This is the first report that characterizes specific inhibitor of Smad3 (SIS3) as a potent and selective inhibitor of Smad3 function. In the reporter assay, the increased luciferase activity of p3TP-lux by the overexpression of constitutively active form of ALK-5 was abrogated by the treatment with SIS3 in a dosedependent manner. Immunoprecipitation revealed that SIS3 attenuated the transforming growth factor (TGF)-␤1-induced phosphorylation of Smad3 and interaction of Smad3 with Smad4. On the other hand, this reagent did not affect the phosphorylation of Smad2. Thereafter, we evaluated the ability of SIS3 in the suppression of the TGF-␤1-induced type I procollagen up-regulation in human dermal fibroblasts. We found that the addition of SIS3 attenuated the effects of TGF-␤1 by reducing the transcriptional activity. SIS3 also inhibited the myofibroblast differentiation of fibroblasts by TGF-␤1. Moreover, we demonstrated that SIS3 completely diminished the constitutive phosphorylation of Smad3 as well as the up-regulated type I collagen expression in scleroderma fibroblasts. Together, our study suggested that SIS3 is a useful tool to evaluate the TGF-␤-regulated cellular mechanisms via selective inhibition of Smad3.Transforming growth factor (TGF)-␤1 plays a critical role in a variety of biological processes, including proliferation, differentiation, extracellular matrix production, and apoptosis. The diverse cellular responses elicited by TGF-␤1 are triggered by the activation of serine/threonine kinase TGF-␤ receptors. On activation by TGF-␤1 or related ligands, signaling from the receptors to the nucleus is mediated by phosphorylation of cytoplasmic mediators called Smads. The receptor-associated Smads, such as Smad2 and Smad3, interact directly with, and are phosphorylated by, activated TGF-␤ receptor type I (Nakao et al., 1997). They are ligandspecific and form, on phosphorylation, heteromeric complexes with Smad4. The latter functions as a common mediator for all Smad pathways. These complexes then are translocated into the nucleus, where they function as transcription factors, possibly in association with other proteins, such as Sp1. The third group of Smad proteins, the inhibitory Smads such as Smad6 or Smad7, prevents phosphorylation and/or nuclear translocation of receptor-associated Smads.TGF-␤1 has been implicated in the development of fibrotic condition, including skin, lung, or liver. Systemic sclerosis or scleroderma is an acquired disorder that typically results in fibrosis of the skin and internal organs. Fibroblasts from affected scleroderma skin cultured in vitro produce excessive amounts of extracellular matrix (ECM), various collagens, mainly type I and III collagens, and display increased transcription of corresponding genes (Hitraya and Jimenez, 1996). Many of the characteristics of scleroderma fibroblasts resemble those of normal fibroblasts stimulated by TGF-␤1 (LeRoy et al., 1989), suggesting that activation of dermal fibroblast in scleroderma may be a result of stimulation by aut...

Connective tissue growth factor (CTGF) is a novel peptide that exhibits platelet-derived growth factor-like activities and is produced by skin fibroblasts after activation with transforming growth factor-beta. Coordinate expression of transforming growth factor-beta followed by CTGF during wound repair suggests a cascade process for control of tissue regeneration. We recently reported a significant correlation between CTGF mRNA expression and histologic sclerosis in systemic sclerosis. To confirm the relation between CTGF and skin fibrosis, we investigated CTGF gene expression in tissue expression in tissue sections from patients with localized scleroderma, keloid, other sclerotic skin disorders using nonradioactive in situ hybridization. In localized scleroderma, the fibroblasts with positive signals for CTGF mRNA were scattered throughout the sclerotic lesions with no preferential distribution around the inflammatory cells or perivascular regions, whereas the adjacent nonaffected dermis was negative for CTGF mRNA. In keloid tissue, the fibroblasts positive for CTGF mRNA were diffusely distributed, especially in the peripheral expanding lesions. In scar tissue, however, the fibroblasts in the fibrotic lesions showed partially positive signals for CTGF mRNA. In eosinophilic fasciitis, nodular fasciitis, and Dupuytren's contracture, CTGF mRNA was also expressed partially in the fibroblasts of the fibrotic lesions. Our findings reinforce a correlation between CTGF gene expression and skin sclerosis and support the hypothesis that transforming growth factor-beta plays an important role in the pathogenesis of fibrosis, as it is the only inducer for CTGF identified to date.

Transforming growth factor (TGF)-beta regulates diverse biologic activities including cell growth, cell death or apoptosis, cell differentiation, and extracellular matrix (ECM) synthesis. TGF-beta is believed to be a key mediator of tissue fibrosis as a consequence of ECM accumulation in pathologic states such as systemic sclerosis. TGF-beta is known to induce the expression of ECM proteins in mesenchymal cells, and to stimulate the production of protease inhibitors that prevent enzymatic breakdown of the ECM. Connective tissue growth factor (CTGF), which is induced by TGF-beta, has been reported to mediate stimulatory actions of TGF-beta ECM synthesis. This review focuses on the possible role of TGF-beta and CTGF in the pathogenesis of fibrosis.

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The role of some growth factors and cytokines in the pathogenesis of systemic sclerosis (SSc) has been suggested. In particular, the contribution of transforming growth factor beta in the progression of skin sclerosis is suspected. Connective tissue growth factor (CTGF) was originally identified in human umbilical vein endothelial cells, and a recent study has revealed that human skin fibroblasts produce CTGF after stimulation with transforming growth factor beta. In the present study, the distribution of CTGF gene expression in tissue sections from patients with SSc was investigated by digoxigenin-labeled in situ hybridization. Strong CTGF mRNA signals were observed in the fibroblasts in sclerotic lesions, especially in the deep dermis, of the skin specimens from patients with SSc, whereas there was no expression in the skin from normal controls. The number of fibroblasts with positive hybridization signals was more abundant in the dermis from the sclerotic stage than in that from the inflammatory stage. Our findings indicate a correlation between CTGF gene expression and skin sclerosis and support the hypothesis that transforming growth factor-beta plays an important role in the pathogenesis of SSc, because transforming growth factor beta is the only inducer for CTGF identified to date.

Scleroderma fibroblasts exhibit numerous phenotypic differences when compared with healthy skin fibroblasts. Some of these differences, in particular overexpression of collagen type I and other extracellular matrix proteins, parallel the effect of transforming growth factor-beta (TGF-beta) on dermal fibroblasts, suggesting that the scleroderma fibroblast phenotype may result from activation of autocrine TGF-beta signaling. To test this hypothesis we examined the role of TGF-beta Type I and Type II receptors in regulating collagen type I transcription. We have shown that overexpression of either Type I or Type II receptors significantly (3-4-fold) increases alpha2 (I) collagen promoter activity in transient transfection assays in dermal fibroblasts. Addition of anti-TGF-beta antibody abolished, whereas addition of plasmin enhanced, the stimulatory effect of receptor overexpression on collagen promoter activity, suggesting that this effect depends on autocrine TGF-beta. Moreover, these cotransfection experiments indicated that expression levels of TGF-beta receptors is a limiting factor in the autocrine regulation of collagen type I transcription by TGF-beta. Comparison of the TGF-beta receptor Type I and Type II mRA expression levels in scleroderma and normal fibroblasts have indicated elevated expression (2-fold) of both receptor types in scleroderma cells, which correlated with increased binding of TGF-beta. Significantly, elevated TGF-beta receptor levels correlated with elevated alpha2 (I) collagen mRNA levels. These results suggest that the elevated production of collagen type I by scleroderma fibroblasts results from overexpression of TGF-beta receptors.

Recently emerging cancer immunotherapies combine the applications of therapeutics to disrupt the immunosuppressive conditions in tumor-bearing hosts. In this study, we found that targeting the proinflammatory cytokine IL6 enhances tumor-specific Th1 responses and subsequent antitumor effects in tumor-bearing mice. IL6 blockade upregulated expression of the immune checkpoint molecule programmed death-ligand 1 (PD-L1) on melanoma cells. This PD-L1 induction was canceled in IFNγ-deficient mice or CD4 T cell-depleted mice, suggesting that CD4 T cell-derived IFNγ is important for PD-L1 induction in tumor-bearing hosts. In some patients with melanoma, however, treatment with the anti-PD-1 antibody nivolumab increased systemic levels of IL6, which was associated with poor clinical responses. This PD-L1 blockade-evoked induction of IL6 was reproducible in melanoma-bearing mice. We found that PD-1/PD-L1 blockade prompted PD-1 macrophages to produce IL6 in the tumor microenvironment. Depletion of macrophages in melanoma-bearing mice reduced the levels of IL6 during PD-L1 blockade, suggesting macrophages are responsible for the IL6-mediated defective CD4 Th1 response. Combined blockade of the mutually regulated immunosuppressive activities of IL6 and PD-1/PD-L1 signals enhanced expression of T cell-attracting chemokines and promoted infiltration of IFNγ-producing CD4 T cells in tumor tissues, exerting a synergistic antitumor effect, whereas PD-L1 blockade alone did not promote Th1 response. Collectively, these findings suggest that IL6 is a rational immunosuppressive target for overcoming the narrow therapeutic window of anti-PD-1/PD-L1 therapy. These findings advance our understanding of IL6-PD1/PD-L1 cross-talk in the tumor microenvironment and provide clues for targeted interventional therapy that may prove more effective against cancer. .

IntroductionRecent accumulating evidence indicates a crucial involvement of macrophage lineage in the pathogenesis of systemic sclerosis (SSc). To analyze the assembly of the monocyte/macrophage population, we evaluated the expression of CD163 and CD204 and various activated macrophage markers, in the inflammatory cells of the skin and in the peripheral blood mononuclear cells (PBMCs) derived from patients with SSc.MethodsSkin biopsy specimens from 6 healthy controls and 10 SSc patients (7 limited cutaneous SSc and 3 diffuse cutaneous SSc) were analyzed by immunohistochemistry using monoclonal antibody against CD68 (pan-macrophage marker), CD163 and CD204. Surface and/or intracellular protein expression of CD14 (marker for monocyte lineage), CD163 and CD204 was analysed by flow cytometry in PBMCs from 16 healthy controls and 41 SSc patients (26 limited cutaneous SSc and 15 diffuse cutaneous SSc). Statistical analysis was carried out using Mann-Whitney U test for comparison of means.ResultsIn the skin from SSc patients, the number of CD163+ cells or CD204+ cells between the collagen fibers was significantly larger than that in healthy controls. Flow cytometry showed that the population of CD14+ cells was significantly greater in PBMCs from SSc patients than that in healthy controls. Further analysis of CD14+ cells in SSc patients revealed higher expression of CD163 and the presence of two unique peaks in the CD204 histogram. Additionally, we found that the CD163+ cells belong to CD14brightCD204+ population.ConclusionsThis is the first report indicating CD163+ or CD204+ activated macrophages may be one of the potential fibrogenic regulators in the SSc skin. Furthermore, this study suggests a portion of PBMCs in SSc patients abnormally differentiates into CD14brightCD163+CD204+ subset. The subset specific to SSc may play an important role in the pathogenesis of this disease, as the source of CD163+ or CD204+ macrophages in the skin.