Gastroenterologia Japonica Copyright 9 1992 by The Japanese Society of Gastroenterology
VoL 27, No. 6 Printed in Japan
Electron microscopic study of hepatitis B virus-associated antigens on the infected liver cell membrane in relation to analysis of immune target antigens in chronic hepatitis B T a k a f u m i SAiTO 1, Tomoteru KAMIMURA 2, Masamichi ISHIBASHI ~, Haruhide SHINZAWA 1, and Tsuneo TAKAHASHI ~
1Second Department of Internal Medicine, Yamagata University School of Medicine, Yamagata, Japan; and :Third Department of Internal Medicine, Niigata University School of Medicine, Niigata, Japan
Summary: To clarify the characteristics of cell surface Pre-S2 Ag, HBcAg and HBeAg immunohistochemically and to explore their relationship with a cellular immune target antigen, 31 liver biopsy specimens from chronic HBV carriers were examined by immunoperoxidase staining. By immune light microscopy, Pre-S2 Ag was detected on the liver cell membrane in 18 (580/0) of the 31 cases, HBcAg in 4 cases (13%) and HBeAg in 4 cases (13%). Pre-S2 Ag frequently showed a honeycomb-like membrane expression pattern which was present regardless of liver inflammation, whereas HBcAg and HBeAg exhibited a scattered membrane expression pattern detected in areas of marked inflammation. Of the 18 cases showing a honeycomb-like Pre-S2 Ag expression, 3 concomitantly showed a scattered membrane expression pattern. Immunoelectron microscopy revealed these two distinct membrane expression patterns. In areas showing a honeycomb-like membrane expression pattern, Pre-S2 Ag was demonstrated in the intercellular space and on the basolateral membranes of hepatocytes, but was not detected on the cell membranes in areas of the intercellular space lacking an immunoreaction. Cytoplasmic expression of Pre-S2 Ag was less extensive in these hepatocytes. These findings suggest that the honeycomb-like membrane expression ofPre-S2 Ag results from attachment of extracellular antigen to the liver cell membrane. In contrast, in areas showing a scattered membrane expression pattern, Pre-S2 Ag, HBcAg and HBeAg were each detected as single-layered linear deposits along the cell membrane, but were absent in the intercellular space. Each antigen was also expressed abundantly in the cytoplasm, and the immunoproducts appeared to fuse with the cell membrane. These findings suggest that the scattered membrane expression of these antigens results from intrahepatic transfer of antigen synthesized in the liver cell to the cell membrane, possibly serving as a target for the host immune-mediated response in connection with inflammation. GastroenterolJpn 1992;27:734-744. Key words:
hepatitis B; hepatitis B virus; immunohistochemistry; target antigen.
Introduction Immunopathological studies have revealed that the m e c h a n i s m of liver cell injury in chronic hepatitis B virus (HBV) infection is mediated by the host's cellular i m m u n e response to infected liver cells 1. In this mechanism, T cell-mediated cell cytotoxicity plays the major role in progres-
sion of liver injury ~. Immunological killing of HBV-infected hepatocytes is thought to be initiated by sensitized cytotoxic T lymphocytes which recognize viral target antigens expressed on the liver cell plasma m e m b r a n e 35. What is the target antigen recognized by T lymphocytes in HBV infection? Expression of HBV-associated antigens on the infected liver cell m e m b r a n e provides an
Received December 5, 1991. Accepted June 26, 1992. Address for correspondence: Takafumi Saito, M.D., The Second Department of Internal Medicine, Yamagata University School of Medicine, Yamagata 990-23, Japan.
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important clue. Although a large number of immunohistochemical studies have been done on HBV-associated antigen expression on the liver cell membrane, antigen expression patterns have not yet been sufficiently characterized. Against this background, the authors carried out an immunohistochemical study of the expression patterns of Pre-S2, HBc and HBe antigens (Ag) on the infected liver cell surface. Immunoelectron microscopy was considered to give new insight into the interpretation of the various expression patterns of these antigens on the liver cell membrane. The aim of the present study was to clarify the characteristics of these surface HBV-associated antigens immunohistochemically and to explore their relationship with a cellular immune target antigen on the basis of their morphological features. Here we report the results of an immunohistochemical study of liver tissues from patients with chronic hepatitis B virus infection. Patients and Methods
Patients Liver biopsy specimens from 31 patients (25 males, 6 females, mean age 34.8+8.8 years, mean+SD) with chronic HBV infection were examined. Liver tissue was obtained by diagnostic needle biopsy, after obtaining informed consent. The histological features were classified according to the international standard criteria6, and included; nonspecific reactive hepatitis (NSRH) in 6 cases, chronic persistent hepatitis (CPH) in 6 cases, chronic active hepatitis (CAH) in 17 cases, and liver cirrhosis in 2 cases. The level of alanine aminotransferase activity (ALT) in serum ranged from 11 to 491 IU/1 (129: [74, 238], Median: [25%, 75% ]), and serum HBeAg was positive in all cases. None of the patients had recieved any immunosuppressive or antiviral therapy before the liver biopsy. Monoclonal antibodies Mouse monoclonal antibodies against hepatitis B pre-S2 antigen (anti-pre-S2), hepatitis B core antigen (anti-HBc), or hepatitis B e antigen (anti-
735
HBe) were purchased from the Institute of Immunology, Co. Ltd., Tokyo, Japan. These ascitic fluids contained specific mouse IgG against each antigen with a protein concentration of 0.5 mg/ml and were used at a dilution of 1:30. Immunohistochemistry An indirect immunoperoxidase staining technique was employed for detection of Pre-S2 Ag, HBcAg, HBeAg on the hepatocyte plasma membrane. Light microscopic study: Liver biopsy specimens were fixed immediately in periodate-lysine4% paraformaldehyde (PLP) solution, embedded in OCT compound (Miles Lab. Inc., Naperville, IL) and quickly frozen with cooled hexane. Sixmicrometer-thick serial cryostat sections were prepared, and washed extensively with phosphatebuffered saline, pH 7.2 (PBS), before immunostaining. After inhibition of endogenous peroxidase activity of the liver tissue with 0.05M periodic acid solution for 10 rain at 4~ the sections were incubated with mouse monoclonal anti-Pre$2, anti-HBc or anti-HBe in a moist chamber overnight at 4~ followed by incubation with peroxidase-conjugated sheep anti-mouse IgG (Fab'2) (Amersham Japan Co. Ltd., Tokyo, Japan) for 3 hours. The reaction products of these antigens were stained with a 0.02% solution of 3,3'diaminobenzidine (DAB) in 0.05M Tris-Hcl buffer, pH 7.2, containing 0.01% hydrogen peroxide. Slides were counterstained with methyl green, dehydrated and mounted. Electron microscopic study: After incubation with the primary and secondary antibodies, as described above, the sections were further fixed in 1% glutaraldehyde in PBS for 10 min and incubated in DAB solution containing 0.01% hydrogen peroxide for 5 min. This was followed by postfixation with 2% osmium tetroxide in PBS for 1 hour, dehydration in a graded ethanol series to 100%, and embedding in Epon 812 in gelatin capsules. Ultrathin sections were observed by electron microscopy without additional staining. As an immunostaining control, sections were incubated with non-immune mouse ascites or PBS instead of the primary antibody in the first
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Figure 1. Immune light microscopy reveals two kinds of expression pattern of Pre-S2 Ag on the ceil membrane. (A) Honeycomb-like membrane expression pattern, connected from cell to cell, is evident regardless of inflammation in the liver. (B) Scattered membrane expression pattern is evident in highly inflammatory areas. Liver cells showing cytoplasmic expression with membranous accentuation are swollen and partially disrupted in areas of piecemeal necrosis, and one of them is surrounded by the inflammatory cellular infiltrates and fibrosis (arrow). Immunoperoxidase; A: x 200, B: x 320.
step of the immunostaining procedure. Furthermore, there was no specific staining in control liver biopsy specimens from patients with HBsAgnegative chronic liver disease. Results
Immune light microscopy Pre-S2 Ag showed a honeycomb-like membrane expression pattern, which was apparently connected from cell to cell. The cytoplasm of cells
showing this membrane expression pattern contained hardly any Pre-S2 Ag (Figure 1A). This expression pattern was specifically observed in the liver. In 3 cases of CAH among 18 cases with a honeycomb-like membrane expression pattern, membrane Pre-S2 Ag-positive cells were also demonstrated with a scattered membrane expression pattern in areas of piecemeal necrosis or high inflammatory activity. In the cells of these 3 cases, expression of Pre-S2 Ag was also detected in submembranous areas of the cytoplasm (Figure 1B).
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Figure 2. The nucleocapsid antigens (HBcAg and HBeAg) are demonstrated with scattered membrane expression in highly inflammatory areas. Membrane HBcAgpositive cells (A) and membrane HBeAgpositive cells (B) are swollen and partially disrupted in areas of piecemeal necrosis. Cytoplasmic, or both cytoplasmic and nuclear expression, are simultaneously detected with membranous accentuation. P: Portal area. Immunoperoxidase; A: x320, B: x 350.
The distribution of membrane HBcAg-positive cells was observed with a scattered membrane expression pattern in areas of high inflammatory activity in the liver. Immunoreaction for HBcAg was also detected in the nucleus and cytoplasm, or only in the cytoplasm, in these hepatocytes
(Figure 2A). The distribution of membrane HBeAg-positive cells was demonstrated with a scattered membrane expression pattern in highly inflammatory areas, as in the case of HBcAg. Membrane
HBeAg-positive cells had both nuclear and cytoplasmic expression, or cytoplasmic expression only (Figure 2B). However, the distribution of membrane HBcAg-positive cells was not always in accordance with that of membrane HBeAg-positive cells when serial sections were examined. The expression of HBV-associated antigens on the infected liver cell membrane in comparison with histological findings is summarized in Table 1. Of the 31 liver specimens, Pre-S2 Ag was detected on the cell membrane in 18 cases (58%), HBcAg in 4
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Table 1. Membrane expression pattern of Pre-S2 Ag, HBcAg and HBeAg in comparison with liver histology
Membrane expression pattern Pre-S2 Ag
Honeycomb-like expression
No. of cases positive (31 )
Histology NSRH (6)
CPH (6)
CAH (17)
LC (2)
18
4
3
10
1
0
0
3
0
0
0
3
1
0
0
3
1
(580/0) Scattered expression
3
(10%) HBcAg
Scattered expression
4
(13o/0) HBeAg
Scattered expression
4 (13%)
NSRH: nonspecific reactive hepatitis, CPH: chronic persistent hepatitis, CAH: chronic active hepatitis, LC: liver cirrhosis.
cases (13%) and HBeAg in 4 cases (130/0). In relation to the histological features, a honeycomb-like membrane expression pattern of Pre-S2 Ag was observed in various types of histology, whereas a scattered membrane expression pattern of Pre-S2 Ag, HBcAg or HBeAg was observed only in cases of marked inflammation such as CAH or LC.
Immunoelectron microscopy (1) Pre-S2 Ag I m m u n e light microscopy revealed two kinds of Pre-S2 Ag expression pattern on the infected liver cell surface. One was a honeycomb-like membrane expression pattern which was present regardless of inflammation in the liver, and the other was a scattered membrane expression pattern detected in areas of marked inflammation. Immunoelectron microscopy revealed a difference of these two distinct Pre-S2 Ag membrane-expression patterns. An area of honeycomb-like membrane expression as revealed by immune light microscopy was selected for immunoelectron microscopic observation. As described previously7, the immunoreaction for Pre-S2 Ag was observed on the liver cell plasma membrane except for the bile canalicular membrane. It was also observed in the intercellular space, but was less extensive in the cytoplasm. A higher magnification view of the lateral surface of an infected hepatocyte is shown in Figure 3. The immunoreaction for Pre-S2 Ag was observed in the intercellular space on the left side
partially indicated by arrows, but it was not present on the right side, as indicated by arrowheads. Pre-S2 Ag was detected on the plasma membrane in areas of the intercellular space showing a positive immunoreaction (left side shown by arrows), but was not detected on the plasma membrane in areas of the intercellular space lacking an immunoreaction (right side shown by arrow heads). Submembranous cytoplasmic expression was not observed. These findings suggest that the honeycomb-like membrane expression of Pre-S2 Ag results from attachment of extracellular antigen to the liver cell membrane. The ultrastructural morphology in areas showing a scattered membrane expression pattern of Pre-S2 Ag by i m m u n e light microscopy is shown in Figure 4. Pre-S2 Ag was detected as a singlelayered linear deposit along the cell membrane, but was absent in the intercellular space. Pre-S2 Ag was also expressed abundantly in the cytoplasm immediately below the membrane, and the immunoproducts appeared to fuse with the cell membrane. (2) HBcAg and HBeAg The electron microscopic expression pattern of HBcAg on the liver cell membrane is shown in Figure 5. Except for the bile canalicular membrane, HBcAg showed a mono-layered linear distribution along the cell membrane. It is noteworthy that HBcAg expression was absent in the intercellular spaces, although it was present on the membrane of adjoining liver cells and abundant in their
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Lwer membrane HBV-assoclated antigens
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Figure 3. Ultrastructural demonstration of the lateral surface of infected hepatocytes showing a honeycomb-like membrane expression pattern. Pre-S2 Ag is present in the intercellular space on the left, but is absent on the right. Both plasma membranes adjacent to the reaction-positive intercellular space (left side, inset), as shown by arrows, are positive for this antigen. In contrast, both plasma membranes unassociated with any immunoreaction in the intercellular space (right side), as indicated by arrow heads, show no reaction. No counterstain; x25000, inset, x60000.
cell organelles. The electron microscopic expression pattern of HBeAg on the liver cell membrane is shown in Figure 6. The expression pattern of HBeAg was fundamentally the same as that of HBcAg; i.e., HBeAg showed a mono-layered linear distribution along the cell membrane and was absent in the intercellular spaces. Similarly, the immunoreaction for HBeAg was abundant in the cell organelles. The reaction product for HBeAg in the positive membrane and that in the cytoplasm immediately below the membrane were occasionally fused. These findings suggest that the scattered membrane expression of Pre-S2 Ag, HBcAg or HBeAg results from intrahepatic transfer of antigens synthesized in the liver cell to the cell membrane.
Discussion
In the present study, Pre-S2 Ag, HBcAg, and HBeAg were all demonstrated on the plasma membrane of infected liver cells at the ultrastructural level, and the morphological features of each antigen expressed on the cell surface were specific. Target antigens for immunological response in the cell-damaging mechanism of hepatitis B have been studied with various methods. In vitro, HBsAg was first revealed as a target antigen by lymphocyte test against HBsAg-coated target cells8. A series of papers have claimed that HBcAg is a target antigen based on the autologous lymphocyte cytotoxicity test 912. Histologically, the expression of HBV-associated antigens on the
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Figure 4. Pre-S2 Ag showing a scattered membrane expression pattern appears as a single-layered linear deposit along the cell membrane and is absent in the intercellular space. Large amounts of reaction product for Pre-S2 Ag are evident in the cytoplasm immediately below the cell membrane, and have partially fused with the membrane. No counterstain; x22000.
liver cell membrane has been detected, and its correlation with target antigens were explored. In earlier studies, HBsAg was considered to be a cellular immune target antigen, because an immunofluorescence study revealed it easily on the liver cell surface with a honeycomb-like membrane expression pattern ~3. HBcAg was also demonstrated on infected liver cell surface, showing a scattered membrane expression pattern immunohistochemically1416, although its incidence seems to be lower because anti-HBc antibody, which is abundant in blood, can mask the antigen membrane expression 17. As a result of analysis of histological findings and viral replication in relation to antigen expression patterns in hepatic lobules by light microscopy, it has been reported that a diffuse, honeycomb-like membrane expression of
HBsAg is associated with viral replication and not with tissue inflammation, and that localization of HBcAg confined to the cytoplasm or membrane is associated with active viral replication and intense inflammation14~6'18. lmmunoelectron microscopy has also demonstrated the expression of HBcAg on the liver cell membrane and attachment of lymphocytes to liver cells with abundant HBcAg ~9. The significance of HBcAg as a target antigen has been indicated. However, HBsAg may also serve as a target antigen under some conditions, because expression of HBsAg on the cell membrane and attachment of CD8-positive lymphocytes to such membranes have been demonstrated by immunoelectron microscopy and immune double staining method 2~ These previous histochemical reports were
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Liver membrane HBV-associated antigens
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Figure 5. HBcAg shows a mono-layered linear distribution along the membranes of adjoining liver cells (arrows) but is absent in the intercellular spaces. Immunoreactions for HBcAg are abundant in the cell organelles. No counterstain; x 14000.
designed to clarify whether HBV-associated antigens are present on the infected liver cell membrane. The present study is the first attempt to analyze the antigen expression patterns on the liver cell membrane and to evaluate target antigens based on the morphological features of membrane-associated antigens. In addition to study of HBsAg and HBcAg, it is necessary to investigate the expression of HBeAg and Pre-S2 Ag on the infected liver cell membrane because of the reported possibility that these antigens may also serve as target antigens 2225. In this study, Pre-S2 Ag was found by light microscopy to show not only the previously reported honeycomb-like membrane expression pattern 7,26, similar to that of HBsAg 13'~4, but also a scattered membrane expression pattern in markedly inflamed areas. By light microscopy, both
HBcAg and HBeAg were detected on the cell surface with a scattered membrane expression pattern in areas of marked inflammation. These antigens were also seen in the cytoplasm and occasionally in the nucleus. There were only a few cases that showed a scattered membrane expression of these antigens. Kojima et al found a scattered membrane expression of HBcAg in only 4 out of the 25 cases 19, and Yamada et al found that of HBeAg in only 2 of 19 cases 27. The frequency of positive scattered membrane expression of these antigens in our study was roughly in agreement with the above reports. It is very difficult to identify a scattered membrane expression of these antigens in a limited number of biopsied specimens histologically. Some reports suggest that immunological response against envelope antigen, including Pre-S2 Ag is closely related to the
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Figure 6. HBeAg shows a mono-layered linear distribution continuously along the cell membrane. Reaction product for HBeAg in the positive membrane and that in the cytoplasm immediately below the membrane have partially fused. Note the absence of immunoreactions for HBeAg in the membrane of the adjoining cell and intercellular space. No counterstain; x 16000.
immunological clearance of HBV in acute hepatitis B 28'29. During acute exacerbation of chronic hepatitis B, a low level of IgM-HBc antibody can be detected in blood concomitantly with a proliferation of HBV and severe acute inflammation occurs in liver. There is a possibility that Pre-S2 Ag might serve as a target antigen under some condition such as acute exacerbation of chronic hepatitis B22. Our present data suggest that Pre-S2 Ag might also serve as a target antigen at a particular stage of chronic hepatitis B since a scattered membrane expression of Pre-S2 Ag can be detected as well as that of HBcAg or HBeAg in areas of marked inflammation. Immunoelectron microscopy revealed these two distinct membrane expression patterns of the antigens, suggesting that the membrane-bound
expression of each antigen is mediated by a different mechanism. Ultrastructural morphology suggested that if the localization pattern of Pre-S2 Ag showed a honeycomb-like membrane expression in hepatic lobules, then the antigen originated from outside liver cells. This type of membrane expression of Pre-S2 Ag was unique to the liver. It was proposed in a previous report that membrane-associated Pre-S2 Ag might originate from an extracellular circulating antigen 7. It has been speculated that Pre-S2 Ag play a role as a receptor for polymerized human serum albumin (pHSA) 3~ and can adhere to hepatocytes specifically by binding to pHSA receptors on the liver cell surface membranes via pHSA in blood. HBeAg did not have a honeycomb-like membrane expression pattern like Pre-
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Liver membrane HBV-assoctated antigens
$2 Ag. This type of membrane expression of HBeAg has not yet been documented, and its molecular mechanism remains unknown. HBeAg may not be able to bind to the membrane by itself, whereas Pre-S2 Ag adheres to the membrane specifically via pHSA. The mechanism responsible for this type of membrane expression was thought to be absorption ofPre-S2 Ag onto the cell-surface membrane from the extracellular space. In liver tissue showing a honeycomb-like membrane expression of Pre-S2 Ag observed in various types of liver histology, this expression pattern does not correlate with inflammatory activity26. On the other hand, if the localization pattern of Pre-S2 Ag showed a scattered membrane expression, then the antigen on the membrane must have been synthesized in the liver cells and then transported to the membrane. HBcAg or HBeAg also showed a similar pattern of scattered membrane expression in hepatic lobules, suggesting that the mechanism responsible for this type of membrane expression was synthesis of these antigens in the liver cell and subsequent intrahepatic transport to the cell-surface membrane. Although ultrastructural evidence for the presence of HBeAg on parts of the plasma membrane has been reported 27, this study illustrated for the first time continuous surface membrane expression of HBeAg, like that of HBcAg. Regions showing such a scattered membrane expression pattern of these antigens had severe lymphocyte infiltration and cell necrosis. The present findings suggest that the scattered membrane expression of Pre-S2 Ag, HBcAg or HBeAg on the infected liver cell surface is of biological significance as a target antigen in connection with inflammation, and that this target antigen expression occurs as a result of its transport within liver cells to the membrane. The authors are most grateful to Dr. Fumihiro Ichida and Dr. Makoto Ishikawa for giving an opportunity to do this work.
References 1. Ferrari C, Penna A, DegliAntoni A, et al. Cellular immune response to hepatitis B virus antigens (An overview). J Hepatol
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1988;7:21-33. 2. Eggink HF, HouthoffHJ, Huitema S, et al. Cellular and humoral immune reactions in chronic active liver disease. Lymphocyte subsets and viral antigens in liver biopsies of patients with acute and chronic hepatitis B. Clin exp Immunol 1984;56:121-128. 3. Thomas HC, Path MRC, Lok ASE The immunopathology of autoimmune and hepatitis B virus-induced chronic hepatitis. Sem Liver Dis 1984;4:36-46. 4. Mondelli M, Eddleston ALWF: Mechanisms of liver cell injury in acute and chronic hepatitis B. Sem Liver Dis 1984;4:47-58. 5. Eddleston ALWF, Mondelli M. Immunopathological mechanisms of liver cell injury in chronic hepatitis B virus infection. J Hepatol 1986;3(Suppl. 2):s17-s23. 6. Bianchi L, Groote JD, Desmet VJ, et al. Acute and chronic hepatitis revisited. Review by an international group, lancet 1977;914919. 7. Saito T, Kamimura T, Asakura H, et al. Ultrastructural localization of Pre-S2 polypeptides in the liver tissues of patients with chronic hepatitis B virus infection. Liver 1989;9:329-337. 8. Alberti A, Realdi G, Bortolotti F, et al. T-lymphocyte cytotoxicity to HBsAg-coated target cells in hepatitis B virus infection. Gut 1977;18:1004-1009. 9. Mondelli M, Vergani GM, Albertl A, et al. Specificity of T lymphocyte cytotoxicity to autologous hepatocytes in chronic hepatitis B virus infection: evidence that T cells are directed against HBV core antigen expressed on hepatocytes. J Immunol 1982;129: 2773-2778. 10. Naumov NV, Mondelli M, Alexander GJM, et al. Relationship between expression of hepatitis B virus antigens in isolated hepatocytes and autologous lymphocyte cytotoxicity in patients with chronic hepatitis B virus infection. Hepatology 1984;4:63-68. 11. Vento S, Hegarty JE, Alberti A, et al. T lymphocyte sensitization to HBcAg and T cell-mediated unresponsiveness to HBsAg in hepatitis B virus-related chronic liver disease. Hepatology 1985; 5:192-197. 12. Mondelli M, Bortolottl F, Pontisso P, et al. Definition of hepatitis B virus (HBV)-specific target antigens recognized by cytotoxic T cells in acute HBV infection. Clin Exp Immuno11987;68:242-250. 13. Ray MB, Desmet VJ, Bradburne AF, et al. Differential distribution of hepatitis B surface antigen and hepatitis B core antigen in the liver of hepatitis B patients. Gastroenterology 1976;71:462-467. 14. Chu CM, Liaw YF. Intrahepatic distribution of hepatitis B surface and core antigens in chronic hepatitis B virus infection. Gastroenterology 1987;92:220-225. 15. Hsu HC, Su IJ, Lai MY, et al. Biologic and prognostic significance of hepatocyte hepatitis B core antigen expressions in the natural course of chronic hepatitis B virus infection. J Hepato11987;5:4550. 16. Suzuki K, Uchida T, Shikata T. Histopathological analysis of chronic hepatitis B virus (HBV) infection in relation to HBV replication. Liver 1987;7:260-270. 17. Trevisan A, Realdi G, Alberti A, et al. Core antigen-specific immunoglobulin G bound to the liver cell membrane in chronic hepatitis B. Gastroenterology 1982;82:218-222. 18. Ramalho F, Brunetto M, Rocca G, et al. Serum markers of hepatitis B virus replication, liver histology and intrahepatic expression of hepatitis B core antigen. J Hepatol 1988;7:14-20. 19. Kojima T, Bloemen J, Desmet VJ. Immune electron microscopic demonstration of hepatitis B core antigen (HBcAg) in liver cell plasma membranes. Liver 1987;7:191-200. 20. Sasaki H, Kojima T, Matsui S, et al. Interaction of lymphocytes
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with hepatocytes containing hepatitis B antigen: ultrastructural demonstration of target antigen and T-cell subsets by the peroxidase antibody technique. Virchows Arch A 1987;411:489-498. Aoyama K, Kojima T, Inoue K, et al. Immunohistochemical investigation of hepatitis B virus associated antigens, HLA antigens and lymphocyte subsets in type B chronic hepatitis. Gastroenterol Jpn 1990;25:41-53. Barnaba V, Franco A, Alberti A, et al. Recognition of hepatitis B virus envelope proteins by liver-infiltrating T lymphocytes in chronic HBV infection. J Immunol 1989;143:2650-2655. Hu KQ, Hao LJ, Zhang YY, et al. Intrahepatic Expression of Pre-S proteins of hepatitis B virus and its possible relation to liver cell necrosis. Am J Gastroenterol 1989;84:1538-1542. Pignatelli M, Waters J, Lever A, et al. Cytotoxic T-cell responses to the nucleocapsid proteins of HBV in chronic hepatitis. Evidence that antibody modulation may cause protracted infection. J Hepatol 1987;4:15-21. Milich DR, McLachlan A, Moriarty A, et al. Immune response to hepatitis B virus core antigen (HBcAg): Localization of T cell recognition sites within HBcAg/HBeAg. J Immunol 1987;139:
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1223-1231. 26. Kakumu S, Arao M, Mizokami M, et al. Pre-S proteins in chronic hepatitis B virus infection: markers of active viral infection. Am J Gastroenterol 1989;84:1250-1254. 27. Yamada G, Takaguchi K, Matsueda K, et al. Immunoelectron microscopic observation of intrahepatic HBeAg in patients with chronic hepatitis B. Hepatology 1990;12:133-140. 28. Budkowska A, Dubreil P, Capel F, et al. Hepatitis B virus pre-S2 gene-encoded antigenic specificity and anti-pre-S2 antibody: Relationship between anti-pre-S2 response and recovery. Hepatology 1986;6:360-368. 29. Budkowska A, Riottot MM, Dubreuil P, et al. Monoclonal antibody recognizing pre-S2 epitope of hepatitis B virus: Characterization of pre-S2 epitope and anti-pre-S2 antibody. J Med Virol 1986;20:111-125. 30. Okamoto H, Imai M, Usuda S, et al. Hemagglutination assay of polypeptide coded by the pre-S region of hepatitis B virus DNA with monoclonal antibody; correlation of pre-S polypeptide with the receptor for polymerized human serum albumin in serums containing hepatitis B antigens. J Immunol 1985;134:1212-1216.
VoL 27, No. 6 Printed in Japan
Electron microscopic study of hepatitis B virus-associated antigens on the infected liver cell membrane in relation to analysis of immune target antigens in chronic hepatitis B T a k a f u m i SAiTO 1, Tomoteru KAMIMURA 2, Masamichi ISHIBASHI ~, Haruhide SHINZAWA 1, and Tsuneo TAKAHASHI ~
1Second Department of Internal Medicine, Yamagata University School of Medicine, Yamagata, Japan; and :Third Department of Internal Medicine, Niigata University School of Medicine, Niigata, Japan
Summary: To clarify the characteristics of cell surface Pre-S2 Ag, HBcAg and HBeAg immunohistochemically and to explore their relationship with a cellular immune target antigen, 31 liver biopsy specimens from chronic HBV carriers were examined by immunoperoxidase staining. By immune light microscopy, Pre-S2 Ag was detected on the liver cell membrane in 18 (580/0) of the 31 cases, HBcAg in 4 cases (13%) and HBeAg in 4 cases (13%). Pre-S2 Ag frequently showed a honeycomb-like membrane expression pattern which was present regardless of liver inflammation, whereas HBcAg and HBeAg exhibited a scattered membrane expression pattern detected in areas of marked inflammation. Of the 18 cases showing a honeycomb-like Pre-S2 Ag expression, 3 concomitantly showed a scattered membrane expression pattern. Immunoelectron microscopy revealed these two distinct membrane expression patterns. In areas showing a honeycomb-like membrane expression pattern, Pre-S2 Ag was demonstrated in the intercellular space and on the basolateral membranes of hepatocytes, but was not detected on the cell membranes in areas of the intercellular space lacking an immunoreaction. Cytoplasmic expression of Pre-S2 Ag was less extensive in these hepatocytes. These findings suggest that the honeycomb-like membrane expression ofPre-S2 Ag results from attachment of extracellular antigen to the liver cell membrane. In contrast, in areas showing a scattered membrane expression pattern, Pre-S2 Ag, HBcAg and HBeAg were each detected as single-layered linear deposits along the cell membrane, but were absent in the intercellular space. Each antigen was also expressed abundantly in the cytoplasm, and the immunoproducts appeared to fuse with the cell membrane. These findings suggest that the scattered membrane expression of these antigens results from intrahepatic transfer of antigen synthesized in the liver cell to the cell membrane, possibly serving as a target for the host immune-mediated response in connection with inflammation. GastroenterolJpn 1992;27:734-744. Key words:
hepatitis B; hepatitis B virus; immunohistochemistry; target antigen.
Introduction Immunopathological studies have revealed that the m e c h a n i s m of liver cell injury in chronic hepatitis B virus (HBV) infection is mediated by the host's cellular i m m u n e response to infected liver cells 1. In this mechanism, T cell-mediated cell cytotoxicity plays the major role in progres-
sion of liver injury ~. Immunological killing of HBV-infected hepatocytes is thought to be initiated by sensitized cytotoxic T lymphocytes which recognize viral target antigens expressed on the liver cell plasma m e m b r a n e 35. What is the target antigen recognized by T lymphocytes in HBV infection? Expression of HBV-associated antigens on the infected liver cell m e m b r a n e provides an
Received December 5, 1991. Accepted June 26, 1992. Address for correspondence: Takafumi Saito, M.D., The Second Department of Internal Medicine, Yamagata University School of Medicine, Yamagata 990-23, Japan.
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Liver membrane HBV-associated antigens
important clue. Although a large number of immunohistochemical studies have been done on HBV-associated antigen expression on the liver cell membrane, antigen expression patterns have not yet been sufficiently characterized. Against this background, the authors carried out an immunohistochemical study of the expression patterns of Pre-S2, HBc and HBe antigens (Ag) on the infected liver cell surface. Immunoelectron microscopy was considered to give new insight into the interpretation of the various expression patterns of these antigens on the liver cell membrane. The aim of the present study was to clarify the characteristics of these surface HBV-associated antigens immunohistochemically and to explore their relationship with a cellular immune target antigen on the basis of their morphological features. Here we report the results of an immunohistochemical study of liver tissues from patients with chronic hepatitis B virus infection. Patients and Methods
Patients Liver biopsy specimens from 31 patients (25 males, 6 females, mean age 34.8+8.8 years, mean+SD) with chronic HBV infection were examined. Liver tissue was obtained by diagnostic needle biopsy, after obtaining informed consent. The histological features were classified according to the international standard criteria6, and included; nonspecific reactive hepatitis (NSRH) in 6 cases, chronic persistent hepatitis (CPH) in 6 cases, chronic active hepatitis (CAH) in 17 cases, and liver cirrhosis in 2 cases. The level of alanine aminotransferase activity (ALT) in serum ranged from 11 to 491 IU/1 (129: [74, 238], Median: [25%, 75% ]), and serum HBeAg was positive in all cases. None of the patients had recieved any immunosuppressive or antiviral therapy before the liver biopsy. Monoclonal antibodies Mouse monoclonal antibodies against hepatitis B pre-S2 antigen (anti-pre-S2), hepatitis B core antigen (anti-HBc), or hepatitis B e antigen (anti-
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HBe) were purchased from the Institute of Immunology, Co. Ltd., Tokyo, Japan. These ascitic fluids contained specific mouse IgG against each antigen with a protein concentration of 0.5 mg/ml and were used at a dilution of 1:30. Immunohistochemistry An indirect immunoperoxidase staining technique was employed for detection of Pre-S2 Ag, HBcAg, HBeAg on the hepatocyte plasma membrane. Light microscopic study: Liver biopsy specimens were fixed immediately in periodate-lysine4% paraformaldehyde (PLP) solution, embedded in OCT compound (Miles Lab. Inc., Naperville, IL) and quickly frozen with cooled hexane. Sixmicrometer-thick serial cryostat sections were prepared, and washed extensively with phosphatebuffered saline, pH 7.2 (PBS), before immunostaining. After inhibition of endogenous peroxidase activity of the liver tissue with 0.05M periodic acid solution for 10 rain at 4~ the sections were incubated with mouse monoclonal anti-Pre$2, anti-HBc or anti-HBe in a moist chamber overnight at 4~ followed by incubation with peroxidase-conjugated sheep anti-mouse IgG (Fab'2) (Amersham Japan Co. Ltd., Tokyo, Japan) for 3 hours. The reaction products of these antigens were stained with a 0.02% solution of 3,3'diaminobenzidine (DAB) in 0.05M Tris-Hcl buffer, pH 7.2, containing 0.01% hydrogen peroxide. Slides were counterstained with methyl green, dehydrated and mounted. Electron microscopic study: After incubation with the primary and secondary antibodies, as described above, the sections were further fixed in 1% glutaraldehyde in PBS for 10 min and incubated in DAB solution containing 0.01% hydrogen peroxide for 5 min. This was followed by postfixation with 2% osmium tetroxide in PBS for 1 hour, dehydration in a graded ethanol series to 100%, and embedding in Epon 812 in gelatin capsules. Ultrathin sections were observed by electron microscopy without additional staining. As an immunostaining control, sections were incubated with non-immune mouse ascites or PBS instead of the primary antibody in the first
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Figure 1. Immune light microscopy reveals two kinds of expression pattern of Pre-S2 Ag on the ceil membrane. (A) Honeycomb-like membrane expression pattern, connected from cell to cell, is evident regardless of inflammation in the liver. (B) Scattered membrane expression pattern is evident in highly inflammatory areas. Liver cells showing cytoplasmic expression with membranous accentuation are swollen and partially disrupted in areas of piecemeal necrosis, and one of them is surrounded by the inflammatory cellular infiltrates and fibrosis (arrow). Immunoperoxidase; A: x 200, B: x 320.
step of the immunostaining procedure. Furthermore, there was no specific staining in control liver biopsy specimens from patients with HBsAgnegative chronic liver disease. Results
Immune light microscopy Pre-S2 Ag showed a honeycomb-like membrane expression pattern, which was apparently connected from cell to cell. The cytoplasm of cells
showing this membrane expression pattern contained hardly any Pre-S2 Ag (Figure 1A). This expression pattern was specifically observed in the liver. In 3 cases of CAH among 18 cases with a honeycomb-like membrane expression pattern, membrane Pre-S2 Ag-positive cells were also demonstrated with a scattered membrane expression pattern in areas of piecemeal necrosis or high inflammatory activity. In the cells of these 3 cases, expression of Pre-S2 Ag was also detected in submembranous areas of the cytoplasm (Figure 1B).
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Figure 2. The nucleocapsid antigens (HBcAg and HBeAg) are demonstrated with scattered membrane expression in highly inflammatory areas. Membrane HBcAgpositive cells (A) and membrane HBeAgpositive cells (B) are swollen and partially disrupted in areas of piecemeal necrosis. Cytoplasmic, or both cytoplasmic and nuclear expression, are simultaneously detected with membranous accentuation. P: Portal area. Immunoperoxidase; A: x320, B: x 350.
The distribution of membrane HBcAg-positive cells was observed with a scattered membrane expression pattern in areas of high inflammatory activity in the liver. Immunoreaction for HBcAg was also detected in the nucleus and cytoplasm, or only in the cytoplasm, in these hepatocytes
(Figure 2A). The distribution of membrane HBeAg-positive cells was demonstrated with a scattered membrane expression pattern in highly inflammatory areas, as in the case of HBcAg. Membrane
HBeAg-positive cells had both nuclear and cytoplasmic expression, or cytoplasmic expression only (Figure 2B). However, the distribution of membrane HBcAg-positive cells was not always in accordance with that of membrane HBeAg-positive cells when serial sections were examined. The expression of HBV-associated antigens on the infected liver cell membrane in comparison with histological findings is summarized in Table 1. Of the 31 liver specimens, Pre-S2 Ag was detected on the cell membrane in 18 cases (58%), HBcAg in 4
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Table 1. Membrane expression pattern of Pre-S2 Ag, HBcAg and HBeAg in comparison with liver histology
Membrane expression pattern Pre-S2 Ag
Honeycomb-like expression
No. of cases positive (31 )
Histology NSRH (6)
CPH (6)
CAH (17)
LC (2)
18
4
3
10
1
0
0
3
0
0
0
3
1
0
0
3
1
(580/0) Scattered expression
3
(10%) HBcAg
Scattered expression
4
(13o/0) HBeAg
Scattered expression
4 (13%)
NSRH: nonspecific reactive hepatitis, CPH: chronic persistent hepatitis, CAH: chronic active hepatitis, LC: liver cirrhosis.
cases (13%) and HBeAg in 4 cases (130/0). In relation to the histological features, a honeycomb-like membrane expression pattern of Pre-S2 Ag was observed in various types of histology, whereas a scattered membrane expression pattern of Pre-S2 Ag, HBcAg or HBeAg was observed only in cases of marked inflammation such as CAH or LC.
Immunoelectron microscopy (1) Pre-S2 Ag I m m u n e light microscopy revealed two kinds of Pre-S2 Ag expression pattern on the infected liver cell surface. One was a honeycomb-like membrane expression pattern which was present regardless of inflammation in the liver, and the other was a scattered membrane expression pattern detected in areas of marked inflammation. Immunoelectron microscopy revealed a difference of these two distinct Pre-S2 Ag membrane-expression patterns. An area of honeycomb-like membrane expression as revealed by immune light microscopy was selected for immunoelectron microscopic observation. As described previously7, the immunoreaction for Pre-S2 Ag was observed on the liver cell plasma membrane except for the bile canalicular membrane. It was also observed in the intercellular space, but was less extensive in the cytoplasm. A higher magnification view of the lateral surface of an infected hepatocyte is shown in Figure 3. The immunoreaction for Pre-S2 Ag was observed in the intercellular space on the left side
partially indicated by arrows, but it was not present on the right side, as indicated by arrowheads. Pre-S2 Ag was detected on the plasma membrane in areas of the intercellular space showing a positive immunoreaction (left side shown by arrows), but was not detected on the plasma membrane in areas of the intercellular space lacking an immunoreaction (right side shown by arrow heads). Submembranous cytoplasmic expression was not observed. These findings suggest that the honeycomb-like membrane expression of Pre-S2 Ag results from attachment of extracellular antigen to the liver cell membrane. The ultrastructural morphology in areas showing a scattered membrane expression pattern of Pre-S2 Ag by i m m u n e light microscopy is shown in Figure 4. Pre-S2 Ag was detected as a singlelayered linear deposit along the cell membrane, but was absent in the intercellular space. Pre-S2 Ag was also expressed abundantly in the cytoplasm immediately below the membrane, and the immunoproducts appeared to fuse with the cell membrane. (2) HBcAg and HBeAg The electron microscopic expression pattern of HBcAg on the liver cell membrane is shown in Figure 5. Except for the bile canalicular membrane, HBcAg showed a mono-layered linear distribution along the cell membrane. It is noteworthy that HBcAg expression was absent in the intercellular spaces, although it was present on the membrane of adjoining liver cells and abundant in their
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Figure 3. Ultrastructural demonstration of the lateral surface of infected hepatocytes showing a honeycomb-like membrane expression pattern. Pre-S2 Ag is present in the intercellular space on the left, but is absent on the right. Both plasma membranes adjacent to the reaction-positive intercellular space (left side, inset), as shown by arrows, are positive for this antigen. In contrast, both plasma membranes unassociated with any immunoreaction in the intercellular space (right side), as indicated by arrow heads, show no reaction. No counterstain; x25000, inset, x60000.
cell organelles. The electron microscopic expression pattern of HBeAg on the liver cell membrane is shown in Figure 6. The expression pattern of HBeAg was fundamentally the same as that of HBcAg; i.e., HBeAg showed a mono-layered linear distribution along the cell membrane and was absent in the intercellular spaces. Similarly, the immunoreaction for HBeAg was abundant in the cell organelles. The reaction product for HBeAg in the positive membrane and that in the cytoplasm immediately below the membrane were occasionally fused. These findings suggest that the scattered membrane expression of Pre-S2 Ag, HBcAg or HBeAg results from intrahepatic transfer of antigens synthesized in the liver cell to the cell membrane.
Discussion
In the present study, Pre-S2 Ag, HBcAg, and HBeAg were all demonstrated on the plasma membrane of infected liver cells at the ultrastructural level, and the morphological features of each antigen expressed on the cell surface were specific. Target antigens for immunological response in the cell-damaging mechanism of hepatitis B have been studied with various methods. In vitro, HBsAg was first revealed as a target antigen by lymphocyte test against HBsAg-coated target cells8. A series of papers have claimed that HBcAg is a target antigen based on the autologous lymphocyte cytotoxicity test 912. Histologically, the expression of HBV-associated antigens on the
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Figure 4. Pre-S2 Ag showing a scattered membrane expression pattern appears as a single-layered linear deposit along the cell membrane and is absent in the intercellular space. Large amounts of reaction product for Pre-S2 Ag are evident in the cytoplasm immediately below the cell membrane, and have partially fused with the membrane. No counterstain; x22000.
liver cell membrane has been detected, and its correlation with target antigens were explored. In earlier studies, HBsAg was considered to be a cellular immune target antigen, because an immunofluorescence study revealed it easily on the liver cell surface with a honeycomb-like membrane expression pattern ~3. HBcAg was also demonstrated on infected liver cell surface, showing a scattered membrane expression pattern immunohistochemically1416, although its incidence seems to be lower because anti-HBc antibody, which is abundant in blood, can mask the antigen membrane expression 17. As a result of analysis of histological findings and viral replication in relation to antigen expression patterns in hepatic lobules by light microscopy, it has been reported that a diffuse, honeycomb-like membrane expression of
HBsAg is associated with viral replication and not with tissue inflammation, and that localization of HBcAg confined to the cytoplasm or membrane is associated with active viral replication and intense inflammation14~6'18. lmmunoelectron microscopy has also demonstrated the expression of HBcAg on the liver cell membrane and attachment of lymphocytes to liver cells with abundant HBcAg ~9. The significance of HBcAg as a target antigen has been indicated. However, HBsAg may also serve as a target antigen under some conditions, because expression of HBsAg on the cell membrane and attachment of CD8-positive lymphocytes to such membranes have been demonstrated by immunoelectron microscopy and immune double staining method 2~ These previous histochemical reports were
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Figure 5. HBcAg shows a mono-layered linear distribution along the membranes of adjoining liver cells (arrows) but is absent in the intercellular spaces. Immunoreactions for HBcAg are abundant in the cell organelles. No counterstain; x 14000.
designed to clarify whether HBV-associated antigens are present on the infected liver cell membrane. The present study is the first attempt to analyze the antigen expression patterns on the liver cell membrane and to evaluate target antigens based on the morphological features of membrane-associated antigens. In addition to study of HBsAg and HBcAg, it is necessary to investigate the expression of HBeAg and Pre-S2 Ag on the infected liver cell membrane because of the reported possibility that these antigens may also serve as target antigens 2225. In this study, Pre-S2 Ag was found by light microscopy to show not only the previously reported honeycomb-like membrane expression pattern 7,26, similar to that of HBsAg 13'~4, but also a scattered membrane expression pattern in markedly inflamed areas. By light microscopy, both
HBcAg and HBeAg were detected on the cell surface with a scattered membrane expression pattern in areas of marked inflammation. These antigens were also seen in the cytoplasm and occasionally in the nucleus. There were only a few cases that showed a scattered membrane expression of these antigens. Kojima et al found a scattered membrane expression of HBcAg in only 4 out of the 25 cases 19, and Yamada et al found that of HBeAg in only 2 of 19 cases 27. The frequency of positive scattered membrane expression of these antigens in our study was roughly in agreement with the above reports. It is very difficult to identify a scattered membrane expression of these antigens in a limited number of biopsied specimens histologically. Some reports suggest that immunological response against envelope antigen, including Pre-S2 Ag is closely related to the
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Figure 6. HBeAg shows a mono-layered linear distribution continuously along the cell membrane. Reaction product for HBeAg in the positive membrane and that in the cytoplasm immediately below the membrane have partially fused. Note the absence of immunoreactions for HBeAg in the membrane of the adjoining cell and intercellular space. No counterstain; x 16000.
immunological clearance of HBV in acute hepatitis B 28'29. During acute exacerbation of chronic hepatitis B, a low level of IgM-HBc antibody can be detected in blood concomitantly with a proliferation of HBV and severe acute inflammation occurs in liver. There is a possibility that Pre-S2 Ag might serve as a target antigen under some condition such as acute exacerbation of chronic hepatitis B22. Our present data suggest that Pre-S2 Ag might also serve as a target antigen at a particular stage of chronic hepatitis B since a scattered membrane expression of Pre-S2 Ag can be detected as well as that of HBcAg or HBeAg in areas of marked inflammation. Immunoelectron microscopy revealed these two distinct membrane expression patterns of the antigens, suggesting that the membrane-bound
expression of each antigen is mediated by a different mechanism. Ultrastructural morphology suggested that if the localization pattern of Pre-S2 Ag showed a honeycomb-like membrane expression in hepatic lobules, then the antigen originated from outside liver cells. This type of membrane expression of Pre-S2 Ag was unique to the liver. It was proposed in a previous report that membrane-associated Pre-S2 Ag might originate from an extracellular circulating antigen 7. It has been speculated that Pre-S2 Ag play a role as a receptor for polymerized human serum albumin (pHSA) 3~ and can adhere to hepatocytes specifically by binding to pHSA receptors on the liver cell surface membranes via pHSA in blood. HBeAg did not have a honeycomb-like membrane expression pattern like Pre-
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Liver membrane HBV-assoctated antigens
$2 Ag. This type of membrane expression of HBeAg has not yet been documented, and its molecular mechanism remains unknown. HBeAg may not be able to bind to the membrane by itself, whereas Pre-S2 Ag adheres to the membrane specifically via pHSA. The mechanism responsible for this type of membrane expression was thought to be absorption ofPre-S2 Ag onto the cell-surface membrane from the extracellular space. In liver tissue showing a honeycomb-like membrane expression of Pre-S2 Ag observed in various types of liver histology, this expression pattern does not correlate with inflammatory activity26. On the other hand, if the localization pattern of Pre-S2 Ag showed a scattered membrane expression, then the antigen on the membrane must have been synthesized in the liver cells and then transported to the membrane. HBcAg or HBeAg also showed a similar pattern of scattered membrane expression in hepatic lobules, suggesting that the mechanism responsible for this type of membrane expression was synthesis of these antigens in the liver cell and subsequent intrahepatic transport to the cell-surface membrane. Although ultrastructural evidence for the presence of HBeAg on parts of the plasma membrane has been reported 27, this study illustrated for the first time continuous surface membrane expression of HBeAg, like that of HBcAg. Regions showing such a scattered membrane expression pattern of these antigens had severe lymphocyte infiltration and cell necrosis. The present findings suggest that the scattered membrane expression of Pre-S2 Ag, HBcAg or HBeAg on the infected liver cell surface is of biological significance as a target antigen in connection with inflammation, and that this target antigen expression occurs as a result of its transport within liver cells to the membrane. The authors are most grateful to Dr. Fumihiro Ichida and Dr. Makoto Ishikawa for giving an opportunity to do this work.
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