Gut, 1976, 17, 837-843
Complement fixing hepatitis B core antigen immune complexes in the liver of patients with HBS antigen positive chronic disease M. RIZZETTO, F. BONINO, 0. CRIVELLI, M. G. CANESE, AND G. VERME From the Department of Gastroenterology, Ospedale Mauriziano Umberto I, Turin, and the Centre for Electron Microscopy, III Chair of Morbid Anatomy, University of Turin, Italy
One hundred and fifty-two biopsies from serologically HBsAg positive and negative patients with liver disease were studied in immunofluorescence for the presence of the surface (HBs) and the core (HBc) antigenic determinants of the hepatitis B virus, of immunoglobulins and complement (C) deposits, and for the capacity to fix human C. Circumstantial evidence is presented suggesting that HBc immune-complexes are a relevant feature in the establishment and progression of chronic HBsAg liver disease. C fixation by liver cells was shown in all HBc positive patients with chronic hepatitis; an active form was present in every case, except two with a persistent hepatitis, an inverse ratio of HBc to C binding fluorescence being noted between active chronic hepatitis and cirrhotic patients. HB, without C fixation was observed in only three patients in the incubation phase of infectious hepatitis. IgG deposits were often found in HBc containing, C fixing nuclei. No C binding or IgG deposits were observed in acute self-limited type B hepatitis, in serologically positive patients with normal liver or minimal histological lesions, with and without HB% cytoplasmic fluorescence in their biopsy, or in serologically negative individuals.
SUMMARY
The serological detection of hepatitis B core antibody (Hoofnagle et al., 1973) and the intrahepatic localisation of hepatitis B core antigen (HBc) by immunofluorescence (Hadziyannis, 1973; Gudat et al., 1975) are recent developments with promising clinical applications in the study of HBsAg positive liver disease. While studying HBc positive liver biopsies (Rizzetto et al., 1976) an attempt was made to set up an indirect immunofluorescence (IFL) test for the detection of core antibodies. This was sometimes impeded by the presence of IgG bound on core positive nuclei, which precluded indirect IFL. When we tried to overcome the problem by using the C fixing capacity of the HBeAg-HBcAb system, it became apparent that many biopsies from HBsAg positive patients were able to stain with anti C3 fluorescent serum independently of HBcAb fixation. These findings are reported and their serological and clinical implications discussed. Received for publication 1 July 1976
Methods PATIENTS AND BIOPSIES
One hundred and forty-six percutaneous or laparoscopic needle biopsies and six operative or post mortem specimens were studied. Eighty patients had no HBsAg in the serum. They included patients with a variety of acute and chronic liver diseases. Seventy-two patients were HBsAg positive: their histological or clinical diagnosis is shown in Table 1. In the HBsAg acute hepatitis group, 21 biopsies were taken four to 22 days after sudden onset of jaundice and after enzyme levels had reached a peak; in three patients admitted for asymptomatic antigenaemia a biopsy was taken five, seven, and eight days before the onset of acute icteric hepatitis, at a time when transaminase levels were normal in two cases and only slightly raised in the third. Histological criteria were those of De Groote et al. (1968). Tissue specimens were divided in two parts, one processed for routine histology, the other frozen, cut 837
M. Rizzetto, F. Bonino, 0. Crivelli, M. G. Canese, and G. Verme
838
Table 1 HBgAg, HBcAg, and HB,Ag associated with HBcAg immunofluorescence in 72 serologically HB8Ag positive patients
Ca++ and Mg++ containing buffer; after 30 minutes, the slides were washed, covered with anti-human Clq, C3 and C4 FITC conjugated sera, washed again and mounted; control slides were covered with the Patients No. of biopsies positive Diagnosis in IFL for tested same serum heated for 30 minutes at 56°C, or (no.) diluted in EDTA-containing buffers and treated as HBsAg HBcAg HBsAg previously described. + HBcAg To study the relations between cells containing the HB virus determinants and those with immuno11 14 Healthy chronic carriers 3 Acute self-limited hepatitis 21 globulins deposits or with C fixing capacity, three Patients incubating acute serial slides from each positive biopsy were cut, 3 3 hepatitis Chronic hepatitis in ether, covered for 30 minutes with fresh fixed 2 10 4 Persistent human serum, washed, and double stained-the 1 6 8 Active Cirrhosis first with FITC anti HBc and RITC anti human C3 4 3 8 Active sera, the second with FITC anti HBc and R1TC 6 1 1 Inactive anti IgG sera, and the third with FITC anti C3 and 2 1 Hepatic fibrosis RITC anti HBs sera. Specimens were examined with a Universal with the cryostat in 4 ,u sections and fixed with ether Zeiss fluorescence microscope, alternating the fluorescein and rodamine excitation wavelength. at room temperature for five minutes. Photographs were taken with an automatic camera applied to the microscope. ANTISERA HBs antiserum was prepared from Behring-Hoescht precipitating antiserum RBB15 by conjugation of ELECTRON MICROSCOPY the gamma globulin fraction with rodamine iso- For the ultrastructural study small blocks of tissue thiocyanate (RITC) (cat. N. 12198, Becton and from patients nos. 3, 12, and 13 (Table 2) were left in cold phosphate buffered 3% glutaraldehyde for Dickinson). HBc antiserum was prepared from the blood of a two hours, post-fixed in 1 % osmium tetroxide, and healthy HBsAg chronic carrier; the IgG fraction, embedded in Araldite (Durcupan ACM Fluka). isolated after chromatography on DE52 (Whatmann) Staining was carried out with uranyl acetate (Watson, 1958) during alcoholic dehydration and lead citrate was conjugated with fluorescein isothiocyanate (Reynolds, 1963) was then applied to the section. (FITC) (BDH Isomer I). The details of preparation and specificities of the antisera have already been described (Rizzetto et al., Results 1976). FITC anti human IgA, IgM, C3, and C4 were obtained from Behring-Hoechst (TKA05, TKC05, IMMUNOFLUORESCENCE RESULTS TKDO5, TKG05); FITC anti human Clq and IFL results are summarised in Tables 1 and 2. No staining of the liver biopsy was observed in RITC anti human IgG and anti human C3 were prepared in the laboratory, by conjugating the the 80 HB5Ag negative patients. In Table 1, HB5Ag positive patients are divided immunoglobulin fractions isolated from BehringHoechst precipitating antisera to human Clq according to the histological or clinical diagnosis and the results with HB% and HBe antisera are (TNI05), IgG (TOB05), and C3 (TEA05). reported. In 11 healthy chronic carriers without liver disease a diffuse cytoplasmic localisation of HBsAg IMMUNOFLUORESCENCE To localise the surface and the core antigens in the was observed in many hepatocytes; no other positive liver, each biopsy was double stained with FITC reaction occurred in this group of biopsies. In the acute self-limited hepatitis (AH) group, the conjugated antiserum against HBc and RITC majority of biopsies did not react with any anticonjugated antiserum against HBs. To detect immunoglobulins or complement fixed serum; in three patients HB5 was seen in the cytoin vivo, biopsies were covered with a drop of FITC plasm of a few isolated liver cells. In three individuals incubating infectious hepatitis (nos. 18, 19, conjugated antihuman IgG, A, M, Clq, C3, C4. To detect the capacity of hepatocytes to fix 20, Table 2) HBe was found in the nuclei of almost human complement in vitro, sections were covered all the hepatocytes in many serial sections, unaccomfor 30 minutes with fresh, AB group human serum, panied by any other fluorescence. Reactions with all antisera were negative in 11 negative for autoantibodies and HBe antibodies in IFL and for HBs antibodies in RIA, diluted in patients in the chronic liver disease group (Table 1). -
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-
Complement fixing hepatitis B core antigen immune complexes in the liver
839
Table 2 Histological and immunofluorescence data in 20 patients with HBcAg in liver biopsy Case Sex
Age
no.
Biopsy no.
Histological
Transaminases
Immunofltorescence
SGOT SGPT (
Complement fixing hepatitis B core antigen immune complexes in the liver of patients with HBS antigen positive chronic disease M. RIZZETTO, F. BONINO, 0. CRIVELLI, M. G. CANESE, AND G. VERME From the Department of Gastroenterology, Ospedale Mauriziano Umberto I, Turin, and the Centre for Electron Microscopy, III Chair of Morbid Anatomy, University of Turin, Italy
One hundred and fifty-two biopsies from serologically HBsAg positive and negative patients with liver disease were studied in immunofluorescence for the presence of the surface (HBs) and the core (HBc) antigenic determinants of the hepatitis B virus, of immunoglobulins and complement (C) deposits, and for the capacity to fix human C. Circumstantial evidence is presented suggesting that HBc immune-complexes are a relevant feature in the establishment and progression of chronic HBsAg liver disease. C fixation by liver cells was shown in all HBc positive patients with chronic hepatitis; an active form was present in every case, except two with a persistent hepatitis, an inverse ratio of HBc to C binding fluorescence being noted between active chronic hepatitis and cirrhotic patients. HB, without C fixation was observed in only three patients in the incubation phase of infectious hepatitis. IgG deposits were often found in HBc containing, C fixing nuclei. No C binding or IgG deposits were observed in acute self-limited type B hepatitis, in serologically positive patients with normal liver or minimal histological lesions, with and without HB% cytoplasmic fluorescence in their biopsy, or in serologically negative individuals.
SUMMARY
The serological detection of hepatitis B core antibody (Hoofnagle et al., 1973) and the intrahepatic localisation of hepatitis B core antigen (HBc) by immunofluorescence (Hadziyannis, 1973; Gudat et al., 1975) are recent developments with promising clinical applications in the study of HBsAg positive liver disease. While studying HBc positive liver biopsies (Rizzetto et al., 1976) an attempt was made to set up an indirect immunofluorescence (IFL) test for the detection of core antibodies. This was sometimes impeded by the presence of IgG bound on core positive nuclei, which precluded indirect IFL. When we tried to overcome the problem by using the C fixing capacity of the HBeAg-HBcAb system, it became apparent that many biopsies from HBsAg positive patients were able to stain with anti C3 fluorescent serum independently of HBcAb fixation. These findings are reported and their serological and clinical implications discussed. Received for publication 1 July 1976
Methods PATIENTS AND BIOPSIES
One hundred and forty-six percutaneous or laparoscopic needle biopsies and six operative or post mortem specimens were studied. Eighty patients had no HBsAg in the serum. They included patients with a variety of acute and chronic liver diseases. Seventy-two patients were HBsAg positive: their histological or clinical diagnosis is shown in Table 1. In the HBsAg acute hepatitis group, 21 biopsies were taken four to 22 days after sudden onset of jaundice and after enzyme levels had reached a peak; in three patients admitted for asymptomatic antigenaemia a biopsy was taken five, seven, and eight days before the onset of acute icteric hepatitis, at a time when transaminase levels were normal in two cases and only slightly raised in the third. Histological criteria were those of De Groote et al. (1968). Tissue specimens were divided in two parts, one processed for routine histology, the other frozen, cut 837
M. Rizzetto, F. Bonino, 0. Crivelli, M. G. Canese, and G. Verme
838
Table 1 HBgAg, HBcAg, and HB,Ag associated with HBcAg immunofluorescence in 72 serologically HB8Ag positive patients
Ca++ and Mg++ containing buffer; after 30 minutes, the slides were washed, covered with anti-human Clq, C3 and C4 FITC conjugated sera, washed again and mounted; control slides were covered with the Patients No. of biopsies positive Diagnosis in IFL for tested same serum heated for 30 minutes at 56°C, or (no.) diluted in EDTA-containing buffers and treated as HBsAg HBcAg HBsAg previously described. + HBcAg To study the relations between cells containing the HB virus determinants and those with immuno11 14 Healthy chronic carriers 3 Acute self-limited hepatitis 21 globulins deposits or with C fixing capacity, three Patients incubating acute serial slides from each positive biopsy were cut, 3 3 hepatitis Chronic hepatitis in ether, covered for 30 minutes with fresh fixed 2 10 4 Persistent human serum, washed, and double stained-the 1 6 8 Active Cirrhosis first with FITC anti HBc and RITC anti human C3 4 3 8 Active sera, the second with FITC anti HBc and R1TC 6 1 1 Inactive anti IgG sera, and the third with FITC anti C3 and 2 1 Hepatic fibrosis RITC anti HBs sera. Specimens were examined with a Universal with the cryostat in 4 ,u sections and fixed with ether Zeiss fluorescence microscope, alternating the fluorescein and rodamine excitation wavelength. at room temperature for five minutes. Photographs were taken with an automatic camera applied to the microscope. ANTISERA HBs antiserum was prepared from Behring-Hoescht precipitating antiserum RBB15 by conjugation of ELECTRON MICROSCOPY the gamma globulin fraction with rodamine iso- For the ultrastructural study small blocks of tissue thiocyanate (RITC) (cat. N. 12198, Becton and from patients nos. 3, 12, and 13 (Table 2) were left in cold phosphate buffered 3% glutaraldehyde for Dickinson). HBc antiserum was prepared from the blood of a two hours, post-fixed in 1 % osmium tetroxide, and healthy HBsAg chronic carrier; the IgG fraction, embedded in Araldite (Durcupan ACM Fluka). isolated after chromatography on DE52 (Whatmann) Staining was carried out with uranyl acetate (Watson, 1958) during alcoholic dehydration and lead citrate was conjugated with fluorescein isothiocyanate (Reynolds, 1963) was then applied to the section. (FITC) (BDH Isomer I). The details of preparation and specificities of the antisera have already been described (Rizzetto et al., Results 1976). FITC anti human IgA, IgM, C3, and C4 were obtained from Behring-Hoechst (TKA05, TKC05, IMMUNOFLUORESCENCE RESULTS TKDO5, TKG05); FITC anti human Clq and IFL results are summarised in Tables 1 and 2. No staining of the liver biopsy was observed in RITC anti human IgG and anti human C3 were prepared in the laboratory, by conjugating the the 80 HB5Ag negative patients. In Table 1, HB5Ag positive patients are divided immunoglobulin fractions isolated from BehringHoechst precipitating antisera to human Clq according to the histological or clinical diagnosis and the results with HB% and HBe antisera are (TNI05), IgG (TOB05), and C3 (TEA05). reported. In 11 healthy chronic carriers without liver disease a diffuse cytoplasmic localisation of HBsAg IMMUNOFLUORESCENCE To localise the surface and the core antigens in the was observed in many hepatocytes; no other positive liver, each biopsy was double stained with FITC reaction occurred in this group of biopsies. In the acute self-limited hepatitis (AH) group, the conjugated antiserum against HBc and RITC majority of biopsies did not react with any anticonjugated antiserum against HBs. To detect immunoglobulins or complement fixed serum; in three patients HB5 was seen in the cytoin vivo, biopsies were covered with a drop of FITC plasm of a few isolated liver cells. In three individuals incubating infectious hepatitis (nos. 18, 19, conjugated antihuman IgG, A, M, Clq, C3, C4. To detect the capacity of hepatocytes to fix 20, Table 2) HBe was found in the nuclei of almost human complement in vitro, sections were covered all the hepatocytes in many serial sections, unaccomfor 30 minutes with fresh, AB group human serum, panied by any other fluorescence. Reactions with all antisera were negative in 11 negative for autoantibodies and HBe antibodies in IFL and for HBs antibodies in RIA, diluted in patients in the chronic liver disease group (Table 1). -
-
-
Complement fixing hepatitis B core antigen immune complexes in the liver
839
Table 2 Histological and immunofluorescence data in 20 patients with HBcAg in liver biopsy Case Sex
Age
no.
Biopsy no.
Histological
Transaminases
Immunofltorescence
SGOT SGPT (
