Analysis of the effect of halofuginone on bleomycin-induced scleroderma

T. Yamamoto and K. Nishioka

Department of Dermatology, Tokyo Medical and Dental University, School of Medicine, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan

SIR, Systemic sclerosis (SSc) is a connective tissue disease with unknown aetiology, which involves not only the skin but also various internal organs [1, 2]. SSc is characterized by (i) excessive production and deposition of extracellular matrix, (ii) injury of endothelial cells, and (iii) an immunological abnormality in which various cytokines or mediators released from inflammatory cells infiltrate the dermis [3]. A number of proinflammatory or fibrogenic cytokines produced by activated cells of the immune system may contribute to extracellular matrix deposition and vascular damage in SSc.

Halofuginone is an alkaloid originally isolated from the plant Dichroa febrifuga. A recent study demonstrated that this agent suppresses the synthesis of collagen in avian skin in vivo [4]. In vitro, halofuginone attenuates collagen synthesis and the expression of the collagen gene in avian and murine skin fibroblasts [5]. Halofuginone specifically inhibits {alpha}1(I) collagen gene expression without affecting the synthesis of other types of collagen, such as types II and III [5, 6]. It also prevents skin fibrosis in two models of scleroderma: murine chronic graft-versus-host disease (cGvHD) and the tight-skin mouse [7].

Bleomycin has been demonstrated to increase collagen synthesis in cultured fibroblasts [811], and an in vivo study also showed that mRNAs for procollagens I and III and fibronectin were selectively up-regulated after instillation of bleomycin [12]. Bleomycin-induced lung injury is a well-known mouse model which resembles human pulmonary fibrosis [1315]. We have recently established a mouse model of scleroderma induced by bleomycin [1619]. In this model, dermal sclerosis mimicking human scleroderma both histologically and biochemically is induced by repeated local injections of bleomycin for 3–4 weeks. In the present study we examined the therapeutic effect of halofuginone on the development of dermal sclerosis induced by bleomycin in this model.

Specific pathogen-free female C3H mice (purchased from Sankyo Labo Service, Tokyo, Japan), aged 6 weeks and weighing 20–25 g, were used. Mice were kept in separate clean rooms.

Mice received intraperitoneal or subcutaneous injections of 100 µl of halofuginone (Stenorol, an anti-coccidial agent; a generous gift from Intervet International, Boxmeer, The Netherlands) (100 µl containing 0.01–1 µg/ml diluted in 0.1% acetone and sterilized with a 0.2 µm filter) simultaneously with subcutaneous injections of bleomycin [100 µl containing 100 µg/ml, diluted in phosphate-buffered saline (PBS)] on the shaved skin of the back on alternate days. Mice in the control group were injected with PBS alone. Mice were killed by cervical dislocation on the day after the final treatment, and the back skins were harvested. Six mice from each group were examined.

Biopsied skin was fixed in 10% formalin solution and embedded in paraffin. The general histological appearance was examined by routine haematoxylin and eosin staining. Dermal thickness was assessed using biopsies of PBS-injected skin as a reference group and was scored to provide a semiquantitative measurement (score 0, <125% of the value for PBS-injected skin; 1, 125–150%; 2, 150–175%; 3, 175–200%; 4, >200%).

For the collagen assay, 6-mm punch biopsy specimens were excised from the shaved back skin (n=6 in each group) and stored at -80°C. Collagen deposition was estimated by determining the total collagen content of the skin using the Sircol Collagen Assay kit (Biocolor, Newtownabbey, Northern Ireland, UK) according to the manufacturer's instructions. The biopsies were homogenized in 0.5 M acetic acid, and 1 ml of Sircol dye reagent (which binds to collagen) was added to each sample, which was then mixed for 30 min. After centrifugation, the pellet was suspended in 1 ml of the alkali reagent included in the kit and assessed colorimetrically at 540 nm by spectrophotometry. Collagen standard solutions were used to construct a standard curve. Results were expressed as percentages compared with mice that received only PBS injections.

Results are expressed as mean±S.D. The Mann–Whitney U-test was used to test for significance. A P value of <0.05 was considered to be significant.

We assessed the inhibitory effect of halofuginone administered together with bleomycin on the induction of dermal sclerosis. In preliminary experiments, mice died within 3 weeks when injected with 1 µg/ml halofuginone either subcutaneously (n=4) or intraperitoneally (n=4). PBS injections produced no dermal sclerosis (Fig. 1AGo), whereas mice treated with bleomycin alone or together with 0.1% acetone showed definite dermal sclerosis characterized by thickened collagen bundles and deposition of amorphous materials in the dermis (Fig. 1BGo). Daily intraperitoneal (n=6) or subcutaneous (n=6) injections of halofuginone (0.01–0.1 µg/ml) together with subcutaneous injections of bleomycin for 3 weeks did not attenuate the dermal sclerosis in the mice (Fig. 1CGo and DGo). Subcutaneous injections of 0.1 µg/ml halofuginone together with bleomycin slightly moderated the dermal sclerosis but did not prevent its occurrence (Fig. 1DGo). The degree of dermal thickness was not significantly reduced by either systemic or local administration of halofuginone (not shown). Analysis of the collagen content in the skin showed that injections of bleomycin together with 0.1% acetone for 3 weeks induced an increase in the collagen content of up to 210±15% compared with PBS treatment. Simultaneous treatment with halofuginone and bleomycin did not produce a significant reduction in the collagen content (not shown). Our results show that neither subcutaneous nor intraperitoneal administration of halofuginone inhibited the dermal sclerosis induced by bleomycin.



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FIG. 1.  Representative photomicrographs of haematoxylin and eosin (HE) staining showing the effect of halofuginone on bleomycin-induced dermal sclerosis. Mice injected with PBS on alternate days for 3 weeks did not show dermal sclerosis (A), whereas mice treated with bleomycin with 0.1% acetone showed definite sclerosis of the skin, with thickened collagen bundles and deposition of homogeneous material (B). Mice treated with bleomycin simultaneously with intraperitoneal (C) or subcutaneous (D) administration of halofuginone (0.1 µg/ml) for 3 weeks had sclerotic lesions similar to those in mice treated with bleomycin without halofuginone (B). Magnification: A,x130; B–D,x170.

 
Halofuginone not only exerts an inhibitory effect in murine fibrosis [7] but also abrogates bleomycin-induced lung fibrosis [20]. On the contrary, our results showed that halofuginone does not prevent bleomycin-induced scleroderma in our model. One possible reason for this is that the treatment period was 3 weeks, which may be a little short. In previous studies, halofuginone was administered for 45–52 days to produce an effect sufficient to reduce cutaneous fibrosis [7] and for 4–6 weeks to prevent lung fibrosis [20]. Another reason is that our method requires frequently repeated treatment with bleomycin (injections on alternate days) to induce dermal sclerosis. Once collagen is synthesized and secreted into the extracellular space, halofuginone can no longer affect its metabolism [5]. In the bleomycin-induced lung injury model, a single administration of bleomycin can cause pulmonary fibrosis. On the contrary, our model of bleomycin-induced dermal sclerosis requires repeated injections of bleomycin. Thus, our method can induce cutaneous sclerosis but not fibrosis. We conclude that halofuginone cannot suppress the development of bleomycin-induced scleroderma following the methods used in this study.

Notes

Correspondence to: T. Yamamoto. Back

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Accepted 29 November 2001





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