Correspondence Dr Chao-Hung Hung, Department of Internal Medicine, Division of Hepatogastroenterology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, 123 Ta Pei Road, Niao Sung 833 Kaohsiung, Taiwan Tel: +886 7 7317123, ext. 8301 Fax: 886 7 7322402 e-mail: email@example.com
Background: Steatosis is recognized as a predictor of the severity as well as the progression of fibrosis in chronic hepatitis C. The mechanisms that cause increased hepatocellular injury associated with steatosis remain largely unknown.
Methods: We studied the correlation of hepatic expression of death receptors: Fas and tumour necrosis factor-α receptor 1 (TNF-R1), and downstream caspase (caspase-3) with hepatic steatosis by immunohistochemical study in chronic hepatitis C and determined the role of nuclear factor-κB (NF-κB).
Results: Ninety patients (49 males and 41 females, mean age of 50.5 ± 10.4 years, genotype 1 or 2) with chronic hepatitis C virus infection were recruited. The factors associated with steatosis grade were body mass index (P=0.004) and fibrosis stage (P=0.034). Moderate/severe steatosis was an independent variable associated with advanced fibrosis stage by stepwise logistic regression analysis. The expression of immunoreactivity for Fas, TNF-R1 and active caspases-3 in liver tissues was significantly correlated with the steatosis grade (P<0.001, P<0.001 and P<0.001 respectively). The extent of active caspases-3 correlated significantly with the expression of Fas (r=0.659, P<0.001) and TNF-R1 (r=0.617, P<0.001). NF-κB p65 expression correlated significantly with the extent of Fas (r=0.405, P<0.001), TNF-R1 (r=0.448, P=0.002) and active caspase-3 (r=0.313, P=0.003), and correlated with steatosis grade (P<0.001) but not with inflammatory and fibrosis scores.
Conclusion: Our observations suggest a mechanism whereby steatosis contributes to the progression of liver injury in chronic hepatitis C through upregulation of death receptors and activation of NF-κB.
Chronic hepatitis C virus (HCV) infection is associated with a wide spectrum of liver injury, ranging from mild hepatitis to cirrhosis. Approximately 20–30% of patients have an accelerated course and will eventually develop cirrhosis over decades (1–4). Factors implicated as the cause of progressive disease include viral factors, older age at infection, male gender and cofactors such as excessive alcohol intake and an elevated body mass index (BMI) (4–12). A number of studies have now demonstrated a significant relationship between steatosis and fibrosis, suggesting that either steatosis or its worsening is a strong independent predictor of the severity as well as the progression of fibrosis (12–15).
The mechanisms by which steatosis may contribute to progression of chronic HCV infection remain largely unknown. One possible explanation is that steatosis enhances hepatocyte apoptosis, which is recognized as an important mechanism in toxic liver injury, and may be associated with liver fibrogenesis and development of cirrhosis (16–18). Apoptosis is prominent in experimental ethanol-induced injury, and has been described recently in patients with non-alcoholic steatohepatitis and alcoholic steatohepatitis (19–22). However, only a few studies have investigated potential mechanistic associations between apoptosis and steatosis in chronic hepatitis C (16–18). Death receptor–ligand interactions are important initiators of apoptosis by the so-called extrinsic pathway. So far, the association of hepatic expression of death receptors: Fas (CD95) and tumour necrosis factor-α receptor 1 (TNF-R1) with steatosis in chronic hepatitis C has not been determined.
Nuclear factor-κB (NF-κB) is a transcription factor that regulates both pro-inflammatory and anti-apoptotic genes and its activation may contribute to HCV-mediated pathogenesis (23–28). Activated NF-κB can also induce genes that regulate Bcl-2 family and caspase function. A recent study has reported that patients with alcoholic hepatitis or non-alcoholic steatohepatitis had a remarkable expression of active NF-κB as compared with controls (22). To date, the role of NF-κB in the disease progression contributed by steatosis in chronic hepatitis C has not been clarified.
In the present study, we aimed to determine the relation of hepatic expression of death receptors (Fas, TNF-R1) and caspase-3 to steatosis in chronic hepatitis C by immunohistochemical analysis. Furthermore, we studied the correlation of hepatic expression of death receptors and caspases-3 with NF-κB.
Patients and methods
From October 2004 to July 2006, a total of 238 consecutive patients with chronic hepatitis C had undergone liver biopsies in our department. They included 90 (37.8%) with no steatosis, 110 (46.2%) with mild steatosis and 38 (16.0%) with moderate/severe steatosis. We randomly selected 30 patients from each steatotic grade for enrolling into this study. The diagnosis of chronic hepatitis C was based on elevated serum alanine aminotransferase (ALT) levels for at least 6 months, histological examination and persistent detectable HCV RNA. Each patient was positive for antibody to HCV (by third-generation enzyme-linked immunosorbent array (ELISA)]. Patients were excluded if they had any evidence of autoimmune hepatitis, markers of hepatitis B surface antigen, hepatitis D or human immunodeficiency virus and a previous history of interferon-based antiviral therapy. Alcohol abuse was defined as a high consumption of alcohol more than 50 g/day (4). The Human Research and Ethics committee (Institutional Review Board) approved this study and informed consent was obtained from each patient involved in this study.
Pathological diagnosis was performed by percutaneous liver biopsies, which were analysed by a single pathologist (F.-Y. K.) unaware of the patients' characteristics. Histological grading and staging were scored according to the modified Knodell histology activity index (HAI), reflecting the degree of hepatic inflammation and fibrosis respectively (29). Steatosis was scored according to the Metavir classification system (30), as follows: 0, no steatosis; mild, involving <10% of hepatocytes with steatosis; moderate, 10–30% of hepatocytes affected; and severe, more than 30% of hepatocytes affected.
Qualitative measurement of hepatitis C virus RNA and hepatitis C virus genotyping
Serum was prepared in a laminar flow bench and frozen at −80 °C. Qualitative detection of HCV RNA was performed by a standardized qualitative reverse transcription-polymerase chain reaction assay (Amplicor™; Roche Diagnostics, Branchburg, NJ, USA), using biotinylated primers for the 5′ noncoding region. The lowest detection of the assay was 100 copies/ml. Genotyping of HCV was carried out by a reverse hybridization assay (Inno-LiPA™ HCV II; Innogenetics N.V., Gent, Belgium) in the HCV-Amplicor products.
Immunohistochemical staining for Fas, tumour necrosis factor-α receptor type 1, caspase-3 and active nuclear factor-κB p65
The immunoCruz® staining system was used in deparaffinized liver tissue according to the manufacturer's instructions (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Briefly, slides were soaked in 3% hydrogen peroxide for 5 min, washed and incubated in serum blocking solution for 20 min. Specimens were then incubated with primary antibodies for 2 h at 37 °C. Tissue samples were probed with mouse monoclonal antibodies reactive to TNF-R1 and NF-κB p65, and rabbit polyclonal anti-Fas receptor and caspase-3 antibodies. After rinsing, specimens were incubated with biotinylated secondary antibody and a horseradish peroxidase–streptavidin complex, for 30 min each. Tissue samples were then colorized with 3,3'-diaminobenzidine substrate, counterstained, mounted and examined. Fas, TNF-R1 and caspase-3 immunohistochemistry was semiquantitatively evaluated using a three-point scoring system [(i) positive staining in <30% of cells/high-power field (× 400); (ii) positive staining in 30–70% of cells/high-power field and (iii) positive staining >70% of cells/high-power field]. NF-κB immunoreactivity was expressed as the number of positive cells/high-power field (× 400). The sections were scored by a single pathologist (F.-Y. K.) who was blinded to the clinical data, laboratory parameters and experiment design. There was disagreement between scores given by the two assessments in <5% of the cases for each immunostaining. Any cases with discrepant scores were reassessed to produce final scores for further analyses. The immunohistochemistry assay was selective for each marker, in that reactive product is not observed in the absence of the respective primary antibody.
Continuous variables were presented as mean ± standard deviation, and discrete variables were expressed as numbers per study population. Differences between groups are analysed using χ2, Fisher's exact, one-way anova and Student's t-tests for statistical analysis where appropriate. Univariate analysis by Spearman's test and stepwise multiple logistic regression were used to identify the variables. A two-sided P value <0.05 was considered to be statistically significant.
Baseline characteristics and factors associated with steatosis and fibrosis
Table 1 shows the baseline characteristics of the 90 patients with chronic hepatitis C. They were 49 males and 41 females, 18–77 years old, with a mean age of 50.5 ± 10.4 years. Twelve per cent (10/81) of patients had a history of diabetes mellitus. Six per cent (5/82) had a history of alcohol abuse previously. Forty-one patients (46%) were infected with genotype 1, whereas 43 (48%) were infected with genotype 2. Genotypes were unknown in six patients. Factors associated with steatosis grade were BMI (P=0.004) and fibrosis stage (P=0.034). By univariate analysis, age (r=0.249, P=0.018), HAI score (0.333, P=0.002), steatosis grade (r=0.233, P=0.027), platelet count (r=−0.569, P<0.001) and ALT (r=0.260, P=0.013) were significant factors associated with advanced fibrosis (≥F2). Stepwise logistic regression analysis showed that moderate/severe steatosis grade [odds ratio (OR) 4.869, 95% confident incidence (CI)=1.607–14.75], platelet count <12 (104/μl) (OR 11.36, 95% CI=2.717–47.62) and ALT≥80 U/L (OR 4.605, 95% CI=1.182–17.94) were independent variables associated with advanced fibrosis (≥2).
Table 1. Baseline characteristics of the 90 patients according to the severity of steatosis
Expression of death receptor (Fas and tumour necrosis factor-α receptor type 1) and active caspse-3 in liver tissues by immunohistochemical staining
As shown in Figure 1, hepatocytes were strongly stained by antibody to Fas in the cytoplasm and the membrane in the severe steatotic liver (a), and were focally stained in the mild steatotic liver (b). In contrast, little obvious cytoplasmic staining was observed in the non-steatotic liver except positive staining at the Kupffer cells or sinusoid endothelial cells (c). Figure 2 shows the representative expression of TNF-R1 with diffuse cytoplasmic staining of hepatocytes in the severe steatotic liver (a); in contrast, little cytoplasmic staining was observed in the non-steatotic liver (c). Figure 3 shows the immunohistochemical detection of active caspase-3. The immunoreactivity was localized to the cytoplasm and most caspase-positive cells show diffuse cytoplasmic staining without obvious apoptotic nuclear morphological features (a).
As shown in Figure 4, the expressions of immunoreactivity for Fas, TNF-R1 and active caspases-3 were significantly correlated with the steatosis grade (P<0.001, P<0.001 and P<0.001). The extent of active caspases-3 correlated significantly with the expression of Fas (r=0.659, P<0.001) and TNF-R1 (r=0.617, P<0.001).
Expression of active nuclear factor-κB p65 and the correlation with Fas, tumour necrosis factor-α receptor type 1 and active caspse-3
Figure 5 shows the nuclear staining of active NF-κB p65 in some hepatocytes and inflammatory cells by immunohistochemical detection (a). In contrast, no obvious staining was observed in the non-steatotic liver (b). In this study, immunoreactivity for NF-κB was analysed as the expression rate because of the low positivity (37%). The positive expression of NF-κB p65 correlated significantly with the extent of Fas (r=0.405, P<0.001), TNF-R1 (r=0.448, P=0.002) and active caspase-3 (r=0.313, P=0.003).
In Figure 6, NF-κB p65 expression correlated with the steatosis grade (P<0.001). Although patients with advanced fibrosis (≥2) had a higher rate of NF-κB p65 expression (52.5 vs. 32.0%, P=0.04), there was no significant difference among the fibrosis stages. There was also no significant difference in the HAI score between NF-κB-positive and -negative subjects (6.9 ± 2.6 vs. 6.5 ± 2.6, P=0.563).
The causes and significance of hepatic steatosis in chronic HCV infection continue to be elucidated. Patients infected with HCV genotype 3 have a higher prevalence and more severe steatosis than those infected with other genotypes (31, 32). A recent study has shown that HCV genotype 3 core protein had a stronger effect on activation of fatty acid synthase in comparison with HCV-1b core, which could contribute to the higher prevalence and severity of steatosis (33). In contrast, classical metabolic risk factors account for the vast majority of cases of steatosis in patients infected by non-genotype 3 HCV strains. In our study population, the majority of patients were infected by genotype 1 or 2. There were no subjects with HCV genotype 3; thus, we could exclude the mechanism by which steatosis is induced by the virus itself through a direct cytopathic effect (31–33).
The mechanisms that cause increased hepatocellular injury in patients with steatosis remain largely unknown. A recent study demonstrated that a significant increase in liver cell apoptosis was seen in liver sections with increasing grade of steatosis (17). In addition, increasing steatosis was associated with decreased Bcl-2 mRNA levels and an increase in the pro-apoptotic Bax/Bcl-2 ratio. These data support an important role of hepatocyte apoptosis in the steatosis-mediated liver injury. Several factors have been associated with hepatocyte apoptosis, including oxidative stress and subsequent mitochondrial dysfunction, which may result from the activation of death receptor-mediated process, involving both the Fas/Fas ligand and the TNF-R1/TNF-α systems. The death pathway leads to the recruitment of initiator caspases such as caspase-8 into a death-inducing signalling complex. Upon binding to signalling molecules, they activate downstream effector caspases such as caspase-3, -6 and -7, which, through substrate cleavage, produce the typical apoptotic alterations.
In this study, we investigated the expression of death receptors: Fas and TNF-R1 in the liver tissues, and their correlation with the steatosis grades in chronic HCV infection. Our data showed that increasing steatosis in chronic hepatitis C was associated significantly with an increased expression of Fas and TNF-R1 in hepatocytes. Likewise, the extent of caspase activation (active caspase-3) correlated with the expression of Fas and TNF-R1 and the degree of steatosis. Taken together, these findings suggest that hepatocyte apoptosis is an important mechanism responsible for the steatosis-mediated liver damage, which is through overexpression of death receptors and activation of downstream caspase.
Nuclear factor-κB is an inducible transcription factor that is controlled by the signal activation cascades. NF-κB controls a number of genes involved in immuno-inflammatory responses, cell cycle progression and inhibition of cell adhesion (23–26). In this study, we found that NF-κB correlated significantly with the expression of death receptor and active caspase-3 in chronic HCV infection. These data support the previous findings that TNF-initiated signalling pathways result in a potent activation of pro-inflammatory gene expression via activation of NF-κB (34). Moreover, more recent evidences revealed that NF-κB might play a role in protecting cells against apoptotic signals (27, 28). Complete inhibition of NF-κB leads to increased hepatocyte apoptosis and worsens liver disease. An in vivo study showed that mice lacking the p65 subunit of NF-κB died embryonically by massive, TNF-mediated apoptotic cell death in the liver (35). In our data, NF-κB p65 expression was associated with the degree of steatosis in chronic HCV infection but not with the degrees of hepatic inflammation and fibrosis. One explanation for this would be that activation of NF-κB is more closely associated with reactive anti-apoptosis than with hepatic inflammation. However, the apparent activation of this transcription factor needs to be further explored as playing a major role in pro-inflammatory pathway or the anti-apoptotic pathway.
The major limitation of our study is the lack of further examination in the protein expression of death receptor and active caspase-3. Immunohistochemistry is the most time-saving and cost-effective way to detect overexpression of antigens, and it offers the advantage of in situ analysis. However, it would have been of value to follow up the immunohistochemistry with a Western blot analysis in the quantification of protein expression. Further studies are still necessary to clarify this point.
In summary, the present results showed the increased expression of death receptors and activation of caspase in HCV-infected liver biopsies with advanced steatosis. Steatosis has been linked to both death receptor expression and fibrosis, although the relative contribution of steatosis and apoptosis to fibrogenesis is yet to be delineated. Further studies are needed to clarify the detailed mechanisms.
This study was supported by National Science Council (Taiwan, Republic of China), grant number: NSC94-2314-B182A-151 to C.-H. H..