This article appears in the following Rheumatology issue: Update in systemic sclerosis [View the issue table of contents]
Pro- and anti-fibrotic effects of TGF-β in scleroderma
1Division of Experimental Pathophysiology and Immunology and 2Laboratory of Autoimmuity, Biocenter, Medical University of Innsbruck, Innsbruck, Austria.
Correspondence to: G. Wick, Division of Experimental Pathophysiology and Immunology, Laboratory of Autoimmunity, Biocenter, Medical University of Innsbruck, Peter-Mayr-Straße 4a, A-6020 Innsbruck, Austria. E-mail: georg.wick{at}i-med.ac.at
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The University of California at Davis 200 and 206 (UCD-200/206) lines of chickens have proven to be the animal model that best reflects the situation in human SSc. We have demonstrated a misbalance of pro-fibrotic (TGF-β1) and anti-fibrotic (TGF-β2 and -β3) TGF-β isoforms as a possible cause for fibrotic alterations in this model. This opens new avenues for diagnosis and therapy for this still intractable condition.
KEY WORDS: Systemic sclerosis, Transforming growth factor-β1, Transforming growth factor-β2, Transforming growth factor-β3, Fibrosis, The University of California at Davis 200 chicken
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Fibrosis, i.e. excessive extracellular matrix (ECM) formation, is a major health problem that receives insufficient attention in basic and clinical research with respect to its aetiology, pathogenesis, diagnosis and therapy.
In principle, fibrosis can occur as a consequence of many different pathological conditions. The most important of these are:
- Fibrosis after tissue damage, e.g. post-operative adhesions, burns, alcoholic and post-infectious liver cirrhosis, among others.
- Fibrosis after inflammatory diseases, e.g. infections, arteriosclerosis, CTDs.
- Fibrosis around foreign body implants, e.g. silicone mammary implants, cardiac pace maker, etc.
- ‘Spontaneous’ fibrosis, e.g. keloids, Dupuytren's contracture, etc.
- Tumours, e.g. neurofibromatosis.
Systemic sclerosis (SSc, Scleroderma) is a paradigmatic CTD characterized by two consecutive pathogenetic stages, i.e. a first inflammatory stage followed by a chronic fibrotic stage, the latter finally leading to death by fibrotic alterations of internal organs, notably the lung.
In contrast to the then current dogma, we have previously shown that the earliest alterations that can be found in SSc are not perivascular mononuclear inflammatory infiltrations but rather consist of apoptosis of microvascular endothelial cells induced by anti-endothelial cell autoantibodies (AECAs) entailing the subsequent inflammatory and finally fibrotic complications [1]. This early endothelial damage is not yet clinically manifest and has, therefore, received little attention in previous investigations. In fact, patients with SSc are usually only seen by their physicians when overt symptoms occur, most often first apparent as characteristic lesions in the skin. In order to elucidate the exact pathogenetic cause of the disease, the use of appropriate animal models is indispensable. Among these, the University of California at Davis 200 and 206 (UCD-200/206) lines of chickens have proven to most closely model the human situation in all histopathological, immunological and clinical aspects of SSc [2]. In addition, the chicken also provides the unique opportunity to study and easily manipulate the embryo in hatching eggs thus allowing for an extended observation period in a given animal.
The University of California at Davis 200 and 206 lines of chickens—a spontaneously occurring model for scleroderma
UCD-200/206 lines of chickens are selectively bred for the clinical symptoms of a scleroderma-like disease. They are not inbred but homozygous at the MHC (B-locus in chickens), the UCD-200 line carrying B15, the 206 line B17.
Members of both lines develop scleroderma-like clinical symptoms in a time-lapse fashion, UCD-206 being slightly more severely affected than UCD-200. Alterations start in the skin within the first week after hatching and then extend to internal organs (notably oesophagus, lung, kidney) such that about 90% of the birds are afflicted at the age of 5 weeks.
Since the rest of the skin of the chicken is feathered, the most impressive lesions are first observed in the comb, characterized by oedema followed by Raynaud's syndrome-like changes, finally leading to complete necrosis, a process called ‘self-dubbing’. Between 3 and 6 weeks, the oesophagus and also other internal organs become involved in the disease process finally leading to severe fibrosis. Due to the fact that the gonads are also affected, breeding of UCD-200/206 chickens is difficult and, therefore, requires special expertise and careful selection of breeders.
As mentioned earlier, we have shown that the first stage of the disease comprises microvascular endothelial apoptosis—a phenomenon that in subsequent experiments could also be verified in humans with SSc. AECA-induced apoptosis is not brought about by complement-mediated cytotoxicity, but by antibody-dependent cellular cytotoxicity (ADCC), mediated via the Fas/Fas-ligand rather than the perforin-granzyme pathway. Endothelial cell apoptosis can also be induced by the passive transfer of AECA into embryos via local application onto the chorioallantoic membrane (CAM) or intravenous injection.
The next stage consists of massive perivascular mononuclear cell infiltration, followed by proliferation of fibroblasts and collagenous and non-collagenous ECM deposition. In addition to the pathohistological hallmarks of SSc, the UCD-200/206 model also presents with serological parameters that are characteristic of the human disease. In addition to AECA, these include ANAs with a centromeric staining pattern in indirect IF as well as anti-phospholipid antibodies.
Among the pro- and anti-fibrotic cytokines produced by the mononuclear infiltrate, TGF-β has received the greatest attention in our laboratory.
Cellular and molecular characteristics of fibrosis in UCD-200 and 206 chickens
Fibrosis is a characteristic hallmark of SSc and the major cause of functional impairment of the affected organs [3]. In UCD-200/206 chickens, fibrosis is most prominent in skin, oesophagus and lung. It is characterized by accumulation of ECM, mainly collagen types I, III and VI, which are produced by activated fibroblasts [4]. As in the human disease, no gross alteration of collagen genes could be demonstrated in chickens, as shown by restriction fragment length polymorphism (RFLP) studies [5]. Analyses by RNase protection assays (RPAs) of UCD-200 skin, oesophagus and lung, revealed two pro-collagen 
2(I) mRNA products, represented by 115 and 180 bp bands, respectively. Compared with healthy controls, the smaller, previously unknown variant was significantly increased in the inflammatory disease stage, suggesting that it might be a marker for or even play a role in the initiation of fibrosis [6]. Recently, we have designed appropriate human sense and anti-sense riboprobes that are homologous to the respective chicken pro2(I) probes. Using these probes to analyse human tissues by RPA also resulted in two bands, 207 and 170 bp in size. Whereas the smaller variant was almost absent in normal healthy skin, it was increased in various fibrotic conditions, such as keloids, Dupuytren's disease and excessive fibrous capsules that develop around silicone mammary implants. Thus, it seems that the smaller pro2(I) variant is a general marker for early fibrotic processes in human patients, too (unpublished data).
The role of TGF-β in avian fibrosis
The expression of type I collagen is regulated by various pro- and anti-fibrotic cytokines and growth factors at the transcriptional level. Among these, TGF-β is thought to be a major player in driving fibrosis and many studies have focussed on its role in the pathogenesis of SSc [7]. There are three TGF-β isoforms, TGF-β1, -β2 and -β3, which use similar signalling pathways and exert overlapping, albeit not identical biological functions. It is undisputed that TGF-β1 can activate fibroblasts and is a potent stimulator of collagen production, but the specific function of the other two TGF-β isoforms in the pathogenesis of SSc remains elusive since results from various studies are contradictory. As in the human disease, TGF-β1 has a pro-fibrotic activity on chicken fibroblasts reflected by enhanced proliferation and increased pro-collagen type I expression. Taking advantage of the UCD-200 model, we showed that TGF-β2, in contrast to general belief, can act as an anti-fibrotic cytokine in the pathogenesis of SSc [8]. Chicken embryonic fibroblasts (CEFs) from UCD-200 express significantly more of the pro-fibrotic pro2(I) mRNA variant and show decreased expression of the canonical 180 bp pro2(I) mRNA transcript compared with fibroblasts from healthy normal White Leghorn (NWL). TGF-β2 and TGF-β3 both reduced the expression of the profibrotic 115 bp pro2(I) variant in UCD-200-CEF to the same levels seen in healthy controls, whereas TGF-β1 increased its expression. Moreover, TGF-β2 also reduced the 180 bp transcript in UCD-200 and NWL-CEF, whereas TGF-β3 reduced the 180 bp band only in NWL, but not in UCD-200-CEF. Interestingly, analysis of cell culture supernatants by ELISA revealed that NWL-CEF produced 4.1 times more TGF-β2 than UCD-200-CEF (Fig. 1). The constitutive overproduction of the pro-fibrotic pro2(I) mRNA variant and the diminished TGF-β2 synthesis found in untreated UCD-200-CEF, suggest that TGF-β2 might be a key cytokine during fibrosis onset.
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0.01 untreated UCD-200-CEF vs untreated NWL-CEF, XXP Conclusions and therapeutic implications
TGF-β plays essential roles in health and disease-regulating cell proliferation and differentiation, immune response, angiogenesis and tissue repair [9]. Thus, various TGF-β isoforms are considered promising therapeutic targets. However, a placebo-controlled Phase I/II trial with anti-TGF-β1 antibody therapy in SSc patients showed no evidence of efficacy, but rather increased morbidity and mortality [10]. This is not too surprising since TGF-β1 not only promotes fibrosis, but also has beneficial effects by inhibiting inflammation. Like TGF-β1, TGF-β2 is anti-inflammatory, but in contrast to TGF-β1, it can also act as an anti-fibrotic cytokine and thus, TGF-β2 may be a promising candidate for SSc therapy, especially during the early inflammatory disease stage. The striking immunological and pathological similarities found between avian and human SSc make the UCD-200/206 model an ideal tool to test such a novel therapeutic approach.
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This work has been continuously supported by the Austrian Research Fund (FWF), by most recent project numbers 14466 (to G.W.) and no. 18726-B05 (to R.S.).
Supplement: This paper forms part of the supplement entitled ‘Update in systemic sclerosis’. This supplement was supported by an unrestricted grant from Encysive.
Disclosure statement: The authors have declared no conflicts of interest.
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- Sgonc R, Dietrich H, Gershwin ME, Colombatti A, Wick G. Genomic analysis of collagen and endogenous virus loci in the UCD-200 and 206 lines of chickens, animal models for scleroderma. J Autoimmunity (1995) 8:763–70.[CrossRef][Web of Science][Medline]
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[Free Full Text] - Denton CP, Merkel PA, Furst DE, et al. Cat-192 Study Group; Scleroderma Clinical Trials Consortium. Recombinant human anti-transforming growth factor beta1 antibody therapy in systemic sclerosis: a multicenter, randomized, placebo-controlled phase I/II trial of CAT-192. Arthritis Rheum (2007) 56:323–33.[CrossRef][Web of Science][Medline]
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