Novel autoantibody to Cu/Zn superoxide dismutase in patients with localized scleroderma

Abnormal production of reactive oxygen species (ROS) induces tissue damage and superoxide dismutase (SOD) that converts superoxide radicals to hydrogen peroxide functions as defense against ROS. Cu/Zn SOD administration has been shown to be effective for various fibrotic conditions by inhibiting the fibrogenic effects of ROS. We hypothesized that autoimmune background in localized scleroderma induced anti-Cu/Zn SOD autoantibodies that inhibited SOD activity and thereby contributed to fibrosis by increasing ROS. ELISA using human purified Cu/Zn SOD revealed that IgG or IgM anti-Cu/Zn SOD Ab was detected in the serum of 89% of localized scleroderma patients, especially 100% of patients with generalized morphea, the severest form of localized scleroderma, but was positive only in the serum of less than 15% of patients with other autoimmune disorders, including systemic sclerosis, systemic lupus erythematosus, dermatomyositis, and autoimmune bullous disorders. The immunoblotting analysis confirmed the presence of IgG anti-Cu/Zn SOD Ab in sera from localized scleroderma patients. Remarkably, anti-Cu/Zn SOD autoantibody could inhibit Cu/Zn SOD enzymatic activity. Collectively, these results indicate that anti-Cu/Zn SOD Ab is a novel, major autoantibody in localized scleroderma, and also suggest that the autoantibody may play a role in the development of fibrosis by directly inhibiting SOD activity.     

Scleroderma

Sclerodermas may occur in two basic forms: localized sleroderma (LSc) and systemic scleroderma (SSc). Pseudoscleroderma as well as overlap syndromes have to be differentiated from these two variants. From the clinical point of view, localized scleroderma can be subdivided into type I = plaque-like LSc (= morphea), type II = linear LSc, and type III = deep LSc. According to the degree of the cutaneous involvement, systemic scleroderma can likewise be classified into type I = sclerodactylia, type II = acrosclerosis, and type III = scleroderma with primary involvement of the trunk (diffuse scleroderma). In LSc, we never find systemic involvement; SSc, in contrast, is almost always associated with Raynaud's phenomenon, changes of the esophagus, as well as an increased titer of antinuclear antibodies (Hep-2 cell test). Only 23% of our patients with LSc showed elevated ANA titers. We present and discuss data of 56 patients with LSc and 52 patients with SSc. Evidence in the literature as well as our own findings suggest that the pathogenesis of LSc is different from that of SSc. The influence of various mediators and cytokines on the collagen metabolism might be regarded as a theoretical approach in order to develop new therapeutic regimens. This is even more important since there is still no efficient mode of treatment for neither localized nor systemic scleroderma.     

Antinucleosome antibody is a major autoantibody in localized scleroderma

BACKGROUND: Localized scleroderma (LSc) exhibits autoimmunity, and antihistone antibody is frequently detected. The major antigens recognized by antihistone antibody are histones H1, H2A and H2B, which are located on the outer side of the nucleosome and are relatively more accessible for antibody binding. Therefore, it has been hypothesized that antihistone antibody is induced by nucleosome or native chromatin as immunogens in LSc. OBJECTIVES: To determine whether antinucleosome antibody is present in patients with LSc. METHODS: Antinucleosome antibody, antihistone antibody and antidouble-stranded DNA (dsDNA) antibody were determined by enzyme-linked immunosorbent assay. Results IgG or IgM antinucleosome antibody was detected more frequently in patients with LSc than was antihistone antibody: in 40 of 49 (82%) vs. 26 of 49 (53%), respectively. No patients had anti-dsDNA antibody. The prevalence of antinucleosome antibody positivity was comparable in the three subgroups of LSc (generalized morphoea, 89%; linear scleroderma, 71%; morphoea, 90%). Patients with systemic lupus erythematosus (SLE) exhibited a similar frequency of antinucleosome antibody positivity (13 of 15, 87%), but their IgG levels of this autoantibody were much higher than those found in patients with LSc. By contrast, IgM antinucleosome antibody levels were normal in patients with SLE, while they were significantly increased in patients with LSc compared with normal controls. Antinucleosome antibody was also detected at lower frequency in patients with systemic sclerosis (five of 20, 25%) or dermatomyositis (five of 15, 33%). Nucleosome-restricted antibodies, i.e. antibodies that react with the whole nucleosome particle but not with its individual components (histones and dsDNA) were also present in 35% of patients with LSc. CONCLUSIONS: Although antinucleosome antibody was not specific to LSc, its high prevalence in LSc indicates that antinucleosome antibody is a major autoantibody in this disease.     

Soluble CD4 and CD8 in serum from patients with localized scleroderma

Localized scleroderma has been shown to be accompanied by various immunologic abnormalities. To obtain functional information on activated CD4+ or CD8+ T cells, we studied the levels of soluble CD4 (sCD4) and soluble CD8 (sCD8) in serum from patients with localized scleroderma. Serum samples were examined by enzyme-linked immunosorbent assay. The samples were obtained from 49 patients in the following three subgroups: 15 patients with generalized morphea, 22 with linear scleroderma, and 12 with morphea. The levels of sCD4 and sCD8 were significantly elevated in patients with generalized morphea. Furthermore, these patients showed significantly higher levels of sCD4 than those with systemic sclerosis (SSc). The frequency of positivity for IgG anti-single-stranded DNA (ssDNA) antibody was significantly higher in localized scleroderma patients with elevated sCD4 levels than in patients with normal sCD4 levels. The frequency of positivity for antinuclear antibodies, IgM antihistone antibodies, IgG anti-ssDNA antibody and rheumatoid factor, and elevated sCD23 levels were significantly higher in localized scleroderma patients with elevated sCD8 levels than in patients with normal sCD8 levels. Our findings suggest that both CD4+ and CD8+ T cells are activated in vivo in generalized morphea and that the immunologic events in generalized morphea are different from those in SSc.     

Involvement of alphavbeta5 integrin in the establishment of autocrine TGF-beta signaling in dermal fibroblasts derived from localized scleroderma

Localized scleroderma (LSc) is a connective tissue disorder limited to skin and subcutaneous tissue, which may share pathogenic processes with systemic sclerosis (SSc). We previously demonstrated that upregulated expression of integrin alphavbeta5 might contribute to autocrine TGF-beta signaling in SSc fibroblasts. Based on these data, we presently focused on alphavbeta5 and assessed its involvement in pathogenesis of LSc. We initially demonstrated that LSc fibroblasts might be activated by the stimulation of autocrine TGF-beta. Consistent with SSc fibroblasts, expression levels of alphavbeta5 were elevated in LSc fibroblasts in vitro and in vivo. Anti-alphavbeta5 antibody partially reversed expression levels of type I procollagen and MMP-1 and constitutive DNA-Smad3 binding in LSc fibroblasts. In LSc fibroblasts pretreated with antisense TGF-beta1, exogenous latent TGF-beta1 stimulation increased expression of type I procollagen in an alphavbeta5-dependent manner. The luciferase activities of TMLC cells, Mv1Lu cells stably expressing a portion of the plasminogen activator inhibitor 1 promoter, co-cultured with LSc fibroblasts were significantly elevated compared with those co-cultured with normal fibroblasts and were significantly reduced in the presence of anti-alphavbeta5 antibody. Anti-alphavbeta5 antibody reversed the myofibroblastic features of LSc fibroblasts. These results indicate that upregulated expression of alphavbeta5 contributes to autocrine TGF-beta signaling in LSc fibroblasts.     

Anti-agalactosyl immunoglobulin G antibodies in localized scleroderma

BACKGROUND: Anti-agalactosyl immunoglobulin G (IgG) antibodies (anti-AG IgG) have been reported to be detected and correlated with disease activity in some collagen diseases. METHOD: Forty-seven serum samples from patients with localized scleroderma were examined using an enzyme-linked immunosorbent assay. RESULTS: Anti-AG IgG were positive in 19% of patients with localized scleroderma. The frequency of anti-AG IgG in generalized morphea was much higher than that in linear scleroderma or that in morphea. There was a significant correlation between anti-AG IgG levels and the number of the sclerotic lesions and between anti-AG IgG levels and the number of involved areas. The levels of anti-AG IgG were significantly higher in patients with antinuclear antibody, antisingle-stranded DNA antibody or rheumatoid factor than in those without. CONCLUSION: Anti-AG IgG can be an indicator of the severity of localized scleroderma.     

Elevated serum BAFF levels in patients with localized scleroderma in contrast to other organ-specific autoimmune diseases

Serum levels of B-cell activating factor belonging to the tumor necrosis factor family (BAFF), a potent B-cell survival factor, are elevated in patients with systemic autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis and systemic sclerosis (SSc). The objective of this study was to determine serum BAFF levels and relate the results to the clinical features in patients with organ-specific autoimmune diseases of the skin, such as localized scleroderma and autoimmune bullous diseases. Serum BAFF levels were examined by enzyme-linked immunosorbent assay in 44 patients with localized scleroderma, 20 with pemphigus vulgaris/pemphigus foliaceus, 20 with bullous pemphigoid and 30 healthy controls. Twenty patients with SSc and 20 with SLE were also examined as disease controls. Serum BAFF levels were elevated in localized scleroderma patients compared with healthy controls. Concerning localized scleroderma subgroups, patients with generalized morphea, the severest form of localized scleroderma, had higher serum BAFF levels than linear scleroderma or morphea patients. The BAFF levels of generalized morphea were comparable with those of SSc or SLE. Furthermore, serum BAFF levels correlated positively with antihistone antibody levels and the severity of skin lesion as well as the number of skin lesions. By contrast, serum BAFF levels were not significantly elevated in patients with pemphigus or pemphigoid. These results suggest that BAFF may be contributing to autoimmunity and disease development in localized scleroderma.     

Dermal mast cells in scleroderma: their skin density, tryptase/chymase phenotypes and degranulation

To determine the distribution, tryptase/chymase phenotypes and degranulation of mast cells (MCs) in the dermis of patients with scleroderma, we examined MC density in the skin of 22 patients with systemic sclerosis (SSc) and 11 with localized scleroderma (LSc). We used antitryptase and antichymase antibodies after Carnoy's fixation. Detailed reports of two representative patients with SSc and LSc are included. In the scleroedematous stage (grade 1) showing oedema in both papillary and reticular dermis with variable homogenization of collagen bundles in the reticular dermis, MC skin density was variable in each specimen although MC skin density, as a whole, was significantly increased as compared with normal skin ( P  < 0.05). In the sclerotic stage (grade 2) characterized by homogenization of collagen bundles in the entire dermis, MC skin density was significantly decreased as compared with normal skin ( P  < 0.005). LSc showed changes similar to those in SSc. The ratio of MC_TC cells (both tryptase- and chymase-positive MC) to MC_T cells (tryptase-positive but chymase-negative MC) was variable in SSc and LSc. MC_T cells were exclusively dominant in three patients with SSc and two with LSc. In a patient with SSc (patient 1) showing remarkable perivascular and interstitial oedema in the upper dermis, MC skin density was increased in the oedematous portion and tryptase-positive granules were distributed in extracellular locations. In another patient with LSc (patient 2), tryptase positivity increased and chymase positivity decreased in both number and intensity as the skin sclerosis progressed. MCs must have variable interactions with the lesional skin in SSc and LSc. The present study suggests that MCs are involved in the development of interstitial oedema.     

Autoantibody against one of the antioxidant repair enzymes, methionine sulfoxide reductase A, in systemic sclerosis: association with pulmonary fibrosis and vascular damage

Systemic sclerosis (SSc) is a connective tissue disease characterized by fibrosis and vascular changes in the skin and internal organs with autoimmune background. It has been suggested that oxidative stress plays an important role in the development of SSc. To determine the prevalence and clinical correlation of autoantibody to methionine sulfoxide reductase A (MSRA), one of the antioxidant repair enzymes, in SSc, serum anti-MSRA autoantibody levels were examined in patients with SSc by enzyme-linked immunosorbent assay using recombinant MSRA. The presence of anti-MSRA antibody was evaluated by immunoblotting. To determine the functional relevance of anti-MSRA antibody in vivo, we assessed whether anti-MSRA antibody was able to inhibit MSRA enzymatic activity. Serum anti-MSRA antibody levels in SSc patients were significantly higher compared to controls and this autoantibody was detected in 33% of SSc patients. Serum anti-MSRA levels were significantly elevated in SSc patients with pulmonary fibrosis, cardiac involvement, or decreased total antioxidant power compared with those without them. Anti-MSRA antibodies also correlated positively with renal vascular damage determined as pulsatility index by color-flow Doppler ultrasonography of the renal interlobar arteries and negatively with pulmonary function tests. Furthermore, anti-MSRA antibody levels correlated positively with serum levels of 8-isoprostane and heat shock protein 70 that are markers of oxidative and cellular stresses. Remarkably, MSRA activity was inhibited by IgG isolated from SSc sera containing IgG anti-MSRA antibody. These results suggest that elevated anti-MSRA autoantibody is associated with the disease severity of SSc and may enhance the oxidative stress by inhibiting MSRA enzymatic activity.     

Anti-DNA topoisomerase II α autoantibodies in Japanese patients with systemic sclerosis

The pathogenesis and etiology of systemic sclerosis (SSc) remain unknown, but the presence of several autoantibodies is recognized as one of its prominent features. The clinical significance of anti-DNA topoisomerase II α antibody (anti-topo II α Ab) remains unknown in Japanese patients with SSc. To determine the prevalence and clinical correlation of anti-topo II α Ab in Japanese patients with SSc. Serum samples were obtained from 103 Japanese patients with SSc. Control serum samples were obtained from 43 healthy Japanese volunteers. Anti-topo II α Abs were determined by enzyme linked-immunosorbent assay.IgG anti-topo II α Ab levels were significantly increased in SSc patients (n=103) compared to normal controls (n=43; P<0.005). IgG or IgM anti-topo II α Ab was detected in 19% (20/103) of SSc patients when absorbance values higher than the mean+2SD of control serum samples were considered positive. By contrast, IgG or IgM anti-topo II α Ab was observed in only 7% (3/43) of healthy individuals. The presence of pulmonary fibrosis was more frequently detected in SSc patients with IgG anti-topo II α Ab than those without the Ab (P<0.05). Moreover, % DLco and % VC were significantly decreased in SSc patients with anti-topo II α Ab relative to those without the Ab (P<0.05 and P<0.01, respectively). The elevated levels of both erythrocyte sedimentation rate and C-reactive protein were also more frequently observed in SSc patients positive for IgG anti-topo II α Ab (P<0.005). The results of the present study indicate that anti-topo II α Ab represent one of the autoantibody specificities detected on SSc patients and may be regarded a serological marker of pulmonary fibrosis in Japanese patients with SSc. This work was supported by a grant-in-aid from the Ministry of Health and Welfare of Japan (to Minoru Hasegawa and Shinichi Sato)     

Antinuclear and anti-single-stranded DNA antibodies in morphea and generalized morphea

The clinical features of localized scleroderma have allowed investigators to distinguish three morphologic variants: morphea, generalized morphea, and linear scleroderma. The latter has been reported to have a higher frequency of antinuclear antibodies and has been associated with antibodies to single-stranded DNA (ssDNA). In this study we determined the frequency of antinuclear antibodies and anti-ssDNA antibodies in 22 patients with morphea or generalized morphea. None had Raynaud's phenomenon or evidence of a systemic connective-tissue disease. Antinuclear antibodies were present in 18% of patients when serum samples were tested on mouse kidney substrate but were found in 50% of HEp-2 cells. The serum samples contained anti-ssDNA antibodies in 59% of the patients, with the highest levels of ssDNA binding observed in patients with generalized morphea. The frequency of antibodies to ssDNA was higher in patients with clinical evidence of active compared with inactive disease. Discordance in immune reactivity indicates that at least three distinctive serum autoantibodies exist in morphea and generalized morphea: anti-ssDNA antibodies and antinuclear antibodies with either homogeneous or speckled immunofluorescence patterns. These findings are similar to those recently described in linear scleroderma and suggest that comparable serum autoantibody abnormalities are present in all the variants of localized scleroderma.     

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 levels of tumor necrosis factor and interleukin-13 are elevated in patients with localized scleroderma

BACKGROUND: The enhanced production of some cytokines may be related to the pathogenesis of localized scleroderma (LSc). OBJECTIVE: To determine whether serum levels of tumor necrosis factor (TNF) and interleukin (IL)-13 are elevated, and whether they correlated with clinical or serological features in patients with LSc. METHODS: Serum levels of TNF and IL-13 were examined by ELISA in 45 patients with LSc and in 20 healthy controls. RESULTS: The frequency of serum TNF detection was significantly higher in patients with LSc (24%) compared with that in healthy controls (0%). The frequency of serum IL-13 detection was also significantly higher in patients with LSc (29%) compared with that in controls (0%). In patients with LSc, elevated TNF levels significantly correlated with the presence of IgM antihistone antibodies, anti-single-stranded DNA antibodies, elevated serum IL-6 levels, the number of linear lesions, and muscle involvement. Elevated IL-13 levels were significantly associated with the number of plaque lesions and the total number of lesions. CONCLUSIONS: These results suggest that TNF and IL-13 may be associated with the development of LSc.     

Function blocking autoantibodies against matrix metalloproteinase-1 in patients with systemic sclerosis

Systemic sclerosis is characterized by fibrosis and systemic autoimmunity; however, roles of autoantibodies in the development of fibrosis remain unknown in systemic sclerosis. The net accumulation of extracellular matrix is dependent on the balance between the synthesis and degradation of extracellular matrix components, the latter process regulated by matrix metalloproteinases. Matrix metalloproteinase-1 (interstitial collagenase-1) can initiate degradation of collagen types I-III that are major extracellular matrix constituents in affected skin of systemic sclerosis. In this study, we tested the hypothesis that systemic autoimmunity in systemic sclerosis induced anti-matrix metalloproteinase-1 autoantibodies that inhibited matrix metallo-proteinase-1 activity, resulting in collagen accumulation. Enzyme-linked immunosorbent assay using human recombinant matrix metalloproteinase-1 revealed that IgG anti-matrix metalloproteinase-1 autoantibody levels were significantly elevated in sera from patients with systemic sclerosis, but not patients with active systemic lupus erythematosus or dermatomyositis, relative to normal controls. IgG anti-matrix metalloproteinase-1 autoantibody levels were significantly higher in patients with diffuse cutaneous systemic sclerosis than those found in patients with limited cutaneous systemic sclerosis. Furthermore, IgG anti-matrix metalloproteinase-1 antibody levels significantly correlated with the extent of fibrosis in the skin, lung, and renal blood vessels. The presence of IgG anti-matrix metalloproteinase-1 autoantibody in sera from systemic sclerosis patients was confirmed by immunoblotting analysis. Remarkably, IgG anti-matrix metalloproteinase-1 autoantibody in sera from systemic sclerosis patients inhibited matrix metalloproteinase-1 collagenase activity. Collectively, the results of this study suggest that anti-matrix metalloproteinase-1 autoantibody contributes to the development of fibrosis by inhibiting matrix metalloproteinase-1 collagenase activity and reducing the extracellular matrix turnover and suggest that the presence of anti-matrix metalloproteinase-1 autoantibody in systemic sclerosis is the link between systemic autoimmunity and fibrosis.     

Double-blind, placebo-controlled study of oral calcitriol for the treatment of localized and systemic scleroderma

BACKGROUND: Various treatments including corticosteroids, nonsteroidal anti-inflammatory drugs, D-penicillamine, interferon gamma, cyclosporine, and cytostatic drugs have been used with limited success in both morphea and systemic sclerosis (SSc). OBJECTIVE: We investigated the effect of treatment with oral calcitriol in patients with localized or systemic scleroderma. METHODS: A randomized, double-blind, placebo-controlled study of 9 months' duration with a 6-month follow-up was performed at the Department of Dermatology. A total of 27 patients (7 patients with SSc and 20 with morphea) were selected on a minimal skin score of 3 for patients with morphea and 12 for those with SSc. Each patient received calcitriol (0.75 microg/day for 6 months plus 1.25 microg/day for 3 months) or placebo for 9 months. Efficacy parameters included skin score, measurement of serum markers of collagen synthesis and degradation and, additional for the patients with SSc, oral aperture measurements, lung function studies, and esophagus motility. RESULTS: The skin score in patients with morphea after 9 months' treatment showed no significant difference between the placebo and calcitriol groups (mean percentage reduction [SD] in skin score in the placebo group was -29.3 [57.9]; in the calcitriol group it was -19.4 [46.6]). The small group of patients with SSc was inadequate to allow us to draw any conclusions regarding efficacy. No significant change was found in the serum markers of collagen metabolism. CONCLUSION: In this study calcitriol was not more effective than placebo in patients with morphea. Because of the small group of patients with SSc treated, no conclusions regarding efficacy in SSc can be drawn.     

Prevalence and characteristics of anti-single-stranded DNA antibodies in localized scleroderma. Comparison with systemic lupus erythematosus

Prevalence, levels, and immunoglobulin classes of anti-single-stranded DNA antibodies were determined by an enzyme-linked immunosorbent assay in 52 patients with localized scleroderma (33 with morphea, four with generalized morphea, and 15 with linear scleroderma), in 60 healthy controls, and, for comparison, in 31 patients with systemic lupus erythematosus. Localized scleroderma revealed a significant prevalence of anti-single-stranded DNA antibodies, mainly characterized by high levels and IgM and IgA isotypes. Comparison of antibody characteristics in different clinical forms of localized scleroderma showed some significant differences (levels and immunoglobulin isotypes). Comparison with systemic lupus erythematosus showed that frequency, high levels, and IgG isotype of anti-single-stranded DNA antibodies significantly prevailed in systemic lupus erythematosus, while the IgM isotype significantly prevailed in localized scleroderma. However, generalized morphea and linear scleroderma did not significantly differ from systemic lupus erythematosus as regards antibody frequency and prevalence of high antibody levels.     

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.     

Steady-state mRNA levels of collagens I, III, fibronectin, and collagenase in skin biopsies of systemic sclerosis patients.

Total RNA was extracted from skin biopsies of nine patients suffering from systemic sclerosis (SSc). Steady-state mRNA levels of collagen alpha 1(I) and alpha 1(III), collagenase, fibronectin, and beta-actin were studied using specific cDNA probes and compared to those of 12 sex- and age-matched healthy individuals. There was a more than three-fold elevation of collagen I mRNA levels in SSc skin compared to controls. No difference was found, however, for collagen III, collagenase, and fibronectin mRNA levels in SSc and control biopsies. The selective increase of collagen alpha 1(I) mRNA levels indicates a specific alteration of fibroblast metabolism in scleroderma. Analysis of mRNA levels in skin biopsies might not only offer a direct approach to the understanding of the pathophysiology of SSc, but also facilitate the monitoring of fibrotic activity in SSc patients during therapeutic trials.     

Constitutive activation of c‐Abl/protein kinase C‐δ/Fli1 pathway in dermal fibroblasts derived from patients with localized scleroderma

Background A noncanonical pathway of transforming growth factor‐β signalling, the c‐Abl/protein kinase C‐δ (PKC‐δ)/Friend leukemia virus integration 1 (Fli1) axis, is a powerful regulator of collagen synthesis in dermal fibroblasts. Objectives To investigate the significance of the c‐Abl/PKC‐δ/Fli1 pathway for the establishment of the profibrotic phenotype in lesional dermal fibroblasts from patients with localized scleroderma (LSc). Methods The activation status of the c‐Abl/PKC‐δ/Fli1 pathway was evaluated by immunoblotting and chromatin immunoprecipitation using cultured dermal fibroblasts from patients with LSc and closely matched healthy controls and by immunostaining on skin sections. The effects of a platelet‐derived growth factor receptor inhibitor AG1296 and gene silencing of c‐Abl on the expression levels of type I collagen were evaluated by immunoblotting. Results The phosphorylation levels of Fli1 at threonine 312 were increased, while the total Fli1 levels and the binding of Fli1 to the COL1A2 promoter were decreased, in cultured LSc fibroblasts compared with cultured normal fibroblasts. Furthermore, in cultured LSc fibroblasts, the expression levels of c‐Abl were elevated compared with cultured normal fibroblasts and PKC‐δ was preferentially localized in the nucleus. These findings were also confirmed in vivo by immunohistochemistry using skin sections. Moreover, gene silencing of c‐Abl, but not AG1296, significantly suppressed the expression of type I collagen in cultured LSc fibroblasts. Conclusions Constitutive activation of the c‐Abl/PKC‐δ/Fli1 pathway at least partially contributes to the establishment of the profibrotic phenotype in LSc dermal fibroblasts, which provides a novel molecular basis to explain the efficacy of imatinib against skin sclerosis in a certain subset of LSc.     

Suggested mechanisms of action of UVA phototherapy in morphea: a molecular study

BACKGROUND: Ultraviolet A (UVA) phototherapy proved to be an efficient line of treatment of scleroderma. The mechanism through which it acts is still not clear. OBJECTIVES: To detect the mechanism of action of UVA phototherapy in morphea through measuring its effect on the levels of different parameters related to collagen metabolism. METHODS: Twenty-one cases of morphea were treated with low-dose broad-band UVA for 20 sessions. Twelve cases received 20 J/cm(2)/session with a cumulative dose of 400 J/cm(2) and nine cases received 10 J/cm(2)/session with a cumulative dose of 200 J/cm(2). The response was assessed clinically every week. Two skin biopsies were taken from the lesional skin of each patient before starting and after the end of therapy. Paraffin sections were examined for quantitative polymerase chain reaction measurement of collagen I, collagen III, collagenase, transforming growth factor-beta (TGF-beta) and interferon gamma (IFNgamma). RESULTS: Eighteen patients reported remarkable softening of the skin lesions, with variable degrees ranging from moderate in 57.1% of them good in 19% to very good response in 9.5%. After treatment, all the studied parameters revealed statistically significant changes. There was a significant decrease in collagen I, collagen III and TGF-beta and a significant increase in collagenase (MMP-1) and IFNgamma. The relative change was found to be greatest in collagenase, followed by IFNgamma then TGF-beta and finally collagen I. The changes in collagen I, collagenase, IFNgamma and TGF-beta were found to increase gradually with the degree of clinical response. In all the parameters studied the relative change was significantly higher in cases treated with 20 J/cm(2)/session in contrast to those treated with 10 J/cm(2)/session although no statistically significant difference could be detected in the clinical response to those doses. CONCLUSIONS: The efficacy of low-dose UVA phototherapy in the treatment of localized scleroderma is mainly obtained by the increased production of MMP-1 and IFNgamma, and to a lesser extent by decreasing TGF-beta and collagen production. Concerning the use of 10 or 20 J/cm(2)/session those effects are dose dependent, but the clinical response does not significantly differ.