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Detection of hepatitis B virus antigens in liver tissue
GASTROENTEROLOGY
1990;99:1248-1253
Detection of Hepatitis B Virus Antigens in Liver Tissue A Relation to Viral Replication Chronic Hepatitis B Infection
and Histology in
NIKOLAI V. NAOUMOV, BERNARD C. PORTMANN, RICHARD S. TEDDER, BRIDGET FERNS, ADRIAN L.W.F. EDDLESTON, GRAEME J. M. ALEXANDER, and ROGER WILLIAMS Liver Unit, King’s College Hospital and School of Medicine Virology, Middlesex Hoipital, iondon, England
and Dentistry, and Department
of
The expression of hepatitis B virus antigens was studied by double staining liver tissue with appropriate antisera and correlated with serum hepatitis B viral DNA and histology in 28 patients with disease related to chronic hepatitis B virus infection. The cellular localization of hepatitis B core and hepatitis B e antigens generally coincided, but there were important differences at a subcellular level. Thus, hepatitis B e antigen was detected in nuclei and/or cytoplasm but strong cytoplasmic hepatitis B e antigen was associated with a high serum hepatitis B viral DNA (P = 0.0017) but not with active liver disease. Hepatitis B core antigen could also be detected in nuclei and/or cytoplasm, but strong cytoplasmic hepatitis B core antigen expression, exceeding that of hepatitis B e antigen, was associated with active liver disease (P = 0.041) and not with serum hepatitis B virus DNA. The proportion of hepatocytes expressing hepatitis B surface antigen correlated inversely with the serum titer (P = O.O017),whereas hepatitis B surface and nucleocapsid antigens were usually expressed independently. The data support the hypothesis that cytoplasmic hepatitis B core antigen and not hepatitis B e antigen is the target for immune system-mediated cytolysis of hepatocytes. Cytoplasmic hepatitis B e antigen is not associated with liver damage but is instead associated with high levels of hepatitis B virus replication.
nucleocapsid and surface antigen expression (l-3) whereas more recent studies based on the subcellular localization of hepatitis B core antigen (HBcAg) emphasized the importance of this antigen as a target for immune system-mediated liver damage (4-6).These histological observations are supported by immunological studies indicating that viral nucleocapsid antigens are the target for cytotoxic T cells (7-9). The synthesis of the nucleocapsid antigens is related to the presence of free HBV DNA in hepatocytes, but the synthesis of the hepatitis B surface antigen (HBsAg) may be related to either free or integrated HBV DNA (10). Two nucleocapsid antigens, HBcAg and hepatitis B e antigen (HBeAg), can be identified in the hepatocytes of chronic HBV carriers with evidence of active viral replication. While a relation between cytoplasmic HBcAg expression and liver damage has been consistently shown (4-61,the subcellular localization of HBeAg and its relation to liver damage are less certain (ll,l2). Furthermore, it is uncertain whether HBcAg and HBeAg are expressed with the same subcellular localization within the same hepatocytes. The purpose of the present study was to evaluate the relationship between HBV antigen expression by double staining using appropriate monoclonal and polyclonal antibodies and to correlate these findings with viral replication and liver histology.
he expression of hepatitis B virus (HBV) antigens in the liver reflects the state of viral replication and may influence the immune response of the host. Early studies highlighted the contrast between HBV
Abbreviations used in this paper: DAB, diaminobenzidine; TBS, T&buffered saline. 0 1990 by the American Gastroenterological Association 0018-5085/90/$3.00
T
October 1996
REPLICATION AND HBV ANTIGENS IN LIVER
Materials and Methods
were purchased from DAK0 Ltd. [High Wycombe, gland]. Rabbit polyclonal anti-HBs (working dilution,
Patients Twenty-eight males seropositive for HBsAg for at least 12 months who underwent a diagnostic liver biopsy were studied (Table 1). Sixteen were seropositive for HBeAg, and 12 were seropositive for the antibody to HBeAg (antiHBe). Hepatitis B virus DNA was identified in the sera of 19 of 28. All patients were seronegative for total antibody to hepatitis delta virus (HDV). Histological examination of the liver showed minimal liver damage in 4, chronic persistent hepatitis in 10, chronic active hepatitis in 4, chronic active hepatitis with cirrhosis in 5, and inactive cirrhosis in 5.
Serology Serum was tested for HBsAg, HBeAg, total antibody to HDV by radioimmunoassay nostics, Wokingham, U.K.) and for HBV quantitative dot-blot technique as previously
Double Staining in Liver Tissue
for Hepatitis
anti-HBe, and (Abbott Diag DNA using a described (13).
B Virus Antigens
Sections of formalin-fixed, paraffin-embedded liver 5 pm thick were prepared to maximize HBcAg detection according to previous recommendations (14.15). Thus, formalin fixation did not exceed 24 hours, and sections were heated before deparaffinisation and kept in xylene for only 10-15 seconds. HBV antigens were sought individually and also in the following combinations: [a] HBcAg and HBsAg, using rabbit polyclonal antibody to HBcAg (anti-HBc) and murine monoclonal antibody to HBsAg (anti-HBs); (b) HBcAg and HBeAg, using rabbit polyclonal anti-HBc and two murine monoclonal antibodies to HBeAg; and (c) HBsAg and HBeAg, using polyclonal anti-HBs and two monoclonal antibodies to HBeAg. After blocking endogenous peroxidase activity, sections were covered with 10% normal goat serum in Tris-buffered saline (TBS), pH 7.4, for 15 minutes to block nonspecific absorption. Appropriately diluted rabbit polyclonal anti-HBc or polyclonal rabbit anti-HBs were applied for 40 minutes at 37°C. The peroxidase reaction was developed with diaminobenzidine (DAB] as a substrate (0.5 mg/mL in TBS plus 0.01% H,O,). After washing in distilled water and TBS, liver sections were then covered with either mouse monoclonal anti-HBe or mouse monoclonal anti-HBs to reveal the second antigen. After 40 minutes at 37’C rabbit anti-mouse antibody was applied for 30 minutes at 37°C. The alkaline phosphatase reaction was developed using naphthol and Fast Red TR salt (Sigma Chemical Co., St. Louis, MO] as described (16). Liver sections were washed, counterstained and mounted in glycerol in TBS.
1249
with
Monoclonal anti-HBs (B5 A7) raised against a common epitope in the a region was diluted l:5 in TBS. Two mouse monoclonal antibodies (E2 and E6) against separate antigenic sites on HBeAg without cross-inhibition (17) were used simultaneously to maximise detection of HBeAg. Biotinylated goat anti-rabbit antibody (working dilution, I:200 with 1% normal goat serum] and Vectastain ABC kit were purchased from Vector Laboratories (Buringame, CA). To ensure that detection of the second HBV antigen was maximal in all sections studied, single staining for HBeAg was performed in each instance using monoclonal antibodies. The number of HBsAg positive hepatocytes stained with monoclonal anti-HBs to detect the second antigen was compared in each instance with the number detected using polyclonal anti-HBs to detect the first antigen. In each case, detection of the second antigen was maximal. Specificity controls included equally diluted normal rabbit antiserum and monoclonal anti-rubella antibody as the primary antibody instead of HBV specific antibody.
Statistics ResuIts were analyzed using Fisher’s exact probability test.
Results Of 19 patients seropositive for HBV DNA, all three HBV antigens (HBcAg, HBeAg, and HBsAg) were detected in the liver of 16, HBcAg and HBsAg but not HBeAg were detected in 1, and HBsAg only was detected in 2. Among 9 patients seronegative for HBV DNA, HBcAg but not HBeAg was identified in occasional hepatocyte nuclei of 2 cases without lobular inflammation, in association with HBsAg; 3 further cases were positive for HBsAg only, and in 4 instances no viral antigens were detected. The number of hepatocytes expressing HBcAg or HBeAg were scored as follows: none, 0; 1% -25%, 1+ ; 26%-50%,
2+;
and
>50%,
Rabbit polyclonal anti-HBc (working dilution, 1:200 plus 10% normal goat serum] and rabbit anti-mouse IgG labeled with alkaline phosphatase [working dilution, ~20)
3t
(Table
2). When
pa-
Table 1. Histological Features and Serum Hepatitis B Virus Markers in the 28 Patients HBeAg/ anti-HBe
hematoxylin,
Antisera
En-
l:40 plus 10% normal goat serum) was purchased from Behringwerke (Marburg, West Germany]. The mouse monoclonal antibodies used were produced by B. Ferns and R. Tedder.
Minimal liver damage (n = 4) Chronic persistent hepatitis U = to1 Chronic active hepatitis (n = 4) Chronic active hepatitis and cirrhosis (n = 5) Inactive cirrhosis (n = 5)
o/4
Serum HBV DNA 4
10/o 4/o
10 4
2/3 O/5
5 0
1250 NAOUMOV
ET AL.
Table 2. Intrahepatic
GASTROENTEROLOGY
Expression
of Hepatitis
Vol. 99, No. 4
B Core and e Antigens in Relation to Serum Hepatitis B Virus DNA and
Histology HBcAg Cytoplasmic
Nuclear
HBV DNA >!iOpg/40~Lserum(n=lO) t50 pg/40 pL serum (n = 7) Chronic persistent hepatitis [n = 8) Chronic active hepatitis [n = 9)
HBeAg Nuclear
Cytoplasmic
0
1+
2+
3+
0
1+
2+
3+
0
1+
2f
3t
0
1+
2+
3+
0 0
1 2
0 3
9 2
3 1
2 2
0 0
5 4
0 1
1 4
1 1
8 1
2 5
1 2
0 0
7 0
0 0
1 1
1 3
6 5
3 1
3 1
0 0
2 7
0 1
2 3
2 1
4 4
2 5
2 1
0 0
4 3
tients were divided according to the level of serum HBV DNA [either >50 or ~50 pg/40 PL serum), those with high serum levels of HBV DNA had stronger expression of nucleocapsid antigens, with more than 50% of hepatocyte nuclei positive for HBcAg and HBeAg compared with patients with low levels of serum HBV DNA (P = 0.0175 and P = 0.01162, respectively; Table 21. Nuclear expression of HBcAg and HBeAg was largely coincident. Strong cytoplasmic expression of HBeAg was found only in patients with high levels of serum HBV DNA (P = 0.0017) and was independent of the severity of liver disease [Table 2). In three patients with high serum levels of HBV DNA, intense cytoplasmic HBeAg gave a “ground-glass” appearance in some hepatocytes (Figure 1). In each instance, staining for HBsAg was negative in these cells. In contrast, cytoplasmic expression of HBcAg was unrelated to serum HBV DNA, but was significantly more prominent in patients with chronic active hepati-
tis compared with those with chronic persistent hepatitis (P = 0.04; Table 2). Hepatitis B surface antigen was detected in the cytoplasm of isolated single cells or in small clusters of hepatocytes. Membranous staining was identified in a small number of hepatocytes only and was associated with high levels of viral replication. When associated with cytoplasmic staining, membranous staining was difficult to distinguish. In 23 cases with HBsAg in the liver in which the serum HBsAg titer was measured simultaneously, it was noted that 6 of 7 patients with clusters of HBsAg-positive hepatocytes usually had a low titer of serum HBsAg (tl:12,800), whereas 14 of 16 patients with isolated HBsAg-positive hepatocytes had higher serum HBsAg titers (>1:25,000; P = 0.0017). Simultaneous detection of HBsAg and HBcAg showed that these antigens were expressed independently in most hepatocytes, with only a few hepatocytes containing both antigens [Figure 2), irrespective of which antigen was most frequently present (Table
Figure I. Liver stained with monoclonal antiNBe from a patient with high levels of HBV DNA in the serum with chronic persistent hepatitis. In this section, HBeAg stains pink. Note prominent nuclear staining and cytoplasmic staining, giving a “ground-glass” appearance (arrow).
REPLICATION
October 1990
AND HBV ANTIGENS
IN LIVER
1251
Figure 2. Liver stained with anti-HBc and anti-HBs from a patient with low levels of HBV DNA in serum and chronic active hepatitis. Both nuclei and cytoplasm show HBcAg (brown stain), whereas HBsAg @ink stain) is present in the cytoplasm of occasional hepatocytes. Rarely, both antigens are expressed in the same cell (arrow).
3). The same relationship was identified between HBsAg and HBeAg (Figure 3). Double staining for HBcAg and HBeAg generally showed coincident expression at the cellular level, but there were differences in the ratio of HBeAg to HBcAg at a subcellular level [Table 3). In patients with high levels of serum HBV DNA, the number of HBeAgpositive cells exceeded the number of HBcAg-positive cells and the ratio of cytoplasmic HBeAg to HBcAg usually exceeded 1 (P = 0.0017), whereas the ratio of cytoplasmic HBcAg to HBeAg usually exceeded 1 in patients with chronic active hepatitis [P = 0.041) (Figure 4). Discussion The data show that HBcAg and HBeAg generally have a coincident cellular expression, but that at a subcellular level there are important differences. Table 3. Relation Between Hepatitis B Virus Antigens,
Strong cytoplasmic HBeAg expression is found in patients with high levels of serum HBV DNA and is associated with nuclear expression of HBcAg and chronic persistent hepatitis-the early, asymptomatic, and immunotolerant phase of chronic HBV infection. In contrast, cytoplasmic HBcAg expression exceeds that of HBeAg in patients with chronic active hepatitis. In a previous study using monoclonal antibodies against HBeAg and HBcAg (181, HBeAg was found in excess. The use of polyclonal anti-HBc in the present study allows more complete detection of HBcAg, and the analysis in relation to viral replication and histology has shown the association between cytoplasmic HBeAg and high levels of viral replication. Nuclear HBeAg expression was a more constant finding in patients with low-level replication because cytoplasmic HBeAg is not present in such cases. The detection of HBcAg in the livers of three patients who were HBeAg-negative in their livers emphasizes that
Viral Replication,
and Histology Expression of HBcAg and HBeAg
Expression of HBsAg and HBcAg HBs > HBc
HBc > HBs
HBc > HBe
HBe > HBc
1
9
4
6
2
5
7
0”
1 2
7 7
3 8
5 lb
HBV DNA >50 pg/40 rL serum (n = 10) HBV DNA ~50 pg/40 PL
serum (n = 7)
Chronic persistent hepatitis (n = 8) Chronic active hepatitis (n = 9) “P = 0.0017. I’P = 0.041.
1252
NAOLJMOV
ET AL.
GASTROENTEROLOGY
Vol. 99, No. 4
Figure 3. Liver stained with antiaBe and anHNBs from a patient with long-standing chronic HBV infection. HBeAg (pink) is largely confined to nuclei, and a number of hepatocytes contain cytoplasmic HBsAg (brown). Coincident expression of both antigens is rare (arrow).
HBcAg is still the best tissue marker of continuing replication at low levels. The correlation between cytoplasmic HBcAg expression and liver damage has also been shown in two other studies (5,6), and these observations are consistent with the findings of the present study and support previous in vitro data that HBcAg, rather than HBeAg, is the likely target for immune system-mediated cytolysis (7.8). The independent expression of HBsAg and HBcAg also supports the view that selective lysis of HBcAg containing hepatocytes during the phase of
Figure 4. Liver stained for HBcAg and HBeAg from a patient with high levels of HBV DNA in serum and chronic persistent hepatitis. HBcAg (brown) is predominantly nuclear and ohscures nuclear HBeAg, whereas HBeAg [pink), rather than HBcAg, is more prominent in the cytoplasm (arrow).
active viral replication favors the selection of HBsAgpositive hepatocytes with integrated HBV DNA (8). The pattern of HBsAg expression in this study is also consistent with recently described changes in HBsAg expression in relation to the natural history of chronic HBV (19) Patients with active viral replication had occasional hepatocytes with cytoplasmic HBsAg, and such cells usually were negative for HBcAg and HBeAg, suggesting that hepatocytes supporting virion production have efficient transport and secretion. Typically, cells with cytoplasmic HBsAg contain inte-
October 1996
grated HBV DNA (20) and clonal expansion of such cells probably explains the clustering of HBsAgpositive hepatocytes in patients without active viral replication (19). The basis for differential expression of HBeAg and HBcAg remains to be resolved. Recent in vitro data suggest that expression of the precore sequence is essential for insertion of nucleocapsid protein into the membrane of the endoplasmic reticulum and secretion of the nucleocapsid protein as HBeAg from hepatocytes into serum (21,22). In the absence of expression of the precore sequence, the nucleocapsid protein accumulates in the cytoplasm as HBcAg. Expression of the precore peptide in hepatocyte cytoplasm in relation to viral replication and liver damage remains to be examined.
References Ray MB, Desmet VJ, Bradburne AF, Desmyter J, Fevery J, De Groote J. Differential distribution of hepatitis B surface antigen and hepatitis B core antigen in liver of hepatitis B patients. Gastroenterology 1976;71:462-467. 2. Gudat F, Bianchi L, Sonnabend W, Thiel G, Aenishaenslin W, Stadler GA. Pattern of core and surface expression in liver tissue reflects state of specific immune response in hepatitis B. Lab Invest 1975;32:1-9. 3. Huang SN. Neurath AR. Immunohistologic demonstration of hepatitis B viral antigens in liver with reference to its significance in liver injury. Lab Invest 1979;46:1-17. 4. Bortolotti F, Alberti A, Cadrobbi P. Rugge M. Armigliato M, Realdi G. Prognostic value of hepatitis B core antigen (HBcAg) expression in the liver of children with chronic hepatitis type B. Liver i985;5:40-47. distribution of hepatitis B 5. Chu CM, Liaw YF. Intrahepatic surface and core antigens in chronic hepatitis B virus infection. Gastroenterology 1987;92:220-225. 6. 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 Hepatol1967;5:45-50. 7. Mondelli M, Mieli-Vergani G. Alberti A, Vergani D, Portmann B. Eddleston ALWF. Williams R. 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 Immunol1982;129:27732778. GJM. Tedder RS, a. Naoumov NV, Mondelli M, Alexander Eddleston ALWF, Williams R. Relationship between expression of hepatitis B virus antigens in isolated hepatocytes and autologous lymphocyte cytotoxicity in patients with chronic hepatitis B virus infection. Hepatology 1964:4:63-68. 9. Pignatelli M, Waters J. Lever A, Iwarson S. Gerety R, Thomas HC. Cytotoxic T cell responses to the nucleocapsid proteins of 1.
REPLICATION AND HBV ANTIGENS IN LIVER
1253
HBV in chronic hepatitis. Evidence that antibody modulation may cause protracted infection. J Hepatol1967:4:15-21. 10. Omata M, Yokosuka 0, Imazeki F, Ito Y, Mori J, Uchiumi K. Okuda K. Correlation of hepatitis B virus DNA and antigens in the liver. A study in chronic liver disease. Gastroenterology 19a7;92:192-196. 11. Trepo C, Vitvitsky L, Neurath
R, Hashimoto N. Schaefer R, Nemoz G, Prnce AM. Detection of e antigen by immunofluorescence in cytoplasm of hepatocytes of HBsAg carriers. Lancet 1976;1:466. 12. Arnold W, Nielson JO, Hardt F, Meyer zum Buschenfelde KH. Localisation of e antigen in nuclei of hepatocytes in HBsAg positive liver diseases. Gut 1977$X994-996. 13. Fagan EA. Guarner P, Perera SDK. et al. Quantitation of hepatitis B virus DNA (HBV DNA) in serum using the spot hybridisation technique and scintillation counting. J Virol Meth 1985;12:251-262. 14. Trevisan A, Gudat F, Busachi
C, Stocklin E. Bianchi L. An improved method for HBcAg demonstration in paraffinembedded liver tissue. Liver 1982;2:331-339. 15. Gowans EJ, Burrell CJ. Widespread presence of cytoplasmic HBcAg in hepatitis B infected liver detected by improved immunochemical methods. J Clin Path01 1985;38:393-398. 16. Cordell JL, Falini B, Erber WN, Ghosh AK, Abdulaziz Z, MacDonald S, Pulford KAF, Stein H, Mason DY. Immunoenzymatic labelling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes]. J Histochem Cytochem 1984; 32:219-228. 17. Ferns BR, Tedder
RS. Monoclonal antibodies to hepatitis Be antigen (HBeAg) derived from hepatitis B core antigen (HBcAg): their use in characterisation and detection of HBeAg. J Gen Virol i984:65:899-908. ia. Mondelli M, Tedder RS, Ferns B. Pontisso P, Realdi G, Alberti A. Diffrential distribution of hepatitis B core and e antigens in hepatocytes: analysis by monoclonal antibodies. Hepatology i988;6:199-204. 19. Hsu HC. Lai MY, Su IJ, Chen DS, Chang MH, Yang PM, Wu CY, Hsieh HC. Correlation of hepatocyte HBsAg expression with virus replication and liver pathology. Hepatology 19aa;a: 749-754. 20. Hadziyannis SJ, Lieberman HM, Arvountzis GG. 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 1963;3:656-662. 21. Ou J, Laub 0, Rutter WJ. Hepatitis B virus gene function: the precore region targets the core antigen to cellular membranes and causes the secretion of the e antigen. Proc Nat1 Acad Sci usAl986;a3:157a-1582. 22. Uy A, Bruss V. Gerlich WH, Kochel HG, Thomssen
R. Precore sequence of hepatitis B virus inducing e antigen and membrane association of the viral core protein. Virology 1986;155:89-96.
Received September 26.1969. Accepted February 231990. Address requests for reprints to: Roger Williams, M.D., Liver Unit, King’s College Hospital, Denmark Hill, London SE5 9RS, England. Dr. Naoumov was supported by the Wellcome Trust.
1990;99:1248-1253
Detection of Hepatitis B Virus Antigens in Liver Tissue A Relation to Viral Replication Chronic Hepatitis B Infection
and Histology in
NIKOLAI V. NAOUMOV, BERNARD C. PORTMANN, RICHARD S. TEDDER, BRIDGET FERNS, ADRIAN L.W.F. EDDLESTON, GRAEME J. M. ALEXANDER, and ROGER WILLIAMS Liver Unit, King’s College Hospital and School of Medicine Virology, Middlesex Hoipital, iondon, England
and Dentistry, and Department
of
The expression of hepatitis B virus antigens was studied by double staining liver tissue with appropriate antisera and correlated with serum hepatitis B viral DNA and histology in 28 patients with disease related to chronic hepatitis B virus infection. The cellular localization of hepatitis B core and hepatitis B e antigens generally coincided, but there were important differences at a subcellular level. Thus, hepatitis B e antigen was detected in nuclei and/or cytoplasm but strong cytoplasmic hepatitis B e antigen was associated with a high serum hepatitis B viral DNA (P = 0.0017) but not with active liver disease. Hepatitis B core antigen could also be detected in nuclei and/or cytoplasm, but strong cytoplasmic hepatitis B core antigen expression, exceeding that of hepatitis B e antigen, was associated with active liver disease (P = 0.041) and not with serum hepatitis B virus DNA. The proportion of hepatocytes expressing hepatitis B surface antigen correlated inversely with the serum titer (P = O.O017),whereas hepatitis B surface and nucleocapsid antigens were usually expressed independently. The data support the hypothesis that cytoplasmic hepatitis B core antigen and not hepatitis B e antigen is the target for immune system-mediated cytolysis of hepatocytes. Cytoplasmic hepatitis B e antigen is not associated with liver damage but is instead associated with high levels of hepatitis B virus replication.
nucleocapsid and surface antigen expression (l-3) whereas more recent studies based on the subcellular localization of hepatitis B core antigen (HBcAg) emphasized the importance of this antigen as a target for immune system-mediated liver damage (4-6).These histological observations are supported by immunological studies indicating that viral nucleocapsid antigens are the target for cytotoxic T cells (7-9). The synthesis of the nucleocapsid antigens is related to the presence of free HBV DNA in hepatocytes, but the synthesis of the hepatitis B surface antigen (HBsAg) may be related to either free or integrated HBV DNA (10). Two nucleocapsid antigens, HBcAg and hepatitis B e antigen (HBeAg), can be identified in the hepatocytes of chronic HBV carriers with evidence of active viral replication. While a relation between cytoplasmic HBcAg expression and liver damage has been consistently shown (4-61,the subcellular localization of HBeAg and its relation to liver damage are less certain (ll,l2). Furthermore, it is uncertain whether HBcAg and HBeAg are expressed with the same subcellular localization within the same hepatocytes. The purpose of the present study was to evaluate the relationship between HBV antigen expression by double staining using appropriate monoclonal and polyclonal antibodies and to correlate these findings with viral replication and liver histology.
he expression of hepatitis B virus (HBV) antigens in the liver reflects the state of viral replication and may influence the immune response of the host. Early studies highlighted the contrast between HBV
Abbreviations used in this paper: DAB, diaminobenzidine; TBS, T&buffered saline. 0 1990 by the American Gastroenterological Association 0018-5085/90/$3.00
T
October 1996
REPLICATION AND HBV ANTIGENS IN LIVER
Materials and Methods
were purchased from DAK0 Ltd. [High Wycombe, gland]. Rabbit polyclonal anti-HBs (working dilution,
Patients Twenty-eight males seropositive for HBsAg for at least 12 months who underwent a diagnostic liver biopsy were studied (Table 1). Sixteen were seropositive for HBeAg, and 12 were seropositive for the antibody to HBeAg (antiHBe). Hepatitis B virus DNA was identified in the sera of 19 of 28. All patients were seronegative for total antibody to hepatitis delta virus (HDV). Histological examination of the liver showed minimal liver damage in 4, chronic persistent hepatitis in 10, chronic active hepatitis in 4, chronic active hepatitis with cirrhosis in 5, and inactive cirrhosis in 5.
Serology Serum was tested for HBsAg, HBeAg, total antibody to HDV by radioimmunoassay nostics, Wokingham, U.K.) and for HBV quantitative dot-blot technique as previously
Double Staining in Liver Tissue
for Hepatitis
anti-HBe, and (Abbott Diag DNA using a described (13).
B Virus Antigens
Sections of formalin-fixed, paraffin-embedded liver 5 pm thick were prepared to maximize HBcAg detection according to previous recommendations (14.15). Thus, formalin fixation did not exceed 24 hours, and sections were heated before deparaffinisation and kept in xylene for only 10-15 seconds. HBV antigens were sought individually and also in the following combinations: [a] HBcAg and HBsAg, using rabbit polyclonal antibody to HBcAg (anti-HBc) and murine monoclonal antibody to HBsAg (anti-HBs); (b) HBcAg and HBeAg, using rabbit polyclonal anti-HBc and two murine monoclonal antibodies to HBeAg; and (c) HBsAg and HBeAg, using polyclonal anti-HBs and two monoclonal antibodies to HBeAg. After blocking endogenous peroxidase activity, sections were covered with 10% normal goat serum in Tris-buffered saline (TBS), pH 7.4, for 15 minutes to block nonspecific absorption. Appropriately diluted rabbit polyclonal anti-HBc or polyclonal rabbit anti-HBs were applied for 40 minutes at 37°C. The peroxidase reaction was developed with diaminobenzidine (DAB] as a substrate (0.5 mg/mL in TBS plus 0.01% H,O,). After washing in distilled water and TBS, liver sections were then covered with either mouse monoclonal anti-HBe or mouse monoclonal anti-HBs to reveal the second antigen. After 40 minutes at 37’C rabbit anti-mouse antibody was applied for 30 minutes at 37°C. The alkaline phosphatase reaction was developed using naphthol and Fast Red TR salt (Sigma Chemical Co., St. Louis, MO] as described (16). Liver sections were washed, counterstained and mounted in glycerol in TBS.
1249
with
Monoclonal anti-HBs (B5 A7) raised against a common epitope in the a region was diluted l:5 in TBS. Two mouse monoclonal antibodies (E2 and E6) against separate antigenic sites on HBeAg without cross-inhibition (17) were used simultaneously to maximise detection of HBeAg. Biotinylated goat anti-rabbit antibody (working dilution, I:200 with 1% normal goat serum] and Vectastain ABC kit were purchased from Vector Laboratories (Buringame, CA). To ensure that detection of the second HBV antigen was maximal in all sections studied, single staining for HBeAg was performed in each instance using monoclonal antibodies. The number of HBsAg positive hepatocytes stained with monoclonal anti-HBs to detect the second antigen was compared in each instance with the number detected using polyclonal anti-HBs to detect the first antigen. In each case, detection of the second antigen was maximal. Specificity controls included equally diluted normal rabbit antiserum and monoclonal anti-rubella antibody as the primary antibody instead of HBV specific antibody.
Statistics ResuIts were analyzed using Fisher’s exact probability test.
Results Of 19 patients seropositive for HBV DNA, all three HBV antigens (HBcAg, HBeAg, and HBsAg) were detected in the liver of 16, HBcAg and HBsAg but not HBeAg were detected in 1, and HBsAg only was detected in 2. Among 9 patients seronegative for HBV DNA, HBcAg but not HBeAg was identified in occasional hepatocyte nuclei of 2 cases without lobular inflammation, in association with HBsAg; 3 further cases were positive for HBsAg only, and in 4 instances no viral antigens were detected. The number of hepatocytes expressing HBcAg or HBeAg were scored as follows: none, 0; 1% -25%, 1+ ; 26%-50%,
2+;
and
>50%,
Rabbit polyclonal anti-HBc (working dilution, 1:200 plus 10% normal goat serum] and rabbit anti-mouse IgG labeled with alkaline phosphatase [working dilution, ~20)
3t
(Table
2). When
pa-
Table 1. Histological Features and Serum Hepatitis B Virus Markers in the 28 Patients HBeAg/ anti-HBe
hematoxylin,
Antisera
En-
l:40 plus 10% normal goat serum) was purchased from Behringwerke (Marburg, West Germany]. The mouse monoclonal antibodies used were produced by B. Ferns and R. Tedder.
Minimal liver damage (n = 4) Chronic persistent hepatitis U = to1 Chronic active hepatitis (n = 4) Chronic active hepatitis and cirrhosis (n = 5) Inactive cirrhosis (n = 5)
o/4
Serum HBV DNA 4
10/o 4/o
10 4
2/3 O/5
5 0
1250 NAOUMOV
ET AL.
Table 2. Intrahepatic
GASTROENTEROLOGY
Expression
of Hepatitis
Vol. 99, No. 4
B Core and e Antigens in Relation to Serum Hepatitis B Virus DNA and
Histology HBcAg Cytoplasmic
Nuclear
HBV DNA >!iOpg/40~Lserum(n=lO) t50 pg/40 pL serum (n = 7) Chronic persistent hepatitis [n = 8) Chronic active hepatitis [n = 9)
HBeAg Nuclear
Cytoplasmic
0
1+
2+
3+
0
1+
2+
3+
0
1+
2f
3t
0
1+
2+
3+
0 0
1 2
0 3
9 2
3 1
2 2
0 0
5 4
0 1
1 4
1 1
8 1
2 5
1 2
0 0
7 0
0 0
1 1
1 3
6 5
3 1
3 1
0 0
2 7
0 1
2 3
2 1
4 4
2 5
2 1
0 0
4 3
tients were divided according to the level of serum HBV DNA [either >50 or ~50 pg/40 PL serum), those with high serum levels of HBV DNA had stronger expression of nucleocapsid antigens, with more than 50% of hepatocyte nuclei positive for HBcAg and HBeAg compared with patients with low levels of serum HBV DNA (P = 0.0175 and P = 0.01162, respectively; Table 21. Nuclear expression of HBcAg and HBeAg was largely coincident. Strong cytoplasmic expression of HBeAg was found only in patients with high levels of serum HBV DNA (P = 0.0017) and was independent of the severity of liver disease [Table 2). In three patients with high serum levels of HBV DNA, intense cytoplasmic HBeAg gave a “ground-glass” appearance in some hepatocytes (Figure 1). In each instance, staining for HBsAg was negative in these cells. In contrast, cytoplasmic expression of HBcAg was unrelated to serum HBV DNA, but was significantly more prominent in patients with chronic active hepati-
tis compared with those with chronic persistent hepatitis (P = 0.04; Table 2). Hepatitis B surface antigen was detected in the cytoplasm of isolated single cells or in small clusters of hepatocytes. Membranous staining was identified in a small number of hepatocytes only and was associated with high levels of viral replication. When associated with cytoplasmic staining, membranous staining was difficult to distinguish. In 23 cases with HBsAg in the liver in which the serum HBsAg titer was measured simultaneously, it was noted that 6 of 7 patients with clusters of HBsAg-positive hepatocytes usually had a low titer of serum HBsAg (tl:12,800), whereas 14 of 16 patients with isolated HBsAg-positive hepatocytes had higher serum HBsAg titers (>1:25,000; P = 0.0017). Simultaneous detection of HBsAg and HBcAg showed that these antigens were expressed independently in most hepatocytes, with only a few hepatocytes containing both antigens [Figure 2), irrespective of which antigen was most frequently present (Table
Figure I. Liver stained with monoclonal antiNBe from a patient with high levels of HBV DNA in the serum with chronic persistent hepatitis. In this section, HBeAg stains pink. Note prominent nuclear staining and cytoplasmic staining, giving a “ground-glass” appearance (arrow).
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Figure 2. Liver stained with anti-HBc and anti-HBs from a patient with low levels of HBV DNA in serum and chronic active hepatitis. Both nuclei and cytoplasm show HBcAg (brown stain), whereas HBsAg @ink stain) is present in the cytoplasm of occasional hepatocytes. Rarely, both antigens are expressed in the same cell (arrow).
3). The same relationship was identified between HBsAg and HBeAg (Figure 3). Double staining for HBcAg and HBeAg generally showed coincident expression at the cellular level, but there were differences in the ratio of HBeAg to HBcAg at a subcellular level [Table 3). In patients with high levels of serum HBV DNA, the number of HBeAgpositive cells exceeded the number of HBcAg-positive cells and the ratio of cytoplasmic HBeAg to HBcAg usually exceeded 1 (P = 0.0017), whereas the ratio of cytoplasmic HBcAg to HBeAg usually exceeded 1 in patients with chronic active hepatitis [P = 0.041) (Figure 4). Discussion The data show that HBcAg and HBeAg generally have a coincident cellular expression, but that at a subcellular level there are important differences. Table 3. Relation Between Hepatitis B Virus Antigens,
Strong cytoplasmic HBeAg expression is found in patients with high levels of serum HBV DNA and is associated with nuclear expression of HBcAg and chronic persistent hepatitis-the early, asymptomatic, and immunotolerant phase of chronic HBV infection. In contrast, cytoplasmic HBcAg expression exceeds that of HBeAg in patients with chronic active hepatitis. In a previous study using monoclonal antibodies against HBeAg and HBcAg (181, HBeAg was found in excess. The use of polyclonal anti-HBc in the present study allows more complete detection of HBcAg, and the analysis in relation to viral replication and histology has shown the association between cytoplasmic HBeAg and high levels of viral replication. Nuclear HBeAg expression was a more constant finding in patients with low-level replication because cytoplasmic HBeAg is not present in such cases. The detection of HBcAg in the livers of three patients who were HBeAg-negative in their livers emphasizes that
Viral Replication,
and Histology Expression of HBcAg and HBeAg
Expression of HBsAg and HBcAg HBs > HBc
HBc > HBs
HBc > HBe
HBe > HBc
1
9
4
6
2
5
7
0”
1 2
7 7
3 8
5 lb
HBV DNA >50 pg/40 rL serum (n = 10) HBV DNA ~50 pg/40 PL
serum (n = 7)
Chronic persistent hepatitis (n = 8) Chronic active hepatitis (n = 9) “P = 0.0017. I’P = 0.041.
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Figure 3. Liver stained with antiaBe and anHNBs from a patient with long-standing chronic HBV infection. HBeAg (pink) is largely confined to nuclei, and a number of hepatocytes contain cytoplasmic HBsAg (brown). Coincident expression of both antigens is rare (arrow).
HBcAg is still the best tissue marker of continuing replication at low levels. The correlation between cytoplasmic HBcAg expression and liver damage has also been shown in two other studies (5,6), and these observations are consistent with the findings of the present study and support previous in vitro data that HBcAg, rather than HBeAg, is the likely target for immune system-mediated cytolysis (7.8). The independent expression of HBsAg and HBcAg also supports the view that selective lysis of HBcAg containing hepatocytes during the phase of
Figure 4. Liver stained for HBcAg and HBeAg from a patient with high levels of HBV DNA in serum and chronic persistent hepatitis. HBcAg (brown) is predominantly nuclear and ohscures nuclear HBeAg, whereas HBeAg [pink), rather than HBcAg, is more prominent in the cytoplasm (arrow).
active viral replication favors the selection of HBsAgpositive hepatocytes with integrated HBV DNA (8). The pattern of HBsAg expression in this study is also consistent with recently described changes in HBsAg expression in relation to the natural history of chronic HBV (19) Patients with active viral replication had occasional hepatocytes with cytoplasmic HBsAg, and such cells usually were negative for HBcAg and HBeAg, suggesting that hepatocytes supporting virion production have efficient transport and secretion. Typically, cells with cytoplasmic HBsAg contain inte-
October 1996
grated HBV DNA (20) and clonal expansion of such cells probably explains the clustering of HBsAgpositive hepatocytes in patients without active viral replication (19). The basis for differential expression of HBeAg and HBcAg remains to be resolved. Recent in vitro data suggest that expression of the precore sequence is essential for insertion of nucleocapsid protein into the membrane of the endoplasmic reticulum and secretion of the nucleocapsid protein as HBeAg from hepatocytes into serum (21,22). In the absence of expression of the precore sequence, the nucleocapsid protein accumulates in the cytoplasm as HBcAg. Expression of the precore peptide in hepatocyte cytoplasm in relation to viral replication and liver damage remains to be examined.
References Ray MB, Desmet VJ, Bradburne AF, Desmyter J, Fevery J, De Groote J. Differential distribution of hepatitis B surface antigen and hepatitis B core antigen in liver of hepatitis B patients. Gastroenterology 1976;71:462-467. 2. Gudat F, Bianchi L, Sonnabend W, Thiel G, Aenishaenslin W, Stadler GA. Pattern of core and surface expression in liver tissue reflects state of specific immune response in hepatitis B. Lab Invest 1975;32:1-9. 3. Huang SN. Neurath AR. Immunohistologic demonstration of hepatitis B viral antigens in liver with reference to its significance in liver injury. Lab Invest 1979;46:1-17. 4. Bortolotti F, Alberti A, Cadrobbi P. Rugge M. Armigliato M, Realdi G. Prognostic value of hepatitis B core antigen (HBcAg) expression in the liver of children with chronic hepatitis type B. Liver i985;5:40-47. distribution of hepatitis B 5. Chu CM, Liaw YF. Intrahepatic surface and core antigens in chronic hepatitis B virus infection. Gastroenterology 1987;92:220-225. 6. 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 Hepatol1967;5:45-50. 7. Mondelli M, Mieli-Vergani G. Alberti A, Vergani D, Portmann B. Eddleston ALWF. Williams R. 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 Immunol1982;129:27732778. GJM. Tedder RS, a. Naoumov NV, Mondelli M, Alexander Eddleston ALWF, Williams R. Relationship between expression of hepatitis B virus antigens in isolated hepatocytes and autologous lymphocyte cytotoxicity in patients with chronic hepatitis B virus infection. Hepatology 1964:4:63-68. 9. Pignatelli M, Waters J. Lever A, Iwarson S. Gerety R, Thomas HC. Cytotoxic T cell responses to the nucleocapsid proteins of 1.
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HBV in chronic hepatitis. Evidence that antibody modulation may cause protracted infection. J Hepatol1967:4:15-21. 10. Omata M, Yokosuka 0, Imazeki F, Ito Y, Mori J, Uchiumi K. Okuda K. Correlation of hepatitis B virus DNA and antigens in the liver. A study in chronic liver disease. Gastroenterology 19a7;92:192-196. 11. Trepo C, Vitvitsky L, Neurath
R, Hashimoto N. Schaefer R, Nemoz G, Prnce AM. Detection of e antigen by immunofluorescence in cytoplasm of hepatocytes of HBsAg carriers. Lancet 1976;1:466. 12. Arnold W, Nielson JO, Hardt F, Meyer zum Buschenfelde KH. Localisation of e antigen in nuclei of hepatocytes in HBsAg positive liver diseases. Gut 1977$X994-996. 13. Fagan EA. Guarner P, Perera SDK. et al. Quantitation of hepatitis B virus DNA (HBV DNA) in serum using the spot hybridisation technique and scintillation counting. J Virol Meth 1985;12:251-262. 14. Trevisan A, Gudat F, Busachi
C, Stocklin E. Bianchi L. An improved method for HBcAg demonstration in paraffinembedded liver tissue. Liver 1982;2:331-339. 15. Gowans EJ, Burrell CJ. Widespread presence of cytoplasmic HBcAg in hepatitis B infected liver detected by improved immunochemical methods. J Clin Path01 1985;38:393-398. 16. Cordell JL, Falini B, Erber WN, Ghosh AK, Abdulaziz Z, MacDonald S, Pulford KAF, Stein H, Mason DY. Immunoenzymatic labelling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes]. J Histochem Cytochem 1984; 32:219-228. 17. Ferns BR, Tedder
RS. Monoclonal antibodies to hepatitis Be antigen (HBeAg) derived from hepatitis B core antigen (HBcAg): their use in characterisation and detection of HBeAg. J Gen Virol i984:65:899-908. ia. Mondelli M, Tedder RS, Ferns B. Pontisso P, Realdi G, Alberti A. Diffrential distribution of hepatitis B core and e antigens in hepatocytes: analysis by monoclonal antibodies. Hepatology i988;6:199-204. 19. Hsu HC. Lai MY, Su IJ, Chen DS, Chang MH, Yang PM, Wu CY, Hsieh HC. Correlation of hepatocyte HBsAg expression with virus replication and liver pathology. Hepatology 19aa;a: 749-754. 20. Hadziyannis SJ, Lieberman HM, Arvountzis GG. 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 1963;3:656-662. 21. Ou J, Laub 0, Rutter WJ. Hepatitis B virus gene function: the precore region targets the core antigen to cellular membranes and causes the secretion of the e antigen. Proc Nat1 Acad Sci usAl986;a3:157a-1582. 22. Uy A, Bruss V. Gerlich WH, Kochel HG, Thomssen
R. Precore sequence of hepatitis B virus inducing e antigen and membrane association of the viral core protein. Virology 1986;155:89-96.
Received September 26.1969. Accepted February 231990. Address requests for reprints to: Roger Williams, M.D., Liver Unit, King’s College Hospital, Denmark Hill, London SE5 9RS, England. Dr. Naoumov was supported by the Wellcome Trust.