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Subcellular localization of hepatitis B core antigen in relation to hepatocyte regeneration in chronic hepatitis B
GASTROENTEROLOGY1995;109:1926-1932
Subcellular Localization of Hepatitis B Core Antigen in Relation to Hepatocyte Regeneration in Chronic Hepatitis B CHIA-MING CHU, CHAU-TING YEH, I-SHYAN SHEEN, and YUN-FAN LIAW Liver Research Unit, ChangGungMemorial Hospital, ChangGungMedical College, Taipei, Taiwan
Background & Aims: To test whether the dominant cytoplasmic expression of hepatitis B core antigen (HBcAg) in active chronic hepatitis B is secondary to liver damage and regeneration, the relationship between subcellular localization of HBcAg, liver inflammatory activity, and hepatocyte regeneration in chronic hepatitis B was studied. Methods: Correlation of the clinical and laboratory data with the topographical distribution of HBcAg was studied in 30 patients. The subcellular localization of HBcAg in relation to hepatocyte cell cycles was studied by double immunostaining of HBcAg and proliferating cell nuclear antigen. Results: Patients with predominant cytoplasmic HBcAg had significantly higher levels of biochemical and histological activities and proliferating cell nuclear antigen expression than patients with predominant nuclear HBcAg. The levels of proliferating cell nuclear antigen expression correlated positively with biochemical and histological activities and degrees of cytoplasmic HBcAg expression but negatively with degrees of nuclear HBcAg expression. Proliferating cell nuclear antigen expression was shown in 49% of hepatocytes with cytoplasmic HBcAg but in only 2% of hepatocytes with nuclear HBcAg. Conclusions: These findings suggested that, following liver damage, the regeneration of surviving hepatocytes might cause the shift of intracellular HBcAg from nucleus to cytoplasm. As a result, the extent of nuclear HBcAg expression reduces with concomitant increase in cytoplasmic HBcAg expression.
' n chronic hepatitis B virus (HBV) infection, the pres,ence of hepatitis B c o r e antigen (HBcAg) in infected liver is usually assumed to indicate active viral replication. 1-v Of note is the fact that HBcAg was predominantly localized in the cytoplasm in patients with active and ongoing hepatitis, but in patients with normal liver histology or minimal histological activity, HBcAg was predominantly distributed in the nucleus. 8-ii These observations have led to the postulation that hepatocytes with cytoplasmic/membranous HBcAg were possible targets for immune hepatocytolysis. 8 However, the mechanism of the differential subcellular localization of HBcAg in infected hepatocytes is still unclear.
It has been shown that the carboxyl-terminal argininerich domain of the HBcAg protein contains a signal for nuclear localization. 12 A recent study by Yeh et al. in two different cell lines indicated that nuclear localization of HBcAg was cell cycle-regulated; in the G0/G1 phases, HBcAg was predominantly localized in the nucleus, and in the S phase, the amount of HBcAg in the nucleus was greatly reduced and HBcAg was found almost entirely in the cytoplasm. 13 The investigators thus speculated that the differential subcellular localization of HBcAg during the natural infection might be related to the cell cycles of the hepatocytes. HBcAg would be localized in the nucleus during the asymptomatic infection when hepatocytes were largely quiescent and arrested in the G0/G1 phases, whereas HBcAg would be detected in the cytoplasm of a significant number of hepatocytes during aggressive hepatitis when liver injury and regeneration caused many hepatocytes to enter cell cycles. This postulation, however, needs to be elucidated in HBV naturally infected human liver. Various kinds of markers for proliferating cells, such as bromodeoxyuridine labeling, 14 Ki-67, i5 DNA polymerase cz,16 and proliferating cell nuclear antigen (PCNA), iv have been developed. In most cases, fresh tissue specimens or frozen sections have to be prepared for these marker analyses. Recently, it has been shown that a PCNA clone (PCl0) could be successfully applied to conventionally fixed and processed tissues, i8'I9 At present, PCNA immunohistochemistry is one of the best markers for retrospective assessment of cell proliferation using tissues that were routinely submitted for diagnostic purposes. The expression of PCNA by hepatocytes has been studied in various acute and chronic liver diseases using formalin-fixed and paraffin-embeded tissues. 2°-25 The results indicated that immunohistochemical staining of PCNA is a reliable marker for proliferating hepatocytes. The present study aimed to investigate the relationship between the subcellular localization ofhepatocyte HBcAg, the liver inflammatory activAbbreviations used in this paper: HAl, histology activity index; PCNA, proliferatingcell nuclearantigen. © 1995 by the AmericanGastroenterologicalAssociation 0016-5085/95/$3.00
December 1995
ity, and the hepatocyte regenerative activity by using P C N A immunohistochemistry in patients with chronic H B V infection. Particular emphasis was made to study the subcellular localization of H B c A g in relation to the hepatocyte cell cycles using double immunostaining of H B c A g and P C N A .
Materials and Methods Patients In a 4-month period, a total of 46 patients who were seropositive for hepatitis B surface antigen (HBsAg) for more than 6 months and undergoing liver biopsy as part of their routine clinical management were studied at our unit. Of these, 30 patients were seroposive for hepatitis B e antigen (HBeAg) and HBV D N A and negative for hepatitis C virus (HCV) and hepatitis D virus (HDV) markers. No patients had documented history of acute HBV infection previously. Sixteen patients presented with constitutional symptoms of chronic liver disease, whereas the other 14 patients were incidentally found to have chronic HBV infection during blood donation or routine check-up. Five patients were presumed to be infected perinatally because of the HBsAg seropositivity of their mothers. The maternal HBsAg status was negative or uncertain in the other 25 patients. No patients were homosexuals or intravenous drug abusers. None had ever received any antiviral or immunomodulatory therapy.
Serological Studies Serum was tested for HBsAg, HBeAg, antibody to HBe, and total antibody against HDV by radioimmunoassay (Abbott Diagnostics, Chicago, IL). Antibodies against HCV were tested by a second-generation enzyme immunoassay (Abbott Diagnostics), and serum HBV D N A was assayed using a dot spot hybridization technique as previously reported. 26
Liver Biopsy and Preparation of the Tissue Sections Percutaneous needle biopsies were performed using a Menghini needle. In general, a 1 - 3-cm biopsy core was available from each patient for study. The liver specimens were fixed in 10% formaldehyde and embeded in paraffin. Histological diagnosis was made according to the standard criteria. 2v In addition, the histopathologic findings were evaluated and numerically scored according to the histology activity index (HAI) scoring system proposed by Knodell et al. 2. The HAI scores were assessed independently by two observers without the knowledge of clinical data, and the interobserver variation was between 0 and 2.
Immunohistochemistry Five-micrometer paraffin sections were cut and collected on poly-L-lysine-coated slides for immunohistochemistry. After deparaffinization through graded ethanol, the sections were washed in phosphate-buffered saline (PBS). Hepatocyte expression of HBcAg was studied by the avidin-
SUBCELLULAR LOCALIZATION OF HBcAg 1927
biotin immunoperoxidase method. The tissue sections were first reacted with 10% normal swine serum for 10 minutes; then a 1:200 dilution of rabbit polyclonal antibody against HBcAg (Dakopatts, Copenhagen, Denmark) was applied for 30 minutes. To inhibit endogenous tissue enzyme activity, the sections were incubated with methyl alcohol containing 0.3% hydroperoxide. Sections were subsequently incubated with a 1:100 dilution of biotin-conjugated swine anti-rabbit immunoglobulins for 15 minutes. After rinsing with PBS, sections were treated with avidin-biotin-peroxidase complex for 15 minutes. The reaction products were developed by incubating for 10 minutes in Tris buffer containing a 1% solution of 3,3diaminobenzidine and 0.03% H202, which results in a brown staining of immunoreactive sites. The expression of PCNA in liver was studied using a three-step immunoalkaline-phospharase (APAAP) method. The tissue sections were first reacted with normal rabbit serum for 10 minutes. The sections then were incubated for 45 minutes with murine monoclonal antibody against PCNA (Clone PC10, Dakopatts; concentration, 25 /~g/mL). After a wash in PBS, sections were incubated for 30 minutes with a 1:20 dilution of rabbit anti-mouse immunoglobulins. After a further wash in PBS, a 1:30 dilution of APAAP immune complexes was applied to the sections for 30 minutes. The alkaline phosphatase reaction was developed using the hexazotized new fuchsin procedure, which results in a red staining of immunoreactive sites. Endogenous alkaline phosphatase activity was blocked by adding levamisole to the substrate solution at a concentration of 1 mmol/L. Control tests consisted of omission of the primary antibody or substitution with nonimmune rabbit or murine immunoglobulin. For the simultaneous detection of HBcAg and PCNA on the hepatocytes, sequential double immunoenzymatic staining was applied. The procedure involved the staining of tissue sections with the avidin-biotin immunoperoxidase method for HBcAg, followed by the immunoalkaline phosphatase method for PCNA. As shown in Figure 1, HBcAg-positive nuclei or cytoplasms are brown, whereas PCNA-positive nuclei are red in color. Double-labeled HBcAg-positive PCNA-positive nuclei revealed a mixed brown and red positivity. To ensure that the detection of HBcAg and PCNA was maximal, single staining of HBcAg or PCNA in serial liver sections was compared with double immunostaining; in each case, the detection of HBcAg and PCNA was optimal.
Quantification Each section was examined under code by two independent observers without the knowledge of clinical and histological diagnosis. The distribution and quantitative expression of hepatocyte HBcAg and the nuclear labeling index for PCNA (%) were determined by random evaluation of at least 1000 hepatocytes with distinct nuclei by using an Olympus microscope (Tokyo, Japan) at a magnification of 400× with an eyepiece graticule. The interobserver variation was between 10% and 15%.
Statistical Methods For paired comparison, the paired Student's t test and the Wilcoxon matched pair signed rank test were used. Data
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patients in group 2a than in group 1. Serum levels of AST and ALT, HAI scores, and PCNA labeling index showed no significant difference between patients in group 2b and group 3. Patients in group 2b and group 3 had significantly higher levels of AST and ALT, HAI scores, and PCNA labeling index than patients in group 1 and group 2a. Further analysis of the PCNA labeling index in these 30 study patients showed that the PCNA labeling index correlated positively with serum levels ofAST (r = 0.68; P < 0.001) and ALT (r = 0.69; P < 0.001), the HAI scores (r = 0.89; P < 0.001), and the degrees of cytoplasmic expression of HBcAg (r = 0.68; P < 0.001), but negatively with the degrees of nuclear expression of HBcAg (r = -0.69; P < 0.001). Double immunoenzymatic staining of HBcAg and PCNA revealed that nearly half of the hepatocytes with cytoplasmic staining of HBcAg coexpressed PCNA, whereas hepatocytes with nuclear staining of HBcAg were rarely PCNA positive (Table 2). Figure 2 shows the double immunostaining of HBcAg and PCNA in patients with chronic persistant hepatitis (A) and chronic active hepatitis (B).
Discussion Figure 1. Double immunostaining of HBcAg and PCNA in chronic hepatitis B as assayed by sequential avidin-biotin peroxidase method for HBcAg followed by immunoalkaline phosphatase method for PCNA (brown, HBcAg; red, PCNA). Note the coexpression of HBcAg and PCNA in a nucleus, which revealed a mixed brown and red positivity (arrow) (original magnification 400×).
are routinely given as mean _+ SEM, except where otherwise stated. A correlation coefficient by linear regression was calculated between the PCNA labeling index and serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), HAI scores, and the degrees of nuclear expression of HBcAg and the degrees of cytoplasmic expression of HBcAg. A statistically significant difference was considered to exist when the P value was <0.05.
Results HBcAg was detectable in the liver from all of the 30 study patients, including 7 with purely nuclear staining (group 1), 17 with mixed nuclear and cytoplasmic staining (group 2), and 6 with purely cytoplasmic staining (group 3). Among group 2 patients, 5 had the expression of HBcAg in the nucleus exceeding in the cytoplasm (group 2a), and the other 12 had the expression of HBcAg in the cytoplasm exceeding in the nucleus (group 2b). Table 1 lists the detailed clinical, laboratory, and immunohistochemical data of the study patients. Serum levels of AST and ALT, HAI scores, and PCNA labeling index were significantly higher in
The results of the present study have confirmed previous observations that there was a close relationship between the topographical distribution of HBcAg and the inflammatory activity of the liver in chronic HBV infection. Patients with predominant cytoplasmic expression of HBcAg had significantly higher biochemical and histological activities than patients with predominant nuclear expression of HBcAg (Table 1). It has been suggested that the natural history of chronic HBV infection could be divided into three sequential phases: (1) the high replicative immune tolerance phase, (2) the low replicative immune clearance phase, and (3) the nonreplicative residual integration phase. 26 The data presented here are in keeping with the results of the previous studies 8-I1 that HBcAg is distributed mainly in the nucleus in the immune tolerance phase with normal liver histology or minimal histological activity and that the extent of nuclear expression of HBcAg decreases with concomitant decrease in cytoplasmic expression of HBcAg in the ensuing immune clearance phase with active and ongoing hepatitis. The mechanism of immune tolerance to HBV is still unclear. It is well known that HBV is not directly cytopathic and that liver cell damage in chronic HBV infection is mediated by cytotoxic T cells directed against viral determinants, most likely the HBV nucleocapsid protein, on the surface of infected hepatocytes in the context of HLA class I antigens. 29-31 Thus, it has been suggested that the shift of intrahepatic HBcAg from
December 1995
SUBCELLULAR LOCALIZATION OF HBcAg 1929
Table 1, Clinical, Laboratory, and Immunohistochemical Data of the Study Patients % of hepatocytes with Age Case no. Group 1 (HBcAg expression exclusively in the nucleus 1 2 3 4 5 6 7 Mean (SEM) Group 2a (HBcAg expression in the nucleus exceeding in the cytoplasm) 1 2 3 4 5 Mean (SEM) Group 2b (HBcAg expression in the cytoplasm exceeding in the nucleus) 1 2 3 4 5 6 7 8 9 10 11 12 Mean (SEM) Group 3 (HBcAg expression exclusively in the cytoplasm) 1 2 3 4 5 6 Mean (SEM)
AST
ALT
(yr)
Sex
(U/L)
(U/L)
Histology
HAl scores
30 38 21 18 21 29 44 29 (4)
F M F M F M M
21 30 37 24 19 20 71 32 (7)
23 54 56 33 20 32 182 57 (21)
CPH CPH CPH CPH CPH CPH CPH
1 2 2 3 1 1 3 1.9 (0.3)
31 20 60 59 30 33 6 34.1 (7.4)
44 41 30 39 24 36 (4)
M F M M F
85 43 49 48 59 57 (8)
131 79 125 71 121 105 (13)
CAH CPH CPH CPH CPH
5 4 4 2 4 3.8 (0.5)
14 30 12 71 47 34.8 (11.0)
8 20 5 25 23 16.2 (4.1)
8 4 5 4 9 6.0 (0.9)
33 54 36 36 16 36 48 31 21 30 40 33 35 (3)
M F M M M M F M M M M F
90 51 77 52 74 241 595 61 276 65 66 186 153 (46)
151 73 245 83 193 325 847 74 629 155 108 230 259 (69)
CAH CAH CAH CAH CAH CAH CAH CAH CAH CPH CPH CAH
5 8 7 6 8 9 12 6 10 4 4 9 7,3 (0.7)
2 2 5 5 6 3 2 2 3 2 10 3 3.8 (0.7)
34 21 24 42 34 31 34 11 14 16 40 55 29.7 (3.7)
26 23 14 20 24 19 30 13 21 18 10 20 19.8 (1.6)
34 27 22 39 35 38 33 (3)
F M F F M M
192 75 46 257 123 387 180 (52)
263 113 82 369 209 613 275 (89)
CAH CAH CPH CAH CAH CAH
8 6 4 9 8 10 7,5 (1.0)
20 18 14 3 20 35 18.3 (4.2)
22 18 6 20 18 29 18.8 (3.1)
Nuclear HBcAg
0 0 0 0 0 0
Cytoplasmic HBcAg
0 0 0 0 0 0 0
PCNA
1 2 2 3 1 1 5 2.1 (0.5)
CAH, chronic active hepatitis; CPH, chronic persistent hepatitis.
nucleus to cytoplasm would enhance the expression of this target viral antigen and render the infected hepatocytes susceptible to the immune attack by the cytotoxic T cells. 8 However, a recent study has shown that the subcellular localization of HBcAg in cultured cell lines is cell cycleregulated. 13 This finding has led to the speculation that the predominant cytoplasmic expression of HBcAg typically observed in patients with active and ongoing hepatitis might be secondary to liver damage and regeneration,
rather than the primary event that triggers the liver damage. To the best of the authors' knowledge, the present investigation is the first study to address this issue of the relationship between the subcellular localization of HBcAg and hepatocyte regeneration using PCNA immunohistochemistry in HBV naturally infected human liver. PCNA is an auxiliary protein of DNA polymerase (5.32 It has been shown that there is a close association between PCNA expression in the nucleus and cell proliferation. The PCNA expression appears in the nucleus
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Table 2. C o e x p r e s s i o n of PCNA in HBcAg-Positive Hepatocytes % of PCNA expression
Patterns of HBcAg staining Pure nuclear expression (n = 7) Nuclear expression > cytoplasmic expression (n = 5) Cytoplasmic expression > nuclear expression (n = 12) Pure cytoplasmic expression (n = 6) Total
Hepatocytes with nuclear HBcAg
Hepatocytes with cytoplasmic HBcAg
2.0 _+_0.3
2.6 + 0.2 a
45.0 + 5,3 a
1.8 + 0.6"
51.0 _+ 4,2 °
2.0 _+ 0.3"
47.8 + 4.9 48.9 + 2.7 °
~P < 0,001.
during the late G1 phase immediately before the onset of DNA synthesis, becomes maximal during the S phase, and declines again during the G2 and M phases. 3~-36 The present results showed that the degrees of PCNA expression in the hepatocytes correlated significantly with the biochemical and histological activities in chronic HBV
infection, suggesting a close association between liver cell damage and hepatocyte regeneration. Patients with predominant cytoplasmic expression of HBcAg showed significantly higher inflammatory and regenerative activity in liver than patients with predominant nuclear expression of HBcAg (Table 1). Furthermore, the levels of PCNA expression correlated positively with the degrees of cytoplasmic expression of HBcAg but negatively with the degrees of nuclear expression of HBcAg. These findings suggested that the differential subcellular localization of HBcAg in chronic HBV infection was closely related to the activity of hepatocyte regeneration. Perhaps the most important finding of the present study is that, as shown in the double immunoenzymatic staining of HBcAg and PCNA, nearly half of the hepatocytes with cytoplasmic expression of HBcAg were PCNA positive, whereas hepatocytes with nuclear expression of HBcAg were rarely PCNA positive. These findings indicated that almost all of the hepatocytes with nuclear expression of HBcAg were quiescent and resting cells; on the contrary, up to half of the hepatocytes with cytoplasmic expression of HBcAg were regenerating and proliferating cells. The present data did not seem to support the previous postulation that intrahepatic shift of HBcAg
Figure 2. Double immunostaining of HBcAg and PCNA in patients with (A) chronic persistent hepatitis and (B) chronic active hepatitis (brown, HBcAg; red, PCNA). (A) Note that there is diffuse nuclear expression of HBcAg and very occassionally coexpression of HBcAg and PCNA. The central rectangle is amplified and shown at the right upper corner. Arrow indicates the nucleus caexpressed HBcAg and PCNA (original magnification 200×). (B) Note that there is diffuse cytoplasmic expression of HBcAg and frequently coexpression of HBcAg and PCNA (original magnification 4 0 0 x ) .
December 1995
from nucleus to cytoplasm is the primary event that triggers the immune-mediated liver injury. 8 In that case, the percent of PCNA expression among hepatocytes with nuclear expression of HBcAg should be similar to that among hepatocytes with cytoplasmic expression of HBcAg unless it is proved that the surviving hepatocytes with cytoplasmic expression of HBcAg have a substantially high proliferation capacity compared with those with nuclear expression of HBcAg. It seems more likely that the predominant cytoplasmic expression of HBcAg in patients with active and ongoing hepatitis might be, at least in part, secondary to the liver cell damageinduced hepatocyte regeneration, as speculated by Yeh et al. 1~ In other words, HBcAg was localized mainly in the nucleus in patients with little or no inflammatory activity. Following liver damage, intracellular HBcAg will shift from nucleus to cytoplasm as the surviving hepatocytes regenerate and enter into the cell cycles, As a result, the extent of nuclear expression of HBcAg decreases with concomitant increase in cytoplasmic expression of HBcAg. In conclusion, the present data have shown that in chronic HBV infection, patients with predominant cytoplasmic expression of HBcAg had significantly higher inflammatory activity in addition to regenerative activity in the liver than patients with predominant nuclear expression of HBcAg. Moreover, almost all of hepatocytes with nuclear expression of HBcAg were resting cells, whereas about half of the hepatocytes with cytoplasmic expression of HBcAg were proliferating cells. These findings indicated that the subcellular localization of hepatocyte HBcAg was cell cycle-regulated and suggested that the predominant cytoplasmic localization of HBcAg in patients with active and ongoing hepatitis might be secondary to liver damage and regeneration.
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6. Hadziyannis SJ, Lieberman HM, Karvountzis GB, Shafritz DA. Analysis of liver disease, nuclear HBcAg, viral replication, and hepatitis B virus DNA in liver and serum of HBeAg vs anti-HBe positive carriers of hepatitis B virus. Hepatology 1983;3:656662. 7. Chu CM, Liaw YF. Membrane staining for hepatitis B surface antigen on hepatocyte: a sensitive and specific marker of active viral replication in hepatitis B. J Clin Pathol 1995;48:470-473. 8. Chu CM, Liaw YF. Intrahepatic distribution of hepatitis B surface and core antigens in chronic hepatitis B virus infection. Hepatocyte with cytoplasmic/membranous hepatitis B core antigen as a possible target for immune hepatocytolysis. Gastroenterology 1987;92:220-225. 9. Yoo JY, Howard R, Waggoner JG, Hoofnagle JH. Peroxidase-antiperoxidase detection of hepatitis B surface and core antigen in liver biopsy specimens from patients with chronic type B hepatitis. J Med Virol 1987;23:273-281. 10. Hsu HC, Su IJ, Lai MY, Chen DS, Chang MH, Chuang SM, Sung JL. Biologic and prognostic significance of hepatocyte hepatitis B core antigen expression in the natural course of chronic hepatitis B virus infection. J Hepatol 1987;5:45-50. 11. Chu CM, Liaw YF. Immunohistological study of intrahepatic expression of hepatitis B core and E antigens in chronic type B hepatitis. J Clin Pathol 1992;45:791-795. 12. Yeh CT, Liaw YF, Ou JH. The arginine-rich domain of hepatitis B virus precore and core proteins contains a signal for nuclear transport. J Virol 1990;64:6141-6147. 13. Yeh CT, Wong SW, FungYK, Ou JH. Cell cycle regulation of nuclear localization of hepatitis B virus core protein. Proc Natl Acad Sci USA 1993;90:6459-6463. 14. Gratzner HG. Monoclonal antibody to 5-bromo- and 5qododeoxyuridine: a new reagent for detection of DNA replication. Science 1982; 218:474-476. 15. Gerdes J, Lemke H, Baisch H, Wacker HH, Schmab U, Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 1984; 133:1710-1715. 16. Tsutsumi Y, Hori S, Onoda N. DNA polymerase alpha. An immunohistochemical marker for proliferating cells in normal and neoplastic human tissues. Am J Clin Pathol 1990;93:643-650. 17. Nakane PK, Moriuehi T, Koji T. Proliferative cell nuclear antigen (PCNA/cyclin): review and some new findings. Acta Histochem Cytochem 1989; 22:105-116. 18. Waseem NH, Lane DP. Monoclonal antibody analysis of the proliferating cell nuclear antigen (PCNA). Structural conservation and the detection of a nucleolar form. J Cell Sci 1990;96:121-129. 19. Hall PA, Levison DA, Woods AI, Yu CCW, Bellock DB, Watkins JA, Barnes DM, Gillett CE, Campleijohn R, Dover R, Waseem NH, Lane DP. Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms. J Pathol 1990; 162:285-294. 20. Wolf HK, Michalopoulos GK. Hepatocyte regeneration in acute fulminant and nonfutminant hepatitis: a study of proliferating cell nuclear antigen expression. Hepatology 1992; 15:707-713. 21. Kayano K, Yasunaga M, Kubota M, Takenaka K, Mori K, Yamashaita A, Kubo Y, Sakaida I, Okita K, Sanuki K. Detection of proliferating hepatocytes by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) in patients with acute liver failure. Liver 1992; 12:132-136. 22. Koukoulis G, Rayner A, Tan KH, Williams R, Portamann B. Immunolocalization of regenerating cells after submassive liver necrosis using PCNA staining. J Pathol 1992; 166:359-368. 23. Kawakita N, Seki S, Sakaguchi H, Yanai A, Kuroki T, Mizoguchi Y, Kobayashi K, Monna T. Analysis of proliferating hepatocytes using a monoclonal antibody against proliferating cell nuclear
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Received May 4, 1995. Accepted August 17, 1995. Address requests for reprints to: Chia-Ming Chu, M.D., Liver Research Unit, Chang Gung Memorial Hospital, 199, Tung Hwa North Road, Taipei, Taiwan 105. Fax: (886) 3-328-2824. Supported by a grant from the National Council of Science, Republic of China (NSC83-O419-B-182A-O02 and NSC84-O419-B-182A001). The authors thank W. C. Shyu and T. M. Wang for excellent technical assistance and M. H. Tsai for preparing the manuscript.
Subcellular Localization of Hepatitis B Core Antigen in Relation to Hepatocyte Regeneration in Chronic Hepatitis B CHIA-MING CHU, CHAU-TING YEH, I-SHYAN SHEEN, and YUN-FAN LIAW Liver Research Unit, ChangGungMemorial Hospital, ChangGungMedical College, Taipei, Taiwan
Background & Aims: To test whether the dominant cytoplasmic expression of hepatitis B core antigen (HBcAg) in active chronic hepatitis B is secondary to liver damage and regeneration, the relationship between subcellular localization of HBcAg, liver inflammatory activity, and hepatocyte regeneration in chronic hepatitis B was studied. Methods: Correlation of the clinical and laboratory data with the topographical distribution of HBcAg was studied in 30 patients. The subcellular localization of HBcAg in relation to hepatocyte cell cycles was studied by double immunostaining of HBcAg and proliferating cell nuclear antigen. Results: Patients with predominant cytoplasmic HBcAg had significantly higher levels of biochemical and histological activities and proliferating cell nuclear antigen expression than patients with predominant nuclear HBcAg. The levels of proliferating cell nuclear antigen expression correlated positively with biochemical and histological activities and degrees of cytoplasmic HBcAg expression but negatively with degrees of nuclear HBcAg expression. Proliferating cell nuclear antigen expression was shown in 49% of hepatocytes with cytoplasmic HBcAg but in only 2% of hepatocytes with nuclear HBcAg. Conclusions: These findings suggested that, following liver damage, the regeneration of surviving hepatocytes might cause the shift of intracellular HBcAg from nucleus to cytoplasm. As a result, the extent of nuclear HBcAg expression reduces with concomitant increase in cytoplasmic HBcAg expression.
' n chronic hepatitis B virus (HBV) infection, the pres,ence of hepatitis B c o r e antigen (HBcAg) in infected liver is usually assumed to indicate active viral replication. 1-v Of note is the fact that HBcAg was predominantly localized in the cytoplasm in patients with active and ongoing hepatitis, but in patients with normal liver histology or minimal histological activity, HBcAg was predominantly distributed in the nucleus. 8-ii These observations have led to the postulation that hepatocytes with cytoplasmic/membranous HBcAg were possible targets for immune hepatocytolysis. 8 However, the mechanism of the differential subcellular localization of HBcAg in infected hepatocytes is still unclear.
It has been shown that the carboxyl-terminal argininerich domain of the HBcAg protein contains a signal for nuclear localization. 12 A recent study by Yeh et al. in two different cell lines indicated that nuclear localization of HBcAg was cell cycle-regulated; in the G0/G1 phases, HBcAg was predominantly localized in the nucleus, and in the S phase, the amount of HBcAg in the nucleus was greatly reduced and HBcAg was found almost entirely in the cytoplasm. 13 The investigators thus speculated that the differential subcellular localization of HBcAg during the natural infection might be related to the cell cycles of the hepatocytes. HBcAg would be localized in the nucleus during the asymptomatic infection when hepatocytes were largely quiescent and arrested in the G0/G1 phases, whereas HBcAg would be detected in the cytoplasm of a significant number of hepatocytes during aggressive hepatitis when liver injury and regeneration caused many hepatocytes to enter cell cycles. This postulation, however, needs to be elucidated in HBV naturally infected human liver. Various kinds of markers for proliferating cells, such as bromodeoxyuridine labeling, 14 Ki-67, i5 DNA polymerase cz,16 and proliferating cell nuclear antigen (PCNA), iv have been developed. In most cases, fresh tissue specimens or frozen sections have to be prepared for these marker analyses. Recently, it has been shown that a PCNA clone (PCl0) could be successfully applied to conventionally fixed and processed tissues, i8'I9 At present, PCNA immunohistochemistry is one of the best markers for retrospective assessment of cell proliferation using tissues that were routinely submitted for diagnostic purposes. The expression of PCNA by hepatocytes has been studied in various acute and chronic liver diseases using formalin-fixed and paraffin-embeded tissues. 2°-25 The results indicated that immunohistochemical staining of PCNA is a reliable marker for proliferating hepatocytes. The present study aimed to investigate the relationship between the subcellular localization ofhepatocyte HBcAg, the liver inflammatory activAbbreviations used in this paper: HAl, histology activity index; PCNA, proliferatingcell nuclearantigen. © 1995 by the AmericanGastroenterologicalAssociation 0016-5085/95/$3.00
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ity, and the hepatocyte regenerative activity by using P C N A immunohistochemistry in patients with chronic H B V infection. Particular emphasis was made to study the subcellular localization of H B c A g in relation to the hepatocyte cell cycles using double immunostaining of H B c A g and P C N A .
Materials and Methods Patients In a 4-month period, a total of 46 patients who were seropositive for hepatitis B surface antigen (HBsAg) for more than 6 months and undergoing liver biopsy as part of their routine clinical management were studied at our unit. Of these, 30 patients were seroposive for hepatitis B e antigen (HBeAg) and HBV D N A and negative for hepatitis C virus (HCV) and hepatitis D virus (HDV) markers. No patients had documented history of acute HBV infection previously. Sixteen patients presented with constitutional symptoms of chronic liver disease, whereas the other 14 patients were incidentally found to have chronic HBV infection during blood donation or routine check-up. Five patients were presumed to be infected perinatally because of the HBsAg seropositivity of their mothers. The maternal HBsAg status was negative or uncertain in the other 25 patients. No patients were homosexuals or intravenous drug abusers. None had ever received any antiviral or immunomodulatory therapy.
Serological Studies Serum was tested for HBsAg, HBeAg, antibody to HBe, and total antibody against HDV by radioimmunoassay (Abbott Diagnostics, Chicago, IL). Antibodies against HCV were tested by a second-generation enzyme immunoassay (Abbott Diagnostics), and serum HBV D N A was assayed using a dot spot hybridization technique as previously reported. 26
Liver Biopsy and Preparation of the Tissue Sections Percutaneous needle biopsies were performed using a Menghini needle. In general, a 1 - 3-cm biopsy core was available from each patient for study. The liver specimens were fixed in 10% formaldehyde and embeded in paraffin. Histological diagnosis was made according to the standard criteria. 2v In addition, the histopathologic findings were evaluated and numerically scored according to the histology activity index (HAI) scoring system proposed by Knodell et al. 2. The HAI scores were assessed independently by two observers without the knowledge of clinical data, and the interobserver variation was between 0 and 2.
Immunohistochemistry Five-micrometer paraffin sections were cut and collected on poly-L-lysine-coated slides for immunohistochemistry. After deparaffinization through graded ethanol, the sections were washed in phosphate-buffered saline (PBS). Hepatocyte expression of HBcAg was studied by the avidin-
SUBCELLULAR LOCALIZATION OF HBcAg 1927
biotin immunoperoxidase method. The tissue sections were first reacted with 10% normal swine serum for 10 minutes; then a 1:200 dilution of rabbit polyclonal antibody against HBcAg (Dakopatts, Copenhagen, Denmark) was applied for 30 minutes. To inhibit endogenous tissue enzyme activity, the sections were incubated with methyl alcohol containing 0.3% hydroperoxide. Sections were subsequently incubated with a 1:100 dilution of biotin-conjugated swine anti-rabbit immunoglobulins for 15 minutes. After rinsing with PBS, sections were treated with avidin-biotin-peroxidase complex for 15 minutes. The reaction products were developed by incubating for 10 minutes in Tris buffer containing a 1% solution of 3,3diaminobenzidine and 0.03% H202, which results in a brown staining of immunoreactive sites. The expression of PCNA in liver was studied using a three-step immunoalkaline-phospharase (APAAP) method. The tissue sections were first reacted with normal rabbit serum for 10 minutes. The sections then were incubated for 45 minutes with murine monoclonal antibody against PCNA (Clone PC10, Dakopatts; concentration, 25 /~g/mL). After a wash in PBS, sections were incubated for 30 minutes with a 1:20 dilution of rabbit anti-mouse immunoglobulins. After a further wash in PBS, a 1:30 dilution of APAAP immune complexes was applied to the sections for 30 minutes. The alkaline phosphatase reaction was developed using the hexazotized new fuchsin procedure, which results in a red staining of immunoreactive sites. Endogenous alkaline phosphatase activity was blocked by adding levamisole to the substrate solution at a concentration of 1 mmol/L. Control tests consisted of omission of the primary antibody or substitution with nonimmune rabbit or murine immunoglobulin. For the simultaneous detection of HBcAg and PCNA on the hepatocytes, sequential double immunoenzymatic staining was applied. The procedure involved the staining of tissue sections with the avidin-biotin immunoperoxidase method for HBcAg, followed by the immunoalkaline phosphatase method for PCNA. As shown in Figure 1, HBcAg-positive nuclei or cytoplasms are brown, whereas PCNA-positive nuclei are red in color. Double-labeled HBcAg-positive PCNA-positive nuclei revealed a mixed brown and red positivity. To ensure that the detection of HBcAg and PCNA was maximal, single staining of HBcAg or PCNA in serial liver sections was compared with double immunostaining; in each case, the detection of HBcAg and PCNA was optimal.
Quantification Each section was examined under code by two independent observers without the knowledge of clinical and histological diagnosis. The distribution and quantitative expression of hepatocyte HBcAg and the nuclear labeling index for PCNA (%) were determined by random evaluation of at least 1000 hepatocytes with distinct nuclei by using an Olympus microscope (Tokyo, Japan) at a magnification of 400× with an eyepiece graticule. The interobserver variation was between 10% and 15%.
Statistical Methods For paired comparison, the paired Student's t test and the Wilcoxon matched pair signed rank test were used. Data
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patients in group 2a than in group 1. Serum levels of AST and ALT, HAI scores, and PCNA labeling index showed no significant difference between patients in group 2b and group 3. Patients in group 2b and group 3 had significantly higher levels of AST and ALT, HAI scores, and PCNA labeling index than patients in group 1 and group 2a. Further analysis of the PCNA labeling index in these 30 study patients showed that the PCNA labeling index correlated positively with serum levels ofAST (r = 0.68; P < 0.001) and ALT (r = 0.69; P < 0.001), the HAI scores (r = 0.89; P < 0.001), and the degrees of cytoplasmic expression of HBcAg (r = 0.68; P < 0.001), but negatively with the degrees of nuclear expression of HBcAg (r = -0.69; P < 0.001). Double immunoenzymatic staining of HBcAg and PCNA revealed that nearly half of the hepatocytes with cytoplasmic staining of HBcAg coexpressed PCNA, whereas hepatocytes with nuclear staining of HBcAg were rarely PCNA positive (Table 2). Figure 2 shows the double immunostaining of HBcAg and PCNA in patients with chronic persistant hepatitis (A) and chronic active hepatitis (B).
Discussion Figure 1. Double immunostaining of HBcAg and PCNA in chronic hepatitis B as assayed by sequential avidin-biotin peroxidase method for HBcAg followed by immunoalkaline phosphatase method for PCNA (brown, HBcAg; red, PCNA). Note the coexpression of HBcAg and PCNA in a nucleus, which revealed a mixed brown and red positivity (arrow) (original magnification 400×).
are routinely given as mean _+ SEM, except where otherwise stated. A correlation coefficient by linear regression was calculated between the PCNA labeling index and serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), HAI scores, and the degrees of nuclear expression of HBcAg and the degrees of cytoplasmic expression of HBcAg. A statistically significant difference was considered to exist when the P value was <0.05.
Results HBcAg was detectable in the liver from all of the 30 study patients, including 7 with purely nuclear staining (group 1), 17 with mixed nuclear and cytoplasmic staining (group 2), and 6 with purely cytoplasmic staining (group 3). Among group 2 patients, 5 had the expression of HBcAg in the nucleus exceeding in the cytoplasm (group 2a), and the other 12 had the expression of HBcAg in the cytoplasm exceeding in the nucleus (group 2b). Table 1 lists the detailed clinical, laboratory, and immunohistochemical data of the study patients. Serum levels of AST and ALT, HAI scores, and PCNA labeling index were significantly higher in
The results of the present study have confirmed previous observations that there was a close relationship between the topographical distribution of HBcAg and the inflammatory activity of the liver in chronic HBV infection. Patients with predominant cytoplasmic expression of HBcAg had significantly higher biochemical and histological activities than patients with predominant nuclear expression of HBcAg (Table 1). It has been suggested that the natural history of chronic HBV infection could be divided into three sequential phases: (1) the high replicative immune tolerance phase, (2) the low replicative immune clearance phase, and (3) the nonreplicative residual integration phase. 26 The data presented here are in keeping with the results of the previous studies 8-I1 that HBcAg is distributed mainly in the nucleus in the immune tolerance phase with normal liver histology or minimal histological activity and that the extent of nuclear expression of HBcAg decreases with concomitant decrease in cytoplasmic expression of HBcAg in the ensuing immune clearance phase with active and ongoing hepatitis. The mechanism of immune tolerance to HBV is still unclear. It is well known that HBV is not directly cytopathic and that liver cell damage in chronic HBV infection is mediated by cytotoxic T cells directed against viral determinants, most likely the HBV nucleocapsid protein, on the surface of infected hepatocytes in the context of HLA class I antigens. 29-31 Thus, it has been suggested that the shift of intrahepatic HBcAg from
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Table 1, Clinical, Laboratory, and Immunohistochemical Data of the Study Patients % of hepatocytes with Age Case no. Group 1 (HBcAg expression exclusively in the nucleus 1 2 3 4 5 6 7 Mean (SEM) Group 2a (HBcAg expression in the nucleus exceeding in the cytoplasm) 1 2 3 4 5 Mean (SEM) Group 2b (HBcAg expression in the cytoplasm exceeding in the nucleus) 1 2 3 4 5 6 7 8 9 10 11 12 Mean (SEM) Group 3 (HBcAg expression exclusively in the cytoplasm) 1 2 3 4 5 6 Mean (SEM)
AST
ALT
(yr)
Sex
(U/L)
(U/L)
Histology
HAl scores
30 38 21 18 21 29 44 29 (4)
F M F M F M M
21 30 37 24 19 20 71 32 (7)
23 54 56 33 20 32 182 57 (21)
CPH CPH CPH CPH CPH CPH CPH
1 2 2 3 1 1 3 1.9 (0.3)
31 20 60 59 30 33 6 34.1 (7.4)
44 41 30 39 24 36 (4)
M F M M F
85 43 49 48 59 57 (8)
131 79 125 71 121 105 (13)
CAH CPH CPH CPH CPH
5 4 4 2 4 3.8 (0.5)
14 30 12 71 47 34.8 (11.0)
8 20 5 25 23 16.2 (4.1)
8 4 5 4 9 6.0 (0.9)
33 54 36 36 16 36 48 31 21 30 40 33 35 (3)
M F M M M M F M M M M F
90 51 77 52 74 241 595 61 276 65 66 186 153 (46)
151 73 245 83 193 325 847 74 629 155 108 230 259 (69)
CAH CAH CAH CAH CAH CAH CAH CAH CAH CPH CPH CAH
5 8 7 6 8 9 12 6 10 4 4 9 7,3 (0.7)
2 2 5 5 6 3 2 2 3 2 10 3 3.8 (0.7)
34 21 24 42 34 31 34 11 14 16 40 55 29.7 (3.7)
26 23 14 20 24 19 30 13 21 18 10 20 19.8 (1.6)
34 27 22 39 35 38 33 (3)
F M F F M M
192 75 46 257 123 387 180 (52)
263 113 82 369 209 613 275 (89)
CAH CAH CPH CAH CAH CAH
8 6 4 9 8 10 7,5 (1.0)
20 18 14 3 20 35 18.3 (4.2)
22 18 6 20 18 29 18.8 (3.1)
Nuclear HBcAg
0 0 0 0 0 0
Cytoplasmic HBcAg
0 0 0 0 0 0 0
PCNA
1 2 2 3 1 1 5 2.1 (0.5)
CAH, chronic active hepatitis; CPH, chronic persistent hepatitis.
nucleus to cytoplasm would enhance the expression of this target viral antigen and render the infected hepatocytes susceptible to the immune attack by the cytotoxic T cells. 8 However, a recent study has shown that the subcellular localization of HBcAg in cultured cell lines is cell cycleregulated. 13 This finding has led to the speculation that the predominant cytoplasmic expression of HBcAg typically observed in patients with active and ongoing hepatitis might be secondary to liver damage and regeneration,
rather than the primary event that triggers the liver damage. To the best of the authors' knowledge, the present investigation is the first study to address this issue of the relationship between the subcellular localization of HBcAg and hepatocyte regeneration using PCNA immunohistochemistry in HBV naturally infected human liver. PCNA is an auxiliary protein of DNA polymerase (5.32 It has been shown that there is a close association between PCNA expression in the nucleus and cell proliferation. The PCNA expression appears in the nucleus
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Table 2. C o e x p r e s s i o n of PCNA in HBcAg-Positive Hepatocytes % of PCNA expression
Patterns of HBcAg staining Pure nuclear expression (n = 7) Nuclear expression > cytoplasmic expression (n = 5) Cytoplasmic expression > nuclear expression (n = 12) Pure cytoplasmic expression (n = 6) Total
Hepatocytes with nuclear HBcAg
Hepatocytes with cytoplasmic HBcAg
2.0 _+_0.3
2.6 + 0.2 a
45.0 + 5,3 a
1.8 + 0.6"
51.0 _+ 4,2 °
2.0 _+ 0.3"
47.8 + 4.9 48.9 + 2.7 °
~P < 0,001.
during the late G1 phase immediately before the onset of DNA synthesis, becomes maximal during the S phase, and declines again during the G2 and M phases. 3~-36 The present results showed that the degrees of PCNA expression in the hepatocytes correlated significantly with the biochemical and histological activities in chronic HBV
infection, suggesting a close association between liver cell damage and hepatocyte regeneration. Patients with predominant cytoplasmic expression of HBcAg showed significantly higher inflammatory and regenerative activity in liver than patients with predominant nuclear expression of HBcAg (Table 1). Furthermore, the levels of PCNA expression correlated positively with the degrees of cytoplasmic expression of HBcAg but negatively with the degrees of nuclear expression of HBcAg. These findings suggested that the differential subcellular localization of HBcAg in chronic HBV infection was closely related to the activity of hepatocyte regeneration. Perhaps the most important finding of the present study is that, as shown in the double immunoenzymatic staining of HBcAg and PCNA, nearly half of the hepatocytes with cytoplasmic expression of HBcAg were PCNA positive, whereas hepatocytes with nuclear expression of HBcAg were rarely PCNA positive. These findings indicated that almost all of the hepatocytes with nuclear expression of HBcAg were quiescent and resting cells; on the contrary, up to half of the hepatocytes with cytoplasmic expression of HBcAg were regenerating and proliferating cells. The present data did not seem to support the previous postulation that intrahepatic shift of HBcAg
Figure 2. Double immunostaining of HBcAg and PCNA in patients with (A) chronic persistent hepatitis and (B) chronic active hepatitis (brown, HBcAg; red, PCNA). (A) Note that there is diffuse nuclear expression of HBcAg and very occassionally coexpression of HBcAg and PCNA. The central rectangle is amplified and shown at the right upper corner. Arrow indicates the nucleus caexpressed HBcAg and PCNA (original magnification 200×). (B) Note that there is diffuse cytoplasmic expression of HBcAg and frequently coexpression of HBcAg and PCNA (original magnification 4 0 0 x ) .
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from nucleus to cytoplasm is the primary event that triggers the immune-mediated liver injury. 8 In that case, the percent of PCNA expression among hepatocytes with nuclear expression of HBcAg should be similar to that among hepatocytes with cytoplasmic expression of HBcAg unless it is proved that the surviving hepatocytes with cytoplasmic expression of HBcAg have a substantially high proliferation capacity compared with those with nuclear expression of HBcAg. It seems more likely that the predominant cytoplasmic expression of HBcAg in patients with active and ongoing hepatitis might be, at least in part, secondary to the liver cell damageinduced hepatocyte regeneration, as speculated by Yeh et al. 1~ In other words, HBcAg was localized mainly in the nucleus in patients with little or no inflammatory activity. Following liver damage, intracellular HBcAg will shift from nucleus to cytoplasm as the surviving hepatocytes regenerate and enter into the cell cycles, As a result, the extent of nuclear expression of HBcAg decreases with concomitant increase in cytoplasmic expression of HBcAg. In conclusion, the present data have shown that in chronic HBV infection, patients with predominant cytoplasmic expression of HBcAg had significantly higher inflammatory activity in addition to regenerative activity in the liver than patients with predominant nuclear expression of HBcAg. Moreover, almost all of hepatocytes with nuclear expression of HBcAg were resting cells, whereas about half of the hepatocytes with cytoplasmic expression of HBcAg were proliferating cells. These findings indicated that the subcellular localization of hepatocyte HBcAg was cell cycle-regulated and suggested that the predominant cytoplasmic localization of HBcAg in patients with active and ongoing hepatitis might be secondary to liver damage and regeneration.
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Received May 4, 1995. Accepted August 17, 1995. Address requests for reprints to: Chia-Ming Chu, M.D., Liver Research Unit, Chang Gung Memorial Hospital, 199, Tung Hwa North Road, Taipei, Taiwan 105. Fax: (886) 3-328-2824. Supported by a grant from the National Council of Science, Republic of China (NSC83-O419-B-182A-O02 and NSC84-O419-B-182A001). The authors thank W. C. Shyu and T. M. Wang for excellent technical assistance and M. H. Tsai for preparing the manuscript.