Monocyte chemoattractant protein 3 as a mediator of fibrosis: Overexpression in systemic sclerosis and the type 1 tight‐skin mouse

Objective

To determine the gene‐expression profile in dermal fibroblasts from type 1 tight‐skin (Tsk1) mice, and to examine the expression and potential fibrotic activity of monocyte chemoattractant protein 3 (MCP‐3) in Tsk1 mouse and human systemic sclerosis (SSc) skin.

Methods

Complementary DNA microarrays (Atlas 1.2) were used to compare Tsk1 fibroblasts with non‐Tsk1 littermate cells at 10 days, 6 weeks, and 12 weeks of age. Expression of MCP‐3 protein was assessed by Western blotting of fibroblast culture supernatants, and localized in the mouse and human skin biopsy samples by immunohistochemistry. Activation of collagen reporter genes by MCP‐3 was explored in transgenic mouse fibroblasts and by transient transfection assays.

Results

MCP‐3 was highly overexpressed by neonatal Tsk1 fibroblasts and by fibroblasts cultured from the lesional skin of patients with early‐stage diffuse cutaneous SSc. Immunolocalization confirmed increased expression of MCP‐3 in the dermis of 4 of 5 Tsk1 skin samples and 14 of 28 lesional SSc skin samples, compared with that in matched healthy mice (n = 5) and human controls (n = 11). Proα2(I) collagen promoter–reporter gene constructs were activated by MCP‐3 in transgenic mice and by transient transfection assays. This response was maximal between 16 and 24 hours of culture and mediated via sequences within the proximal promoter. The effects of MCP‐3 could be diminished by a neutralizing antibody to transforming growth factor β.

Conclusion

We demonstrate, for the first time, overexpression of MCP‐3 in early‐stage SSc and in Tsk1 skin, and suggest a novel role for this protein as a fibrotic mediator activating extracellular matrix gene expression in addition to promoting leukocyte trafficking. This chemokine may be an important early member of the cytokine cascade driving the pathogenesis of SSc.

     

A modified model of graft‐versus‐host–induced systemic sclerosis (scleroderma) exhibits all major aspects of the human disease

Objective

Diffuse systemic sclerosis (SSc; scleroderma) is a debilitating disease characterized by excessive dermal fibrosis with later progression to internal organs. In addition to the fibrotic component, major aspects of the disease include vascular or circulatory involvement and immune dysregulation evidenced by inflammatory cells in affected tissues and production of autoantibodies. Many animal models resembling this disease have been studied, including genetic models in mice and chickens, challenge with chemicals such as bleomycin or vinyl chloride to induce fibrosis, and models of graft‐versus‐host (GVH)–induced disease using certain strains of mice with differences in minor histocompatibility loci. The present studies were undertaken to determine if alteration of the induction of GVH‐induced scleroderma could result in a model that more fully represented the human condition.

Methods

Disease was induced by injection of spleen cells from B10.D2 mice into BALB/c mice deficient in mature T and B cells (recombination‐activating gene 2 targeted). Dermal thickening, collagen deposition, vasoconstriction, and parameters of immunity were analyzed.

Results

Similar to the human disease, this modified GVH model of SSc demonstrated evidence of dermal thickening, particularly in the extremities, progressive fibrosis of internal organs, vasoconstriction and altered expression of vascularity markers in skin and internal organs, early immune activation, inflammation in skin and internal organs, and autoantibody generation.

Conclusion

This modified model of GVH‐induced SSc exhibits all major components of human disease and is likely to contribute to better understanding of the disease mechanisms and, ultimately, improved treatments for patients.

     

Loss of β1 integrin in mouse fibroblasts results in resistance to skin scleroderma in a mouse model

Objective

Activated adhesive signaling is a hallmark of fibroblasts isolated from the scars of scleroderma (systemic sclerosis) lesions. Beta‐1 integrin plays a key role in adhesive signaling. The aim of the present study was to examine the role of β1 integrin in a mouse model of skin scleroderma using mice bearing a fibroblast‐specific deletion of β1 integrin.

Methods

Cutaneous sclerosis was induced by subcutaneous injection of bleomycin. Control groups were treated with phosphate buffered saline. Mice bearing a fibroblast‐specific deletion of β1 integrin and control mice were investigated. Dermal thickness, collagen production, and the number of α‐smooth muscle actin–positive cells were determined. The quantity of the collagen‐specific amino acid hydroxyproline was also measured.

Results

Bleomycin treatment induced marked cutaneous thickening and fibrosis in control mice. Conversely, the deletion of β1 integrin resulted in resistance to bleomycin‐induced fibrosis.

Conclusion

Expression of β1 integrin by fibroblasts is required for fibrogenesis. Inhibition of β1 integrin may be a viable method to alleviate the development of cutaneous sclerosis.

     

Activation of a fibroblast‐specific enhancer of the Proα2(I) collagen gene in tight‐skin mice

Objective

Reporter transgenes were introduced into the type 1 tight‐skin (Tsk1/+) mouse model of scleroderma to test the hypothesis that fibroblast‐specific genetic programs are activated in fibrosis.

Methods

Transgenes harboring upstream fragments of the 5′ flanking region of the mouse proα2(I) collagen gene (Col1a2), linked to a 400‐bp minimal Col1a2 promoter driving an Escherichia coli β‐galactosidase (LacZ) reporter gene, were introduced into Tsk1/+ mice by breeding. Expression of these transgenes, which function as lineage‐specific markers of fibroblast differentiation, was compared between the Tsk‐LacZ mice and non‐Tsk littermates. Responsiveness of these constructs to the profibrotic cytokine, transforming growth factor β1 (TGFβ1), was investigated by transient transfection of reporter constructs in tissue‐culture cells.

Results

There was significant activation of reporter genes harboring the upstream enhancer in Tsk1/+ mice starting from 1 week of age. This was maximal at 6 weeks old (mean ± SD 237 ± 24% of non‐Tsk controls; P= 0.001). Recombinant TGFβ1 significantly activated reporter genes regulated by the upstream enhancer in transient transfection, and Tsk‐LacZ fibroblasts showed elevated LacZ expression in tissue culture.

Conclusion

These data suggest that activating signals in Tsk1/+ mice may act via fibroblast‐specific regulatory elements within the murine Col1a2 gene. Although TGFβ has been implicated in the pathogenesis of fibrosis, and reporter genes regulated by the upstream enhancer appear to be TGFβ responsive in vitro, our results suggest that fibroblast‐specific pathways may also be involved.

     

Reduction of CpG‐induced arthritis by suppressive oligodeoxynucleotides

Objective

Bacterial DNA contains immunostimulatory CpG motifs that cause inflammation when injected into the knee joints of normal mice. We examined whether synthetic oligodeoxynucleotides (ODN) that suppress CpG‐induced immune responses prevent CpG‐induced arthritis.

Methods

CpG, suppressive, and/or control ODN were injected into the knees of BALB/c mice. Joint swelling and inflammation were evaluated by physical measurement, by histologic analysis of joint tissue, and by magnetic resonance imaging.

Results

Immunostimulatory CpG DNA induced local arthritis, characterized by swelling of the knee joints, the presence of inflammatory cell infiltrates, the perivascular accumulation of mononuclear cells, and hyperplasia of the synovial lining. Administering suppressive (but not control) ODN reduced the manifestations and severity of arthritis up to 80%.

Conclusion

Suppressive ODN may be useful for the prevention or treatment of arthritis induced by bacterial DNA.

     

Increased resistance to collagen‐induced arthritis in CD44‐deficient DBA/1 mice

Objective

To study the role of CD44, the principal hyaluronan (HA) receptor, in experimental arthritis.

Methods

We generated CD44 gene deficiency in arthritis‐susceptible DBA/1LacJ mice to study the role of CD44 directly in collagen‐induced arthritis (CIA). Wild‐type and CD44‐deficient mice were immunized with chicken type II collagen, and the onset and severity of CIA were monitored up to day 64. The immune status of immunized mice was determined at the end of the experiments. Cell transfer experiments were performed to monitor lymphocyte traffic to the inflamed joints.

Results

Mice homozygous for the CD44 mutation developed normally and showed no phenotypic defects. Although they showed a normal response to immunization with type II collagen and had Th1/Th2 ratios comparable with those in wild‐type animals, CD44‐deficient mice exhibited significant reductions in both the incidence and severity of CIA. This was accompanied by altered serum levels of HA, reduced expression of L‐selectin, and a delayed entry of intravenously injected CD44‐deficient donor lymphocytes into the arthritic joints of recipient mice.

Conclusion

While CD44 is not essential for morphogenesis and autoimmunity, this cell surface receptor seems to play an important role in the development of arthritis, most likely by directing leukocyte traffic to the site of inflammation.

     

Targeting ADAM‐17/notch signaling abrogates the development of systemic sclerosis in a murine model

Objective

Systemic sclerosis (SSc) is characterized by the fibrosis of various organs, vascular hyperreactivity, and immunologic dysregulation. Since Notch signaling is known to affect fibroblast homeostasis, angiogenesis, and lymphocyte development, we undertook this study to investigate the role of the Notch pathway in human and murine SSc.

Methods

SSc was induced in BALB/c mice by subcutaneous injections of HOCl every day for 6 weeks. Notch activation was analyzed in tissues from mice with SSc and from patients with scleroderma. Mice with SSc were either treated or not treated with the γ‐secretase inhibitor DAPT, a specific inhibitor of the Notch pathway, and the severity of the disease was evaluated.

Results

As previously described, mice exposed to HOCl developed a diffuse cutaneous SSc with pulmonary fibrosis and anti–DNA topoisomerase I antibodies. The Notch pathway was hyperactivated in the skin, lung, fibroblasts, and splenocytes of diseased mice and in skin biopsy samples from patients with scleroderma. ADAM‐17, a proteinase involved in Notch activation, was overexpressed in the skin of mice and patients in response to the local production of reactive oxygen species. In HOCl‐injected mice, DAPT significantly reduced the development of skin and lung fibrosis, decreased skin fibroblast proliferation and ex vivo serum‐induced endothelial H₂O₂ production, and abrogated the production of anti–DNA topoisomerase I antibodies.

Conclusion

Our results show the pivotal role of the ADAM‐17/Notch pathway in SSc following activation by reactive oxygen species. The inhibition of this pathway may represent a new treatment of this life‐threatening disease.

     

Investigation of sensory neurogenic components in a bleomycin‐induced scleroderma model using transient receptor potential vanilloid 1 receptor– and calcitonin gene‐related peptide–knockout mice

Objective

Along with their classic afferent function (nociception), capsaicin‐sensitive transient receptor potential vanilloid 1 (TRPV1) receptor–expressing sensory nerve terminals exert local and systemic efferent activities. Activation of TRPV1 causes sensory neuropeptide release, which modulates the inflammation process. The aim of the present study was to examine the role of this modulatory role of TRPV1 receptor and that of calcitonin gene‐related peptide (CGRP) in bleomycin‐induced scleroderma, using transgenic mice.

Methods

Cutaneous sclerosis was induced with daily subcutaneous injections of bleomycin for 30 days. Control groups were treated with phosphate buffered saline (PBS). TRPV1 receptor gene–deficient (TRPV1^−/−) mice and CGRP‐knockout (CGRP^−/−) mice and their wild‐type (WT) counterparts were investigated. A composite sclerosis score was calculated on the basis of thickening, leukocyte infiltration, and the amount/orientation of collagen bundles. Dermal thickness and the number of α‐smooth muscle actin (α‐SMA)–positive cells were also determined. The quantity of the collagen‐specific amino acid hydroxyproline was measured by spectrophotometry.

Results

Bleomycin treatment induced marked cutaneous thickening and fibrosis compared with that observed in control mice treated with PBS. The composite sclerosis score was 18% higher, dermal thickness was 19% higher, the number of α‐SMA–positive cells was 47% higher, and the amount of hydroxyproline was 57% higher in TRPV1^−/− mice than in their WT counterparts. Similarly, the composite sclerosis score was 47% higher, dermal thickness was 29% higher, the number of α‐SMA–positive cells was 76% higher, and the amount of hydroxyproline was 30% higher in CGRP^−/− mice than in the respective WT groups.

Conclusion

These results suggest that activation of the TRPV1 receptor by mediators of inflammation induces sensory neuropeptide release, which might exert protective action against fibrosis. We confirmed the protective role of CGRP in the development of cutaneous sclerosis.

     

Suppressive oligonucleotides protect against collagen‐induced arthritis in mice

Objective

To examine whether systemic administration of oligonucleotides (ODNs), known to inhibit the production of proinflammatory cytokines, alters host susceptibility to collagen‐induced arthritis (CIA), a murine model of rheumatoid arthritis (RA).

Methods

CIA was induced by injecting DBA/1 mice with type II collagen (CII) in Freund's complete adjuvant, followed 3 weeks later by CII in Freund's incomplete adjuvant. The effect of suppressive ODNs on the incidence and severity of disease was monitored, as were immune correlates of CIA.

Results

Suppressive ODNs administered during the inductive phase of CIA significantly reduced the incidence and severity of arthritis. Treatment with suppressive ODNs significantly decreased serum titers of pathogenic IgG anti‐CII autoantibodies and interferon‐γ production by collagen‐reactive T cells.

Conclusion

Suppressive ODNs may be of therapeutic value in the treatment of RA, and potentially other autoimmune diseases.

     

Thymic Stromal Lymphopoietin Is Up‐Regulated in the Skin of Patients With Systemic Sclerosis and Induces Profibrotic Genes and Intracellular Signaling That Overlap With Those Induced by Interleukin‐13 and Transforming Growth Factor β

Objective

To explore the expression of thymic stromal lymphopoietin (TSLP) in patients with diffuse cutaneous systemic sclerosis (dcSSc) and compare its effects in vivo and in vitro with those of interleukin‐13 (IL‐13) and transforming growth factor β (TGFβ).

Methods

Skin biopsy specimens from patients with dcSSc (n = 14) and healthy controls (n = 13) were analyzed by immunohistochemistry and immunofluorescence for TSLP, TSLP receptor, CD4, CD8, CD31, and CD163 markers. Wild‐type, IL‐4Rα1–, and TSLP‐deficient mice were treated with TGFβ, IL‐13, poly(I‐C), or TSLP by osmotic pump. Human fibroblasts and peripheral blood mononuclear cells (PBMCs) were stimulated with TGFβ, IL‐13, poly(I‐C), or TSLP. Microarray analysis and quantitative polymerase chain reaction were performed to determine gene expression, and protein levels of phospho‐Smad2 and macrophage marker CD163 were tested.

Results

TSLP was highly expressed in the skin of dcSSc patients, more strongly in perivascular areas and in immune cells, and was produced mainly by CD163+ cells. The skin of TSLP‐treated mice showed up‐regulated clusters of gene expression that overlapped strongly with those in IL‐13– and TGFβ‐treated mice. TSLP up‐regulated specific genes, including CXCL9, proteasome, and interferon (IFN)–regulated genes. TSLP treatment in IL‐4Rα1–deficient mice promoted similar cutaneous inflammation as in wild‐type mice, though TSLP‐induced arginase 1, CCL2, and matrix metalloproteinase 12 messenger RNA levels were blocked. In PBMCs, TSLP up‐regulated tumor necrosis factor α, Mx‐1, IFNγ, CXCL9, and mannose receptor 1 gene expression. TSLP‐deficient mice treated with TGFβ showed less fibrosis and blocked expression of plasminogen activator inhibitor 1 and osteopontin 1. Poly(I‐C)–treated mice showed high levels of cutaneous TSLP.

Conclusion

TSLP is highly expressed in the skin of dcSSc patients and interacts in a complex manner with 2 other profibrotic cytokines, TGFβ and IL‐13, strongly suggesting that it might promote SSc fibrosis directly or indirectly by synergistically stimulating profibrotic genes, or production of these cytokines.

     

Suppression of skin and kidney disease by inhibition of spleen tyrosine kinase in lupus‐prone mice

Objective

Spleen tyrosine kinase (Syk) is involved in membrane‐mediated signaling in various cells, including immune cells. It is overexpressed in T cells from patients with systemic lupus erythematosus (SLE), and its inhibition has been shown to improve T cell function as well as to improve disease manifestations in (NZB × NZW)F₁ lupus‐prone mice and in patients with rheumatoid arthritis. While clinical trials examining Syk inhibition in patients with SLE are being considered, the aim of our experiments was to determine whether the therapeutic effects of Syk inhibition extend to other strains of lupus‐prone mice and whether they result in improvement in skin disease and modification of established disease.

Methods

Female MRL/lpr or BAK/BAX mice were studied. Starting either at age 4 weeks (before disease) or at age 16 weeks (after established disease) and continuing for up to 16 weeks, mice were fed chow containing the Syk inhibitor R788 or control chow.

Results

We found that inhibition of Syk in MRL/lpr and BAK/BAX mice prevented the development of skin disease and significantly reduced established skin disease. Similarly, Syk inhibition reduced the size of the spleen and lymph nodes, suppressed the development of renal disease, and suppressed established renal disease. Discontinuation of treatment resulted in extended suppression of skin disease for at least 8 weeks and suppression of renal disease for 4 weeks.

Conclusion

Syk inhibition suppresses the development of lupus skin and kidney disease in lupus‐prone mice, suppresses established disease in lupus‐prone mice, and may represent a valuable treatment for patients with SLE.

     

Large particle hyaluronic acid for the treatment of facial lipoatrophy in HIV‐positive patients: 3‐year follow‐up study

Objectives

Facial lipoatrophy can be a stigmatizing side effect of antiretroviral (AVR) treatment for HIV‐infected patients. We sought to evaluate the long‐term efficacy and safety of a new formulation of hyaluronic acid that can be injected in larger amounts and into deeper skin layers during 3 years of follow‐up.

Methods

Twenty patients received injections of Restylane SubQ^™. Refill treatment was offered at 12 and 24 months. Treatment effects were evaluated using ultrasound, the Global Aesthetic Improvement Scale, visual analogue scale (VAS) and the Rosenberg self‐esteem scale.

Results

Seventeen patients remained at 36 months. Mean (± standard deviation) total cutaneous thickness increased from 6 ± 1 mm at baseline to 12 ± 1 mm (P<0.001) at 36 months. Response rate (total cutaneous thickness >10 mm) was 70%. Fifteen patients classified their facial appearance as very much or moderately improved. VAS increased from 39 ± 25 to 70 ± 20 (P<0.05) and higher self‐esteem scores were reported. Local swelling and tenderness after treatment was common. Persistent papules found in several patients after treatment were removed effectively with hyaluronidase injections. Three patients, treated only at baseline, still had higher total cutaneous thickness scores at 36 months.

Conclusions

Our results indicate that a large particle hyaluronic acid formulation is a durable and well‐tolerated dermal filler for treating HIV‐positive patients with facial lipoatrophy.

     

Angiotensin receptor blockers suppress antigen‐specific T cell responses and ameliorate collagen‐induced arthritis in mice

Objective

The renin–angiotensin system plays an important role in the regulation of cardiovascular, renal, and endocrine functions. Recent studies have demonstrated that angiotensin II has proinflammatory effects that may contribute to the pathogenesis of immune‐mediated diseases. We used the collagen‐induced arthritis (CIA) model to investigate the influence of angiotensin II receptor blockers (ARBs) on antigen‐specific immune responses and determine whether ARBs have preventive or therapeutic effects on the development of arthritis.

Methods

We administered ARBs (olmesartan, candesartan, and telmisartan) to mice and evaluated antigen‐specific T cell proliferation and cytokine production following immunization with ovalbumin (OVA) or type II collagen in Freund's complete adjuvant (CFA) or aluminum hydroxide (alum). Next, we induced CIA in DBA/1 mice and administered olmesartan. The severity and incidence of arthritis were scored according to clinical manifestations, and joint tissue sections were examined histopathologically.

Results

ARBs severely suppressed lymphocyte proliferation and interferon‐γ production in mice immunized with OVA or type II collagen in CFA. Olmesartan also suppressed lymphocyte proliferation in mice immunized with ovalbumin in alum. In the CIA model, olmesartan reduced the mean arthritis score and the incidence of severe arthritis, even when it was administered only after disease onset. Histopathologic findings for joint destruction were improved in olmesartan‐treated mice.

Conclusion

ARBs suppressed antigen‐specific immune responses for Th1 and Th2 in vivo. Furthermore, olmesartan suppressed the development of severe arthritis and joint destruction in the CIA model. These findings suggest that ARBs may have therapeutic potential in rheumatoid arthritis.

     

Germinal center reaction in the joints of mice with collagen‐induced arthritis: An animal model of lymphocyte activation and differentiation in arthritic joints

Objective

To establish an animal model and provide a basis for investigating the role of germinal center (GC) reaction in autoimmune arthritis.

Methods

DBA/1 mice were immunized with bovine type II collagen (CII) to elicit collagen‐induced arthritis (CIA). Sections of arthritic joints were examined by in situ immunohistochemical studies, and purified cells from affected joints were subjected to flow cytometry analysis.

Results

De novo GC reaction was induced in the arthritic joints of male DBA/1 mice by immunization with bovine CII. In comparison with GCs formed in lymphoid tissues, such as spleen and lymph nodes, we found that these GCs formed in the joint tissues of mice with CIA were morphologically typical, as determined by immunohistologic and flow cytometric assays.

Conclusion

The local immune responses in murine CIA induced ectopic GC formation, as observed in the synovial tissues of patients with rheumatoid arthritis. This system will allow for the first time the direct study of the role of the GC reaction in autoimmune arthritis in an animal model.

     

Increased bone density and resistance to ovariectomy‐induced bone loss in FoxP3‐transgenic mice based on impaired osteoclast differentiation

Objective

Immune activation triggers bone loss. Activated T cells are the cellular link between immune activation and bone destruction. The aim of this study was to determine whether immune regulatory mechanisms, such as naturally occurring Treg cells, also extend their protective effects to bone homeostasis in vivo.

Methods

Bone parameters in FoxP3‐transgenic (Tg) mice were compared with those in their wild‐type (WT) littermate controls. Ovariectomy was performed in FoxP3‐Tg mice as a model of postmenopausal osteoporosis, and the bone parameters were analyzed. The bones of RAG‐1^–/– mice were analyzed following the adoptive transfer of isolated CD4+CD25+ T cells. CD4+CD25+ T cells and CD4+ T cells isolated from FoxP3‐Tg mice and WT mice were cocultured with monocytes to determine their ability to suppress osteoclastogenesis in vitro.

Results

FoxP3‐Tg mice developed higher bone mass and were protected from ovariectomy‐induced bone loss. The increase in bone mass was found to be the result of impaired osteoclast differentiation and bone resorption in vivo. Bone formation was not affected. Adoptive transfer of CD4+CD25+ T cells into T cell–deficient RAG‐1^–/– mice also increased the bone mass, indicating that Treg cells directly affect bone homeostasis without the need to engage other T cell lineages.

Conclusion

These data demonstrate that Treg cells can control bone resorption in vivo and can preserve bone mass during physiologic and pathologic bone remodeling.

     

Cappuccino mutation in an autoimmune‐prone strain of mice suggests a role of platelet function in the progression of immune complex crescentic glomerulonephritis

Objective

Crescent formation in the renal glomerulus is a typical manifestation of progressive glomerulopathy associated with fatal renal failure; therefore, its prevention is of clinical importance. Little is known about the pathogenic mechanism for crescent formation. This study was undertaken in an attempt to identify the events that are critical for crescent formation in immune complex crescentic glomerulonephritis (CGN) by analyzing a novel mutant strain of mice.

Methods

A spontaneous mutant strain of mice was isolated from the autoimmune‐prone strain EOD, which stably develops fatal CGN. The mutant phenotypes were assessed histopathologically, hematologically, and immunologically. The mutation was searched for with positional cloning using microsatellite markers.

Results

Compared with wild‐type EOD (WT‐EOD) mice, mutant EOD (mut‐EOD) mice showed marked improvement in CGN in conjunction with an improvement in spontaneous mortality. In WT‐EOD mice, an inverse correlation between blood urea nitrogen concentration and blood platelet count and massive accumulation of platelets in the glomerulus were evident, suggesting that an accumulation of platelets in the glomerulus contributes to the progression of CGN. The mutant platelets showed an abnormal aggregation in response to collagen and thrombin, associated with a bleeding tendency in mut‐EOD mice. Genetic analysis revealed a deleterious mutation in the cappuccino gene (cno), which encodes a protein that belongs to a complex called the biogenesis of lysosome‐related organelle complex 1 and is profoundly involved in platelet function. Morphologic examination revealed a partial defect in dense body formation in the δ‐granule of platelets.

Conclusion

The present findings suggest that platelet functions have a critical role in crescent formation in autoimmune GN.

     

Mutant fibrillin 1 from tight skin mice increases extracellular matrix incorporation of microfibril‐associated glycoprotein 2 and type I collagen

Objective

Skin fibrosis in the TSK mouse, a model of skin fibrosis seen in systemic sclerosis (SSc), is caused by a large in‐frame duplication in the Fbn1 gene, tsk‐Fbn1. We investigated whether tsk‐Fbn1 might cause dermal fibrosis by affecting Fbn1 and associated extracellular matrices. We also studied whether deposition of microfibril‐associated glycoprotein 2 (MAGP‐2), a protein that is associated with fibrillin 1, was altered in the skin of patients with SSc.

Methods

An in vitro model of the TSK mouse was created by conditionally expressing tsk‐Fbn1 in mouse embryonic fibroblasts (MEFs). Cell cultures were examined by immunofluorescence and Western and Northern blotting to determine the effect of tsk‐Fbn1 on the structure, expression, and deposition of fibrillin 1 (Fbn‐1), type I collagen, and MAGP‐2. The skin of TSK mice and SSc patients was analyzed by immunohistochemistry for MAGP‐2 expression.

Results

Expression of tsk‐Fbn1 in cultured MEF cells altered the morphology of Fbn‐1 fibers and increased the deposition of type I collagen into the extracellular matrix (ECM) without concomitantly changing messenger RNA expression, secretion, or processing of type I procollagen. Moreover, MEF cells expressing tsk‐Fbn1 showed increased MAGP‐2 matrix. MAGP‐2 was increased in the dermis of TSK mice. Fibrotic SSc skin also showed higher levels of MAGP‐2 in the dermis than nonfibrotic SSc skin and normal skin.

Conclusion

Tsk‐Fbn1 altered ECM organization and caused fibrosis by affecting the deposition of MAGP‐2 or other Fbn‐1–associated proteins. Alterations in microfibril structure or deposition might contribute to fibrosis in SSc.

     

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.

     

CCN2 is required for bleomycin‐induced skin fibrosis in mice

Objective

No therapy for fibrotic disease is available. The proadhesive matricellular protein connective tissue growth factor CCN2 is a marker of fibrotic cells; however, the specific role of CCN2 in connective tissue biology in general and in fibrogenesis in particular is unclear. The aim of this study was to assess whether adult mice bearing a smooth muscle cell/fibroblast–specific deletion of CCN2 are resistant to bleomycin‐induced skin scleroderma.

Methods

Cutaneous fibrosis was induced in mice by subcutaneous injection of bleomycin. Untreated control groups were injected with phosphate buffered saline. Mice bearing a fibroblast/smooth muscle cell–specific deletion of CCN2 were investigated for changes in dermal thickness, collagen content, and the number of α‐smooth muscle actin (α‐SMA)–positive cells. Dermal fibroblasts were isolated to assess whether the induction of collagen and α‐SMA messenger RNA in response to transforming growth factor β (TGFβ) was impaired.

Results

The loss of CCN2 resulted in resistance to bleomycin‐induced skin fibrosis. In response to bleomycin, wild‐type mice possessed, but CCN2‐deficient mice lacked, abundant α‐SMA–expressing myofibroblasts within fibrotic lesions. Fibroblast responses to TGFβ, a potent inducer of myofibroblast differentiation, were not affected. Collectively, these results indicate that CCN2 is essential for bleomycin‐induced skin fibrosis, likely due to a defect in myofibroblast recruitment.

Conclusion

These data indicate that therapeutic strategies that involve blocking CCN2 in vivo may be of benefit in combating fibrotic skin disease.