Effect of mast cell chymase inhibitor on the development of scleroderma in tight-skin mice

Abstract

1 Although the pathogenesis of scleroderma is not fully understood, activation of connective-tissue-type mast cells (CTMCs) has been implicated in various fibrotic diseases. 2 Our previous study showed that the number of CTMCs was markedly increased during fibrous proliferation in the skin of a scleroderma model, namely tight-skin (Tsk) mice. Because mast cells express numerous bioactive factors, such as cytokines, growth factors, proteases, and others, it is crucial to identify the primary factors that may be involved in the pathogenesis of scleroderma. Our previous study also showed that a CTMC-specific protease, chymase-4, was selectively upregulated in accordance with the development of skin fibrosis in Tsk mice. 3 To further elucidate the role of chymase secreted from CTMCs, we evaluated the therapeutic effects of a synthetic chymase-specific inhibitor, SUN-C8257, on the development of skin fibrosis in Tsk mice. SUN-C8257 (50 mg kg-1 day-1) was administered via intraperitoneal injection in 13-week-old Tsk mice for a period of 2 weeks. 4 Treatment with SUN-C8257 significantly reduced chymase activity by 43% and the chymase-4 mRNA level by 47%, and also decreased the thickness of the subcutaneous fibrous layer of Tsk mice by 42% compared with that of Tsk mice injected with vehicle. 5 Furthermore, immunohistochemical analysis revealed that transforming growth factor (TGF)-beta1 staining in the fibrous layer of Tsk skin was markedly reduced by the treatment with SUN-C8257. This chymase inhibitor may prevent the chymase-dependent pathway that activates the latent TGF-beta1 in fibrous tissue, and may exhibit beneficial effects that inhibit the development of fibrosis. 6 In conclusion, our results strongly support the assumption that CTMC-derived chymase may play a key role in the pathogenesis of scleroderma.

Figure 1

Effect of a chymase inhibitor on the skin fibrous proliferation in Tsk and control C57B/6J mice. Skin fibrosis was analyzed with Azan-stained sections from a vehicle-treated Tsk mouse (a), SUN-C8257-treated Tsk mouse (b), and vehicle-treated control C57BL/6J (c). The blue color staining indicates the presence of collagen. In vehicle-treated Tsk mouse, thick blue-stained fibrous layer was detected beneath panniculus carnosus muscle layer (a), whereas fibrous layer in SUN-C8257-treated Tsk mouse was thinner than that in vehicle-treated Tsk mouse (b). The fibrous layer in control C57BL/6J mice was obviously thin (c). F: fibrous layer, M: panniculus carnosus muscle layer. Scale bar=100 μm.

Figure 2

Effect of a chymase inhibitor on the skin fibrous proliferation and the number of skin mast cells in Tsk and control C57B/6J mice. Thickness of the subcutaneous fibrous layer (a) and the number of skin mast cells (b) of Tsk and control C57BL/6J mice were measured after 2 weeks treatment with vehicle or SUN-C8257. White bars indicate the data of vehicle-treated mice (Vehicle) and black bars indicate the data of mice treated with SUN-C8257 (SUN). Vertical bars represent s.e.m. ^*P<0.05. NS=not significant.

Figure 3

Effect of a chymase inhibitor on chymase activity and chymase-4 mRNA level in the skin tissues of Tsk and control C57B/6J mice. Chymase activity (a) and chymase-4 mRNA level (b) of Tsk and control C57BL/6J mice were measured after 2 weeks treatment with vehicle or SUN-C8257. White bars indicate the data of vehicle-treated mice (Vehicle) and black bars indicate the data of mice treated with SUN-C8257 (SUN). Vertical bars represent s.e.m. ^*P<0.05. NS=not significant.

Figure 4

Effect of a chymase inhibitor on immunohistochemical staining of TGF-beta1. Histological cross-sections of the skin tissues from vehicle-treated Tsk mouse (a), SUN-C8257-treated Tsk mouse (b), and vehicle-treated control C57BL/6J mouse (c) were incubated with antibody against TGF-beta1, and then the tissue-bound antibody was detected with the DAKO ENVISION kit and DAB substrate. Sections were then counterstained with hematoxylin. The brown color staining indicates the presence of TGF-beta1. F: fibrous layer; M: panniculus carnosus muscle layer. Scale bar=100 μm.

Figure 5

Immunohistochemical localization of TGF-beta1 and chymase. Histological cross-sections of skin tissues from vehicle-treated Tsk mouse were incubated with polyclonal antibody against TGF-beta1 or chymase. The antibody against TGF-beta1 was detected with the DAKO ENVISION kit and DAB substrate. The brown color staining indicates the presence of TGF-beta1 (a). To identify mast cells, adjacent section to that in part (a) was stained with toluidine blue. On reacting with toluidine blue, mast cells showed metachromasia, which is in red-purple color (b). The antibody against chymase was detected with the DAKO ENVISION kit and TrueBlue peroxidase substrate. Sections were then counterstained with eosin. Positive staining of chymase was revealed by dark blue color (c). To identify mast cells, adjacent section to that in part (c) was stained with toluidine blue. Mast cells were stained with red-purple color (d). Arrows point to mast cells. Scale bar=50 μm.