lung cancer. FEBS Lett 579:2873–7.[CrossRef][Web of Science][Medline]
  34. Lafon-Cazal M, Adjali O, Galeotti N, et al. (2003) Proteomic analysis of astrocytic secretion in the mouse. Comparison with the cerebrospinal fluid proteome. J Biol Chem 278:24438–48.
  35. Schroder E, Willis AC, Ponting CP. (1998) Porcine natural-killer-enhancing factor-B: oligomerisation and identification as a calpain substrate in vitro. Biochim Biophys Acta 1383:279–91.[CrossRef][Medline]
  36. Jin DY, Chae HZ, Rhee SG, Jeang KT. (1997) Regulatory role for a novel human thioredoxin peroxidase in NF-kappaB activation. J Biol Chem 272:30952–61.[Abstract/Free Full Text]
  37. Rahman I, Biswas SK, Kode A. (2006) Oxidant and antioxidant balance in the airways and airway diseases. Eur J Pharmacol 533:222–39.[CrossRef][Web of Science][Medline]
  38. Simonini G, Pignone A, Generini S, Falcini F, Cerinic MM. (2000) Emerging potentials for an antioxidant therapy as a new approach to the treatment of systemic sclerosis. Toxicology 155:1–15.[CrossRef][Medline]
  39. Shingu M, Yoshioka K, Nobunaga M, Yoshida K. (1985) Human vascular smooth muscle cells and endothelial cells lack catalase activity and are susceptible to hydrogen peroxide. Inflammation 9:309–20.[CrossRef][Web of Science][Medline]
  40. Peng SL, Fatenejad S, Craft J. (1997) Scleroderma: a disease related to damaged proteins? Nat Med 3:276–8.[CrossRef][Web of Science][Medline]
  41. Kinnula VL, Lehtonen S, Kaarteenaho-Wiik R, et al. (2002) Cell specific expression of peroxiredoxins in human lung and pulmonary sarcoidosis. Thorax 57:157–64.[Abstract/Free Full Text]
Submitted 23 October 2006; revised version accepted 3 January 2007.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
46/5/790    most recent
kem010v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (5)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Iwata, Y.
Right arrow Articles by Sato, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Iwata, Y.
Right arrow Articles by Sato, S.
Related Collections
Right arrow Systemic Sclerosis
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?