Chemokine receptors CCR2 and CX3CR1 regulate skin fibrosis in the mouse model of cytokine-induced systemic sclerosis.

BACKGROUND: Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive accumulation of extracellular matrix (ECM), and develop under the influence of certain cytokines. We previously established a mouse model of skin fibrosis induced by exogenous application of cytokines. We have revealed that both the number of macrophages and the levels of macrophage chemoattractant protein-1 (MCP-1) mRNA positively correlate with the extent of skin fibrosis. Macrophages can be divided into two subsets, the first expressing CCR2, and the second expressing CX3CR1. OBJECTIVE: To elucidate the role of skin infiltrating macrophages based on CCR2 and CX3CR1 in this cytokine-induced murine fibrosis model. METHODS: We examined the amounts of collagen deposited in granulation tissues, the numbers of macrophages and the levels of several mRNA in wild type (WT) mice, CCR2(-/-) mice, and CX3CR1(-/-) mice during injections of transforming growth factor-ß (TGF-ß) followed by injections of connective tissue growth factor (CTGF). RESULTS: TGF-ß injection increased the expressions of MCP-1, fractalkine, CCR2 and CX3CR1 mRNA in WT mice. The overproduction of collagen induced by TGF-ß was significantly reduced by CCR2 deficiency, while collagen contents induced by CTGF were restored to wild-type levels. In contrast, overproduction of collagen in CX3CR1-deficient mice decreased nearly 50% by both TGF-ß and CTGF stimulations. CONCLUSION: The involvement of CCR2/MCP-1 interaction (CCR2-dependent loop) was during the TGF-ß phase. In contrast, the fractalkine/CX3CR1 interaction contributes to the initiation of fibrosis by TGF-ß and its maintenance by CTGF. Collectively, two subsets of macrophages both cooperatively and independently play important roles in the development of fibrosis.

Role of connective tissue growth factor and its interaction with basic fibroblast growth factor and macrophage chemoattractant protein-1 in skin fibrosis.

Activation of the immune system and abnormal growth of skin fibroblasts cause systemic sclerosis. Growth factors have various biological activities, including mediation of immune reactions. The growth factor family includes basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta), and connective tissue growth factor (CTGF). CTGF, an important downstream mediator of TGF-beta in fibrosis, has been suggested to play a specific role in fibrotic disorders. We have directed our attention to the role of CTGF in sustaining skin fibrosis. To better understand its effects in vivo, we established an animal model of skin fibrosis induced by exogenous application of growth factors. In this model, bFGF transiently induced subcutaneous fibrosis. Simultaneous injection of bFGF and CTGF increased skin fibrosis compared with a single injection of bFGF. Serial injections of bFGF for 3 days followed by CTGF for 4 days, or of CTGF followed by bFGF, did not cause skin fibrosis but simultaneous injections increased macrophage chemoattractant protein-1 (MCP-1) mRNA expression levels. To further define the mechanisms of skin fibrosis in vivo, bFGF and CTGF were injected simultaneously into MCP-1 knockout mice, resulting in decreased collagen levels in granulation tissues on day 8. The number of inflammatory cells, such as mast cells, macrophages and lymphocytes, was significantly decreased in MCP-1 knockout mice compared with wild-type mice. These results suggest that bFGF induces collagen production by stimulating skin fibroblasts and CTGF cooperates with bFGF. Our results indicate that the induction of MCP-1 is necessary for infiltration of inflammatory cells.

Neutralizing monoclonal antibody to human connective tissue growth factor ameliorates transforming growth factor-beta-induced mouse fibrosis.

Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive accumulation of extracellular matrix (ECM) and are understood to develop under the influence of fibrogenic growth factors. To better understand the detailed mechanisms of persistent fibrosis in SSc, we have previously established an animal model of skin fibrosis induced by exogenous application of growth factors. In this model, transforming growth factor-beta (TGF-beta) transiently induced subcutaneous fibrosis and serial injections of connective tissue growth factor (CTGF) after TGF-beta caused persistent fibrosis. These results suggest that CTGF plays an important role in the development of persistent skin fibrosis and that CTGF may be a potential and specific therapeutic target in skin fibrosis. Therefore, the aim of the current study is to develop a neutralizing monoclonal antibody against human CTGF. We also investigated the neutralizing effect of the antibodies in our animal model. Firstly, by using the DNA immunization method, we developed a panel of anti-CTGF antibodies recognizing the native conformation of human CTGF. Next, to examine the anti-fibrosing effects of these antibodies, newborn B6 mice received subcutaneous injections of TGF-beta for 3 days with either anti-CTGF neutralizing antibodies or control purified immunoglobulin. Anti-CTGF antibodies significantly reduced skin fibrosis and collagen contents compared with the control group. These results suggest that our anti-CTGF antibodies are capable of blocking the development of skin fibrosis at least partially and these anti-CTGF neutralizing antibodies may be useful as the feasible strategy to treat skin fibrotic diseases as SSc.

Connective tissue growth factor causes persistent proalpha2(I) collagen gene expression induced by transforming growth factor-beta in a mouse fibrosis model.

Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive production of extracellular matrix (ECM) and understood to develop under the influence of certain growth factors. Connective tissue growth factor (CTGF) is a cysteine-rich mitogenic peptide that is implicated in various fibrotic disorders and induced in fibroblasts after activation with transforming growth factor-beta (TGF-beta). To better understand the mechanisms of persistent fibrosis seen in SSc, we previously established an animal model of skin fibrosis induced by exogenous application of growth factors. In this model, TGF-beta transiently induced subcutaneous fibrosis and serial injections of CTGF after TGF-beta caused persistent fibrosis. To further define the mechanisms of skin fibrosis induced by TGF-beta and CTGF in vivo, we investigated in this study, the effects of growth factors on the promoter activity of the proalpha2 (I) collagen (COL1A2) gene in skin fibrosis. For this purpose, we utilized transgenic reporter mice harboring the -17 kb promoter sequence of the mouse COL1A2 linked to either a firefly luciferase gene or a bacterial beta-galactosidase gene. Serial injections of CTGF after TGF-beta resulted in a sustained elevation of COL1A2 mRNA expression and promoter activity compared with consecutive injection of TGF-beta alone on day 8. We also demonstrated that the number of fibroblasts with activated COL1A2 transcription was increased by serial injections of CTGF after TGF-beta in comparison with the injection of TGF-beta alone. Furthermore, the serial injections recruited mast cells and macrophages. The number of mast cells reached a maximum on day 4 and remained relatively high up to day 8. In contrast to the kinetics of mast cells, the number of macrophages was increased on day 4 and continued to rise during the subsequent consecutive CTGF injections until day 8. These results suggested that CTGF maintains TGF-beta-induced skin fibrosis by sustaining COL1A2 promoter activation and increasing the number of activated fibroblasts. The infiltrated mast cells and macrophages may also contribute to the maintenance of fibrosis.