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Rheumatology Advance Access originally published online on January 3, 2008
Rheumatology 2008 47(2):172-175; doi:10.1093/rheumatology/kem344
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Effectiveness of etanercept in bleomycin-induced experimental scleroderma

S. S. Koca1, A. Isik1, I. H. Ozercan2, B. Ustundag3, B. Evren1 and K. Metin3

1Department of Rheumatology, 2Department of Pathology and 3Department of Biochemistry, Faculty of Medicine, Firat University, Elazig, Turkey.

Correspondence to: S. S. Koca, Firat University, Firat Tip Merkezi, Ic Hastaliklari AD./Romatoloji BD., 23119-Elazig, Turkey. E-mail: kocassk{at}yahoo.com


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Objectives. To evaluate the effects of etanercept and thalidomide in the mouse model of bleomycin-induced scleroderma (BLM-IS).

Methods. This study involved four groups (n = 8 mice in each group). Dermal sclerosis was induced by repeated subcutaneous injections of BLM (10 µg) for 4 weeks in BALB/c mice. Control group received only phosphate-buffered saline. The second group received only BLM; the third and fourth groups were also given an intraperitoneal injection of 100 µg etanercept or 150 mg/kg thalidomide, respectively.

Results. BLM increased serum TGF-β1, tissue hydroxyproline levels and expression of {alpha}-smooth muscle actin ({alpha}-SMA), and dermal fibrosis was histopathologically prominent. Although thalidomide had no significant effect, etanercept caused decreases in levels of serum TGF-β1, tissue hydroxyproline and number of {alpha}-SMA-positive cells.

Conclusion. Inhibition of TNF-{alpha} with etanercept in BLM-IS was resulted in a significant reduction of the dermal sclerosis, collagen accumulation and the number of infiltrating myofibroblastic cells. TNF-{alpha} may play a key role in the progression of BLM-IS and TNF-{alpha} antagonists may be useful in the management of scleroderma.

KEY WORDS: Scleroderma, Bleomycin, Etanercept, Animal model


   Introduction
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 Abstract
 Introduction
Methods
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 Discussion
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Scleroderma is an autoimmune disease, characterized by progressive fibrosis of the skin and internal organs including lung and gastrointestinal tract. Several growth factors and cytokines that are partly released from inflammatory cells infiltrating affected tissues have been suggested to play a central role in the initiation and the development of fibrosis in scleroderma [1] Cytokines regulate immune response that is likely to affect fibroblast functions [2, 3]. The infiltration of inflammatory cells into the affected tissues is a process highly regulated by expression of chemokines, and modulated by soluble functional forms of cell adhesion molecules [3]. It is well known that the level of TGF-β, a profibrotic cytokine, increases in scleroderma [1]. TGF-β causes increased production of {alpha}-smooth muscle actin ({alpha}-SMA) [4, 5]. It has been also shown that TNF-{alpha} participates in the activation of vascular endothelium, regulation of immune response and metabolism of the connective tissue by modulation of fibroblastic function. Scleroderma patients exhibit both systemic and local increase of TNF-{alpha} levels and consecutively these increases contribute to progression in scleroderma, development of fibrosing alveolitis and skin fibrosis [6]. Increased TNF-{alpha} in scleroderma has been reported in clinical and experimental studies [7–11]. Moreover, it has been reported that the level of TNF-{alpha} correlates with the disease activity in scleroderma [9]. There are also studies reporting the effective improvement of bleomycin-induced pulmonary fibrosis (BLM-IPF) after anti-TNF-{alpha} and soluble receptor of TNF treatments [12, 13].

The exact pathogenesis of scleroderma has not been fully understood yet, despite the known data and ongoing studies about it. Therefore, the therapy of scleroderma is not at a satisfactory level as no highly effective agent is available. The aims of the present study were to evaluate pathogenic roles of TNF-{alpha}, and therapeutic effects of etanercept, an anti-TNF-{alpha} agent and thalidomide, an immunomodulator agent, in the mouse model of BLM-induced scleroderma (BLM-IS). A mouse model for scleroderma has been established previously by repeated local injections of BLM [4].


   Methods
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 Abstract
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 Methods
Results
 Discussion
 References
 
Animal model for scleroderma and administration of etanercept and thalidomide
Specific pathogen-free BALB/c mice (6 weeks old, female), weighing 20–25 g, were used. They were randomly classified into four groups (n = 8 in each group). Dorsal skin was shaved for subcutaneous injections. Subcutaneous injections were performed by a 26 gauge needle, every day for 4 weeks. BLM, etanercept, and thalidomide were dissolved or diluted in phosphate-buffered saline (PBS) and sterilized by filtration (0.2 µm filter). The first group was taken as the control one and received only 100 µl PBS. The other three groups received 100 µl (10 µg) BLM, subcutaneously as described by Yamamoto et al. [14]. The third group received additional intraperitoneal 100 µg etanercept three times in a week and the fourth group received additional intraperitoneal 150 mg/kg/day thalidomide every day, as reported by Karrow et al. [15]. Approval of the ethics committee of Firat University was obtained.

Histopathology and histochemistry
Animals were sacrificed by cervical dislocation under anaesthesia with ketamine hydrochloride on the day following the final applications, and the dorsal skins were harvested for further examinations. The skin specimens were cut into two parts; one part was fixed with 10% formalin solution, and the other was stored immediately at –80°C for tissue hydroxyproline (OH-proline) content assay. The skin specimens embedded in paraffin were sectioned at 4 µm thickness using a microtome. They were then stained with haematoxylin and eosin (H&E) and Van-Gieson. Dermal thickness (measured from the epidermal–dermal junction to dermal–fat junction) was determined from five randomly selected sites of two or more skin sections in each animal by x100 magnification. They were examined under an Olympus BX-50 light microscope.

The numbers of {alpha}-SMA-positive fibroblastic cells were counted using an ocular grid under a light microscope of x400 high-power field (HPF) from five randomly selected fields in each specimen, and the mean number was counted after it was stained with streptavidin biotin technique (Actin Muscle Ab-6 [MSA06] Neomarkers).

Collagen deposition was estimated by determining the total OH-proline content of the skin. The stored skin specimens were washed with normal saline and dried in an oven at 100°C for 72 h, then hydrolysed with 12 N hydrochloric acid at 130°C for 3 h according to the method of Woessner [16]. After neutralizing with sodium hydroxide, the hydrolysates were diluted with distilled water. OH-proline in the hydrolysates was assessed colorimetrically at 560 nm with p-dimethylaminobenzaldehyde. OH-proline contents were measured and expressed as milligrams per gram of dry tissue.

Enzyme-linked immunosorbent assay of serum cytokines
Blood samples were taken by cardiac puncture and sera were obtained after centrifugation at 3000 r.p.m. for 10 min and stored at –20°C until the day of the analyses. Serum TNF-{alpha}, IL-6 and TGF-β1 levels were measured using appropriate commercial kits (Biosource International, Camarillo, CA, USA) by the enzyme-linked immunosorbent assay method.

Statistical analysis
Data were presented as mean ± S.D. and for comparisons between the groups, Kruskal–Wallis one-way analysis of variance and for dual comparisons Mann–Whitney U-test were used. Statistical evaluations were performed using the SPSS package program, version 11.0. A P-value of < 0.05 was considered to be statistically significant.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Injections of PBS did not induce dermal fibrosis (Fig. 1A). However, injections of BLM increased serum TGF-β1 (P < 0.05) and tissue OH-proline levels (P < 0.05) and expression of {alpha}-SMA (P < 0.001), compared with the control PBS-treated skin (Table 1). BLM injections increased serum TNF-{alpha} and IL-6 levels by approximately 2-fold. However, these increases were not significant (Table 1) and moreover, resulted in histologically prominent dermal fibrosis (Fig. 1B). H&E examinations revealed definite dermal sclerosis characterized by thickened collagen bundles with cellular infiltrates (Fig. 1B) that mimics scleroderma. Increased deposition of collagen in the thickened skin was determined by Van Gieson staining.


Figure 1
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  		FIG. 1. Histopathological evaluation of skin sections in BALB/c mice (H&E stain, x100). (A) Histopathological appearances of PBS-administered mice were normal. (B) Mice treated with BLM demonstrated definite dermal sclerosis with thickened collagen bundles in the thickened dermis. (C) Mice treated with BLM and etanercept did not show the latter signs at this level. (D) Mice treated with BLM and thalidomide did not show significant improvements.

 

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  		TABLE 1. Effects of etanercept and thalidomide applications on levels of serum cytokines, tissue OH-proline level and histopathological findings in BALB/c mice groups

 
Etanercept treatment decreased serum TGF-β1 level and tissue OH-proline content in the skin and number of {alpha}-SMA-positive cells (P< 0.05, for all, Table 1, Fig. 2). H&E examination revealed decreased dermal sclerosis (P < 0.05) and inflammatory cell infiltration in the etanercept-treated group (Fig. 1C). However, thalidomide did not have any significant effect on any of the study parameters in this mouse model (Table 1, Fig. 1D). Although thalidomide treatment decreased serum IL-6 and TGF-β1 levels, these reductions were not significant (Table 1).


Figure 2
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  		FIG. 2. Effects of different treatment applications on the number of {alpha}-SMA-positive cells in BALB/c mice groups. BLM application increased {alpha}-SMA-positive cell counts (P < 0.001). Etanercept treatment reduced the number of {alpha}-SMA-positive cells (P < 0.05). HPF, high-power field (x400).

 

   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In the present study, the anti-sclerotic effect of anti-TNF-{alpha} treatment on BLM-induced dermal sclerosis was examined in BALB/c mouse model. The mouse model of BLM-IS has been well documented recently [4, 17]. Daily BLM injections induced increased expressions of TGF-β1 and {alpha}-SMA, leading to dermal fibrosis in the present study. Moreover, OH-proline contents in the BLM-treated skin were significantly higher than those in the control PBS-treated skin.

The effects of different cytokines have been shown on this mouse model of scleroderma [4, 17, 18]. BLM has been shown to induce synthesis and release of IL-4 and IL-6 [18]. Administration of anti-TGF-β antibody has been reported to be effective in inhibiting dermal sclerosis and BLM-IPF in experimental animal models [18, 19] and TNF-{alpha} has been shown to play an important role in the induction of BLM-IPF [12]. In the present study, repeated BLM injections for 4 weeks increased the levels of cytokines and also infiltration of the inflammatory cells.

Scleroderma is an inflammatory disease in which cytokines play pathogenic roles [1, 2, 7–11] as in BLM-induced mouse model [4, 17, 18]. It has been shown that the levels of IL-4, IL-12, IL-13, TNF-{alpha} and sIL-6R are high and serum levels of IL-6 and IL-10 correlate with total skin thickness score in patients with scleroderma [10]. Increased IL-6 and TNF-{alpha} levels have been reported in another clinical study [11]. It has been shown that TNF-{alpha} participates in the activation of vascular endothelium-derived mediators, regulation of immune response and metabolism of connective tissue by the modulation of fibroblastic function. Scleroderma patients exhibit both systemic and local increase in TNF-{alpha} level [7–11]. Moreover, it has been reported that the level of TNF-{alpha} correlates with the disease activity in scleroderma [9]. It has also been shown that treatment with anti-TNF-{alpha} agents causes reduction in BLM-IPF [12, 13]. Inhibition of TNF-{alpha} with infliximab in patients with collagen vascular disease has been reported to stabilize the progression of pulmonary fibrosis [20].

Anti-TNF-{alpha} agents are effective and frequently used in the treatment of a variety of inflammatory diseases [21, 22]. It has been suggested that anti-TNF-{alpha} agents might also be efficient in the treatment of scleroderma [22]. Decreased serum cytokines such as IL-1β and IL-6 and reduced expressions of adhesion molecules have been reported in RA patients who have been treated with etanercept [23]. Adhesion molecules are necessary for the migration of inflammatory cells to the extracellular area, and they have prominent roles in the pathogenesis of scleroderma [3]. In the present study, etanercept improved BLM-induced skin fibrosis.

TNF-{alpha} increases the release of endothelin-1 and expression of adhesion molecules such as E-selectin, ICAM-1 and VCAM-1 from endothelial cells. Additionally, it stimulates the proliferation of fibroblasts via TGF-β [24]. Treatment with an anti-TNF-{alpha} agent might improve the skin fibrosis in this mouse model not only by its direct effects but also by decreasing TGF-β level. Anyhow, anti-TGF-β antibody has also been reported to decrease the formation of myofibroblasts [18]. In the present study, administration of etanercept decreased TGF-β1 level.

Thalidomide has also been shown to reduce lipopolysaccharide-stimulated TNF-{alpha}, IL-8, IL-12p40 and IL-18 release from alveolar macrophages in idiopathic pulmonary fibrosis patients [25]. Settles et al. [26] have reported that thalidomide might significantly reduce cell adhesion molecule expression such as ICAM-1, E-selectin and LFA-1. In another report [27], thalidomide has inhibited nuclear factor {kappa}-B (NF-{kappa}B) transcriptional activity induced by TNF-{alpha} and has suppressed TGF-β1-induced {alpha}-SMA expression and collagen deposition in hepatic stellate cells.

Thalidomide has been shown to be effective in the treatment of dermal signs of graft-vs-host disease (GVHD) [28]. Moreover, in another clinical study [29] including relatively smaller sample size, a 12-week thalidomide treatment has successfully relieved the clinical signs and has provided histopathological improvement. In that study, these effects have been suggested to be due to its immunomodulatory effects. However, thalidomide did not affect either histopathological finding of BLM-induced fibrosis or serum levels of cytokines in our study. The duration of thalidomide administration in the present study might be insufficient to provide immunomodulation.

In conclusion, our results indicate that in the BLM-IS, inhibition of TNF-{alpha} resulted in a significant reduction of the severity of the dermal sclerosis, collagen accumulation and the number of infiltrating myofibroblastic cells. These data suggest that TNF-{alpha} might play a key role in the progression of BLM-IS and that the treatment with an anti-TNF-{alpha} agent might be efficient in the treatment of scleroderma.

Formula

Disclosure statement: The authors have declared no conflicts of interest.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Submitted 25 July 2007; revised version accepted 19 November 2007.
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