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Similar increased serum dipeptidyl peptidase IV activity in chronic hepatitis C and other viral infections
Journal of Clinical Virology 27 (2003) 59 /68 www.elsevier.com/locate/jcv
Similar increased serum dipeptidyl peptidase IV activity in chronic hepatitis C and other viral infections T. Andrieu a, V. Thibault a, I. Malet a, J. Laporte a, B. Bauvois b, H. Agut a, A. Cahour a,* a
Laboratoire de Virologie, CERVI, UPRES EA 2387, Groupe Hospitalier Pitie´-Salpeˆtrie`re, 75651 Paris Cedex 13, France b Unite´ 365 INSERM, Institut Curie, 75248 Paris Cedex 05, France Accepted 19 July 2002
Abstract Background: Dipeptidyl peptidase IV is a transmembrane enzyme widely expressed in many cell types, but also present as a soluble form in biological fluids. Its abnormal activity is sometimes associated with liver disease related pathologies. Objectives: The aim of this study was to evaluate the clinical relevance of changes in serum DPPIV activity in hepatitis C and other viral infections. Study design: DPPIV activity was assessed by using a microplate-based colorimetric assay on serum from 88 subjects: 12 healthy uninfected controls, 10 patients with primary biliary cirrhosis (PBC) as a reference group, 36 HCV-infected patients, and patients suffering from viral infections of different etiologies. Levels of DPPIV activity were compared with: (1) those of other serum biochemical parameters such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma glutamyl transpeptidase (GGT), and bilirubin concentrations; and (2) criteria representative of liver histological status. Results: Compared with healthy subjects, DPPIV activity was significantly increased during viral infections and in PBC (P B/0.01). In HCV-infected patients, the median activity (interquartile range, IQR), 29.78 IU/l (24.66 /35.95), differed significantly (P B/0.05) from that of controls: 21.42 (19.76 /24.93). No correlation was observed between DPPIV activity and either ALT, AST, bilirubin, or the stage of liver fibrosis and necroinflammatory activity, although GGT was moderately correlated (r / 0.58, P B/0.05). Conclusions: Although we confirmed an elevation of serum DPPIV activity in PBC, it seems to be a non-specific phenomenon common to viral infections. The absence of correlation between serum DPPIV and markers of liver disease in HCV-infected patients, suggests that this activity originates not only from the liver, but also from other sources such as peripheral blood cells involved in the control of viral infections. # 2002 Elsevier Science B.V. All rights reserved. Keywords: DPPIV/CD26; HCV; Viral infection; Fibrosis; Cholestasis; Pathophysiology
1. Introduction * Corresponding author. Tel.: /33-1-45-82-62-98; fax: /331-45-82-63-14 E-mail address: [email protected] (A. Cahour).
Dipeptidyl peptidase IV (DPPIV/CD26) is a transmembrane protein expressed in a large variety of different cell types including lymphocytes,
1386-6532/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 5 3 2 ( 0 2 ) 0 0 1 2 8 - 2
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kidney, liver and epithelial cells (Heike et al., 1988; Vanhoof et al., 1992). Peptidase activity of DPPIV (EC 3.4.14.5) resides in its ectodomain and is specific of polypeptides with either L-proline or Lalanine at the penultimate amino-terminal position (Torimoto et al., 1992). As an integral membrane glycoprotein capable of transducing intracellular signals, DPPIV has been implicated in the regulation of cell growth, apoptosis and differentiation (Fleischer, 1994; De Meester et al., 1999; Iwata and Morimoto, 1999; Antczak et al., 2001a). Most of these functional properties have been elucidated in T cell proliferation through T-cell receptors (TCR) (Hegen et al., 1990; Kameoka et al., 1993; Tanaka et al., 1993). Dysregulated expression of DPPIV is observed in various human malignancies, suggesting its value as a clinical marker (Antczak et al., 2001b). Moreover, DPPIV is located in the canalicular membranes of hepatocytes (McCaughan et al., 1990), and its expression is aberrant in liver cirrhosis (Matsumoto et al., 1992; Lakatos et al., 2000) and hepatocellular carcinoma (Stecca et al., 1997). Given its altered distribution in liver diseases, DPPIV could play an important role in the maintenance of liver architecture by interacting with extracellular matrix (ECM) proteins, and/or in the tissue destruction/ regeneration process. To date, little is known about the potential functions of DPPIV in liver tissue. Harada et al. (1995) observed a significant upregulation of DPPIV in a HepG2 cell line stably transfected with HCV non-structural polyprotein. Gaetaniello et al. (1998) demonstrated that DPPIV/CD26 occupancy delivers a potent apoptotic signal in a human hepatoma cell line. Although DPPIV exists as a soluble form in body fluids including plasma, serum and urine (Chikuma et al., 1990; Maes et al., 1994; Rakoczi et al., 1995), its physiological significance and origin remain unclear. Recently, most of serum DPPIV activity (95%) has been demonstrated to be associated with CD26 (Durinx et al., 2001). DPPIV activity may vary considerably with pathological conditions (Iwata and Morimoto, 1999; McCaughan et al., 2000). Accordingly, elevated serum enzyme activity has been reported in cirrhosis (Matsumoto et al., 1992; Lakatos et al., 2000) and primary biliary cirrhosis (PBC) (Laka-
tos et al., 1999), and more recently, in chronic hepatitis C where a negative correlation with the response to interferon therapy has been observed (Firneisz et al., 2001). Chronic hepatitis C, like most of chronic liver diseases, is characterized by continuing hepatocellular injury, inflammation and cirrhosis. The aim of this study was to assess whether modifications of serum DPPIV activity in patients with chronic HCV infection are related to disease progression, thus providing an alternative to liver biopsy for the noninvasive evaluation of liver injury in these patients.
2. Patients and methods 2.1. Patients and healthy controls All patients with liver diseases were followed up in the Liver and Gastroenterology Clinics of the Pitie´-Salpeˆtrie`re Hospital. Thirty-six treatmentnaive patients with chronic hepatitis C were selected according to the presence in sera of: (1) anti-HCV antibodies detected using two third generation immunoassays (Monolisa HCV plus, SANOFI, BioRad; and HCV 3.0, ABBOTT, Axsym); and (2) HCV RNA detected by RTPCR (Amplicor HCV, Roche Diagnostic Systems). All subjects were negative for HBsAg and anti-HIV antibodies. Ten patients with PBC, 10 patients chronically infected with HBV (defined by the persistence of HBsAg for /6 months), 10 patients with acute HAV (defined by the presence of IgM anti-HAV antibodies), and 10 patients with primary EBV infection (defined by VCA specific M immunoglobulins) were also included in the study. Finally, 12 age-matched (30 /50 years old) healthy subjects (7 males, 5 females), seronegative for HCV, HBV, and HIV, served as controls with normal liver function. Serum samples were taken from all patients and stored at /80 8C until analyzed. 2.2. Liver biopsies Liver biopsies were graded according to the Metavir scoring system (Bedossa and Poynard,
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1996): A being defined as the grade of necroinflammatory activity, from A0 (no activity) to A3 (severe activity); and F indicating the stage of liver fibrosis, from F0 (no fibrosis) to F4 (cirrhosis).
2.3. Biochemical assays Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma glutamyl transpeptidase (GGT) activities, and bilirubin concentrations were routinely measured by autoanalyzer. Upper limits of normal were defined as 35 IU/l, 30 IU/l, 32 IU/l, and 17 mmol/l for ALT, AST, GGT and total bilirubin, respectively.
2.4. DPPIV activity assay DPPIV was measured spectrophotometrically according to Bauvois (1990) with minor modifications necessary to a microplate-based technique adapted to serum. The enzymatic reaction results in the hydrolysis of the chromogenic substrate Gly-Pro-para -Nitroanilide (Gly-Pro-pNA) (Sigma) to liberate para-Nitroaniline (pNA). Briefly, in each well of round-bottomed 96-well ELISA plates (Beckman), 5 ml of serum were incubated with 2 ml of 3 mM (1 mg/ml) Gly-PropNA and 13 ml of 100 mM Hepes buffer, pH 7.6, containing 0.12 M NaCl, 5 mM KCl, 1.2 mM MgSO4 and 8 mM glucose. After an incubation time of 30 min at 37 8C, the reaction was stopped by the addition of 180 ml of distilled water. The yield of pNA was measured by absorbance at 405 nm. The amount of pNA was quantified by reference to a standard curve prepared with pNA (Sigma). Results were expressed as specific DPPIV activity that produces 1 mmole of pNA per minute under the assay conditions. Specificity of the activity measured was evaluated by performing the reaction in presence of 1 mM diprotin A (Sigma), the inhibitor of DPPIV. Remaining DPPIV activity was then expressed as the percentage of the control activity without the inhibitor.
Fig. 1. Boxplots of serum DPPIV activity in the control group and patients with PBC and those infected with HCV. The lines at the top, bottom, and the middle of the box correspond to the 75, 25, and 50th percentiles, respectively. The bars from the top and the bottom of the box extend to the 90 and 10th percentiles, respectively. The circles indicate the outlier cases. Hatched boxes depict healthy controls (HC) values, and grey boxes values of HCV-infected patients. PBC, primary biliary cirrhosis. S (P B/0.05) for statistical significance versus control, according to Mann-Whitney test.
2.5. Statistics All statistical calculations were performed using version 5.0 of STATVIEW software. Since the distribution of most data was non-Gaussian, the nonparametric Mann-Whitney U test was used for comparison between groups, and the nonparametric Kruskal-Wallis H test for comparison between several groups. For both tests, a P B/ 0.05 was considered statistically significant. Results are graphically represented by boxplots and expressed as medians and IQRs. Spearman rank correlation tests were used to examine the correlation between various parameters.
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Table 1 Comparison between serum DPPIV activity of healthy controls and groups of patients with diseases of different etiology
PBC, primary biliary cirrhosis; HCV, hepatits C virus; HAV, hepatitis A virus; EBV, epstein-barr virus; HBV, hepatitis B Virus. Median (50%) DPPIV activity (IU/l). b IQR, interquartile ranges (25 /75th percentiles). c Calculated as a percentage ratio to healthy controls value HC, healthy controls. a
3. Results
serum DPPIV activity does not appear to be a specific indicator of HCV infection.
3.1. Serum DPPIV activity in serum of HCVinfected patients
3.2. Correlation of serum DPPIV activity with severity of liver injury in chronic hepatitis C
Serum DDPIV activities from healthy controls individuals without any biological disorder and virus infection (healthy controls) were compared to those of patients with either chronic hepatitis C or PBC. Serum DPPIV activity was significantly increased in HCV-infected patients (median 29.78 U/l, range 24.66 /35.95) compared to healthy controls (median 21.42, range 19.76 /24.93) (P B/ 0.01) (Fig. 1 and Table 1). DPPIV activities were also significantly higher in patients with PBC, a disease of non-viral etiology (median 39.96, range 29.88 /49.48) than in uninfected controls (P B/ 0.01) (Fig. 1 and Table 1). Therefore, increased
To determine the clinical significance of increased serum DPPIV activity in HCV-infected individuals, the relationship between DPPIV activity and histological features was assessed. Although all HCV-infected patient groups showed a significant increase in their serum DDPIV activity level compared to healthy controls (P B/ 0.05) (Fig. 2A and B), no correlation was found with either their liver inflammatory activity score (graded from A0 to A2) or their stage of fibrosis (from F0 to F4). Moreover, serum DPPIV activity was not correlated with liver cytolysis, reflected by ALT and AST activities in the HCV-infected
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Fig. 2. Association of serum DPPIV activity with severity of chronic hepatitis C: (A) histological grade of necroinflammatory activity defined according to A index in Materials and Methods (M and M), (B) with stage of fibrosis scaled from 0 to 4 as described in M and M, (C) regression analysis correlating DPPIV and serum ALT activities, (D) regression analysis correlating DPPIV and AST activities. For (A) and (B) box plots of serum DPPIV activity are represented as described in Fig. 1. Hatched boxes depict healthy controls (HC) and grey boxes values of HCV-infected patients. *S (P B/0.05), significant; **NS, non-significant, according to Mann-Whitney test.
group (Fig. 2C and D). These data indicate that serum DPPIV activity is not related to liver damage in chronic hepatitis C. 3.3. Elevated serum DPPIV activity in diseases of different viral etiology In order to assess any relationship between abnormal levels of serum DPPIV and liver impair-
ment, DPPIV activity was further evaluated in sera of patients infected either with a hepatotropic virus i.e. HBV (n /10), HAV (n /10), or HCV (n /36; already studied above), or with EBV as a non-hepatotropic virus. In all cases, DPPIV activity was increased compared to that of healthy controls (Fig. 3), with a significant difference (P B/ 0.01) for HCV-, HAV- and EBV-infected groups (Table 1). Therefore, elevated DPPIV activity does
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Fig. 3. Boxplots of serum DPPIV activity for patients infected with different viruses: HCV, hepatitis C virus; HBV, hepatitis B virus; HAV, hepatitis A virus; EBV, epstein-barr virus. Boxplots of serum DPPIV enzyme activity are represented as described in Fig. 1. Hatched boxes depict healthy controls (HC) and grey boxes values of HCV-infected patients.
not appear to be exclusively related to viral hepatotropism; indeed, the highest median activity was obtained in the group with acute EBV infection. Moreover, as depicted in Fig. 3, the highest order of magnitude for raised DDPIV activity was mostly observed for the HAV and EBV groups, representative of viral infection in the acute phase, with a relative augmentation of about 70% (Table 1). Conversely, for both chronic HCV and HBV infections, a moderate increase was noticed, with relative augmentations of 40 and 20%, respectively. Therefore, the range of increase of serum DPPIV activity might be a more perti-
Fig. 4. Regression analysis correlating (A) DPPIV and GGT activities, and (B) DPPIV activity and bilirubin level, for the whole population within which each group can be identified according to different symbols.
Table 2 Correlation between serum DPPIV activity and GGT activity or bilirubin level GGT activity (IU/l)
Bilirubin concentration (mmol/l)
Groups of patientsa
Number of patients
Median (IQRb)
Pc
Number of patients
Median (IQR)
P
Whole population PBC HCV HBV HAV EBV
53 5 25 10 8 5
53 (30.75 /174.25) 170 (125.25 /657.25) 47 (29.25 /102.5) 19 (14 /53) 236.5 (87 /282.5) 35 (32.25 /151)
S (P B/0.0001) S (P /0.0476) S (P /0.0243) NS NS NS
47 6 19 10 8 5
12 (9 /29) 16 (11.25 /26.75) 10 (8.25 /12.75) 10 (8 /29) 79.5 (34.5 /135) 29 (14 /44.25)
NS NS NS NS NS NS
S, significant; NS, non-significant. GGT, gamma glutamyl transpeptidase. a Abbreviations for different groups of patients as described in Table 1. b IQR, interquartile ranges (25 /75th percentiles). c Significance of correlations estimated by Spearman Rank correlation test.
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nent indicator of the temporal stage (acute versus chronic) of any viral disease, rather than a marker of viral hepatotropism. 3.4. Serum DPPIV activity as a potent marker of cholestasis In normal human liver, DPPIV is predominantly confined to the bile canalicular domain of hepatocytes with a characteristic pattern of acinar zonal distribution (McCaughan et al., 1990). As previously described in rats with PBC and patients with various liver diseases (Perner et al., 1999), serum DPPIV activity could be an indicator of cholestasis that is associated with progressive destruction of bile ducts and results in alterations of hepatobiliary transport (Kullak-Ublick et al., 2000; Poupon et al., 2000). To establish whether a correlation exists between DPPIV activity and cholestasis in all patients included in the study, parallel measurements of DPPIV, GGT, ALT and bilirubin were performed (Table 2). According to previous reports (Lakatos et al., 1999; Perner et al., 1999), higher values for serum DPPIV activity than for healthy controls were obtained for patients with PBC, usually characterized by cholestasis (Fig. 1). In addition, in the whole patient population, DPPIV activity was significantly correlated with serum GGT (P B/0.0001, r /0.31), indicating that serum DPPIV activity could reflect cholestasis (Table 2 and Fig. 4A). However, when each group was examined separately, modulations in correlation between DPPIV activity and GGT were observed such as a significant increase for PBC and hepatitis C infection, and a slight raise of activity in HBV infection, although not reaching significance (Table 2). Moreover, no correlation between DPPIV activity and bilirubin level was observed, neither in the whole population (P / 0.05) (Fig. 4B) nor in each group of patients (Table 2). This observation argues against the use of DPPIV as a surrogate marker of cholestasis.
4. Discussion Membrane and/or soluble forms of DPPIV already represent clinical markers in some diseases
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(Iwata and Morimoto, 1999; McCaughan et al., 2000). Sources of DPPIV in serum may include release from T cells and non-lymphoid tissues, mainly the liver (McCaughan et al., 1990). Elevated serum DPPIV activity has been reported in PBC (Lakatos et al., 1999) and cirrhosis (Matsumoto et al., 1992; Lakatos et al., 2000). The current study shows that serum DPPIV activity is definitely higher in HCV-infected patients than in healthy controls. However, this elevated activity does not appear to be specific for HCV infection, since a similar increase was observed in sera from patients infected with HAV, HBV or EBV, which had not previously been documented. The role of soluble DPPIV in biological fluids remains poorly understood, but is likely multifactorial. In this study, the essential question was to determine whether the dysregulation observed in hepatitis C played a contributory role in the progression of the disease process. Given the predominant location of DPPIV in the bile canalicular domain at the acinar pole of hepatocytes (McCaughan et al., 1990), the possibility of DDPIV shedding after bile duct injury had to be considered in viral hepatitis. As recently, reported (Lakatos et al., 1999), we found that DPPIV activity was dramatically increased in the sera of patients with PBC, possibly accounting mostly for cholestasis. Moreover, the good correlation obtained for HCV-infected patients between serum DPPIV and GGT activities would have suggested the same tendency to cholestasis, except that no correlation was found with bilirubin. Accordingly, on further examination of respective values of markers of cholestasis for all patients, when both GGT and bilirubin are elevated, it appeared that only a few of them could be considered as cholestatic (data not shown). Alternatively, if resulting from liver injury, increased DPPIV activity in hepatitis C may result from cytolysis of hepatocytes rather than that of biliary epithelial cells. Yet, we did not find any correlation between DPPIV and ALT or AST levels in HCV-infected patients. Moreover, DPPIV levels did not correlate with the stage of liver fibrosis or necroinflammatory activity. Therefore, it seems unlikely that cytolysis alone could explain serum DPPIV activity. The following explanation
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could be proposed for these unexpected data. Liver fibrogenesis is mostly characterized by remodeling of the ECM, which is mediated at least in part by activated hepatic stellate cells (Friedman, 1997; Poynard et al., 2000). The activation of hepatic stellate cells leads to overexpression of fibroblast activation protein (FAP), a membrane-bound protein exhibiting an enzyme activity closely related to that of DPPIV (Levy et al., 1999). Of note is that DPPIV activity has never been found to be associated with stellate cells, although there is evidence indicating that DPPIV is involved in the interaction between hepatocytes and ECM proteins (Piazza et al., 1989). Thus, the lack of correlation between DPPIV activity and stage of fibrosis could reflect the involvement of FAP in that process, rather than DPPIV, although no enzyme activity has yet been reported for the soluble form of FAP. Moreover, it cannot be excluded that more complex mechanisms exist inside the liver tissue. Indeed, as recently, described for patients chronically infected with HCV and treated with interferon alfa, an increased DPPIV activity has been observed, indicative of liver injury (Firneisz et al., 2001). Although antiviral therapy appears to be the only parameter differing between this study and ours, it does not seem to account for the discrepancy between conclusions drawn in each case. These authors described that nonresponders to interferon had an elevated DPPIV activity equivalent to that of treatment-naive patients, whereas responders exhibited levels equivalent to healthy controls, likely due to elimination of the virus. Thus, antiviral treatment may provide an additional argument favoring the direct implication of HCV infection in the observed dysregulation of DPPIV. In contrast, our study indicates that this change in activity is not restricted to HCV, since a notable enhancement was observed in other viral infections. Therefore, the increase of DPPIV activity may originate from the immune response to viral infection, which has to be considered apart from liver damage. First, DDPIV is probably released from activated T lymphocytes by enzymatic cleavage of surface protein (Kasahara et al., 1984). Host immune responses have been reported to
play an important role in the pathogenesis of hepatic injury associated with chronic HCV infection (Cerny and Chisari, 1999; Bertoletti and Maini, 2000; Rehermann, 2000). HCV-specific CD8 cytotoxic T lymphocytes have been described to be present in a highly activated state as main components among non-lymphoid liver cells (Cooper et al., 1999; He et al., 1999). This suggests a dynamic process, necessary to maintain a pool of recruited lymphoid cells in the liver, and responsible for the release of large amounts of proinflammatory cytokines over years during chronic infection. We speculate that such a phenomenon involved in the liver destruction associated with HCV infection, results in an increased release of DPPIV. However, the absence of any correlation between liver inflammation and serum DDPIV activity does not support this hypothesis. Therein, measurement of DPPIV in the peripheral circulation may not faithfully reflect events occurring within the liver. Indeed, intrahepatic recruitment and activation of lymphocytes play a major role in liver damage either directly or by stimulating other cells such as hepatic stellate cells (Valiante et al., 2000). Second, serum DPPIV may originate from shedding from T circulating lymphocytes occurring upon T cell immune stimulation following viral infection. This possibility is unlikely to be predominant for HCV infection as the T cell immune response is weak during the chronic phase of the disease, compared to that observed in the acute phase (Rehermann and Chisari, 2000). Therefore, this phenomenon might more likely be responsible for the increase in DPPIV activity observed in both the HAV- and EBV-infected groups included in this study during the acute stage of infection. Such an immunological component could account for the significant higher activity (P B/0.05) for these two groups considered together (36.68 IU/l; IQR, 28.37 /44.79), than that observed for chronic HBV and HCV infections (28.19 IU/l; IQR, 24.10 /36.00). In conclusion, increased production of serum DPPIV appears to be multifactorial. It could partly account for the pathophysiology of cholestasis in the case of chronic hepatitis C, but to a lesser extent for other viral infections for which an immunological component is suggested to have a
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role, principally during the acute phase of infection. Further studies are needed to elucidate the origin of serum DPPIV in liver diseases and to gain insight into its implication in the disease process.
Acknowledgements We thank Bruno Rabanel for technical assistance and Dr Robert P. Myers for editing the manuscript. We are very grateful to Dr Ingrid De Meester for useful criticism, and to Profs. Pierre Opolon and Thierry Poynard for having kindly provided us, together with Miche`le Fonfrede, with the necessary clinical data from the patients included in the study. This work was supported in part by the Ministe`re de l’Education Nationale, de la Recherche et de la Technologie: programme de Recherche Fondamentale en Microbiologie et Maladies Infectieuses et Parasitaires (re´seau national des He´patites), the Association pour la Recherche contre le Cancer (contrat N85619), and the Association Claude Bernard.
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Poynard T, Ratziu V, Benmanov Y, Martino VD, Bedossa P, Opolon P. Fibrosis in patients with chronic hepatitis C: detection and significance. Semin Liver Dis 2000;20:47 / 55. Rakoczi G, Takacs L, Jakabfi P, Kunos I, Selmeci L, Gyuris T, et al. Increased urinary dipeptidyl peptidase IV activity in extrahepatic biliary atresia. Lancet 1995;345:864 /5. Rehermann B. Interaction between the hepatitis C virus and the immune system. Semin Liver Dis 2000;20:127 /41. Rehermann B, Chisari FV. Cell mediated immune response to the hepatitis C virus. Curr Top Microbiol Immunol 2000;242:299 /325. Stecca BA, Nardo B, Chieco P, Mazziotti A, Bolondi L, Cavallari A. Aberrant dipeptidyl peptidase IV (DPP IV/ CD26) expression in human hepatocellular carcinoma. J Hepatol 1997;27:337 /45. Tanaka T, Kameoka J, Yaron A, Schlossman SF, Morimoto C. The costimulatory activity of the CD26 antigen requires dipeptidyl peptidase IV enzymatic activity. Proc Natl Acad Sci USA 1993;90:4586 /90. Torimoto Y, Dang NH, Tanaka T, Prado C, Schlossman SF, Morimoto C. Biochemical characterization of CD26 (dipeptidyl peptidase IV): functional comparison of distinct epitopes recognized by various anti-CD26 monoclonal antibodies. Mol Immunol 1992;29:183 /92. Valiante NM, D’Andrea A, Crotta S, Lechner F, Klenerman P, Nuti S, et al. Life, activation and death of intrahepatic lymphocytes in chronic hepatitis C. Immunol Rev 2000;174:77 /89. Vanhoof G, De Meester I, van Sande M, Scharpe S, Yaron A. Distribution of proline-specific aminopeptidases in human tissues and body fluids. Eur J Clin Chem Clin Biochem 1992;30:333 /8.
Similar increased serum dipeptidyl peptidase IV activity in chronic hepatitis C and other viral infections T. Andrieu a, V. Thibault a, I. Malet a, J. Laporte a, B. Bauvois b, H. Agut a, A. Cahour a,* a
Laboratoire de Virologie, CERVI, UPRES EA 2387, Groupe Hospitalier Pitie´-Salpeˆtrie`re, 75651 Paris Cedex 13, France b Unite´ 365 INSERM, Institut Curie, 75248 Paris Cedex 05, France Accepted 19 July 2002
Abstract Background: Dipeptidyl peptidase IV is a transmembrane enzyme widely expressed in many cell types, but also present as a soluble form in biological fluids. Its abnormal activity is sometimes associated with liver disease related pathologies. Objectives: The aim of this study was to evaluate the clinical relevance of changes in serum DPPIV activity in hepatitis C and other viral infections. Study design: DPPIV activity was assessed by using a microplate-based colorimetric assay on serum from 88 subjects: 12 healthy uninfected controls, 10 patients with primary biliary cirrhosis (PBC) as a reference group, 36 HCV-infected patients, and patients suffering from viral infections of different etiologies. Levels of DPPIV activity were compared with: (1) those of other serum biochemical parameters such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma glutamyl transpeptidase (GGT), and bilirubin concentrations; and (2) criteria representative of liver histological status. Results: Compared with healthy subjects, DPPIV activity was significantly increased during viral infections and in PBC (P B/0.01). In HCV-infected patients, the median activity (interquartile range, IQR), 29.78 IU/l (24.66 /35.95), differed significantly (P B/0.05) from that of controls: 21.42 (19.76 /24.93). No correlation was observed between DPPIV activity and either ALT, AST, bilirubin, or the stage of liver fibrosis and necroinflammatory activity, although GGT was moderately correlated (r / 0.58, P B/0.05). Conclusions: Although we confirmed an elevation of serum DPPIV activity in PBC, it seems to be a non-specific phenomenon common to viral infections. The absence of correlation between serum DPPIV and markers of liver disease in HCV-infected patients, suggests that this activity originates not only from the liver, but also from other sources such as peripheral blood cells involved in the control of viral infections. # 2002 Elsevier Science B.V. All rights reserved. Keywords: DPPIV/CD26; HCV; Viral infection; Fibrosis; Cholestasis; Pathophysiology
1. Introduction * Corresponding author. Tel.: /33-1-45-82-62-98; fax: /331-45-82-63-14 E-mail address: [email protected] (A. Cahour).
Dipeptidyl peptidase IV (DPPIV/CD26) is a transmembrane protein expressed in a large variety of different cell types including lymphocytes,
1386-6532/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 5 3 2 ( 0 2 ) 0 0 1 2 8 - 2
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kidney, liver and epithelial cells (Heike et al., 1988; Vanhoof et al., 1992). Peptidase activity of DPPIV (EC 3.4.14.5) resides in its ectodomain and is specific of polypeptides with either L-proline or Lalanine at the penultimate amino-terminal position (Torimoto et al., 1992). As an integral membrane glycoprotein capable of transducing intracellular signals, DPPIV has been implicated in the regulation of cell growth, apoptosis and differentiation (Fleischer, 1994; De Meester et al., 1999; Iwata and Morimoto, 1999; Antczak et al., 2001a). Most of these functional properties have been elucidated in T cell proliferation through T-cell receptors (TCR) (Hegen et al., 1990; Kameoka et al., 1993; Tanaka et al., 1993). Dysregulated expression of DPPIV is observed in various human malignancies, suggesting its value as a clinical marker (Antczak et al., 2001b). Moreover, DPPIV is located in the canalicular membranes of hepatocytes (McCaughan et al., 1990), and its expression is aberrant in liver cirrhosis (Matsumoto et al., 1992; Lakatos et al., 2000) and hepatocellular carcinoma (Stecca et al., 1997). Given its altered distribution in liver diseases, DPPIV could play an important role in the maintenance of liver architecture by interacting with extracellular matrix (ECM) proteins, and/or in the tissue destruction/ regeneration process. To date, little is known about the potential functions of DPPIV in liver tissue. Harada et al. (1995) observed a significant upregulation of DPPIV in a HepG2 cell line stably transfected with HCV non-structural polyprotein. Gaetaniello et al. (1998) demonstrated that DPPIV/CD26 occupancy delivers a potent apoptotic signal in a human hepatoma cell line. Although DPPIV exists as a soluble form in body fluids including plasma, serum and urine (Chikuma et al., 1990; Maes et al., 1994; Rakoczi et al., 1995), its physiological significance and origin remain unclear. Recently, most of serum DPPIV activity (95%) has been demonstrated to be associated with CD26 (Durinx et al., 2001). DPPIV activity may vary considerably with pathological conditions (Iwata and Morimoto, 1999; McCaughan et al., 2000). Accordingly, elevated serum enzyme activity has been reported in cirrhosis (Matsumoto et al., 1992; Lakatos et al., 2000) and primary biliary cirrhosis (PBC) (Laka-
tos et al., 1999), and more recently, in chronic hepatitis C where a negative correlation with the response to interferon therapy has been observed (Firneisz et al., 2001). Chronic hepatitis C, like most of chronic liver diseases, is characterized by continuing hepatocellular injury, inflammation and cirrhosis. The aim of this study was to assess whether modifications of serum DPPIV activity in patients with chronic HCV infection are related to disease progression, thus providing an alternative to liver biopsy for the noninvasive evaluation of liver injury in these patients.
2. Patients and methods 2.1. Patients and healthy controls All patients with liver diseases were followed up in the Liver and Gastroenterology Clinics of the Pitie´-Salpeˆtrie`re Hospital. Thirty-six treatmentnaive patients with chronic hepatitis C were selected according to the presence in sera of: (1) anti-HCV antibodies detected using two third generation immunoassays (Monolisa HCV plus, SANOFI, BioRad; and HCV 3.0, ABBOTT, Axsym); and (2) HCV RNA detected by RTPCR (Amplicor HCV, Roche Diagnostic Systems). All subjects were negative for HBsAg and anti-HIV antibodies. Ten patients with PBC, 10 patients chronically infected with HBV (defined by the persistence of HBsAg for /6 months), 10 patients with acute HAV (defined by the presence of IgM anti-HAV antibodies), and 10 patients with primary EBV infection (defined by VCA specific M immunoglobulins) were also included in the study. Finally, 12 age-matched (30 /50 years old) healthy subjects (7 males, 5 females), seronegative for HCV, HBV, and HIV, served as controls with normal liver function. Serum samples were taken from all patients and stored at /80 8C until analyzed. 2.2. Liver biopsies Liver biopsies were graded according to the Metavir scoring system (Bedossa and Poynard,
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1996): A being defined as the grade of necroinflammatory activity, from A0 (no activity) to A3 (severe activity); and F indicating the stage of liver fibrosis, from F0 (no fibrosis) to F4 (cirrhosis).
2.3. Biochemical assays Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma glutamyl transpeptidase (GGT) activities, and bilirubin concentrations were routinely measured by autoanalyzer. Upper limits of normal were defined as 35 IU/l, 30 IU/l, 32 IU/l, and 17 mmol/l for ALT, AST, GGT and total bilirubin, respectively.
2.4. DPPIV activity assay DPPIV was measured spectrophotometrically according to Bauvois (1990) with minor modifications necessary to a microplate-based technique adapted to serum. The enzymatic reaction results in the hydrolysis of the chromogenic substrate Gly-Pro-para -Nitroanilide (Gly-Pro-pNA) (Sigma) to liberate para-Nitroaniline (pNA). Briefly, in each well of round-bottomed 96-well ELISA plates (Beckman), 5 ml of serum were incubated with 2 ml of 3 mM (1 mg/ml) Gly-PropNA and 13 ml of 100 mM Hepes buffer, pH 7.6, containing 0.12 M NaCl, 5 mM KCl, 1.2 mM MgSO4 and 8 mM glucose. After an incubation time of 30 min at 37 8C, the reaction was stopped by the addition of 180 ml of distilled water. The yield of pNA was measured by absorbance at 405 nm. The amount of pNA was quantified by reference to a standard curve prepared with pNA (Sigma). Results were expressed as specific DPPIV activity that produces 1 mmole of pNA per minute under the assay conditions. Specificity of the activity measured was evaluated by performing the reaction in presence of 1 mM diprotin A (Sigma), the inhibitor of DPPIV. Remaining DPPIV activity was then expressed as the percentage of the control activity without the inhibitor.
Fig. 1. Boxplots of serum DPPIV activity in the control group and patients with PBC and those infected with HCV. The lines at the top, bottom, and the middle of the box correspond to the 75, 25, and 50th percentiles, respectively. The bars from the top and the bottom of the box extend to the 90 and 10th percentiles, respectively. The circles indicate the outlier cases. Hatched boxes depict healthy controls (HC) values, and grey boxes values of HCV-infected patients. PBC, primary biliary cirrhosis. S (P B/0.05) for statistical significance versus control, according to Mann-Whitney test.
2.5. Statistics All statistical calculations were performed using version 5.0 of STATVIEW software. Since the distribution of most data was non-Gaussian, the nonparametric Mann-Whitney U test was used for comparison between groups, and the nonparametric Kruskal-Wallis H test for comparison between several groups. For both tests, a P B/ 0.05 was considered statistically significant. Results are graphically represented by boxplots and expressed as medians and IQRs. Spearman rank correlation tests were used to examine the correlation between various parameters.
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Table 1 Comparison between serum DPPIV activity of healthy controls and groups of patients with diseases of different etiology
PBC, primary biliary cirrhosis; HCV, hepatits C virus; HAV, hepatitis A virus; EBV, epstein-barr virus; HBV, hepatitis B Virus. Median (50%) DPPIV activity (IU/l). b IQR, interquartile ranges (25 /75th percentiles). c Calculated as a percentage ratio to healthy controls value HC, healthy controls. a
3. Results
serum DPPIV activity does not appear to be a specific indicator of HCV infection.
3.1. Serum DPPIV activity in serum of HCVinfected patients
3.2. Correlation of serum DPPIV activity with severity of liver injury in chronic hepatitis C
Serum DDPIV activities from healthy controls individuals without any biological disorder and virus infection (healthy controls) were compared to those of patients with either chronic hepatitis C or PBC. Serum DPPIV activity was significantly increased in HCV-infected patients (median 29.78 U/l, range 24.66 /35.95) compared to healthy controls (median 21.42, range 19.76 /24.93) (P B/ 0.01) (Fig. 1 and Table 1). DPPIV activities were also significantly higher in patients with PBC, a disease of non-viral etiology (median 39.96, range 29.88 /49.48) than in uninfected controls (P B/ 0.01) (Fig. 1 and Table 1). Therefore, increased
To determine the clinical significance of increased serum DPPIV activity in HCV-infected individuals, the relationship between DPPIV activity and histological features was assessed. Although all HCV-infected patient groups showed a significant increase in their serum DDPIV activity level compared to healthy controls (P B/ 0.05) (Fig. 2A and B), no correlation was found with either their liver inflammatory activity score (graded from A0 to A2) or their stage of fibrosis (from F0 to F4). Moreover, serum DPPIV activity was not correlated with liver cytolysis, reflected by ALT and AST activities in the HCV-infected
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Fig. 2. Association of serum DPPIV activity with severity of chronic hepatitis C: (A) histological grade of necroinflammatory activity defined according to A index in Materials and Methods (M and M), (B) with stage of fibrosis scaled from 0 to 4 as described in M and M, (C) regression analysis correlating DPPIV and serum ALT activities, (D) regression analysis correlating DPPIV and AST activities. For (A) and (B) box plots of serum DPPIV activity are represented as described in Fig. 1. Hatched boxes depict healthy controls (HC) and grey boxes values of HCV-infected patients. *S (P B/0.05), significant; **NS, non-significant, according to Mann-Whitney test.
group (Fig. 2C and D). These data indicate that serum DPPIV activity is not related to liver damage in chronic hepatitis C. 3.3. Elevated serum DPPIV activity in diseases of different viral etiology In order to assess any relationship between abnormal levels of serum DPPIV and liver impair-
ment, DPPIV activity was further evaluated in sera of patients infected either with a hepatotropic virus i.e. HBV (n /10), HAV (n /10), or HCV (n /36; already studied above), or with EBV as a non-hepatotropic virus. In all cases, DPPIV activity was increased compared to that of healthy controls (Fig. 3), with a significant difference (P B/ 0.01) for HCV-, HAV- and EBV-infected groups (Table 1). Therefore, elevated DPPIV activity does
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Fig. 3. Boxplots of serum DPPIV activity for patients infected with different viruses: HCV, hepatitis C virus; HBV, hepatitis B virus; HAV, hepatitis A virus; EBV, epstein-barr virus. Boxplots of serum DPPIV enzyme activity are represented as described in Fig. 1. Hatched boxes depict healthy controls (HC) and grey boxes values of HCV-infected patients.
not appear to be exclusively related to viral hepatotropism; indeed, the highest median activity was obtained in the group with acute EBV infection. Moreover, as depicted in Fig. 3, the highest order of magnitude for raised DDPIV activity was mostly observed for the HAV and EBV groups, representative of viral infection in the acute phase, with a relative augmentation of about 70% (Table 1). Conversely, for both chronic HCV and HBV infections, a moderate increase was noticed, with relative augmentations of 40 and 20%, respectively. Therefore, the range of increase of serum DPPIV activity might be a more perti-
Fig. 4. Regression analysis correlating (A) DPPIV and GGT activities, and (B) DPPIV activity and bilirubin level, for the whole population within which each group can be identified according to different symbols.
Table 2 Correlation between serum DPPIV activity and GGT activity or bilirubin level GGT activity (IU/l)
Bilirubin concentration (mmol/l)
Groups of patientsa
Number of patients
Median (IQRb)
Pc
Number of patients
Median (IQR)
P
Whole population PBC HCV HBV HAV EBV
53 5 25 10 8 5
53 (30.75 /174.25) 170 (125.25 /657.25) 47 (29.25 /102.5) 19 (14 /53) 236.5 (87 /282.5) 35 (32.25 /151)
S (P B/0.0001) S (P /0.0476) S (P /0.0243) NS NS NS
47 6 19 10 8 5
12 (9 /29) 16 (11.25 /26.75) 10 (8.25 /12.75) 10 (8 /29) 79.5 (34.5 /135) 29 (14 /44.25)
NS NS NS NS NS NS
S, significant; NS, non-significant. GGT, gamma glutamyl transpeptidase. a Abbreviations for different groups of patients as described in Table 1. b IQR, interquartile ranges (25 /75th percentiles). c Significance of correlations estimated by Spearman Rank correlation test.
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nent indicator of the temporal stage (acute versus chronic) of any viral disease, rather than a marker of viral hepatotropism. 3.4. Serum DPPIV activity as a potent marker of cholestasis In normal human liver, DPPIV is predominantly confined to the bile canalicular domain of hepatocytes with a characteristic pattern of acinar zonal distribution (McCaughan et al., 1990). As previously described in rats with PBC and patients with various liver diseases (Perner et al., 1999), serum DPPIV activity could be an indicator of cholestasis that is associated with progressive destruction of bile ducts and results in alterations of hepatobiliary transport (Kullak-Ublick et al., 2000; Poupon et al., 2000). To establish whether a correlation exists between DPPIV activity and cholestasis in all patients included in the study, parallel measurements of DPPIV, GGT, ALT and bilirubin were performed (Table 2). According to previous reports (Lakatos et al., 1999; Perner et al., 1999), higher values for serum DPPIV activity than for healthy controls were obtained for patients with PBC, usually characterized by cholestasis (Fig. 1). In addition, in the whole patient population, DPPIV activity was significantly correlated with serum GGT (P B/0.0001, r /0.31), indicating that serum DPPIV activity could reflect cholestasis (Table 2 and Fig. 4A). However, when each group was examined separately, modulations in correlation between DPPIV activity and GGT were observed such as a significant increase for PBC and hepatitis C infection, and a slight raise of activity in HBV infection, although not reaching significance (Table 2). Moreover, no correlation between DPPIV activity and bilirubin level was observed, neither in the whole population (P / 0.05) (Fig. 4B) nor in each group of patients (Table 2). This observation argues against the use of DPPIV as a surrogate marker of cholestasis.
4. Discussion Membrane and/or soluble forms of DPPIV already represent clinical markers in some diseases
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(Iwata and Morimoto, 1999; McCaughan et al., 2000). Sources of DPPIV in serum may include release from T cells and non-lymphoid tissues, mainly the liver (McCaughan et al., 1990). Elevated serum DPPIV activity has been reported in PBC (Lakatos et al., 1999) and cirrhosis (Matsumoto et al., 1992; Lakatos et al., 2000). The current study shows that serum DPPIV activity is definitely higher in HCV-infected patients than in healthy controls. However, this elevated activity does not appear to be specific for HCV infection, since a similar increase was observed in sera from patients infected with HAV, HBV or EBV, which had not previously been documented. The role of soluble DPPIV in biological fluids remains poorly understood, but is likely multifactorial. In this study, the essential question was to determine whether the dysregulation observed in hepatitis C played a contributory role in the progression of the disease process. Given the predominant location of DPPIV in the bile canalicular domain at the acinar pole of hepatocytes (McCaughan et al., 1990), the possibility of DDPIV shedding after bile duct injury had to be considered in viral hepatitis. As recently, reported (Lakatos et al., 1999), we found that DPPIV activity was dramatically increased in the sera of patients with PBC, possibly accounting mostly for cholestasis. Moreover, the good correlation obtained for HCV-infected patients between serum DPPIV and GGT activities would have suggested the same tendency to cholestasis, except that no correlation was found with bilirubin. Accordingly, on further examination of respective values of markers of cholestasis for all patients, when both GGT and bilirubin are elevated, it appeared that only a few of them could be considered as cholestatic (data not shown). Alternatively, if resulting from liver injury, increased DPPIV activity in hepatitis C may result from cytolysis of hepatocytes rather than that of biliary epithelial cells. Yet, we did not find any correlation between DPPIV and ALT or AST levels in HCV-infected patients. Moreover, DPPIV levels did not correlate with the stage of liver fibrosis or necroinflammatory activity. Therefore, it seems unlikely that cytolysis alone could explain serum DPPIV activity. The following explanation
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could be proposed for these unexpected data. Liver fibrogenesis is mostly characterized by remodeling of the ECM, which is mediated at least in part by activated hepatic stellate cells (Friedman, 1997; Poynard et al., 2000). The activation of hepatic stellate cells leads to overexpression of fibroblast activation protein (FAP), a membrane-bound protein exhibiting an enzyme activity closely related to that of DPPIV (Levy et al., 1999). Of note is that DPPIV activity has never been found to be associated with stellate cells, although there is evidence indicating that DPPIV is involved in the interaction between hepatocytes and ECM proteins (Piazza et al., 1989). Thus, the lack of correlation between DPPIV activity and stage of fibrosis could reflect the involvement of FAP in that process, rather than DPPIV, although no enzyme activity has yet been reported for the soluble form of FAP. Moreover, it cannot be excluded that more complex mechanisms exist inside the liver tissue. Indeed, as recently, described for patients chronically infected with HCV and treated with interferon alfa, an increased DPPIV activity has been observed, indicative of liver injury (Firneisz et al., 2001). Although antiviral therapy appears to be the only parameter differing between this study and ours, it does not seem to account for the discrepancy between conclusions drawn in each case. These authors described that nonresponders to interferon had an elevated DPPIV activity equivalent to that of treatment-naive patients, whereas responders exhibited levels equivalent to healthy controls, likely due to elimination of the virus. Thus, antiviral treatment may provide an additional argument favoring the direct implication of HCV infection in the observed dysregulation of DPPIV. In contrast, our study indicates that this change in activity is not restricted to HCV, since a notable enhancement was observed in other viral infections. Therefore, the increase of DPPIV activity may originate from the immune response to viral infection, which has to be considered apart from liver damage. First, DDPIV is probably released from activated T lymphocytes by enzymatic cleavage of surface protein (Kasahara et al., 1984). Host immune responses have been reported to
play an important role in the pathogenesis of hepatic injury associated with chronic HCV infection (Cerny and Chisari, 1999; Bertoletti and Maini, 2000; Rehermann, 2000). HCV-specific CD8 cytotoxic T lymphocytes have been described to be present in a highly activated state as main components among non-lymphoid liver cells (Cooper et al., 1999; He et al., 1999). This suggests a dynamic process, necessary to maintain a pool of recruited lymphoid cells in the liver, and responsible for the release of large amounts of proinflammatory cytokines over years during chronic infection. We speculate that such a phenomenon involved in the liver destruction associated with HCV infection, results in an increased release of DPPIV. However, the absence of any correlation between liver inflammation and serum DDPIV activity does not support this hypothesis. Therein, measurement of DPPIV in the peripheral circulation may not faithfully reflect events occurring within the liver. Indeed, intrahepatic recruitment and activation of lymphocytes play a major role in liver damage either directly or by stimulating other cells such as hepatic stellate cells (Valiante et al., 2000). Second, serum DPPIV may originate from shedding from T circulating lymphocytes occurring upon T cell immune stimulation following viral infection. This possibility is unlikely to be predominant for HCV infection as the T cell immune response is weak during the chronic phase of the disease, compared to that observed in the acute phase (Rehermann and Chisari, 2000). Therefore, this phenomenon might more likely be responsible for the increase in DPPIV activity observed in both the HAV- and EBV-infected groups included in this study during the acute stage of infection. Such an immunological component could account for the significant higher activity (P B/0.05) for these two groups considered together (36.68 IU/l; IQR, 28.37 /44.79), than that observed for chronic HBV and HCV infections (28.19 IU/l; IQR, 24.10 /36.00). In conclusion, increased production of serum DPPIV appears to be multifactorial. It could partly account for the pathophysiology of cholestasis in the case of chronic hepatitis C, but to a lesser extent for other viral infections for which an immunological component is suggested to have a
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role, principally during the acute phase of infection. Further studies are needed to elucidate the origin of serum DPPIV in liver diseases and to gain insight into its implication in the disease process.
Acknowledgements We thank Bruno Rabanel for technical assistance and Dr Robert P. Myers for editing the manuscript. We are very grateful to Dr Ingrid De Meester for useful criticism, and to Profs. Pierre Opolon and Thierry Poynard for having kindly provided us, together with Miche`le Fonfrede, with the necessary clinical data from the patients included in the study. This work was supported in part by the Ministe`re de l’Education Nationale, de la Recherche et de la Technologie: programme de Recherche Fondamentale en Microbiologie et Maladies Infectieuses et Parasitaires (re´seau national des He´patites), the Association pour la Recherche contre le Cancer (contrat N85619), and the Association Claude Bernard.
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