Circulating levels of active transforming growth factor ß1 are reduced in diffuse cutaneous systemic sclerosis and correlate inversely with the modified Rodnan skin score

M. Dziadzio, R. E. Smith, D. J. Abraham, C. M. Black and C. P. Denton

Centre for Rheumatology and Connective Tissue Diseases, Royal Free and University College Medical School, University College, London, UK.

Correspondence to: C. P. Denton, Centre for Rheumatology, Royal Free and University College Medical School, University College London, Rowland Hill Street, London NW3 2PF, UK. E-mail: c.denton{at}medsch.ucl.ac.uk


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Objectives. To determine the relationship between clinical features and circulating levels of active transforming growth factor (TGF) ß1 in the major subsets of systemic sclerosis (SSc).

Methods. In a cross-sectional study cases of diffuse cutaneous SSc (dose) (n=27) or limited cutaneous SSc (dose) (n=20) were compared with healthy controls (n=22). Active and total TGFß1 was measured in serum and plasma by a high-sensitivity enzyme-linked immunosorbent assay.

Results. There were no significant differences between levels of total serum TGFß1. However, cases of dcSSc had lower levels of active TGFß1 than cases of lcSSc or controls. In addition, more cases of dcSSc (18/27; 66%, P<0.025) had no detectable active TGFß1 than controls (7/22, 32%) or lcSSc (7/20, 35%). In dcSSc, serum active TGFß1 levels correlated negatively with skin score and positively with disease duration.

Conclusions. Contrary to expectation, levels of active TGFß1 are reduced in dcSSc and this correlates with two variables known to associate with disease activity, shorter duration and more extensive skin sclerosis. This suggests that active TGFß1 may be sequestered in active involved SSc skin and that serum levels are reduced despite strong evidence implicating TGFß isoforms in the pathogenesis of fibrosis. Our findings may have implications for systemic TGFß-trapping therapies in this disease.

KEY WORDS: Scleroderma, Systemic sclerosis, Biological markers, Severity of illness index, Transforming growth factor beta


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Transforming growth factor ß1 (TGFß1) modulates cell proliferation, promotes mesenchymal cell differentiation with enhanced deposition of extracellular matrix and has immunosuppressive properties [1]. The specific action of TGFß1 on a particular cell depends on differential expression of its receptors and allows for a precise response in differing cellular environments [2].

A substantial body of evidence implicates altered activity of TGFß1 in the pathogenesis of systemic sclerosis (SSc) [3, 4]. Persuasive support comes from the profound effects of TGFß1 on fibroblasts inducing a fibrogenic phenotype and promoting differentiation of myofibroblasts [5]. Other evidence is provided by studies reporting increased expression of TGFß ligand in many experimental models of fibrosis and indirectly from a number of studies that demonstrate perturbed TGFß signalling in SSc fibroblasts [6, 7]. In addition, it is a potent immunosuppressive cytokine and an important product of regulatory lymphocyte subpopulations [8]. Consistent with this broad range of important biological properties, TGFß1 bioactivity is tightly regulated [9]. This regulation includes a non-covalent association with latency associated peptide (LAP) to form biologically inactive latent TGFß complex. Dissociation from LAP is necessary for biological activity and mechanisms of activation may differ according to physiological or pathological context [10]. Serum and plasma levels of TGFß isoforms have been measured in several diseases [2] but the results have been conflicting. One limitation has been the discrimination between latent and active forms; another potential confounder is that serum levels may be difficult to interpret due to release of TGFß from platelets ex vivo. Consequently, the significance of circulating levels of this profibrotic factor remains unclear [11]. Levels of TGFß1 in SSc have previously been measured using low-sensitivity assays, with discordant results [12–18]. Interpretation is confounded as some assays detect TGFß1 in its biologically inactive latent form and are not specific for active 25 kDa protein. Clinical heterogeneity and a lack of the standardized assessment of clinical variables has limited interpretation of these earlier studies [19].

We have examined serum and plasma levels of TGFß1 in well-characterized patients with SSc and investigated correlation(s) with clinical variables as well as with disease activity and severity indices [20, 21].


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Study subjects
Patients were recruited from the out-patient clinic at the Rheumatology Department, Royal Free Hospital, over a 24-week period. Forty-seven patients with systemic sclerosis (SSc) and 22 healthy controls (HC) were enrolled into the study. The Royal Free Hospital Local Research Ethics Committee approved the study and all subjects provided informed consent to participate.

Patients fulfilled the American College of Rheumatology (formerly the American Rheumatism Association) preliminary criteria for the classification of SSc [22]. Twenty-seven patients (20 female, 7 male) had diffuse cutaneous systemic sclerosis (dcSSc) and 20 patients (16 female, 4 male) had limited cutaneous systemic sclerosis (lcSSc) [23]. Disease duration was defined as the time from onset of the first non-Raynaud's manifestation of SSc. Clinical assessment of skin sclerosis was performed concurrently with blood sampling: skin score was measured by the modified Rodnan skin scoring technique [24]. The presence of antinuclear antibodies, anticentromere antibodies, antidouble-stranded DNA (dsDNA) antibodies, antiextractable nuclear antigen (ENA) antibodies including anti-Scl-70, anti-PM-Scl, anti-nRNP, anti-Jo-1, anti-Ro and anti-La, anti-Sm and anti-polymerase I and III antibodies as well as rheumatoid factor was determined by validated assays. Patients were screened for major visceral complications of SSc according to current standard practice including pulmonary function tests, ECG, Doppler echocardiography and calculated creatinine clearance. When these basic tests were abnormal, further investigation by high-resolution CT scan (HRCT) or right heart catheterization was performed. Pulmonary hypertension (PHT) was defined by mean pulmonary artery pressure (PAP) above 25 mmHg at rest or 30 mmHg on exercise. Lung fibrosis was determined by HRCT, renal involvement by creatinine clearance of less than 60 ml/min or history of scleroderma renal crisis, and gastrointestinal tract involvement was determined by typical history or abnormal investigations prompted by symptoms. Skeletal muscle involvement was determined by creatine kinase (CPK) greater than four times the upper limit of normal. Disease activity and severity were determined according to the guidelines formulated recently [21]. Briefly, in the assessment of disease activity, the following parameters were evaluated and scored: modified Rodnan skin score, presence of scleredema, any recent worsening of the cutaneous/vascular/cardiopulmonary involvement, active digital ulcers, presence of arthritis, diffusing lung capacity for carbon monoxide (DLCO) <80%, erythrocyte sedimentation rate (ESR) >30 and low levels of serum complement. Scoring was different for each of those parameters with the total score ranging from 0 to 10. The disease was considered active if the score was ≥3. The assessment of disease severity was based on the evaluation of nine organs/systems: general, peripheral vascular, skin, joints and tendons, muscles, gastrointestinal tract, lung, heart and kidneys. The severity of the involvement of each organ was evaluated following guidelines and scored from 0–4, where 0 was normal or no involvement, 1 mild, 2 moderate, 3 severe and 4 end-stage. The single scores were summarized and the final value denoted disease severity.

Control samples were obtained from 22 healthy volunteers (Table 1).


View this table:
[in this window]
[in a new window]
 
TABLE 1. Characteristics of healthy controls and scleroderma patients

 
Blood sampling
Blood samples were taken from each subject in the morning. Serum and plasma samples were obtained by centrifugation of whole blood at 3000 g for 10 min and aliquots were stored at –20°C until assayed. Levels of TGFß1 were measured using a high-sensitivity sandwich immunoassay specific for the active ligand (Promega, WI, USA), according to the manufacturer's protocol. Each sample was assayed in triplicate.

In brief, the 96-well microtitre plates were coated with 100 µl per well of anti-TGFß1 monoclonal antibody diluted in carbonate coating buffer (concentration 1 µl/ml), which binds soluble TGFß1. After incubation overnight at 4°C, TGFß1 standard (Promega) or test samples (dilution 1:50) were added to the wells and reacted for 90 min at room temperature. The plates were then incubated with 1 µl/ml of biotinylated anti-TGFß1 polyclonal antibody (Promega) for 2 h at room temperature and subsequently with 10 µl/ml of horseradish peroxidase (HRP) conjugated antibody for 2 h. The colour reaction was induced by the addition of substrate solution (tetramethylbenzidine/hydrogen peroxide) and was stopped with 100 µl 1 N HCl. An automated microplate reader was used to measure the optical density (OD) at a wavelength of 450 nm. Between each of these steps the plates were washed five times with TBST wash buffer. Baseline and acid-treated samples for each subject were analysed. For the measurements of naturally occurring active TGFß1, serum or plasma samples were diluted 1:50 in the sample buffer provided. For total TGFß1 levels, an aliquot of the sample was diluted 1:5 in Dulbecco's phosphate buffered saline and treated with 1 N HCl for 15 min (obtaining a sample pH of approximately 2.6); subsequently samples were neutralized with 1 N NaOH to a pH of approximately 7.6. Acid-treated standard TGFß1 supplied at a concentration of 1 µg/ml was used as a calibration standard at concentrations ranging from 15.6 to 1000 ng/l.

Statistical analysis
Medians and interquartile ranges were used to express summary statistics. The Kruskal–Wallis test was used to compare outcome between several groups, and the Mann–Whitney test for differences between two groups. Active TGFß1 data are skewed and a logarithmic transformation was carried out in order to normalize them. Linear association between variables was explored using the method of least squares. The square of the correlation coefficient, R, is reported and confidence intervals on the slope of the lines presented. Correlations between active TGFß1 and disease duration and skin score were sought using log10 transformed active TGFß1 values. Group differences between the detectable and undetectable concentrations of TGFß1 were tested using {chi}2 test. Differences were considered significant if P ≤ 0.05.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Demographic and clinical features of study cohort
These are summarized in Tables 1 and 2. The healthy control group was younger than both patient groups (P = 0.001), but lcSSc and dcSSc groups were age-matched. Gender differences were ignored in all analyses as there was a 36:11 F:M ratio in the study cohort, reflecting the expected female preponderance for SSc. Five of 27 patients with dcSSc and seven of 20 patients with lcSSc had isolated pulmonary hypertension. Thirteen of 27 dcSSc patients and eight of 20 lcSSc patients had lung fibrosis. There was a non-linear relationship between skin score and time from diagnosis for the dcSSc group; (R2 = 0.4995, P<0.00006) reflecting the fact that in the first few years of the disease skin scores are at their highest.


View this table:
[in this window]
[in a new window]
 
TABLE 2. Scleroderma activity and severity scores [20, 21]

 
Circulating TGFß1 levels
The median, range and interquartile range of the serum TGFß1 concentrations (µg/l) are shown in Table 3. Single patient measurements are shown in Fig. 1a and b. Total levels of TGFß1 were detected in all HC and SSc samples. We did not detect circulating levels of active TGFß1 in 7/22 of HC, 7/20 of lcSSc and 18/27 of dcSSc ({chi}2 = 7.39, P<0.025). The samples with undetectable levels of TGFß1 were analysed for the second time at lower dilution (1:10) and the levels remained undetectable in all samples. Whilst total TGFß1 levels were marginally higher in dcSSc than lcSSc or HC, these differences did not reach significance (P<0.08 HC vs dcSSc). The values for active TGFß1 were skewed and therefore a logarithmic transformation (log10) was carried out. Zero values, evenly spread across the groups (n = 5 in HC, n = 6 in lcSSc and n = 4 in dcSSc) were excluded from the final analysis and from the graphical representation of the data (Fig. 1a). The Kruskal–Wallis test was significant (P<0.01) and dcSSc was significantly different from HC (P<0.01) and lcSSc (P<0.02). For comparison, plasma TGFß1 levels reflected those of contemporary serum samples but were, on average, 30% lower than in serum in all groups, which is in keeping with published reports [16].


View this table:
[in this window]
[in a new window]
 
TABLE 3. Serum TGFß1 concentrations

 


View larger version (16K):
[in this window]
[in a new window]
 
FIG. 1. Circulating levels of total or active TGFß1 in the major systemic sclerosis subsets. (a) Active TGFß1. This is a logarithmic plot. The short horizontal lines are the group median values. The dotted horizontal line represents the limit of detection for the assay (for explanation see Results section). (b) Total TGFß1. The short horizontal lines are the group median values; all values are above the threshold of detection.

 
In dcSSc (but not in lcSSc), there was a negative correlation between skin score and log10 circulating serum active TGFß1 (R2 = 0.194, P<0.04) and also a positive correlation between log10 circulating serum active TGFß1 levels and disease duration (R2 = 0.198, P<0.04) (Fig. 2). There was a positive correlation between total serum TGFß1 and disease duration in lcSSc (R2 = 0.229, P<0.05) but not in dcSSc (Fig. 3). Confidence intervals were calculated for the slopes of those plots in Figs 2 and 3, where a significant relationship was found. For active TGFß1 in dcSSc vs skin score, the slope was –0.038 [confidence interval (CI) –0.072 to –0.004]. The slopes for active TGFß1 in dcSSc vs disease duration was 0.093/yr (CI 0.011 to 0.174) and total TGFß1 in lcSSc vs disease duration was 3.10 µg/ml/yr (CI 0.40–5.80). No correlation was found between total TGFß1 and skin score (Fig. 3). Also, no correlation was found between the circulating levels of either active TGFß1 or total TGFß1 and disease activity or severity scores in both dcSSc and lcSSc. No difference in concentration of either active TGFß1 or total TGFß1 was observed when patients were categorized as those with PHT or those without PHT as well as those with or without lung fibrosis. Finally, there was no correlation between active or total TGFß1 and age in any of the groups (HC, lcSSc and dcSSc).



View larger version (14K):
[in this window]
[in a new window]
 
FIG. 2. Circulating log10 active TGFß1 correlates with both disease duration and skin score in diffuse cutaneous systemic sclerosis. The inverse correlation with skin score (R2 = 0.194, P<0.04) and the positive correlation with disease duration (R2 = 0.198, P<0.04) both reflect the fact the TGFß1 levels are low in the first few years of the disease when skin scores are at their highest.

 


View larger version (16K):
[in this window]
[in a new window]
 
FIG. 3. Total TGFß1 levels do not correlate with disease duration or skin sclerosis score in diffuse cutaneous systemic sclerosis. Circulating total TGFß1 increases with disease duration in limited scleroderma (R2 = 0.229, P<0.05 (top right)), but there are no other relationships of note.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we have used a high-sensitivity enzyme-linked immunosorbent assay (ELISA) to measure circulating levels of TGFß1 in patients with SSc and in healthy controls (HC). We have determined and compared active and total TGFß1 levels; the latter obtained after serum acidification, and compared then with clinical features. We found low or undetectable levels of active TGFß1 in early dcSSc, which correlated inversely with skin score. In contrast, levels of active TGFß1 in lcSSc did not differ from HC; there were no significant differences in total TGFß1 between the groups. The presence of substantial levels of active TGFß1 in HC in this study and in other reports [15–17] suggests that it is not detrimental and raises the possibility that TGFß1 may be important, for example, as a modulator of immune cell function. Low serum levels of active TGFß1 in early dcSSc and the inverse correlation with modified Rodnan skin score have clinical relevance in the context of the evaluation of the extent of skin sclerosis. Skin score constitutes a key clinical parameter for assessing disease severity and predicting prognosis: severe skin involvement has been associated with poor prognosis, decreased survival and a greater risk of developing scleroderma renal crisis, pulmonary fibrosis and myocardial disease [25].

The biological significance of our results should be interpreted in the context of the physiology of TGFß1. Important potential sources of circulating TGFß1 are activated macrophages, T lymphocytes, platelets and endothelial cells. Active TGFß1 is generated from latent TGFß1 complex at sites of injury by a combination of proteolysis and low pH. In human serum TGFß1 is bound covalently by alpha-2 macroglobulin ({alpha}2M) in an inactive form; the interaction of TGFß1 with {alpha}2M accounts for the latency of serum TGFß [26]. It has been suggested that the majority of serum TGFß in the form of a covalent complex with {alpha}2M may represent TGFß targeted for clearance via the {alpha}2M receptor whereas the non-covalent complex might reflect a protected serum pool of potentially active TGFß to be carried and released at target sites [26]. Active TGFß1 is rapidly cleared by mechanisms which include binding to {alpha}2M, decorin and soluble TGFß receptors, followed by its clearance via the liver [2, 27, 28]. In the context of systemic sclerosis, in vitro protein and mRNA expression studies have revealed that SSc fibroblasts do not secrete more TGFß than normal cells [29]; it has been shown that overexpression of either type I or type II TGFß receptors significantly increases alpha2(I) collagen promoter activity in transient transfection assays in dermal fibroblasts [30]. Addition of anti-TGFß antibody abolished the stimulatory effect of receptor overexpression on collagen promoter activity. Comparison of TGFß receptor type I and type II mRNA expression levels in SSc and normal fibroblasts have shown elevated (2-fold) expression of both receptor types in SSc cells, which correlated with increased binding of TGFß and with elevated alpha2(I) collagen mRNA levels [30, 31].

A plausible explanation for our findings is that increased binding sites or altered turnover of active TGFß1 may reduce the level of active cytokine in dcSSc. There are several reports suggesting increased expression of TGFß receptors in SSc [30–32]. In addition, a number of potential TGFß-interacting proteins such as fibrillin or decorin are present at elevated levels in SSc, and extracellular matrix binding sites are likely to be increased [33–35]. Whether this is a primary pathogenetic process or a bystander phenomenon is unclear. However, it is interesting to speculate that in the active phase of early dcSSc and in cases with extensive skin involvement, an increase in cell surface and extracellular matrix TGFß binding sites may sequester active TGFß1 as it becomes released from the latent complex. This may augment the matrix stimulating activity of TGFß1 and explain why relatively low levels are detectable in SSc since bound TGFß1 may not be readily accessible for immunolocalization; low circulating level of TGFß1 may reduce endogenous immunosuppressive activity and contribute to the increased immunological activity in early dcSSc that is a hallmark of this disease subset.

Previous studies of circulating serum or plasma TGFß levels in SSc [12–18] have generated discordant results. For example, Falanga and Julien [12] did not detect any difference in the levels of circulating plasma TGFß between SSc patients and healthy controls; they reported, however, increased TGFß binding to SSc fibroblasts in culture and immunocytochemically detectable TGFß in inflammatory infiltrates from SSc skin biopsy samples, and it has recently been reported that mononuclear cells in dcSSc produce increased amounts of TGFß1 in vitro [36]. Keystone et al. [13] found elevated serum TGFß in patients with dcSSc and lcSSc; correlations with clinical variables have not been analysed in this abstract. Higley et al. [14] found no significant difference in plasma concentrations of active TGFß1 between SSc patients and HC, using a low sensitivity ELISA assay (1 µg/l). In the same study, significantly higher plasma levels were observed in patients with morphoea when compared with HC and SSc; a similar trend of high values was also observed in patients with primary Raynaud's. The authors reported technical difficulties in TGFß activation so total levels of TGFß1 were not estimated. Snowden et al. [15] measured active TGFß levels in plasma from 39 SSc, nine primary Raynaud's phenomenon patients and 60 HC: active TGFß1 was detected in six out of 39 SSc patients only and in none of the other samples. No significant correlation was found between the concentrations of TGFß1 and the pattern of cutaneous or visceral involvement, or the levels of N-terminal peptide of type III procollagen (PIIINP). Giacomelli et al. [16] looked for any correlation between the in vitro spontaneous and phytohemagglutinin (PHA)-induced production of TGFß1 by peripheral blood mononuclear cells (PBMC) of SSc patients and total serum and plasma levels of TGFß1 in the same SSc subject and in HC. No significant difference in total TGFß1 levels in plasma, serum or supernatants from unstimulated and stimulated cultures of PBMC was found. Sato et al. [17] measured serum levels of TGFß1 in 32 SSc patients and 20 controls using a commercial ELISA kit (R&D); it is unclear whether the authors measured active or total levels of TGFß1. TGFß1 was found in all samples, with no difference between SSc and HC; no correlation between serum TGFß1 and modified Rodnan skin score was found in that study. Finally, Choi et al. [18], who found increased serum levels of vascular endothelial growth factor (VEGF) in SSc, reported positive correlation of VEGF with serum TGFß levels. No numerical TGFß concentrations or clinical correlations are described in that paper. In summary, marked variability between the studies is likely to reflect disease heterogeneity, different methodologies employed to study TGFß levels and bioactivity and the complex nature of TGFß secretion and activation. Differences in study designs, samples, techniques, antibodies and lack of consensus in describing clinical details make any attempt at systematic review or meta-analysis of these data complicated. Finally, although a contemporaneous evaluation of the active TGFß1 levels in the affected SSc skin and its serological measurements appears attractive, this approach has some limitations, in particular substantial technical issues concerning the reliable detection of TGFß1 ligand in lesional skin and the discrimination between active and latent forms of this growth factor. In fact a number of experiments have been performed with various results [12, 14, 30].

There are no conclusive data in the literature on any correlation between TGFß1 serum concentrations and age in both healthy subjects and various disease groups. Grainger et al. [37] showed no correlation between active TGFß and age in either healthy controls or subjects with atherosclerosis, whereas Stefoni et al. [38] reported a weak negative correlation between total TGFß1 and age in healthy subjects and in patients on haemodialysis. The latter study did not, however, evaluate active TGFß1. In our study we found no correlation between either active or total TGFß1 levels and age; our healthy controls were slightly younger than scleroderma patients and yet their serum TGFß1 levels were lower than those found in the dcSSc group, which would be in contrast with previous findings [38]. Also, any potential age effect would not negate our findings of lower active TGFß1 in dcSSc compared with lcSSc as these groups were very well matched for age.

Difficulties in accurate measurements of active TGFß are related to its complex regulation and short half-life in biological fluids (2–3 min). Latent TGFß is present in large amounts in platelets, and therefore inaccurate sample handling may lead to the release and activation of latent TGFß into plasma from platelets during separation and storage or, in case of serum, during clotting, leading to falsely high concentrations of active TGFß1. In fact, significantly higher levels of TGFß1 are found in the serum when compared with plasma, which may reflect the release of TGFß1 from activated platelets during clot formation [16, and our data]. Therefore, careful and quick sample preparation is mandatory for reliable serological assays of TGFß. Finally, antibodies not specific for active 25 kDa TGFß1 peptide have been used in older assays, detecting also latent TGFß1; at present, highly sensitive and specific immunoassays for active 25kDa TGFß1 are being developed.

In summary, detailed assessment and comparison with clinical features may further elucidate the role of circulating pro-fibrotic factors as markers or mediators of disease in SSc. The potential significance of low levels of TGFß1 and the relevance of our findings to systemic TGFß-trapping strategies in early stage dcSSc justify further study, including prospective longitudinal assessment of circulating levels of active TGFß1 in SSc.


    Acknowledgments
 
Dr Gisela Lindahl provided valuable help with ELISA measurements. We acknowledge financial support from the Raynaud's and Scleroderma Association, the Scleroderma Society and the Arthritis Research Campaign.

C. Black has received hororaria and research support from Genzyme Inc., Actelion Pharmaceuticals and Cambridge Antibody Technology. C. P. Denton has received hororaria and research support from Genzyme Inc., Actelion Pharmaceuticals and Cambridge Antibody Technology. The other authors have declared no conflicts of interest.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Roberts AB, Sporn MB. Physiological actions and clinical applications of transforming growth factor-ß. Growth Factors 1993;8:1–9.[ISI][Medline]
  2. Blobe GC, Schiemann WP, Lodish HF. Role of transforming growth factor beta in human disease. N Engl J Med 2000;342:1350–8.[Free Full Text]
  3. Black CM, Denton CP. Systemic sclerosis and related disorders in adults and children. In: Maddison PG, Isenberg DA, Woo P, Glass DN, eds. Oxford Textbook of Rheumatology, 2nd Edn, Vol. 2. Oxford: Oxford University Press, 1998:1217–47.
  4. Denton CP, Abraham DJ. Transforming growth factor-beta and connective tissue growth factor: cytokines in scleroderma pathogenesis. Curr Opin Rheumatol 2001;13:506–11.
  5. Varga J, Rosenbloom J, Jimenez SA. Transforming growth factor-ß causes a persistent increase in steady-state amounts of type I and type III collagen and fibronectin mRNAs in normal human dermal fibroblasts. Biochemistry 1987;297:597–604.[CrossRef]
  6. Varga J. Antifibrotic therapy in scleroderma: extracellularly activated fibroblasts? Curr Rheumatol Rep 2004;6:164–70.[Medline]
  7. Smith EA, LeRoy EC. A possible role for transforming growth factor beta in systemic sclerosis. J Invest Dermatol 1990;95:125S–127S.[CrossRef][Medline]
  8. Wahl SM, Hunt DA, Wong HL et al. Transforming growth factor ß is a potent immunosuppressive that inhibits IL-1-dependent lymphocyte proliferation. J Immunol 1988;140:3026–32.[Abstract/Free Full Text]
  9. Annes JP, Munger JS, Rifkin DB. Making sense of latent TGFß activation. J Cell Sci 2003;116:217–24.[Abstract/Free Full Text]
  10. Taipale J, Miyazana K, Heldin CH, Keski-Oja J. Latent transforming growth factor ß associates to fibroblasts extracellular matrix via latent transforming growth factor ß binding protein. J Cell Biol 1994;124:171–81.[Abstract]
  11. LeRoy EC, Smith EA, Kahaleh MB, Trojanowska M, Silver RM. A strategy for determining the pathogenesis of systemic sclerosis. Is transforming growth factor beta the answer? Arthritis Rheum 1989;32:817–25.[ISI][Medline]
  12. Falanga V, Julien J. Observations in the potential role of transforming growth factor-ß in cutaneous fibrosis: systemic sclerosis. In: Piez K, Sporn M, eds. Transforming Growth Factor-betas. Chemistry, Biology and Therapeutics. New York: New York Academy of Science. 1990:161–71.
  13. Keystone E, Lok C, Appleton B, Narenden N, Lee P, Paige C. Elevated serum levels of TGFß in patients with scleroderma. Arthritis Rheum 1992;35S:206 (abstract).
  14. Higley H, Persichitte K, Chu S, Waegell W, Vancheeswaran R, Black C. Immunocytochemical localisation and serologic detection of transforming growth factor beta-1. Arthritis Rheum 1994;37:278–88.[ISI][Medline]
  15. Snowden N, Coupes B, Herrick A, Illingworth K, Jayson MIV, Brenchley PEC. Plasma TGFß in systemic sclerosis: a cross-sectional study. Ann Rheum Dis 1994;53:763–7.[Abstract]
  16. Giacomelli R, Cipriani P, Danese C et al. Peripheral blood mononuclear cells of patients with systemic sclerosis produce increased amounts of interleukin 6, but not transforming growth factor beta-1. J Rheumatol 1996;23:291–6.[ISI][Medline]
  17. Sato S, Hasegawa M, Takehara K. Serum levels of interleukin-6 and interleukin-10 correlate with total skin thickness score in patients with systemic sclerosis. J Dermatol Sci 2001;27:140–6.[CrossRef][ISI][Medline]
  18. Choi J, Min D, Lacho M et al. Elevated vascular endothelial growth factor in systemic sclerosis. J Rheumatol 2003;30:1529–33.[ISI][Medline]
  19. McHugh NJ, Distler O, Giacomelli R, Riemekasten G. Non organ based laboratory markers in systemic sclerosis. Clin Exp Rheumatol 2003; 21(Suppl. 29):S32–S38.
  20. Valentini G, Medsger TA Jr, Silman AJ, Bombardieri S. Conclusion and identification of the core set of variables to be used in clinical investigations. Clin Exp Rheumatol 2003;21(Suppl. 29):S47–S56.
  21. Appendix. Manual of signs, symptoms, methods and procedures for the assessment of the patient with SSc. Clin Exp Rheumatol 2003;21(Suppl. 29):S49–S56.
  22. Subcommittee for Scleroderma Criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis Rheum 1980;23:581–90.[ISI][Medline]
  23. LeRoy EC, Black CM, Fleishmajer R et al. Scleroderma (systemic sclerosis): classification, subsets and pathogenesis. J Rheumatol 1988;15:202–5.[ISI][Medline]
  24. Clements P, Lachenbrush P, Seibold JR et al. Skin thickness score in systemic sclerosis: an assessment of interobserver variability in 3 independent studies. J Rheumatol 1993;20:1892–6.[ISI][Medline]
  25. Clements PJ, Hurwitz EL, Wong WK et al. Skin thickness score as a predictor and correlate of outcome in systemic sclerosis: high-dose versus low-dose penicillamine trial. Arthritis Rheum 2000;43:2445–54.[CrossRef][ISI][Medline]
  26. O’Connor-McCourt MD, Wakefield LM. Latent transforming growth factor ß in serum. J Biol Chem 1987;262:14090–9.[Abstract/Free Full Text]
  27. Pentinen RP, Kobayashi S, Bornstein P. Transforming growth factor ß increases mRNA for matrix proteins both in the presence and in the absence of changes in mRNA stability. Proc Natl Acad Sci USA 1988;85:1105–8.[Abstract/Free Full Text]
  28. Heldin CH, Miyazono K, Dijke P. TGFß signalling from cell membrane to nucleus through SMAD proteins. Nature 1997;390:465–71.[CrossRef][ISI][Medline]
  29. Clark RAF, Nielsen LD, McPherson JM. Collagen matrices express unstimulated and TGFß—stimulated fibroblast synthesis of collagen. J Cell Biol 1987;105:212A.
  30. Kawakami T, Ihn H, Xu W, Smith E, LeRoy C, Trojanowska M. Increased expression of TGFß receptors by scleroderma fibroblasts: evidence for contribution of autocrine TGFß signalling to scleroderma phenotype. J Invest Dermatol 1998;110:47–51.[CrossRef][ISI][Medline]
  31. Ihn H, Yamane K, Kubo M, Takaki K. Blockade of endogenous transforming growth factor beta signalling prevents up-regulated collagen synthesis in scleroderma fibroblasts: association with increased expression of transforming growth factor beta receptors. Arthritis Rheum 2002;44:474–80.[ISI]
  32. Miyazono K. TGFß receptors and signal transduction. Int J Hematol 1997;65:97–104.[CrossRef][ISI][Medline]
  33. Kissin EY, Lemaire R, Korn JH, Lafyatis R. Transforming growth factor ß induces fibroblast fibrillin-1 matrix. Arthritis Rheum 2002;46:3000–9.[CrossRef][ISI][Medline]
  34. Isogai Z, Ono RN, Ushiro S et al. Latent transforming growth factor beta-binding protein 1 interacts with fibrillin and is a microfibril-associated protein. J Biol Chem 2003;278:2750–7.[Abstract/Free Full Text]
  35. Vuorio T, Kahari VM, Black C, Vuorio E. Expression of osteonectin, decorin, and transforming growth factor-ß in fibroblasts cultured from patients with systemic sclerosis and morphoea. J Rheumatol 1991;18:247–51.[ISI][Medline]
  36. Hasegawa M, Sato S, Takehara K. Augmented production of transforming growth factor-beta by cultured peripheral blood mononuclear cells from patients with systemic sclerosis. Arch Dermatol Res 2004;296:89–93.[ISI][Medline]
  37. Grainger DJ, Kemp PR, Metcalfe JC et al. The serum concentration of active transforming growth factor ß is severely depressed in advanced atherosclerosis. Nat Med 1995;1:74–9.[CrossRef][ISI][Medline]
  38. Stefoni S, Cianciolo G, Donati G et al. Low TGFß1 serum levels are a risk factor for atherosclerotic disease in ESRD patients. Kidney Int 2002;61:324–35.[CrossRef][ISI][Medline]
Submitted 15 March 2005; revised version accepted 1 August 2005.



This Article
Abstract
Full Text (PDF)
All Versions of this Article:
44/12/1518    most recent
kei088v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Dziadzio, M.
Articles by Denton, C. P.
PubMed
PubMed Citation
Articles by Dziadzio, M.
Articles by Denton, C. P.
Related Collections
Systemic Sclerosis