Serum levels of soluble programmed death‐1 and programmed death ligand‐1 in systemic sclerosis: Association with extent of skin sclerosis

The interaction of programmed death‐1 (PD‐1) with its ligand, programmed death ligand‐1 (PD‐L1), has been considered to play a key role in the negative regulation of immune responses. Patients with diffuse cutaneous systemic sclerosis (SSc) had higher levels of soluble PD‐1 (sPD‐1) than those with limited cutaneous SSc and healthy individuals. Serum sPD‐1 levels positively correlated with the severity of skin sclerosis. In contrast, serum sPD‐L1 levels were significantly increased in patients with SSc compared with healthy individuals. Moreover, serum sPD‐L1 levels were not associated with the extent of skin sclerosis and were elevated not only in patients with diffuse cutaneous SSc, but also in those with limited cutaneous SSc. These results suggested that serum sPD‐1 levels may increase in patients with SSc and correlate with the severity of skin sclerosis. PD‐1/PD‐L1 interaction may contribute to the development of skin sclerosis in SSc.     

Clinical significance of serum soluble T‐cell immunoglobulin and mucin domain 3 levels in systemic sclerosis: Association with disease severity

T‐cell immunoglobulin and mucin domain 3 (TIM‐3) has been thought to play a crucial role in the negative regulation of immune responses. Here, we examined the levels of serum soluble TIM‐3 (sTIM‐3) in patients with systemic sclerosis (SSc) and evaluated the results with respect to the clinical features of the disease. Patients with diffuse cutaneous SSc (dcSSc) had higher levels of sTIM‐3 than those with limited cutaneous SSc and healthy individuals. Serum sTIM‐3 levels were positively correlated with the severity of skin sclerosis in early phase dcSSc. Moreover, serum sTIM‐3 levels were increased more often in patients with renal crisis and cardiac involvement than in those with normal sTIM‐3 levels. These results suggest that serum sTIM‐3 levels may be increased in patients with early phase dcSSc and associated with cardiac involvement and renal crisis. Measurement of serum sTIM‐3 may be useful for risk stratification in the early stage of the disease.     

Serum interleukin‐34 levels in patients with systemic sclerosis: Clinical association with interstitial lung disease

Interleukin (IL)‐34 is a hematopoietic cytokine promoting proliferation and differentiation of macrophages. Because abnormal activation of macrophages is involved in the development of systemic sclerosis (SSc), we investigated serum IL‐34 levels in patients with SSc. Serum IL‐34 levels were significantly increased in diffuse cutaneous SSc compared with limited cutaneous SSc and healthy controls, while there were no significant differences between limited cutaneous SSc and healthy controls. In addition, SSc patients with increased serum IL‐34 levels more often had interstitial lung disease (ILD) than those with normal levels. Moreover, in SSc patients, serum IL‐34 levels negatively correlated with the percentage of predicted vital capacity, while they positively correlated with ground‐glass opacity score and fibrotic score on chest computed tomography. Collectively, increased serum IL‐34 levels were associated with greater frequency and severity of ILD in SSc patients. Serum IL‐34 levels may be a useful serological marker for SSc‐associated ILD.     

Possible association of decreased serum CXCL14 levels with digital ulcers in patients with systemic sclerosis

CXCL14 serves as a chemoattractant for activated macrophages, immature dendritic cells and natural killer cells, as well as an antiangiogenic factor by preventing the migration of endothelial cells. CXCL14 also exerts an inhibitory effect on the CXCL12/CXCR4 signaling pathway, which is involved in the maintenance of T‐helper (Th)2 bias, and promotes Th1 immune response under the physiological and pathological conditions. Because CXCL14‐mediated biological processes seem to be involved in the development of systemic sclerosis (SSc), which is characterized by Th2/Th17‐skewed immune polarization and impaired neovascularization, we investigated the clinical correlation of serum CXCL14 levels in patients with this disease. Serum CXCL14 levels were significantly decreased in SSc patients compared with healthy individuals and in diffuse cutaneous SSc patients relative to limited cutaneous SSc patients. SSc patients with digital ulcers had serum CXCL14 levels significantly lower than those without. Furthermore, i.v. cyclophosphamide pulse significantly increased serum CXCL14 levels as compared with the baseline in SSc patients with interstitial lung disease successfully treated with this therapy. These results indicate that decreased CXCL14 expression may contribute to the maintenance of Th2‐skewed immune polarization and dysregulated neovascularization, both of which underlie the developmental process of SSc.     

Anti‐topoisomerase I antibody levels as serum markers of skin sclerosis in systemic sclerosis

The aim of the present study was to clarify the clinical significance of anti‐topoisomerase I antibody (Ab) levels in Japanese patients with systemic sclerosis (SSc). Using immunoprecipitation assays and enzyme‐linked immunoassay (ELISA), anti‐topoisomerase I Ab was detected in 53 SSc patients who visited Kanazawa University Hospital between 2001 and 2010. In these patients, the association between serum anti‐topoisomerase I Ab levels measured with ELISA and clinical features were compared using univariate analysis and multiple regression analysis. There were significantly positive correlations between anti‐topoisomerase I Ab levels and the modified Rodnan total skin thickness score (MRSS) and skin thickness progression rate, and a significantly negative correlation with disease duration. On the other hand, anti‐topoisomerase I Ab levels were not significantly associated with other clinical features including lung involvement. In a longitudinal study, anti‐topoisomerase I Ab levels were decreased significantly in patients that had decreased MRSS, but not in patients that had unchanged or increased MRSS. There was a significantly positive association between anti‐topoisomerase I Ab levels and MRSS and a significantly negative association with disease duration by multiple regression analysis. Our findings suggest that serum levels of anti‐topoisomerase I Ab reflect the severity of skin sclerosis in patients with SSc.     

Serum levels of interleukin‐18‐binding protein isoform a: Clinical association with inflammation and pulmonary hypertension in systemic sclerosis

Systemic sclerosis (SSc) is a chronic autoimmune inflammatory disease characterized by extensive tissue fibrosis and various vascular complications. A wealth of evidence suggests the substantial contribution of pro‐inflammatory cytokines to the development of SSc, but the role of interleukin (IL)‐18 signaling in this disease still remains elusive. To address this issue, we herein determined serum levels of IL‐18‐binding protein isoform a (IL‐18BPa), a soluble decoy receptor for IL‐18, by enzyme‐linked immunosorbent assay in 57 SSc patients and 20 healthy controls and evaluated their clinical correlation. Serum IL‐18BPa levels were higher in SSc patients than in healthy controls, while comparable between diffuse cutaneous SSc and limited cutaneous SSc patients. Although serum IL‐18BPa levels were not associated with dermal and pulmonary fibrotic parameters in SSc patients, there was a significant positive correlation between serum IL‐18BPa levels and right ventricular systolic pressure estimated by echocardiography. Furthermore, in 24 SSc patients who underwent right heart catheterization, serum IL‐18BPa levels positively correlated with mean pulmonary arterial pressure. As for systemic inflammatory markers, significant positive correlations of circulating IL‐18BPa levels with erythrocyte sedimentation rate and C‐reactive protein were noted. These results suggest that the inhibition of IL‐18 signaling by IL‐18BPa may be involved in the development of pulmonary vascular involvement leading to pulmonary hypertension and modulate the systemic inflammation in SSc.     

Serum levels of interleukin‐1α in patients with systemic sclerosis

Systemic sclerosis (SSc) is an autoimmune systemic connective tissue disorder characterized by sclerotic change of the skin and multiple internal organs. Although the pathogenesis of this disorder is still unknown, overproduction of extracellular matrix proteins, including collagens and fibronectin, and aberrant immune activation may be involved in the mechanism. Interleukin (IL)‐1 is one of the key regulators of inflammatory response. IL‐1 is also involved in regulating connective tissue remodeling and cellular differentiation of epithelial and ectodermal cells. There are three major members of the IL‐1 family: IL‐1α, IL‐1β and IL‐1 receptor antagonist. IL‐1α was first described as a factor derived from keratinocytes that stimulates thymocyte proliferation. IL‐1α plays a crucial role in procollagen production by fibroblasts derived from patients with SSc. The present study was undertaken to investigate the serum levels of IL‐1α in patients with SSc. Serum samples were obtained from 66 Japanese patients with SSc and 19 healthy controls. Levels of serum IL‐1α were measured with a specific enzyme‐linked immunoassay kit. Mean serum levels were significantly higher in SSc patients than in those healthy control subjects. Moreover, contracture of phalanges was found at a significantly lower prevalence in SSc patients with elevated serum IL‐1α levels than those with normal levels. These results suggest that IL‐1α may play a role in the pathogenesis of SSc.     

Serum heparanase levels: A protective marker against digital ulcers in patients with systemic sclerosis

Heparanase is an endo‐β‐D‐glucuronidase cleaving heparan sulfate side‐chains of heparin sulfate proteoglycans, which is involved in wound healing, inflammation, neovascularization and tumor progression through the degradation and remodeling of the extracellular matrix and the release of sequestered pro‐angiogenic factors. Because heparanase‐mediated biological processes seem to be involved in the development of systemic sclerosis (SSc), we investigated the clinical correlation of serum heparanase levels in patients with this disease. Serum heparanase levels were significantly higher in SSc patients than in healthy individuals, while comparable between diffuse cutaneous SSc and limited cutaneous SSc subgroups. On the other hand, SSc patients with digital ulcers had serum heparanase levels significantly lower than those without. These results suggest that serum heparanase levels may be elevated in SSc patients reflecting the contribution of heparanase‐dependent biological processes to the development of SSc. SSc patients with high serum heparanase levels may be protected from the development of digital ulcers due to the increased release of sequestered pro‐angiogenic factors such as vascular endothelial growth factor. Therefore, serum heparanase levels may serve as a protective marker against digital ulcers in SSc patients.     

Serum levels of tenascin‐C in collagen diseases

Tenascins are a family of large multimetric extracellular matrix (ECM) proteins. Among them, large molecular weight variant tenascin‐C is known to be specifically expressed in pathological conditions. However, no link between tenascin‐C and collagen diseases has been established. The aim of our study was to determine the serum tenascin‐C levels in patients with various collagen diseases, and to evaluate the possibility that serum levels of tenascin‐C can be a useful marker for collagen diseases, correlating with the pathogenesis. Serum tenascin‐C levels of 33 patients with scleroderma (SSc), 10 patients with scleroderma spectrum disorder (SSD), 15 patients with localized scleroderma (LSc), 12 patients with dermatomyositis (DM), 10 patients with systemic lupus erythematosus (SLE) and 15 healthy controls were measured with specific enzyme‐linked immunosorbent assays. Serum tenascin‐C levels were significantly elevated in patients with SSc, SSD and LSc than in healthy controls. Significantly higher total skin thickness score or higher incidence of pitting scars/ulcers and diffuse pigmentation were observed in SSc patients with elevated tenascin‐C levels than in those with normal levels. Our study suggests that serum tenascin‐C levels are increased in fibrotic conditions, and that tenascin‐C contributes to the pathogenesis of vascular damage as well as fibrosis in SSc patients. Clarifying the role of tenascin‐C in the pathogenesis of collagen diseases may lead to a new therapeutic approach.     

Serum omentin levels: A possible contribution to vascular involvement in patients with systemic sclerosis

Adipokines have been shown to be potentially involved in various pathological processes of systemic sclerosis (SSc), including inflammation, vasculopathy and fibrosis, through their pleiotropic effects. Omentin is a member of the adipokines, and has a protective effect against vascular inflammation and pathological remodeling leading to atherosclerosis as well as a vasodilatory effect. To assess the potential role of omentin in the development of SSc, we determined serum omentin levels by enzyme‐linked immunosorbent assay in 66 SSc and 21 control subjects and evaluated their clinical correlation. Serum omentin levels were significantly decreased in diffuse cutaneous SSc patients compared with limited cutaneous SSc patients, while comparable between total SSc patients and healthy controls. In diffuse cutaneous (dc)SSc, patients with a disease duration of 5 years or less had serum omentin levels significantly lower than those with a disease duration of more than 5 years. In total SSc, serum omentin levels were significantly higher in patients with elevated right ventricular systolic pressure than in the others, while serum omentin levels did not correlate with fibrotic and systemic inflammatory parameters. These results suggest that a loss of omentin‐dependent protection against vascular inflammation and remodeling may be related to pathological vascular events of early dcSSc. The elevation of serum omentin levels may serve as a marker of vascular involvement leading to pulmonary arterial hypertension in SSc, which is possibly due to the compensatory induction of omentin against the increased pulmonary vascular tone.     

Clinical significance of serum retinol binding protein‐4 levels in patients with systemic sclerosis

Background Retinol binding protein‐4 (RBP‐4) is a member of adipocytokines, which is potentially associated with fibrosis, vasodilation, and angiogenesis in addition to insulin resistance. Objective To investigate the clinical significance of serum RBP4 levels in patients with systemic sclerosis (SSc), which is a systemic autoimmune disease characterized by fibrosis and vasculopathy. Methods Serum RBP4 levels were determined by enzyme‐linked immunosorbent assay in 62 SSc patients and 19 healthy controls. Results Similar to patients with chronic kidney disease, serum RBP4 levels inversely correlated with estimated glomerular filtration rate in SSc patients with renal dysfunction. Therefore, analyses were carried out by excluding SSc patients with estimated glomerular filtration rate <60 mL/min/1.73 m². Serum RBP4 levels were significantly lower in diffuse cutaneous SSc (dcSSc) than in control subjects [median (25–75 percentile); 25.8 μg/mL (19.6–47.0) vs. 43.1 μg/mL (31.7–53.4), P < 0.05], while there was no significant difference between limited cutaneous SSc (lcSSc) [28.0 μg/mL (25.4–43.3)] and control subjects. In both of dcSSc and lcSSc, patients with Raynaud’s phenomenon had RBP4 levels significantly lower than those without. Furthermore, serum RBP4 levels inversely correlated with pulmonary function test results in dcSSc and with right ventricular systolic pressure in lcSSc. Conclusion Decreased RBP4 levels are associated with the prevalence of Raynaud’s phenomenon in dcSSc and lcSSc, with the severity of interstitial lung disease in dcSSc, and with the degree of pulmonary vascular involvement in lcSSc, suggesting the possible contribution of RBP4 to the pathological events in this disorder.     

Serum levels of ADAM12‐S: possible association with the initiation and progression of dermal fibrosis and interstitial lung disease in patients with systemic sclerosis

Background A disintegrin and metalloprotease (ADAM) 12 is one of the metalloproteinase‐type ADAMs and possesses extracellular metalloprotease and cell‐binding functions. ADAM12 is expressed in two alternative forms, such as a membrane‐anchored form (ADAM12‐L) and a short secreted form (ADAM12‐S). Objective To investigate the clinical significance of serum ADAM12‐S levels in systemic sclerosis (SSc). Methods Serum ADAM12‐S levels were determined by a specific enzyme‐linked immunosorbent assay in 61 SSc patients and 18 healthy controls. Results Serum ADAM12‐S levels were significantly increased in diffuse cutaneous SSc (dcSSc) patients than in healthy controls (0.417 ± 0.389 vs. 0.226 ± 0.065 ng/mL; P < 0.05), while being comparable between limited cutaneous SSc (0.282 ± 0.258 ng/mL) and healthy controls. Serum ADAM12‐S levels significantly elevated in dcSSc patients with disease duration of ≤6 years (0.537 ± 0.449 ng/mL, P < 0.05), but not in dcSSc with disease duration of >6 years (0.225 ± 0.049 ng/mL), compared to healthy controls. Furthermore, in dcSSc patients with disease duration of ≤6 years, serum ADAM12‐S levels correlated positively with modified Rodnan total skin thickness score, ground glass score, and serum C‐reactive protein values, while showed inverse correlation with fibrosis score. Conclusion Elevated serum ADAM12‐S levels are associated with elevated serum inflammatory marker, severity of skin fibrosis, and activity of interstitial lung disease in dcSSc, suggesting the possible contribution of ADAM12‐S to the pathological events in this disorder.     

A possible contribution of elevated serum clusterin levels to the inhibition of digital ulcers and pulmonary arterial hypertension in systemic sclerosis

Clusterin has been demonstrated to be one of the anti-inflammatory and anti-apoptotic factors, suggesting the potential to be involved in the development of systemic sclerosis (SSc). The aim of this study is to determine serum clusterin levels and their clinical associations in patients with SSc. Serum clusterin levels were examined by enzyme-linked immunosorbent assay in 61 SSc patients and 24 healthy individuals. Serum clusterin levels were significantly elevated in SSc patients compared with healthy individuals (P < 0.001). Among SSc patients, there were no differences in serum clusterin levels between those with limited cutaneous SSc (n = 30) and those with diffuse cutaneous SSc (n = 31). SSc patients with raised clusterin levels had digital ulcers and pulmonary arterial hypertension less often than those with normal clusterin levels. The results show that the serum clusterin levels were increased in patients with SSc, and associated with a lower frequency of digital ulcers and pulmonary arterial hypertension. Clusterin could be a protective factor against the development of digital ulcers and pulmonary arterial hypertension in this disease and as such would be a possible therapeutic target.     

Association of anti‐RNA polymerase III antibody and malignancy in Japanese patients with systemic sclerosis

Patients with systemic sclerosis (SSc) have an increased risk of malignancy compared with the general population. Recently, SSc patients with anti‐RNA polymerase III antibody have been reported to have an increased risk of malignancy as compared with those with other disease‐specific autoantibodies in US, European and Australian populations. Therefore, we studied the relationship between disease‐specific autoantibodies and malignancy in 261 Japanese SSc patients. The prevalence of malignancy was significantly higher in patients with anti‐RNA polymerase III antibody (7/22, 31.8%) than in those with anti‐topoisomerase I antibody (2/82, 2.4%) and in those with anticentromere antibody (8/137, 5.8%). Importantly, among seven patients with anti‐RNA polymerase III antibody and malignancy, three patients (42.9%) developed malignancy from 6 months before to 12 months after SSc onset. Thus, malignancy complication in Japanese SSc patients with anti‐RNA polymerase III antibody is as high as that in other races, suggesting that SSc patients with anti‐RNA polymerase III antibody share the same pathological process among different ethnic groups.     

Augmented production of soluble CD93 in patients with systemic sclerosis and clinical association with severity of skin sclerosis

Background The cell surface protein CD93, expressed on endothelial and myeloid cells, mediates phagocytosis, inflammation and cell adhesion. A soluble form of CD93 (sCD93) is released during inflammation. Objectives To determine the serum sCD93 level and its association with clinical parameters in patients with systemic sclerosis (SSc). Methods Serum sCD93 levels were examined by enzyme‐linked immunosorbent assay in 59 patients with SSc, 24 patients with systemic lupus erythematosus and 47 healthy individuals. The expression of CD93 in skin tissues was examined immunohistochemically. In a retrospective longitudinal study, sera from 11 patients with SSc were analysed. Results Serum sCD93 levels were increased in patients with SSc compared with healthy individuals (P < 0·001). Patients with diffuse cutaneous SSc showed greater levels of sCD93 than those with limited cutaneous SSc (P < 0·01) or systemic lupus erythematosus (P < 0·01). Serum sCD93 levels correlated positively with the severity of skin sclerosis. Strong CD93 immunostaining was observed on endothelial cells in lesional skin tissues. In the longitudinal study, sCD93 levels decreased in parallel with improvement in skin sclerosis. Conclusions Serum sCD93 levels are increased in patients with SSc and correlate with the severity and activity of skin sclerosis. CD93 may contribute to the development of skin fibrosis in SSc.     

Possible pro‐inflammatory role of heparin‐binding epidermal growth factor‐like growth factor in the active phase of systemic sclerosis

Heparin‐binding epidermal growth factor (EGF)‐like growth factor (HB‐EGF) is a member of the EGF family growth factors, which affects multiple aspects of the wound healing process such as epithelialization, wound contraction and angiogenesis. In our study, we measured the serum HB‐EGF levels of 51 systemic sclerosis (SSc) patients, which showed a significant increase compared with those of 20 normal subjects. Further analysis revealed a positive correlation between the HB‐EGF level and pulmonary ground‐glass score but no correlation between the former and pulmonary fibrosis score. Other findings include: a significant increase of serum sialylated carbohydrate antigen KL‐6 levels and significant shortness of disease duration in the diffuse cutaneous SSc patients with elevated HB‐EGF levels; and significantly higher HB‐EGF levels in the presence of Raynaud's phenomenon, in that of telangiectasia, and in the absence of contracture of phalanges in all SSc patients. We then evaluated HB‐EGF mRNA levels of fibroblasts harvested from skin samples of the SSc patients and those of foreskin‐derived fibroblasts treated with transforming growth factor‐β, both of which were significantly higher than each control. In conclusion, we speculate that HB‐EGF plays a pro‐inflammatory role in the active skin and lung lesions of SSc.     

No association of atherosclerosis with digital ulcers in Japanese patients with systemic sclerosis: Evaluation of carotid intima‐media thickness and plaque characteristics

Patients with systemic sclerosis (SSc) usually develop Raynaud's phenomenon, persistent digital ischemia and sometimes develop digital ulcers (DU). Several studies have reported an association of carotid artery atherosclerosis with SSc by evaluating carotid intima‐media thickness (IMT) in SSc patients. However, none of those studies analyzed the association between DU and carotid artery atherosclerosis in SSc patients. We examined the association of carotid artery atherosclerosis with digital ulcers by comparing SSc patients with (n = 48, 29.5%) and without (n = 206, 70.5%) DU. The demographic and clinical features of the SSc patients showed that young age, male sex, anti‐topoisomerase I antibody positivity, severe skin sclerosis, interstitial lung disease complication and cardiac involvements were significantly prevalent in patients with DU. In addition, diffuse cutaneous type, anti‐RNA polymerase III antibody positivity and severe skin sclerosis are more frequent in SSc patients with DU at the extensor surface of joints than SSc patients with DU at the digital tip. There were no differences in serum lipid level, carotid IMT or plaque score between SSc patients with and without DU, suggesting that atherosclerotic changes are not primarily involved in the development of DU.