Gut 2000;46:255–259 255 Liver infiltrating T lymphocytes express interferon ã and inducible nitric oxide synthase in chronic Gut: first published as 10.1136/gut.46.2.255 on 1 February 2000. Downloaded from hepatitis C virus infection S Schweyer, S Mihm, H J Radzun, H Hartmann, A Fayyazi Abstract cytokine, interferon ã (IFN-ã), has been Background—Pathogenesis of hepatitis C suggested to promote the hepatic pathology.5–7 virus (HCV) associated liver injury is Animal models of experimentally induced thought to be due to the host antiviral hepatitis support this hypothesis—for example, immune response. Using a quantitative, in a model of transgenic mice expressing IFN-ã competitive RT-PCR technique, we re- under the control of a liver specific promoter, cently showed that expression of inter- the animals developed a chronic hepatitis.8 feron ã (IFN-ã) and IFN-ã inducible type Furthermore, IFN-ã has been shown to be the of nitric oxide synthase (iNOS) is in- principal mediator of the hepatic inflammatory creased in homogenised liver tissue of process induced by syngeneic lymphocytes patients with chronic HCV infection. against hepatitis B surface antigen (HBsAg) Aims—To determine the cellular origin of expressed in the liver of transgenic mice.9 IFN-ã and iNOS expression and to exam- However, the mechanism of IFN-ã associated ine the hypothesis that T cell derived liver injury is still unclear. IFN-ã secretion induces iNOS in hepato- IFN-ã has been shown to upregulate the cytes in chronic HCV infection. inducible isoform of nitric oxide synthase Methods—By applying a non-radioactive (iNOS) in monocytes/macrophages, resulting in situ hybridisation method combined in NO production.10 As a gaseous free radical, with indirect immunofluorescence, 33 monocyte/macrophage derived NO may de- liver biopsy specimens from patients with stroy infected as well as non-infected host chronic HCV infection were studied for cells.11 Moreover, it is known that stimulated cellular expression of IFN-ã and iNOS hepatocytes can express iNOS.12 On the mRNA. assumption that an IFN-ã dependent induc- Results—In chronic HCV infection, both tion of iNOS in hepatocytes results in NO pro- IFN-ã and iNOS gene expression were sig- duction which plays a dual proinflammatory http://gut.bmj.com/ nificantly increased. IFN-ã and iNOS and antiviral role in hepatitis C, we analysed mRNA were observed in CD3+ lym- liver biopsy specimens of patients chronically phocytes infiltrating portal tracts and infected with HCV. One part of each biopsy hepatic lobules, but not in hepatocytes. was fixed in formaldehyde and embedded in Conclusions—Results are consistent with paraYn wax for histopathological evaluation. previous reports that IFN-ã and iNOS The other part was homogenised and analysed transcripts are elevated in chronic HCV for the expression of IFN-ã and iNOS by a on September 25, 2021 by guest. Protected copyright. infection. In contrast to the hypothesis, quantitative, competitive reverse transcription IFN-ã expressing T cells do not induce polymerase chain reaction (RT-PCR).13 Al- Department of though it could be shown that in HCV infected Pathology, Division of iNOS in hepatocytes, but probably in T Pathology, cells. T lymphocytes expressing IFN-ã patients hepatic iNOS expression is upregu- Georg-August and/or iNOS have the potential to partici- lated in an IFN-ã dependent manner, the level University, Göttingen, pate in autocrine and paracrine pathways of transcription of iNOS did not correlate to Germany that may contribute to the pathobiology of liver injury, but correlated to hepatic HCV S Schweyer chronic hepatitis C. RNA content. This new evidence was not con- H J Radzun sistent with our assumption and raised the A Fayyazi (Gut 2000;46:255–259) question whether iNOS is really produced in Department of Keywords: hepatitis C; interferon type II; nitric oxide hepatocytes. To map the cellular origin of Internal Medicine, synthase; in situ hybridisation IFN-ã and iNOS mRNA, the formalin fixed, Division of paraYn wax embedded counterpart of liver Gastroenterology and biopsy specimens of the same HCV infected Hepatitis C virus (HCV) is responsible for over Endocrinology patients were studied by non-radioactive in situ S Mihm 90% of what was previously called non-A hybridisation combined with immunofluores- H Hartmann non-B hepatitis, most cases of post-transfusion 1 cence. Correspondence to: hepatitis, and many sporadic infections. Al- Dr S Schweyer, though more than 50% of infected patients Universitätsklinikum develop a chronic hepatitis,2 little is known Göttingen, Abteilung about the HCV associated mechanism(s) lead- Abbreviations used in this paper: ALT, alanine Pathologie, 3 aminotransferase; DIH, drug induced hepatitis; Robert-Koch-Strasse 40, ing to liver destruction. We and others have D-37075 Göttingen, shown that the immune cells and mediators HbsAg, hepatitis B surface antigen; HBV, hepatitis B Germany. virus; HCV, hepatitis C virus; IFN-ã, interferon ã; involved in the process of virus elimination are iNOS, inducible nitric oxide synthase; ISH, in situ 4–7 Accepted for publication responsible for hepatocellular injury. In this hybridisation; PBC, primary biliary cirrhosis; Th1, T 18 August 1999 respect, the secretion of the T helper 1 (Th1) helper 1. 256 Schweyer, Mihm, Radzun, et al Materials and methods lymphoma, who underwent liver biopsy as a PATIENTS, TISSUE SAMPLES, AND part of lymphoma staging. HISTOPATHOLOGICAL EVALUATION Tissue samples were fixed in 4% formalde- Gut: first published as 10.1136/gut.46.2.255 on 1 February 2000. Downloaded from Thirty three liver biopsy specimens including hyde and embedded in paraYn wax. Sections three explanted livers from patients with (5–10 µm thick) were mounted on slides coated chronic HCV infection were studied (17 with 2% 3-aminopropyltriethoxy-silane (Sigma, women, 16 men; aged 33–65 years, mean Munich, Germany) dissolved in acetone. After 45.2). Chronic HCV infection was diagnosed deparaYnisation, sections were stained histo- histopathologically according to established chemically (haematoxylin + eosin, and tri- criteria14 as well as by the presence of anti-HCV chrome), and by applying in situ hybridisation antibodies and HCV RNA in sera and/or by combined with indirect immunofluorescence. elevated serum aspartate aminotransferase and A modified form of the Histology Activity alanine aminotransferase (ALT) activities ob- Index designed by Knodell and colleagues15 served over a period longer than six months. served to assess grading and staging of chronic Serum aminotransferase activities ranged from HCV infection, as described previously.16 In 10 to 108 U/l (mean 43 U/l) for aspartate liver biopsy specimens, the prevalence of aminotransferase, and from 12 to 250 U/l necroinflammatory changes was graded as (mean 81 U/l) for ALT. mild in 40% (n=13), moderate in 45% (n=15), Liver biopsy specimens from patients with and severe hepatitis in 15% (n=5) of patients. chronic hepatitis B (HBV; n=3), primary Architectural alterations (staging) were graded biliary cirrhosis (PBC; n=1), drug induced as absent or mild fibrosis in 46% (n=15), mod- hepatitis (DIH; n=2), or without any hepatic erate fibrosis in 27% (n=9), and notable disorder (n=10) served as controls. Biopsy fibrosis/cirrhosis in 27% (n=9) of patients. specimens without pathological changes were Histopathological evaluation was performed obtained from patients with malignant without knowledge of the biochemical or clini- cal data. NON-RADIOACTIVE IN SITU HYBRIDISATION For preparation of riboprobes, cDNA frag- ments derived from the 3' terminal region of the human IFN-ã (position 381–600 of the coding DNA) and hepatic iNOS gene (position 2406–2658 of the coding DNA) were sub- cloned into pBluescript II KS+ (Stratagene, California, USA). The subcloned fragments served as templates for in vitro transcription of digoxigenin 11-dUTP labelled antisense and http://gut.bmj.com/ sense probes which were produced by virtue of T3 or T7 RNA polymerase according to the manufacturer’s instructions (Boehringer, Mann- heim, Germany). In situ hybridisation (ISH) was then performed on deparaYnised sections as described by Breitschopf et al.17 Briefly, sec- tions were digested with proteinase K (10 µg/ml) and incubated overnight with digoxi- on September 25, 2021 by guest. Protected copyright. genin 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 buVered saline (TBS; 50 mM Tris-HCl, 150 mM NaCl, pH 7,5) containing 0.1% (vol/vol) Tween 20 (Boeh- ringer) and 1% fetal calf serum (CC Pro, Neu- stadt, Germany), and incubated (two hours at room temperature) with a sheep alkaline phos- phatase conjugated polyclonal antibody F(ab)2 fragment against digoxigenin (Boehringer). Signal was detected using 5-bromo-4- chloro-3-indolyl phosphate as a substrate and nitro blue tetrazolium as a coupler (Boe- hringer). Liver biopsy specimens were counter- stained with Mayer’s haematoxylin and mounted in Aquamount. Figure 1 Non-radioactive in situ hybridisation for the Samples were then examined by using light antisense cRNA probe of IFN-ã on liver specimens from patients chronically infected with HCV: (A) combined with microscopy. Positive cells showed strong cyto- immunofluoresecent labelling for T cells (CD3); (B) on the plasmic staining around the clearly demarcated same section. Open arrows denote IFN-ã mRNA expressing nuclei. Liver sections from PBC served as con- CD3+ T cells. In situ hybridisation carried out by applying labelled sense cRNA probe of IFN-ã was negative (C). trols for ISH experiments with IFN-ã cRNA Original magnification × 400. probes, and skin biopsy specimens from IFN-ã and iNOS in chronic HCV infection 257 normal skin and psoriatic lesions for ISH Fluorescent Mounting Medium (Dako, Ham- experiments with iNOS cRNA probes.18 19 burg, Germany) and examined using fluores- Each tissue sample was also stained with cence microscopy.