Signaling pathways regulating intercellular adhesion molecule 1 expression by endothelin 1: Comparison with interleukin‐1β in normal and scleroderma dermal fibroblasts

Objective

Endothelin 1 (ET‐1) has been implicated in the pathogenesis of fibrotic and inflammatory diseases, including scleroderma. In addition to modulating vascular tone and extracellular matrix turnover, ET‐1 up‐regulates cell surface adhesion molecules including intercellular adhesion molecule 1 (ICAM‐1), which is key to cell–cell and cell–matrix adhesion and leukocyte infiltration. This study was undertaken to delineate the signal transduction pathways utilized by ET‐1 and compare them with those adopted by proinflammatory cytokine interleukin‐1β (IL‐1β) in normal and scleroderma dermal fibroblasts.

Methods

Protein expression induced by ET‐1 and IL‐1β on normal dermal fibroblasts, with or without signaling inhibitors, was detected by enzyme‐linked immunosorbent assay, while messenger RNA (mRNA) levels were analyzed by LightCycler polymerase chain reaction. Expression of protein kinase Cδ (PKCδ) and PKCϵ protein in normal dermal fibroblasts and scleroderma dermal fibroblasts was determined by Western blotting, and PKCϵ involvement in ET‐1 signaling was confirmed through transfection of an ICAM‐1 promoter construct into murine PKCϵ^−/− fibroblasts. NF‐κB activation was confirmed via electrophoretic mobility supershift assay, and analysis of the ICAM‐1 promoter region was achieved via transfection of deletion constructs into human dermal fibroblasts.

Results

In normal dermal fibroblasts, ET‐1 induced ICAM‐1 mRNA and surface protein expression in a dose‐ and time‐dependent manner via both receptor subtypes, ET_A and ET_B; antagonism of both abolished the ET‐1 response. MEK was involved in the signaling cascade, but phosphatidylinositol 3‐kinase and p38 MAPK were not. Key to the cascade was activation of NF‐κB, achieved by ligation of either receptor subtype. PKCϵ activation led to downstream activation of MEK and, in part, NF‐κB. IL‐1β signaling required NF‐κB and MEK activation, along with activation of PKCδ. ET‐1 and IL‐1β each utilized the same ICAM‐1 promoter region and the same NF‐κB site at –157 bp. Responses to ET‐1 and IL‐1β differed in scleroderma dermal fibroblasts, with ET‐1 sensitivity decreasing and IL‐1β responses remaining intact. Expression of PKCϵ and PKCδ in scleroderma dermal fibroblasts was also altered.

Conclusion

The findings of this study indicate that differences in sensitivity to ET‐1 and IL‐1β in scleroderma dermal fibroblasts may be explained by altered expression of the PKC isoforms and cytokine receptors.

     

Interleukin‐13–producing CD8+ T cells mediate dermal fibrosis in patients with systemic sclerosis

Objective

Fibrosis is a major contributor to morbidity and mortality in systemic sclerosis (SSc). T cells are the predominant inflammatory infiltrate in affected tissue and are thought to produce cytokines that drive the synthesis of extracellular matrix (ECM) proteins by fibroblasts, resulting in excessive fibrosis. We have previously shown that aberrant interleukin‐13 (IL‐13) production by peripheral blood effector CD8+ T cells from SSc patients correlates with the extent of skin fibrosis. The present study was undertaken to investigate the role of IL‐13 production by CD8+ T cells in dermal fibrosis, an early and specific manifestation of SSc.

Methods

ECM protein production by normal dermal fibroblasts cocultured with SSc CD8+ T cell supernatants was determined by quantitative polymerase chain reaction and Western blotting. Skin‐homing receptor expression and IL‐13 production by CD8+ T cells in the peripheral blood of SSc patients were measured by flow cytometry. IL‐13+ and CD8+ cells in sclerotic skin were identified by immunohistochemistry.

Results

IL‐13–producing circulating CD8+ T cells from patients with SSc expressed skin‐homing receptors and induced a profibrotic phenotype in normal dermal fibroblasts, which was inhibited by an anti–IL‐13 antibody. High numbers of CD8+ T cells and IL‐13+ cells were found in the skin lesions of SSc patients, particularly during the early inflammatory phase of the disease.

Conclusion

These findings show that IL‐13–producing CD8+ T cells are directly involved in modulating dermal fibrosis in SSc. The demonstration that CD8+ T cells homing to the skin early in the course of SSc are associated with accumulation of IL‐13 is an important mechanistic contribution to the understanding of the pathogenesis of dermal fibrosis in SSc and may represent a potential target for therapeutic intervention.

     

Systemic sclerosis Th2 cells inhibit collagen production by dermal fibroblasts via membrane‐associated tumor necrosis factor α

Objective

In systemic sclerosis (SSc; scleroderma), T cells infiltrate organs undergoing fibrotic changes and may participate in dysregulated production of collagen by fibroblasts. The objective of this study was to functionally characterize T cells infiltrating skin lesions in early SSc and investigate their capacity to affect production of type I collagen and interstitial collagenase (matrix metalloproteinase 1 [MMP‐1]) by dermal fibroblasts.

Methods

Four‐color cytometric analysis was used to characterize subset distribution and production of interferon‐γ (IFNγ) and interleukin‐4 (IL‐4) in T cell lines generated from the skin of patients with SSc. T cell clones were generated, and their capacity to modulate collagen and MMP‐1 production by fibroblasts derived from patients with SSc and from normal individuals was assessed. Neutralizing reagents were used to identify T cell mediators involved in fibroblast modulation.

Results

The skin of individuals with early‐stage SSc contained T cells preferentially producing high levels of IL‐4. Cloned CD4+ Th2‐like cells inhibited collagen production by normal fibroblasts. Th2 cell‐dependent inhibition was, at least in part, contact‐dependent, was essentially mediated by tumor necrosis factor α (TNFα), and was dominant over the enhancement induced by profibrotic IL‐4 and transforming growth factor β cytokines. The simultaneous induction of MMP‐1 production confirmed the specificity of these observations. To be inhibitory, Th2 cells required activation by CD3 ligation. Th2 cells were less potent than were Th1 cells in inhibiting collagen production by normal fibroblasts via cell‐to‐cell interaction, and SSc fibroblasts were resistant to inhibition.

Conclusion

These findings indicate that, despite their production of IL‐4, Th2 cells reduce type I collagen synthesis by dermal fibroblasts because of the dominant effect of TNFα, and suggest that strategies based on TNFα blockade aimed at controlling fibrosis in SSc may be unwise.

     

Monocyte chemoattractant protein 1 released from glycosaminoglycans mediates its profibrotic effects in systemic sclerosis via the release of interleukin‐4 from T cells

Objective

Monocyte chemoattractant protein 1 (MCP‐1; CCL2) has been implicated in the pathogenesis of fibrotic diseases and is up‐regulated in patients with systemic sclerosis (SSc). The aim of the present study was to examine the mechanisms by which MCP‐1 mediates its profibrotic effects in the setting of SSc.

Methods

The expression of receptors for MCP‐1 on dermal fibroblasts was analyzed by real‐time polymerase chain reaction and fluorescence‐activated cell sorting. The ability of extracellular matrix proteins to bind and release MCP‐1 was quantified by enzyme‐linked immunosorbent assay. Th0 cells were isolated using a magnetic‐activated cell sorting system and were stimulated twice in the presence of MCP‐1. The synthesis of collagen was measured using the Sircol collagen assay kit.

Results

The glycosaminoglycan chondroitin sulfate, but not fibronectin or collagens, bound and released MCP‐1 in a time‐dependent manner. MCP‐1 that was released from chondroitin sulfate induced the differentiation of interleukin‐4 (IL‐4)–producing T cells in a dose‐dependent manner. In turn, dermal fibroblasts from patients with SSc expressed IL‐4 receptor, and stimulation with IL‐4 significantly increased the production of collagen in dermal fibroblasts. In contrast, CCR2a and CCR2b, as well as D6 and US28 (other potential receptors of MCP‐1), were not detectable in SSc and normal fibroblasts, and their expression was not induced by platelet‐derived growth factor, IL‐1β, or IL‐4. In addition, MCP‐1 had no direct effects on collagen production by fibroblasts.

Conclusion

MCP‐1 has no direct effects on dermal fibroblasts but contributes to fibrosis in patients with SSc by inducing the differentiation of IL‐4–producing T cells. Because MCP‐1 has both proinflammatory and profibrotic effects, pharmacologic targeting of MCP‐1 could be a promising therapeutic approach in SSc.

     

Chemokine expression by systemic sclerosis fibroblasts: Abnormal regulation of monocyte chemoattractant protein 1 expression

Objective

Chemokines are important mediators in the chemoattraction of leukocytes to sites of inflammation. This study investigated the potential contribution of systemic sclerosis (SSc) fibroblasts to chemokine production and its potential relevance to the pathogenesis of SSc.

Methods

The expression of messenger RNA (mRNA) for different C‐C and C‐X‐C chemokines by SSc and normal fibroblasts was studied by RNase protection assay. Monocyte chemoattractant protein 1 (MCP‐1) protein production was analyzed by enzyme‐linked immunosorbent assay. The chemotactic effect of fibroblast‐derived MCP‐1 on monocytic cells was analyzed in a transmigration assay. Nuclear factor κB (NF‐κB) and activator protein 1 (AP‐1) activation in fibroblasts was studied by electromobility shift analysis. MCP‐1 expression in SSc skin sections was studied by immunohistochemistry.

Results

Among all chemokine genes studied, only MCP‐1 and interleukin‐8 mRNA were expressed by nonstimulated normal and SSc fibroblasts. SSc fibroblasts displayed increased constitutive expression of MCP‐1 mRNA and protein and showed a blunted response to oxidative stress. Increased MCP‐1 production was associated with higher chemotactic activity for monocytic cells. Increased NF‐κB or AP‐1 activation was not responsible for the constitutive overexpression of MCP‐1 by SSc fibroblasts. In SSc skin sections, MCP‐1 expression was detected in fibroblasts, keratinocytes, and mononuclear cells, whereas it was undetectable in normal skin.

Conclusion

SSc fibroblasts display a specific pattern of chemokine gene expression that is characterized by constitutively increased and abnormally regulated expression of MCP‐1 in vitro. MCP‐1 is also expressed in lesional skin and can participate in the pathogenesis of SSc.

     

Transforming growth factor β induces fibroblast fibrillin‐1 matrix formation

Objective

Fibrillin, an extracellular matrix protein implicated in dermal fibrosis, is increased in the reticular dermis of systemic sclerosis (SSc) skin. We undertook this study to investigate the hypothesis that transforming growth factor β (TGFβ) or other cytokines regulate fibrillin matrix formation by normal and SSc fibroblasts. We further investigated the mechanism of TGFβ‐induced fibrillin fibrillogenesis and its relationship to myofibroblasts.

Methods

Fibrillin and fibronectin matrix deposition and α‐smooth muscle actin expression by fibroblast cultures from normal and SSc skin treated with TGFβ or other cytokines were analyzed by immunofluorescence. Supernatant and extracellular matrix from normal and SSc fibroblasts treated with or without TGFβ were evaluated by Western blot and Northern blot for fibrillin protein and messenger RNA (mRNA) expression, respectively.

Results

Immunofluorescence demonstrated increased fibrillin matrix formation by normal and scleroderma fibroblasts after TGFβ treatment. Other cytokines, including tumor necrosis factor α, interleukin‐1β (IL‐1β), IL‐4, granulocyte–macrophage colony‐stimulating factor, and platelet‐derived growth factor, did not affect fibrillin fibrillogenesis. Fibrillin matrix formed in proximity to myofibroblasts and independently of up‐regulation of fibronectin matrix or cell number. Western blot analysis of extracellular matrix confirmed increased fibrillin after TGFβ stimulation of normal or scleroderma fibroblasts. However, TGFβ did not alter the expression of either soluble fibrillin protein or fibrillin mRNA.

Conclusion

Our data show that TGFβ induces fibrillin protein incorporation into the extracellular matrix without affecting fibrillin gene expression or protein synthesis, suggesting that fibrillin matrix assembly is regulated extracellularly. TGFβ might increase fibrillin matrix by activating myofibroblasts. Such TGFβ‐mediated effects could account for the increased fibrillin matrix observed in SSc skin.

     

Interleukin‐17A+ Cell Counts Are Increased in Systemic Sclerosis Skin and Their Number Is Inversely Correlated With the Extent of Skin Involvement

Objective

Levels of interleukin‐17A (IL‐17A) have been found to be increased in synovial fluid from individuals with systemic sclerosis (SSc). This study was undertaken to investigate whether IL‐17A–producing cells are present in affected SSc skin, and whether IL‐17A exerts a role in the transdifferentiation of myofibroblasts.

Methods

Skin biopsy samples were obtained from the involved skin of 8 SSc patients and from 8 healthy control donors undergoing plastic surgery. Immunohistochemistry and multicolor immunofluorescence techniques were used to identify and quantify the cell subsets in vivo, including IL‐17A+, IL‐4+, CD3+, tryptase‐positive, α‐smooth muscle actin (α‐SMA)–positive, myeloperoxidase‐positive, and CD1a+ cells. Dermal fibroblast cell lines were generated from all skin biopsy samples, and quantitative polymerase chain reaction, Western blotting, and solid‐phase assays were used to quantify α‐SMA, type I collagen, and matrix metalloproteinase 1 (MMP‐1) production by the cultured fibroblasts.

Results

IL‐17A+ cells were significantly more numerous in SSc skin than in healthy control skin (P = 0.0019) and were observed to be present in both the superficial and deep dermis. Involvement of both T cells and tryptase‐positive mast cells in the production of IL‐17A was observed. Fibroblasts positive for α‐SMA were found adjacent to IL‐17A+ cells, but not IL‐4+ cells. However, IL‐17A did not induce α‐SMA expression in cultured fibroblasts. In the presence of IL‐17A, the α‐SMA expression induced in response to transforming growth factor β was decreased, while MMP‐1 production was directly enhanced. Furthermore, the frequency of IL‐17A+ cells was higher in the skin of SSc patients with greater severity of skin fibrosis (lower global skin thickness score).

Conclusion

IL‐17A+ cells belonging to the innate and adaptive immune system are numerous in SSc skin. IL‐17A participates in inflammation while exerting an inhibitory activity on myofibroblast transdifferentiation. These findings are consistent with the notion that IL‐17A has a direct negative‐regulatory role in the development of dermal fibrosis in humans.

     

Rosiglitazone induces interleukin‐1 receptor antagonist in interleukin‐1β–stimulated rat synovial fibroblasts via a peroxisome proliferator–activated receptor β/δ–dependent mechanism

Objective

To study the potency of 2 peroxisome proliferator–activated receptor γ (PPARγ) agonists, 15‐deoxy‐Δ^12,14‐prostaglandin J₂ (15‐deoxy‐PGJ₂) and rosiglitazone, to modulate the expression of interleukin‐1 receptor antagonist (IL‐1Ra) in rat synovial fibroblasts.

Methods

Levels of messenger RNA for IL‐1Ra and PPAR isotypes (α, β/δ, γ) were assessed by real‐time polymerase chain reaction in rat synovial fibroblasts exposed to 10 ng/ml of IL‐1β. PPAR levels were assessed by Western blotting and secreted IL‐1Ra levels by immunoassay. The potency of PPARγ agonists and the PPARβ/δ agonist GW‐501516 on IL‐1Ra levels was tested in the range of 1–10 μM and at 100 pM, respectively. The contribution of PPARγ to the effects of rosiglitazone on IL‐1Ra secretion was examined either by its overexpression or by inhibition using wild‐type or dominant‐negative constructs and the antagonist GW‐9662 (10 μM), respectively. The dominant‐negative strategy was also performed to investigate the possible contribution of PPARβ/δ and NF‐κB activation.

Results

IL‐1β–induced IL‐1Ra production was increased by 10 μM rosiglitazone but was reduced dose‐dependently by 15‐deoxy‐PGJ₂. Both agonists lowered IL‐1β secretion, but rosiglitazone alone reduced the imbalance of IL‐1β/IL‐1Ra toward basal levels. Enhancement of IL‐1β–induced IL‐1Ra production by rosiglitazone was not affected by PPARγ overexpression or by its inhibition with dominant‐negative PPARγ or GW‐9662. Inhibition of NF‐κB was also ineffective against rosiglitazone but abolished the stimulating effect of IL‐1β on IL‐1Ra. All PPAR isotypes were expressed constitutively in rat synoviocytes, but PPARγ decreased dramatically upon IL‐1β exposure, whereas PPARβ/δ remained stable. Dominant‐negative PPARβ/δ abolished the enhancement of IL‐1Ra by rosiglitazone, whereas GW‐501516 reproduced the effect of rosiglitazone on IL‐1Ra secretion.

Conclusion

Rosiglitazone stimulates IL‐1Ra production by a PPARβ/δ mechanism in activated rat synovial fibroblasts, further contributing to its potential antiarthritic properties and opening new perspectives for the modulation of inflammatory genes by specific PPAR agonists in articular cells.

     

Treatment with rapamycin prevents fibrosis in tight‐skin and bleomycin‐induced mouse models of systemic sclerosis

Objective

Rapamycin, a novel macrolide immunosuppressive drug, is increasingly used as an agent for posttransplant immunosuppression and treatment of autoimmune disease. The molecular mechanism related to rapamycin‐mediated immunosuppression is that rapamycin binds to FK‐506 binding protein 12, and the formed complex inhibits the function of the mammalian target of rapamycin (mTOR), which in turn reduces protein phosphorylation, cell cycle progression, and cytokine production. The aim of this study was to examine the effect of rapamycin against the development of fibrosis and autoimmunity in 2 different types of systemic sclerosis (SSc) model mice.

Methods

Tight skin (TSK/+) mice and bleomycin‐ induced SSc model mice were used to evaluate the effect of rapamycin on fibrosis and immunologic abnormalities. Furthermore, the antifibrotic effect of rapamycin was assessed using TSK/+ mouse fibroblasts.

Results

Treatment with rapamycin reduced skin fibrosis of TSK/+ mice and skin and lung fibrosis of bleomycin‐induced SSc model mice. The production of fibrogenic cytokines, such as interleukin‐4 (IL‐4), IL‐6, IL‐17, and transforming growth factor β1, was attenuated by rapamycin. Hypergammaglobulinemia and anti–topoisomerase I antibody production were also reduced by rapamycin treatment in TSK/+ mice. In addition, mTOR expression levels were increased in TSK/+ mouse fibroblasts compared with those in wild‐type mouse fibroblasts. Rapamycin treatment inhibited proliferation and collagen production of TSK/+ mouse fibroblasts in a dose‐dependent manner.

Conclusion

This study is the first to show that rapamycin has a significant inhibitory effect on fibrosis in both TSK/+ and bleomycin‐induced SSc model mice. These results suggest that rapamycin might be an attractive candidate for clinical trials in SSc patients.

     

Oxidative stress in scleroderma: Maintenance of scleroderma fibroblast phenotype by the constitutive up‐regulation of reactive oxygen species generation through the NADPH oxidase complex pathway

Objective

To explore the role of reactive oxygen species (ROS) in the in vitro activation of skin fibroblasts from patients with systemic sclerosis (SSc).

Methods

Fibroblasts were obtained from involved skin of patients with limited or diffuse SSc. Oxidative activity imaging in living cells was carried out using confocal microscopy. Levels of O₂⁻ and H₂O₂ released from fibroblasts were estimated by the superoxide dismutase (SOD)–inhibitable cytochrome c reduction and homovanilic acid assays, respectively. To verify NADPH oxidase activation, the light membrane of fibroblasts was immunoblotted with an anti‐p47^phox–specific antibody. Fibroblasts were stimulated with various cytokines and growth factors to determine whether any of these factors modulate ROS generation. Cell proliferation was estimated by ³H‐thymidine incorporation. Northern blot analysis was used to study α1 and α2 type I collagen gene expression.

Results

Unstimulated skin fibroblasts from SSc patients released more O₂⁻ and H₂O₂ in vitro through the NADPH oxidase complex pathway than did normal fibroblasts, since incubation of SSc fibroblasts with diphenylene iodonium, a flavoprotein inhibitor, suppressed the generation of ROS. This suppression was not seen with rotenone, a mitochondrial oxidase inhibitor, or allopurinol, a xanthine oxidase inhibitor. Furthermore, the cytosolic component of NADPH oxidase, p47^phox, was translocated to the plasma membrane of resting SSc fibroblasts. A transient increase in ROS production was induced in normal but not in SSc fibroblasts by interleukin‐1β (IL‐1β), platelet‐derived growth factor type BB (PDGF‐BB), transforming growth factor β1 (TGFβ1), and H₂O₂. Treatment of normal and SSc fibroblasts with tumor necrosis factor α (TNFα), IL‐2, IL‐4, IL‐6, IL‐10, interferon‐α (IFNα), IFNγ, granulocyte–macrophage colony‐stimulating factor (GM‐CSP), G‐CSF, or connective tissue growth factor (CTGF) had no effect on ROS generation. Constitutive ROS production by SSc fibroblasts was not inhibited when these cells were treated with catalase, SOD, IL‐1 receptor antagonist, or antibodies blocking the effect of TGFβ1, PDGF‐BB, and other agonists (IL‐4, IL‐6, TNFα, CTGF). In contrast, treatment of SSc fibroblasts with the membrane‐permeant antioxidant N‐acetyl‐L ‐cysteine inhibited ROS production, and this was accompanied by decreased proliferation of these cells and down‐regulation of α1(I) and α2(I) collagen messenger RNA.

Conclusion

The constitutive intracellular production of ROS by SSc fibroblasts derives from the activation of an NADPH oxidase–like system and is essential to fibroblast proliferation and expression of type I collagen genes in SSc cells. Our results also exclude O₂⁻, H₂O₂, IL‐1β, TGFβ1, PDGF‐BB, IL‐4, IL‐6, TNFα, or CTGF as mediators of a positive, autocrine feedback mechanism of ROS generation.

     

Expression of interleukin‐21 receptor in epidermis from patients with systemic sclerosis

Objective

To examine the expression and regulation of interleukin‐21 (IL‐21) and IL‐21 receptor (IL‐21R) in patients with systemic sclerosis (SSc; scleroderma).

Methods

Skin biopsy specimens were obtained from 23 patients with SSc and 15 healthy controls. IL‐21/IL‐21R messenger RNA (mRNA) was quantified using real‐time polymerase chain reaction (PCR). The expression pattern of IL‐21/IL‐21R was analyzed by in situ hybridization and Western blotting. Stimulation experiments were performed with cultured dermal fibroblasts from patients with SSc and healthy controls as well as with keratinocytes, using IL‐1β, platelet‐derived growth factor BB, monocyte chemoattractant protein 1, transforming growth factor β, and IL‐21. The SCID‐hu skin mouse model was used for in vivo experiments.

Results

IL‐21R mRNA was detected in all biopsy specimens from patients with SSc and controls, with a 4.7‐fold increase observed in SSc samples. In situ hybridization and immunohistochemical analysis showed an up‐regulation of IL‐21R in samples of epidermis from SSc patients, whereas no signal was detected in skin specimens from healthy controls. These results were confirmed in vitro, in that cultured keratinocytes expressed significant levels of IL‐21R, whereas no signal was observed in fibroblasts. Interestingly, mRNA for IL‐21 could not be detected by real‐time PCR and in situ hybridization. Various concentrations of key cytokines in the pathogenesis of SSc did not stimulate the expression of IL‐21R mRNA in cultured keratinocytes and dermal fibroblasts. In the SCID mouse transplantation model, the overexpression of IL‐21R mRNA in SSc keratinocytes remained unchanged after transplantation.

Conclusion

The up‐regulation of IL‐21R in keratinocytes indicates that, similar to fibroblasts and endothelial cells, the expression pattern is altered in SSc. Moreover, the expression of IL‐21R appears to be independent of key cytokines that are operant in SSc.

     

Regulation of interleukin‐1β–induced chemokine production and matrix metalloproteinase 2 activation by epigallocatechin‐3‐gallate in rheumatoid arthritis synovial fibroblasts

Objective

To evaluate the efficacy of epigallocatechin‐3‐gallate (EGCG), a potent antiinflammatory molecule, in regulating interleukin‐1β (IL‐1β)–induced production of the chemokines RANTES (CCL5), monocyte chemoattractant protein 1 (MCP‐1/CCL2), epithelial neutrophil–activating peptide 78 (ENA‐78/CXCL5), growth‐regulated oncogene α (GROα/CXCL1), and matrix metalloproteinase 2 (MMP‐2) activity in rheumatoid arthritis (RA) synovial fibroblasts.

Methods

Fibroblasts obtained from RA synovium were grown, and conditioned medium was obtained. Cell viability was determined by MTT assay. RANTES, MCP‐1, ENA‐78, and GROα produced in culture supernatants were measured by enzyme‐linked immunosorbent assay. MMP‐2 activity was analyzed by gelatin zymography. Western blotting was used to study the phosphorylation of protein kinase C (PKC) isoforms and nuclear translocation of NF‐κB.

Results

EGCG was nontoxic to RA synovial fibroblasts. Treatment with EGCG at 10 μM or 20 μM significantly inhibited IL‐1β–induced ENA‐78, RANTES, and GROα, but not MCP‐1 production in a concentration‐dependent manner. EGCG at 50 μM caused a complete block of IL‐1β–induced production of RANTES, ENA‐78, and GROα, and reduced production of MCP‐1 by 48% (P < 0.05). Zymography showed that EGCG blocked constitutive, IL‐1β–induced, and chemokine‐mediated MMP‐2 activity. Evaluation of signaling events revealed that EGCG preferentially blocked the phosphorylation of PKCδ and inhibited the activation and nuclear translocation of NF‐κB in IL‐1β–treated RA synovial fibroblasts.

Conclusion

These results suggest that EGCG may be of potential therapeutic value in inhibiting joint destruction in RA.

     

Decreased susceptibility to Fas‐induced apoptosis of systemic sclerosis dermal fibroblasts

Objective

To determine whether dysregulated apoptosis of systemic sclerosis (SSc) fibroblasts contributes to progressive fibrosis by promoting fibroblast longevity.

Methods

We examined the pattern of fibroblast proliferation and apoptosis in SSc skin lesions and the susceptibility of cultured SSc dermal fibroblasts to apoptosis. Skin biopsy samples from SSc patients and control subjects were used to establish fibroblast cultures and were examined histologically. In skin sections, apoptosis was examined by TUNEL, and proliferation by immunostaining for proliferating cell nuclear antigen. Susceptibility of fibroblasts to apoptosis induced in vitro by different stimuli was studied by TUNEL. Expression of Bcl‐2, Bcl‐x, and Bax proteins in cultured fibroblasts was studied by Western blotting.

Results

Proliferation of dermal fibroblasts was not observed in normal skin but was present in skin from patients with SSc and other inflammatory skin diseases. Apoptosis of fibroblasts in SSc fibrotic skin lesions was not observed. In vitro, SSc fibroblasts were specifically resistant to apoptosis induced by Fas receptor stimulation but had normal susceptibility to apoptosis induced by nonspecific stimuli (protein kinase inhibition or serum withdrawal). Decreased susceptibility to Fas stimulation was not caused by decreased levels of surface Fas receptor. In SSc fibroblasts, quiescence induced by confluence and serum starvation was followed by an abnormal down‐regulation of proapoptotic Bax protein. Up‐regulation of the Bax:Bcl‐2 ratio in SSc fibroblasts by Bcl‐2 antisense oligonucleotides restored their susceptibility to Fas‐mediated apoptosis.

Conclusion

Our findings suggest that abnormal apoptotic regulation in fibroblasts can contribute to the pathogenesis of progressive fibrosis in SSc. Modulation of Bcl‐2–related proteins appears to be a potential target for the development of apoptosis‐based antifibrotic strategies.