Archives of Dermatological Research

, Volume 305, Issue 1, pp 17–23

Increased production of soluble inducible costimulator in patients with diffuse cutaneous systemic sclerosis

Authors

    • Department of Dermatology, Faculty of MedicineThe University of Tokyo
  • Yoshihide Asano
    • Department of Dermatology, Faculty of MedicineThe University of Tokyo
  • Shinji Noda
    • Department of Dermatology, Faculty of MedicineThe University of Tokyo
  • Kaname Akamata
    • Department of Dermatology, Faculty of MedicineThe University of Tokyo
  • Naohiko Aozasa
    • Department of Dermatology, Faculty of MedicineThe University of Tokyo
  • Takashi Taniguchi
    • Department of Dermatology, Faculty of MedicineThe University of Tokyo
  • Takehiro Takahashi
    • Department of Dermatology, Faculty of MedicineThe University of Tokyo
  • Yohei Ichimura
    • Department of Dermatology, Faculty of MedicineThe University of Tokyo
  • Tetsuo Toyama
    • Department of Dermatology, Faculty of MedicineThe University of Tokyo
  • Hayakazu Sumida
    • Department of Dermatology, Faculty of MedicineThe University of Tokyo
  • Yoshihiro Kuwano
    • Department of Dermatology, Faculty of MedicineThe University of Tokyo
  • Yayoi Tada
    • Department of Dermatology, Faculty of MedicineThe University of Tokyo
  • Makoto Sugaya
    • Department of Dermatology, Faculty of MedicineThe University of Tokyo
  • Takafumi Kadono
    • Department of Dermatology, Faculty of MedicineThe University of Tokyo
  • Shinichi Sato
    • Department of Dermatology, Faculty of MedicineThe University of Tokyo
Original Paper

DOI: 10.1007/s00403-012-1292-7

Cite this article as:
Yanaba, K., Asano, Y., Noda, S. et al. Arch Dermatol Res (2013) 305: 17. doi:10.1007/s00403-012-1292-7

Abstract

Inducible costimulator (ICOS) is crucial for T cell proliferation, production of various cytokines, and T cell-dependent B-cell responses. To determine the serum soluble ICOS (sICOS) level and its association with clinical parameters in patients with systemic sclerosis (SSc), serum sICOS level was examined by enzyme-linked immunosorbent assay in 38 patients with SSc and 24 healthy individuals. The expression of ICOS and ICOS ligand in skin was examined immunohistochemically. There was no significant difference in serum sICOS level between patients with SSc and healthy individuals. Patients with diffuse cutaneous SSc had higher levels of sICOS than those with limited cutaneous SSc (P < 0.05) or healthy individuals (P < 0.05). Serum sICOS level correlated positively with the severity of skin sclerosis. Patients with SSc and elevated sICOS level more often had interstitial lung disease and decreased vital capacity than those with normal sICOS level. The serum sICOS level was significantly greater in patients with early phase SSc than those with late phase SSc. ICOS and ICOS ligand immunostaining were observed on infiltrating dermal mononuclear cells in lesional skin tissue. These results suggest that the serum level of sICOS is increased in patients with diffuse cutaneous SSc and correlates with the severity and activity of skin sclerosis and interstitial lung disease. ICOS may contribute to the development of SSc. In addition, measurement of serum sICOS level in patients with early SSc may offer an important means for further evaluation of SSc disease severity.

Keywords

Systemic sclerosisICOSFibrosisELISAImmunohistochemistry

Introduction

Systemic sclerosis (SSc) is a heterogeneous disorder characterized by excessive fibrosis and microvascular damage of the skin and various internal organs. Although the pathogenesis of SSc remains unclear, a number of immunologic abnormalities have been detected suggesting that SSc has an autoimmune background [32]. Cutaneous mononuclear cell infiltrates in early SSc skin lesions are mostly activated T cells, which are also increased in lung interstitium and bronchoalveolar fluid for patients with SSc and active interstitial lung disease [32]. Furthermore, cytokines or growth factors regulate SSc induction by stimulating the synthesis of extracellular matrix components, which may injure endothelial cells and modulate leukocyte function [32]. These infiltrating activated T cells are likely to release cytokines, chemokines, or growth factors, which play a crucial part in the initiation and development of fibrosis in SSc.

Inducible costimulator (ICOS), a member of the CD28 family of costimulatory molecules, is expressed in activated T cells, but not in naïve T cells [18]. ICOS specifically binds to ICOS ligand (ICOSL, also known as B7-related protein and B7 homologous protein), which is constitutively expressed on antigen-presenting cells such as B-cells and macrophages [22]. ICOS-mediated costimulation is crucial for T cell proliferation, production of various cytokines, and T cell-dependent B-cell responses [6, 22, 29]. In addition, previous studies have suggested that the interaction of ICOS with ICOSL plays an important role in autoimmunity. ICOS expression on T cells is increased in patients with systemic lupus erythematosus and correlates with disease severity [11, 15], while blockade of the ICOS–ICOSL interaction ameliorates systemic lupus erythematosus in murine models [5, 12]. Blockade of the ICOS–ICOSL interaction has been shown to reduce the severity of rheumatoid arthritis in mice [9, 13, 26]. Furthermore, ICOS-deficient mice develop attenuated skin and lung fibrosis in a model of SSc [30]. Therefore, we hypothesized that ICOS may play a role in the pathogenesis of SSc. In the present study, we examined the level of serum soluble ICOS (sICOS) in patients with SSc and evaluated the results with respect to clinical features. In addition, we assessed ICOS and ICOSL expression in lesional skin from patients with SSc by immunohistochemical analysis.

Materials and methods

Patients

The study protocol was approved by the University of Tokyo and University of Tokyo Hospital (Tokyo, Japan), and informed consent was obtained from all patients. Serum samples were obtained from 38 Japanese patients with SSc [37 women and 1 man; mean age 56 (range 25–74) years]. All patients fulfilled the criteria for SSc proposed by the American College of Rheumatology [28]. Patients were classified according to the system proposed by LeRoy et al. [14]: 24 patients had limited cutaneous SSc (lcSSc) and 14 had diffused cutaneous SSc (dcSSc). Anti-topoisomerase I (topo I) antibodies (Abs) were present in 17 patients, anti-centromere Abs (ACA) in 10, anti-U1RNP Abs in 2, and anti-U3RNP Abs in 1. No patients were positive for anti-RNA polymerase I and III Abs. The remaining 8 patients were negative for all these Abs. The mean disease duration was 6.3 ± 8.0 (range 0.2–30) years. Duration was calculated from the time of onset of the first clear clinical manifestation of SSc (excluding Raynaud’s phenomenon). None of the patients had received corticosteroids or other immunosuppressants. Twenty-four age- and sex-matched healthy Japanese individuals were included in the study as healthy controls. Fresh venous blood samples were centrifuged shortly after clot formation. All samples were stored at −70 °C until use.

Clinical assessment

During their first visit, all patients underwent physical examination, complete medical histories were obtained, and laboratory tests were performed. Organ system involvement was defined as previously described [25, 27], lung: bibasilar fibrosis on chest radiography and high-resolution computed tomography; esophagus: hypomotility shown by barium radiography; heart: pericarditis, congestive heart failure, or arrhythmias requiring treatment; kidney: malignant hypertension and rapidly progressive renal failure with no other explanation; joint: inflammatory polyarthralgias or arthritis; and muscle: proximal muscle weakness and elevated serum creatine kinase level. Pulmonary fibrosis was defined as bibasilar interstitial fibrosis on chest high-resolution computed tomography. In addition, a pulmonary function test, including vital capacity (VC) and diffusion capacity for carbon monoxide (DLco), was evaluated to examine the severity of interstitial lung disease. DLco <70 % and VC <80 % of predicted normal values were considered abnormal.

Patients with SSc who were smokers or who had respiratory disorders that might have affected DLco or VC were excluded from this study. Pulmonary artery pressure was estimated by Doppler echocardiogram. The modified Rodnan skin score (mRSS) was measured as the sum of skin thickness measurements determined by palpation on a scale of 0–3 in 17 body areas [3].

Measurement of serum sICOS

Fresh venous blood samples were drawn into pyrogen-free blood collection tubes without additives, immediately immersed in melting ice, and allowed to clot for 1 h before centrifugation. All serum samples were stored at −70 °C until use. Serum sICOS level was measured with a specific enzyme-linked immunosorbent assay (ELISA) kit (Uscn life science Inc., Wuhan, China), according to the manufacturer’s protocol. Each sample was tested in duplicate. The detection limit of this assay was 7.1 pg/ml.

Histological analysis

Skin biopsy samples were taken from the dorsal aspect of the mid-forearm of eight female patients with dcSSc and eight healthy female volunteers. Sections were stained with hematoxylin and eosin. ICOS and ICOSL immunostaining in skin tissue was determined using a rabbit anti-human ICOS polyclonal Ab and a rabbit anti-human ICOSL polyclonal Ab (abcam, Cambridge, UK), respectively. Rabbit IgG (Sigma-Aldrich, St. Louis, MO) was used as a control for nonspecific ICOS and ICOSL staining.

Statistical analysis

Data are presented as the mean ± standard deviation (SD). The Kruskal–Wallis test was used to compare ICOS levels between groups, Fisher’s exact probability test was used to compare frequencies, and Bonferroni’s test was used for multiple comparisons. Spearman’s rank correlation coefficient was used to examine the relationship between two continuous variables. A probability (P) value <0.05 was considered significant.

Results

Serum sICOS level in patients with SSc

The mean serum sICOS level at first visit was comparable between patients with SSc (712.3 ± 404.3 pg/ml) and healthy individuals (628.6 ± 178.1 pg/ml) (Fig. 1). With respect to the SSc subgroups, the mean sICOS level in patients with dcSSc (903.1 ± 434.1 pg/ml) was significantly increased compared to that in patients with lcSSc (600.9 ± 348.3 pg/ml) and in healthy individuals (P < 0.05 for both). There was no significant difference in serum sICOS levels between patients with lcSSc and healthy individuals. Consistent with these results, serum sICOS level in patients with SSc and anti-topo I Abs were significantly higher than those in patients with SSc and ACA (Fig. 2). Thus, the serum sICOS level was increased in patients with dcSSc, but not in patients with lcSSc.
https://static-content.springer.com/image/art%3A10.1007%2Fs00403-012-1292-7/MediaObjects/403_2012_1292_Fig1_HTML.gif
Fig. 1

Serum soluble inducible costimulator (sICOS) level in patients with systemic sclerosis (SSc), diffuse cutaneous SSc (dcSSc), and limited cutaneous SSc (lcSSc), and in healthy individuals (Control). Serum ICOS level was determined using a specific enzyme-linked immunosorbent assay (ELISA). Horizontal lines indicate the mean value in each group. The dashed line indicates the cut-off value (mean ± 2 standard deviations of the values from healthy individuals)

https://static-content.springer.com/image/art%3A10.1007%2Fs00403-012-1292-7/MediaObjects/403_2012_1292_Fig2_HTML.gif
Fig. 2

sICOS level in SSc patients with anti-topoisomerase I antibodies (Topo I), those with anti-centromere antibodies (ACA), those with anti-nuclear antibodies other than Topo I and ACA, and those without anti-nuclear antibodies (Negative), and in healthy individuals (Control). Serum ICOS level was determined using a specific ELISA. Horizontal lines indicate the mean value in each group. The dashed line indicates the cut-off value (mean ± 2 standard deviations of the values from healthy individuals)

Clinical correlation with serum sICOS level

Clinical and laboratory parameters obtained at the first evaluation were compared between patients with SSc and increased sICOS levels, and those with SSc and normal sICOS levels. Values greater than the mean +2 SDs (984.7 pg/ml) of control serum samples were considered increased in this study. An increased sICOS level was observed in 18 % (7/38) of all patients with SSc, 36 % (5/14) of patients with dcSSc, and 8 % (2/24) of patients with lcSSc. Patients with SSc and an increased sICOS level more frequently had dcSSc than those with a normal sICOS level (71 vs. 29 %, P < 0.05) (Table 1). Consistent with an association between higher sICOS levels and dcSSc, patients with SSc and increased sICOS level showed significantly greater mRSS values than those with normal sICOS level (14.5 ± 7.4 vs. 5.7 ± 4.4, P < 0.01). Furthermore, the sICOS level correlated positively with mRSS (P < 0.01, r = 0.46) (Fig. 3). In addition, the prevalence of interstitial lung disease and decreased %VC in patients with SSc and elevated sICOS level was significantly higher than in those with normal sICOS level (86 vs. 42 %, P < 0.05, 57 vs. 19 %, P < 0.05, respectively). Thus, sICOS level correlated not only with the extent of skin sclerosis but also with the severity of interstitial lung disease in SSc.
Table 1

Clinical and laboratory findings in patients with SSc with elevated serum sICOS level at the initial visit

 

Elevated sICOS (n = 7)

Normal sICOS (n = 31)

Age at onset, years (mean ± SD)

47 ± 11

52 ± 13

Male:female

1:6

0:31

Duration, years (mean ± SD)

3.6 ± 5.1

6.9 ± 8.5

mRSS, points (mean ± SD)

14.5 ± 7.4**

5.7 ± 4.4

Clinical features (%)

 dcSSc

71*

29

 lcSSc

29*

71

 Pitting scars/ulcers

29

35

 Contracture of phalanges

43

42

 Diffuse pigmentation

29

39

 Telangiectasia

43

32

Organ involvement (%)

 Interstitial lung disease

86*

42

 Decreased %VC

57*

19

 Decreased %DLco

57

35

 Pulmonary hypertension

14

19

 Esophagus

57

42

 Heart

0

3

 Kidney

0

6

 Joint

29

29

 Muscle

14

10

Laboratory findings (%)

 Anti-topo I Ab

57

42

 ACA

14

29

 Increased IgG

43

13

 Increased ESR

71

58

 Increased CRP

43

39

Unless otherwise stated, values are expressed as percentage

mRSS modified Rodnan total skin score, dcSSc diffuse cutaneous systemic sclerosis, lcSSc limited cutaneous SSc, VC vital capacity, DLco diffusion capacity for carbon monoxide, topo I topoisomerase I, Ab antibody, ACA anti-centromere Abs, ESR erythrocyte sedimentation rate, CRP C-reactive protein

P < 0.05, ** P < 0.01 versus patients with SSc and normal serum sICOS level

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Fig. 3

Correlation between serum sICOS level and modified Rodnan skin score (mRSS) value and %VC in patients with SSc. Serum ICOS level was determined using a specific ELISA. The dashed lines indicate the cut-off values (mean ± 2 standard deviations of the values from healthy individuals)

Patients with SSc and elevated serum sICOS level had shorter disease duration than those with normal sICOS level (3.6 ± 5.1 vs. 6.9 ± 8.5 years), although the difference was not statistically significant. Moreover, patients with SSc and a disease duration of <5 years showed significantly increased serum sICOS level compared to those with a disease duration of ≥5 years (P < 0.05) (Fig. 4). Thus, an increased serum sICOS level was associated with shorter disease duration in patients with SSc.
https://static-content.springer.com/image/art%3A10.1007%2Fs00403-012-1292-7/MediaObjects/403_2012_1292_Fig4_HTML.gif
Fig. 4

Correlation between disease duration (< 5 and ≥5 years) and serum sICOS level in patients with SSc. Serum ICOS level was determined using a specific ELISA. The dashed lines indicate the cut-off values (mean ± 2 standard deviations of the values from healthy individuals)

Immunohistochemical staining for ICOS and ICOSL in skin from patients with dcSSc

The expression of ICOS and ICOSL in the skin was assessed by immunohistochemical analysis. Immunostaining for ICOS and ICOSL was not observed in skin from healthy individuals (Fig. 5). By contrast, in lesional skin from patients with dcSSc, ICOS and ICOSL expression was detected in infiltrating dermal mononuclear cells. Thus, augmented immunostaining of ICOS and ICOSL was found in lesional skin from patients with dcSSc.
https://static-content.springer.com/image/art%3A10.1007%2Fs00403-012-1292-7/MediaObjects/403_2012_1292_Fig5_HTML.jpg
Fig. 5

Representative histologic sections of normal skin and lesional skin tissue from patients with dcSSc; the sections were stained with hematoxylin and eosin, anti-ICOS antibody, and anti-ICOSL antibody. Magnification × 250

Discussion

To our knowledge, this is the first study to evaluate serum sICOS level in patients with SSc. Mean sICOS level was increased in patients with dcSSc, but not in those with lcSSc (Fig. 1). sICOS levels were significantly higher in patients with SSc and anti-topo I Abs than those with SSc and ACA (Fig. 2). An increased sICOS level was consistently associated with greater severity of skin fibrosis, and greater frequency and severity of interstitial lung disease (Fig. 3; Table 1). sICOS levels were significantly greater in patients with early phase SSc than in those with late phase SSc (Fig. 4). Furthermore, strong ICOS and ICOSL immunostaining was observed in infiltrating dermal mononuclear cells in sclerotic skin from patients with dcSSc (Fig. 5). These results suggest that ICOS may play an important role in the development of skin sclerosis and interstitial lung disease in patients with SSc.

SSc is generally considered to be a T-helper (Th) 2-dominant autoimmune disease. Serum levels of interleukin (IL)-4 and IL-13, representative Th2 cytokines, are significantly increased in patients with SSc compared to healthy individuals [8, 10, 20], whereas serum IL-4 level gradually decrease as skin sclerosis improves [17]. By contrast, the serum level of IL-12, a Th1-inducing cytokine, is lower in the early phase of SSc than in healthy individuals but increases in the late phase of disease [17], suggesting that a shift from the Th2 to the Th1 response may be associated with improvement of SSc. Previous studies have suggested that ICOS expression is stronger in Th2 cells than in Th1 cells [4, 18]. Furthermore, ICOS stimulation induces IL-4 production, and leads to Th2 polarization [18, 21]. Thus, increased sICOS and enhanced ICOS–ICOSL interaction may contribute to Th2 polarization in SSc. Further, studies are required to examine the association between enhanced ICOS and Th2 polarization in SSc.

Follicular helper T cells express high levels of ICOS [2]. These cells localize to the B-cell follicles in secondary lymphoid organs, and provide cognate help to B-cells, which constitutively express ICOSL, leading to somatic hypermutation, immunoglobulin class switching, and germinal center formation [2, 31]. ICOS or ICOSL deficiency results in decreased numbers of follicular helper T cells, impaired germinal center formation, and diminished T cell-dependent Ab response [1, 16, 19]. Autoantibodies are found in more than 90 % of patients with SSc, and these autoantibodies react to various intracellular components such as DNA topoisomerase I, centromeres, RNA polymerases, U1RNP, and U3RNP [23]. Hypergammaglobulinemia and polyclonal B-cell hyperactivity have been detected in patients with SSc [7]. Moreover, serum anti-topoisomerase I Ab levels correlate positively with disease severity and activity in patients with SSc [24]. Therefore, ICOS–ICOSL interaction between follicular helper T cells and B-cells in secondary lymphoid organs may be enhanced in patients with SSc, leading to increased serum sICOS levels. Further studies are needed to clarify the role of ICOS-expressing follicular helper T cells in SSc.

There are several limitations of the present study that should be considered. First, the population was relatively small; a larger study is essential to confirm our results. Second, because all patients with SSc and healthy individuals in the present study were Japanese, additional studies are needed to verify these findings in other ethnic groups. Third, the precise mechanism by which ICOS contributes to the pathogenesis of SSc has not been clarified. Forth, mRSS values in patients with SSc and normal sICOS level were relatively low. There might be a bias in the selection of patients. Fifth, we have assessed the severity of skin fibrosis only by mRSS. Further studies evaluating skin fibrosis by quantifying the hydroxyproline content in the lesional skin is required. Nevertheless, our findings suggest that the measurement of sICOS in patients with SSc may offer an important means for further evaluation of SSc disease severity.

Acknowledgments

This work was supported by a grant for Research on Intractable Diseases from the Ministry of Health, Labor and Welfare of Japan.

Conflict of interest

The authors have no conflicts of interest.

Copyright information

© Springer-Verlag Berlin Heidelberg 2012