We have established a mouse model for scleroderma induced by repeated local injections of bleomycin (BLM). Daily injection of BLM at a dose of >10 microg per ml for 4 wk induced histologic changes of dermal sclerosis, but not fibrosis, with thickened and homogenous collagen bundles and cellular infiltrates in BALB/C mice, whereas clinical signs of scleroderma were not apparent. In addition, lung fibrosis was also induced preceding the cutaneous changes. Sclerotic changes were not found in other sites of the skin distant from the injection site. Dermal sclerosis could also be induced by injecting BLM only every other day. The sclerotic changes of the dermis were sustained after ceasing BLM applications for at least 6 wk. Mast cells gradually increased in number as the sclerotic changes developed. Marked degranulation of mast cells was observed with elevated histamine release. The amount of hydroxyproline in skin was significantly increased at 4 wk of BLM treatment as compared with that in untreated or phosphate-buffered saline-treated mice. Anti-nuclear antibody was detected in serum of BLM-treated mice. Transforming growth factor-beta1 mRNA was detected at an early phase, while transforming growth factor-beta2 mRNA was strongly expressed at 4 wk when the sclerotic features were prominent. These results suggest that dermal sclerosis induced by BLM closely resembles systemic sclerosis both histologically and biochemically. Our mouse model can provide a powerful tool of inducing dermal sclerosis to examine the pathogenesis and the therapeutic approach of scleroderma.

Transforming growth factor-beta (TGF-beta) is a potent stimulus of connective tissue accumulation, and is implicated in the pathogenesis of scleroderma and other fibrotic disorders. Smad3 functions as a key intracellular signal transducer for profibrotic TGF-beta responses in normal skin fibroblasts. The potential role of Smad3 in the pathogenesis of scleroderma was investigated in Smad3-null (Smad3(-/-)) mice using a model of skin fibrosis induced by subcutaneous injections of bleomycin. At early time points, bleomycin-induced macrophage infiltration in the dermis and local TGF-beta production were similar in Smad3(-/-) and wild-type mice. In contrast, at day 28, lesional skin from Smad3(-/-) mice showed attenuated fibrosis, lower synthesis and accumulation of collagen, and reduced collagen gene transcription in situ, compared to wild-type mice. Connective tissue growth factor and alpha-smooth muscle actin expression in lesional skin were also significantly attenuated. Electron microscopy revealed an absence of small diameter collagen fibrils in the dermis from bleomycin-treated Smad3(-/-) mice. Compared to fibroblasts derived from wild-type mice, Smad3(-/-) fibroblasts showed reduced in vitro proliferative and profibrotic responses elicited by TGF-beta. Together, these results indicate that ablation of Smad3 is associated with markedly altered fibroblast regulation in vivo and in vitro, and confers partial protection from bleomycin-induced scleroderma in mice. Reduced fibrosis is due to deregulated fibroblast function, as the inflammatory response induced by bleomycin was similar in wild-type and Smad3(-/-) mice.

Transforming growth factor-␤ (TGF-␤

Transforming growth factor-beta is responsible for triggering a cascade of events leading to fibrosis in scleroderma. The Smads are intracellular signal transducers recently shown to mediate fibroblast activation and other profibrotic responses elicited by transforming growth factor-betain vitro. To understand better the involvement of Smads in the pathogenesis of fibrosis, we examined Smad expression and activation in situ in a murine model of scleroderma. Bleomycin injections induced striking dermal infiltration with macrophages by 3 d, and progressive fibrosis by 2 wk. Infiltrating macrophages and resident fibroblasts expressed Smad3, the positive mediator for transforming growth factor-beta responses. Importantly, in bleomycin-injected skin, fibroblasts showed predominantly nuclear localization of Smad3 and intense staining for phospho-Smad2/3. Furthermore, phosphorylated Smad2/3 in fibroblasts was detected even after the resolution of inflammation. Expression of Smad7, the endogenous inhibitor of transforming growth factor-beta/Smad signaling, was strongly induced in dermal cells by transforming growth factor-beta, but not by bleomycin injections. Collectively, these results indicate that bleomycin-induced murine scleroderma is associated with rapid and sustained induction of transforming growth factor-beta/Smad signaling in resident dermal fibroblasts. Despite apparent activation of the intracellular transforming growth factor-beta signaling pathway in the lesional dermis, the expression of transforming growth factor-beta-inducible Smad7 was not upregulated. In light of the critical function of Smad7 as an endogenous inhibitor of Smad signaling that restricts the duration and magnitude of transforming growth factor-beta responses, and as a mediator of apoptosis, relative Smad7 deficiency observed in the present studies may account for sustained activation of transforming growth factor-beta/Smad signaling in lesional tissues. These findings raise the possibility that Smads plays an important part in the pathogenesis of fibrosis, and may therefore represent targets for selective anti-fibrotic interventions.

Objective. Transforming growth factor ␤ (TGF␤) induces profibrotic responses in normal fibroblasts, and plays a fundamental role in the pathogenesis of fibrosis in scleroderma (systemic sclerosis [SSc]). The intensity of cellular responses elicited by cytokines is modulated by transcriptional coactivators such as the histone acetylase p300. The objective of these studies was to delineate the physiologic role of p300 in Smaddependent profibrotic responses elicited by TGF␤.Methods. Ectopic p300 was transiently expressed in normal dermal fibroblasts. Cellular p300 levels were suppressed using p300-specific ribozymes. The regulation of gene expression was examined by transient transfection assays, Northern blotting, and immunoblot analysis. The expression of p300 in normal and scleroderma fibroblasts was evaluated by confocal microscopy and immunoblotting, and p300 levels in skin from mice with experimental scleroderma were assessed by immunohistochemistry.Results. In normal fibroblasts, TGF␤ induced an increase in the levels of p300. Forced expression of ectopic p300 in these cells dramatically enhanced the magnitude of TGF␤ responses, whereas selective depletion of p300 using ribozyme resulted in abrogation of TGF␤-induced collagen synthesis and promoter activity. Furthermore, TGF␤ lost its ability to induce Smaddependent transcription in p300-depleted fibroblasts. These responses could be fully rescued with ectopic p300. Abrogation of Smad-mediated TGF␤ signaling was not due to alterations in the levels or the liganddependent phosphorylation or intracellular trafficking of endogenous Smads. Immunohistochemical analysis demonstrated substantially increased p300 expression in lesional skin from mice with chronic graft-versushost disease, an animal model of scleroderma. Furthermore, levels of p300 were 2-3-fold higher in cultured fibroblasts derived from SSc patients than in fibroblasts from matched normal controls.Conclusion. These results establish, for the first time, that the coactivator histone acetylase p300, itself a target of TGF␤ regulation, is an essential component of the cellular TGF␤ signal transduction pathways mediating stimulation of collagen synthesis in fibroblasts.

Contact hypersensitivity (CHS) is thought to be mainly associated with the activation of T helper type 1 (Th1) cells. However, there is also evidence that Th2 cells or Th2 cytokines play a role in the development of CHS. To analyze the functional contribution of Th2 cytokines interleukin (IL)-4 and IL-13, signal transducer and activator of transcription 6 (STAT6)-deficient (STAT6−/−) and wild-type (wt) control C57BL/6 mice were contact sensitized with 5% 2,4,6-trinitrochlorobenzene (TNCB), 0.5% 2,4-dinitrofluorobenzene, or 5% 4-ethoxyl methylene-2-phenyl-2-oxazolin-5-one, and any skin reactions were examined. Ear swelling was significantly reduced with a delayed peak response in STAT6−/− mice compared with wt mice.A histological analysis revealed that the infiltration of both eosinophils and neutrophils in the skin challenged after 24 h in STAT6−/− mice decreased substantially compared with that in wt mice. The expression of Th2 cytokines (IL-4, IL-5) in TNCB-challenged skin tissues and the supernatants from T cells stimulated by 2,4,6-trinitrobenzene sulfonate–modified spleen cells, as well as the immunoglobulin (Ig)E and IgG1 response after challenge, were also profoundly reduced in STAT6−/− mice, whereas the expression of interferon γ was the same in STAT6−/− and wt mice after challenge. Furthermore, adoptive transfer experiments revealed that STAT6−/− mice induced CHS after injection of lymph node cells obtained from sensitized wt mice. Our data suggest that the STAT6 signal plays a critical role in the induction phase of CHS.

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Scar and fibrosis are often the end result of mechanical injury and inflammatory diseases. One chemokine that is repeatedly linked to fibrotic responses is monocyte chemoattractant protein-1 (MCP-1). We utilized a murine fibrosis model that produces dermal lesions similar to scleroderma to evaluate collagen fibrillogenesis in the absence of MCP-1. Dermal fibrosis was induced by subcutaneous injection of bleomycin into the dorsal skin of MCP-1-/- and wild-type C57BL/6 mice. After 4 weeks of daily injections, bleomycin treatment led to thickened collagen bundles with robust inflammation in the lesional dermis of wild-type mice. In contrast, the lesional skin of MCP-1-/- mice exhibited a dermal architecture similar to phosphate-buffered saline (PBS)-injected control and normal skin, with few inflammatory cells. Ultrastructural analysis of the lesional dermis from bleomycin-injected wild-type mice revealed markedly abnormal arrangement of collagen fibrils, with normal large diameter collagen fibrils replaced by small collagen fibrils of 41.5 nm. In comparison, the dermis of bleomycin-injected MCP-1-/- mice displayed a uniform pattern of fibril diameters that was similar to normal skin (average diameter 76.7 nm). The findings implicate MCP-1 as a key determinant in the development of skin fibrosis induced by bleomycin, and suggest that MCP-1 may influence collagen fiber formation in vivo.