Soluble tumor necrosis factor receptors in chronic hepatitis C: a correlation with histological fibrosis and activity

Journal of Hepatology 1999; 30:185-191 Printed in Denmark • All rights reserved Munksgaard. Copenhagen

Copyright © European Association for the Stu@ of the Liver 1999 Journal of Hepatology ISSN 0168-8278

Soluble tumor necrosis factor receptors in chronic hepatitis C: a correlation with histological fibrosis and activity Herv6 Zylberberg 1., Anne-C6cile Rimaniol 2., Stanislas Pol 1, Annie Masson 2, Donat De Groote 3, Pierre Berthelot 1, Jean-Franqois Bach 2, Christian Br6chot ~'4'5 and Flora Zavala 2 1Unit~ d'HOpatologie, Hdpital Necker, 21NSERM U-25, Hdpital Necker, 4 INSERM U-370, Hdpital Necker, 5Hybridotest lnstitut Pasteur, Paris, France, and 3Biosource Europe SA., Zoning Industriel, Fleurus, Belgium

Background~Aims: Tumor necrosis factor-alpha (TNF) is a mediator of inflammation and cellular immune response. Soluble TNF receptors (sTNFR) sTNF-R55 and sTNF-R75, which compete with cellular receptors for the binding of TNF, have been detected at high levels in infectious diseases including human immunodeficiency virus and HBV infection. In order to investigate the activation of the TNF system in HCV infection, we have analyzed the balance between TNF and sTNF-R in 60 HCV-infected subjects according to their clinical, biological, virological and histological characteristics. Methods: Serum TNF, sTNF-R55 and sTNF-R75 levels were determined by ELISA before any therapy and were compared to a control group of 60 healthy subjects and a group of 34 HBV-infected patients. Results: Mean TNF levels were 50.5_+4.5 pg/ml in HCV patients, and undetectable (<5 pg/ml) in the control subjects, sTNF-R55 and sTNF-R75 levels were significantly higher in HCV-infected patients than in the controls: 2.88_+0.14 ng/ml vs 1.30_+0.05, (p=0.0001), and 9.54_+0.58 ng/ml vs 4.19_+016, (p= 0.0001), respectively, sTNF-R55 and TNF-a levels in HCV patients were not significantly different from levels in HBV patients, sTNF-R75 levels were slightly lower than in HBV patients (9.54_+0.58 vs 11.4_+0.79

ng/ml, p=0.03). In contrast to other infectious diseases, there was no correlation between levels of sTNF-R and TNE sTNF-R75 but not TNF levels were correlated with aminotransferases levels (17= 0.0001 and p=0.0015 for aspartate and alanine aminotransferase, respectively), while sTNF-R55 levels were significantly correlated only with aspartate aminotransferase levels (p=0.003). sTNF-R75 levels were significantly correlated with the Metavir activity index (p=0.01), and sTNF-R55 and sTNF-R75 levels were significantly higher in patients with vs. without cirrhosis (3.22+0.21 vs. 2.54--.0.17 ng/ml (p<0.02) and 11.6_+0.86 vs. 7.5_+0.53 ng/ml (p<0.001), respectively), sTNF-R55, sTNF-R75 and TNF levels were not correlated with viral load, genotype or response to interferon therapy. Conclusions: Levels of soluble TNF receptors, and particularly sTNF-R75, are significantly correlated with the severity of the disease but not with virological parameters such as quantitative viremia and genotype. High TNF-R production could thus suggest that HCV-related liver disease involves immunological mechanisms, including activation of the TNF system.

MOR NECROSISfactor a (TNF-a) is a mediator of nflammation and cellular immune response produced primarily by activated monocytes, and Kupffer cells (1,2). This cytokine exhibits a wide range of bio-

logical properties, including cytotoxicity against tumors and virus-infected cells, direct antiviral activity, and stimulation of numerous immune effector cells through binding to specific cellular receptors (TNF-R), TNF-R55 and TNF-R75 (3,4). Extracellular domains of TNF receptors can be shed by proteolytic cleavage, giving rise to soluble TNF receptors (sTNF-R55 and sTNF-R75), which retain their ability to bind circulating TNF-a and inhibit its biological activity by preventing its binding to cellular re-

Received 23 February; revised 17 August; accepted 8 September 1998

Correspondence: Herv6 Zylberberg, Unit6 d'H6patologie, H6pital Necker, 149 rue de S~vres, F-75015 Paris, France. Tel: +33 1 444943 32. Fax: 33 1 444943 30. * HZ and ACR have equally contributed to this work.

Key words: Hepatitis C virus; Liver biopsy; Tumor necrosis factor; Tumor necrosis factor receptors.

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ceptors (5-7). Various cytokines, among which is TNE can induce sTNF-R shedding, which therefore represent circulating markers of the degree of the inflammatory reaction with higher stability than TNF-a itself. Accordingly, soluble forms of both TNF-R are present in the serum of healthy subjects and increased circulating levels of sTNF-R55 and sTNF-R75 have been reported in both inflammatory and infectious diseases, including cancer (8), autoimmune diseases such as lupus (9), rheumatoid polyarthritis (10), meningococcal infection (11), sepsis (12) or human immunodeficiency virus (HIV) infection (13-16), suggesting a direct or indirect implication of the TNF system in the pathophysiology of these diseases. The TNF/TNF-receptor system has been reported to play a role in hepatitis B infection (17) in which the mechanisms of liver injury are related to immune cellular response against hepatitis B antigens expressed by infected hepatocytes. Because the pathogenicity of hepatitis C virus (HCV) infection is still debated (18) and the implication of TNF-a in liver injury in chronic hepatitis C has not been clearly demonstrated, the aim of this study was to investigate the relationship between TNFa together with the soluble forms of receptors sTNFR55 and sTNF-R75, and clinical, biological, virological and histological characteristics of HCV-infected patients.

Patients and Methods Patients A m o n g our cohort of 1620 anti-HCV positive subjects, we randomly selected 60 viremic i m m u n o c o m p e t e n t patients (defined by absence of HIV infection, organ transplantation, hemodialysis or other situations of immunosuppression such as hemopathy or drug-related immunosuppression) including 30 subjects with chronic hepatitis and 30 with cirrhosis, respectively. There were 41 males and 19 females, with a m e a n age o f 4 8 _+ 12 (range 22 to 74 ) years. They had not received any antiviral or i m m u n o m o d u l a t o r y treatments in the preceding 6 months. All patients were anti-HCV positive by the second-generation ELISA assay and were viremic as assessed by serum H C V R N A determination. H C V infection was related to blood transfusions in 36%, intravenous drug usage in 20%, and was of u n k n o w n etiology in 44%. Genotype distribution was as follows: 1: 60%; 2: 7%; 3: 20%; others: 13%. After blood collection, all patients were treated with standard alpha-2b recombinant interferon (IFN-a) treatment: 3 M U IntronA subcutaneously three times weekly for 6 or 12 months. Responders were classically defined as patients who normalized transaminase activities within the first 3 m o n t h s of treatment (n = 30, 50%): long-term complete responders (n = 14, 23%) still had n o r m a l transaminase activities during therapy and at least 6 m o n t h s after therapy, while relapsers (n = 16, 27%) increased transaminase activities after cessation of treatment. Non-responders were defined as those patients who did not normalize transaminase activities during therapy (n = 30, 50%). Six treated patients were prospectively analyzed. Blood samples were collected before, during (every 3 months) and 6 to 12 m o n t h s after the end of therapy. A group of 34 i m m u n o c o m p e t e n t HBV-infected patients was analyzed. They were 26 males and 8 females with a mean age of 41 _+13

186

(range 18 to 79). They had not received any antiviral or i m m u n o m o d ulatory treatments in the preceding 6 months. Fourteen had cirrhosis, 26 (61%) had detectable HBV-DNA and 17 (50%) were HBVAg positive.

Methods Histological analysis. Liver biopsy samples were embedded in paraffin. Sections were stained with hematoxylin and eosin and Perls stain. Chronic liver disease was classified as chronic hepatitis or cirrhosis. For an accurate evaluation of the activity of the liver disease, a semiquantitative scoring system was blindly used according to the Metavir scoring method. This method scores necroinflammatory activity (A0 to A3) and fibrosis (F0 to F4).

Virological analysis. Serological anti-HCV assays." Serum antibodies to HCV were detected with the second-generation HCV enzymelinked immunosorbent assay (Raritan, New Jerse>: USA), according to the manufacturer's instructions. Qualitative detection of serum H C V RNA: Briefly, R N A was extracted from 150 Ill of serum, reverse transcribed to c D N A and amplified by polymerase chain reaction (PCR) as previously described by a standard nested PCR method with primers located in the 5' non-coding region. Amplified products were stained with ethidium bromide and hybridized with a radiolabeled oligonucleotide probe within the amplified sequence. Quantitative detection of serum H C V RNA." Quantitative viremia was evaluated by the b - D N A test (Quantiplex, Chiron Corporation, Emeryville, California, USA) using the manufacturer's protocol. This sandwich assay with signal amplification is based on specific hybridization of synthetic oligonucleotides to the 5' untranslated and core regions of HCV R N A in a crude serum lysate. The b D N A assay is quantitative and gives results in numbers of copies of HCV genome equivalents per milliliter (Eq/ml). Quantitative viremia was also evaluated in some patients by a quantitative PCR using the commercially available Monitor c~ test (Roche Diagnostic Systems. Neuilly sur Seine, France). Determination of HCVgenotype: This analysis was based on a restriction fragment length polymorphism assay (RFLP). Briefly, c D N A s were subjected to an amplification, and three aliquots of the PCR products were digested by three specific restriction endonucteases, BstN1, BstU 1 and Sau3a, according to the manufacturer's conditions (Biolabs). The fragments were electrophoresed and detected by ethidium bromide staining during the comparison with a standard-size D N A marker (100 bp ladder-Promega). Their sizes were then used to determine the different genotypes 1, 2, 3 and 4. 5 and the subtypes la and lb. sTNF-R and TNF-a assays. Blood (5 ml) was collected in sterile vacu u m tubes without additives. After centrifugation (600 g, 10 min), serum was stored in 200-i,1 aliquots at -70°C. s T N F - R and T N F - a were assayed in duplicate in serum samples which were thawed only once. TNF-sR55, TNF-sR75 and T N F - a were measured with commercial ELISA methods (Biosource Europe SA, Fleurus, Belgium), according to the manufacturer's instructions. The detection limit was 50, 100 and 5 pg/ml, for sTNF-R55, sTNF-R75 and T N F - a , respectively. s T N F - R to T N F - a molar ratio was defined as follows: [sTNF-R] (pg/ml) x Molar mass T N F [TNF-a] (pg/ml) x Molar mass s T N F - R

Statistical andlysis Data were analyzed with StatView T M SE+ software (Abacus Concept, Inc., Berkeley. CA, USA). Values are given as mean_+SE. The statistical analysis was performed using nonparametric tests (Mann-Whitney U and Kruskal-Wallis tests). Correlation analysis

Soluble TNF receptors in HCV infection

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Serum sTNF-R and TNF-a levels in HCV-infected subjects: comparison with healthy subjects and HB Vinfected subjects sTNF-R levels were significantly higher in the 60 HCVinfected patients than in the 60 healthy controls (sTNF-R55:2.88___0.14 ng/ml vs. 1.30_+0.05, p = 0.0001, and sTNF-R75:9.54___0.58 ng/ml vs. 4.19___016, p=0.0001) (Fig. 1). The mean level of TNF-a was 50.5+__4.5 pg/ml in HCV-infected patients and undetectable in controls (<5 pg/ml). As previously described (17), sTNF-Rs and TNF-a levels were significantly higher in HBV-infected patients than in control subjects (sTNF-R55:2.97___0.11 ng/ml; sTNF-R75:11.4--+0.79 ng/ml, TNF: 47.7___10 pg/ml, p=0.0001), sTNF-R55 and TNF-a levels were not statistically different in HCV patients vs HBV patients, circulating sTNF-R75 levels were lower in HCVinfected patients than in HBV-infected patients (p= 0.03). The sTNF-R55/TNF-~ and sTNF-R75/TNF-~ molar ratios in HCV-infected patients were 32-+ 17.3 and 79.3-- 35, respectively (mean-+ SE of individual ratios), with a strong correlation between circulating levels of sTNF-R55 and sTNF-R75 in HCV-infected patients (p=0.0001). No significant relationship was observed between circulating levels of sTNF-R and TNF-a (p= 0.08 and p=0.09 for sTNF-R55 and sTNF-R75, respectively).

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(B) and TNF-a (C) in healthy controls (n=60), in hepatitis B virus-infected patients (n=34) and in hepatitis C virus-infected patients (n=60). Horizontal bars denote mean values. Statistical significance (p) was evaluated by Mann-Whitney U-test.

Serum levels of sTNF-R and TNF-a and the severity of H C V disease A strong correlation was observed between sTNF-R75 levels and aminotransferase levels (p=0.0001 and p = 0.0015 for aspartate aminotransferase (AST) and alanine aminotransferase (ALT), respectively), while sTNF-R55 levels were only significantly correlated with AST levels (p=0.003). Neither TNF-a levels, nor sTNF-R/TNF molar ratio were correlated with transaminase levels (Table 1).

TABLE 1 Correlation between serum levels of sTNF-R55, sTNF-R75, T N F - a and sTNF-R/TNF ratio and aminotransferase level in HCV-infected patients

AST ALT

sTNF-R55

sTNF-R75

TNF-a

sTNF-R55/TNF-a

sTNF-R75/TNF-a

0.30** 0.017

0.48**** 0.33**

0.13 0.16

0.28 0.30

0.41 0.24

Values represent correlation coefficient (R). p-values were calculated using Spearman rank-regression analysis. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

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Levels of sTNF-R75, but not sTNF-R55, increased with the Metavir activity score (8.62+0.86 ng/ml, 9.65_+0.87 and 12.38+1.16 for groups A1, A2 and A3, respectively, p=0.01) (Fig. 2). TNF-a levels also increased with the overall Metavir activity score (p= 0.03); interestingly, the significance was higher when comparing group A3 vs A1 and A2 (46+5.88 pg/ml, 36.8+6.7 pg/ml and 65.5+11.2 pg/ml for groups A1, A2 and A3 respectively, p=0.02). 188

sTNF-R55 and sTNF-R75 levels were significantly elevated in patients with cirrhosis as compared to patients without cirrhosis (3.22+0.21 vs. 2.54_+0.17 ng/ ml, p=0.016 for sTNF-R55 and 11.6_+0.86 vs. 7.5_+0.53 ng/ml, p=0.0002 for sTNF-R75, respectively). Circulating levels of TNF-a were not significantly different in patients with and without cirrhosis (55.5-+6.9 vs 45.8_+6.14 pg/ml, respectively) (Fig. 3). We found no differences in sTNF-R/TNF molar ratio between the two groups of patients: 32.5_+17 vs. 31_+15.5 and 87.7+52.3 vs 68.7_+36.2 for sTNF-R55/ TNF and sTNF-R75/TNE respectively. Serum levels of s T N F - R and TNF-a and virological characteristics in HCV-infeeted patients We found no correlation between circulating levels of sTNF-R55, sTNF-R75 and TNF-cz and the levels of HCV RNA in serum of 36 subjects (p=0.92; p=0.92 and p=0.81, respectively) (data not shown). We found no significant differences between pretreatment levels of sTNF-Rs, TNF-a or sTNF-Rs/TNF-a and genotype distribution (data not shown). Serum levels of s T N F - R and TNF-a and the severity of H B V disease We compared sTNF-R and TNF levels with clinical, virological and histological characteristics of HBV-infected patients. We found a correlation between sTNFR75 levels and aminotransferase levels (p=0.0003 and p=0.003 for AST and ALT, respectively), sTNF-R75 levels increased with Metavir activity score (A0: 7.94_+0.85, AI: 10.80_+1.4, A2: 9.93_+0.77, A3: 16.34 + 1.47 ng/ml, p=0.0073), and with fibrosis index (F0: 8.33_+2, FI: 8.97_+1, F2: 12.1_+2.5, F3: 8.76+0.9, F4:15_+1.2 ng/ml, p=0.009), sTNF-R55 and TNF-a levels were not correlated with these parameters. We found no correlation between circulating levels of sTNF-R55, sTNF-R75 and TNF-a and serum level of HBV DNA and presence of HBeAg (data not shown). Serum levels of s T N F - R and TNF-a and the response to therapy in H C V patients Circulating sTNF-Rs and TNF-a levels were measured 1 day before treatment with IFN-a in the 60 HCV patients. We investigated whether circulating TNF and/ or sTNF-R could be predictive of the response to interferon treatment. We found no significant differences in levels of sTNF-R55, sTNF-R75, TNF-a or sTNF-R/ TNF molar ratio between the patients who failed to respond to IFN-a treatment (n=30) and those who exhibited a sustained response (n= 14) or a primary response (n=30, including long-term responders and relapsers).

Soluble TNF receptors in HCV infection

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Moreover, we did not find significant variation in sTNF-R level during therapy (measurements were performed every 3 months during and 12 months after therapy) in six subjects including responders (two longterm responders and two relapsers) and non-responders (n=2) (data not shown).

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Fig. 3. Serum concentration of sTNF-R55 (A), sTNF-R75 (B) and TNF-ct (C) in chronic hepatitis C patients without (n=30) and with cirrhosis (n=30). Horizontal bars denote mean values. Statistical significance (p) was evaluated by Mann- Whitney U-test.

The mechanisms of pathogenesis in chronic hepatitis C are still a matter of debate. The relative contribution of viral cytopathic effect and immune-related lesions in this setting remains unclear. TNF-a has been involved in the pathogenesis of a diversity of liver conditions including viral hepatitis (19). The precise stimulus responsible for enhanced TNF-a production in HCV infection has not been clearly defined. TNF-a may be generated during the inflammatory reaction that follows the immune recognition of viral antigens, and/or by infected cells, in response to intracellular viral compounds. A moderate but significant increase of circulating TNF-a levels in patients with A3 vs. A1 and A2 Metavir activity scores was observed. However, TNF-a levels did not correlate with viral load. The short half-life of TNF-a in biological fluids may in part overshadow the implication of TNF-a in HCV-disease. It has been proposed that circulating sTNF-R levels reflect activation of the TNF system, elevated levels persisting longer than those of TNF itself (20). The most likely mechanism for the release of soluble TNF receptors is a proteolytic processing, which leads to shedding of soluble TNF receptors from the cell membranes (21). Soluble TNF receptors are released by activated neutrophils, mononuclear blood cells and fibroblasts (22,23), in response to diverse activators, including bacterial products, HIV and cytokines (IL-10, IL-2, GM-CSF) (24-27). Interestingly, TNF-a itself is also a potent inducer of sTNF-R by mononuclear blood cells (28,29). A strong correlation between circulating levels of sTNF-R and TNF-a has been reported in various diseases, including HIV infection (30), or chronic renal failure (31). The fact that we did not find a correlation between TNF and soluble TNF receptors in the serum of HCV-infected patients suggests that sTNF-R production is not restricted to TNF-a production, but could be induced either by a network of cytokines or directly by viral infection independently of the resulting TNF-a production, as we previously demonstrated for HIV infection (25). Moreover, it is conceivable that cells other than blood cells may be responsible for TNF-R shedding, as suggested by the enhanced expression of TNF-R on hepatocytes reported in various inflammatory liver dis189

H. Zylberberg et al.

eases, including chronic hepatitis B, chronic hepatitis C, autoimmune and alcoholic hepatitis (32). Furthermore, sTNF-R could also be directly released by necrosis of hepatocytes. Soluble TNF-R, and particularly sTNF-R75 levels, were strongly correlated with the severity of the disease assessed by aminotransferase levels, histological activity and fibrosis, but not with quantitative viremia nor with genotype distribution. Because the fibrotic scores in our randomly selected group of HCV patients happened to be either minimal, or severe, i.e. with cirrhosis, this did not allow us to establish a strict correlation between sTNF-R levels and the degree of fibrosis. However, a significant increase was found for both sTNF-R55 and sTNF-R75 levels when comparing patients with vs. without cirrhosis. Soluble TNF-R55 levels were not correlated with the Metavir activity score. This discrepancy between the two soluble receptor types may originate in the fact that the Metavir scoring method does not discriminate between the necrotic and the inflammatory processes that take place in liver disease. Although T N F bioactivity including necrosis is preferentially mediated through TNF-R55 and may play a role in sTNF-R55 release, sTNF-R75 is released at much higher levels than sTNF-R55 by inflammatory cytokines, possibly resulting in an overestimation of the inflammatory vs. necrotic process through the soluble T N F receptor system, with this scoring method. Our results in HBV-infected patients, showing that sTNF-R75 levels were significantly enhanced in association with hepatic inflammation and fibrosis, but not with hepatitis B virus replication, are in accordance with previous results (17). Altogether, these results suggest that sTNF-R75 particularly may be a marker of severity in both HCV- and HBV-related liver diseases. Soluble TNF-R55 and TNF-R75 are present in serum of HCV-infected patients in a 30- and 80-fold excess over TNF-a, respectively. As the sTNF-R concentration must be 500- to 1000-fold higher than the TNF-c~ level to block 50% of T N F - a bioactivity (unpublished observations and (33)), circulating sTNF-R levels in the patients studied would not have been adequate to neutralize free circulating T N F bioactivity. These observations, together with the absence of correlation between sTNF-R/TNF-a ratio and activity of the disease suggest that the relationship between circulating sTNF-R levels and disease activity is not solely related to bioactive TNF-a, but may also result from the activation of other immune mediators. Little is known about the mechanisms determining the response to interferon-alpha in HCV infection. Re190

cently, Larrea et al. (34) reported that: 1. pretreatment levels of T N F - a m R N A in peripheral blood mononuclear cells and liver cells were significantly higher in HCV-infected patients who failed to respond to IFNa than in cases exhibiting sustained complete response; 2. the highest values of T N F - a transcripts were observed in patients with genotype lb (34), which is known to be associated with IFN-a resistance (35). Since we did not find any relation between pre-therapeutic circulating levels of TNF-a and response to IFN-a, TNF-a gene expression but not short-lived circulating T N F - a might be indicative of the response to IFN-a in HCV infection. Moreover, at variance with HBV infection, where elevated serum levels of sTNFR before interferon therapy were found to predict a successful response to treatment (17), we did not find any relationship between pre-therapeutic circulating levels of sTNF-R in HCV-infected patients and the response to IFN-a treatment. In addition, in contrast to a previous report (36), we did not find any increase in sTNF-R levels under therapy. These observations could reflect a major difference in pathophysiology for HCV and HBV infections, and, particularly, in viral clearance during IFN therapy: HBV clearance under IFN therapy is closely related to cellular immune response as reflected by increasing levels of aminotransferases before seroconversion, whereas antiviral effect is more probably involved in HCV infection. In conclusion, we found that elevated circulating sTNF-R present in HCV-infected patients were significantly correlated with liver injury. This relationship is independent of circulating TNF-a levels. High TNFR production could thus reflect an intense immune response and/or liver injury during HCV infection. Involvement of cellular immune response in this setting remains to be established.

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