Increased serum soluble CD40 levels in patients with systemic sclerosis

OBJECTIVE: To determine serum levels of soluble CD40 (sCD40) and clinical association in patients with systemic sclerosis (SSc). METHODS: Serum sCD40 levels were examined by ELISA in 49 patients with SSc, 15 patients with systemic lupus erythematosus, and 26 healthy individuals. sCD40 levels in plasma samples, which were obtained at the same time, were also determined. SSc patients were grouped into 22 patients with limited cutaneous SSc (lcSSc) and 27 patients with diffuse cutaneous SSc (dcSSc). RESULTS: There was no significant difference between sCD40 levels of sera and those of plasma. Serum sCD40 levels were significantly elevated in patients with SSc compared to patients with systemic lupus erythematosus and controls (p < 0.001). Serum sCD40 levels were higher in patients with lcSSc than in those with dcSSc (p <0.001). There was no correlation between sCD40 and sCD40 ligand levels in patients with SSc. CONCLUSION: Elevated serum sCD40 levels were associated with lcSSc. These results suggest that the blockade of CD40/CD40 ligand interaction could be a potential therapeutic strategy in SSc.     

Clinical significance of serum levels of matrix metalloproteinase-13 in patients with systemic sclerosis

OBJECTIVES: To investigate the clinical significance of serum matrix metalloproteinase-13 (MMP-13) levels in patients with systemic sclerosis (SSc). METHODS: Serum MMP-13 levels were determined by using a peptide substrate cleavage assay in 20 patients with diffuse cutaneous SSc (dcSSc), 20 with limited cutaneous SSc (lcSSc) and 10 normal controls. RESULTS: The serum MMP-13 levels in patients with dcSSc or lcSSc were significantly lower than those in normal controls (53.4 +/- 14.1 vs 73.2 +/- 11.5 ng/ml, P < 0.0005; 59.4 +/- 14.8 vs 73.2 +/- 11.5 ng/ml, P < 0.005, respectively), but there was no significant difference in the serum MMP-13 levels between patients with dcSSc and those with lcSSc. Disease duration prior to the diagnosis was significantly shorter in SSc patients with decreased serum MMP-13 levels than in those with normal levels (3.0 +/- 2.2 vs 8.6 +/- 7.6 yr, P < 0.0005). In addition, serum MMP-13 levels were moderately correlated with the duration of the disease (r = 0.451, P < 0.05). Though there was no significant difference in the frequencies of pulmonary fibrosis or reduced %DLco (diffusing capacity of lung for carbon monoxide), the frequency of reduced %VC (vital capacity) was significantly greater in patients with decreased serum MMP-13 levels than in those with normal levels (73 vs 24%, P < 0.05). CONCLUSIONS: Matrix metalloproteinase-13 may be involved in the fibrotic process of SSc, especially in the initiation of fibrosis. The serum MMP-13 levels may serve as a useful marker for the severity of pulmonary fibrosis in patients with SSc.     

Clinical significance of soluble CD31 in patients with systemic sclerosis (SSc): association with limited cutaneous SSc.

OBJECTIVE: To determine serum levels of soluble CD31 (sCD31) and its clinical associations in patients with systemic sclerosis (SSc). METHODS: Serum sCD31 levels from 70 patients with SSc were examined by ELISA. For a longitudinal study, 64 sera from 17 SSc patients were analyzed (followup: 0.4-3.9 yrs). RESULTS: Serum sCD31 levels were elevated in patients with SSc (n = 70) compared with healthy controls (n = 20) and patients with systemic lupus erythematosus (n = 15). Serum sCD31 levels were higher in patients with limited cutaneous SSc (lSSc; n = 37) than those with diffuse cutaneous SSc (n = 33). Patients with elevated sCD31 levels had pulmonary fibrosis and decreased percentage vital capacity (%VC) less frequently than those with normal sCD31 levels. sCD31 levels correlated positively with %VC in patients with SSc. This association of elevated sCD31 levels with the lower frequency of pulmonary involvement and better %VC was still observed when analyzed among ISSc patients alone. The elevation of sCD31 was associated with shorter disease duration in patients with lSSc. In a longitudinal study, 75% of patients with SSc showed increased sCD31 levels only transiently in the early phase of the disease. Serum sCD31 levels remained normal during followup in all patients with normal sCD31 levels at the first visit. CONCLUSION: Elevated sCD31 levels were associated with ISSc with relatively early onset and lower frequency and severity of pulmonary fibrosis. These results suggest that sCD31 would be a protective factor for the development of skin sclerosis and pulmonary fibrosis in SSc, since sCD31 has an antiinflammatory effect by inhibiting CD31 mediated transendothelial migration of leukocytes.     

Circulating collagen metabolites in systemic sclerosis. Differences between limited and diffuse form and relationship with pulmonary involvement

OBJECTIVE: To study collagen metabolites in systemic sclerosis (SSc) and their relationship with clinical manifestations of the disease. METHODS: Forty-eight SSc patients, 13 with a diffuse form (dcSSc), 23 with a limited form (lcSSc) and 12 with suspected SSc not fulfilling the ACR criteria, and 31 healthy controls were examined. Serum concentrations of aminoterminal type III procollagen peptide (PIIINP), aminoterminal and carboxyterminal type I procollagen peptides (PINP and PICP) and cross-linked carboxyterminal telopeptide of collagen I (ICTP) were determined by radioimmunoassay. RESULTS: Increased serum concentrations of ICTP were found in SSc patients compared with controls. Distinctly higher levels of ICTP were observed in dcSSc than in lcSSc. High serum ICTP was correlated with skin score and acute phase reactants, and with reduced pulmonary function. Serum PIIINP concentration was elevated in both lcSSc and dcSSc. CONCLUSION: Augmented collagen catabolism accompanies the increased collagen synthesis in SSc. Serum ICTP concentration is a marker of this feature and also reflects clinical severity.     

Elevated levels of circulating CD44 in patients with systemic sclerosis: association with a milder subset

OBJECTIVE: To determine serum levels of soluble CD44 (sCD44), one of the adhesion molecules that regulate the migration of leucocytes, and clinical associations of these levels in patients with systemic sclerosis (SSc). METHODS: Serum sCD44 levels were examined by enzyme-linked immunosorbent assay. RESULTS: Serum sCD44 levels were elevated in SSc patients compared with normal controls. Serum sCD44 levels were higher in patients with limited cutaneous SSc than in those with diffuse cutaneous SSc. Patients with elevated sCD44 levels had pulmonary fibrosis less frequently than those with normal sCD44 levels. Serum sCD44 levels remained elevated during the follow-up in almost all patients with elevated levels at their first visit, whereas they remained normal in all patients with normal levels. CONCLUSION: Elevated sCD44 levels were associated with a relatively mild subset of SSc. These results suggest that CD44 could be a potential therapeutic target in SSc.     

Circulating levels of active transforming growth factor beta1 are reduced in diffuse cutaneous systemic sclerosis and correlate inversely with the modified Rodnan skin score

OBJECTIVES: To determine the relationship between clinical features and circulating levels of active transforming growth factor (TGF) beta1 in the major subsets of systemic sclerosis (SSc). METHODS: In a cross-sectional study cases of diffuse cutaneous SSc (dose) (n = 27) or limited cutaneous SSc (dose) (n = 20) were compared with healthy controls (n = 22). Active and total TGFbeta1 was measured in serum and plasma by a high-sensitivity enzyme-linked immunosorbent assay. RESULTS: There were no significant differences between levels of total serum TGFbeta1. However, cases of dcSSc had lower levels of active TGFbeta1 than cases of lcSSc or controls. In addition, more cases of dcSSc (18/27; 66%, P < 0.025) had no detectable active TGFbeta1 than controls (7/22, 32%) or lcSSc (7/20, 35%). In dcSSc, serum active TGFbeta1 levels correlated negatively with skin score and positively with disease duration. CONCLUSIONS: Contrary to expectation, levels of active TGFbeta1 are reduced in dcSSc and this correlates with two variables known to associate with disease activity, shorter duration and more extensive skin sclerosis. This suggests that active TGFbeta1 may be sequestered in active involved SSc skin and that serum levels are reduced despite strong evidence implicating TGFbeta isoforms in the pathogenesis of fibrosis. Our findings may have implications for systemic TGFbeta-trapping therapies in this disease.     

Serum TIMP-1, TIMP-2, and MMP-1 in patients with systemic sclerosis, primary Raynaud's phenomenon, and in normal controls

BACKGROUND: Excess tissue matrix accumulates in systemic sclerosis (SSc), accounting for both visceral and dermal fibrosis. It is suggested that decreased serum levels of matrix metalloproteinases (MMPs) or increased levels of tissue inhibitors of matrix metalloproteinases (TIMPs) may account for this matrix accumulation. OBJECTIVE: To measure serum levels of tissue inhibitors of metalloproteinases, TIMP-1, TIMP-2, and collagenase-1 (MMP-1), in patients with diffuse cutaneous systemic sclerosis (dcSSc), limited cutaneous systemic sclerosis (lcSSc), primary Raynaud's phenomenon (RP), and in normal controls. METHODS: Serum samples from patients with dcSSc (n=83), lcSSc (n=87), RP (n=80), and normal controls (n=98) were analysed using enzyme linked immunosorbent assays (ELISAs) for total TIMP-1, TIMP-2, and MMP-1. Results from each assay were analysed by the Kruskal-Wallis test. Dunn's multiple comparison post-test was then applied between groups. RESULTS: TIMP-1 levels were significantly raised in dcSSc and lcSSc groups compared with the RP group and normal controls (p<0.01 to p<0.001). In the dcSSc group, TIMP-1 levels were significantly higher in early disease (<2 years) than in late stage disease (>4 years) (p<0.05). This was not found for the lcSSc group. Serum TIMP-2 and MMP-1 levels in dcSSc and lcSSc did not differ significantly from those in normal controls. Increased levels of TIMPs were not convincingly associated with organ disease. No assay result correlated with autoantibody status (anti-topoisomerase 1 (anti-Scl-70), anticentromere antibody, or anti-RNA polymerase). No significant differences in serum TIMP-1, TIMP-2, or MMP-1 levels were shown in the RP group compared with normal controls. CONCLUSIONS: Raised TIMP-1 levels in the SSc groups support the hypothesis that matrix accumulation occurs in SSc at least in part owing to decreased degradation. Moreover, the variation in TIMP-1 levels between the early and late disease stages of dcSSc seems to reflect the early progressive course of dermal fibrosis seen clinically. The expected reduction in serum MMP-1 levels in the SSc groups was not found. This suggests that tissue matrix accumulation is due to increased inhibitors rather than to decreased MMPs.     

Elevated levels of eosinophil major basic protein in the sera of patients with systemic sclerosis

Objective. To examine eosinophil activation, as reflected by evidence of eosinophil degranulation in the blood and affected tissues, in patients with diffuse and limited cutaneous forms of systemic sclerosis (SSc). Methods. Levels of the eosinophil-derived major basic protein (MBP), a marker of eosinophil degranulation, were determined in sera from 46 SSc patients, from patients with rheumatoid arthritis and giant cell arteritis, and from healthy volunteers, and in bronchoalveolar lavage fluid from 4 SSc patients. Extracellular tissue deposition of MBP was evaluated in biopsy specimens from affected skin or lung of 11 SSc patients. Results. Patients with diffuse cutaneous SSc (dcSSc) had elevated serum MBP levels compared with normal individuals (mean ± SD 762 ± 271 ng/ml versus 534 ± 144 ng/ml; P = 0.0004). MBP levels were positively correlated with the extent of cutaneous involvement, and negatively correlated with pulmonary function and duration of disease (r = –0.20). By immunohistochemical analysis, modest extracellular MBP deposition could be demonstrated in involved skin in 7 of 10 biopsy specimens, and MBP staining was prominent in affected lung tissues in 2 patients. Conclusion. Eosinophil degranulation appears to be increased in some patients with dcSSc, as indicated by elevated serum levels of MBP and extracellular accumulation of MBP in the lung. Eosinophil granule proteins may contribute to the development of cutaneous and pulmonary fibrosis in SSc.     

Pulmonary capillary endothelial dysfunction in early systemic sclerosis

OBJECTIVE: Pulmonary capillary endothelium-bound angiotensin-converting enzyme (PCEB-ACE) activity is a sensitive and quantifiable index of endothelial function in vivo. Systemic sclerosis (SSc) is characterized by endothelial damage and excess collagen formation, causing mainly pulmonary hypertension (PH) in the limited cutaneous SSc (lcSSc) subset and interstitial lung disease with pulmonary interstitial fibrosis (PIF) in the diffuse cutaneous SSc (dcSSc) subset. This study was undertaken to investigate the hypothesis that PCEB-ACE activity is reduced early in SSc, in the absence of PH or PIF. METHODS: Applying indicator-dilution techniques, we measured single-pass transpulmonary hydrolysis and percent metabolism (%M) of a synthetic ACE substrate and calculated functional capillary surface area (FCSA) in 25 SSc patients and 11 controls. Substrate hydrolysis and %M reflect ACE activity per capillary; FCSA reflects ACE activity per vascular bed. RESULTS: PCEB-ACE activity was decreased in both SSc subsets. Among patients without PH, substrate hydrolysis and %M were decreased in patients with lcSSc and more profoundly in those with dcSSc; loss of FCSA normalized to body surface area (FCSA/BSA) was observed in dcSSc, but not in lcSSc. High-resolution computed tomography of the lung, performed in all SSc patients, revealed no correlation between substrate %M, hydrolysis, or FCSA/BSA and the degree of PIF; 5 dcSSc and 5 lcSSc patients with no detectable PIF exhibited decreases in hydrolysis and %M, while FCSA/BSA was decreased only in dcSSc. CONCLUSION: Depression of PCEB-ACE activity, indicating pulmonary endothelial dysfunction, occurs early in SSc, in the absence of PH or PIF, and is more pronounced, at this early pulmonary disease stage, in dcSSc than in lcSSc.     

Elevated circulating CD40L concentrations in patients with systemic sclerosis

OBJECTIVE: B cell activation, fibrosis, and expression of adhesion molecules on endothelial cells are regulated by soluble CD40L (sCD40L)/CD40 interactions. Since these effects are characteristic in patients with systemic sclerosis (SSc), serum concentrations of sCD40L were determined in patients with SSc. METHODS: Fifty-two Japanese patients with SSc were examined. They were grouped into 24 patients with limited cutaneous SSc (lSSc) and 28 with diffuse cutaneous SSc (dSSc). Serum sCD40L levels were examined by ELISA. As a disease control, serum samples from 20 patients with systemic lupus erythematosus (SLE) were also examined. In addition, a retrospective longitudinal study was performed in 71 serum samples from 18 patients with SSc. RESULTS: Serum sCD40L levels were elevated in SSc patients compared with healthy controls (p < 0.001). Levels of sCD40L in patients with SSc were higher than in patients with SLE (p < 0.001) that had elevated sCD40L levels compared with healthy controls. Among SSc subsets, there were no differences in sCD40L levels between lSSc and dSSc. sCD40L levels correlated positively with C-reactive protein levels in SSc patients (p < 0.0001, r = 0.449). In a cross-sectional study and a longitudinal study, serum sCD40L levels in dSSc patients were persistently elevated, although those in lSSc patients were temporarily elevated at the early phase of the disease process. CONCLUSION: Patients with SSc exhibited elevated sCD40L levels that may correlate with disease activity. These results suggest that CD40/CD40L interactions may be potential therapeutic targets in SSc.     

Disease subsets, antinuclear antibody profile, and clinical features in 127 French and 247 US adult patients with systemic sclerosis

OBJECTIVE: To investigate the specificities of antinuclear antibodies (ANA) associated with systemic sclerosis (SSc) disease classification and internal organ involvement among patients with SSc of different origins (European and American). METHODS: Serum samples from 374 adult patients diagnosed with SSc were studied: 127 French patients (Paris) were compared with 247 US patients (Pittsburgh). Patients were classified into diffuse cutaneous (dc) and limited cutaneous (lc) SSc subsets. Antibodies associated with SSc were determined by protein and/or RNA immunoprecipitation, indirect immunofluorescence, and immunodiffusion. RESULTS: SSc classification differed significantly in the 2 cohorts: lcSSc and overlap patients with lcSSc combined made up 76% of the French series versus 52% of the US group (p < 0.0001). The frequency of anti-RNA polymerase III antibody was significantly increased in US patients compared with French patients (p < 0.0001). The frequency of anti-topoisomerase I (topo I) antibody was significantly increased among French patients (p < 0.0048). Anti-topo I-positive French SSc patients were less likely to have dcSSc (38% vs 65%) and more likely to have milder disease than US anti-topo I-positive patients. The French dcSSc patients had lower proportions of joint/tendon manifestations and renal crisis (7% vs 17%), but more often had radiographic evidence of pulmonary fibrosis (57% vs 30%). French lcSSc patients had a lower frequency of pulmonary arterial hypertension than US lcSSc patients (9% vs 31%; p = 0.002). CONCLUSION: There are disease classification and SSc-related serum autoantibody differences between French and American patients with SSc. These differences help to explain variations in clinical features reported from different geographic regions.     

Serum levels of heat shock protein 70, a biomarker of cellular stress, are elevated in patients with systemic sclerosis: association with fibrosis and vascular damage

OBJECTIVE: To determine the clinical significance of heat shock protein (Hsp) 70, a sensitive biomarker for monitoring cellular stress, in systemic sclerosis (SSc), we investigated the prevalence and clinical correlation of serum Hsp70 levels in SSc patients. METHODS: Serum Hsp70 levels were examined in 48 patients with SSc by enzyme-linked immunosorbent assay. RESULT: Serum Hsp70 levels were significantly elevated in SSc patients compared to normal controls (n=30), and were similar between patients with diffuse cutaneous SSc (n=26) and those with limited cutaneous SSc (n=22). Serum Hsp70 levels were elevated in 27% of total SSc patients with 30% of diffuse cutaneous SSc patients and 23% of limited cutaneous SSc patients. Hsp70 levels were significantly increased in SSc patients with pulmonary fibrosis or contracture of phalanges compared with those without pulmonary fibrosis or contracture of phalanges. Serum Hsp70 levels correlated positively with modified Rodnan total skin thickness score, renal vascular resistance, serum levels of monocyte chemotactic protein-1, C-reacting protein, and serum levels of 8-isoprostane. CONCLUSION: Serum Hsp70 levels were increased in SSc patients and were associated with pulmonary fibrosis, skin sclerosis, renal vascular damage, oxidative stress, and inflammation. These results suggest that Hsp70 is a useful serological marker for evaluating cellular stresses and the disease severity in SSc.     

Clinical subsets, skin thickness progression rate, and serum antibody levels in systemic sclerosis patients with anti-topoisomerase I antibody

OBJECTIVE: To describe the clinical and laboratory features and natural history of the disease in systemic sclerosis (SSc; scleroderma) patients with anti-topoisomerase I (anti-topo I) antibody who have different skin thickness progression rates (STPRs). METHODS: SSc patients (n = 212) who were anti-topo I antibody positive were divided into 5 subgroups based on STPRs. Skin thickness was measured using the modified Rodnan skin thickness score (MRSS). Anti-topo I IgG antibody levels were determined. RESULTS: Sixty patients who were anti-topo I antibody positive had diffuse cutaneous SSc (dcSSc) with rapid progression, 82 had dcSSC with intermediate progression, and 29 had dcSSc with slow progression, 14 had limited cutaneous SSc (lcSSc) that became dcSSc, and 27 had lcSSc that did not change throughout. Patients beginning with lcSSc were younger at disease onset and had longer disease duration when diagnosed as having SSc. Interstitial lung disease was common and was equally distributed across the subgroups. Renal crisis occurred most often in patients with rapid progression (22%) and was absent in lcSSc patients. Cardiac involvement was most frequent in the dcSSc subgroups. Both kidney and heart disease occurred most often within 3 years after the onset of skin thickening. The 10-year cumulative survival rate was <40% for patients with rapid and intermediate progression. Renal and cardiac causes of death were disproportionately frequent in these 2 subgroups. Anti-topo I antibody levels correlated with the STPR and the MRSS. CONCLUSION: Anti-topo I antibody-positive patients with SSc with a rapid STPR have reduced survival rates, primarily due to early and often fatal renal and cardiac involvement. Anti-topo I antibody levels parallel the MRSS at the first visit and the STPR. This information is important for managing physicians and researchers planning clinical trials involving patients with early dcSSc.     

Clinical and laboratory manifestations of systemic sclerosis (scleroderma) in Black South Africans.

A retrospective study of systemic sclerosis (SSc) in Blacks attending a tertiary hospital on the Witwatersrand, South Africa, was undertaken. The female:male ratio of the 63 patients was 4.6:1 and the mean age of onset of SSc was 36.1 yr. Four of the 11 males were ex-goldminers and nine females resided close to goldmines. Forty-one patients had diffuse cutaneous SSc (dcSSc), 18 had limited cutaneous SSc (lcSSc) and four were unclassified. Overall, 56% had pulmonary fibrosis, 37% had myositis and 98% were antinuclear antibody (ANA) positive, with a notable absence of anti-centromere antibodies. Subset comparisons showed myositis and a reduced forced vital capacity to be significantly more common with dcSSc than lcSSc. The only significant sex differences were that arthralgia/arthritis was more common in women, while calcinosis occurred more frequently in men. Seven of the eight known deaths occurred in patients with dcSSc. These findings, particularly the age of disease onset, predominance of the dcSSc subset, inflammatory features of myositis and a raised erythrocyte sedimentation rate, and absence of anti-centromere antibodies, are similar to those reported previously in African-Americans.     

Serum levels of monocyte chemotactic protein-3/CCL7 are raised in patients with systemic sclerosis: association with extent of skin sclerosis and severity of pulmonary fibrosis

OBJECTIVE: To determine serum levels of monocyte chemotactic protein-3 (MCP-3) and its clinical associations in patients with systemic sclerosis (SSc). METHODS: Serum MCP-3 levels from 69 patients with SSc were examined by ELISA. RESULTS: Serum MCP-3 levels were raised in patients with SSc (n = 69) compared with healthy controls (n = 28). Patients with diffuse cutaneous SSc (n = 36) had higher levels of serum MCP-3 than those with limited cutaneous SSc (n = 33). Patients with raised MCP-3 levels had pulmonary fibrosis and decreased vital capacity (VC) more often than those with normal MCP-3 levels. MCP-3 levels correlated positively with the extent of skin fibrosis, and inversely with %VC and carbon monoxide transfer factor (Tlco) in patients with SSc. CONCLUSION: MCP-3 levels were increased in patients with SSc, and correlated with the extent of skin sclerosis and the severity of pulmonary fibrosis. These results suggest that MCP-3 may have a role in the development of fibrosis in SSc.     

Anti-fibrillarin antibodies in systemic sclerosis

OBJECTIVES: To investigate the nature and extent of organ involvement in anti-fibrillarin antibody (AFA)-positive patients within a UK systemic sclerosis (SSc) population. METHODS: We investigated 1026 consecutive patients with SSc. AFA was identified by the characteristic clumpy nucleolar and coilin body pattern of staining in interphase cells and staining of fibrillarin in metaphase cells by indirect immunofluorescence using HEp-2 cells. Identity of the 34-kDa fibrillarin protein was confirmed by immunoprecipitation from [(35)S]methionine-labelled HeLa cell extract. RESULTS: AFA was detected in 42 patients (4.1%) with early disease onset (mean age 36 yr). Sixteen (38%) patients had limited cutaneous (lcSSc) and 26 (62%) diffuse cutaneous SSc (dcSSc). All eight Afro-Caribbean patients with AFA had dcSSc whereas the Caucasians were equally divided between dcSSc and lcSSc. Within the dcSSc subgroup, 54% had myositis, 35% had pulmonary hypertension, 15% had cardiac involvement and 23% had renal involvement. CONCLUSIONS: AFA identifies young SSc patients with frequent internal organ involvement, especially pulmonary hypertension, myositis and renal disease. In contrast to previous reports, AFA was not restricted to dcSSc patients in Caucasians.     

Antihistone Antibodies in Systemic Sclerosis

Objective. To determine the prevalence and clinical significance of antihistone antibodies (AHA) in systemic sclerosis (SSc). Methods. Serum samples from patients with limited cutaneous SSc (n = 44), diffuse cutaneous SSc (dcSSc; n = 48), and other SSc-related disorders (n = 22) were examined by enzyme-linked immunosorbent assay and immunoblotting for AHA. Results. AHA were demonstrated in 29% of the 92 SSc patients and in 44% of those with dcSSc. The presence of AHA correlated with severe pulmonary fibrosis in those with dcSSc. Immunoblotting revealed that the predominant antigen was histone H1. Conclusion. AHA might be a serologic indicator of the severity of pulmonary fibrosis in SSc.