J Clin Pathol 1989;42:1065-1069

Immunolocalisation of a interferon in liver diseaseC G G SUTHERLAND, A M T AL-BADRI, A G HOWATSON, M A FARQUHARSON,A K FOULIS

From the Department ofPathology, Royal Infirmary, Glasgow, Scotland

SUMMARY The expression ofimmunoreactive a interferon was examined in 78 liver biopsy specimensusing an indirect immunoperoxidase technique. Biopsy specimens included cases of acute viralhepatitis, chronic active hepatitis, primary biliary cirrhosis, alcoholic hepatitis, large bile ductobstruction and normal liver. Kupffer cells were positive for a interferon in all cases. Hepatocyteswere negative for a interferon in normal liver but in acute viral hepatitis were positive in perivenularand necrotic areas. Hepatocytes were positive in periportal areas, associated with piecemeal necrosis,in chronic active hepatitis and primary biliary cirrhosis, and were positive in perivenular areas inalcoholic hepatitis and large bile duct obstruction.The unexpected finding of a interferon in hepatocytes in non-viral liver disease indicates that the

presence of this substance in liver cells cannot be taken as a specific marker of viral infection.

Hepatitis B (HBV) virus is not directly cytopathic andT cell mediated cytolysis is considered to be the meansof viral elimination in HBV infection. The recognitionof infected cells by cytotoxic T lymphocytes dependson the association between viral determinants andclass I major histocompatibility complex (MHC)molecules. The interferons are known to induce anincrease of synthesis and display of MHC class Imolecules, and it has been proposed that during acuteHBV infection, interferon promotes hepatocyte MHCclass I display, which increases the possibility ofimmune attack by cytotoxic T cells. There is indirectevidence to support this hypothesis. In uncomplicatedacute HBV infection plasma interferon concentrationsrise,' and increased hepatocyte expression of (2microglobulin (a subunit of HLA class I antigen) hasbeen shown.2 Recently a technique for the detection ofimmunoreactive a interferon (IFN-a) in formalinfixed, paraffin wax embedded tissue has beendeveloped.3 We used this technique on a series of liverbiopsy specimens to investigate the expression of ainterferon in viral hepatitis and in other liver diseasesto ascertain ifits expression by hepatocytes is a specificindicator of viral infection.

Material and methods

Seventy eight percutaneous and wedge liver biopsyspecimens from nine cases of acute viral hepatitis, 14

Accepted for publication 25 May 1989

of chronic active hepatitis, 14 of primary biliarycirrhosis, 15 of alcoholic hepatitis and 14 of extra-hepatic biliary obstruction and 12 patients withoutliver disease were selected from our files. Biopsyspecimens had been fixed in either buffered formalinor formol-saline.

Sections 4 um thick were cut from the paraffin waxblocks and mounted on slides coated with poly-L-lysine. They were stained using an indirect immuno-peroxidase technique with a sheep anti-a interferonantiserum (gift from Dr A Meager, National Instituteof Biological Standards and Control, Potter's Bar,Hertfordshire). Peroxidase conjugated swine anti-sheep (Serotec, Oxford, England) was used as a bridgeand diaminobenzidine was the substrate. The sheepanti-a-interferon antiserum (H51) was raised withhuman lymphoblastoid interferon (Hu IFN-alpha LyNamalwa (Wellferon), Wellcome ResearchLaboratories, Beckenham, Kent) as antigen which wasgreater than 80% pure with respect to interferonprotein. In a viral culture inhibition assay the anti-serum neutralised all preparations containing a inter-feron4 but did not neutralise y interferon.5 The antigenreacted with a interferon but not with recombinant (3interferon (Triton Biosciences, California USA) onimmunoblot analysis.

Diluted antiserum and normal sheep serum used incontrol studies were absorbed with a mixture ofguineapig and porcine liver powders (Sigma, Dorset,England) before their use in the indirect immuno-peroxidase technique and this reduced non-specificbinding to human tissues.

1065

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A section of each case was stained with normalsheep serum as a negative control and in addition allpositive staining of liver sections was abolished whensections were stained with H51 antiserum which hadbeen preabsorbed with Wellferon. Sections from thecases ofchronic active hepatitis and alcoholic hepatitiswere also stained using an indirect immunoperoxidasetechnique with a goat antibody to hepatitis B surfaceantigen (Dako Ltd, High Wycombe, Buckingham-shire).3

Results

All examples of normal liver showed positive stainingof Kupffer cells. Hepatocytes and bile duct epitheliumwere consistently negative (fig 1).

ACUTE VIRAL HEPATITIS

As patients with viral hepatitis are now biopsied veryrarely most of these biopsy specimens were from theera before serological identification ofHBsAg becamepossible. The serological state was unknown, but ineach case the history and histological features were

strongly indicative of viral hepatitis. All cases showedstrong hepatocyte staining for a interferon in theperivenular areas (fig 2). This pattern was particularly

easy to recognise in the cases showing spotty necrosiswhere bridging and massive necrosis had not disruptedthe architecture. There were also scanty positivelystaining hepatocytes throughout the lobules but onlyin relation to inflammation or necrosis.

CHRONIC ACTIVE HEPATITIS

Two patients were known to be HBsAg positive whilein the remaining 12 there was no history of hepatitis Binfection and no immunohistochemical staining forHBsAg in the biopsy specimens, all of which showedthe histological features of chronic active hepatitiswith portal and periportal inflammation, erosion ofthe limiting plate, piecemeal necrosis and a greater orlesser degree of fibrosis. Although inflammation was

severe in all biopsy specimens, there was a range inseverity ofpiecemeal necrosis from relatively scanty toextremely severe and widespread. All 14 cases showedspecific staining for a interferon and this took the formof cytoplasmic staining of hepatocytes related topiecemeal necrosis (fig 3). Where disruption of thelimiting plate was identified, associated hepatocyteswere always positive. Similarly, isolated hepatocyteswere invariably positive. Scanty positively staininghepatocytes within the lobules were also identified insix cases, and these hepatocytes were adjacent to

1066

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1067

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inflammatory cells within the lobules. The stainingpattern in the two HBsAg positive cases was indistin-guishable from the rest. In one case there was positivestaining for a interferon in bile duct epithelium.

PRIMARY BILIARY CIRRHOSISFourteen cases of primary biliary cirrhosis with stage1-4 disease activity were studied. Eleven cases werestage 2 or 3 and showed a variable degree ofpiecemealnecrosis. In all cases most hepatocytes were negativefor x interferon except in the presence ofinflammatorydisruption of the limiting plates, and all 11 cases withpiecemeal necrosis showed a pattern ofIFN-a stainingsimilar to that observed in chronic active hepatitis.Hepatocytes distant from areas of piecemeal necrosiswere usually negative as were hepatocytes associatedwith lobular inflammation. Hepatocytes adjacent toportal tracts where there was intense inflammation butno piecemeal necrosis were occasionally positive butthe extent of staining was much less conspicuous thanthat in chronic active hepatitis. Proliferating bileductules were occasionally positive but not normalbile ducts.

ALCOHOLIC HEPATITISFifteen biopsy specimens of alcoholic liver disease

varying from mild to severe alcoholic hepatitis withoutcirrhosis were examined. All cases were negative whenstained with anti-HBsAg. Hepatocytes showingpositive staining for a interferon were present in all$pecimens. In eight biopsy specimens staining wasconcentrated maximally in perivenular areas andoccurred in hepatocytes which looked essentiallynonnal as well as those which exhibited degenerativefeatures (fig 4). In seven cases positively stainedhepatocytes occurred in a pan-lobular distribution orin scanty hepatocytes dispersed throughout the lobule.Steatotic hepatocytes were frequently positive and inthese cases the cytoplasmic staining was identified atthe periphery of the cell, displaced by the cytoplasmiclipid. Although there was extensive a interferon stain-ing of inflammatory cells, staining of hepatocytes didnot seem to be specifically related to inflammation.Bile duct epithelium was negative.

LARGE BILE DUCT OBSTRUCTIONTwelve biopsy specimens contained hepatocytes withpositive a interferon staining, and in all of these it wasmaximal in perivenular zones. In four cases there wasalso focal hepatocyte staining in the periportal areasassociated with intense portal tract inflammation. In12 biopsy specimens there was also focal positivestaining in inflamed bile ducts.

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Immunolocalisation ofa interferon in liver diseaseDiscussion

The use of this technique has already shown that innormal liver Kupffer cells contain immunoreactive ainterferon.6 Our study has shown that Kupffer cellswere also positive for IFN-a in all other groups ofliverdisease examined. This disagrees with the findings of aprevious study which used immunofluorescence local-isation ofIFN-a in liver tissue infected with hepatitis Bvirus and showed its presence only in mononuclearcells of the lymphocyte/plasma cell series, fibroblasts,and polymorphonuclear leucocytes. Kupffer cells andhepatocytes were invariably negative.7 In that studythe IFN-a anti-sera satisfied usual immunofluoresencespecificity controls, but abolition of positive stainingby preabsorption of antibody with IFN-a was notattempted. Our own preabsorption control studiesstrongly support the specificity of the H51 antibody.

Furthermore, there is increasing evidence in animalsthat macrophages can be induced to produce inter-ferons.' Cells of the mononuclear phagocyte system,including Kupffer cells, have been shown to containimmunoreactive IFN-a using the H5 1 antibody, and ithas been suggested that these cells might be the sourceof the low concentrations ofendogenous IFN-a foundin physiological conditions.6We have shown that in all groups of liver disease

studied, hepatocytes could be identified which werepositive for IFN-a. The distribution of positivehepatocytes generally related to areas of hepatocytedamage. Our finding of IFN-a, mainly in perivenularareas and associated with bridging necrosis in acuteviral hepatitis and in periportal zones and areas oflobular inflammation in chronic active hepatitis, issimilar to the reported distribution off2 microglobulinin HBV infection.2 Similarly, in non-viral liver diseasethe localisation of IFN-a staining was seen in areas ofhepatocyte damage or degeneration.

Using the techniques described here, we can onlyspeculate as to whether hepatocytes actually synthe-sise IFN-a or are engaged in its uptake from surround-ing cells. Non-specific uptake of IFN-a by degenerat-ing hepatocytes is obviously a possible explanation forpositive hepatocyte staining. In the cases of alcoholicliver disease and large bile duct obstruction, however,positively stained hepatocytes were found in areasquite unrelated to the presence of inflammatory cells.This indicates that hepatocytes containing IFN-a are

1069not merely a consequence of increased local concen-trations of IFN-a in areas of inflammation.The cuboidal lining epithelium ofthe choroid plexus

has been shown to be strongly positive for immuno-reactive IFN-a using the same immunohistochemicaltechnique as that- used in the present study,6 andrecently the presence ofIFN-a mRNA has been shownin these cells, indicating that cells outwith the mono-nuclear phagocyte and leucocyte series have a role inthe synthesis of IFN-aL (Howatson et al, unpublishedobservations). Application of in situ hybridisationtechniques to the study of interferon synthesis in theliver would clearly be of interest.

Interferons are known to have a wide range ofbiological effects and these, in addition to antiviralactivity and effects on MHC expression, includeantigrowth activity and effects on cellular differen-tiation.9 Interferon may have a role in liver diseasebeyond that currently proposed in viral hepatitis.References

1 Levin S, Hahn T. Interferon system in acute viral hepatitis. Lancet1982;i:592-4.

2 Nagafuchi Y, Scheuer PJ. Expression of B2 microglobulin onhepatocytes in acute and chronic type B hepatitis. Hepatology1986;6:20-3.

3 Foulis AK, Farquharson MA, Meager A. Immunoreactive alpha-interferon in insulin-secreting B cells in type I diabetes mellitus.Lancet 1987;ii: 1423-7.

4 Exley T, Parti S, Barwick S, Meager A. A comparison of theneutralising properties of monoclonal and polyclonalantibodies to human interferon alpha. J Gen Virol 1984;65:2277-80.

5 Meager A. Antibodies against interferons: characterisation ofinterferons and immunoassays. In: Clemens MJ, Morris AG,Gearing AJH, eds. Lymphokines and interferons-a practicalapproach. Oxford: IRL Press, 1987:105-27.

6 Khan NU-D, Pulford KAF, Farquharson MA, et al. Thedistribution of immunoreactive interferon-alpha in normalhuman tissues. Immunology 1989;66:201-6.

7 Jilbert AR, Burrell CJ, Gowans EJ, Hertzog PJ, Linnane AW,Marmion BP. Cellular localisation of alpha-interferon inhepatitis B virus-infected liver tissue. Hepatology 1986;6:957-61.

8 De Maeyer E, De Maeyer-Guignard J. Macrophages as interferonproducers and interferons as modulators of macrophageactivity. In: De Maeyer E, De Maeyer-Guignard J, eds.Interferon and other regulatory cytokines. Chichester: Wiley &Sons, 1987:194-220.

9 Pestka S, Langer JA, Zoon KC, Samuel CE. Interferons and theiractions. Ann Rev Biochem 1987;56:727-77.

Requests for reprints to: Dr C G G Sutherland, Departmentof Pathology, Royal Infirmary, Glasgow G4 OSF, Scotland.