Early dcSSc Research Articles

Peripheral T cells from patients with early systemic sclerosis kill autologous fibroblasts in co-culture: is T-cell response aimed to play a protective role?

Oligoclonal T-cell infiltrates have been detected in the skin of patients with early dcSSc. Peripheral T cells from patients with early dcSSc co-cultured with autologous fibroblasts have been found to expand the same T-cell clonotypes found in the affected skin. Here, we characterize oligoclonally expanded T lymphocytes and investigate functional changes occurring in early-dcSSc co-cultured T lymphocytes and fibroblasts. Peripheral T lymphocytes from five patients with early (<3-year duration) dcSSc were co-cultured with the autologous fibroblasts obtained by punch biopsy of involved skin. T-cell clonotypes expanded in co-cultures were found to be alphabeta(+) and HLA-DR(+), and to promote the apoptosis of autologous fibroblasts. Fibroblasts up-regulated Fas and underwent apoptosis that paired with the expression of Fas ligand (Fasl) on CD4(+) T cells. Finally, the addition of a blocking anti-Fas antibody to the co-cultures resulted in a marked reduction of fibroblast apoptosis, suggesting a critical role of Fas/Fasl engagement in mediating apoptosis in co-cultured fibroblasts. In the co-culture supernatants, we found TGF-beta, IL-1beta, IL-6 and IL-8, cytokines that are known to promote fibrosis in SSc. The same results were registered in each co-culture. Taken together, these data suggest that T-cell response in SSc may also represent an attempt of the immune system to kill fibroblasts, cells likely expressing (auto)antigens, although the overall outcome of the T-cell response contributes to sustain inflammatory loops leading to fibrosis.

Tendon friction rubs in early diffuse systemic sclerosis: prevalence, characteristics and longitudinal changes in a randomized controlled trial

To characterize the baseline tendon friction rubs (TFRs) in early dcSSc and to evaluate the association of change in TFR over 6 and 12 months with changes in modified Rodnan skin score (MRSS) and HAQ-Disability Index (HAQ-DI) over 12 and 24 months, respectively. We analysed data from the d-Pen study, a 2-year study in early dcSSc (< or =18 months from first non-Raynaud's symptom). TFR was scored as present/absent at seven anatomical sites at baseline and every 6 months thereafter. Multivariable linear regression models assessed associations between TFR and change in MRSS, and change in the HAQ-DI, over 12 and 24 months, respectively. Covariates included baseline TFR, change in the TFR over 6 and 12 months, age, sex, duration of SSc, MRSS, and tender joint count and swollen joint count (SJC). Forty-nine (37%) of 134 patients had TFR at baseline, 50% had resolution of their TFR, whereas 21% developed new TFRs. Patients with baseline TFRs were likely to be Caucasian (86 vs 58%) and had a higher HAQ-DI score (P = 0.008). In regression analyses, change in TFR (P = 0.04) and baseline MRSS (P = 0.03) predicted change in MRSS over a 12-month period (Model R(2 )= 0.14). For the HAQ-DI model, independent predictors were change in TFR at 6 months (P = 0.008) and baseline SJC (P = 0.04, Model R(2 )= 0.19). Results were similar for 24-month models. We document the presence of TFR very early in the course of dcSSc. Changes in TFR over 6 and 12 months predict changes in MRSS and HAQ-DI over 12 and 24 months, respectively.

Dermal tissue and cellular expression of fibrillin-1 in diffuse cutaneous systemic sclerosis

To assess dermal expression and fibroblast production of fibrillin-1 (FBN-1) in SSc. In vivo analysis of microfibrillar network was performed using EM from affected and unaffected skin biopsy specimens of dcSSc patients (n = 5) compared with healthy controls (n = 2). FBN-1 matrix deposition and organization by dermal fibroblast cultures from dcSSc (n = 6), healthy (n = 5) and Marfan (n = 4) controls was analysed in vitro by IF with or without TGF-beta activation. Finally, production of FBN-1 by cultured dermal fibroblasts was evaluated by western blot (WB) and real-time PCR. We observed a striking decrease of tissue microfibrillar network in the dermis of SSc patients compared with healthy controls affecting both clinically involved and uninvolved skin. In cultures, SSc dermal fibroblasts displayed no apparent in vitro alteration of synthesis, secretion and organization of microfibril network. The WB and real-time PCR analyses showed similar FBN-1 amounts in matrix and FBN1 gene expression in SSc and healthy controls. We observed a striking decrease of in vivo microfibrillar network in clinically affected and unaffected skin in early dcSSc patients. This does not relate to an inability of SSc dermal fibroblasts to produce, secrete and organize microfibrils in vitro. Therefore, the disturbances of microfibrils in SSc may be a secondary event to matrix remodelling that occurs in this disease.

Increased Frequency and Compromised Function of T Regulatory Cells in Systemic Sclerosis (SSc) Is Related to a Diminished CD69 and TGFβ Expression

BackgroundRegulatory T cells (Tregs) are essential in the control of tolerance. Evidence implicates Tregs in human autoimmune conditions. Here we investigated their role in systemic sclerosis (SSc).Methods/Principal FindingsPatients were subdivided as having limited cutaneous SSc (lcSSc, n = 20) or diffuse cutaneous SSc (dcSSc, n = 48). Further subdivision was made between early dcSSc (n = 24) and late dcSSc (n = 24) based upon the duration of disease. 26 controls were studied for comparison. CD3+ cells were isolated using FACS and subsequently studied for the expression of CD4, CD8, CD25, FoxP3, CD127, CD62L, GITR, CD69 using flow cytometry. T cell suppression assays were performed using sorted CD4CD25highCD127- and CD4CD25lowCD127high and CD3+ cells. Suppressive function was correlated with CD69 surface expression and TGFβ secretion/expression. The frequency of CD4+CD25+ and CD25highFoxP3highCD127neg T cells was highly increased in all SSc subgroups. Although the expression of CD25 and GITR was comparable between groups, expression of CD62L and CD69 was dramatically lower in SSc patients, which correlated with a diminished suppressive function. Co-incubation of Tregs from healthy donors with plasma from SSc patients fully abrogated suppressive activity. Activation of Tregs from healthy donors or SSc patients with PHA significantly up regulated CD69 expression that could be inhibited by SSc plasma.Conclusions/SignificanceThese results indicate that soluble factors in SSc plasma inhibit Treg function specifically that is associated with altered Treg CD69 and TGFβ expression. These data suggest that a defective Treg function may underlie the immune dysfunction in systemic sclerosis.

The Pronounced Th17 Profile in Systemic Sclerosis (SSc) Together with Intracellular Expression of TGFβ and IFNγ Distinguishes SSc Phenotypes

BackgroundSystemic sclerosis (SSc) is an autoimmune disease where controversy on Th1/Th2 balance dominates. We investigated whether the recently discovered Th17 pattern was present in SSc.Methodology and Principal FindingsPatients were subdivided as having limited cutaneous SSc (lcSSc, n = 12) or diffuse cutaneous SSc (dcSSc, n = 24). A further arbitrary subdivision was made between early dcSSc (n = 11) and late dcSSc (n = 13) based upon the duration of disease. As a comparator group 14 healthy controls were studied. CD3+ cells were isolated using FACS and subsequently studied for the expression of CD4, CD8, CD25, CD45Ro, CD45Ra, IL-23, GITR, CD69 and intracellular expression of IL-17, TGFβ and IFNγ using flow cytometry. Levels of IL-17, IL-6, IL-1α and IL-23 were measured using Bioplex assays. SSc patients had more and more activated CD4+ cells. In addition, CD4, CD45Ro and CD45Ra cells from all SSc patients highly expressed the IL23R, which was associated with a higher IL-17 expression as well. In contrast, IFNγ and TGFβ were selectively up regulated in SSc subsets. In line with these observation, circulating levels of IL-17 inducing cytokines IL-6, IL-23 and IL-1α were increased in all or subsets of SSc patients.Conclusion and SignificanceThe combination of IL-17, IFNγ and TGFβ levels in CD45Ro and CD45Ra cells from SSc patients is useful to distinguish between lSSc, ldSSc or edSSc. Blocking Th17 inducing cytokines such as IL-6 and IL-23 may provide a useful tool to intervene in the progression of SSc.

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

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). 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. 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. 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.

Can CCL2 serum levels be used in risk stratification or to monitor treatment response in systemic sclerosis?

Objective: The chemokine CCL2 has been consistently found to be up-regulated in systemic sclerosis. To explore the potential value of serum CCL2 measurement in disease assessment, we have compared CCL2...

Recombinant human anti–transforming growth factor β1 antibody therapy in systemic sclerosis: A multicenter, randomized, placebo‐controlled phase I/II trial of CAT‐192

To evaluate CAT-192, a recombinant human antibody that neutralizes transforming growth factor beta1 (TGFbeta1), in the treatment of early-stage diffuse cutaneous systemic sclerosis (dcSSc). Patients with SSc duration of <18 months were randomly assigned to the placebo group or to 1 of 3 CAT-192 treatment groups: 10 mg/kg, 5 mg/kg, 0.5 mg/kg. Infusions were given on day 0 and weeks 6, 12, and 18. The primary objective of this study was to evaluate the safety, tolerability, and pharmacokinetics of CAT-192. Secondary outcomes included the modified Rodnan skin thickness score (MRSS), the Scleroderma Health Assessment Questionnaire, assessment of organ-based disease, serum levels of soluble interleukin-2 receptor, collagen propeptides (N propeptide of type I [PINP] and type III collagen), and tissue levels of messenger RNA for procollagens I and III and for TGFbeta1 and TGFbeta2. Forty-five patients were enrolled. There was significant morbidity and mortality, including 1 death in the group receiving 0.5 mg/kg of CAT-192 and 3 deaths in the group receiving 5 mg/kg of CAT-192. There were more adverse events and more serious adverse events in patients receiving CAT-192 than in those receiving placebo, although these events were not more frequent in the high-dose treatment group. The MRSS improved in all groups during the study, but there was no evidence of a treatment effect for CAT-192. Improvement in the MRSS correlated with the disease duration (r = -0.54, P = 0.0008). Changes in the PINP level from baseline correlated with changes in the MRSS (r = 0.37, P = 0.027). We report the first evaluation of a systemically administered and repeatedly dosed anti-TGFbeta1 drug. In this pilot study, CAT-192, in doses up to 10 mg/kg, showed no evidence of efficacy. The utility of clinical and biochemical outcome measures and the feasibility of multicenter trials of early dcSSc were confirmed.

Low‐dose intravenous cyclophosphamide in systemic sclerosis: an open prospective efficacy study in patients with early diffuse disease

Objective: To investigate the efficacy of a treatment with low‐dose intravenous cyclophosphamide (CYC) and low‐dose prednisone in early diffuse cutaneous systemic sclerosis (dcSSc).Methods: Patients with dcSSc and a disease duration <24 months consecutively admitted to a tertiary centre underwent a prospective 1‐year study. They were treated with i.v. CYC 500 mg/pulses, 10 mg prednisone equivalent, and supportive therapy. Modified Rodnan skin score (mRss), Health Assessment Questionnaire – Disability Index (HAQ‐DI), forced vital capacity (FVC), and diffusing lung capacity for CO (DLCO) were assessed as outcome measures. In addition, the nine Medsger severity scale scores were evaluated.Results: mRss and DLCO significantly improved at both 6 (p = 0.002 and 0.012, respectively) and 12 months (p = 0.002 and 0.003, respectively). HAQ‐DI showed a nearly significant reduction at 12 months (p = 0.06). Medsger's severity scores also improved for general condition (p = 0.001), peripheral vascular (p = 0.05), skin (p = 0.02), joint/tendon (p = 0.001), muscle (p = 0.05), and lung (p = 0.02). No treatment interruption was needed.Conclusions: This preliminary study suggests a role for low‐dose i.v. CYC in the treatment of early dcSSc. Controlled studies are warranted.

Chemokine receptor CCR2 expression by systemic sclerosis fibroblasts: Evidence for autocrine regulation of myofibroblast differentiation

To investigate expression of the chemokine receptor CCR2 on key cell types involved in the pathogenesis of systemic sclerosis (SSc) and to assess the potential for autocrine activation of SSc dermal fibroblasts via CCL2/CCR2. Chemokine receptor expression in skin biopsy tissues and explanted dermal fibroblasts from a well-characterized cohort of SSc patients was examined using immunohistochemistry and flow cytometry techniques. Autocrine regulation of the expression of fibrotic markers in CCR2+ SSc fibroblast cell lines was assessed using specific ligand or receptor antagonists. We identified strong CCR2 expression in skin biopsy samples of early-stage diffuse cutaneous SSc (dcSSc), but not late-stage dcSSc or limited cutaneous SSc. Double labeling confirmed up-regulation of CCL2/CCR2 on myofibroblasts, pericytes, lymphocytes, macrophages, and endothelial cells. Explanted dermal fibroblasts from early dcSSc tissues expressed CCR2 and CXCR2 in 55% and 66% of cell strains, respectively. There was no expression in control fibroblasts. CCR2+ fibroblasts demonstrated a profibrotic phenotype, with overexpression of alpha-smooth muscle actin (alpha-SMA), connective tissue growth factor (CTGF), and CCL2. Flow cytometric analysis identified a subset of CCR2+ SSc fibroblasts expressing the myofibroblast marker alpha-SMA. In these cultures, specific inhibition of CCL2 or CCR2 attenuated the overexpression of alpha-SMA, but not CTGF or plasminogen activator inhibitor 1. Our results show that CCR2 is up-regulated in early dcSSc on cell types known to be activated in the disease, which is consistent with a key role in SSc pathogenesis. CCR2 expression on SSc fibroblasts appears to regulate the expression of CCL2 and alpha-SMA. Our findings suggest potential autocrine regulation of key profibrotic properties via a CCL2/CCR2 loop in early-stage dcSSc.

Upregulation of Monocyte Chemoattractant Protein-1 in Early Stage Diffuse Scleroderma

Fibroblast derived monocyte chemoattractant protein1 (MCP1 or CCU) is a candidate mediator that may link inflammatory and fibrotic processes in scleroderma (SSc) pathogenesis. This study examines the relationship between MCP-1 ligand and receptor expression and stage and subset of scleroderma. METHODS: Serum samples and skin biopsies were examined from 54 patients with SSc and 12 healthy controls. 20 had early (within 3 years from onset) diffuse cutaneous SSc (dcSSc), 14 late dcSSc and 20 limited cutaneous SSc (1cSSc). Levels of MCP-1 in serum were measured by commercial ELISA. Expression of MCP-1 and its major receptor CCRZ in snap-frozen skin biopsies was determined by immunohistochemistry. Fibroblasts were cultured from a randomly selected subgroup of early dcSSc and culture supernatants examined by western blot. Chemokine receptor expression (CCR2, CXCRZ and CCRS) on fibroblasts was determined by flow cytometry, comparing receptor-specific antibody binding with an isotype matched control. RESULTS: MCP1 serum levels (meansem) were significantly elevated in SSc patients (342&4 pg/ml) compared to controls (132i26pg/ml, p=0.002) and were highest in the early dcSSc subset (433i47 pg/ml) compared to late dcSSc (307+_41 pg/ml) and lcSSc (276228 pg/ml). Western Blot analysis of cultured fibroblasts supernatants confirmed substantial overproduction (meanksem) of MCP-I by cells from early dcSSc (n=8) (RDU=37.5+11.2) compared with normal fibroblasts (RDU=8.3d2.0) ( ~ 3 . 0 0 2 ) . lmmunohistochemistry showed strong expression of MCP1 and CCRZ in skin from all patients with early stage dcSSc. but expression in late stage dcSSc and lcSSc was weaker or absent. By flow cytometry CCRZ was detectable only on early-stage dcSSc, both normal and SSc fibroblasts expressed CXCR2 whereas CCR5 was not detected. CONCLUSION: High serum levels of MCP-I in early diffuse disease, overproduction by fibroblasts cultured from early-stage dcSSc biopsies and CCR2 expression on fibroblasts confirm upregulation of the MCP-I ligand-receptor axis in SSc and suggest that chemokine antagonism may be a logical therapeutic strategy.

D-penicillamine in systemic sclerosis? Yes!

The use of D-penicillamine in systemic sclerosis (SSc) has been controversial. We have reviewed the major published studies on this drug in SSc with diffuse cutaneous (dc) involvement and summarized our own recent experience in dcSSc patients treated with and without D-penicillamine. We conclude that D-penicillamine favourably alters the natural history of skin involvement in dcSSc, even when used in low dose. Furthermore, recurrence of diffuse skin change after discontinuation of D-penicillamine and improvement in skin thickening after reinitiation of the drug support its effectiveness. We believe that the rheumatologic community should use D-penicillamine in patients with early dcSSc.

Cytokine production and serum levels in systemic sclerosis

Serum levels of various cytokines, tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL1-β), and interleukin 2 (IL2), and of soluble IL2 receptors (sIL2R) were determined in 30 patients with definite systemic sclerosis (SSc). Spontaneous and lipopolysaccharide- or mitogen-induced production of the cytokines, TNF-α, IL1-β, and IFN-γ, by peripheral blood mononuclear cells (PBMNC) of these SSc patients was measured by immunoassays. The patients were divided into three groups: 12 with limited cutaneous disease (lcSSc), 7 with diffuse cutaneous disease (dcSSc) <3 years duration, and 11 with dcSSc >3 years duration. None were treated with cytotoxic drugs or biologic response modifiers. Sera of patients with SSc had elevated sIL2R levels, and only low levels of IL2 (1–2 U/ml) were detected in 10 29 sera tested. Spontaneous production of TNF-α and IL1-β by PBMNC of patients with SSc (829 pg/ml ± 215 SEM and 728 pg/ml ± 186, respectively) was significantly higher than that by normal PBMNC obtained from 30 volunteers (25 ± 10 and 34 ± 6 pg/ml, respectively) and tested at the same time as patients' PBMNC. The largest increases in spontaneous release of TNF-α or IL1-β were seen in patients with early dcSSc. No significant difference in spontaneous IFN-γ production by patient or control PBMNC was detected. On the other hand, the mean level of mitogen-induced IFN-γ production by PBMNC was significantly depressed in patients with SSc (103 U/ml ± 18 vs 255 ± 33 U/ml in controls). In vitro-induced production of TNF-α or IL1-β by patients' PBMNC was comparable to that of normal PBMNC. These data indicate that in vivo-activated PBMNC of patients with SSc spontaneously secrete excessive amounts of fibrogenic cytokines, which are involved in modulation of connective tissue synthesis.