Systemic Sclerosis Of Recent Onset Research Articles

Global gene expression analysis of systemic sclerosis myofibroblasts demonstrates a marked increase in the expression of multiple NBPF genes

Myofibroblasts are the key effector cells responsible for the exaggerated tissue fibrosis in Systemic Sclerosis (SSc). Despite their importance to SSc pathogenesis, the specific transcriptome of SSc myofibroblasts has not been described. The purpose of this study was to identify transcriptome differences between SSc myofibroblasts and non-myofibroblastic cells. Alpha smooth muscle actin (α-SMA) expressing myofibroblasts and α-SMA negative cells were isolated employing laser capture microdissection from dermal cell cultures from four patients with diffuse SSc of recent onset. Total mRNA was extracted from both cell populations, amplified and analyzed employing microarrays. Results for specific genes were validated by Western blots and by immunohistochemistry. Transcriptome analysis revealed 97 differentially expressed transcripts in SSc myofibroblasts compared with non-myofibroblasts. Annotation clustering of the SSc myofibroblast-specific transcripts failed to show a TGF-β signature. The most represented transcripts corresponded to several different genes from the Neuroblastoma Breakpoint Family (NBPF) of genes. NBPF genes are highly expanded in humans but are not present in murine or rat genomes. In vitro studies employing cultured SSc dermal fibroblasts and immunohistochemistry of affected SSc skin confirmed increased NBPF expression in SSc. These results indicate that SSc myofibroblasts represent a unique cell lineage expressing a specific transcriptome that includes very high levels of transcripts corresponding to numerous NBPF genes. Elevated expression of NBPF genes in SSc myofibroblasts suggests that NBPF gene products may play a role in SSc pathogenesis and may represent a novel therapeutic target.

O30 Long non-coding RNA HOTAIR induces beta catenin expression in systemic sclerosis through EZH2 dependent repression of Axin-2

Abstract Background Fibroblasts explanted from affected tissues in systemic sclerosis (SSc) maintain their pro-fibrotic phenotype in vitro. This includes increased secretion of collagens and other extracellular matrix proteins and increased proportion of α-Smooth Muscle Actin (α-SMA) positive cells (myofibroblasts). It has been previously shown that among their profibrotic features, myofibroblasts display activation of WNT signalling, which is linked to a decreased basal expression of AXIN2. Here we aimed to determine whether specific long non-coding RNA (lncRNAs) expressed in myofibroblasts could drive the epigenetically stable decreased expression of Axin 2 in vitro. Methods Full thickness skin biopsies were surgically obtained from the forearms of twelve adult patients with SSc of recent onset. Fibroblasts were isolated and cultured in monolayers and protein and RNA extracted from the fibroblast cultures. Laser capture was performed to isolate cells expressing or not α-SMA as a marker of myofibroblast differentiation. LncRNA HOTAIR was overexpressed in healthy dermal fibroblasts by lentiviral induction. EZH2 was blocked in cultured fibroblasts with the specific inhibitor GSK126. Results HOTAIR expression was increased in SSc patients’ skin (100 fold) and in SSc explanted fibroblasts (5 fold; p < 0.001 for both). Further, laser captured α-SMA expressing fibroblasts expressed in average 2.5 fold higher HOTAIR RNA levels compared to α -SMA negative cells from the same donors (P < 0.05). In vitro, lentiviral induced stable overexpression of HOTAIR in healthy dermal fibroblasts led to their profibrotic activation, including significantly increased expression of Col1A1 and α-SMA both at mRNA and protein levels (2.8 and 1.8 fold respectively, p < 0.05). We further showed that HOTAIR-induced profibrotic activation was due to EZH2 dependent spread of H3k27me3 methylation marker, as demonstrated by complete inhibition by treatment with GSK126. HOTAIR driven EZH2 histone methylation suppressed the expression of Axin 2 in SSc fibroblasts. The reduced Axin 2 levels led to stabilisation of beta catenin and WNT signalling pathway activation. Conclusion Here we show that the epigenetically stable activation of SSc dermal fibroblasts is due to HOTAIR. We also identified a major downstream target of HOTAIR is Axin-2. The results of these studies identify a new venue to modulate fibroblasts biology which could inform clinical research to resolve chronic fibrosis and re-establish tissue homeostasis in SSc. Disclosures C.W. Wasson None. G. Abignano None. R. Ross None. F. Del Galdo Consultancies; AstraZeneca, GSK, Boehringer-Ingelheim, Actelion, Capella Biosciences, Chemomab.

Pharmacological stress, rest perfusion and delayed enhancement cardiac magnetic resonance identifies very early cardiac involvement in systemic sclerosis patients of recent onset.

To evaluate occult cardiac involvement in asymptomatic systemic sclerosis (SSc) patients by pharmacological stress, rest perfusion and delayed enhancement cardiac magnetic resonance (CMR), for a very early identification of patients at higher risk of cardiac-related mortality. Sixteen consecutive patients with definite SSc, fulfilling the American College of Rheumatology/European League Against Rheumatism 2013 classification criteria in less than 1 year from the onset of Raynaud's phenomenon, underwent pharmacological stress, rest perfusion and delayed enhancement CMR. At enrollment, no patient showed signs and/or symptoms suggestive for cardiac involvement. No patient showed traditional cardiovascular risk factors. Both the 12-lead electrocardiogram examination and echocardiographic evaluation did not show any alterations in our cohort. Stress perfusion defects of left ventricle were detected in six out of 16 (37.5%) patients and these defects did not match with the coronary flow distribution. The results showed the presence of two different patterns of stress perfusion defects: sub-endocardial and/or a midmyocardial. The presence of stress perfusion defects did not correlate with any clinical feature of enrolled patients. Myocardial stress perfusion defects may be detected early by pharmacological stress perfusion CMR, a reliable and sensitive technique for the noninvasive evaluation of SSc heart disease, in patients with SSc of recent onset. These defects seem to be independent from traditional risk factors and associated comorbidities, suggesting they are a specific hallmark of the disease.

Brief Report: Successful pregnancies but a higher risk of preterm births in patients with systemic sclerosis: An Italian multicenter study

To assess fetal and maternal outcomes in women with systemic sclerosis (SSc). Prospectively collected data on 99 women with SSc from 25 Italian centers were analyzed retrospectively. Women with SSc were observed during 109 pregnancies (from 2000 to 2011), and outcomes were compared to those in the general obstetric population (total of 3,939 deliveries). The maternal age at conception was a mean ± SD 31.8 ± 5.3 years, and the median disease duration at conception was 60 months (range 2-193 months). SSc patients, compared to the general obstetric population, had a significantly increased frequency of preterm deliveries (25% versus 12%) and severe preterm deliveries (<34 weeks of gestation) (10% versus 5%), intrauterine growth restriction (6% versus 1%), and babies with very-low birth weight (5% versus 1%). Results of multivariable analysis showed that corticosteroid use was associated with preterm deliveries (odds ratio [OR] 3.63, 95% confidence interval [95% CI] 1.12-11.78), whereas the use of folic acid (OR 0.30, 95% CI 0.10-0.91) and presence of anti-Scl-70 antibodies (OR 0.26, 95% CI 0.08-0.85) were protective. The disease remained stable in most SSc patients, but there were 4 cases of progression of disease within 1 year from delivery, all in anti-Scl-70 antibody-positive women, 3 of whom had a disease duration of <3 years. Women with SSc can have successful pregnancies, but they have a higher-than-normal risk of preterm delivery, intrauterine growth restriction, and babies with very-low birth weight. Progression of the disease during or after pregnancy is rare, but possible. High-risk multidisciplinary management should be standard for these patients, and pregnancy should be avoided in women with severe organ damage and postponed in women with SSc of recent onset, particularly if the patient is positive for anti-Scl-70 antibodies.

A Prospective Observational Study of Mycophenolate Mofetil Treatment in Progressive Diffuse Cutaneous Systemic Sclerosis of Recent Onset

A prospective observational study of mycophenolate mofetil (MMF) treatment in patients with diffuse progressive cutaneous systemic sclerosis (SSc) of recent onset. Twenty-five previously untreated consecutive patients with recent-onset (< 24 mo) diffuse progressive cutaneous SSc received MMF as the only disease-modifying therapy. Modified Rodnan skin score (mRSS) and affected body surface area (BSA) were compared from initiation of MMF to study end. Pulmonary function tests performed at the same institution before therapy and at study end were available in 15 patients. Histopathology and real-time PCR assessment of fibrosis-related gene expression were performed before and after treatment in skin biopsies from 3 patients. At 18.2 ± 8.73 months of MMF therapy (median 2000 mg/day) the mRSS decreased from 24.56 ± 8.62 to 14.52 ± 10.9 (p = 0.0004) and the affected BSA from 36% ± 16% to 14% ± 13.3% (p = 0.00001). Pulmonary function tests remained stable from initiation of MMF to the end of the study. Skin histopathology showed a remarkable reduction in accumulation of fibrotic tissue. Real-time PCR of skin biopsies demonstrated a marked decrease in expression of fibrosis-related genes. Patients with diffuse progressive cutaneous SSc of recent onset treated with MMF experienced marked improvement in skin involvement and stabilization of pulmonary function. Skin biopsies from 3 patients demonstrated histopathological improvement and decreased expression of fibrosis-related genes.

Perceived Functioning Has Ethnic-specific Associations in Systemic Sclerosis: Another Dimension of Personalized Medicine

To measure self-reported physical and mental functioning and associated clinical features at study entry in 3 ethnic groups with systemic sclerosis (SSc). Sixty Hispanic, 39 African American, and 104 Caucasian patients with recent-onset SSc (< 5 yrs) were assessed for perceived physical and mental functioning, using the Medical Outcomes Study Short Form-36 (SF-36) and Scleroderma-Health Assessment Questionnaire (Scleroderma-HAQ). Socioeconomic, demographic, clinical, immunologic, immunogenetic, behavioral, and psychological variables (Interpersonal Support Evaluation List, ISEL; Illness Behavior Questionnaire, IBQ; and Arthritis Helplessness Index, AHI) were analyzed by linear regression models for associations with SF-36 and mHAQ scores as dependent variables. Perceived physical functioning scores had ethnic-specific associations with AHI > fatigue scores > IBQ > clinical variables (hypertension, skin score, and percentage predicted DLCO). Scleroderma-HAQ scores had ethnic-specific associations with IBQ > AHI scores > most clinical and laboratory variables. Decreased mental component summary (MCS) scores associated with AHI > ISEL. Ethnic-specific immunogenetic variables HLA-DQB1*0202 (Caucasian) and HLA-DRB 1*11 (African American), and HLA-DQA1*0501 (Hispanic) also associated with MCS. Antinuclear autoantibodies, anti-topoisomerase I, and RNA polymerases I and III also demonstrated associations with functioning in African American and Hispanic groups. Clinical, psychosocial, and immunogenetic variables had ethnic-specific associations with perceived physical and mental functioning. Consideration of ethnic-specific psychological and behavioral support in designing more personalized, relevant therapeutic interventions for the patient may improve therapeutic efficacy in SSc.

A retrospective randomly selected cohort study of D-penicillamine treatment in rapidly progressive diffuse cutaneous systemic sclerosis of recent onset

A retrospective randomly selected cohort study of D-penicillamine treatment in rapidly progressive diffuse cutaneous systemic sclerosis of recent onset

A retrospective randomly selected cohort study of D-penicillamine treatment in rapidly progressive diffuse cutaneous systemic sclerosis of recent onset

A retrospective randomly selected cohort study of D-penicillamine treatment in rapidly progressive diffuse cutaneous systemic sclerosis of recent onset

A retrospective randomly selected cohort study of D-penicillamine treatment in rapidly progressive diffuse cutaneous systemic sclerosis of recent onset

Several uncontrolled studies in systemic sclerosis have shown that D-penicillamine may cause improvement in skin sclerosis, decrease the rate of new visceral organ involvement, and improve overall survival. To undertake a single-centre retrospective randomly selected cohort study to examine the effects of D-penicillamine treatment on skin and visceral organ involvement in patients with rapidly progressive systemic sclerosis of recent onset. Eighty-four patients with diffuse cutaneous systemic sclerosis who had received D-penicillamine within 24 months of clinically detectable onset of skin sclerosis were randomly selected from the systemic sclerosis cohort followed at the Scleroderma Center of Thomas Jefferson University. Employing a previously described severity scale, disease severity and skin involvement were compared from initiation of D-penicillamine to end of study and a correlated matched t-test was used to establish statistical significance. At a mean+/-SD duration of D-penicillamine therapy of 29.2+/-5.5 months and at a median dose of 750 mg per day statistically significant improvement in skin (P<0.01) and cardiac, pulmonary and renal involvement (P<0.05) was observed. At last follow-up, 17 (20%) patients were still receiving D-penicillamine, 25 (30%) had discontinued it owing to disease improvement, and 18 (21%) had discontinued it owing to side-effects. In a population of patients with diffuse cutaneous systemic sclerosis, with progressive disease of recent onset, D-penicillamine treatment at a median dose of 750 mg per day caused a statistically significant reduction in skin involvement and improvement of renal, cardiac and pulmonary involvement.

The role of DMARDs in systemic sclerosis therapy

The aim of this review is to evaluate the evidence for disease-modifying anti-rheumatic drugs (DMARDs) for treatment of systemic sclerosis (SSc). In the previously published trials, DMARD therapy was usually initiated for severe skin thickening, organ involvement and alveolitis. These studies suggest beneficial effects of methotrexate, azathioprine, ciclosporine A and cyclophosphamide therapy in SSc patients. However, many of these data were derived from retrospective analyses with low numbers of patients, short-term follow-up and often without an appropriate control group. Finally, some of these studies led to inconsistent results. At the present time there is no DMARD therapy of proven efficacy in SSc. Immunosuppressive therapy should only be considered in patients with early diffuse disease, overlap syndromes or pulmonary fibrosis. Current expert recommendations suggest a therapy with methotrexate for skin thickening or cyclophosphamide for acute alveolitis. However, more clinical trials with larger numbers of patients with recent onset SSc are needed.

Inhibition of systemic sclerosis dermal fibroblast type I collagen production and gene expression by simvastatin

To examine whether statins are capable of modulating collagen gene expression in cultured systemic sclerosis dermal fibroblasts. Cultured dermal fibroblasts from 3 patients with diffuse systemic sclerosis of recent onset were treated with 5 microM and 10 microM of simvastatin for 3 or 4 days. Morphologic features, cytotoxicity, and type I collagen production and messenger RNA (mRNA) levels in the fibroblasts were examined. The effects of mevalonate, geranylgeranyl pyrophosphate (GGPP), and farnesyl pyrophosphate (FPP), which are lipids downstream from the hydroxymethylglutaryl-coenzyme A block, were also examined. Transient transfections with COL1A1 promoter-reporter constructs and electrophoretic gel mobility shift assays were utilized to examine COL1A1 transcription and Sp1 and CCAAT-box binding factor (CBF) binding. Simvastatin did not cause morphologic changes or cytotoxicity in the fibroblasts, even after 4 days of treatment. Type I collagen production and mRNA levels showed a potent and dose-related inhibition following 3 and 4 days of treatment. The inhibition of collagen gene expression by simvastatin was completely reversed by mevalonate and GGPP, but not by FPP. The statin effects occurred at the transcriptional level and involved the proximal COL1A1 promoter region encompassing -174 bp. A significant reduction in Sp1 and CBF binding activity was also found in simvastatin-treated cells. Simvastatin is a powerful inhibitor of type I collagen gene expression in normal and systemic sclerosis fibroblasts. The pleiotropic protective effects of statins on various endothelial and immune cell functions in conjunction with their potent inhibitory effects on type I collagen gene expression suggest that statins may be effective therapeutic agents in systemic sclerosis.

Constitutively phosphorylated Smad3 interacts with Sp1 and p300 in scleroderma fibroblasts

To elucidate the role of transforming growth factor-beta (TGF-beta)/Smad signalling in the increased expression of the collagen gene in systemic sclerosis (SSc) fibroblasts. Dermal fibroblasts from seven patients with diffuse SSc of recent onset and from seven healthy individuals were studied. The expression levels of Smad2, Smad3 and Smad4 proteins were determined by immunoblotting. Smad3 phosphorylation and the interaction of Smad3 with Sp1 or p300 were analysed using immunoprecipitation. The effects of overexpression of Smad proteins or Sp1 on the human alpha2(I) collagen gene transcription were investigated with chloramphenicol acetyltransferase (CAT) assays using the -772 COL1A2/CAT construct. Constitutive increased Smad3 phosphorylation was detected in SSc fibroblasts compared with normal fibroblasts. Increased interaction of Smad3 with Sp1 as well as p300 was also detected in SSc fibroblasts. The overexpression of Smad3 caused an increase of up to 5-fold in COL1A2 promoter activity in normal fibroblasts, while Smad3 caused a small increase in COL1A2 promoter activity in SSc fibroblasts. However, neither Smad2 nor Smad4 caused significant effects in COL1A2 promoter activity in normal fibroblasts or SSc fibroblasts. The overexpression of Sp1 caused further increase in COL1A2 promoter activity stimulated by TGF-beta in normal fibroblasts, but did not change COL1A2 promoter activity in the presence of TGF-beta in SSc fibroblasts. The combined overexpression of Smad3 and Sp1 significantly enhanced TGF-beta response in normal fibroblasts, but less markedly in SSc fibroblasts. These results suggested that SSc fibroblasts are less sensitive to exogenous TGF-beta stimulation because they are already activated by the autocrine TGF-beta loop.

High-dose immunosuppressive therapy and autologous progenitor cell transplantation for systemic sclerosis

High-dose immunosuppressive therapy aimed at immunoablation, given together with autologous stem cell transplantation, has resulted in prolonged (>3 years) improvements of skin thickening and functional ability, together with a stabilization of pulmonary function in two-thirds of treated patients with severe systemic sclerosis. Transplant-related mortality occurred in 17% of the first cohort of 41 patients reported to the European Group for Blood and Marrow Transplantation, which included cases from the United States, but this figure has dropped to 8.7% in a recent retrospective analysis of 57 transplants performed in Europe. Similar outcomes were reported in multicentre studies from the United States of 19 patients, and from France of 12 patients, with improvements in skin scores and functional assessments, together with a stabilization of internal organ dysfunction. Based on the results of these phase I/II studies, multicentre prospective randomized trials are being planned or are in progress in Europe and the United States employing uniform patient entry criteria and outcome parameters. The aim of these studies is to compare the clinical benefits and safety of transplant regimens versus conventional chemotherapy in patients with severe systemic sclerosis who are at risk of life-threatening organ failure and premature mortality. Eligibility criteria include a diagnosis of diffuse systemic sclerosis of recent onset and major organ (heart, kidney, lungs) involvement according to predefined criteria. The prospective randomized trials address two issues related to the treatment of severe systemic sclerosis: is intensive immunosuppressive therapy superior to conventional therapy, and can self-tolerance be re-established?

Increased transcriptional activities of transforming growth factor beta receptors in scleroderma fibroblasts.

To investigate the molecular mechanism of the overexpression of transforming growth factor beta receptors (TGF(beta)Rs) in dermal fibroblasts from patients with systemic sclerosis (SSc). Dermal fibroblasts from 7 patients with diffuse SSc of recent onset and from 7 healthy individuals were studied. The expression of TGF(beta)R type I (TGF(beta)RI), TGF(beta)RII, and type I collagen proteins in dermal fibroblasts was determined by immunoblotting. TGF(beta)RI, TGF(beta)RII, and alpha2(I) collagen messenger RNA (mRNA) were evaluated by Northern blot analysis. The transcriptional activities of the TGF(beta)RI and TGF(beta)RII genes were examined by luciferase assay. SSc fibroblasts expressed increased levels of TGF(beta)RI and TGF(beta)RII protein and mRNA, as well as increased levels of type I collagen protein and alpha2(I) collagen mRNA. Moreover, the half-lives of TGF(beta)RI and TGF(beta)RII mRNA in SSc fibroblasts did not change compared with those in control dermal fibroblasts. The promoter activities of the TGF(beta)RI and TGF(beta)RII genes were both significantly increased in SSc fibroblasts compared with those in control fibroblasts. Calphostin C, a specific inhibitor of protein kinase C (PKC), inhibited TGF(beta)RI promoter activity in SSc fibroblasts, and LY294002, an inhibitor of phosphoinositide 3-kinase (PI 3-kinase), inhibited TGF(beta)RII promoter activity in SSc fibroblasts. Moreover, calphostin C and LY294002 inhibited the up-regulation of TGF(beta)RI and TGF(beta)RII mRNA, respectively, in SSc fibroblasts. These results suggest that increased levels of TGF(beta)Rs in SSc fibroblasts play a role in excessive collagen production, and that up-regulation of TGF(beta)R expression might occur at the transcriptional levels. PKC and/or PI 3-kinase might contribute to the up-regulation of TGF(beta)R expression in SSc fibroblasts.

Th2-oriented profile of male offspring T cells present in women with systemic sclerosis and reactive with maternal major histocompatibility complex antigens.

To characterize the cytokine production profile of male-offspring T cells reactive against maternal major histocompatibility complex (MHC) antigens present in the peripheral blood and/or skin from women with systemic sclerosis (SSc). T cell clones were generated from peripheral blood and/or skin biopsy specimens from 3 women with SSc of recent onset and from peripheral blood from 3 healthy women, all of whom had 1 male child. All clones were screened for their proliferative response in vitro to maternal MHC antigens by measuring (3)H-thymidine uptake and for their expression of Y chromosome by using fluorescence in situ hybridization. The concentrations of interferon-gamma and interleukin-4 (IL-4) released by T cell clones in response to maternal MHC antigens were evaluated in culture supernatants, using appropriate enzyme-linked immunosorbent assays. Thirty-nine of 202 T cell clones generated from women with SSc and 11 of 312 from healthy women proliferated in vitro in response to maternal MHC antigens. Seven MHC-reactive T cell clones obtained from women with SSc and 1 obtained from healthy women exhibited the Y chromosome, thus indicating that the clones were derived from T cells of male offspring. All clones generated from male-offspring T cells of SSc women (but not from those of healthy women) produced significantly higher levels of IL-4 in response to stimulation with maternal MHC antigens than did all other clones generated from the same women. The other clones proliferated in response to maternal or allogeneic MHC antigens but did not exhibit the Y chromosome. Male-offspring T cells that are present in the blood and skin of women with SSc and react with maternal MHC antigens exhibit a Th2-oriented profile, supporting the possibility that a chronic graft-versus-host reaction attributable to long-term microchimerism plays a pathogenic role in SSc.

Identification of elements in the promoter region of the α1(I) procollagen gene involved in its up-regulated expression in systemic sclerosis

Objective To identify regulatory elements in the promoter region of the α1(I) procollagen gene (COL1A1) involved in the transcriptional activation of this gene in systemic sclerosis (SSc), and to identify the transcription factors interacting with these regulatory elements. Methods Dermal fibroblasts from 6 patients with diffuse SSc of recent onset and from 6 healthy individuals were studied. The transcriptional regulation of COL1A1 was examined by transient transfections with deletion constructs containing portions of the COL1A1 promoter. The DNA binding activity of nuclear proteins recognizing the regulatory regions in the COL1A1 promoter was examined by gel mobility shift assays. A procedure was established to allow the quantitative determination of the amount of DNA binding proteins interacting with the COL1A1 promoter, employing DNA binding protein and DNA titration experiments analyzed by gel mobility shift assays. Results Maximal chloramphenicol acetyltransferase activity was observed with a -174-bp to +42-bp COL1A1 promoter construct in both normal and SSc cells; however, the activity driven by this construct was 70-260% higher in SSc fibroblasts. Most of the transcriptional activity of the COL1A1 promoter was contained in a minimal promoter region encompassing -174 bp to -84 bp. Electrophoretic mobility shift assays performed within oligonucleotides corresponding to the regions spanning -129/-107 bp and -104/-78 bp of the COL1A1 promoter revealed marked increases in the intensities of DNA-protein complexes formed with both oligonucleotides in nuclear extracts prepared from each of the SSc cell lines in comparison with normal fibroblasts. Competition experiments showed that each of these regions contained elements recognized by Sp1 and nuclear factor 1 (NF-1) binding proteins. A quantitative determination of DNA binding activity recognizing the Sp1 binding element within the -129/-107-bp region showed that it was 23.6 nM in SSc fibroblasts compared with 6.9 nM in normal fibroblasts. Conclusion The results demonstrate that a short region in the proximal promoter of COL1A1 containing 2 tandem NF-1/Sp1 elements displays up-regulated transcriptional activity in SSc fibroblasts, and that SSc fibroblasts contain 3.4-fold greater DNA binding activity recognizing these elements than normal cells.

Identification of elements in the promoter region of the ?1(I) procollagen gene involved in its up-regulated expression in systemic sclerosis

To identify regulatory elements in the promoter region of the alpha1(I) procollagen gene (COL1A1) involved in the transcriptional activation of this gene in systemic sclerosis (SSc), and to identify the transcription factors interacting with these regulatory elements. Dermal fibroblasts from 6 patients with diffuse SSc of recent onset and from 6 healthy individuals were studied. The transcriptional regulation of COL1A1 was examined by transient transfections with deletion constructs containing portions of the COL1A1 promoter. The DNA binding activity of nuclear proteins recognizing the regulatory regions in the COL1A1 promoter was examined by gel mobility shift assays. A procedure was established to allow the quantitative determination of the amount of DNA binding proteins interacting with the COL1A1 promoter, employing DNA binding protein and DNA titration experiments analyzed by gel mobility shift assays. Maximal chloramphenicol acetyltransferase activity was observed with a -174-bp to +42-bp COL1A1 promoter construct in both normal and SSc cells; however, the activity driven by this construct was 70-260% higher in SSc fibroblasts. Most of the transcriptional activity of the COL1A1 promoter was contained in a minimal promoter region encompassing -174 bp to -84 bp. Electrophoretic mobility shift assays performed with oligonucleotides corresponding to the regions spanning -129/-107 bp and -104/-78 bp of the COL1A1 promoter revealed marked increases in the intensities of DNA-protein complexes formed with both oligonucleotides in nuclear extracts prepared from each of the SSc cell lines in comparison with normal fibroblasts. Competition experiments showed that each of these regions contained elements recognized by Sp1 and nuclear factor 1 (NF-1) binding proteins. A quantitative determination of DNA binding activity recognizing the Sp1 binding element within the -129/-107-bp region showed that it was 23.6 nM in SSc fibroblasts compared with 6.9 nM in normal fibroblasts. The results demonstrate that a short region in the proximal promoter of COL1A1 containing 2 tandem NF-1/Sp1 elements displays up-regulated transcriptional activity in SSc fibroblasts, and that SSc fibroblasts contain 3.4-fold greater DNA binding activity recognizing these elements than normal cells.

Identification of fetal DNA and cells in skin lesions from women with systemic sclerosis.

Systemic sclerosis is a disease of unknown origin which often occurs in women after their childbearing years. It has many clinical and histopathological similarities to chronic graft-versus-host disease. Recent studies indicate that fetal stem cells can survive in the maternal circulation for many years post partum. This finding suggests that fetal cells persisting in the maternal circulation or tissues could be involved in the pathogenesis of systemic sclerosis by initiating a graft-versus-host reaction. We used the polymerase chain reaction (PCR) to identify Y-chromosome sequences in DNA extracted from peripheral-blood cells and skin lesions from women with systemic sclerosis of recent onset. To confirm the PCR findings, we used fluorescence in situ hybridization of peripheral-blood cells and cells within chronic inflammatory-cell infiltrates in biopsy specimens of affected skin. Y-chromosome sequences were found in DNA from peripheral-blood cells in 32 of 69 women with systemic sclerosis (46 percent), as compared with 1 of 25 normal women (4 percent, P<0.001), and in T lymphocytes from 3 women with systemic sclerosis who had male offspring. Furthermore, Y-chromosome sequences were identified in skin-biopsy specimens from 11 of 19 women with systemic sclerosis (58 percent); 9 of the 11 were known to have carried male fetuses. Nucleated cells containing Y chromosomes were detected by fluorescence in situ hybridization in paraffin-embedded sections of skin lesions from all seven women we tested whose skin-biopsy specimens contained Y-chromosome sequences. Fetal antimaternal graft-versus-host reactions may be involved in the pathogenesis of systemic sclerosis in some women.

Transcriptional activation of the α1(1) procollagen gene in systemic sclerosis dermal fibroblasts. Role of intronic sequences

To investigate the transcriptional regulation of the alpha 1(I) procollagen gene (COL1A1) in cultured dermal fibroblasts from patients with diffuse systemic sclerosis (SSc) of recent onset and to evaluate the role that intronic sequences may play in the upregulated expression of COL1A1 in SSc dermal fibroblasts. Dermal fibroblasts from 6 patients with diffuse SSc of recent onset and from 3 healthy individuals were studied. The steady-state levels of alpha 1(I) procollagen messenger RNA were evaluated by Northern hybridization analysis, and the transcriptional regulation of COL1A1 was examined by transient transfection experiments with deletion constructs containing portions of COL1A1 promoter (with 5' end points at -5.3 kb, -2.3 kb, and -804 bp and 3' end point at +42 bp) ligated to the chloramphenicol acetyltransferase (CAT) reporter gene. To examine the role of intronic sequences, constructs containing, in addition to the COL1A1 promoter, a portion of the first intron (+380 bp to +1,440 bp) cloned in front of the CAT gene were transfected. The efficiency of transfections was normalized relative to the net amount of CAT plasmid actually transfected into recipient cells, determined by a modified Southern hybridization procedure. Maximal CAT activity was observed with constructs extending from -804 bp to +42 bp in both normal and SSc fibroblasts. However, the activity driven by this construct was 80-110% higher in SSc fibroblasts. The CAT activity driven by a construct with a 5' end point at -5.3 kb was only 15-20% higher in SSc cells, and the CAT activity driven by a construct with a 5' end point -2.3 kb was 35-45% higher in SSc fibroblasts. The CAT activity driven by the -804-bp promoter construct was increased up to 4-fold in SSc fibroblasts in comparison with normal cells when the intronic segment spanning +380 bp to +1,440 bp was included in the transfected construct. The results directly demonstrate the transcriptional activation of COL1A1 in dermal fibroblasts from SSc patients. The data also indicate that first-intron sequences of COL1A1 are required for maximal transcriptional activity of the collagen gene and may play an important role in the up-regulation of its expression in SSc fibroblasts.

Mononuciear Cellular Infiltrates in Clinically Involved Skin from Patients with Systemic Sclerosis of Recent Onset Predominantly Consist of Monocytes/Macrophages

Systemic sclerosis (SSc) is a generalized autoimmune disorder characterized by immunological abnormalities, microvascular dysfunction, and tissue fibrosis. This study evaluated the inflammatory processes occurring in skin of 7 patients with SSc of recent onset (average disease duration of 10 +/- 3 months) to assess the involvement of monocytes/macrophages during the early stages of SSc. All SSc skin biopsies displayed inflammatory microvascular endothelial cell activation and fibrosis. Increased numbers of infiltrating inflammatory leukocytes were present in affected skin of recent onset SSc (p < 0.01) mainly consisting of CD14-positive monocytes/macrophages (p < 0.02). CD3 T lymphocytes were only slightly elevated in SSc skin (84 +/- 39) compared to normal (51 +/- 12), but the differences were statistically not significant. The ratio of CD14/CD3 cells was substantially higher in affected skin of recent onset SSc (4.0 +/- 2.0) than in normal skin (1.4 +/- 0.5, p < 0.01). Monocytes/macrophages, therefore, are the predominant infiltrating mononuclear cell in skin lesions of recent-onset SSc. These results strongly suggest that CD14-positive monocytes/macrophages play an important role during the early stages of SSc pathogenesis.