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Hepatology Research 2014; 44: 1286–1298
doi: 10.1111/hepr.12304
Original Article
Soluble intracellular adhesion molecule, M30 and M65 as serum markers of disease activity and prognosis in cholestatic liver diseases Gerald Denk,1* Ahmed-Jawid Omary,1* Florian Paul Reiter,1 Simon Hohenester,1 Ralf Wimmer,1 Stefan Holdenrieder2 and Christian Rust3 1 Department of Medicine II – Grosshadern, University of Munich, 3Department of Medicine I, Krankenhaus Barmherzige Brüder, Munich, and 2Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
Aim: Hepatic apoptosis is involved in the pathogenesis of immune-mediated liver diseases such as autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). The aim of our study was to quantify distinct markers of apoptosis in sera of patients with AIH, PBC and PSC, and to evaluate correlation with markers of disease activity and prognosis.
of patients with immune-mediated liver diseases, whereas DNAse activity was reduced. Interestingly, patients with advanced PSC presented with higher levels of sICAM, M30 and M65 than patients with mild PSC. Regression analysis revealed correlations between serum levels of sICAM, M30 and M65 with the Mayo Risk Score for PSC, and of M65 with the Mayo Risk Score for PBC.
Methods: Sera of patients with AIH, PBC and PSC, and of healthy controls were collected and distinct cell death markers were quantified using a bead-based multiplex enzyme linked immunosorbent assay (soluble intracellular adhesion molecule [sICAM], macrophage migration inhibitory factor [MIF], soluble Fas [sFas], plasminogen activator inhibitor 1 [PAI-1]) or single enzyme-linked immunosorbent assays (DNAse, M30, M65).
Conclusion: Concentrations of the serum markers of apoptosis sFas and M30 and of the marker of total cell death M65 are elevated in patients with immune-mediated liver diseases, whereas activity of DNAse is reduced. In patients with PSC, sICAM, M30 and M65 may serve as indicators for disease activity and prognosis.
Results: In comparison with healthy controls, the apoptotic markers sFas, sICAM (only in PSC patients), M30 and the cell death marker M65 were substantially elevated in sera
INTRODUCTION
A
UTOIMMUNE HEPATITIS (AIH), primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are the most frequent immune-mediated liver diseases in humans.1 Autoimmune hepatitis is a chronic, progressive hepatitis that predominantly affects the interface of the liver lobule. Serologically, aminotransferases and immuno-
Correspondence: Dr Gerald Denk, Department of Medicine II – Klinikum Grosshadern, University of Munich, Marchioninistr. 15, D-81377 München, Germany. Email: gerald.denk@med .uni-muenchen.de *These authors contributed equally to this study. Received 27 August 2013; revision 12 January 2014; accepted 16 January 2014.
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Key words: M30, M65, primary biliary cirrhosis, primary sclerosing cholangitis, soluble Fas, soluble intracellular adhesion molecule
globulin (Ig)G are elevated and distinct autoantibodies can be found (antinuclear autoantibodies and antismooth muscle autoantibodies in type 1 AIH; liver– kidney–microsomal autoantibodies type 1 in type 2 AIH; soluble liver antigen autoantibodies in type 1 as well as in type 2 AIH).2,3 Whereas the prognosis of untreated AIH is unfavorable, adequate immunosuppressive therapy results in long-term survival of most patients.2 Similar to AIH, the chronic cholestatic liver disease PBC mainly affects women. Serum markers of cholestasis (γ-glutamyltransferase and alkaline phosphatase [AP]) as well as serum IgM are elevated. Antimitochondrial autoantibodies (AMA; particularly subtype M2) are the hallmark of PBC and can be found in up to 95% of patients.3,4 Inflammatory alterations of the small bile ducts and cholestasis dominate the
© 2014 The Japan Society of Hepatology
Hepatology Research 2014; 44: 1286–1298
histopathological aspect of PBC, finally leading to liver cirrhosis if left untreated.4 Ursodeoxycholic acid (UDCA; 13–15 mg/kg p.o. daily) is the only established therapy of PBC. Consequent administration of UDCA results in favorable effects in a majority of patients, when therapy is started at an early stage and in patients with a good biochemical response.5 Primary sclerosing cholangitis is an infrequent chronic cholestatic disorder that is characterized by inflammatory and subsequent fibrotic alterations of the intra- and extrahepatic biliary tree. PSC is associated with male sex and inflammatory bowel disease, especially ulcerative colitis.5–7 In addition to elevations of biochemical parameters of cholestasis, PSC is characterized by strictures and multisegmental dilatations of the bile ducts with magnetic resonance cholangiography (MRC) or conventional X-ray cholangiography (ERC).5–7 Although the exact molecular pathways have been only partially unravelled, apoptosis is regarded as a major pathomechanism in immune-mediated liver diseases.1 Basically, there are two major pathways to induce apoptotic cell death. The extrinsic pathway is initiated by binding to death receptors that are located on the surface of the hepatocytes. In contrast, the intrinsic pathway comprises mitochondrial breakdown.1,8 In both pathways, caspases are activated and cleave structures of apoptotic cells resulting in the release of distinct markers of apoptosis such as nucleosomes, Fas (CD95), Fas-ligands, cytokeratin 18 (M65), and fragments thereof (M30).1 Furthermore, pro-inflammatory cytokines such as the macrophage migration inhibitory factor (MIF) or adhesion molecules such as intracellular adhesion molecule (ICAM; CD54) may be of pathophysiological importance.1 Nucleosomes are complexes of DNA and histone proteins, that are released into blood from cells undergoing apoptosis or oncosis.9 Among other mechanisms, DNAse is involved in clearance of nucleosomes from blood.9,10 It was our aim to identify distinct serum markers of apoptosis in patients with immune-mediated liver diseases (AIH, PBC, and PSC) and to evaluate correlation with liver tests and prognostic scores in order to assess their potential as markers of disease activity.
METHODS Patients
S
ERA OF 33 patients with AIH, of 52 patients with PBC and of 37 patients with PSC were collected after written informed consent was obtained. Inclusion criteria for patients with AIH were a minimum of 18 years of
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age with a confirmed diagnosis of AIH. Diagnosis was made according to the modified AIH score of Alvarez et al.11 Inclusion criteria for patients with PBC were a minimum of 18 years of age with a confirmed diagnosis of PBC (stage I–IV). The diagnosis was made based on the current clinical practice guideline of the European Association for the Study of the Liver (EASL):5 Elevation of AMA of 1:40 or more or positivity for AMA-M2 and elevation of serum AP for a minimum of 6 months. Inclusion criteria for patients with PSC were a minimum of 18 years of age with a confirmed diagnosis of PSC. The diagnosis was made based on the current EASL clinical practice guideline5 with presence of characteristic bile duct lesions on MRC or ERC. Exclusion criteria were: age of less than 18 years; no capacity to consent; no consent; infection with hepatitis B or C virus; presence of hemochromatosis, Wilson’s disease or α1-antitrypsin deficiency; excessive intake of alcohol (>30 g daily in men, >20 g daily in women); and suspicion of toxic hepatopathy or vascular liver disease. Sera of 20 healthy volunteers served as controls. For a more detailed description of the patients, please refer to Table S1.
Study protocol Blood was taken from each patient. Quantity of distinct apoptotic markers (soluble ICAM [sICAM], macrophage migration inhibitory factor [MIF], soluble Fas [sFas] and plasminogen activator inhibitor 1 [PAI-1]) were determined by multiplex enzyme-linked immunosorbent assays (ELISA). Quantities of M30 and M65 and reduction of DNAse activity were determined by separate ELISA. In addition, disease-related clinical and demographic data were collected from each patient. The data from the assays were screened for correlation with the stage of disease to draw conclusions in respect to disease activity and prognosis.
Ethics The study was approved by the medical ethics committee of the University of Munich and written informed consent was obtained from all patients participating in this study.
Analysis of apoptotic markers in serum Quantification of concentrations of the apoptotic markers sFas, sICAM, MIF and PAI-1 was performed with the bead-based multiplex ELISA Human Sepsis/ Apoptosis Lincoplex Kit from Millipore (Billerica, MA, USA) as described previously.12 Quantification of reduction of DNAse activity in serum was performed with the
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Figure 1 The apoptotic markers soluble Fas (sFas), soluble intracellular adhesion molecule (sICAM), plasminogen activator inhibitor 1 (PAI-1) and M30, and the cell death marker M65 are elevated in sera of patients with immune-mediated liver diseases (autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis; n = 118–122) when compared with healthy controls (n = 20). In contrast, activity of DNAse is reduced in these patients. Comparison by non-parametric Wilcoxon–Mann– Whitney U-test.
© 2014 The Japan Society of Hepatology
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solid phase ELISA from Orgentec Diagnostics (Mainz, Germany) as described previously.10 As this assay measures reduction of DNAse activity in percentage, higher reduction stands for lower DNAse activity. Concentration of M30, caspase-dependent Asp396 cleaved cytokeratin 18 fragments, that are an indicator of apoptosis, was determined with the M30 Apoptosense ELISA from Peviva (Bromma, Sweden) as described recently.10 Concentration of M65, namely, soluble cytokeratin 18, a marker of total cell death, was determined with the
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M65 EpiDeath ELISA from Peviva according to the manufacturer’s instructions.
Statistics Statistical analysis was performed with the BiAS statistics software (Goethe University, Frankfurt, Germany) and the software SPSS Statistics version 20 (IBM, Armonk, NY, USA). Comparisons of two groups were calculated with the non-parametric Wilcoxon–Mann–Whitney U-test. For comparisons of multiple groups, the
Table 1 Concentrations and activities of distinct apoptotic markers in sera of patients with immune-mediated liver diseases (AIH, PBC, PSC) and controls
DNAse reduction of activity (%)
Controls n = 20
AIH n = 33
PBC n = 52
PSC n = 37
11.3 1 3.4
23.2 1 12.2 P < 0.001
17.1 1 8.7 P < 0.005 P < 0.05
2.4 1 0.9
3.6 1 5.5 n.s.
3.3 1 3.1 n.s. n.s.
2.7 1 0.9
7.5 1 3.8 P < 0.0001
5.6 1 2.6 P < 0.0001 P < 0.05
204.2 1 214.1
223.0 1 183.5 n.s.
227.4 1 219.9 n.s. n.s.
93.0 1 18.3
115.5 1 58.4
115.2 1 51.7
16.5 1 6.6 P < 0.01 P < 0.05 n.s. 2.9 1 4.7 n.s. n.s. n.s. 5.8 1 2.0 P < 0.0001 n.s. n.s. 278.1 1 249.2 P < 0.05 n.s. n.s. 125.3 1 50.3
n.s.
n.s. n.s.
n.s. n.s. n.s.
Controls n = 20
AIH n = 33
PBC n = 50
PSC n = 35
139.0 1 15.9
449.4 1 557.7 P < 0.000005
284.0 1 198.2 P < 0.000005 n.s.
155.6 1 18.1
959.8 1 1551.6 P < 0.000001
547.8 1 530.9 P < 0.000005 n.s.
299.7 1 211.8 P < 0.000005 n.s. n.s. 675.3 1 563.6 P < 0.000005 n.s. n.s.
vs control vs AIH vs PBC
MIF (pg/mL) vs control vs AIH vs PBC sFas (pg/mL) vs control vs AIH vs PBC sICAM (pg/mL) vs control vs AIH vs PBC PAI-1 (pg/mL) vs control vs AIH vs PBC
M30 (U/L) vs control vs AIH vs PBC M65 (U/L) vs control vs AIH vs PBC
Results are presented as means1 standard deviation. Statistics are given as p vs. controls (Kruskal–Wallis test, multiple comparisons according to Dunn with Bonferroni–Holm corrections). AIH, autoimmune hepatitis; MIF, macrophage migration inhibitory factor; n.s., not significant; PAI-1; plasminogen activator inhibitor 1; PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis; sFas, soluble Fas; sICAM, soluble intracellular adhesion molecule.
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Figure 2 Soluble intracellular adhesion molecule (sICAM) concentrations in sera of patients with various immune-mediated liver diseases (autoimmune hepatitis [AIH], primary biliary cirrhosis [PBC], primary sclerosing cholangitis [PSC]) of different stage and disease activity as indicated by distinct markers of liver cell integrity (bilirubin, alkaline phosphatase, aspartate transaminase). Patients with advanced PSC presented with higher levels of sICAM than patients with compensated PSC. AIH, n = 33; PBC, n = 52; PSC, n = 37. Comparisons were done by the Kruskal–Wallis test combined with a post-hoc test for multiple comparisons of pairs according to Dunn with modifications according to Bonferroni–Holm. ULN, upper limit of normal.
© 2014 The Japan Society of Hepatology
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Kruskal–Wallis test was used combined with a post-hoc test for multiple comparisons of pairs according to Dunn with modifications according to Bonferroni– Holm. Dot plots demonstrate the single values, median, and 25% and 75% quartiles. The correlations of scores and of apoptotic markers were calculated by linear regression analysis after logarithmic transformation to achieve a normal distribution of data.
RESULTS
I
N COMPARISON WITH healthy controls the apoptotic markers sFas and sICAM were substantially elevated in sera of patients with immune-mediated liver diseases (AIH, PBC, PSC), 6.2 1 2.9 versus 2.7 1 0.9 pg/mL (P < 0.0001) and 241.6 1 219.8 versus 204.2 1 214.1 pg/mL (P < 0.05), respectively (Fig. 1). Similarly, the concentrations of the apoptotic markers PAI-1 and M30, and of the cell death marker M65 were higher in sera of patients with immune-mediated liver diseases than in sera of controls, 118.3 1 52.9 versus 93.0 1 18.3 pg/mL (P < 0.05), 334.9 1 346.0 versus 139.0 1 15.9 U/L (P < 0.000001), and 700.8 1 947.6 versus 155.6 1 18.1 U/L (P < 0.000001), respectively (Fig. 1). In contrast, reduction of DNAse activity was
higher in sera of patients with immune-mediated liver disease than in sera of healthy controls, 18.6 1 9.6 versus 11.3 1 3.4% (P < 0.0001) (Fig. 1), whereas the serum concentration of the apoptotic marker MIF did not differ significantly between healthy controls and patients with immune-mediated liver diseases. Interestingly, patients with AIH were characterized by higher reduction of DNAse activity than patients with PSC or PBC (both P < 0.05) (Table 1). To stratify the patients in respect to disease activity, they were subdivided into different groups according to the elevation of defined markers of liver cell integrity (bilirubin, aspartate transaminase [AST], AP): normal (2 ULN). Interestingly, patients with advanced PSC, characterized by substantial elevations of these markers, presented with higher levels of sICAM than patients with compensated PSC, characterized by normal markers, an observation that has not been published before to the best of our knowledge. These observations were less pronounced in patients with AIH and largely absent in patients with PBC (Fig. 2). In contrast, the apoptotic serum marker sFas, that was substantially elevated in immune-mediated liver diseases in general
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Figure 3 Concentration of the apoptotic marker M30 in sera of patients with various immune-mediated liver diseases (autoimmune hepatitis [AIH], primary biliary cirrhosis [PBC], primary sclerosing cholangitis [PSC]) of different stage and disease activity as indicated by distinct markers of liver cell integrity (bilirubin, alkaline phosphatase, aspartate transaminase). M30, that was increased in AIH, PBC and PSC, was an indicator for disease activity, when quantified as activity of aspartate transaminase. AIH, n = 33; PBC, n = 50; PSC, n = 35. Comparisons were done by the Kruskal–Wallis test combined with a post-hoc test for multiple comparisons of pairs according to Dunn with modifications according to Bonferroni–Holm. ULN, upper limit of normal.
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(Fig. 1), was not (in AIH and PBC) or only a weak (in PSC) indicator for disease activity (see Fig. S1). To the best of our knowledge, this is the first time that an elevation of Fas in serum of patients with PSC was observed. Another novel observation is the reduction of DNAse activity in patients with immune-mediated liver diseases, an enzyme that is involved in nucleosomal degradation, when compared with healthy controls (Table 1). In contrast, there were substantial elevations of the apoptotic marker M30 and of the cell death marker M65 in patients with immune-mediated liver diseases (Table 1). Whereas a reduction of DNAse activity was no indicator of disease activity (Fig. S2), serum levels of M30 and M65 were correlated with increased AST activity (Figs 3,4). In accordance with these observations, there was a stronger correlation for sICAM than for sFas with the revised Mayo Risk Score for PSC, r2 = 0.44 (P < 0.0001) versus r2 = 0.23 (P < 0.005) (Fig. 5), a model to estimate survival in PSC based on distinct variables such as patient age, bilirubin and albumin level and activity of AST in serum, and history of variceal bleeding.13 Furthermore, serum levels of M30, r2 = 0.39 (P < 0.0001), and
of M65, r2 = 0.49 (P < 0.0001), correlated with the revised Mayo Risk Score for PSC (Fig. 5). This suggests that sICAM, M30 and M65, but not sFas, may serve as indicators for disease activity and prognosis in patients with PSC. In contrast, for patients with PBC, there was a relevant correlation only between M65, r2 = 0.45 (P < 0.001), and the Mayo Risk Score for PBC (Fig. 5), a model to predict survival in PBC based on distinct parameters such as patient age, bilirubin and albumin level, prothrombin time and presence of peripheral edema.14
DISCUSSION
A
POPTOSIS OF HEPATOCYTES and cholangiocytes is a key mechanism in the pathogenesis of immunemediated liver diseases.15–17 Therefore, serum markers of apoptosis may be valuable and convenient parameters to predict prognosis and to assess activity in these diseases. Whereas markers of apoptosis have been extensively investigated in liver tissue,1,17–19 there are no established markers in peripheral blood and only limited data is available.1
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Figure 4 Concentration of the cell death marker M65 in sera of patients with various immune-mediated liver diseases (autoimmune hepatitis [AIH], primary biliary cirrhosis [PBC], primary sclerosing cholangitis [PSC]) of different stage and disease activity as indicated by distinct markers of liver cell integrity (bilirubin, alkaline phosphatase, aspartate transaminase). M65, that was increased in AIH, PBC and PSC, was an indicator for disease activity, when quantified as activity of aspartate transaminase. AIH, n = 33; PBC, n = 50; PSC, n = 35. Comparisons were done by the Kruskal–Wallis test combined with a post-hoc test for multiple comparisons of pairs according to Dunn with modifications according to Bonferroni–Holm. ULN, upper limit of normal.
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As reviewed by Kremer et al.,1 there are substantial restrictions for a clinical use of apoptotic markers in peripheral blood such as: (i) absent specificity; (ii) uncertain chronological decline of serum levels/ activities; and (iii) competing pathomechanisms of cell death such as necrosis or necroptosis. Nevertheless, there have been attempts to establish apoptotic markers for clinical practice of immune-mediated liver diseases.20–22 In accordance with previous reports, the apoptotic markers sFas21 and sICAM (the latter in our study only in PSC)21,22 were elevated in sera of patients with immunemediated liver diseases when compared with healthy controls reflecting the higher rate of apoptosis under these pathological conditions. sICAM is the soluble form of the cell adhesion molecule ICAM (CD54),12 that seems to be involved in the pathogenesis of autoimmune hepatitis,1,23 and sFas is the soluble form of Fas (CD95), a receptor on the cell surface, that is an inducer of cellular apoptosis.12 Interestingly, the concentration of the apoptotic marker PAI-1 was increased in the total collective of patients with immune-mediated liver diseases, an observation that has been made earlier in PBC patients
undergoing orthotopic liver transplantation24 and in patients with advanced liver diseases of different etiology.25,26 These early descriptions focused on the hemostaseological aspects of this observation and not on apoptosis. Fitch et al.27 found less PAI-1 antigen in cirrhotic livers, comprising also advanced PBC, and discussed the possibility that this could be caused by an increased release of PAI-1 from hepatocytes. PAI-1 has been discussed both as antifibrotic28 as well as fibrogenic factor29 in various animal models. As a modulator of the plasmin system, PAI-1 is also a regulator of apoptosis.12 Although the pathophysiological role of PAI-1 remains unclear, an elevation of this marker seems to be associated with advanced liver disease. We observed that in patients with PSC, the apoptotic marker sICAM correlated with parameters of disease activity. Most importantly, sICAM correlated with the revised Mayo Risk Score for PSC,13 highlighting its potential use as a marker of prognosis. An elevation of ICAM in sera of patients with PSC was already described earlier,22,30 but no significant correlation with prognostic parameters such as serum bilirubin, AP, AST and albumin was observed, potentially due to the limited number of 19 PSC patients30 versus the 37 in our study,
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Figure 5 Regression analysis revealed correlations for soluble intracellular adhesion molecule (sICAM), M30, and M65 with the Mayo Risk Score for primary sclerosing cholangitis (PSC). n = 35–37, and for M65 with the Mayo Risk Score for PBC, n = 50. Linear regression analysis. , 95% confidence interval (data); , 95% confidence interval (regression).
or evaluated.22 Our observation is in line with a study of Bloom et al.31 In this study, ICAM-1 was expressed in the biliary epithelium in five of seven liver biopsies from patients with advanced PSC (histopathological stage 3 or 4), but not in nine liver biopsies from patients with early PSC (stage 1 or 2).31 As described earlier in patients with non-alcoholic steatohepatitis, graft-versus-host disease of the liver, acute liver failure, primary graft dysfunction and alcoholic liver cirrhosis,1,32–35 M30, namely, caspase-cleaved cytokeratin 18 fragments, was also elevated in patients with AIH, PBC and PSC pointing to the pathophysiological relevance of apoptosis in these diseases.
© 2014 The Japan Society of Hepatology
Whereas the elevation of the cell death marker M65, namely, soluble cytokeratin 18, in immune-mediated liver diseases is a novel observation, elevations of M30 have been described before in a rather heterogenous collective of 23 patients with biliary and autoimmune liver diseases (i.e. PBC, PSC, secondary biliary cirrhosis and AIH).36 However, probably due to the limited number of patients, no statistical significance was reached. Moreover, M65 (in PSC and PBC) and M30 (only in PSC) may serve as prognostic indicators due to their correlation with the respective Mayo Risk Scores. In addition, we were able to observe significant reductions of DNAse activity in all three immune-mediated liver
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diseases. This is in line with an earlier report,37 because DNAse activity is important for the degradation of nucleosomes in blood.9 In summary, concentrations and activities of several serum markers of apoptosis are altered in patients with immune-mediated liver diseases. They may be promising tools for disease surveillance and prediction of individual prognosis, but prospective studies are necessary to establish them for clinical practice.
REFERENCES 1 Kremer AE, Rust C, Eichhorn P, Beuers U, Holdenrieder S. Immune-mediated liver diseases: programmed cell death ligands and circulating apoptotic markers. Expert Rev Mol Diagn 2009; 9: 139–56. 2 Mieli-Vergani G, Vergani D. Autoimmune hepatitis. Nat Rev Gastroenterol Hepatol 2011; 8: 320–9. 3 Invernizzi P, Lleo A, Podda M. Interpreting serological tests in diagnosing autoimmune liver diseases. Semin Liver Dis 2007; 27: 161–72. 4 Bhandari BM, Bayat H, Rothstein KD. Primary biliary cirrhosis. Gastroenterol Clin North Am 2011; 40: 373–86. 5 EASL Clinical Practice Guidelines: management of cholestatic liver diseases. J Hepatol 2009; 51: 237–67. 6 Milkiewicz P, Wunsch E. Primary sclerosing cholangitis. Recent Results Cancer Res 2011; 185: 117–33. 7 Beuers U, Kullak-Ublick GA, Pusl T, Rauws ER, Rust C. Medical treatment of primary sclerosing cholangitis: a role for novel bile acids and other (post-)transcriptional modulators? Clin Rev Allergy Immunol 2009; 36: 52–61. 8 Guicciardi ME, Gores GJ. Apoptosis as a mechanism for liver disease progression. Semin Liver Dis 2010; 30: 402–10. 9 Holdenrieder S, Stieber P. Clinical use of circulating nucleosomes. Crit Rev Clin Lab Sci 2009; 46: 1–24. 10 Stoetzer OJ, Fersching DM, Salat C et al. Prediction of response to neoadjuvant chemotherapy in breast cancer patients by circulating apoptotic biomarkers nucleosomes, DNAse, cytokeratin-18 fragments and survivin. Cancer Lett 2013; 336: 140–8. 11 Alvarez F, Berg PA, Bianchi FB et al. International Autoimmune Hepatitis Group Report: review of criteria for diagnosis of autoimmune hepatitis. J Hepatol 1999; 31: 929–38. 12 Fersching DM, Nagel D, Siegele B et al. Apoptosis-related biomarkers sFAS, MIF, ICAM-1 and PAI-1 in serum of breast cancer patients undergoing neoadjuvant chemotherapy. Anticancer Res 2012; 32: 2047–58. 13 Kim WR, Therneau TM, Wiesner RH et al. A revised natural history model for primary sclerosing cholangitis. Mayo Clin Proc 2000; 75: 688–94. 14 Dickson ER, Grambsch PM, Fleming TR, Fisher LD, Langworthy A. Prognosis in primary biliary cirrhosis: model for decision making. Hepatology 1989; 10: 1–7.
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15 Kahraman A, Gerken G, Canbay A. Apoptosis in immunemediated liver diseases. Dig Dis 2010; 28: 144–9. 16 Malhi H, Gores GJ. Cellular and molecular mechanisms of liver injury. Gastroenterology 2008; 134: 1641–54. 17 Fox CK, Furtwaengler A, Nepomuceno RR, Martinez OM, Krams SM. Apoptotic pathways in primary biliary cirrhosis and autoimmune hepatitis. Liver 2001; 21: 272–9. 18 Seki S, Kitada T, Iwai S et al. Immunohistochemical detection of Fas and apoptosis in type-1 autoimmune hepatitis. Hepatogastroenterology 2003; 50: 1274–7. 19 Iwata M, Harada K, Hiramatsu K et al. Fas ligand expressing mononuclear cells around intrahepatic bile ducts coexpress CD68 in primary biliary cirrhosis. Liver 2000; 20: 129–35. 20 Pelli N, Floreani A, Torre F et al. Soluble apoptosis molecules in primary biliary cirrhosis: analysis and commitment of the Fas and tumour necrosis factorrelated apoptosis-inducing ligand systems in comparison with chronic hepatitis C. Clin Exp Immunol 2007; 148: 85–9. 21 Tortorella C, Sacco R, Orlando P, Salerno MT, Schiraldi O, Antonaci S. sICAM-1, sCD95 and sCD95L levels in chronic liver diseases of different etiology. Immunopharmacol Immunotoxicol 2000; 22: 19–33. 22 Thomson AW, Satoh S, Nussler AK et al. Circulating intercellular adhesion molecule-1 (ICAM-1) in autoimmune liver disease and evidence for the production of ICAM-1 by cytokine-stimulated human hepatocytes. Clin Exp Immunol 1994; 95: 83–90. 23 Lapierre P, Beland K, Alvarez F. Pathogenesis of autoimmune hepatitis: from break of tolerance to immunemediated hepatocyte apoptosis. Transl Res 2007; 149: 107–13. 24 Segal H, Cottam S, Potter D, Hunt BJ. Coagulation and fibrinolysis in primary biliary cirrhosis compared with other liver disease and during orthotopic liver transplantation. Hepatology 1997; 25: 683–8. 25 Leiper K, Croll A, Booth NA, Moore NR, Sinclair T, Bennett B. Tissue plasminogen activator, plasminogen activator inhibitors, and activator-inhibitor complex in liver disease. J Clin Pathol 1994; 47: 214–17. 26 Tran-Thang C, Fasel-Felley J, Pralong G, Hofstetter JR, Bachmann F, Kruithof EK. Plasminogen activators and plasminogen activator inhibitors in liver deficiencies caused by chronic alcoholism or infectious hepatitis. Thromb Haemost 1989; 62: 651–3. 27 Fitch P, Bennett B, Booth NA, Croll A, Ewen SW. Distribution of plasminogen activator inhibitor in normal liver, cirrhotic liver, and liver with metastases. J Clin Pathol 1994; 47: 218–21. 28 von Montfort C, Beier JI, Kaiser JP et al. PAI-1 plays a protective role in CCl4-induced hepatic fibrosis in mice: role of hepatocyte division. Am J Physiol Gastrointest Liver Physiol 2010; 298: G657–66.
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29 Hu PF, Chen H, Zhong W et al. Adenovirus-mediated transfer of siRNA against PAI-1 mRNA ameliorates hepatic fibrosis in rats. J Hepatol 2009; 51: 102–13. 30 Adams DH, Mainolfi E, Burra P et al. Detection of circulating intercellular adhesion molecule-1 in chronic liver diseases. Hepatology 1992; 16: 810–14. 31 Bloom S, Fleming K, Chapman R. Adhesion molecule expression in primary sclerosing cholangitis and primary biliary cirrhosis. Gut 1995; 36: 604–9. 32 Yilmaz Y, Dolar E, Ulukaya E et al. Soluble forms of extracellular cytokeratin 18 may differentiate simple steatosis from nonalcoholic steatohepatitis. World J Gastroenterol 2007; 14 (13): 837–44. 33 Younossi ZM, Jarrar M, Nugent C et al. A novel diagnostic biomarker panel for obesity-related nonalcoholic steatohepatitis (NASH). Obes Surg 2008; 18: 1430–7. 34 Luft T, Conzelmann M, Benner A et al. Serum cytokeratin-18 fragments as quantitative markers of epithelial apoptosis in liver and intestinal graft-versus-host disease. Blood 2007; 15 (110): 4535–42. 35 Hetz H, Hoetzenecker K, Hacker S et al. Caspase-cleaved cytokeratin 18 and 20 S proteasome in liver degeneration. J Clin Lab Anal 2007; 21: 277–81. 36 Yagmur E, Trautwein C, Leers MP, Gressner AM, Tacke F. Elevated apoptosis-associated cytokeratin 18 fragments (CK18Asp386) in serum of patients with chronic liver diseases indicate hepatic and biliary inflammation. Clin Biochem 2007; 40: 651–5. 37 Holdenrieder S, Eichhorn P, Beuers U et al. Nucleosomal DNA fragments in autoimmune diseases. Ann N Y Acad Sci 2006; 1075: 318–27.
SUPPORTING INFORMATION
A
DDITIONAL SUPPORTING INFORMATION may be found in the online version of this article at the publisher’s website:
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Figure S1 Soluble Fas (sFas) concentrations in sera of patients with various immune-mediated liver diseases (autoimmune hepatitis [AIH], primary biliary cirrhosis [PBC], primary sclerosing cholangitis [PSC]) of different stage and disease activity as indicated by distinct markers of liver cell integrity (bilirubin, alkaline phosphatase, aspartate transaminase). The apoptotic serum marker sFas, that was substantially elevated in immunemediated liver diseases in general, was no (in AIH and PBC) or only a weak (in PSC) indicator for disease activity. AIH, n = 33; PBC, n = 52; PSC, n = 37. Comparisons were done by the Kruskal–Wallis test combined with a post-hoc test for multiple comparisons of pairs according to Dunn with modifications according to Bonferroni–Holm. ULN, upper limit of normal. Figure S2 Reduction of DNAse activity in sera of patients with various immune-mediated liver diseases (autoimmune hepatitis [AIH], primary biliary cirrhosis [PBC], primary sclerosing cholangitis [PSC]) of different stage and disease activity as indicated by distinct markers of liver cell integrity (bilirubin, alkaline phosphatase, aspartate transaminase). The apoptotic serum marker DNAse, that was reduced in AIH, PBC and PSC was no indicator for disease activity. AIH, n = 33; PBC, n = 52; PSC, n = 37. Comparisons were done by the Kruskal–Wallis test combined with a post-hoc test for multiple comparisons of pairs according to Dunn with modifications according to Bonferroni–Holm. ULN, upper limit of normal. Table S1 Characteristics of patients included.
Hepatology Research 2014; 44: 1286–1298
doi: 10.1111/hepr.12304
Original Article
Soluble intracellular adhesion molecule, M30 and M65 as serum markers of disease activity and prognosis in cholestatic liver diseases Gerald Denk,1* Ahmed-Jawid Omary,1* Florian Paul Reiter,1 Simon Hohenester,1 Ralf Wimmer,1 Stefan Holdenrieder2 and Christian Rust3 1 Department of Medicine II – Grosshadern, University of Munich, 3Department of Medicine I, Krankenhaus Barmherzige Brüder, Munich, and 2Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
Aim: Hepatic apoptosis is involved in the pathogenesis of immune-mediated liver diseases such as autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). The aim of our study was to quantify distinct markers of apoptosis in sera of patients with AIH, PBC and PSC, and to evaluate correlation with markers of disease activity and prognosis.
of patients with immune-mediated liver diseases, whereas DNAse activity was reduced. Interestingly, patients with advanced PSC presented with higher levels of sICAM, M30 and M65 than patients with mild PSC. Regression analysis revealed correlations between serum levels of sICAM, M30 and M65 with the Mayo Risk Score for PSC, and of M65 with the Mayo Risk Score for PBC.
Methods: Sera of patients with AIH, PBC and PSC, and of healthy controls were collected and distinct cell death markers were quantified using a bead-based multiplex enzyme linked immunosorbent assay (soluble intracellular adhesion molecule [sICAM], macrophage migration inhibitory factor [MIF], soluble Fas [sFas], plasminogen activator inhibitor 1 [PAI-1]) or single enzyme-linked immunosorbent assays (DNAse, M30, M65).
Conclusion: Concentrations of the serum markers of apoptosis sFas and M30 and of the marker of total cell death M65 are elevated in patients with immune-mediated liver diseases, whereas activity of DNAse is reduced. In patients with PSC, sICAM, M30 and M65 may serve as indicators for disease activity and prognosis.
Results: In comparison with healthy controls, the apoptotic markers sFas, sICAM (only in PSC patients), M30 and the cell death marker M65 were substantially elevated in sera
INTRODUCTION
A
UTOIMMUNE HEPATITIS (AIH), primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are the most frequent immune-mediated liver diseases in humans.1 Autoimmune hepatitis is a chronic, progressive hepatitis that predominantly affects the interface of the liver lobule. Serologically, aminotransferases and immuno-
Correspondence: Dr Gerald Denk, Department of Medicine II – Klinikum Grosshadern, University of Munich, Marchioninistr. 15, D-81377 München, Germany. Email: gerald.denk@med .uni-muenchen.de *These authors contributed equally to this study. Received 27 August 2013; revision 12 January 2014; accepted 16 January 2014.
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Key words: M30, M65, primary biliary cirrhosis, primary sclerosing cholangitis, soluble Fas, soluble intracellular adhesion molecule
globulin (Ig)G are elevated and distinct autoantibodies can be found (antinuclear autoantibodies and antismooth muscle autoantibodies in type 1 AIH; liver– kidney–microsomal autoantibodies type 1 in type 2 AIH; soluble liver antigen autoantibodies in type 1 as well as in type 2 AIH).2,3 Whereas the prognosis of untreated AIH is unfavorable, adequate immunosuppressive therapy results in long-term survival of most patients.2 Similar to AIH, the chronic cholestatic liver disease PBC mainly affects women. Serum markers of cholestasis (γ-glutamyltransferase and alkaline phosphatase [AP]) as well as serum IgM are elevated. Antimitochondrial autoantibodies (AMA; particularly subtype M2) are the hallmark of PBC and can be found in up to 95% of patients.3,4 Inflammatory alterations of the small bile ducts and cholestasis dominate the
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histopathological aspect of PBC, finally leading to liver cirrhosis if left untreated.4 Ursodeoxycholic acid (UDCA; 13–15 mg/kg p.o. daily) is the only established therapy of PBC. Consequent administration of UDCA results in favorable effects in a majority of patients, when therapy is started at an early stage and in patients with a good biochemical response.5 Primary sclerosing cholangitis is an infrequent chronic cholestatic disorder that is characterized by inflammatory and subsequent fibrotic alterations of the intra- and extrahepatic biliary tree. PSC is associated with male sex and inflammatory bowel disease, especially ulcerative colitis.5–7 In addition to elevations of biochemical parameters of cholestasis, PSC is characterized by strictures and multisegmental dilatations of the bile ducts with magnetic resonance cholangiography (MRC) or conventional X-ray cholangiography (ERC).5–7 Although the exact molecular pathways have been only partially unravelled, apoptosis is regarded as a major pathomechanism in immune-mediated liver diseases.1 Basically, there are two major pathways to induce apoptotic cell death. The extrinsic pathway is initiated by binding to death receptors that are located on the surface of the hepatocytes. In contrast, the intrinsic pathway comprises mitochondrial breakdown.1,8 In both pathways, caspases are activated and cleave structures of apoptotic cells resulting in the release of distinct markers of apoptosis such as nucleosomes, Fas (CD95), Fas-ligands, cytokeratin 18 (M65), and fragments thereof (M30).1 Furthermore, pro-inflammatory cytokines such as the macrophage migration inhibitory factor (MIF) or adhesion molecules such as intracellular adhesion molecule (ICAM; CD54) may be of pathophysiological importance.1 Nucleosomes are complexes of DNA and histone proteins, that are released into blood from cells undergoing apoptosis or oncosis.9 Among other mechanisms, DNAse is involved in clearance of nucleosomes from blood.9,10 It was our aim to identify distinct serum markers of apoptosis in patients with immune-mediated liver diseases (AIH, PBC, and PSC) and to evaluate correlation with liver tests and prognostic scores in order to assess their potential as markers of disease activity.
METHODS Patients
S
ERA OF 33 patients with AIH, of 52 patients with PBC and of 37 patients with PSC were collected after written informed consent was obtained. Inclusion criteria for patients with AIH were a minimum of 18 years of
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age with a confirmed diagnosis of AIH. Diagnosis was made according to the modified AIH score of Alvarez et al.11 Inclusion criteria for patients with PBC were a minimum of 18 years of age with a confirmed diagnosis of PBC (stage I–IV). The diagnosis was made based on the current clinical practice guideline of the European Association for the Study of the Liver (EASL):5 Elevation of AMA of 1:40 or more or positivity for AMA-M2 and elevation of serum AP for a minimum of 6 months. Inclusion criteria for patients with PSC were a minimum of 18 years of age with a confirmed diagnosis of PSC. The diagnosis was made based on the current EASL clinical practice guideline5 with presence of characteristic bile duct lesions on MRC or ERC. Exclusion criteria were: age of less than 18 years; no capacity to consent; no consent; infection with hepatitis B or C virus; presence of hemochromatosis, Wilson’s disease or α1-antitrypsin deficiency; excessive intake of alcohol (>30 g daily in men, >20 g daily in women); and suspicion of toxic hepatopathy or vascular liver disease. Sera of 20 healthy volunteers served as controls. For a more detailed description of the patients, please refer to Table S1.
Study protocol Blood was taken from each patient. Quantity of distinct apoptotic markers (soluble ICAM [sICAM], macrophage migration inhibitory factor [MIF], soluble Fas [sFas] and plasminogen activator inhibitor 1 [PAI-1]) were determined by multiplex enzyme-linked immunosorbent assays (ELISA). Quantities of M30 and M65 and reduction of DNAse activity were determined by separate ELISA. In addition, disease-related clinical and demographic data were collected from each patient. The data from the assays were screened for correlation with the stage of disease to draw conclusions in respect to disease activity and prognosis.
Ethics The study was approved by the medical ethics committee of the University of Munich and written informed consent was obtained from all patients participating in this study.
Analysis of apoptotic markers in serum Quantification of concentrations of the apoptotic markers sFas, sICAM, MIF and PAI-1 was performed with the bead-based multiplex ELISA Human Sepsis/ Apoptosis Lincoplex Kit from Millipore (Billerica, MA, USA) as described previously.12 Quantification of reduction of DNAse activity in serum was performed with the
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Figure 1 The apoptotic markers soluble Fas (sFas), soluble intracellular adhesion molecule (sICAM), plasminogen activator inhibitor 1 (PAI-1) and M30, and the cell death marker M65 are elevated in sera of patients with immune-mediated liver diseases (autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis; n = 118–122) when compared with healthy controls (n = 20). In contrast, activity of DNAse is reduced in these patients. Comparison by non-parametric Wilcoxon–Mann– Whitney U-test.
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solid phase ELISA from Orgentec Diagnostics (Mainz, Germany) as described previously.10 As this assay measures reduction of DNAse activity in percentage, higher reduction stands for lower DNAse activity. Concentration of M30, caspase-dependent Asp396 cleaved cytokeratin 18 fragments, that are an indicator of apoptosis, was determined with the M30 Apoptosense ELISA from Peviva (Bromma, Sweden) as described recently.10 Concentration of M65, namely, soluble cytokeratin 18, a marker of total cell death, was determined with the
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M65 EpiDeath ELISA from Peviva according to the manufacturer’s instructions.
Statistics Statistical analysis was performed with the BiAS statistics software (Goethe University, Frankfurt, Germany) and the software SPSS Statistics version 20 (IBM, Armonk, NY, USA). Comparisons of two groups were calculated with the non-parametric Wilcoxon–Mann–Whitney U-test. For comparisons of multiple groups, the
Table 1 Concentrations and activities of distinct apoptotic markers in sera of patients with immune-mediated liver diseases (AIH, PBC, PSC) and controls
DNAse reduction of activity (%)
Controls n = 20
AIH n = 33
PBC n = 52
PSC n = 37
11.3 1 3.4
23.2 1 12.2 P < 0.001
17.1 1 8.7 P < 0.005 P < 0.05
2.4 1 0.9
3.6 1 5.5 n.s.
3.3 1 3.1 n.s. n.s.
2.7 1 0.9
7.5 1 3.8 P < 0.0001
5.6 1 2.6 P < 0.0001 P < 0.05
204.2 1 214.1
223.0 1 183.5 n.s.
227.4 1 219.9 n.s. n.s.
93.0 1 18.3
115.5 1 58.4
115.2 1 51.7
16.5 1 6.6 P < 0.01 P < 0.05 n.s. 2.9 1 4.7 n.s. n.s. n.s. 5.8 1 2.0 P < 0.0001 n.s. n.s. 278.1 1 249.2 P < 0.05 n.s. n.s. 125.3 1 50.3
n.s.
n.s. n.s.
n.s. n.s. n.s.
Controls n = 20
AIH n = 33
PBC n = 50
PSC n = 35
139.0 1 15.9
449.4 1 557.7 P < 0.000005
284.0 1 198.2 P < 0.000005 n.s.
155.6 1 18.1
959.8 1 1551.6 P < 0.000001
547.8 1 530.9 P < 0.000005 n.s.
299.7 1 211.8 P < 0.000005 n.s. n.s. 675.3 1 563.6 P < 0.000005 n.s. n.s.
vs control vs AIH vs PBC
MIF (pg/mL) vs control vs AIH vs PBC sFas (pg/mL) vs control vs AIH vs PBC sICAM (pg/mL) vs control vs AIH vs PBC PAI-1 (pg/mL) vs control vs AIH vs PBC
M30 (U/L) vs control vs AIH vs PBC M65 (U/L) vs control vs AIH vs PBC
Results are presented as means1 standard deviation. Statistics are given as p vs. controls (Kruskal–Wallis test, multiple comparisons according to Dunn with Bonferroni–Holm corrections). AIH, autoimmune hepatitis; MIF, macrophage migration inhibitory factor; n.s., not significant; PAI-1; plasminogen activator inhibitor 1; PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis; sFas, soluble Fas; sICAM, soluble intracellular adhesion molecule.
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Figure 2 Soluble intracellular adhesion molecule (sICAM) concentrations in sera of patients with various immune-mediated liver diseases (autoimmune hepatitis [AIH], primary biliary cirrhosis [PBC], primary sclerosing cholangitis [PSC]) of different stage and disease activity as indicated by distinct markers of liver cell integrity (bilirubin, alkaline phosphatase, aspartate transaminase). Patients with advanced PSC presented with higher levels of sICAM than patients with compensated PSC. AIH, n = 33; PBC, n = 52; PSC, n = 37. Comparisons were done by the Kruskal–Wallis test combined with a post-hoc test for multiple comparisons of pairs according to Dunn with modifications according to Bonferroni–Holm. ULN, upper limit of normal.
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Kruskal–Wallis test was used combined with a post-hoc test for multiple comparisons of pairs according to Dunn with modifications according to Bonferroni– Holm. Dot plots demonstrate the single values, median, and 25% and 75% quartiles. The correlations of scores and of apoptotic markers were calculated by linear regression analysis after logarithmic transformation to achieve a normal distribution of data.
RESULTS
I
N COMPARISON WITH healthy controls the apoptotic markers sFas and sICAM were substantially elevated in sera of patients with immune-mediated liver diseases (AIH, PBC, PSC), 6.2 1 2.9 versus 2.7 1 0.9 pg/mL (P < 0.0001) and 241.6 1 219.8 versus 204.2 1 214.1 pg/mL (P < 0.05), respectively (Fig. 1). Similarly, the concentrations of the apoptotic markers PAI-1 and M30, and of the cell death marker M65 were higher in sera of patients with immune-mediated liver diseases than in sera of controls, 118.3 1 52.9 versus 93.0 1 18.3 pg/mL (P < 0.05), 334.9 1 346.0 versus 139.0 1 15.9 U/L (P < 0.000001), and 700.8 1 947.6 versus 155.6 1 18.1 U/L (P < 0.000001), respectively (Fig. 1). In contrast, reduction of DNAse activity was
higher in sera of patients with immune-mediated liver disease than in sera of healthy controls, 18.6 1 9.6 versus 11.3 1 3.4% (P < 0.0001) (Fig. 1), whereas the serum concentration of the apoptotic marker MIF did not differ significantly between healthy controls and patients with immune-mediated liver diseases. Interestingly, patients with AIH were characterized by higher reduction of DNAse activity than patients with PSC or PBC (both P < 0.05) (Table 1). To stratify the patients in respect to disease activity, they were subdivided into different groups according to the elevation of defined markers of liver cell integrity (bilirubin, aspartate transaminase [AST], AP): normal (2 ULN). Interestingly, patients with advanced PSC, characterized by substantial elevations of these markers, presented with higher levels of sICAM than patients with compensated PSC, characterized by normal markers, an observation that has not been published before to the best of our knowledge. These observations were less pronounced in patients with AIH and largely absent in patients with PBC (Fig. 2). In contrast, the apoptotic serum marker sFas, that was substantially elevated in immune-mediated liver diseases in general
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Figure 3 Concentration of the apoptotic marker M30 in sera of patients with various immune-mediated liver diseases (autoimmune hepatitis [AIH], primary biliary cirrhosis [PBC], primary sclerosing cholangitis [PSC]) of different stage and disease activity as indicated by distinct markers of liver cell integrity (bilirubin, alkaline phosphatase, aspartate transaminase). M30, that was increased in AIH, PBC and PSC, was an indicator for disease activity, when quantified as activity of aspartate transaminase. AIH, n = 33; PBC, n = 50; PSC, n = 35. Comparisons were done by the Kruskal–Wallis test combined with a post-hoc test for multiple comparisons of pairs according to Dunn with modifications according to Bonferroni–Holm. ULN, upper limit of normal.
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Figure 3 Continued
(Fig. 1), was not (in AIH and PBC) or only a weak (in PSC) indicator for disease activity (see Fig. S1). To the best of our knowledge, this is the first time that an elevation of Fas in serum of patients with PSC was observed. Another novel observation is the reduction of DNAse activity in patients with immune-mediated liver diseases, an enzyme that is involved in nucleosomal degradation, when compared with healthy controls (Table 1). In contrast, there were substantial elevations of the apoptotic marker M30 and of the cell death marker M65 in patients with immune-mediated liver diseases (Table 1). Whereas a reduction of DNAse activity was no indicator of disease activity (Fig. S2), serum levels of M30 and M65 were correlated with increased AST activity (Figs 3,4). In accordance with these observations, there was a stronger correlation for sICAM than for sFas with the revised Mayo Risk Score for PSC, r2 = 0.44 (P < 0.0001) versus r2 = 0.23 (P < 0.005) (Fig. 5), a model to estimate survival in PSC based on distinct variables such as patient age, bilirubin and albumin level and activity of AST in serum, and history of variceal bleeding.13 Furthermore, serum levels of M30, r2 = 0.39 (P < 0.0001), and
of M65, r2 = 0.49 (P < 0.0001), correlated with the revised Mayo Risk Score for PSC (Fig. 5). This suggests that sICAM, M30 and M65, but not sFas, may serve as indicators for disease activity and prognosis in patients with PSC. In contrast, for patients with PBC, there was a relevant correlation only between M65, r2 = 0.45 (P < 0.001), and the Mayo Risk Score for PBC (Fig. 5), a model to predict survival in PBC based on distinct parameters such as patient age, bilirubin and albumin level, prothrombin time and presence of peripheral edema.14
DISCUSSION
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POPTOSIS OF HEPATOCYTES and cholangiocytes is a key mechanism in the pathogenesis of immunemediated liver diseases.15–17 Therefore, serum markers of apoptosis may be valuable and convenient parameters to predict prognosis and to assess activity in these diseases. Whereas markers of apoptosis have been extensively investigated in liver tissue,1,17–19 there are no established markers in peripheral blood and only limited data is available.1
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Figure 4 Concentration of the cell death marker M65 in sera of patients with various immune-mediated liver diseases (autoimmune hepatitis [AIH], primary biliary cirrhosis [PBC], primary sclerosing cholangitis [PSC]) of different stage and disease activity as indicated by distinct markers of liver cell integrity (bilirubin, alkaline phosphatase, aspartate transaminase). M65, that was increased in AIH, PBC and PSC, was an indicator for disease activity, when quantified as activity of aspartate transaminase. AIH, n = 33; PBC, n = 50; PSC, n = 35. Comparisons were done by the Kruskal–Wallis test combined with a post-hoc test for multiple comparisons of pairs according to Dunn with modifications according to Bonferroni–Holm. ULN, upper limit of normal.
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As reviewed by Kremer et al.,1 there are substantial restrictions for a clinical use of apoptotic markers in peripheral blood such as: (i) absent specificity; (ii) uncertain chronological decline of serum levels/ activities; and (iii) competing pathomechanisms of cell death such as necrosis or necroptosis. Nevertheless, there have been attempts to establish apoptotic markers for clinical practice of immune-mediated liver diseases.20–22 In accordance with previous reports, the apoptotic markers sFas21 and sICAM (the latter in our study only in PSC)21,22 were elevated in sera of patients with immunemediated liver diseases when compared with healthy controls reflecting the higher rate of apoptosis under these pathological conditions. sICAM is the soluble form of the cell adhesion molecule ICAM (CD54),12 that seems to be involved in the pathogenesis of autoimmune hepatitis,1,23 and sFas is the soluble form of Fas (CD95), a receptor on the cell surface, that is an inducer of cellular apoptosis.12 Interestingly, the concentration of the apoptotic marker PAI-1 was increased in the total collective of patients with immune-mediated liver diseases, an observation that has been made earlier in PBC patients
undergoing orthotopic liver transplantation24 and in patients with advanced liver diseases of different etiology.25,26 These early descriptions focused on the hemostaseological aspects of this observation and not on apoptosis. Fitch et al.27 found less PAI-1 antigen in cirrhotic livers, comprising also advanced PBC, and discussed the possibility that this could be caused by an increased release of PAI-1 from hepatocytes. PAI-1 has been discussed both as antifibrotic28 as well as fibrogenic factor29 in various animal models. As a modulator of the plasmin system, PAI-1 is also a regulator of apoptosis.12 Although the pathophysiological role of PAI-1 remains unclear, an elevation of this marker seems to be associated with advanced liver disease. We observed that in patients with PSC, the apoptotic marker sICAM correlated with parameters of disease activity. Most importantly, sICAM correlated with the revised Mayo Risk Score for PSC,13 highlighting its potential use as a marker of prognosis. An elevation of ICAM in sera of patients with PSC was already described earlier,22,30 but no significant correlation with prognostic parameters such as serum bilirubin, AP, AST and albumin was observed, potentially due to the limited number of 19 PSC patients30 versus the 37 in our study,
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Figure 5 Regression analysis revealed correlations for soluble intracellular adhesion molecule (sICAM), M30, and M65 with the Mayo Risk Score for primary sclerosing cholangitis (PSC). n = 35–37, and for M65 with the Mayo Risk Score for PBC, n = 50. Linear regression analysis. , 95% confidence interval (data); , 95% confidence interval (regression).
or evaluated.22 Our observation is in line with a study of Bloom et al.31 In this study, ICAM-1 was expressed in the biliary epithelium in five of seven liver biopsies from patients with advanced PSC (histopathological stage 3 or 4), but not in nine liver biopsies from patients with early PSC (stage 1 or 2).31 As described earlier in patients with non-alcoholic steatohepatitis, graft-versus-host disease of the liver, acute liver failure, primary graft dysfunction and alcoholic liver cirrhosis,1,32–35 M30, namely, caspase-cleaved cytokeratin 18 fragments, was also elevated in patients with AIH, PBC and PSC pointing to the pathophysiological relevance of apoptosis in these diseases.
© 2014 The Japan Society of Hepatology
Whereas the elevation of the cell death marker M65, namely, soluble cytokeratin 18, in immune-mediated liver diseases is a novel observation, elevations of M30 have been described before in a rather heterogenous collective of 23 patients with biliary and autoimmune liver diseases (i.e. PBC, PSC, secondary biliary cirrhosis and AIH).36 However, probably due to the limited number of patients, no statistical significance was reached. Moreover, M65 (in PSC and PBC) and M30 (only in PSC) may serve as prognostic indicators due to their correlation with the respective Mayo Risk Scores. In addition, we were able to observe significant reductions of DNAse activity in all three immune-mediated liver
Hepatology Research 2014; 44: 1286–1298
diseases. This is in line with an earlier report,37 because DNAse activity is important for the degradation of nucleosomes in blood.9 In summary, concentrations and activities of several serum markers of apoptosis are altered in patients with immune-mediated liver diseases. They may be promising tools for disease surveillance and prediction of individual prognosis, but prospective studies are necessary to establish them for clinical practice.
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29 Hu PF, Chen H, Zhong W et al. Adenovirus-mediated transfer of siRNA against PAI-1 mRNA ameliorates hepatic fibrosis in rats. J Hepatol 2009; 51: 102–13. 30 Adams DH, Mainolfi E, Burra P et al. Detection of circulating intercellular adhesion molecule-1 in chronic liver diseases. Hepatology 1992; 16: 810–14. 31 Bloom S, Fleming K, Chapman R. Adhesion molecule expression in primary sclerosing cholangitis and primary biliary cirrhosis. Gut 1995; 36: 604–9. 32 Yilmaz Y, Dolar E, Ulukaya E et al. Soluble forms of extracellular cytokeratin 18 may differentiate simple steatosis from nonalcoholic steatohepatitis. World J Gastroenterol 2007; 14 (13): 837–44. 33 Younossi ZM, Jarrar M, Nugent C et al. A novel diagnostic biomarker panel for obesity-related nonalcoholic steatohepatitis (NASH). Obes Surg 2008; 18: 1430–7. 34 Luft T, Conzelmann M, Benner A et al. Serum cytokeratin-18 fragments as quantitative markers of epithelial apoptosis in liver and intestinal graft-versus-host disease. Blood 2007; 15 (110): 4535–42. 35 Hetz H, Hoetzenecker K, Hacker S et al. Caspase-cleaved cytokeratin 18 and 20 S proteasome in liver degeneration. J Clin Lab Anal 2007; 21: 277–81. 36 Yagmur E, Trautwein C, Leers MP, Gressner AM, Tacke F. Elevated apoptosis-associated cytokeratin 18 fragments (CK18Asp386) in serum of patients with chronic liver diseases indicate hepatic and biliary inflammation. Clin Biochem 2007; 40: 651–5. 37 Holdenrieder S, Eichhorn P, Beuers U et al. Nucleosomal DNA fragments in autoimmune diseases. Ann N Y Acad Sci 2006; 1075: 318–27.
SUPPORTING INFORMATION
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DDITIONAL SUPPORTING INFORMATION may be found in the online version of this article at the publisher’s website:
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Hepatology Research 2014; 44: 1286–1298
Figure S1 Soluble Fas (sFas) concentrations in sera of patients with various immune-mediated liver diseases (autoimmune hepatitis [AIH], primary biliary cirrhosis [PBC], primary sclerosing cholangitis [PSC]) of different stage and disease activity as indicated by distinct markers of liver cell integrity (bilirubin, alkaline phosphatase, aspartate transaminase). The apoptotic serum marker sFas, that was substantially elevated in immunemediated liver diseases in general, was no (in AIH and PBC) or only a weak (in PSC) indicator for disease activity. AIH, n = 33; PBC, n = 52; PSC, n = 37. Comparisons were done by the Kruskal–Wallis test combined with a post-hoc test for multiple comparisons of pairs according to Dunn with modifications according to Bonferroni–Holm. ULN, upper limit of normal. Figure S2 Reduction of DNAse activity in sera of patients with various immune-mediated liver diseases (autoimmune hepatitis [AIH], primary biliary cirrhosis [PBC], primary sclerosing cholangitis [PSC]) of different stage and disease activity as indicated by distinct markers of liver cell integrity (bilirubin, alkaline phosphatase, aspartate transaminase). The apoptotic serum marker DNAse, that was reduced in AIH, PBC and PSC was no indicator for disease activity. AIH, n = 33; PBC, n = 52; PSC, n = 37. Comparisons were done by the Kruskal–Wallis test combined with a post-hoc test for multiple comparisons of pairs according to Dunn with modifications according to Bonferroni–Holm. ULN, upper limit of normal. Table S1 Characteristics of patients included.
