Materials and methods

PATIENTS, TISSUE SAMPLES, AND HISTOPATHOLOGICAL EVALUATION

Thirty three liver biopsy specimens including three explanted livers from patients with chronic HCV infection were studied (17 women, 16 men; aged 33–65 years, mean 45.2). Chronic HCV infection was diagnosed histopathologically according to established criteria14 as well as by the presence of anti-HCV antibodies and HCV RNA in sera and/or by elevated serum aspartate aminotransferase and alanine aminotransferase (ALT) activities observed over a period longer than six months. Serum aminotransferase activities ranged from 10 to 108 U/l (mean 43 U/l) for aspartate aminotransferase, and from 12 to 250 U/l (mean 81 U/l) for ALT.

Liver biopsy specimens from patients with chronic hepatitis B (HBV; n=3), primary biliary cirrhosis (PBC; n=1), drug induced hepatitis (DIH; n=2), or without any hepatic disorder (n=10) served as controls. Biopsy specimens without pathological changes were obtained from patients with malignant lymphoma, who underwent liver biopsy as a part of lymphoma staging.

Tissue samples were fixed in 4% formaldehyde and embedded in paraffin wax. Sections (5–10 μm thick) were mounted on slides coated with 2% 3-aminopropyltriethoxy-silane (Sigma, Munich, Germany) dissolved in acetone. After deparaffinisation, sections were stained histochemically (haematoxylin + eosin, and trichrome), and by applying in situ hybridisation combined with indirect immunofluorescence.

A modified form of the Histology Activity Index designed by Knodell and colleagues15 served to assess grading and staging of chronic HCV infection, as described previously.16 In liver biopsy specimens, the prevalence of necroinflammatory changes was graded as mild in 40% (n=13), moderate in 45% (n=15), and severe hepatitis in 15% (n=5) of patients. Architectural alterations (staging) were graded as absent or mild fibrosis in 46% (n=15), moderate fibrosis in 27% (n=9), and notable fibrosis/cirrhosis in 27% (n=9) of patients.

Histopathological evaluation was performed without knowledge of the biochemical or clinical data.

NON-RADIOACTIVE IN SITU HYBRIDISATION

For preparation of riboprobes, cDNA fragments derived from the 3′ terminal region of the human IFN-γ (position 381–600 of the coding DNA) and hepatic iNOS gene (position 2406–2658 of the coding DNA) were subcloned into pBluescript II KS+ (Stratagene, California, USA). The subcloned fragments served as templates for in vitro transcription of digoxigenin 11-dUTP labelled antisense and sense probes which were produced by virtue of T3 or T7 RNA polymerase according to the manufacturer's instructions (Boehringer, Mannheim, Germany). In situ hybridisation (ISH) was then performed on deparaffinised sections as described by Breitschopfet al.17 Briefly, sections were digested with proteinase K (10 μg/ml) and incubated overnight with digoxigenin labelled riboprobes at 55°C. Slides were then washed in 1% sodium dodecyl sulphate (SDS) in 2× saline sodium citrate (SSC) (15 minutes at 55°C), and 1% SDS in 1× SSC (20 minutes at room temperature). Finally, they were washed in Tris buffered saline (TBS; 50 mM Tris-HCl, 150 mM NaCl, pH 7,5) containing 0.1% (vol/vol) Tween 20 (Boehringer) and 1% fetal calf serum (CC Pro, Neustadt, Germany), and incubated (two hours at room temperature) with a sheep alkaline phosphatase conjugated polyclonal antibody F(ab)₂fragment against digoxigenin (Boehringer).

Signal was detected using 5-bromo-4-chloro-3-indolyl phosphate as a substrate and nitro blue tetrazolium as a coupler (Boehringer). Liver biopsy specimens were counterstained with Mayer's haematoxylin and mounted in Aquamount.

Samples were then examined by using light microscopy. Positive cells showed strong cytoplasmic staining around the clearly demarcated nuclei. Liver sections from PBC served as controls for ISH experiments with IFN-γ cRNA probes, and skin biopsy specimens from normal skin and psoriatic lesions for ISH experiments with iNOS cRNA probes.18 19 Each tissue sample was also stained with equivalent quantities of labelled sense riboprobes.

To evaluate ISH data, sections were studied by two pathologists. The number of labelled infiltrating cells was determined per section in four high power visual fields (original magnification × 400) of portal tracts and hepatic lobules.

INDIRECT IMMUNOFLUORESCENCE

The polyclonal antibody against CD3, and the monoclonal antibody against CD20 (clone L26) were obtained from Dako (Hamburg, Germany). The monoclonal antibody CD68 (clone Ki-M1P) recognising all subpopulations of monocytes/macrophages was kindly provided by the Department of Pathology, University of Kiel, Germany.20The primary antibodies were applied at a working dilution of: CD3, 1/50; CD20, 1/20; CD68, 1/2000.

Indirect immunofluorescence was performed immediately after ISH. Sections were incubated for two hours with the primary antibodies. The samples were then incubated with a FITC labelled goat antimouse IgG (working dilution 1/50) for one hour (Dako, Hamburg, Germany). Finally, they were mounted with Fluorescent Mounting Medium (Dako, Hamburg, Germany) and examined using fluorescence microscopy.

For controls, sections from all samples were stained using the above procedures, but omitting the primary or secondary antibodies.

STATISTICS

The non-parametric Mann-Whitney U test and Spearman rank correlation test were applied for the comparison of mean values and for calculation of correlation coefficients, respectively. A value of p<0.01 was considered to indicate statistically significant differences.

Results

CELLULAR LOCALISATION OF HEPATIC IFN-γ EXPRESSION

In liver specimens from HCV infected patients, the number of IFN-γ mRNA expressing cells (mean (SD) per 4× high power field) was 35.4 (8.54). IFN-γ mRNA was found to be restricted to mononuclear cells infiltrating portal tracts and hepatic lobules. These cells were characterised as T cells by applying indirect immunofluorescence for CD3 antigen (fig 1A, B). No specific signal could be detected in control samples hybridised with the sense IFN-γ cRNA probe (fig 1C).

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Figure 1

Non-radioactive in situ hybridisation for the antisense cRNA probe of IFN-γ on liver specimens from patients chronically infected with HCV: (A) combined with immunofluoresecent labelling for T cells (CD3); (B) on the same section. Open arrows denote IFN-γ mRNA expressing CD3+ T cells. In situ hybridisation carried out by applying labelled sense cRNA probe of IFN-γ was negative (C). Original magnification × 400.

In specimens from HBV infected IFN-γ mRNA was found in 36 (5) mononuclear cells, and in those from patients with PBC in 55 mononuclear cells; 4 (1) mononuclear cells in cases of drug induced hepatitis and 0.7 (0.6) mononuclear cells in specimens without a hepatic disorder were positively stained.

The expression of IFN-γ was significantly augmented in specimens from HCV infected patients, compared with liver specimens without a pathological disorder (p<0.0001, Mann-Whitney U test; fig 3). The number of IFN-γ expressing cells within the hepatic infiltrate was not correlated to sex (p=0.7534, r=0.0867, Spearman rank correlation), age (p=0.4126,r=0.1672), serum ALT activity (p=0.1880,r=0.2633), or to the degree of fibrosis (p=0.0544, r=0.3846) (data not shown).

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Figure 3

Liver biopsy specimens from patients with chronic HCV infection (n=33) and individuals without any hepatic disorder (n=10) were studied for IFN-γ and iNOS mRNA by applying non-radioactive in situ hybridisation.

CELLULAR LOCALISATION OF HEPATIC iNOS EXPRESSION

In liver specimens from chronic HCV infected patients the count of iNOS mRNA positive cells was 32.1 (8.2). These cells were characterised as CD3+ T cells infiltrating portal tracts and hepatic lobules (fig 2A, B). By contrast, Ki-M1P+ infiltrating or sessile (Kupffer cells) macrophages, hepatocytes (fig 2C, D), CD20+ B lymphocytes, or vascular endothelium did not reveal any iNOS mRNA expression. No specific signal could be detected in control samples hybridised with the sense iNOS cRNA probe (fig 2E).

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Figure 2

Non-radioactive in situ hybridisation for antisense cRNA probe of iNOS on liver specimens from patients chronically infected with HCV (A,C), combined with immunofluoresecent labelling for T cells (CD3) (B) or macrophages (CD68) (D) on the same sections. Open arrows denote iNOS mRNA expressing cells (A,C) which were characterised as CD3+ lymphocytes (B). CD68+ infiltrating macrophages within portal tracts, Kupffer cells (arrowheads), and hepatocytes (filled arrow) do not reveal any staining for iNOS mRNA (D). In situ hybridisation carried out by applying labelled sense cRNA probe of iNOS was negative (C). Original magnification × 400.

In specimens from HBV infected patients iNOS mRNA was found in 39 (4) mononuclear cells and in those from patients with PBC in 58 mononuclear cells, whereas 3.5 (0.5) mononuclear cells in cases of drug induced hepatitis and 1.6 (0.6) mononuclear cells in specimens without a hepatic disorder were stained positively for iNOS mRNA.

The expression of iNOS was found to be significantly augmented in specimens from HCV infected patients, compared with liver specimens without a pathological disorder (p<0.0001, Mann-Whitney U test; fig3). The number of iNOS expressing cells within the hepatic infiltrate was not correlated to sex (p=0.2780,r=0.2993, Spearman rank correlation), age (p=0.8559, r=0.0371), serum ALT activity (p=0.453, r=0.1499), or to the degree of fibrosis (p=0.4504, r=0.1509) (data not shown).

Discussion

In this study high numbers of IFN-γ and iNOS expressing mononuclear cells were detected in chronic HCV infection. Both IFN-γ and iNOS transcripts were restricted to CD3+ T lymphocytes infiltrating portal tracts and hepatic lobules. In cases of chronic HBV infection and PBC similar amounts of IFN-γ and iNOS transcripts were shown in mononuclear cells, whereas only a few IFN-γ and iNOS expressing mononuclear cells were found in DIH and in liver specimens without any pathological disorder. These results indicate that IFN-γ and iNOS may be involved in T cell mediated hepatic injuries such as viral hepatitis and PBC, but not in those caused by drugs or toxins.

Several studies have pointed to the role of IFN-γ in hepatic pathology in general,8 9 and in the development of HCV associated hepatocellular injury in particular.5-7 21 In an autocrine manner, IFN-γ has been shown to act as a growth factor for the proliferation and differentiation of helper and cytotoxic T lymphocytes^22 23 which attack and lyse hepatocytes presenting viral peptides.24 Nevertheless, in the majority of cases T lymphocytes fail to eradicate viruses harboured in hepatocytes. Previously we reported that in such cases hepatic iNOS expression rises in positive correlation to HCV RNA content.13 Hepatocytes, macrophages, and B lymphocytes have been shown to express iNOS following stimulation with bacterial products (for example, lipopolysaccharide), cytokines (for example, IFN-γ), or after viral infections (for example, Epstein-Barr virus).12 25 26 In situ evidence for the expression of iNOS mRNA in liver biopsy specimens from HBV and HCV infected patients was recently published by Majano et al.27 By applying ISH these authors showed that hepatocytes, vascular endothelium, and infiltrating mononuclear cells express iNOS transcripts.

It is difficult to reconcile published data with our results showing that in liver biopsy specimens from HCV infected patients neither hepatocytes nor endothelial cells express iNOS mRNA. It may be speculated that the shorter iNOS cRNA probe (252 bases instead of 700 bases) and the higher hybridisation temperature (55°C instead of 45°C) used in the present work produced more stringent hybridisation conditions.28 Subsequently it is possible that such stringency helped to differentiate mRNAs of highly homologous proteins, thereby not producing signals within hepatocytes and endothelial cells, but showing iNOS mRNA in CD3+ T cells.

Many reports have shown that activated T cells express iNOS.29-31 The subsequent NO production has been shown to: inhibit Th1 cell proliferation29; suppress interleukin 2 secretion³⁰; and switch Th1 to Th2 type lymphocytes.31 These findings suggest that NO acts as a self regulatory molecule limiting the Th1 immune response, thus downregulating local inflammation.32 Whether iNOS plays a similar suppressive role in the course of Th1 associated liver injuries such as viral hepatitis or PBC remains questionable.

Taken together, our results show that the amounts of IFN-γ and iNOS transcripts are elevated in chronic HCV infection. Considering the high expression of IFN-γ and iNOS in HBV infection and also in PBC, the expression of these immunomodulatory molecules does not seem to be specific for HCV infection and HCV related liver damage. However, they may contribute to the pathobiology of Th1 mediated liver injuries such as HCV infection.