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CIRCULATING TRANSFORMING GROWTH FACTOR β1AS AN INDICATOR OF HEPATIC FUNCTION IMPAIRMENT IN LIVER CIRRHOSIS
doi:10.1006/cyto.1999.0660, available online at http://www.idealibrary.com on
CIRCULATING TRANSFORMING GROWTH FACTOR 1 AS AN INDICATOR OF HEPATIC FUNCTION IMPAIRMENT IN LIVER CIRRHOSIS Robert Flisiak, Barbara Pytel-Krolczuk, Danuta Prokopowicz In the liver, transforming growth factor (TGF) -1 is primarily responsible for activation of fat-storing cells, which are the main source of extracellular matrix proteins. Their deposition play a key role in the development of liver cirrhosis. The aim of this study was to evaluate plasma TGF-1 in patients with different stages of liver cirrhosis and its possible use as an indicator of liver function impairment. TGF-1 was measured in the plasma of 40 patients with liver cirrhosis. To estimate possible effect of liver insufficiency on plasma TGF-1, patients were divided into three groups: A, B and C, univocal with Child-Pugh classes. Normal values were collected from 13 healthy volunteers. Liver cirrhosis resulted in a significant increase of plasma concentration of TGF-1 (39.33.8 ng/ml), which doubled normal values (18.31.6 ng/ml). The highest concentrations were observed in alcoholic patients (44.44.7 ng/ml). TGF-1 level increased depending on the degree of liver insufficiency, demonstrated by a significant positive correlation with Child-Pugh score (r=0.591). Values in group A were similar to normal, but were significantly elevated in groups B and C. These findings suggest posible use of plasma TGF-1 measurement as an indicator of liver function impairment and possible marker of hepatic fibrosis progression in cirrhotic patients. 2000 Academic Press
Transforming growth factor 1 (TGF-1) is a member of a cytokine family with multifunctional biological activity. However, there are three fundamental activities: modulation (mostly supression) of cell proliferation, immunosupression and enhancement the deposition of extracellular matrix. The most widely studied functions of TGF-1 have been fibrosis and wound healing.1 In the liver, TGF-1 has pathophysiological significance primarily with respect to hepatic fibrogenesis, regulation of liver cell growth, tumour development and induction of hepatocellular apoptosis.2 Hepatocyte injury is responsible for the release of numerous cytokines. TGF-1 plays a principal role among them related mainly to activation of fat-storing (Ito) cells. Their transformation into myofibroblasts, which are the main source of extracellular matrix proteins, is an Department of Infectious Diseases, Medical Academy of Białystok, Białystok, Poland Correspondence to: Dr Robert Flisiak, Department of Infectious Diseases, Medical Academy of Białystok, 15-540 Białystok, Zurawia str. 14, Poland; E-mail: [email protected] Received 30 July 1999; received in revised form 22 November 1999; accepted for publication 28 December 1999 2000 Academic Press 1043–4666/00/060677+05 $35.00/0 KEY WORDS: cytokines/liver cirrhosis/transforming rowth factor/ TGF CYTOKINE, Vol. 12, No. 6 (June), 2000: pp 677–681
essential step in the initiation of liver fibrosis.3–5 As was demonstrated recently oxidative stress accompanying hepatocytes damage is associated with the upregulation of TGF-.6 TGF-1 enhances the deposition of extracellular matrix proteins through promotion of synthesis and inhibition of degradation.1,7 Longlasting stimulation of fat-storing cells, resulting from continuous hepatocytes injury and superabundance of profibrogenic factors such as TGF-1, can lead to the development of liver cirrhosis. The aim of this study was to evaluate plasma TGF-1 in patients with different stages of liver cirrhosis (LC) and its possible use as an indicator of liver function impairment degree.
RESULTS Liver cirrhosis resulted in significant increase of plasma concentration of TGF-1 (39.33.8 ng/ml), which doubled normal values (18.31.6 ng/ml). The highest values were observed in ALC patients (44.44.7 ng/ml). However, concentration in the HLC group (33.56.0 ng/ml) was also significantly elevated (Fig. 1). There was no statistical significance between both groups. 677
678 / Flisiak et al.
CYTOKINE, Vol. 12, No. 6 (June, 2000: 677–681)
**
50 *
*
TGF-β1 (ng/ml)
40 30 20 10 0
Control group n = 13
LC n = 40
ALC n = 21
HLC n = 19
Figure 1. Mean concentration of plasma TGF-1 in cirrhotic patients (LC), as well as in respect to etiology of the disease (ALC and HLC). Asterisks indicate statistical significance in comparison with normal values obtained in control group (*P<0.05; **P<0.001).
TABLE 1. Correlation expressed through r-value and its significance between biochemical indices of liver sufficiency and TGF-1 concentration in all cirrhotic patients as well as in both groups
Bilirubin [mg%] Prothrombin index [%] Albumin [mg%] ALT [U/l] AST [U/l] ALP [U/l] GGT [U/l] Score (Child-Pugh)
LC n=40
ALC n=21
HLC n=19
0.42* 0.29 0.27 0.01 0.12 0.09 0.23 0.59**
0.52* 0.46* 0.67* 0.01 0.10 0.40 0.44 0.70**
0.38 0.32 0.07 0.06 0.21 0.09 0.15 0.59*
*P<0.05; **P<0.001.
Comparison performed between TGF-1 and different biochemical indices of liver injury revealed significant correlation with serum bilirubin level. However this was true only with respect to alcoholic patients, who additionally demonstrated significant negative correlation with values of prothrombin index and albumin concentration (Table 1). TGF-1 level analysed in all cirrhotic patients increased depending on the degree of liver insufficiency demonstrated through a significant positive correlation with Child-Pugh score (r=0.591; P<0.001), (Fig. 2). It was most apparent in ALC patients, however significance was also demonstrated in the HLC group (Table 1). As shown in Figure 3 results obtained in group A were similar to normal, whereas in group B and C they were significantly higher. This effect was not agedependent, because there were no statistical differences between age in particular groups (A: 48.52.5, B: 53.83.3, C: 54.13.0). Analysis of individual results revealed that values exceeding upper normal range are predominantly associated with advanced liver insufficiency (Figure 3). This observation was confirmed by
Figure 2. Correlation between plasma TGF-1 and score values of Child-Pugh classification in all cirrhotic patients.
calculation of a positive predictive value of results exceeding upper normal range that reached 96%.
DISCUSSION The possible reason for elevated plasma TGF-1 in cirrhotic patients is increased fibrotic activity in the liver and enhanced intercellular signalling related to inflammation. Some other bioactive substances involved in this process, such as prostanoids, have previously been evaluated as a possible prognostic marker of liver cirrhosis developmen.7 According to De Bleser et al.8 TGF-1 is the most abundant isoform in both normal and fibrotic liver. In normal liver TGF-1, TGF-2 and TGF-3 are expressed strongly by Kupffer cells, fat-storing cells and endothelial cells. In fibrotic liver, expression of all TGF- isoforms increases, and they are also expressed in bile duct epithelial cells.9 Conversely hepatocytes are also recognized as the important cellular source of latent TGF-1 in liver.10 As demonstrated by Knittel et al.11 TGF-1 is the most important fibrogenic mediator acting both on primary culture as well as on activated fat-storing cells, through increase of collagen types I and III, fibronectin, undulin and proteoglycangene expression. TGF-1 also dose-dependently increased synthesis and secretion of tenascin, laminin, entactin, collagen type IV and 2-macroglobulin.11 The initial step in the upregulation of TGF- can be related to oxidative stress associated with hepatocyte injury.6 According to Tiggelman et al.12, the stimulatory effect of TGF- on collagen synthesis by fat-storing cells is observed in vitro at concentration of 10 ng/ml. In our study the level of circulating TGF-1 was four-fold higher, and in the liver tissue it is expected to
TGF-1 and hepatic function in liver cirrhosis / 679
140 130 120 110
TGF-β1 (ng/ml)
100 90 80 70 60 50 40 30 20 10 0 A 20.7 ± 2.7 ng/ml n=8
B 33.2 ± 3.7 ng/ml n = 20
C 61.8 ± 7.6 ng/ml n = 12
Figure 3. Individual and mean values of plasma TGF-1 concentration in patients classified with respect to degree of liver insufficiency as Child-Pugh A, B or C class.
be much higher still. The highest level observed in ALC group can be explained, as demonstrated recently, by high expression of all TGF- isoforms in alcoholic liver disease.15 Moreover, there is a link between increased ethanol-inducible cytochrome P450 2E1 expression and enhanced TGF-1 expression.14 In patients infected with HBV, expression of some viral antigens can induce expression of TGF-115 The possibility that TGF-1 plays a role in the pathogenesis of viral B hepatitis was raised following research carried out in patients with acute liver failure, who had plasma TGF-1 on a similar level to our cirrhotic patients.16 Additionally, TGF-1 determined in healthy people from this study was similar to observed in our control group. A similar rate of plasma TGF-1 in healthy and cirrhotic patients was also demonstrated by Shirai et al.17 However, the highest values were observed in patients with giant hepatic hemangioma (113 ng/ml) and autoimmune hepatitis.19,20 Only a few papers have been dedicated to the prognostic role of TGF-1 in liver diseases. Tsai et al.20 demonstrated a correlation between urinary TGF-1 and circulating amino terminal pro-peptides of type III pro-collagen levels as a fibrosis marker in patients with chronic liver diseases. In another clinical study performed in patients with chronic hepatitis C there was no correlation between TGF-1 and clinical variables, liver biochemistry (ALT) or virological parameters (HCV-RNA level, genotype), but there was a correlation between total TGF-1 and histological fibrosis
score.21 This study is consistent with our findings, because we also found no correlation between the plasma TGF-1 level and majority of biochemical indices of hepatocytes injury or cholestasis. Exceptions were observed in alcoholic patients, who had significant impairment of some synthetic liver functions. The relationship between these biochemical indices of liver function and plasma TGF-1 level is probably indirect, and can be related to secondary hepatocellular metabolic alterations resulting from enhanced fibrosis. The difference related to the etiology of the disease could be a result of endotoxemia, which is often associated with alcoholic liver disease and has been recently recognized as a strong induce of TGF expression.22 However comparison of TGF-1 concentration with scored degree of liver insufficiency, including clinical and biochemical indices, revealed significant correlation irrespective of the disease etiology. These findings suggest the possible use of plasma TGF-1 measurement as an indicator of liver function impairment. However long-term follow-up of patients involved in this study is necessary for the final evaluation of TGF-1 as a possible marker of hepatic fibrosis progression in cirrhotic patients.
MATERIALS AND METHODS Patients TGF-1 was measured in the plasma of 40 patients with liver cirrhosis (14 females and 26 males; mean age:
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CYTOKINE, Vol. 12, No. 6 (June, 2000: 677–681)
TABLE 2. Child-Pugh scoring criterions for evaluation of liver insufficiency, adapted from Pugh et al.23 Score
Bilirubin [mg%] Albumin [g%] Prothrombin index [%] Encephalopathy Ascites
1
2
3
<2.0 >3.5 >70 no no
2.0–3.0 2.8–3.5 40–70 1 or 2 Easy to treat
>3.0 <2.8 <40 3 or 4 Difficult to treat
53.12.0). Alcohol-related liver cirrhosis (ALC group) was diagnosed in 21 patients, whereas the role of hepatitis B virus (HBV) as an ethiologic factor was established in the remaining 19 patients (HLC group). To estimate a possible association between degree of liver insufficiency and TGF-1 plasma concentration, patients were also divided into three groups: A, B and C, univocal with Child-Pugh classes. Normal values of plasma TGF-1 concentrations were collected from 13 healthy volunteers (control group), with mean age (47.51.7), similar to LC patients.
Diagnosis Diagnosis of liver cirrhosis required clinical and biochemical signs of chronic hepatocellular dysfunction, accompanied by the presence of fibrosis and nodular regeneration at liver biopsy, performed with Hepafix system (Braun ). Alcoholic liver cirrhosis (ALC group) was recognized in patients negative for HBsAg and anti-HCV, who demonstrated regular alcohol consumption in a daily dose over 80 g for at least 2 years. In contrast, liver cirrhosis related to HBV infection (HLC-group) was diagnosed in patients who abstained from alcohol, but revealed a presence of serological indices of HBV infection (HBsAg, HBeAg, anti-HBc) found with EIA (Abbott, USA).
Degree of liver insufficiency This was established according to Child-Pugh classification.23 Ascites, encephalopathy, prothrombin index, as well as concentrations of bilirubin and albumin were evaluated for this purpose (Table 2). Patients were scored as follows: 5–6 as class (group) A, 7–9 as class (group) B, and 10–15 as class (group) C.24
TGF-1 measurement Venous blood was collected on ice using vacutainer tubes with EDTA as an anti-coagulant and centrifuged at 1000g within 30 min of collection. Obtained plasma was additionally centrifuged at 10 000 g for 10 min at 2–8C for complete platelet removal. To activate latent TGF-1 to immunoreactive TGF-1, 0.1 ml of the mixture, consisting of 2.5 N acetic acid and 10 M urea, was added to 0.1 ml of platelet-poor plasma and incubated for 10 min at room temperature. Acidified sample was neutralized by adding 0.1 ml of mixture consisted of 2.7 N NaOH and 1 M HEPES. Activated plasma samples were diluted four-fold and assayed in duplicate with the quantitative sandwich enzyme immunoassay (EIA) technique using recombinant human TGF-
soluble receptor type II (TRII) as a solid phase precoated onto a microplate (R&D Systems Inc., Minneapolis, MN, USA). Standards and samples were pipetted into the wells and any TGF-1 present was bound by the immobilized receptor. After washing away any unbound substances, polyclonal antibodies specific for TGF-1, conjugated to horseradish peroxidase, were added to the wells. Following a wash to remove any unbound antibody–enzyme reagent, a substrate solution consisting of stabilized hydrogen peroxide and tetramethylbenzidine was added to the wells. Colour developed in proportion to the amount of TGF-1 bound in the initial step. The reaction was stopped by addition of 2 N sulphuric acid and optical density was read with a microtitre plate photometer Stat Fax 2100 (Alab, Poland) at 450 nm and corrected by substraction of readings at 540 nm. The concentration of TGF-1 in a sample was determined by interpolation from a standard curves, prepared with standard samples supplied by manufacturer.
Statistical methods All data were collected and analysed with Microsoft Excel 97 software (Microsoft Corporation). Values were expressed as meanstandard error of the mean (SEM). The significance of the difference was calculated by two- tailed Student’s t-test. For correlation analysis the Pearson product moment correlation was used and linear regression was performed. Values of P<0.05 were considered to be significant.
REFERENCES 1. Gressner AM (1995) Cytokines and cellular crosstalk involved in the activation of fat-storing cells. J Hepatol 22 (Suppl. 2):28–36. 2. Gressner AM, Lahme B, Mannherz HG, Polzar B (1997) TGF--mediated hepatocellular apoptosis by rat and human hepatoma cells and primary rat hepatocytes. J Hepatol 26:1079– 1092. 3. Flisiak R, Li J, Baraona E, Lieber CS (1993) Effects of ethanol on prostanoids production by liver fat-storing cells. Hepatology 18:153–159. 4. Friedman SL (1993) The cellular basis of hepatic fibrosis; mechanism and treatment strategies. N Engl J Med 328: 1828–1835. 5. Ramadori G (1991) The stellate cell (Ito-cell, fat-storing cell, lipocyte, perisinusoidal cell) of the liver. New insights into pathophysiology of an intriguing cell. Virchows Archiv B Cell Pathol 61:147–158. 6. Garcia-Trevijano ER, Iraburu MJ, Fontana L, Dominguez-Rosales JA, Auster A, Covarrubias-Pinedo A, Rojkind M (1999) Transforming growth factor beta1 induces the expression of 1(I) procollagen mRNA by a hydrogen peroxide-C/EBPdependent mechanism in rat hepatic stellate cells. Hepatology 29:960–970. 7. Pinzani M, Marra F, Carloni V, Gentilini A, De Franco R, Caligiuri A, Sali C (1994) Inflammatory mediators and fat-storing cell proliferation. In Surrenti C, Casini A, Milani S, Pinzani M (eds) Fat-storing Cells and Liver Fibrosis, Kluwer Academic Publishers, Dordrecht, pp 222–235. 8. De Bleser PJ, Niki T, Rogiers V, Geerts A (1997) Transforming growth factor-beta gene expression in normal and fibrotic rat liver. J Hepatol 26:886–893. 9. Baer HU, Friess H, Abou-Shady M, Berberat P, Zimmermann A, Gold LI, Korc M, Bu¨ chler MW (1998) Transforming growth factor betas and their receptors in human liver cirrhosis. Eur J Gastroenterol Hepatol 10:1031–1039.
TGF-1 and hepatic function in liver cirrhosis / 681 10. Gressner AM, Wulbrand U (1997) Variation of immunocytochemical expression of transforming growth factor (TGF)- in hepatocytes in culture and liver slices. Cell Tissue Res 287:143–152. 11. Knittel T, Janneck T, Muller L, Fellmer P, Ramadori G (1996) Transforming growth factor 1-regulated gene expression of Ito cells. Hepatology 24:352–360. 12. Tiggelman AM, Boers W, Linthorst C, Sala M, Chamuleau RA (1995) Collagen synthesis by human liver (myo)fibroblasts in culture: evidence for a regulatory role of IL-1 , IL-4, TGF and IFN . J Hepatol 23:307–317. 13. Santos RM, Norton P, Degli-Esposti S, Zern MA (1998) TGF-beta isoforms in alcoholic liver disease. J Gastroenterol 33:383–389. 14. Fang C, Lindros KO, Badger TM, Ronis MJ, IngelmanSundberg M (1998) Zonated expression of cytokines in rat liver: effect of chronic ethanol and the cytochrome P450 2E1 inhibitor, chlormethiazole. Hepatology 27:1304–1310. 15. Yoo YD, Ueda H, Park K, Flanders KC, Lee YI, Jay G, Kim SJ (1996) Regulation of transforming growth factor-beta 1 expression by the hepatitis B virus (HBV) X transactivator. Role in HBV pathogenesis. J Clin Invest 97:388–395. 16. Miwa Y, Ellis AJ, Hughes RD, Langley PG, Wendon JA, Williams R (1996) Effect of ELAD liver support on plasma HGF and TGF-beta 1 in acute liver failure. Int J Artif Organs 19:240–244. 17. Shirai Y, Kawata S, Tamura S, Ito N, Tsushima H, Takaishi K, Kiso S, Matsuzawa Y (1994) Plasma transforming growth factor- 1 in patients with hepatocellular carcinoma. Comparison with chronic liver diseases. Cancer 73:2275–2279.
18. Ito N, Kawata S, Tsushima H, Tamura S, Kiso S, Takami S, Igura T, Monnden M, Matsuzawa Y (1997) Increased circulating transforming growth factor 1 in a patient with giant hepatic hemangioma: possible contribution to an impaired immune function. Hepatology 25:93–96. 19. Bayer EM, Herr W, Kanzler S, Waldmann C, Meyer Zum Bu¨ schenfelde KH, Dienes HP, Lohse AW (1998) Transforming growth factor-beta1 in autoimmune hepatitis: correlation of liver tissue expression and serum levels with disease activity. J Hepatol 28:803–811. 20. Tsai JF, Jeng JE, Chuang LY, Chang WY, Tsai JH (1997) Urinary transforming growth factor beta1 levels in hepatitis C virus-related chronic liver disease: correlation between high levels and severity of disease. Hepatology 25:1141–1146. 21. Nelson DR, Gonzalez-Peralta RP, Qian K, Xu Y, Marousis CG, Davis GL, Lau JY (1997) Transforming growth factor- 1 in chronic hepatitis C. J Viral Hepat 4:29–35. 22. Ja¨ rvela¨ inen HA, Fang C, Ingelman-Sundberg M, Lindros KO (1999) Effect of chronic coadministration of endotoxin and ethanol on rat liver pathology and proinflammatory and antiinflammatory cytokines. Hepatology 29:1503–1510. 23. Pugh RNH, Murray-Lyon IM, Dawson JL, Pietroni MC, Williams R (1973) Transection of the oesophagus for bleeding oesophageal varices. Brit J Surg 60:646–652. 24. Flisiak R, Prokopowicz D (1997) Plasma iPGE2 and i6-keto PGF1 in the course of liver cirrhosis. Prostaglandins 53:11–20.
CIRCULATING TRANSFORMING GROWTH FACTOR 1 AS AN INDICATOR OF HEPATIC FUNCTION IMPAIRMENT IN LIVER CIRRHOSIS Robert Flisiak, Barbara Pytel-Krolczuk, Danuta Prokopowicz In the liver, transforming growth factor (TGF) -1 is primarily responsible for activation of fat-storing cells, which are the main source of extracellular matrix proteins. Their deposition play a key role in the development of liver cirrhosis. The aim of this study was to evaluate plasma TGF-1 in patients with different stages of liver cirrhosis and its possible use as an indicator of liver function impairment. TGF-1 was measured in the plasma of 40 patients with liver cirrhosis. To estimate possible effect of liver insufficiency on plasma TGF-1, patients were divided into three groups: A, B and C, univocal with Child-Pugh classes. Normal values were collected from 13 healthy volunteers. Liver cirrhosis resulted in a significant increase of plasma concentration of TGF-1 (39.33.8 ng/ml), which doubled normal values (18.31.6 ng/ml). The highest concentrations were observed in alcoholic patients (44.44.7 ng/ml). TGF-1 level increased depending on the degree of liver insufficiency, demonstrated by a significant positive correlation with Child-Pugh score (r=0.591). Values in group A were similar to normal, but were significantly elevated in groups B and C. These findings suggest posible use of plasma TGF-1 measurement as an indicator of liver function impairment and possible marker of hepatic fibrosis progression in cirrhotic patients. 2000 Academic Press
Transforming growth factor 1 (TGF-1) is a member of a cytokine family with multifunctional biological activity. However, there are three fundamental activities: modulation (mostly supression) of cell proliferation, immunosupression and enhancement the deposition of extracellular matrix. The most widely studied functions of TGF-1 have been fibrosis and wound healing.1 In the liver, TGF-1 has pathophysiological significance primarily with respect to hepatic fibrogenesis, regulation of liver cell growth, tumour development and induction of hepatocellular apoptosis.2 Hepatocyte injury is responsible for the release of numerous cytokines. TGF-1 plays a principal role among them related mainly to activation of fat-storing (Ito) cells. Their transformation into myofibroblasts, which are the main source of extracellular matrix proteins, is an Department of Infectious Diseases, Medical Academy of Białystok, Białystok, Poland Correspondence to: Dr Robert Flisiak, Department of Infectious Diseases, Medical Academy of Białystok, 15-540 Białystok, Zurawia str. 14, Poland; E-mail: [email protected] Received 30 July 1999; received in revised form 22 November 1999; accepted for publication 28 December 1999 2000 Academic Press 1043–4666/00/060677+05 $35.00/0 KEY WORDS: cytokines/liver cirrhosis/transforming rowth factor/ TGF CYTOKINE, Vol. 12, No. 6 (June), 2000: pp 677–681
essential step in the initiation of liver fibrosis.3–5 As was demonstrated recently oxidative stress accompanying hepatocytes damage is associated with the upregulation of TGF-.6 TGF-1 enhances the deposition of extracellular matrix proteins through promotion of synthesis and inhibition of degradation.1,7 Longlasting stimulation of fat-storing cells, resulting from continuous hepatocytes injury and superabundance of profibrogenic factors such as TGF-1, can lead to the development of liver cirrhosis. The aim of this study was to evaluate plasma TGF-1 in patients with different stages of liver cirrhosis (LC) and its possible use as an indicator of liver function impairment degree.
RESULTS Liver cirrhosis resulted in significant increase of plasma concentration of TGF-1 (39.33.8 ng/ml), which doubled normal values (18.31.6 ng/ml). The highest values were observed in ALC patients (44.44.7 ng/ml). However, concentration in the HLC group (33.56.0 ng/ml) was also significantly elevated (Fig. 1). There was no statistical significance between both groups. 677
678 / Flisiak et al.
CYTOKINE, Vol. 12, No. 6 (June, 2000: 677–681)
**
50 *
*
TGF-β1 (ng/ml)
40 30 20 10 0
Control group n = 13
LC n = 40
ALC n = 21
HLC n = 19
Figure 1. Mean concentration of plasma TGF-1 in cirrhotic patients (LC), as well as in respect to etiology of the disease (ALC and HLC). Asterisks indicate statistical significance in comparison with normal values obtained in control group (*P<0.05; **P<0.001).
TABLE 1. Correlation expressed through r-value and its significance between biochemical indices of liver sufficiency and TGF-1 concentration in all cirrhotic patients as well as in both groups
Bilirubin [mg%] Prothrombin index [%] Albumin [mg%] ALT [U/l] AST [U/l] ALP [U/l] GGT [U/l] Score (Child-Pugh)
LC n=40
ALC n=21
HLC n=19
0.42* 0.29 0.27 0.01 0.12 0.09 0.23 0.59**
0.52* 0.46* 0.67* 0.01 0.10 0.40 0.44 0.70**
0.38 0.32 0.07 0.06 0.21 0.09 0.15 0.59*
*P<0.05; **P<0.001.
Comparison performed between TGF-1 and different biochemical indices of liver injury revealed significant correlation with serum bilirubin level. However this was true only with respect to alcoholic patients, who additionally demonstrated significant negative correlation with values of prothrombin index and albumin concentration (Table 1). TGF-1 level analysed in all cirrhotic patients increased depending on the degree of liver insufficiency demonstrated through a significant positive correlation with Child-Pugh score (r=0.591; P<0.001), (Fig. 2). It was most apparent in ALC patients, however significance was also demonstrated in the HLC group (Table 1). As shown in Figure 3 results obtained in group A were similar to normal, whereas in group B and C they were significantly higher. This effect was not agedependent, because there were no statistical differences between age in particular groups (A: 48.52.5, B: 53.83.3, C: 54.13.0). Analysis of individual results revealed that values exceeding upper normal range are predominantly associated with advanced liver insufficiency (Figure 3). This observation was confirmed by
Figure 2. Correlation between plasma TGF-1 and score values of Child-Pugh classification in all cirrhotic patients.
calculation of a positive predictive value of results exceeding upper normal range that reached 96%.
DISCUSSION The possible reason for elevated plasma TGF-1 in cirrhotic patients is increased fibrotic activity in the liver and enhanced intercellular signalling related to inflammation. Some other bioactive substances involved in this process, such as prostanoids, have previously been evaluated as a possible prognostic marker of liver cirrhosis developmen.7 According to De Bleser et al.8 TGF-1 is the most abundant isoform in both normal and fibrotic liver. In normal liver TGF-1, TGF-2 and TGF-3 are expressed strongly by Kupffer cells, fat-storing cells and endothelial cells. In fibrotic liver, expression of all TGF- isoforms increases, and they are also expressed in bile duct epithelial cells.9 Conversely hepatocytes are also recognized as the important cellular source of latent TGF-1 in liver.10 As demonstrated by Knittel et al.11 TGF-1 is the most important fibrogenic mediator acting both on primary culture as well as on activated fat-storing cells, through increase of collagen types I and III, fibronectin, undulin and proteoglycangene expression. TGF-1 also dose-dependently increased synthesis and secretion of tenascin, laminin, entactin, collagen type IV and 2-macroglobulin.11 The initial step in the upregulation of TGF- can be related to oxidative stress associated with hepatocyte injury.6 According to Tiggelman et al.12, the stimulatory effect of TGF- on collagen synthesis by fat-storing cells is observed in vitro at concentration of 10 ng/ml. In our study the level of circulating TGF-1 was four-fold higher, and in the liver tissue it is expected to
TGF-1 and hepatic function in liver cirrhosis / 679
140 130 120 110
TGF-β1 (ng/ml)
100 90 80 70 60 50 40 30 20 10 0 A 20.7 ± 2.7 ng/ml n=8
B 33.2 ± 3.7 ng/ml n = 20
C 61.8 ± 7.6 ng/ml n = 12
Figure 3. Individual and mean values of plasma TGF-1 concentration in patients classified with respect to degree of liver insufficiency as Child-Pugh A, B or C class.
be much higher still. The highest level observed in ALC group can be explained, as demonstrated recently, by high expression of all TGF- isoforms in alcoholic liver disease.15 Moreover, there is a link between increased ethanol-inducible cytochrome P450 2E1 expression and enhanced TGF-1 expression.14 In patients infected with HBV, expression of some viral antigens can induce expression of TGF-115 The possibility that TGF-1 plays a role in the pathogenesis of viral B hepatitis was raised following research carried out in patients with acute liver failure, who had plasma TGF-1 on a similar level to our cirrhotic patients.16 Additionally, TGF-1 determined in healthy people from this study was similar to observed in our control group. A similar rate of plasma TGF-1 in healthy and cirrhotic patients was also demonstrated by Shirai et al.17 However, the highest values were observed in patients with giant hepatic hemangioma (113 ng/ml) and autoimmune hepatitis.19,20 Only a few papers have been dedicated to the prognostic role of TGF-1 in liver diseases. Tsai et al.20 demonstrated a correlation between urinary TGF-1 and circulating amino terminal pro-peptides of type III pro-collagen levels as a fibrosis marker in patients with chronic liver diseases. In another clinical study performed in patients with chronic hepatitis C there was no correlation between TGF-1 and clinical variables, liver biochemistry (ALT) or virological parameters (HCV-RNA level, genotype), but there was a correlation between total TGF-1 and histological fibrosis
score.21 This study is consistent with our findings, because we also found no correlation between the plasma TGF-1 level and majority of biochemical indices of hepatocytes injury or cholestasis. Exceptions were observed in alcoholic patients, who had significant impairment of some synthetic liver functions. The relationship between these biochemical indices of liver function and plasma TGF-1 level is probably indirect, and can be related to secondary hepatocellular metabolic alterations resulting from enhanced fibrosis. The difference related to the etiology of the disease could be a result of endotoxemia, which is often associated with alcoholic liver disease and has been recently recognized as a strong induce of TGF expression.22 However comparison of TGF-1 concentration with scored degree of liver insufficiency, including clinical and biochemical indices, revealed significant correlation irrespective of the disease etiology. These findings suggest the possible use of plasma TGF-1 measurement as an indicator of liver function impairment. However long-term follow-up of patients involved in this study is necessary for the final evaluation of TGF-1 as a possible marker of hepatic fibrosis progression in cirrhotic patients.
MATERIALS AND METHODS Patients TGF-1 was measured in the plasma of 40 patients with liver cirrhosis (14 females and 26 males; mean age:
680 / Flisiak et al.
CYTOKINE, Vol. 12, No. 6 (June, 2000: 677–681)
TABLE 2. Child-Pugh scoring criterions for evaluation of liver insufficiency, adapted from Pugh et al.23 Score
Bilirubin [mg%] Albumin [g%] Prothrombin index [%] Encephalopathy Ascites
1
2
3
<2.0 >3.5 >70 no no
2.0–3.0 2.8–3.5 40–70 1 or 2 Easy to treat
>3.0 <2.8 <40 3 or 4 Difficult to treat
53.12.0). Alcohol-related liver cirrhosis (ALC group) was diagnosed in 21 patients, whereas the role of hepatitis B virus (HBV) as an ethiologic factor was established in the remaining 19 patients (HLC group). To estimate a possible association between degree of liver insufficiency and TGF-1 plasma concentration, patients were also divided into three groups: A, B and C, univocal with Child-Pugh classes. Normal values of plasma TGF-1 concentrations were collected from 13 healthy volunteers (control group), with mean age (47.51.7), similar to LC patients.
Diagnosis Diagnosis of liver cirrhosis required clinical and biochemical signs of chronic hepatocellular dysfunction, accompanied by the presence of fibrosis and nodular regeneration at liver biopsy, performed with Hepafix system (Braun ). Alcoholic liver cirrhosis (ALC group) was recognized in patients negative for HBsAg and anti-HCV, who demonstrated regular alcohol consumption in a daily dose over 80 g for at least 2 years. In contrast, liver cirrhosis related to HBV infection (HLC-group) was diagnosed in patients who abstained from alcohol, but revealed a presence of serological indices of HBV infection (HBsAg, HBeAg, anti-HBc) found with EIA (Abbott, USA).
Degree of liver insufficiency This was established according to Child-Pugh classification.23 Ascites, encephalopathy, prothrombin index, as well as concentrations of bilirubin and albumin were evaluated for this purpose (Table 2). Patients were scored as follows: 5–6 as class (group) A, 7–9 as class (group) B, and 10–15 as class (group) C.24
TGF-1 measurement Venous blood was collected on ice using vacutainer tubes with EDTA as an anti-coagulant and centrifuged at 1000g within 30 min of collection. Obtained plasma was additionally centrifuged at 10 000 g for 10 min at 2–8C for complete platelet removal. To activate latent TGF-1 to immunoreactive TGF-1, 0.1 ml of the mixture, consisting of 2.5 N acetic acid and 10 M urea, was added to 0.1 ml of platelet-poor plasma and incubated for 10 min at room temperature. Acidified sample was neutralized by adding 0.1 ml of mixture consisted of 2.7 N NaOH and 1 M HEPES. Activated plasma samples were diluted four-fold and assayed in duplicate with the quantitative sandwich enzyme immunoassay (EIA) technique using recombinant human TGF-
soluble receptor type II (TRII) as a solid phase precoated onto a microplate (R&D Systems Inc., Minneapolis, MN, USA). Standards and samples were pipetted into the wells and any TGF-1 present was bound by the immobilized receptor. After washing away any unbound substances, polyclonal antibodies specific for TGF-1, conjugated to horseradish peroxidase, were added to the wells. Following a wash to remove any unbound antibody–enzyme reagent, a substrate solution consisting of stabilized hydrogen peroxide and tetramethylbenzidine was added to the wells. Colour developed in proportion to the amount of TGF-1 bound in the initial step. The reaction was stopped by addition of 2 N sulphuric acid and optical density was read with a microtitre plate photometer Stat Fax 2100 (Alab, Poland) at 450 nm and corrected by substraction of readings at 540 nm. The concentration of TGF-1 in a sample was determined by interpolation from a standard curves, prepared with standard samples supplied by manufacturer.
Statistical methods All data were collected and analysed with Microsoft Excel 97 software (Microsoft Corporation). Values were expressed as meanstandard error of the mean (SEM). The significance of the difference was calculated by two- tailed Student’s t-test. For correlation analysis the Pearson product moment correlation was used and linear regression was performed. Values of P<0.05 were considered to be significant.
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