Clini. exp. Immuznol. (1976) 25, 396-402.
Liver cell surface localization of hepatitis B antigen and of immunoglobulins in acute and chronic hepatitis and in liver cirrhosis A. ALBERTI,* G. REALDI,* F. TREMOLADA* & G. P. SPINAt *Istituto di Medicina Clinica, Patologia andt Istituto di Patologia Speciale Chirurgica, University of Padua, Padua, Italy
(Received 1 May 1976) SUMMARY
This paper describes immunofluorescence studies on liver cell surface localization of hepatitis B surface antigen (HBsAg) and of IgG in acute and chronic hepatitis and in cirrhosis. In acute hepatitis B, HBsAg was found at the surface of hepatocytes in an early phase of the disease, but not during the recovery. This finding is consistent with the hypothesis that immune reactions to HBsAg may be responsible for the liver cell lysis. In HBsAg-positive chronic hepatitis and cirrhosis the antigen was found in the cytoplasm, but not on the surface of the hepatocytes, while in HBsAg-negative cases the antigen could not be detected in the liver cells. Both in HBsAg-positive and in HBsAg-negative chronic active hepatitis (CAH) and cryptogenic cirrhosis, IgG bound to the membrane of the hepatocytes could be detected, suggesting a role of antibodies in the pathogenesis of the disease.
INTRODUCTION It has been postulated that immunopathological reactions play an important role in the pathogenesis of acute and chronic hepatitis. Both heteroimmune reactions to virus-associated antigens and autoimmune reactions to liver cell membrane antigens must be considered (Popper & Paronetto, 1972). It has been suggested that cellular immune reactions to HBsAg may be responsible for the liver cell damage in HBsAg-positive acute and chronic hepatitis (Dudley, Fox & Sherlock, 1972; Eddleston & Williams, 1974). An important requirement of this hypothesis would be the finding of HBsAg on the surface of infected liver cells. Cell-mediated autoimmune reactions to liver specific antigens can be detected in patients with chronic active hepatitis or cryptogenic cirrhosis, and the frequency of positive reactions in the HBsAg-positive and HBsAg-negative cases is similar (Meyer zum Biuschenfelde, Knolle & Berger, 1974; Reed et al., 1974). A common autoimmune process may be involved in the pathogenesis of the liver cell damage in both groups of patients, but the effector mechanism is not yet clarified. Cell-mediated immune reactions to liver specific proteins are well documented, but an increasing interest in humoral immunity is rising from the detection of IgG bound to the surface of hepatocytes of rabbits with experimentally induced CAH (Hopf & Meyer zum Biischenfelde, 1974), as well as of patients with CAH and signs of inflammatory activity (Arnold et al., 1975; Hopf et al., 1975). In a preliminary report we have described HBsAg at the surface of hepatocytes of patients with acute hepatitis B (Alberti et al., 1975). This paper reports further studies on liver cell surface localization of HBAg and of IgG in acute and chronic liver diseases. Correspondence: Dr A. Alberti, Instituto di Medicina Clinica, Patologia Medica, University of Padua, via Giustiniani 1, 35100 Padua, Italy.
396
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HBsAg and IgG on liver cell surface MATERIALS AND METHODS
Patients. Eighteen patients with acute hepatitis B were studied; nine of whom were in an early phase of the disease with increasing transaminase levels, and nine at a later stage in the illness. Twenty-seven patients with chronic hepatitis or cirrhosis were studied: five of them had chronic persistent hepatitis, nine CAH, seven cryptogenic cirrhosis and six alcoholic cirrhosis. None of them had received immunosuppressive treatment in the last 6 months. Five subjects without liver disease or immunological abnormalities were tested as controls. Liver tissue. Specimens of liver tissue were obtained by needle or surgical biopsy. A portion of each specimen was immediately transferred into 5 ml EMBA (Eagle's medium/Serva no. 47370 A plus 2-5% bovine albumin/Behringwerke, Marburg) pH 7-4 at 370C and treated for immunofluorescence studies. The remaining portion of the biopsy was processed for routine histopathological examination. Preparation ofFITC-conjugated antisera. Specific anti-HBsAg antiserum (human IgG) obtained from a healthy carrier of high titre precipitating hepatitis B antibody, and commercial anti-human IgG antiserum from rabbit (Behringwerke, Marburg) were FITC conjugated according to Arnold & von Mayersbach (1972) and absorbed twice with acetone-dried rabbit liver powder (Sigma). Normal human and rabbit IgG were fluorescein-conjugated in the same manner. Direct immunofluorescence for liver cell surface localization of HBsAg and IgG. Surface localization of HBsAg and IgG was investigated using a direct immunofluorescence antibody technique on vital liver cells isolated in suspension according to Hopf, Meyer zum Buschenfelde & Freudenberg (1974) with some modifications. This technique involves the preparation of living suspensions ofisolated hepatocytes with intact membrane structure and the detection by immunofluorescence ofsurface antigens only. Briefly, biopsy specimens were gently minced in EMBA at 370C in a Petri dish and the resulting cell suspension TABLE 1. Surface and intracellular localization of HBsAg and IgG in the liver cells of patients with acute hepatitis B
Immunofluorescence Surface localization* Intracellular localizationt
Patient HBsAg SMA
HBsAg
IgG
HBsAg
0/
0/
0/
Biopsy within the 1st week of the disease 1 + 0 + +(6) 2 0 +(15) +(10) + 3 0 0 0 + + 4 + +(27) 0 5 + 0 + +(18) 6 0 0 + + 7 0 0 0 + 8 0 + + +(14) 9 + +(22) + +(14) Biopsy after 4-5 weeks of illness 1 0 0 0 0 2 0 0 0 0 3 0 0 0 0 4 0 0 + + 5 0 0 0 -+ 6 0 0 0 0 7 0 + 0 0 8 0 + 0 0 9 0 0 + 0
n.t. n.t. 0 + cyt(19) n.t. 0 n.t.
+cyt(9) +cyt(30) 0 0 n.t. 0 0 0 n.t. 0 0
IgG
n.t. n.t. 0 0 n.t.
0 n.t.
0 0 0 0 n.t. 0 0 0 n.t. 0 0
n.t. = Not tested. HBsAg= hepatitis B antigen in serum detected at the time ofliver biopsy; SMA=smooth muscle antibodies (titre< 1:20); cyt=cytoplasmatic pcsitivity; the percentage of positive cells is given in parentheses. * Detected on vital liver cells. t Detected on air-dried liver cells.
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was filtered through nylon wool and washed twice with EMBA at 370C (190 g for 5 min). The cell pellet was resuspended in 0-2 ml EMBA+0-2 ml FITC-conjugated anti-HBs or anti-IgG antiserum and incubated at 370C for 30 min in a moist chamber. The cells were then washed three times in EMBA at 370C (190 g for 5 min), mounted in liquid medium and examined by fluorescence microscopy. Only living hepatocytes, as detected by phase contrast microscopy, were examined for surface fluorescence. Direct immunofluorescence for liver intracellular localization of HBsAg and IgG. In order to investigate intracellular localization of HBsAg and IgG, liver cells isolated in suspension as described were smeared on slides using a cytocentrifuge (700 rev/min for 10 min) and air-dried before being incubated with FITC-conjugated anti-HBs or anti-IgG antiserum at 370C for 45 min. The stained slides were washed three times in PBS, mounted in glycerine and examined by fluorescence microscopy.
Specificity controls. The fluorescence was considered to be specific when blocked with an unlabelled antiserum in the inhibition test. Further controls were performed on vital liver cell suspensions using FITC-conjugated normal human or rabbit IgG in order to investigate the possibility that living hepatocytes may absorb immunoglobulins passively, or by microphagocytosis under these experimental conditions. Smooth-muscle antibodies. All sera were tested for smooth-muscle antibodies by the indirect fluorescent antibody technique (Johnson, Holborow & Glynn, 1965). Serological tests for HBsAg. All the sera were tested for the presence of HBsAg by counterelectrophoresis and by radioimmunoprecipitation according to methods previously described (Realdi et al., 1974).
RESULTS Acute hepatitis B The results obtained in eighteen patients with acute hepatitis B are summarized in Table 1. Of the nine patients studied in an early phase of the disease, all had HBsAg in the serum and six of them were positive for HBsAg at the liver cell surface. The fluorescence showed a non-homogeneous granular pattern (Fig. 1) and the number of positive cells ranged from 6-27%. A surface localization of IgG was found in two cases, both positive also for HBsAg. Intracellular localization of HBsAg and IgG was investigated in five patients; a granular cytoplasmatic fluorescence for HBsAg was found in three of them (Fig. 2). Of the nine patients studied during the recovery phase, HBsAg was still present in the serum of three, while the remaining six patients had already cleared the antigen. However, all nine were negative for HBsAg and IgG both in relation to the surface and in the cytoplasm of hepatocytes.
Chronic hepatitis and cirrhosis The results obtained in chronic hepatitis and in cirrhosis are summarized in Table 2. HBsAg was not detected at the liver cell surface in any of the twenty-seven patients (ten HBAg-positive, seventeen
FIG. 1. Surface localization of HBsAg on vital liver cells isolated from biopsies of patients with acute hepatitis B and treated with an anti-HBsAg FITC-conjugated antiserum. The fluorescence shows a non-homogeneous granular pattern with granules of different size (a-b), sometimes only at a portion of the membrane (c).
(Magnification x 378.)
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399
FIG. 2. Cytoplasmatic granular fluorescence in air-dried isolated liver cells from patients with HBsAg-positive acute (a) and chronic active (b-c) hepatitis after treatment with an anti-HBsAg FITC-conjugated antiserum. (Magnification x 240.)
FIG. 3. Surface localization of IgG on vital liver cells isolated from the biopsy of a patient with chronic active hepatitis and treated with anti-human IgG FITC-conjugated antiserum. In A and B the same liver cells on a different focus. The fluorescence shows a fine granular pattern (a) and typical membrane appearance (b). (Magnification x 378.)
HBAg-negative in serum) but was found in the cytoplasm of hepatocytes in all nine seropositive cases studied. Nuclear fluorescence was not found in any case although it sometimes happened that cells positive for HBsAg in the cytoplasm showed fluorescent granules in the region of the nucleus, probably caused by a layer of cytoplasm covering the nucleus (Fig. 2). IgG were detected at the surface of liver cells in seven out of nine patients with CAH, in three of seven with cryptogenic cirrhosis, but never in chronic persistent hepatitis or in alcoholic cirrhosis. The fluorescence occurred in a fine granular pattern, more homogeneous than that which we have observed for surface HBsAg in acute hepatitis (fig. 3). Ofthese ten cases with IgG bound to the hepatocyte surface five showed HBsAg in the serum as well as in the cytoplasm of the hepatocytes.
A. Alberti et al.
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TABLE 2. Surface and intracellular localization of HBsAg and IgG in the liver cells of patients with chronic hepatitis and cirrhosis
Immunofluorescence
Surface localization* Intracellular localizations Patient HBsAg SMA
HBsAg
Chronic persistent hepatitis 1 0 0 2 0 + 0 3 + 4 0 + 0 5 + Chronic active hepatitis 1 0 + 2 + 0 3 0 + 0 4 + 0 5 + 6 + + 7 0 0 0 8 + 0 9 + Cryptogenic cirrhosis 1 0 0 2 0 0 0 0 3 0 + 4 0 0 5 0 0 6 0 7 0 Alcoholic cirrhosis 1 0 0 0 2 0 0 0 3 0 4 + 0 0 5 0 6 +
IgG
HBsAg
0 0 0 0 0
0 0 0 0 0
0 +cyt(6)
0 0 0 0
+(8) +(12) 0 +(15) +(5) +(18) +(26) 0 +(15)
+cyt(18) 0
0 0 0
0 0 0 0 0 0 0 0 0
0 +(6)
0 0 0
0 0 0 0 0 0
0 0 0
+(1I) 0 0
+(21) 0
n.t. n.t.
+cyt(10)
n.t.
+cyt(l1) +cyt(14) +cyt(5) n.t. 0
+cyt(15) 0 0 0 n.t.
0 0 n.t.
0 0 0 +cyt(3) 0
+cyt(l1)
IgG
0 0 n.t. n.t. 0 0 0 n.t. 0 0 0 n.t. 0 0 0 0 0 n.t. 0 0 n.t. 0 0 0 0 0 0
n.t. = Not tested. HBsAg= hepatitis B antigen in serum detected at the time of liver biopsy SMA= smooth muscle antibodies (titre < 1:20); cyt= cytoplasmatic positivity; the percentage of positive cells is given in parentheses. * Detected on vital liver cells. t Detected on air-dried liver cells.
Specificity controls All the positive results could be inhibited by unlabelled antisera. Patients without liver disease always gave negative results both for surface and for intracellular HBsAg and IgG. Using FITC-conjugated normal IgG, we could not detect either passive binding or microphagocytosis of IgG by living isolated liver cells, either in patients or in controls. The presence of smooth muscle antibodies, which cross-react with the liver cell membrane (Trenchev, Sneyd & Holborow, 1974), did not correlate with the finding of IgG bound to the hepatocyte surface (Tables 1 and 2).
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401
DISCUSSION Viruses can produce pathological changes in several ways. Many viruses destroy the cells in which they grow, whereas in other instances multiplication of the virus by itself produces little tissue damage and immunopathological reactions are largely responsible for the associated disease. Immunopathological reactions associated with virus infections have different mechanisms. Lysis of infected cells can be related to antibody-mediated cytotoxicity and cell-mediated cytotoxicity as well as antibody dependent cellmediated cytotoxicity. In all these cases virus specific antigens must be present on the plasma membrane of the infected cells. On the other hand, normal cells can be lysed by complexes of virus antigen and antibody (Allison, 1974). A direct cytolytic effect of HBsAg is unlikely since the largest amount of antigen is found in carriers without evidence of liver disease and the smallest amount in massive necrotic viral hepatitis (Hadziyannis et al., 1972; Krawczynski et al., 1972). It can then be postulated that immunopathological reactions against virus-infected cells are largely responsible for the disease. So far, HBAg has been demonstrated in nuclei of hepatocytes (Millman et al., 1969), in the cytoplasm of hepatocytes (Hadziyannis et al., 1972) and in mesenchymal cells in the liver as well as in several extrahepatic tissues (Nowoslawski et al., 1972) using immunofluorescence and electronmicroscopy. However, the presence of HBsAg within the hepatocyte is unlikely to be important in the immune reactions to this antigen which are thought to cause the liver cell damage. The possibility of surface localization of HBsAg must therefore be considered. Using isolated vital liver cells we localized HBsAg to the surface of hepatocytes by direct fluorescence in patients with acute hepatitis B when studied in an early phase of the disease before the peak in the transaminase levels. The high frequency of cellular immune reactions to HBsAg detected in this period of the disease (Gerber et al., 1974) and the disappearance of the antigen from the surface as well as from the cytoplasm of the hepatocytes in a later phase, as demonstrated in our patients studied during the recovery from acute hepatitis B, support the hypothesis that these immune reactions may be effective in the lysis of infected liver cells as well as in the clearance of HBsAg from the liver. Further studies are required to clarify whether the HBsAg detected on the hepatocyte surface is an expression of intracellular virus associated proteins or is a consequence of binding of circulating antigen. In our cases the former possibility is supported by the detection of HBsAg in the cytoplasm in the same cases who were positive at the cell surface. Finally, a possible role of antibodies and of antigen-antibody complexes must be considered since in two cases we could detect both HBsAg and IgG at the cell surface. In patients with chronic liver disease cellular immune reactions to HBsAg are not common (Ito et al., 1972; Reed et al., 1974). On the other hand in chronic hepatitis and in cirrhosis, whether seropositive or seronegative, we could never detect the antigen at the liver cell surface, although HBsAg was found in the cytoplasm of hepatocytes in all the seropositive cases studied. Therefore it seems that the immunological reactions causing the disease in the patients are not directed against HBsAg. However, cell-mediated autoimmune reactions to liver specific membrane antigens are detected in CAH and in cryptogenic cirrhosis, independently of the presence of HBsAg, and these reactions are considered to play a role in the pathogenesis of the disease (Miller et al., 1972; Meyer zum Buschenfelde et al., 1974). We found IgG bound to the liver cell surface in both these two types of liver disease. So far, it has not been possible to establish whether they are autoantibodies to liver specific membrane proteins or antibodies to virus-induced neoantigens. In any case, it may be that they play an important role in liver injury. In a recent report Thomson et al. (1974) described in vitro cytotoxicity to rabbit liver cells by lymphocytes of patients with CAH. The cytotoxicity in this system was not T cell-mediated and a K-cell effect in the presence of antibodies was postulated (Eddleston & Williams, 1974). In conclusion, our results support the hypothesis that cellular immune reactions to HBsAg may be responsible for liver cell lysis in acute hepatitis B but that such a mechanism of damage seems to be unlikely in HBsAg-positive chronic liver disease. In CAH and in cryptogenic cirrhosis a better characterization of IgG bound to the liver cell surface is necessary in order to clarify the importance of an antibody-mediated cellular cytotoxicity in the pathogenesis of liver cell injury.
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Dr A. Alberti was visiting fellow at the laboratory of Professor K. H. Meyer zum Buschenfelde (University of Mainz, Germany) during 1974. The Authors want to thank Professor K. H. Meyer zum Buschenfelde for the kind revision of the manuscript. This work was supported in part by a grant (no. 74.00239.04) from Consiglio Nazionale delle Ricerche, Rome (Italy).
REFERENCES
ALBERTI, A., REALDI, G., TREMOLADA, F. & CADROBBI, P. (1975) HBAg on liver cell surface in viral hepatitis. Lancet, i, 346. ALLISON, A.C. (1974) Immunopathological mechanisms associated wvith virus infections. Advances in the Biosciences, volume 12 (ed by G. Rasp&), p. 347. Pergamon Press, Vieweg. ARNOLD, W. & VON MAYERSBACH, H. (1972) Changes in the solubility of immunoglobulins after fluorescent labelling and its influence on immunofluorescent techniques. J. Histochem. Cytochem. 12, 975. ARNOLD, W., MEYER ZUM BUSCHENFELDE, K.I., HOPF, U. & KORDBARLAG, C. (1975) Untersuchungen zur Hepatitis-B Antigen (HBAg)-fixation an peripheren Lymphocyten und isolierten Leberzellen bei Patienten mit entziindlichen lebererkrankungen. K/in. Wschr. 53, 231. DUDLEY, F.J., Fox, R.A. & SHERLOCK, S. (1972) Cellular immunity and hepatitis associated, Australia antigen liver disease. Lancet, i, 723. EDDLESTON, A.L.W.F. & WILLIAMS, R. (1974) Inadequate antibody response to HBAg or suppressor T-cell defect in development of active chronic hepatitis. Lancet, ii, 1543. GERBER, M1.J., PHUANGSAB, A., VITTAL, S.V., DOURDOUREKAS, D., STEIGMANN, F. & CLOWDUS, B.F. (1974) Cell-mediated immune response to Hepatitis B Antigen in patients with liver disease. Amer. J. dig. Disease, 19, 637. HADZIYANNIS, S., MIOUSSOUROS, A., VISSOULIS, C. & AFROUDAKIS, A. (1972) Cytoplasmic localization of Australia antigen in the liver. Lancet, i, 976. HOPF, U., ARNOLD, W., MEYER ZUM BUSCHENFELDE, K.H., FORSTER, E. & BOLTE, J.P. (1975) Studies on the pathogenesis of chronic inflammatory liver diseases: membranefixed IgG on isolated hepatocytes from patients. Clin. exp. Immunol. 22, 1. HOPF, U. & MEYER ZUM BOSCHENFELDE, K.H. (1974) Studies on the pathogenesis of experimental chronic active hepatitis in rabbits. II. Demonstration of immunoglobulin on isolated hepatocytes. Brit. 5. exp. Path. 55, 509. HOPF, U., MEYER ZUM BUSCHENFELDE, K.H. & FREUDENBERG, J. (1974) Liver specific antigens of different species. II. Localization of a membrane antigen at cell surface of isolated hepatocytes. Clin. exp. Immunol. 16, 117. ITO, K., NAKAGAWA, J., OKIMOTO, J. & NAKANO, H. (1972)
Chronic hepatitis migration inhibition of leucocytes in the presence of Australia antigen. New Engl. 7. Med. 286, 1005. JOHNSON, G.D., HOLBOROW, E.J. & GLYNN, L.E. (1965) Antibody to smooth muscle in patients with liver disease. Lancet, ii, 878. KRAWCZYNSKI, K., NAZAREWICZ, T., BRZOSKO, W. & NOWOSLAWSKI, A. (1972) Cellular localization of hepatitisassociated antigen in the liver of patients with different forms of hepatitis. _A. infect. Dis. 126, 372. MEYER ZUM BUSCHENFELDE, K.H., KNOLLE, J. & BERGER, J. (1974) Cellulare Immunoreaktionen gegenuber homologen leberspezifischen Antigenen (HLP) bei chronischen Leberentzundungen. K/in. Wschr. 52, 246. MILLER, J., MITCHELL, C.G., EDDLESTON, A.L.W.F., SMITH, M.G.M., REED, W.D. & WILLIAMS, R. (1972) Cell-mediated immunity to a human liver specific antigen in patients with active chronic hepatitis and primary biliary cirrhosis. Lancet, ii, 296. MILLMAN, I., ZAVATONE, V., GERSTLEY, B.J.S. & BLUMBERG, B.S. (1969) Australia antigen detected in the nuclei of liver cells of patients with viral hepatitis by fluorescent antibody technique. Nature (Lond.), 222, 181. NOWOSLAWSKI, A., KRAWCZYNSKI, K., BRZOSKO, W.J. & MADALINSKI, K. (1972) Tissue localization of Australia antigen immune complexes in acute and chronic hepatitis and liver cirrhosis. Amer. Y. Path. 68, 31. POPPER, H. & PARONETTO, F. (1972) Immunologic hepatic injury. Ver. Dtsch. Ges. inn. Med. 78, 790.
REALDI, G., ALBERTI, A., RUDE, L., TREMOLADA, F., BORTOLOTTI, F. & FATTOVICH, G. (1974) Metodo radioimmunologico ed elettrosineresi nello studio dell'antigene Australia (HBAg) e dell'anticorpo (HBAc). Acta med. patav. 34, 92. REED, W.D., LEE, W.M., EDDLESTON, A.L.W.F., MITCHELL, C.G., ZUCKERMAN, A.J. & WILLIAMS, R. (1974) Cellmediated immunity to hepatitis B antigen in antigennegative active chronic hepatitis. Gut, 15, 341. THOMSON, A.D., COCHRANE, M.A.G., MCFARLANE, I.G., EDDLESTON, A.L.W.F. & WILLIAMS, R. (1974) Lymphocyte cytotoxicity to isolated hepatocytes in chronic active hepatitis. Nature: New Biol. 252, 721. TRENCHEV, R., SNEYD, P. & HOLBOROW, E.J. (1974) Immunofluorescent tracing of smooth muscle contractile protein antigens in tissue other than smooth muscle. Clin. exp. Immunol. 16, 125.
Liver cell surface localization of hepatitis B antigen and of immunoglobulins in acute and chronic hepatitis and in liver cirrhosis A. ALBERTI,* G. REALDI,* F. TREMOLADA* & G. P. SPINAt *Istituto di Medicina Clinica, Patologia andt Istituto di Patologia Speciale Chirurgica, University of Padua, Padua, Italy
(Received 1 May 1976) SUMMARY
This paper describes immunofluorescence studies on liver cell surface localization of hepatitis B surface antigen (HBsAg) and of IgG in acute and chronic hepatitis and in cirrhosis. In acute hepatitis B, HBsAg was found at the surface of hepatocytes in an early phase of the disease, but not during the recovery. This finding is consistent with the hypothesis that immune reactions to HBsAg may be responsible for the liver cell lysis. In HBsAg-positive chronic hepatitis and cirrhosis the antigen was found in the cytoplasm, but not on the surface of the hepatocytes, while in HBsAg-negative cases the antigen could not be detected in the liver cells. Both in HBsAg-positive and in HBsAg-negative chronic active hepatitis (CAH) and cryptogenic cirrhosis, IgG bound to the membrane of the hepatocytes could be detected, suggesting a role of antibodies in the pathogenesis of the disease.
INTRODUCTION It has been postulated that immunopathological reactions play an important role in the pathogenesis of acute and chronic hepatitis. Both heteroimmune reactions to virus-associated antigens and autoimmune reactions to liver cell membrane antigens must be considered (Popper & Paronetto, 1972). It has been suggested that cellular immune reactions to HBsAg may be responsible for the liver cell damage in HBsAg-positive acute and chronic hepatitis (Dudley, Fox & Sherlock, 1972; Eddleston & Williams, 1974). An important requirement of this hypothesis would be the finding of HBsAg on the surface of infected liver cells. Cell-mediated autoimmune reactions to liver specific antigens can be detected in patients with chronic active hepatitis or cryptogenic cirrhosis, and the frequency of positive reactions in the HBsAg-positive and HBsAg-negative cases is similar (Meyer zum Biuschenfelde, Knolle & Berger, 1974; Reed et al., 1974). A common autoimmune process may be involved in the pathogenesis of the liver cell damage in both groups of patients, but the effector mechanism is not yet clarified. Cell-mediated immune reactions to liver specific proteins are well documented, but an increasing interest in humoral immunity is rising from the detection of IgG bound to the surface of hepatocytes of rabbits with experimentally induced CAH (Hopf & Meyer zum Biischenfelde, 1974), as well as of patients with CAH and signs of inflammatory activity (Arnold et al., 1975; Hopf et al., 1975). In a preliminary report we have described HBsAg at the surface of hepatocytes of patients with acute hepatitis B (Alberti et al., 1975). This paper reports further studies on liver cell surface localization of HBAg and of IgG in acute and chronic liver diseases. Correspondence: Dr A. Alberti, Instituto di Medicina Clinica, Patologia Medica, University of Padua, via Giustiniani 1, 35100 Padua, Italy.
396
397
HBsAg and IgG on liver cell surface MATERIALS AND METHODS
Patients. Eighteen patients with acute hepatitis B were studied; nine of whom were in an early phase of the disease with increasing transaminase levels, and nine at a later stage in the illness. Twenty-seven patients with chronic hepatitis or cirrhosis were studied: five of them had chronic persistent hepatitis, nine CAH, seven cryptogenic cirrhosis and six alcoholic cirrhosis. None of them had received immunosuppressive treatment in the last 6 months. Five subjects without liver disease or immunological abnormalities were tested as controls. Liver tissue. Specimens of liver tissue were obtained by needle or surgical biopsy. A portion of each specimen was immediately transferred into 5 ml EMBA (Eagle's medium/Serva no. 47370 A plus 2-5% bovine albumin/Behringwerke, Marburg) pH 7-4 at 370C and treated for immunofluorescence studies. The remaining portion of the biopsy was processed for routine histopathological examination. Preparation ofFITC-conjugated antisera. Specific anti-HBsAg antiserum (human IgG) obtained from a healthy carrier of high titre precipitating hepatitis B antibody, and commercial anti-human IgG antiserum from rabbit (Behringwerke, Marburg) were FITC conjugated according to Arnold & von Mayersbach (1972) and absorbed twice with acetone-dried rabbit liver powder (Sigma). Normal human and rabbit IgG were fluorescein-conjugated in the same manner. Direct immunofluorescence for liver cell surface localization of HBsAg and IgG. Surface localization of HBsAg and IgG was investigated using a direct immunofluorescence antibody technique on vital liver cells isolated in suspension according to Hopf, Meyer zum Buschenfelde & Freudenberg (1974) with some modifications. This technique involves the preparation of living suspensions ofisolated hepatocytes with intact membrane structure and the detection by immunofluorescence ofsurface antigens only. Briefly, biopsy specimens were gently minced in EMBA at 370C in a Petri dish and the resulting cell suspension TABLE 1. Surface and intracellular localization of HBsAg and IgG in the liver cells of patients with acute hepatitis B
Immunofluorescence Surface localization* Intracellular localizationt
Patient HBsAg SMA
HBsAg
IgG
HBsAg
0/
0/
0/
Biopsy within the 1st week of the disease 1 + 0 + +(6) 2 0 +(15) +(10) + 3 0 0 0 + + 4 + +(27) 0 5 + 0 + +(18) 6 0 0 + + 7 0 0 0 + 8 0 + + +(14) 9 + +(22) + +(14) Biopsy after 4-5 weeks of illness 1 0 0 0 0 2 0 0 0 0 3 0 0 0 0 4 0 0 + + 5 0 0 0 -+ 6 0 0 0 0 7 0 + 0 0 8 0 + 0 0 9 0 0 + 0
n.t. n.t. 0 + cyt(19) n.t. 0 n.t.
+cyt(9) +cyt(30) 0 0 n.t. 0 0 0 n.t. 0 0
IgG
n.t. n.t. 0 0 n.t.
0 n.t.
0 0 0 0 n.t. 0 0 0 n.t. 0 0
n.t. = Not tested. HBsAg= hepatitis B antigen in serum detected at the time ofliver biopsy; SMA=smooth muscle antibodies (titre< 1:20); cyt=cytoplasmatic pcsitivity; the percentage of positive cells is given in parentheses. * Detected on vital liver cells. t Detected on air-dried liver cells.
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was filtered through nylon wool and washed twice with EMBA at 370C (190 g for 5 min). The cell pellet was resuspended in 0-2 ml EMBA+0-2 ml FITC-conjugated anti-HBs or anti-IgG antiserum and incubated at 370C for 30 min in a moist chamber. The cells were then washed three times in EMBA at 370C (190 g for 5 min), mounted in liquid medium and examined by fluorescence microscopy. Only living hepatocytes, as detected by phase contrast microscopy, were examined for surface fluorescence. Direct immunofluorescence for liver intracellular localization of HBsAg and IgG. In order to investigate intracellular localization of HBsAg and IgG, liver cells isolated in suspension as described were smeared on slides using a cytocentrifuge (700 rev/min for 10 min) and air-dried before being incubated with FITC-conjugated anti-HBs or anti-IgG antiserum at 370C for 45 min. The stained slides were washed three times in PBS, mounted in glycerine and examined by fluorescence microscopy.
Specificity controls. The fluorescence was considered to be specific when blocked with an unlabelled antiserum in the inhibition test. Further controls were performed on vital liver cell suspensions using FITC-conjugated normal human or rabbit IgG in order to investigate the possibility that living hepatocytes may absorb immunoglobulins passively, or by microphagocytosis under these experimental conditions. Smooth-muscle antibodies. All sera were tested for smooth-muscle antibodies by the indirect fluorescent antibody technique (Johnson, Holborow & Glynn, 1965). Serological tests for HBsAg. All the sera were tested for the presence of HBsAg by counterelectrophoresis and by radioimmunoprecipitation according to methods previously described (Realdi et al., 1974).
RESULTS Acute hepatitis B The results obtained in eighteen patients with acute hepatitis B are summarized in Table 1. Of the nine patients studied in an early phase of the disease, all had HBsAg in the serum and six of them were positive for HBsAg at the liver cell surface. The fluorescence showed a non-homogeneous granular pattern (Fig. 1) and the number of positive cells ranged from 6-27%. A surface localization of IgG was found in two cases, both positive also for HBsAg. Intracellular localization of HBsAg and IgG was investigated in five patients; a granular cytoplasmatic fluorescence for HBsAg was found in three of them (Fig. 2). Of the nine patients studied during the recovery phase, HBsAg was still present in the serum of three, while the remaining six patients had already cleared the antigen. However, all nine were negative for HBsAg and IgG both in relation to the surface and in the cytoplasm of hepatocytes.
Chronic hepatitis and cirrhosis The results obtained in chronic hepatitis and in cirrhosis are summarized in Table 2. HBsAg was not detected at the liver cell surface in any of the twenty-seven patients (ten HBAg-positive, seventeen
FIG. 1. Surface localization of HBsAg on vital liver cells isolated from biopsies of patients with acute hepatitis B and treated with an anti-HBsAg FITC-conjugated antiserum. The fluorescence shows a non-homogeneous granular pattern with granules of different size (a-b), sometimes only at a portion of the membrane (c).
(Magnification x 378.)
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399
FIG. 2. Cytoplasmatic granular fluorescence in air-dried isolated liver cells from patients with HBsAg-positive acute (a) and chronic active (b-c) hepatitis after treatment with an anti-HBsAg FITC-conjugated antiserum. (Magnification x 240.)
FIG. 3. Surface localization of IgG on vital liver cells isolated from the biopsy of a patient with chronic active hepatitis and treated with anti-human IgG FITC-conjugated antiserum. In A and B the same liver cells on a different focus. The fluorescence shows a fine granular pattern (a) and typical membrane appearance (b). (Magnification x 378.)
HBAg-negative in serum) but was found in the cytoplasm of hepatocytes in all nine seropositive cases studied. Nuclear fluorescence was not found in any case although it sometimes happened that cells positive for HBsAg in the cytoplasm showed fluorescent granules in the region of the nucleus, probably caused by a layer of cytoplasm covering the nucleus (Fig. 2). IgG were detected at the surface of liver cells in seven out of nine patients with CAH, in three of seven with cryptogenic cirrhosis, but never in chronic persistent hepatitis or in alcoholic cirrhosis. The fluorescence occurred in a fine granular pattern, more homogeneous than that which we have observed for surface HBsAg in acute hepatitis (fig. 3). Ofthese ten cases with IgG bound to the hepatocyte surface five showed HBsAg in the serum as well as in the cytoplasm of the hepatocytes.
A. Alberti et al.
400
TABLE 2. Surface and intracellular localization of HBsAg and IgG in the liver cells of patients with chronic hepatitis and cirrhosis
Immunofluorescence
Surface localization* Intracellular localizations Patient HBsAg SMA
HBsAg
Chronic persistent hepatitis 1 0 0 2 0 + 0 3 + 4 0 + 0 5 + Chronic active hepatitis 1 0 + 2 + 0 3 0 + 0 4 + 0 5 + 6 + + 7 0 0 0 8 + 0 9 + Cryptogenic cirrhosis 1 0 0 2 0 0 0 0 3 0 + 4 0 0 5 0 0 6 0 7 0 Alcoholic cirrhosis 1 0 0 0 2 0 0 0 3 0 4 + 0 0 5 0 6 +
IgG
HBsAg
0 0 0 0 0
0 0 0 0 0
0 +cyt(6)
0 0 0 0
+(8) +(12) 0 +(15) +(5) +(18) +(26) 0 +(15)
+cyt(18) 0
0 0 0
0 0 0 0 0 0 0 0 0
0 +(6)
0 0 0
0 0 0 0 0 0
0 0 0
+(1I) 0 0
+(21) 0
n.t. n.t.
+cyt(10)
n.t.
+cyt(l1) +cyt(14) +cyt(5) n.t. 0
+cyt(15) 0 0 0 n.t.
0 0 n.t.
0 0 0 +cyt(3) 0
+cyt(l1)
IgG
0 0 n.t. n.t. 0 0 0 n.t. 0 0 0 n.t. 0 0 0 0 0 n.t. 0 0 n.t. 0 0 0 0 0 0
n.t. = Not tested. HBsAg= hepatitis B antigen in serum detected at the time of liver biopsy SMA= smooth muscle antibodies (titre < 1:20); cyt= cytoplasmatic positivity; the percentage of positive cells is given in parentheses. * Detected on vital liver cells. t Detected on air-dried liver cells.
Specificity controls All the positive results could be inhibited by unlabelled antisera. Patients without liver disease always gave negative results both for surface and for intracellular HBsAg and IgG. Using FITC-conjugated normal IgG, we could not detect either passive binding or microphagocytosis of IgG by living isolated liver cells, either in patients or in controls. The presence of smooth muscle antibodies, which cross-react with the liver cell membrane (Trenchev, Sneyd & Holborow, 1974), did not correlate with the finding of IgG bound to the hepatocyte surface (Tables 1 and 2).
HBsAg and IgG on liver cell surface
401
DISCUSSION Viruses can produce pathological changes in several ways. Many viruses destroy the cells in which they grow, whereas in other instances multiplication of the virus by itself produces little tissue damage and immunopathological reactions are largely responsible for the associated disease. Immunopathological reactions associated with virus infections have different mechanisms. Lysis of infected cells can be related to antibody-mediated cytotoxicity and cell-mediated cytotoxicity as well as antibody dependent cellmediated cytotoxicity. In all these cases virus specific antigens must be present on the plasma membrane of the infected cells. On the other hand, normal cells can be lysed by complexes of virus antigen and antibody (Allison, 1974). A direct cytolytic effect of HBsAg is unlikely since the largest amount of antigen is found in carriers without evidence of liver disease and the smallest amount in massive necrotic viral hepatitis (Hadziyannis et al., 1972; Krawczynski et al., 1972). It can then be postulated that immunopathological reactions against virus-infected cells are largely responsible for the disease. So far, HBAg has been demonstrated in nuclei of hepatocytes (Millman et al., 1969), in the cytoplasm of hepatocytes (Hadziyannis et al., 1972) and in mesenchymal cells in the liver as well as in several extrahepatic tissues (Nowoslawski et al., 1972) using immunofluorescence and electronmicroscopy. However, the presence of HBsAg within the hepatocyte is unlikely to be important in the immune reactions to this antigen which are thought to cause the liver cell damage. The possibility of surface localization of HBsAg must therefore be considered. Using isolated vital liver cells we localized HBsAg to the surface of hepatocytes by direct fluorescence in patients with acute hepatitis B when studied in an early phase of the disease before the peak in the transaminase levels. The high frequency of cellular immune reactions to HBsAg detected in this period of the disease (Gerber et al., 1974) and the disappearance of the antigen from the surface as well as from the cytoplasm of the hepatocytes in a later phase, as demonstrated in our patients studied during the recovery from acute hepatitis B, support the hypothesis that these immune reactions may be effective in the lysis of infected liver cells as well as in the clearance of HBsAg from the liver. Further studies are required to clarify whether the HBsAg detected on the hepatocyte surface is an expression of intracellular virus associated proteins or is a consequence of binding of circulating antigen. In our cases the former possibility is supported by the detection of HBsAg in the cytoplasm in the same cases who were positive at the cell surface. Finally, a possible role of antibodies and of antigen-antibody complexes must be considered since in two cases we could detect both HBsAg and IgG at the cell surface. In patients with chronic liver disease cellular immune reactions to HBsAg are not common (Ito et al., 1972; Reed et al., 1974). On the other hand in chronic hepatitis and in cirrhosis, whether seropositive or seronegative, we could never detect the antigen at the liver cell surface, although HBsAg was found in the cytoplasm of hepatocytes in all the seropositive cases studied. Therefore it seems that the immunological reactions causing the disease in the patients are not directed against HBsAg. However, cell-mediated autoimmune reactions to liver specific membrane antigens are detected in CAH and in cryptogenic cirrhosis, independently of the presence of HBsAg, and these reactions are considered to play a role in the pathogenesis of the disease (Miller et al., 1972; Meyer zum Buschenfelde et al., 1974). We found IgG bound to the liver cell surface in both these two types of liver disease. So far, it has not been possible to establish whether they are autoantibodies to liver specific membrane proteins or antibodies to virus-induced neoantigens. In any case, it may be that they play an important role in liver injury. In a recent report Thomson et al. (1974) described in vitro cytotoxicity to rabbit liver cells by lymphocytes of patients with CAH. The cytotoxicity in this system was not T cell-mediated and a K-cell effect in the presence of antibodies was postulated (Eddleston & Williams, 1974). In conclusion, our results support the hypothesis that cellular immune reactions to HBsAg may be responsible for liver cell lysis in acute hepatitis B but that such a mechanism of damage seems to be unlikely in HBsAg-positive chronic liver disease. In CAH and in cryptogenic cirrhosis a better characterization of IgG bound to the liver cell surface is necessary in order to clarify the importance of an antibody-mediated cellular cytotoxicity in the pathogenesis of liver cell injury.
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A. Alberti et al.
Dr A. Alberti was visiting fellow at the laboratory of Professor K. H. Meyer zum Buschenfelde (University of Mainz, Germany) during 1974. The Authors want to thank Professor K. H. Meyer zum Buschenfelde for the kind revision of the manuscript. This work was supported in part by a grant (no. 74.00239.04) from Consiglio Nazionale delle Ricerche, Rome (Italy).
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