Increased cutaneous T-cell-attracting chemokine levels in sera from patients with systemic sclerosis
I. Hayakawa1,
M. Hasegawa1,
T. Matsushita1,
K. Yanaba1,
M. Kodera1,
K. Komura1,
K. Takehara1 and
S. Sato1,2
1 Department of Dermatology, Kanazawa University Graduate School of Medical Science, Kanazawa and 2 Department of Dermatology, Nagasaki University Graduate School of Biomedical Science, Nagasaki, Japan.
Correspondence to: M. Hasegawa, Department of Dermatology, Kanazawa University Graduate School of Medical Science, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan. E-mail: minoruha{at}derma.m.kanazawa-u.ac.jp
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Abstract
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Objective. We sought to assess serum cutaneous T-cell-attracting chemokine (CTACK) levels and CTACK expression levels in skin from patients with systemic sclerosis (SSc), and determine whether serum CTACK levels correlate with clinical features in SSc patients.
Methods. Serum samples were obtained from 73 SSc patients, 32 patients with systemic lupus erythematosus and 26 patients with dermatomyositis. Serum CTACK levels were determined by enzyme-linked immunoabsorbent assay. CTACK mRNA expression in sclerotic skin was assessed by real-time reverse transcriptionpolymerase chain reaction.
Results. Serum CTACK levels were significantly increased in patients with diffuse cutaneous SSc (dSSc; n=32) and those with limited cutaneous SSc (lSSc; n=41) compared with normal controls (n=31; P<0.05 and P<0.0005, respectively). The presence of calcinosis and muscle involvement was more frequently detected in SSc patients with elevated CTACK levels (P<0.05 and P<0.05, respectively). Elevated C-reactive protein levels were also observed more frequently in SSc patients with increased CTACK levels (P<0.05). CTACK mRNA expression levels in the sclerotic skin of SSc patients were augmented. In a longitudinal study, serum CTACK levels were generally decreased during the follow-up.
Conclusions. The increased serum CTACK levels and enhanced skin CTACK expression in SSc patients suggest that CTACK is related to the inflammation associated with SSc.
KEY WORDS: Systemic sclerosis, Cutaneous T cell-attracting chemokine, CTACK
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Introduction
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Systemic sclerosis (SSc) is a systemic inflammatory disease characterized by activation of circulating T lymphocytes, increased levels of circulating mediators of inflammation, the production of autoantibodies, and typical local inflammatory infiltrates [15]. In its early stages, SSc is characterized by both endothelial damage [6] and perivascular infiltrations with accumulation of lymphocytes [710]. In the skin, these lymphocytes predominantly consist of CD4+ T cells that express increased amounts of activation markers and adhesion molecules on their surfaces [7, 11, 12]. Recent investigations have identified many potential molecules involved in the regulation of recruitment of leucocytes to the inflammatory regions. Chemokines, a protein superfamily of structurally related small secreted proteins, have been shown to regulate the trafficking of distinct leucocyte subsets into peripheral tissues [13, 14]. Cutaneous T-cell-attracting chemokine (CTACK; CCL27) is a member of the CC chemokine family and a functional ligand for CC chemokine receptor (CCR) 10. It is predominantly expressed in skin and attracts CCR10-expressing skin-homing memory T cells [15].
Although the pathogenesis of SSc remains unclear, it is particularly important to understand the mechanisms regulating lymphocyte recruitment to the skin. In this study, we examined serum CTACK levels and their clinical correlation in SSc patients. Furthermore, we assessed the CTACK mRNA expression levels in the sclerotic skin from SSc patients.
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Materials and methods
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Patients
Serum samples were obtained from 73 Japanese SSc patients (63 females and 10 males). The age (mean±S.D.) of these patients was 45±17 yr. All patients fulfilled the criteria proposed by the American College of Rheumatology [16]. SSc patients were grouped according to the classification system proposed by LeRoy et al. [17]: 32 patients (23 females and nine males, age 44±16 yr) had diffuse cutaneous SSc (dSSc) and 41 patients (40 females and one male, age 46±16 yr) had limited cutaneous SSc (lSSc). The disease duration of dSSc patients and lSSc patients was 2.9±5.4 and 9.0±9.7 yr, respectively. None of the SSc patients were treated with steroids, D-penicillamine or immunosuppressive therapy. Antinuclear antibodies (ANAs) were determined by indirect immunofluorescence using HEp-2 cells as substrate and autoantibody specificity was further assessed by enzyme-linked immunosorbent assay (ELISA) specific for antibodies (Abs) against topoisomerase I, centromere or U1 RNP. Anti-topoisomerase I Ab was positive for 25 (dSSc 18, lSSc 7), anticentromere Ab for 25 (dSSc 1, lSSc 24) and anti-U1 RNP Ab for three (all lSSc). Seventeen patients (dSSc 11, lSSc 6) had ANA, but their specificities were not identified by autoantibody-specific ELISA. Of these 11 dSSc patients, two had anti-U3 RNP Ab and six had anti-RNA polymerases I and III Ab by immunoprecipitation; however, autoantibody specificities in the remaining three dSSc patients were not still identified by immunoprecipitation. Of the six lSSc patients with unknown autoantibody specificity by ELISA, one patient had anti 7-2 RNA Ab, although autoantibody specificities in the remaining five lSSc patients were not recognized by immunoprecipitation. Two dSSc patients and one lSSc patient were negative for ANAs. In the longitudinal study, 20 patients with SSc were analysed. Eleven patients (eight females and three males, age 53±16 yr) had dSSc and nine patients (eight females and one male, age 50±17 yr) had lSSc. The disease duration of dSSc patients and lSSc patients was 3.6±4.0 and 8.3±6.3 yr, respectively. At their first visit, none of SSc patients had been treated with steroids, D-penicillamine or other immunosuppressive therapy. During the follow-up, eight dSSc patients received low-dose steroid (prednisolone 520 mg/day). One dSSc patient received low-dose steroid and low-dose D-penicillamine (100300 mg/day), while the remaining two dSSc patients received low-dose steroid combined with cyclophosphamide pulse therapy. Five lSSc patients received low-dose steroid, while the remaining four lSSc patients did not receive oral steroid. Thirty-two patients with systemic lupus erythematosus (SLE; 29 females and three males; age 33±12 yr) who fulfilled the American College of Rheumatology criteria [18] were included in this study. Disease activity was assessed by the SLE disease activity index (SLEDAI) score [19]. Twenty-seven patients had a SLEDAI
10 and were considered relatively inactive. The remaining five patients, with SLEDAI >10, were considered active. At the time of blood drawing, six patients (19%) suffered from active skin disease, six (19%) from florid glomerulonephritis, two (6%) from CNS involvement, one (3%) from vasculitis and seven (22%) from arthritis, and 13 (34%) had lymphopenia or neutropenia. Concerning the type of skin lesion, four patients had chronic cutaneous lupus erythematosus, one patient had subacute cutaneous lupus erythematosus and one patient had acute cutaneous lupus erythematosus. In addition, 26 patients with dermatomyositis (DM; 19 females and seven males; age 41±10 yr) who fulfilled Bohan and Peter criteria [20, 21] were included in this study. Five out of 26 had active myositis. Seven patients had skin lesions: one patient had Gottron's papules and six patients had poikiloderma. None of the patients with SLE or DM received any treatment, including oral steroids and other immunosuppressive drugs. Control serum samples were obtained from 31 age- and gender-matched healthy Japanese volunteers (25 females and six males, age 41±14 yr). Fresh venous blood samples were centrifuged shortly after clot formation. All samples were stored at 70°C prior to use.
Serum samples and clinical and laboratory data were obtained at the same time. Patients underwent a clinical assessment, and involvement of their organ systems was investigated. Skin score was measured using the modified Rodnan total skin thickness score (modified Rodnan TSS) [22]. 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). The modified Rodnan TSS was derived by summation of the scores from all 17 areas (range 051). Organ system involvement was defined as described previously with some modifications [23, 24]: oesophagus = hypomotility shown by barium radiography; pulmonary fibrosis = bibasilar interstitial fibrosis on chest radiogram and high-resolution computed tomogram; pulmonary hypertension = clinical evidence of pulmonary hypertension and increased mean pulmonary arterial pressure (>40 mmHg) documented by echocardiography, in the absence of severe pulmonary interstitial fibrosis, as described previously [25]; joint = inflammatory polyarthralgias or arthritis; heart = pericarditis, congestive heart failure, or arrhythmias requiring treatment; kidney = malignant hypertension and rapidly progressive renal failure with no other explanation; and 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 VC and DLco were <80 and <75%, respectively, of the predicted normal values, these results were considered abnormal. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were considered elevated when the value was higher than 20 mm/h and 0.5 mg/dl, respectively. The protocol was approved by the Kanazawa University Graduate School of Medical Science and informed consent was obtained from all patients.
ELISA for CTACK
The serum CTACK levels were measured with specific ELISA using recombinant human CTACK protein (R&D Systems, Minneapolis, MN, USA), according to the manufacturer's protocol. Briefly, 96-well plates (EIA/RIA; Coster, Cambridge, USA) were coated overnight at room temperature with 2 µg/ml of mouse anti-human CTACK Ab. After washing, plates were blocked for 1 h at room temperature in phosphate buffered saline containing 1% bovine serum albumin and 5% sucrose. Recombinant human CTACK and serum samples were added in triplicate, and the plates were incubated for 2 h at room temperature. After washing, the plates were incubated with biotinylated goat anti-human CTACK Ab (50 ng/ml) for 2 h at room temperature and then with streptavidinperoxidase for 20 min at room temperature. Samples were developed with tetramethylbenzidine and H2O2. Reactions were stopped by adding 1 mol/l H2SO4, and the plates were read at 450 nm.
RNA isolation and real-time polymerase chain reaction
Total RNA was isolated from frozen tissue of sclerotic skin from five patients with SSc (three females and two males; age 47±10 yr) and five healthy subjects (three females and two males; age 41±15 yr) with RNeasy spin columns (Qiagen, Crawley, UK). Three patients (one female and two males, age 44±13 yr) had dSSc and two patients (two females, age 50±1 yr) had lSSc. The disease duration of dSSc patients and lSSc patients was 1.0±0.9 and 9±5.8 yr, respectively. Total RNA from each sample was reverse-transcribed into cDNA. Expression of CTACK was analysed using a real-time polymerase chain reaction (PCR) quantification method, according to the manufacture's instructions (Applied Biosystems, Foster City, CA, USA). Sequence-specific primers and probes were designed by Pre-Developed TaqMan Assay Reagents or Assay-On-Demand (Applied Biosystems). Real-time PCR (40 cycles of denaturing at 92°C for 15 s and annealing at 60°C for 60 s) was performed on ABI Prism 7000 Sequence Detector (Applied Biosystems). Glyceraldehyde-3-phosphate was used to normalize mRNA. Relative expression of real-time PCR products was determined by using the 
Ct method [26] to compare target gene and housekeeping gene (GAPDH) mRNA expression. One of the control samples was chosen as a calibrator sample.
Statistical analysis
Statistical analysis was performed using the Mann-Whitney U test for determining the level of significance of differences between sample means, 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 less than 0.05 was considered statistically significant.
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Results
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Serum CTACK levels by ELISA
CTACK levels in serum samples from patients with connective tissue disorders and normal controls were assessed by ELISA (Fig. 1). In the total number of patients with SSc, serum CTACK levels were elevated compared with SLE patients, DM patients and normal controls (P<0.0001, P<0.05 and P<0.005, respectively). Regarding the SSc subgroups, serum CTACK levels were significantly elevated in dSSc patients compared with SLE patients and normal controls (P<0.005 and P<0.05, respectively). Similarly, lSSc patients exhibited mean CTACK levels that were significantly higher than those of patients with SLE (P<0.0001) or DM (P<0.01) and normal controls (P<0.0005). No significant difference in serum CTACK levels was observed between dSSc and lSSc. By contrast, serum CTACK levels in patients with SLE or DM were similar to those in normal controls. Serum CTACK levels in DM patients tended to be higher than those found in patients with SLE or normal controls (P = 0.129 and P = 0.571, respectively).

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FIG. 1. Serum CTACK levels in patients with dSSc, lSSc, SLE or DM and healthy controls (CTL). Serum CTACK levels were determined by ELISA. A broken line indicates the cut-off value (mean±2 S.D. of normal controls). Horizontal lines indicate the mean values for each group.
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Clinical correlation in SSc classified according to serum CTACK level
Absorbance values higher than the mean + 2 S.D. of control serum samples were considered to be elevated in this study (Fig. 1). Elevated serum CTACK levels were observed in 23% (17/73) of all SSc patients. Regarding the subsets of SSc, serum CTACK levels were increased in 19% (6/32) of dSSc patients, while 34% (11/32) of lSSc patients had an elevated serum CTACK level. Increased serum CTACK levels were detected in 6% (2/32) of SLE and 12% (3/26) of DM patients. By contrast, increased serum CTACK levels were detected in 3% (1/31) of normal controls. Concerning the clinical correlation, the presence of calcinosis was more frequently detected in SSc patients with elevation of serum CTACK levels than those without the elevation (P<0.05, Table 1). The involvement of muscle was more frequently observed in SSc patients with elevated CTACK levels than in those without elevated CTACK levels (P<0.05, Table 1). Moreover, CRP levels were significantly elevated in SSc patients with elevation of CTACK levels than in those without this elevation (P<0.05, Table 1). In SLE patients, the elevation of CTACK levels was not associated with any other clinical and serological features, including the presence of skin lesion, CRP levels, anti-DNA antibodies and complement factors (data not shown). In addition, no association was found in DM patients between the elevation of CTACK levels and CRP levels, the presence of skin lesion, or the presence of active myositis (data not shown).
Longitudinal study of serum CTACK levels
To assess changes in serum CTACK levels, serum samples from 20 patients with SSc were examined twice during an interval of 612 months (Fig. 2). None of these patients had received any treatment at their first visit. Seven out of 20 SSc patients exhibited elevated serum CTACK levels at their first visit (Fig. 2A). Serum CTACK levels in four out of seven SSc patients with high serum CTACK levels at first visit decreased to normal levels during the follow-up. Their disease duration at initial visit was 4.4±4.6 yr. Skin sclerosis was improving in all of the four patients during the follow-up (modified Rodnan TSS; from 15.5±9.0 at first visit to 7.0±2.4). Two patients received oral low-dose steroid alone, whereas remaining two patients received low-dose steroid and cyclophosphamide pulse therapy during the follow-up. Serum CTACK levels in three of seven SSc patients with high serum CTACK levels at their first visit remained high during the follow-up (Fig. 2A). Their disease duration was 2.9±3.4 yr. After the initial visit, treatment with low-dose steroid alone was started in all of these patients. In two out of three SSc patients, skin sclerosis improved (modified Rodnan TSS; from 17.5±0.7 at first visit to 11.0±2.8). Only one patient exhibited remarkable elevated serum CTACK levels during the follow-up. This patient developed subacute deterioration of interstitial pneumonitis, which resulted in death, while her skin sclerosis did not changed during the follow-up (modified Rodnan TSS 6).

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FIG. 2. Changes in serum CTACK levels during the follow-up period. SSc patients were divided into two groups: (A) SSc with high serum CTACK levels at first visit, and (B) those with normal serum CTACK levels at first visit, as described in Materials and methods.
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Serum CTACK levels at first visit were normal in 13 out of 20 SSc patients (Fig. 2B). Their disease duration was slightly longer (6.4±6.3 yr) than that in the seven patients with high serum CTACK levels at the first visit described above, suggesting that these patients might show elevated serum CTACK levels in the earlier phase of the disease. In general, 12 out of 13 SSc patients with normal serum CTACK levels remained with normal levels. In nine out of 12 SSc patients, skin sclerosis improved (modified Rodnan TSS; from 15.6±12.0 at first visit to 11.0±2.9). Six patients received oral low-dose steroid alone. Only one patient was treated with low-dose D-penicillamine in addition to steroid, whereas the remaining two patients received neither steroid nor D-penicillamine. One out of 12 patients exhibited progression of skin sclerosis (modified Rodnan TSS; from 9 at first visit to 12), who received low-dose steroids. Skin sclerosis did not change during the follow-up in the remaining two patients with no steroid treatment. In contrast, one patient, who received low-dose steroids during the follow-up, exhibited slightly elevated CTACK levels during the observation period, did not develop any new organ involvement and showed stable skin sclerosis (modified Rodnan TSS emained at 19).
Expression of CTACK mRNA in sclerotic skin
We examined CTACK mRNA expression in sclerotic skin from SSc patients by real-time RT-PCR. CTACK mRNA levels in SSc patients were significantly (5.8-fold) higher than those in healthy control subjects (4.89±1.15 vs 0.84±0.52, P<0.01; Fig. 3). Thus, CTACK mRNA expression levels in lesional skin from SSc patients were increased.
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Discussion
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In the present study, serum CTACK levels were significantly elevated in SSc patients compared with normal controls. The elevation of serum CTACK levels was associated with the presence of calcinosis, muscle involvement and serum CRP elevation. Moreover, CTACK mRNA expression levels in sclerotic skin from SSc patients were increased. Previous studies reported that patients with atopic dermatitis or psoriasis vulgaris showed elevated serum CTACK levels [27, 28]. Serum CTACK levels overlap considerably among SSc, other connective tissue diseases, atopic dermatitis and psoriasis vulgaris. Although the elevation of serum CTACK was not specific to SSc, these results suggest that it is related to the inflammation associated with SSc. To clarify the relationship between serum CTACK levels and disease activity, we assessed the changes in serum CTACK levels in SSc patients. Our longitudinal analysis demonstrated that serum CTACK levels were generally decreased during the follow-up period. Most SSc patients with high serum CTACK levels at first visit showed decreased serum CTACK levels during the follow-up period when treatment with low-dose steroid or low-dose steroid combined with cyclophosphamide pulse therapy was started. Therefore, the reduction in serum CTACK might be caused by steroid treatment. Consistent with this, it was reported that CTACK was down-modulated by glucocorticosteroids in vivo [15]. Moreover, steroids are sometimes used to treat the inflammatory phase of early dSSc [29]. However, despite treatment with low-dose steroid, serum CTACK levels were not decreased in one patient with high serum CTACK levels at first visit. This patient developed subacute deterioration of interstitial pneumonitis, which resulted in death. Furthermore, serum CTACK levels remained normal independently of treatment with steroid in SSc patients with normal serum CTACK levels at first visit. Collectively, these results suggest that steroid is not the only factor associated with decreasing serum CTACK levels, and also suggest that serum CTACK levels may reflect the disease activity of SSc. However, it should be noted that serum CTACK levels increased in one patient who did not develop any new organ involvement and showed stable skin sclerosis.
In vitro data has revealed that CTACK is expressed in the skin but not in other organs [30]. A recent report has suggested that immobilization of CTACK on dermal extracellular matrix and fibroblasts may sustain a chemokine gradient directing skin-infiltrating lymphocytes from perivascular pockets to a subepidermal location [15]. Calcinosis was more frequently seen in skin from SSc patients who exhibited serum elevated CTACK levels. Its prevalence is thought to correlate with the severity and duration of SSc. Abnormalities of muscle biopsy in SSc patients include the presence of focal mononuclear cellular infiltrates. The pathogenesis of calcinosis and myositis in SSc and other connective tissue disorders, such as DM, is not understood. In this study, serum CTACK levels in DM patients tended to be higher than SLE and normal controls. Moreover the elevation of CRP levels as the biochemical sign of inflammation in SSc patients with elevated serum CTACK levels may be caused by the activity of myositis. Therefore, these results suggest that CTACK may be associated with the occurrence of calcinosis and myositis. Further studies will be required to clarify a role of CTACK in clinical manifestation of SSc.
CTACK attracts skin-homing T cells of both Th1 and Th2 subtypes expressing CCR10. Circulating T cells positive for skin-homing cutaneous lymphocyte-associated antigen (CLA+), dermal microvascular endothelial cells and fibroblasts expressed CCR10 on their cell surface [15]. CLA defines a subset of circulating memory T cells that selectively localize in cutaneous sites, mediated in part by the interaction of CLA with its vascular ligand E-selectin. A recent report has suggested that CTACK is secreted into the papillary dermis, immobilized on the extracellular matrix and displayed on the surface of endothelial cells [15]. In our study, mRNA expression levels of CTACK in the skin of patients with SSc were increased. It has been suggested that CTACK is induced in cultured keratinocytes by the serum thymus and activation-regulated chemokine (TARC) and tumour necrosis factor
(TNF-
) [31]. Previously, we reported that serum TARC and TNF-
levels were elevated in SSc patients [32, 33]. Elevated levels of TNF-
and soluble TNF receptors have also been reported in the skin, bronchoalveolar lavage and other tissue from SSc patients [3335]. Therefore, these observations suggest that CTACK may play a role in the regulation of the immune response involved in the disease process, although the roles of CTACK in the development of SSc remain unknown.
The authors have declared no conflicts of interest.
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Submitted 14 December 2004;
revised version accepted 25 February 2005.