Serum soluble CTLA-4 levels are increased in diffuse cutaneous systemic sclerosis

S. Sato, M. Fujimoto1, M. Hasegawa, K. Komura, K. Yanaba, I. Hayakawa, T. Matsushita and K. Takehara

Department of Dermatology, Kanazawa University Graduate School of Medical Science, Kanazawa and 1 Department of Regenerative Medicine, Research Institute, International Medical Center of Japan, Tokyo, Japan.

Correspondence to: S. Sato, Department of Dermatology, Kanazawa University Graduate School of Medical Science, 13–1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan. E-mail: s-sato{at}med.kanazawa-u.ac.jp


    Abstract
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. To determine serum levels of soluble cytotoxic T-lymphocyte associated molecule-4 (sCTLA-4) and clinical association in patients with systemic sclerosis (SSc).

Methods. Serum sCTLA-4 levels from 32 patients with diffuse cutaneous SSc (dSSc) and 27 patients with limited cutaneous SSc (lSSc) were examined by enzyme-linked immunosorbent assay (ELISA). For a longitudinal study, 211 sera from 30 SSc patients were analysed (follow-up 2.1–7.0 yr).

Results. Serum sCTLA-4 levels were elevated in dSSc patients compared with normal controls (n = 41), lSSc patients and patients with active systemic lupus erythematosus (SLE) (n = 23). By contrast, sCTLA-4 levels in patients with lSSc or SLE were normal. SSc patients with elevated sCTLA-4 levels had a shorter disease duration and more frequent presence of digital pitting scars, contracture of phalanges, diffuse pigmentation, pulmonary fibrosis and decreased percentage vital capacity (%VC) than those with normal sCTLA-4 levels. sCTLA-4 levels correlated positively with the extent of skin fibrosis, serum IgG levels and anti-topoisomerase I antibody levels. In a longitudinal study, sCTLA-4 levels decreased on a parallel with improvement of skin sclerosis in five dSSc patients. Skin sclerosis did not improve in two of six dSSc patients with high sCTLA-4 levels throughout the follow-up, while the remaining four patients showed improvement of skin sclerosis.

Conclusion. These results suggest that sCTLA-4 correlates with disease severity and activity of SSc and that sCTLA-4 plays a role in immunological abnormalities of SSc, since sCTLA-4 may augment humoral immune responses as well as T-cell responses by interfering with B7-CTLA-4 interactions that induce negative signals in T and B cells.

KEY WORDS: Cytotoxic T-lymphocyte associated molecule-4, Soluble form, Diffuse cutaneous systemic sclerosis, Autoantibody


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Systemic sclerosis (SSc) is a connective tissue disease characterized by excessive extracellular matrix deposition in the skin and other visceral organs. A variety of immunological abnormalities of T and B lymphocytes have been detected in SSc [1]. Autoantibodies are positive in over 90% of patients and these autoantibodies react to various intracellular components, such as deoxyribonucleic acid (DNA) topoisomerase I, centromere, ribonucleic acid (RNA) polymerases, U1RNP and U3RNP [2]. In addition, hyper-{gamma}-globulinaemia and polyclonal B-cell hyperactivity are detected in SSc patients [3, 4]. Cutaneous mononuclear cell infiltrates in SSc are mainly activated T cells which are also increased in the lung interstitium and bronchoalveolar fluids from SSc patients with active lung disease [1]. Furthermore, there is evidence of circulating T-cell activation in SSc [5]. The activated T cells may promote fibrosis and endothelial damage, probably through the production of soluble mediators or cytotoxic effects in patients with SSc [1, 6].

T-cell activation requires the delivery of at least two signals to T cells by the antigen-presenting cell [7]. One signal is provided by the ligation of the T-cell receptor by the complex of antigen and major histocompatibility complex, while the second co-stimulatory signal is generated by the interaction between CD28 on T cells and its ligands on the antigen-presenting cell, B7.1 (CD80) and B7.2 (CD86) [7]. Cytotoxic T-lymphocyte associated molecule-4 (CTLA-4, CD152), a molecule homologous to CD28, is expressed on activated T cells and binds with higher affinity to B7.1 and B7.2 molecules [7, 8]. CTLA-4 can engage inhibitory signal transduction machinery, including the phosphatase SHP-2, which opposes the action of kinases downstream of CD3 and CD28 [7, 8]. This negative signal provided by CTLA-4 results in the down-regulation of T-cell responses and induction of apoptosis and immunological anergy [7–9]. CTLA-4 is also expressed on activated B cells and has a negative role for humoral immune responses [10]. Recent studies have revealed an alternative transcript of the CTLA4 gene encoding a protein that lacks a transmembrane region and probably represents a native soluble form of CTLA-4 (sCTLA-4) [11]. sCTLA-4 is a functional receptor, since the amino acid residues critical to B7 molecule binding are intact [11] and sCTLA-4 immunoreactivity is inhibited by its binding to B7.1 [12]. Several reports have shown that sCTLA-4 levels are elevated in sera from patients with some autoimmune disorders, such as autoimmune thyroid disease [12], systemic lupus erythematosus (SLE) [13] and myasthenia gravis [14]. The abnormal activation of lymphocytes in SSc suggests the possible defect in the inhibitory molecules including CTLA-4; however, sCTLA-4 levels have not previously been investigated in SSc. In this study, we examined serum sCTLA-4 levels and their clinical correlation in patients with SSc. Furthermore, we performed a retrospective longitudinal study of sCTLA-4 levels in some of these SSc patients to determine whether or not sCTLA-4 levels are associated with disease activity.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Serum samples
Blood samples were obtained from 59 Japanese SSc patients (52 females and 7 males; age 50±17 yr). All patients fulfilled the criteria proposed by the American College of Rheumatology [15]. These patients were grouped according to the classification system proposed by LeRoy et al. [16]: 32 patients (30 females and 2 males; age 52±13 yr) had limited cutaneous SSc (lSSc) and 27 patients (22 females and 5 males; age 48±20 yr) had diffuse cutaneous SSc (dSSc). The disease duration of lSSc and dSSc patients was 6.9±8.0 and 3.4±5.6 yr respectively. The duration of the disease was calculated from time of onset of the first clinical event (other than Raynaud's phenomenon) that was a clear manifestation of SSc. None of the SSc patients was treated with corticosteroid, D-penicillamine or other immunosuppressive therapy at their first visit.

Antinuclear antibody (Ab) was determined by indirect immunofluorescence using HEp-2 cells as the substrate and autoantibody specificities were further assessed by enzyme-linked immunsorbent assay (ELISA) and immunoprecipitation. Specifically, anti-centromere Ab was determined by the presence of a discrete speckled pattern with indirect immunofluorescence and was confirmed by ELISA using human recombinant CENP-B as antigen (Medical & Biological Laboratories, Nagoya, Japan). Anti-topoisomerase I Ab was determined by ELISA using human recombinant topoisomerase I as antigen (Medical & Biological Laboratories) as previously described [17] and was further confirmed by immunoprecipitation when the Ab titre was low by ELISA. Anti-centromere Ab was positive for 25 patients, anti-topoisomerase I Ab for 21, anti-U1RNP Ab for two, anti-U3RNP Ab for one, anti-RNA polymerase Ab for six and autoantibody with unknown specificities for three. The remaining one patient was negative for autoantibody. Patients with anti-centromere Ab were grouped into 22 (88%) patients with lSSc and three (12%) with dSSc, whereas patients with anti-topoisomerase I Ab were classified into five (24%) patients with lSSc and 16 (76%) with dSSc.

Twenty-three SLE patients (19 females and 4 males; age 35±17 yr), who fulfilled the American College of Rheumatology criteria [18], were also examined. These patients had active SLE as determined by the SLE Disease Activity Index [19]. Five patients with SLE had been treated with low-dose steroids (prednisolone 5–20 mg/day), although none of the patients had received other immunosuppressive therapy. Forty-one age- and sex-matched healthy Japanese individuals (36 females and 5 males; age 49±15 yr) were used as normal controls. These healthy individuals were working in our hospital and were considered normal by annual health check.

For a retrospective longitudinal study, patients whose serum samples were taken more than four times were analysed. They included 211 serum samples from 30 SSc patients (27 females and 3 males; age 52±17 yr) out of the 59 SSc patients assessed for sCTLA-4 levels at initial visit. These patients were classified into 10 lSSc patients (all females; age 56±9 yr) and 20 dSSc patients (17 females and 3 males; age 50±20 yr). The disease duration of patients with lSSc and dSSc at their first visit was 6.5±4.9 and 2.2±2.1 years respectively. These patients had been followed up for 4.8±1.5 (2.1–7.0) yr and serum samples were collected 6.6±2.0 (4–10) times during the follow-up period. At their first visit, none of the patients had been treated with steroids, D-penicillamine or other immunosuppressive therapy. During the follow-up, 17 dSSc patients received low-dose oral steroid (prednisolone 5–20 mg/day), while the remaining three dSSc patients did not receive oral steroid. Ten dSSc patients received low-dose D-penicillamine (100–300 mg/day) after their first visit. Treatment with steroids or D-penicillamine was not started in any lSSc patients, and none of the SSc patients received any other immunosuppressive therapy throughout the follow-up period. Fresh venous blood samples were centrifuged shortly after clot formation. All samples were stored at –70°C prior to use. The protocol was approved by the Kanazawa University Graduate School of Medical Science, and all study subjects provided informed consent according to the Declaration of Helsinki.

Clinical assessment
Complete medical histories, physical examinations and laboratory tests were conducted for all patients at their first visit, with limited evaluations during follow-up visits. Skin score was measured by scoring technique of the modified Rodnan total skin thickness score (modified Rodnan TSS) [20]. The 17 anatomical areas were rated as 0 (normal skin thickness), 1+ (mild but definite skin thickening), 2+ (moderate skin thickening) and 3+ (severe skin thickening) and the modified Rodnan TSS was derived by summation of the scores from all 17 areas (range 0–51). Organ system involvement was defined as described previously [21, 22]: lung = bibasilar fibrosis on chest radiography and high-resolution computed tomography; oesophagus = hypomotility shown by barium radiography; joint = inflammatory polyarthralgias or arthritis; heart = pericarditis, congestive heart failure or arrhythmias requiring treatment; kidney = malignant hypertension and rapidly progressive renal failure without any other explanation; muscle = proximal muscle weakness and elevated serum creatine kinase. Pulmonary function, including vital capacity (VC) and diffusion capacity for carbon monoxide (DLCO), was also tested. When the DLCO and VC were <75% and <80%, respectively, of the predicted normal values, they were considered to be abnormal.

ELISA
Specific ELISA kits were used for measuring serum sCTLA-4 levels (MedSystems Diagnostics, Vienna, Austria), according to the manufacturer's protocol. Each sample was tested in duplicate. Levels of anti-centromere or anti-topoisomerase I Abs were assessed using specific ELISA (Medical & Biological Laboratories).

Statistical analysis
Statistical analysis was performed using the Mann–Whitney U-test for comparison of sCTLA-4 levels, 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. Analysis of variance (ANOVA) was used to determine the treatment effect on serum sCTLA levels in a longitudinal study. A P value of less than 0.05 was considered statistically significant. All data are shown as means±S.D.


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Serum sCTLA-4 levels in SSc
Serum sCTLA-4 levels were significantly elevated in dSSc patients compared with normal controls (P<0.001), lSSc patients (P<0.01) and active SLE patients (P<0.01, Fig. 1). In contrast, there was no significant difference in sCTLA-4 levels between lSSc patients and normal controls. Patients with active SLE had mean sCTLA-4 levels that were similar to those found in normal controls, although several SLE patients exhibited high sCTLA-4 levels. sCTLA-4 levels did not significantly differ between SLE patients with steroid treatment and those without steroid treatment (data not shown).



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FIG. 1. Serum levels of sCTLA-4 in patients with dSSc, lSSc or SLE and normal controls (Control) at the first evaluation. Serum sCTLA-4 levels were determined by a specific ELISA. The short bar indicates the mean value in each group. A broken line indicates the cut-off value (mean + 2 S.D. of the control samples).

 
Values higher than the mean + 2 S.D. (26.5 ng/ml) of the control serum samples were considered to be elevated in this study. Elevated sCTLA-4 levels were observed in 27% (16/59) of all SSc patients. Regarding the subsets of SSc, serum sCTLA-4 levels were increased in 56% (15/27) of dSSc patients, while only 3% (1/32) of lSSc patients had elevated sCTLA-4 levels. Increased sCTLA-4 levels were detected in 13% (3/23) of SLE patients. By contrast, none of normal controls showed elevated sCTLA-4 levels. Thus, sCTLA-4 levels were elevated in dSSc patients.

Clinical correlation of sCTLA-4 levels in SSc
Clinical and laboratory parameters obtained at the first evaluation were compared between SSc patients with elevated serum sCTLA-4 levels and those with normal sCTLA-4 levels (Table 1). SSc patients with elevated sCTLA-4 levels had shorter disease duration (P<0.05) and more frequent presence of dSSc (P<0.0001), digital pitting scars (P<0.01), contracture of phalanges (P<0.01), diffuse pigmentation (P<0.05), pulmonary fibrosis (P<0.05) and decreased %VC (P<0.01) than those with normal sCTLA-4 levels. Consistent with the association of elevated sCTLA-4 levels with dSSc, sCTLA-4 levels correlated positively with modified Rodnan TSS at the first evaluation (P<0.01, Fig. 2). Furthermore, sCTLA-4 levels correlated positively with serum IgG levels (P<0.05) and anti-topoisomerase I Ab levels determined by ELISA at the first evaluation (P<0.01, Fig. 2). However, sCTLA-4 levels did not significantly correlate with serum levels of IgA, IgM, CH50, C3, C4 and C-reactive protein, anti-centromere Ab levels by ELISA and erythrocyte sedimentation rates (data not shown). Thus, elevated sCTLA-4 levels were associated with early dSSc, the severity of SSc and serum levels of IgG and anti-topoisomerase I Ab.


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TABLE 1. Clinical and laboratory data of patients with SSc showing elevated serum sCTLA-4 levels at the first evaluation. Values of clinical features and organ involvement are percentages

 


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FIG. 2. The correlation of sCTLA-4 levels against modified Rodnan TSS, serum IgG levels and anti-topoisomerase I Ab levels in patients with SSc at the first evaluation. Serum levels of sCTLA-4 and anti-topoisomerase I Ab were determined by a specific ELISA.

 
A longitudinal study of sCTLA-4 levels in SSc
To assess changes in serum sCTLA-4 levels over time, 211 serum samples from 30 SSc patients (20 dSSc and 10 lSSc patients) were analysed. None of these patients had received any treatment at their first visit. Eleven out of 20 dSSc patients exhibited elevated sCTLA-4 levels at their first visit. Serum sCTLA-4 levels in five of these 11 dSSc patients were decreasing during the follow-up of 5.3±1.7 yr (Fig. 3A). Their disease duration at the initial visit was 1.8±1.0 yr. Serum sCTLA-4 levels at the final evaluation were within normal range in four of the five patients. Skin sclerosis was improving in all the patients during the follow-up (modified Rodnan TSS 19.4±0.9 at first visit to 7.4±2.8 at the final evaluation). During the follow-up three patients received oral low-dose steroid alone, one patient received low-dose D-penicillamine and steroid and one patient received neither D-penicillamine nor steroid (open circle in Fig. 3A).



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FIG. 3. Serial changes of serum sCTLA-4 levels during the follow-up period in dSSc patients with elevated sCTLA-4 levels at their first visit (A and B), dSSc patients with normal sCTLA-4 levels at their first visit (C) and lSSc patients with normal sCTLA-4 levels at their first visit (D). Serum sCTLA-4 levels were determined by a specific ELISA. The dashed lines indicate the cut-off value. The closed and open symbols represent patients treated with and without oral low-dose steroid, respectively.

 
Serum sCTLA-4 levels in six of the 11 dSSc patients with high sCTLA-4 levels at first visit remained high during the follow-up of 3.8±1.5 yr (Fig. 3B), although some patients transiently showed normal sCTLA-4 levels during the follow-up. Their disease duration was 2.0±1.5 yr. After initial visit, treatment with steroid alone was started in two of the six patients, whereas treatment with steroid and D-penicillamine was started in two patients. The remaining two patients received neither steroid nor D-penicillamine throughout the follow-up period (open circles in Fig. 3B). In the two patients without steroid or D-penicillamine treatment, skin sclerosis did not improve at the final evaluation, while it improved in the remaining four patients (18.7±2.3 at first visit to 9.3±4.6 at the final evaluation).

Serum sCTLA-4 levels at first visit were normal in nine out of 20 dSSc patients (Fig. 3C). Only one patient exhibited transiently elevated sCTLA-4 levels during the follow-up. However, in general, sCTLA-4 levels were decreasing within the normal range during the follow-up of 4.6±1.4 yr. Their disease duration was slightly longer (2.7±2.7 yr) than that in the two patient groups described above, suggesting that these patients might show elevated sCTLA-4 levels in the earlier phase of the disease. All the patients showed the improvement of skin sclerosis during the follow-up (14.2±5.0 at first visit to 7.4±4.2 at the final evaluation). All the patients received oral low-dose steroid after first visit and seven patients received low-dose D-penicillamine in addition to steroid. In all dSSc patients, treatment with steroid alone or that with steroid and D-penicillamine did not significantly affect sCTLA-4 levels. Finally, all 10 lSSc patients examined in this study exhibited normal sCTLA-4 levels at the first visit that stayed within normal range throughout the follow-up period of 5.2±1.4 yr (Fig. 3D). Thus, sCTLA-4 levels generally decreased on a parallel with the improvement in skin sclerosis, but skin sclerosis did not improve in some patients with high sCTLA-4 levels throughout the follow-up.


    Discussion
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 Abstract
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 Patients and methods
 Results
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 References
 
The present study is the first to reveal that serum sCTLA-4 levels were elevated in dSSc patients but not lSSc patients. The elevation of sCTLA-4 levels was consistently associated with the greater extent of skin fibrosis, the higher frequency of digital vascular damage and contracture of phalanges and the greater frequency and severity of pulmonary fibrosis. Although several SLE patients showed high sCTLA-4 levels, sCTLA-4 levels in total patients with active SLE did not differ from those in normal controls. A previous study reported that SLE patients showed elevated sCTLA-4 levels [13]. Although the reasons for this discrepancy remain unclear, it may be due to the difference in the patient population and treatment: the previous study included inactive SLE patients (~20%) and SLE patients treated with immunosuppressive therapy other than steroids (35%) [13], while they were excluded in this study. Although the elevation of sCTLA-4 was not specific to SSc, these results indicate that it correlates with the disease severity of SSc.

It was previously thought that the risk of internal organ involvement in patients with dSSc increased in parallel with disease duration [23]. However, clinical trials have revealed that skin involvement improves spontaneously, even in untreated patients [24]. Furthermore, a recent study has demonstrated that severe organ damage occurs within the first 3 yr of disease onset in most dSSc patients and progression to severe skin thickening seldom occurs after 5 or 6 yr [23]. Collectively, these observations indicate that SSc is not progressive but has the distinct earlier phase of higher disease activity. Therefore, our finding that SSc patients with elevated sCTLA-4 levels exhibited shorter disease duration (mean 2 yr) suggests that sCTLA-4 levels correlate with the disease activity of SSc. To further clarify the relationship between sCTLA-4 levels and disease activity, we performed longitudinal analysis of sCTLA-4 levels in early dSSc patients with mean disease duration of 2.2 yr.

Our longitudinal analysis demonstrated that as skin sclerosis improved, sCTLA-4 levels were generally decreased. Interestingly, in two dSSc patients who did not show an improvement of skin sclerosis, sCTLA-4 levels did not decrease during the observation period. Since almost all dSSc patients whose sCTLA levels were decreasing during the follow-up received treatment with low-dose steroid, the reduction of sCTLA-4 levels might be caused by steroid treatment. However, despite no treatment, sCTLA-4 levels were decreasing in one patient with high sCTLA-4 levels at first visit. In addition, sCTLA-4 levels were not decreased in all patients following steroid treatment, since 4 of 6 dSSc patients with elevated sCTLA-4 levels throughout the follow-up period also received steroid treatment. Furthermore, a previous and the present studies showed that steroid did not have significant effect on sCTLA-4 levels in SLE [13]. These results suggest that steroid is not the only factor for decreasing sCTLA-4 levels. Therefore, it is possible that sCTLA-4 levels may reflect the disease activity of SSc. However, it should be noted that sCTLA-4 levels did not decrease in several dSSc patients whose skin sclerosis improved.

sCTLA-4 is thought to play a potentially important role in the regulation of immune responses, since it is functionally able to bind with the B7 molecule, thereby interfering with the binding of CD28 and/or CTLA-4 [11, 12, 14, 25]. sCTLA-4 could either inhibit or enhance the immune response and this different effect of sCTLA-4 might depend on the activation status of T cells involved [11–14, 25]. At the initiation of the immune response when T cells do not express the transmembrane form of CTLA-4 (mCTLA-4), sCTLA-4 may block B7–CD28 interactions, interfering with T-cell activation. Consistently, recombinant sCTLA-4 is able to inhibit mixed leucocyte reactions in vitro [11]. On the other hand, during the ongoing immune responses when T cells express mCTLA-4 on their cell surface, sCTLA-4 may block B7–mCTLA-4 interactions, resulting in augmented and sustained T-cell responses. Furthermore, since mCTLA-4 on T cells induces immunological anergy, sCTLA-4 may inhibit induction anergy, leading to the enhanced autoreactivity [9]. Consistent with this, blocking the mCTLA-4-B7 interaction by anti-CTLA-4 monoclonal Ab exacerbates experimental allergic encephalomyelitis when injected during the development of the disease [26]. The finding that sCTLA-4 appeared to correlate with the disease severity and activity of SSc suggests that sCTLA-4 blocks B7–mCTLA-4 interactions and thereby enhances T-cell activation and autoreactivity. Alternatively, it is also possible that the elevation of sCTLA-4 levels plays a protective role via interference with B7–CD28 interactions [14]. Further studies will be required to clarify a pathological role of sCTLA-4 in the development of SSc.

The CD28/CTLA-4 and B7 system has been suggested to play a role in the development of fibrosis. In a bleomycin-induced lung fibrosis model, CD28-deficient mice show markedly reduced lung fibrosis, which is associated with the decreased production of cytokines and chemokines relevant to fibrosis [27]. Since the elevation of sCTLA-4 levels was associated with lung fibrosis in SSc, increased sCTLA-4 levels in SSc may augment CD28-mediated T-cell co-stimulation by inhibiting mCTLA-4–B7 interaction, which may result in the development of fibrosis. In addition, B7.2 expression is up-regulated on alveolar macrophages from SSc patients with lung fibrosis. Several studies have shown that elevated B7.2 expression on alveolar macrophages is associated with activation of T helper 2 (Th2) cytokines that generally increase extracellular matrix deposition [28, 29]. Elevation of sCTLA-4 levels in SSc may also enhance Th2 response by blocking mCTLA-4–B7 interaction. Although the mechanisms of sCTLA-4 in the development of SSc remain unknown, these observations suggest that sCTLA-4 may play a role in the regulation of immune responses involved in the development of fibrosis in SSc.

In the current study, serum levels of IgG and anti-topoisomerase I Ab correlated positively with sCTLA-4 levels, suggesting that sCTLA-4 is involved in the abnormal humoral immune responses of SSc. Consistently, CTLA-4-deficient mice exhibit the expansion of B-cell population with activated phenotype and 100-fold increase in serum IgG levels [30, 31]. In addition, mCTLA-4 is expressed on activated B cells, and mCTLA-4 engagement by Ab inhibits IgG production by B cells [10]. Therefore, the increased IgG levels in CTLA-4-deficient mice are considered to be due to lack of CTLA-4-mediated negative signals delivered to helper T cells or B cells [10]. These results suggest that sCTLA-4 plays a role in hyper-{gamma}-globulinaemia and autoantibody production in SSc. Collectively, although the functional significance of sCTLA-4 remains unknown in this study, our results suggest that sCTLA-4 may be related to the immunological abnormalities and disease expression associated with SSc.


    Acknowledgments
 
We thank Ms M. Matsubara for technical assistance.

The authors have declared no conflicts of interest.


    References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 29 March 2004; revised version accepted 15 June 2004.



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