The
Journal of Pathology Vol. 118 No. 1 T H E L O CA L I SA T I O N O F HEPATITIS B A N T I G E N A N D I M M U N O G L O B U L I N G I N LIVER TISSUE: A N I M M U N O FL U O RE SCE N CE , L I G H T A N D E L E C T R O N MICROSCOPIC STUDY C. M. Roos, THEAM. FELTKAMP-VROOM AND A. W. HELDER Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratory of Experimental and Clinical Immunology, University of Amsterdam, Amsterdam, the Netherlands
PLATES1-111 THREEkinds of particles occurring in serum as well as in nuclei and cytoplasm of hepatocytes from patients positive for the hepatitis B antigen (HBAg) have been described (Bayer, Blumberg and Werner, 1968; Dane, Cameron and Briggs, 1970; Ahmed, Huang and Spence, 1971 ;Huang, 1971 ;Gerber, Schaffner and Paronetto, 1972; Nowoslawski et al., 1972). The 42 nm, so-called Dane particles (Dane et al., Nowoslawski et al.) with an electron-dense core of 27 nm present in the serum, are supposed to be the complete, infectious virion. Huang et al. (1972) suggest that the c. 23 nm particles found in the nuclei of hepatocytes represent the core of such Dane particles. The smaller, 20 nm, spherical and tubular structures present in serum and in cytoplasm of hepatocytes, possibly consist of virus-coat proteins (Stein, Fairnaru and Stein, 1972). The intranuclear particles are found in a great number, while the cytoplasmic particles are very sparse in the hepatocytes of HBAg positive liver tissue specimens. Notwithstanding the ultrastructural features of HBAg in liver tissue, Edgington and Ritt (1971) and Haziyannis et al. (1972) could not demonstrate nuclear fluorescence in their studies with anti-HBAg sera, while others (Millman et al., 1969; Coyne et al., 1970; Nowoslawski et al., 1970; Nielsen and Elling, 1971 ; Kater, Borst-Eilers and v. Gorp, 1972) obtained nuclear as well as cytoplasmic fluorescence. -
Received 27 Sept. 1974; accepted 7 Jan. 1975. Requests for reprints should be addressed to Thea M. Feltkamp-Vroom, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, P.O.B. 9190, Amsterdam, the Netherlands. 1. PATH.-VOL.
118 (1976)
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C. M. ROOS, THEA M. FELTKAMP-VROOM AND A. W. HELDER
In the present communication the results are described of an immunofluorescence and electron microscopic study on the localisation of hepatitis B antigen in liver tissue specimens, as well as the light microscopic characteristics of these liver tissue specimens. Since the results of the investigations for the presence of immunoglobulin, fibrinogen and complement, routinely performed in our laboratory, seemed promising for a possible relationship between their localisation and that of hepatitis B antigen, these results are also mentioned. MATERIALS AND METHODS Human liver tissue specimens Liver tissue specimens obtained by surgical or needle biopsy, or at necropsy from 43 HBAg seropositive patients and from 22 seronegative individuals, either healthy or suffering from various liver diseases, were snap-frozen in liquid nitrogen. The needle biopsy specimens were frozen and cut according to the technique of Feltkamp-Vroom and Boode (1970). Antisera Antisera used as first layer in the indirect immunofluorescence method for the demonstration of human IgG, IgM, IgA, Clq, C3c and fibrinogen, were prepared in rabbits. They were tested for specificity with immunoelectrophoretic, agar double diffusion and tanned red cell agglutination techniques. A rabbit anti-HBAg serum (RaHBAg) was raised by hyperimmunisation with HBAg isolated from HBAg-positive serum, according to the method described by Duimel, Brummelhuis and Krijnen (1972). After isolation the material was purified by isopycnic ultracentrifugation on a cesium chloride density gradient. Fractions with a density of 1.17 through 1.23 g/ml were pooled. Electron microscopic investigation of this preparation revealed mainly small spherical 20 MI particles. Tubular structures were seldom observed while the larger Dane particles were not found. After absorption with dried HBA-negative human plasma and with human blood group 0 erythrocytes no antibodies against serum proteins nor heteroagglutinins were detectable in this RaHBAg. The titre was 1 : 526 as determined in the Ouchterlony technique and 1 : 1,OOO,OOO in the passive haemagglutination technique. It produced, in a dilution of 1 : 30, a strong fluorescence in HBAg-positive liver tissue specimens with hardly any background fluorescence. Absorption of this antiserum with human HBAg-positive sem abolished the speciiic fluorescence, while absorption with HBAg-negative serum or normal acetone-dried human liver powder did not influence the fluorescence intensity. A very weak fluorescence of HBAg-positive liver sections was noted, when these sections were treated with human serum containingantibodies against HBAg prior to the incubation with RaHBAg; pre-incubation with normal human serum did not decrease the specific HBAg fluorescence. The RaHBAg was found to contain antibodies against two subspecificities (a and d) of hepatitis B antigen. Normal rabbit serum used as first layer, served as a negative control. A horse anti-rabbit immunoglobulin serum conjugated to fluorescein-isothiocyanate according to The and Feltkamp (1970), was used as second layer. This fluorescent antiserum was characterised by a protein concentration of 12 mglml, an agar block titration titre (Feltkamp, 1970) of 1 : 32 and a molar F/P ratio of 1-8, freed from proteins with F/P ratios of < 1 and > 4. It was used in a final dilution of 1 :70.
Immunofluorescence technique The indirect imrnunofluorescence technique was applied. Cryostat sections of 4 pm thickness were cut at -2O"C, air-dried and washed with phosphate buffered saline, 0 . 0 1 ~ ~ pH 7.2 (PBS) for 30 min. at 20°C. After drying the sections were k e d in acetone for 10 min. at 20°C, air-dried and washed with PBS for 30 min. at 20°C. Sections were then incubated
ANTIGEN AND ANTIBODY IN LIVER
3
with the first layer antisera for 30 min., washed with PBS for 30 min., incubated with the fluorescent serum for 30 min., washed again with PBS for 30 min., mounted in a glycerin-PBS mixture and stored at -2OoC. They were read within 18 hr in a Leitz Orthoplan fluorescence microscope with incident illumination, as described by Ploem (1967) and Hijmans et al. (1970). A Xenon lamp (Osram XBO 150 W) was used for excitation. Light microscopy
Cryostat sections of 4pm thickness were fixed in a formaldehyde-macrodexmixture and stained with hematoxylin and azophloxin, periodic acid-Schiff, A n n and Gomori reticdin. The liver biopsy findings were classified as follows: normal when no obvious alterations were observed; chronic persistent and active (aggressive)hepatitis according to the criteria of the international group of de Groote ef al. (1968); acute hepatic necrosis when many hepatocytes were destroyed, the Kupffer cells swollen and a variable number of inflammatory cells present. Electron microscopy As some needle liver tissue specimens were so small that they only sufficed for the immunofluorescence studies, the specimens of only 15 HBAg seropositive patients could be investi-
gated. Small fragments were cut off from the specimens stored in liquid nitrogen, immersed in Karnovsky's fixative (Karnovsky, 1965) at 2OoC, fixed for periods ranging from 2 br to 7 days at 4"C, post-fixed in a 1 per cent. osmium tetroxidesolutioninphosphatebuffer(pH 7.4) according to Millonig (1961) and embedded in Epon. Ultrathin sections of two tissue blocks were cut on a Reichert ultramicrotome, stained with uranyl acetate for 20 min. and lead hydroxide for 10 min. and examined in a Philips EM 300 electron microscope.
RESULTS The liver tissue specimens of 42 out of the 43 HBAg seropositive patients showed a specific fluorescence with RaHBAg in the cytoplasm of the hepatocytes. The liver specimens of the 22 HBAg seronegative individuals-healthy persons or patients with various liver diseases-showed no fluorescence with RaHBAg. Three out of these 22 individuals had been HBAg seropositivein the past, but were seronegative at the moment of the liver biopsy. Three distinct fluorescence patterns were observed in the cytoplasm of hepatocytes in HBAg-positive liver specimens: 1. Diffuse pattern (fig. I), characterised by a fine fluorescence along the cell surface or in the peripheral zone of the cytoplasm of all hepatocytes. 2. Focal pattern (fig. 2), characterised by a bright homogenous fluorescence of the entire cytoplasm of the hepatocytes with a " punched out " nucleus. These hepatocytes were found in groups, while the surrounding liver parenchyma did not show fluorescence. Dilution of the RaHBAg revealed that this type of fluorescence was caused by confluence of fine fluorescent granules in the cytoplasm. 3. Solitary pattern (fig. 3), characterised by a coarse granular, sometimes confluent fluorescence, disseminated in the cytoplasm of some cells without nuclear fluorescence. These fluorescent cells were situated singly throughout the liver parenchyma.
4
C. M . ROOS, THEA M. FELTKAMP-VROOM AND A . W. HELDER
A combination of two of these three patterns was repeatedly observed in the liver specimens. No difference was seen between centrolobular and periportal areas. In some cases there was also bright fluorescence of the cytoplasm of a few sinusoidal lining cells, probably Kupffer cells. Mononuclear inflammatory cells, if present, never showed fluorescence. The focal pattern was mainly found in liver tissue without perceptible parenchymatous alterations while the solitary pattern was seen especially in the biopsy specimens with acute necrosis. The diffuse pattern was found in cases with chronic persistent as well as in cases with chronic active hepatitis (table). Immunoglobulins and complement were present in a perisinusoidal localisation in all liver tissue specimens, both normal and diseased (fig. 4). Fibrinogen was not found in any of the specimens. With three different rabbit anti-human IgG sera a fine granular nuclear fluorescence was observed in a variable number of hepatocytes in 22 out of the 42 HBAg positive liver tissue specimens (figs. 4 and 5). Some out of these 22 liver specimens demonstrated also a weak nuclear Clq and C3c fluorescence. Serum of seven patients with this nuclear TABLE Relationship between fluorescence patterns and morphological changes in HBAgpositive liver tissue specimens I
I
Light Microscopy
Number studied
Normal Chronic persistent hepatitis Chronic active hepatitis Acute hepatic necrosis
21 12 4
*
5
Fluorescence pattern FocalTDirISoy 5 1 1 0
8* 1*
11 7 0
1*
1 2 4
Combination of patterns as indicated by arrows.
IgG fluorescence was available for antinuclear antibody determination :only one of these seven patients was positive. In the five HBAg-positive cases with normal liver tissue (table) nuclear fluorescence could not be demonstrated, nor in the four cases with the solitary pattern. Electron microscopic examination revealed c. 23 nm spherical particles in the nuclei of the hepatocytes in eight of the 15 liver specimens studied (fig. 6). AJl these eight liver specimens also showed nuclear IgG fluorescence. No such fluorescence was found in the seven liver specimens without intranuclear particles. Ultrastructural particles in the dilated endoplasmic reticulum of the cytoplasm of the hepatocytes in HBAg positive liver tissue as described by Stein et al. 1972) were found in only two cases and only in a small number.
DISCUSSION Contradictory results have been reported from studies on the localisation of HBAg by means of the immunofluorescence technique: only cytoplasmic fluorescence on the one hand (Edgington and Ritt, 1971 ; Hadziyannis et al.,
ANTIGEN AND ANTIBODY IN LIVER
5
1972), and both nuclear and cytoplasmic fluorescence on the other hand (Millman et al., 1969; Coyne et al., 1970; Nowoslawski et al., 1970; Gerber et al., 1972; Kater et al., 1972.) An explanation for these divergent findings might be that the antisera used differed in origin (animal or human) as well as in specificity. Moreover, there are distinct hepatitis B antigenic determinants ; not only are antibodies found directed against the subspecificities a, d and y on the virus-outer coat material, but also antibodies directed against the virus-core (Almeida et al., 1971;Brzosko et al., 1973; Hoofnagle et al., 1973). The anti-hepatitis B antigen serum used in our study was raised in rabbits immunised with a HBAg preparation in which mainly small spherical particles and no Dane particles were found. It could be expected, therefore, that in this RaHBAg only anticoat material would be present. As the serum pool used for the isolation of HBAg appeared to be positive for the antigenic subspecificities a and d, the RaHBAg should contain antibodies against these subspecificities. This was confirmed by precipitation techniques, which also showed that the antibodies against the d antigen were stronger than those against the a antigen. It is therefore possible that the one seropositive patient, whose liver tissue was negative for HBAg in our immunofluorescence study, had been infected with hepatitis B virus with a and y antigenic subspecificities. This seems the more likely, as in a recent study the liver specimens of three patients with the HBAg subspecificity ay showed no fluorescence of the cytoplasm of hepatocytes using this RaHBAg (Ten Kate, Feltkamp-Vroom and Helder, in preparation). All other HBAg seropositive patients showed only a cytoplasmic localisation of HBAg with the immunofluorescence technique. Nuclear fluorescence of hepatocytes with our RaHBAg was not observed. These results seem to support the suggestions of Huang et al. (1972) and Stein et al. (1972) that viruscore material is found in the nuclei and virus-coat proteins in the cytoplasm. Since we found only few ultrastructural particles in the cytoplasm of hepatocytes in two seropositive patients, the bright HBAg fluorescence of the cytoplasm cannot be explained by these structures. In the eight liver tissue specimens, in which intranuclear spherical particles probably representing virus-core material were found, nuclear fluorescence with anti-IgG sera was present. Since the RaHBAg and the three different rabbit anti-human IgG sera did not produce reactions of identity with human HBAgpositive serum in the Ouchterlony technique, it is highly unlikely that these anti-IgG sera prepared against IgG isolated from HBAg-negative serum pools, contain antibodies against core material of Dane particles. The finding of ultrastructural particles in hepatocyte nuclei of those liver tissue specimens, in which nuclear IgG and sometimes a small amount of Clq and C3c was demonstrated, suggest the presence of virus antigen/IgG complexes in these nuclei. In the passive haemagglutination technique using only the " virus-coat system " no antibody against HBAg could be found in our HBAg-positive patients. The absence of this antibody can be explained by the excess of virus-coat antigen in persistent HB antigenaemia. In the study of Hoofnagle et al., however, none of the chronic HBAg carriers had antibodies against virus-coat material, but in
6
C. M. ROOS, THEA M. FELTKAMP-VROOM AND A. W. HELDER
10 per cent. of these patients antibodies against virus-core material were demonstrated. Therefore, anti-core antibody fixation in vivo might occur in the nuclei of hepatocytes containing core material, similarly to the in-vivo nuclear anti-nuclear antibody fixation in skin, liver and renal tissue from systemic lupus erythematosus patients (Tan and Kunkel, 1966; FeltkampVroom, unpublished observations). We have no explanation for the finding, that in only eight out of the 15 HBAg-positive patients with chronic persistent or active hepatitis, intranuclear particles of c. 23 nm were present. From this observation it is clear that electron microscopy of liver biopsy specimens is by itself insufficient for the diagnosis of hepatitis B. The strong cytoplasmic fluorescence in a focal pattern and the peripheral fluorescence of all hepatocytes in a diffuse pattern suggest an uninhibited production of virus-coat protein in chronic hepatitis B. This uninhibited production of antigenic material is in agreement with the great amount of HBAg in the serum of patients with persistent antigenaemia and chronic liver disease. According to Dudley et al. (1972) and to Sutnick (1974) this uninhibited HBAg production might be due to a decrease of the cellular immune defence of these patients. The small number of cells with cytoplasmic fluorescence in HBAgpositive patients with acute liver necrosis in contrast to the large number of fluorescent cells in patients with chronic hepatitis might then be the result of different immune reactions of the individual patient on the hepatitis B antigen. When specific antisera directed against virus-core, virus-coat and coat-subspecificity antigens become available, more insight will be gained about the localisation of the distinct HB antigens in liver tissue from HBAg-positive patients. SUhlhlARY
Liver tissue specimens from 43 HBAg seropositive patients and 22 HBAg seronegative individuals were investigated for the presence of HBAg with the immunofluorescence technique. Histological diagnoses were established with light microscopy on routinely stained frozen liver sections. The liver specimens of 42 out of the 43 HBAg-positive patients showed positive fluorescence. Three distinct fluorescence patterns could be observed : diffuse, focal and solitary. The diffuse pattern appeared to occur particularly in chronic aggressive or persistent hepatitis, the focal pattern was seen only in the group of patients without obvious alterations and the solitary pattern especially in acute hepatic necrosis. Fifteen out of the 42 HBAg-positive liver specimens were investigated with the electron microscope. Eight out of these 15 specimens revealed c. 23 nm nuclear particles. In these eight cases nuclear IgG was observed with the immunofluorescence technique as well as in 14 other specimens, which could not be studied with the electron microscope. The presence of immune complexes consisting of virus-core antigen and IgG in the nuclei of hepatocytes is suggested. Dr B. Houwen, Academic Hospital, Groningen, typed the anti-hepatitis B antigen serum for subspecificity. The authors thank Ir P. Duimel for the isolation of hepatitis B antigen
ANTIGEN AND ANTIBODY IN LIVER
7
and Miss M. van der Giessen for the preparation of the anti-immunoglobulin and anticomplement component sera. The technical assistance of Mr H. J. Ruis and Mrs c. de Graaff-Reitsma as well as the secretarial assistance of Miss H. R. Hertroijs is gratefully acknowledged.
REFERENCES AHMED,M. N., HUANG,S. N., AND SPENCE, L. 1971. Australia antigen in hepatitis. Arch. Path., 92, 66. J. D., RUBENSTEIN, D., AND SCOTT,E. J. 1971. New antigen-antibody system in ALMEIDA, Australia-antigen positive hepatitis. Lancet, 2, 1225. B. S.,AND WERNER, N. 1968. Particles associated with Australia BAYER, M. E., BLUMBERG, antigen in the sera of patients with leukaemai, Down’s syndrome and hepatitis. Nature (Lond.),218, 1957. BRZOSKO, W. J., MADALINSKI, K., KRAWCZYNSKI, K., AND NOWOSLAWSKI, A. 1973. Duality of hepatitis B antigen and its antibody. I. Immunofluorescence studies. J. Infect. Dis.,127, 424. COYNE,V. E., MILLMAN, I., GERDA,J., GERSTLY, B. J. S., LONDON, TH., SUTNICK, A., AND BLUMBERG, B. S. 1970. The localization of Australia antigen by immunofluorescence. J. Exp. Med., 131, 307. C. H., AND BRIGGS, M. 1970. Virus-like particles in serum of DANE,D. S., CAMERON, patients with Australia-antigen-associated hepatitis. Lancet 1, 695. S. 1972. Cellular immunity and hepatitisDUDLEY,F. J., Fox, R. A., AND SHERLOCK, associated, Australia-antigen liver disease. Lancet, 1, 723. H. G. J., AND KRIJNEN,H. W. 1972. The purification of DUIMEL, W. J., BRUMMELHUIS, hepatitis-associated antigen (HAA/SH/AU) from human serum and preparation of a sheep anti-HAA serum. Vox Sang.,23, 249. , J. 1971. Intrahepatic expression of serum hepatitis virus EDGINGTON, T. S., AND R I T ~D. associated antigens. J. Exp. Med., 134, 871. J. H. M. 1970. An embedding and sectioning FELTKAMP-VROOM, THEAM., AND BOODE, technique for immunohistochemical studies of minute specimens of tissue. J. Clin. Path., 23, 188. FELTKAMP, T. E. W. 1970. Titration of conjugates. In Standardization in immunofluorescence, ed. by E. J. Holborow, Blackwell Sci. Publ., Oxford, p. 189. M. A., SCHAFFNER, F., AND PARONETTO. F. 1972. Immuno-electron microscopy of GERBER, hepatitis. Proc. SOC.Exp. Biol.,140, 1334. DE GROOTE, J., DESMET,V. J., GEDIGK,P., KORB,G., POPPER,H., POTJLSEN, H., SCHEWR, P. J., SCHMID,M., THALER, H., AND UEHLINGER, E. 1968. A classification of chronic hepatitis. Lancet, 2, 626. HADZIYANNIS, ST., VISSOULIS,CH., Moussomos, A., AND AFROUDAKIS, A. 1972. Cytoplasmic localization of Australia antigen in the liver. Lancet, 1, 976. J. H., GERETY, R. J., AND BARKER, L. F. 1973. Antibody to hepatitis B-virus HOOFNAGLE, core in man. Lancet, 2, 869. S. N. 1971. Hepatitis associated antigen hepatitis. Amer J. Path., 64, 483. HUANG, HUANG, S. N., MILLMAN, I., O’CONNELL, A., ARONOFF, A., GAULT,H., AND BLUMBERG, B. S. 1972. Virus-like particles in Australia antigen associated hepatitis. An immunoelectron microscopic study of human liver. Amer. J. Path., 67, 453. H. R. E., BANGSMA, A. P. M., AND PLOEM, J. S . 1970. Performance HIJMANS, W., SCHUIT, testing of fluorescent antisera against human immunoglobulins. In Standardization in immunofluorescence, ed. by E. J. Holborow, Blackwell Sci. Publ., Oxford, p. 193. M. J. 1965. A formaldehyde-glutaraldehydefixative of high osmolality for use KARNOVSKY, in electron microscopy. J. Cell Biol., 27, 137a. KATER, L., BORST-EILERS, E., AND GORP,L. H. M. V. 1972. Immunopathological studies on liver biopsies. Folia Med. N e d , 15, 214.
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MILLMAN,I., ZAVATONE, V., GERSTLY,B. J.S., AND BLUMBERG, B. S. 1969. Australia antigen detected in the nuclei of liver cells of patients with viral hepatitis by the fluorescent antibody technique. Nature, 222, 181. MILLONIG, G. 1961. Advantages of phosphate buffer for solutions in fixation. J. appl. Physiol., 32, 16. NIELSEN, J. O., AND ELLING,P. 1971. Investigations of liver biopsies for Australia antigen by immunofluorescence technique. Clin.Exp. Immunol.,9, 699. NOWOSLAWSKI, A., BROZOSKO, W. J., MADALINSKI, K., AND KRAWCZYNSKI, K. 1970. Cellular localization of Australia antigen in the liver of patients with lymphoproliferative disorders. Lancet, 1, 494. NOWOSLAWSKI, A., KRAWCZYNSKI, K., BRZOSKO, W., AND MALADINSKI, K. 1972. Tissue localization of Australia antigen immune complexes in acute and chronic hepatitis and liver cirrhosis. Amer. J . Path., 68, 31. PLOEM, J. S. 1967. The use of a vertical illuminator with interchangeable dichroic mirrors for fluorescence microscopy with incident light. Z. Win. Mikr., 68, 129. STEIN,O., FAIRNARU, M., AND STEIN,Y. 1972. Visualization of virus-like particles in endoplasmic reticulum of hepatocytes of Australia antigen carriers. Lab. Invest., 26, 262. SUTNICK,A. 1. 1974. Australia antigen and the immune response in human diseases. J. Allergy Clin.Immunol., 53, 42. TAN,E. M., AND KUNKEL,H. G. 1966. An immunofluorescent study of skin lesions in systemic lupus erythematosus. Arthr. and Rheum., 9, 37. THE,T. H., AND FELTKAMP, T. E. W. 1970. Conjugation of fluorescein isothiocyanate to antibodies. 11. A reproducible method. Immunology, 18, 875.
ROOS,
FELTKAMP-VRCOM AND HELDER
PLATE
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ANTIGEN AND ANTIBODY IN LIVER
FIG.1.---Fluorescence of a liver section incubated with RaHBAg, showing a diffuse pattern. x 250.
FIG.2.--Fluorescence of a liver section incubated with RaHBAg, showing a focal pattern. x 250.
Roos, FELTKAMP-VROOM AND HELDER
PLATEI1
ANTIGEN AND ANTIBODY IN LIVER
FIG.3.--FIuorescence of a liver section incubated with RaHBAg, showing a solitary pattern. x 500.
FIG.4.--Fluorescence of a liver section incubated with rabbit anti-human IgG, showing a perisinusoidal pattern and fluorescence of several nuclei. x 250.
Roos, FELTKAMP-VROOM AND HELDER
PLATE111
ANTIGEN AND ANTIBODY IN LIVER
FIG. 5.-Liver
section incubated with rabbit anti-human IgG, showing bright granular nuclear fluorescence. x 500.
FIG.6.-Electron micrograph of nucleus of hepatocyte, showing small c. 23 nm particles. x 90,000.
Journal of Pathology Vol. 118 No. 1 T H E L O CA L I SA T I O N O F HEPATITIS B A N T I G E N A N D I M M U N O G L O B U L I N G I N LIVER TISSUE: A N I M M U N O FL U O RE SCE N CE , L I G H T A N D E L E C T R O N MICROSCOPIC STUDY C. M. Roos, THEAM. FELTKAMP-VROOM AND A. W. HELDER Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratory of Experimental and Clinical Immunology, University of Amsterdam, Amsterdam, the Netherlands
PLATES1-111 THREEkinds of particles occurring in serum as well as in nuclei and cytoplasm of hepatocytes from patients positive for the hepatitis B antigen (HBAg) have been described (Bayer, Blumberg and Werner, 1968; Dane, Cameron and Briggs, 1970; Ahmed, Huang and Spence, 1971 ;Huang, 1971 ;Gerber, Schaffner and Paronetto, 1972; Nowoslawski et al., 1972). The 42 nm, so-called Dane particles (Dane et al., Nowoslawski et al.) with an electron-dense core of 27 nm present in the serum, are supposed to be the complete, infectious virion. Huang et al. (1972) suggest that the c. 23 nm particles found in the nuclei of hepatocytes represent the core of such Dane particles. The smaller, 20 nm, spherical and tubular structures present in serum and in cytoplasm of hepatocytes, possibly consist of virus-coat proteins (Stein, Fairnaru and Stein, 1972). The intranuclear particles are found in a great number, while the cytoplasmic particles are very sparse in the hepatocytes of HBAg positive liver tissue specimens. Notwithstanding the ultrastructural features of HBAg in liver tissue, Edgington and Ritt (1971) and Haziyannis et al. (1972) could not demonstrate nuclear fluorescence in their studies with anti-HBAg sera, while others (Millman et al., 1969; Coyne et al., 1970; Nowoslawski et al., 1970; Nielsen and Elling, 1971 ; Kater, Borst-Eilers and v. Gorp, 1972) obtained nuclear as well as cytoplasmic fluorescence. -
Received 27 Sept. 1974; accepted 7 Jan. 1975. Requests for reprints should be addressed to Thea M. Feltkamp-Vroom, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, P.O.B. 9190, Amsterdam, the Netherlands. 1. PATH.-VOL.
118 (1976)
1
A
2
C. M. ROOS, THEA M. FELTKAMP-VROOM AND A. W. HELDER
In the present communication the results are described of an immunofluorescence and electron microscopic study on the localisation of hepatitis B antigen in liver tissue specimens, as well as the light microscopic characteristics of these liver tissue specimens. Since the results of the investigations for the presence of immunoglobulin, fibrinogen and complement, routinely performed in our laboratory, seemed promising for a possible relationship between their localisation and that of hepatitis B antigen, these results are also mentioned. MATERIALS AND METHODS Human liver tissue specimens Liver tissue specimens obtained by surgical or needle biopsy, or at necropsy from 43 HBAg seropositive patients and from 22 seronegative individuals, either healthy or suffering from various liver diseases, were snap-frozen in liquid nitrogen. The needle biopsy specimens were frozen and cut according to the technique of Feltkamp-Vroom and Boode (1970). Antisera Antisera used as first layer in the indirect immunofluorescence method for the demonstration of human IgG, IgM, IgA, Clq, C3c and fibrinogen, were prepared in rabbits. They were tested for specificity with immunoelectrophoretic, agar double diffusion and tanned red cell agglutination techniques. A rabbit anti-HBAg serum (RaHBAg) was raised by hyperimmunisation with HBAg isolated from HBAg-positive serum, according to the method described by Duimel, Brummelhuis and Krijnen (1972). After isolation the material was purified by isopycnic ultracentrifugation on a cesium chloride density gradient. Fractions with a density of 1.17 through 1.23 g/ml were pooled. Electron microscopic investigation of this preparation revealed mainly small spherical 20 MI particles. Tubular structures were seldom observed while the larger Dane particles were not found. After absorption with dried HBA-negative human plasma and with human blood group 0 erythrocytes no antibodies against serum proteins nor heteroagglutinins were detectable in this RaHBAg. The titre was 1 : 526 as determined in the Ouchterlony technique and 1 : 1,OOO,OOO in the passive haemagglutination technique. It produced, in a dilution of 1 : 30, a strong fluorescence in HBAg-positive liver tissue specimens with hardly any background fluorescence. Absorption of this antiserum with human HBAg-positive sem abolished the speciiic fluorescence, while absorption with HBAg-negative serum or normal acetone-dried human liver powder did not influence the fluorescence intensity. A very weak fluorescence of HBAg-positive liver sections was noted, when these sections were treated with human serum containingantibodies against HBAg prior to the incubation with RaHBAg; pre-incubation with normal human serum did not decrease the specific HBAg fluorescence. The RaHBAg was found to contain antibodies against two subspecificities (a and d) of hepatitis B antigen. Normal rabbit serum used as first layer, served as a negative control. A horse anti-rabbit immunoglobulin serum conjugated to fluorescein-isothiocyanate according to The and Feltkamp (1970), was used as second layer. This fluorescent antiserum was characterised by a protein concentration of 12 mglml, an agar block titration titre (Feltkamp, 1970) of 1 : 32 and a molar F/P ratio of 1-8, freed from proteins with F/P ratios of < 1 and > 4. It was used in a final dilution of 1 :70.
Immunofluorescence technique The indirect imrnunofluorescence technique was applied. Cryostat sections of 4 pm thickness were cut at -2O"C, air-dried and washed with phosphate buffered saline, 0 . 0 1 ~ ~ pH 7.2 (PBS) for 30 min. at 20°C. After drying the sections were k e d in acetone for 10 min. at 20°C, air-dried and washed with PBS for 30 min. at 20°C. Sections were then incubated
ANTIGEN AND ANTIBODY IN LIVER
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with the first layer antisera for 30 min., washed with PBS for 30 min., incubated with the fluorescent serum for 30 min., washed again with PBS for 30 min., mounted in a glycerin-PBS mixture and stored at -2OoC. They were read within 18 hr in a Leitz Orthoplan fluorescence microscope with incident illumination, as described by Ploem (1967) and Hijmans et al. (1970). A Xenon lamp (Osram XBO 150 W) was used for excitation. Light microscopy
Cryostat sections of 4pm thickness were fixed in a formaldehyde-macrodexmixture and stained with hematoxylin and azophloxin, periodic acid-Schiff, A n n and Gomori reticdin. The liver biopsy findings were classified as follows: normal when no obvious alterations were observed; chronic persistent and active (aggressive)hepatitis according to the criteria of the international group of de Groote ef al. (1968); acute hepatic necrosis when many hepatocytes were destroyed, the Kupffer cells swollen and a variable number of inflammatory cells present. Electron microscopy As some needle liver tissue specimens were so small that they only sufficed for the immunofluorescence studies, the specimens of only 15 HBAg seropositive patients could be investi-
gated. Small fragments were cut off from the specimens stored in liquid nitrogen, immersed in Karnovsky's fixative (Karnovsky, 1965) at 2OoC, fixed for periods ranging from 2 br to 7 days at 4"C, post-fixed in a 1 per cent. osmium tetroxidesolutioninphosphatebuffer(pH 7.4) according to Millonig (1961) and embedded in Epon. Ultrathin sections of two tissue blocks were cut on a Reichert ultramicrotome, stained with uranyl acetate for 20 min. and lead hydroxide for 10 min. and examined in a Philips EM 300 electron microscope.
RESULTS The liver tissue specimens of 42 out of the 43 HBAg seropositive patients showed a specific fluorescence with RaHBAg in the cytoplasm of the hepatocytes. The liver specimens of the 22 HBAg seronegative individuals-healthy persons or patients with various liver diseases-showed no fluorescence with RaHBAg. Three out of these 22 individuals had been HBAg seropositivein the past, but were seronegative at the moment of the liver biopsy. Three distinct fluorescence patterns were observed in the cytoplasm of hepatocytes in HBAg-positive liver specimens: 1. Diffuse pattern (fig. I), characterised by a fine fluorescence along the cell surface or in the peripheral zone of the cytoplasm of all hepatocytes. 2. Focal pattern (fig. 2), characterised by a bright homogenous fluorescence of the entire cytoplasm of the hepatocytes with a " punched out " nucleus. These hepatocytes were found in groups, while the surrounding liver parenchyma did not show fluorescence. Dilution of the RaHBAg revealed that this type of fluorescence was caused by confluence of fine fluorescent granules in the cytoplasm. 3. Solitary pattern (fig. 3), characterised by a coarse granular, sometimes confluent fluorescence, disseminated in the cytoplasm of some cells without nuclear fluorescence. These fluorescent cells were situated singly throughout the liver parenchyma.
4
C. M . ROOS, THEA M. FELTKAMP-VROOM AND A . W. HELDER
A combination of two of these three patterns was repeatedly observed in the liver specimens. No difference was seen between centrolobular and periportal areas. In some cases there was also bright fluorescence of the cytoplasm of a few sinusoidal lining cells, probably Kupffer cells. Mononuclear inflammatory cells, if present, never showed fluorescence. The focal pattern was mainly found in liver tissue without perceptible parenchymatous alterations while the solitary pattern was seen especially in the biopsy specimens with acute necrosis. The diffuse pattern was found in cases with chronic persistent as well as in cases with chronic active hepatitis (table). Immunoglobulins and complement were present in a perisinusoidal localisation in all liver tissue specimens, both normal and diseased (fig. 4). Fibrinogen was not found in any of the specimens. With three different rabbit anti-human IgG sera a fine granular nuclear fluorescence was observed in a variable number of hepatocytes in 22 out of the 42 HBAg positive liver tissue specimens (figs. 4 and 5). Some out of these 22 liver specimens demonstrated also a weak nuclear Clq and C3c fluorescence. Serum of seven patients with this nuclear TABLE Relationship between fluorescence patterns and morphological changes in HBAgpositive liver tissue specimens I
I
Light Microscopy
Number studied
Normal Chronic persistent hepatitis Chronic active hepatitis Acute hepatic necrosis
21 12 4
*
5
Fluorescence pattern FocalTDirISoy 5 1 1 0
8* 1*
11 7 0
1*
1 2 4
Combination of patterns as indicated by arrows.
IgG fluorescence was available for antinuclear antibody determination :only one of these seven patients was positive. In the five HBAg-positive cases with normal liver tissue (table) nuclear fluorescence could not be demonstrated, nor in the four cases with the solitary pattern. Electron microscopic examination revealed c. 23 nm spherical particles in the nuclei of the hepatocytes in eight of the 15 liver specimens studied (fig. 6). AJl these eight liver specimens also showed nuclear IgG fluorescence. No such fluorescence was found in the seven liver specimens without intranuclear particles. Ultrastructural particles in the dilated endoplasmic reticulum of the cytoplasm of the hepatocytes in HBAg positive liver tissue as described by Stein et al. 1972) were found in only two cases and only in a small number.
DISCUSSION Contradictory results have been reported from studies on the localisation of HBAg by means of the immunofluorescence technique: only cytoplasmic fluorescence on the one hand (Edgington and Ritt, 1971 ; Hadziyannis et al.,
ANTIGEN AND ANTIBODY IN LIVER
5
1972), and both nuclear and cytoplasmic fluorescence on the other hand (Millman et al., 1969; Coyne et al., 1970; Nowoslawski et al., 1970; Gerber et al., 1972; Kater et al., 1972.) An explanation for these divergent findings might be that the antisera used differed in origin (animal or human) as well as in specificity. Moreover, there are distinct hepatitis B antigenic determinants ; not only are antibodies found directed against the subspecificities a, d and y on the virus-outer coat material, but also antibodies directed against the virus-core (Almeida et al., 1971;Brzosko et al., 1973; Hoofnagle et al., 1973). The anti-hepatitis B antigen serum used in our study was raised in rabbits immunised with a HBAg preparation in which mainly small spherical particles and no Dane particles were found. It could be expected, therefore, that in this RaHBAg only anticoat material would be present. As the serum pool used for the isolation of HBAg appeared to be positive for the antigenic subspecificities a and d, the RaHBAg should contain antibodies against these subspecificities. This was confirmed by precipitation techniques, which also showed that the antibodies against the d antigen were stronger than those against the a antigen. It is therefore possible that the one seropositive patient, whose liver tissue was negative for HBAg in our immunofluorescence study, had been infected with hepatitis B virus with a and y antigenic subspecificities. This seems the more likely, as in a recent study the liver specimens of three patients with the HBAg subspecificity ay showed no fluorescence of the cytoplasm of hepatocytes using this RaHBAg (Ten Kate, Feltkamp-Vroom and Helder, in preparation). All other HBAg seropositive patients showed only a cytoplasmic localisation of HBAg with the immunofluorescence technique. Nuclear fluorescence of hepatocytes with our RaHBAg was not observed. These results seem to support the suggestions of Huang et al. (1972) and Stein et al. (1972) that viruscore material is found in the nuclei and virus-coat proteins in the cytoplasm. Since we found only few ultrastructural particles in the cytoplasm of hepatocytes in two seropositive patients, the bright HBAg fluorescence of the cytoplasm cannot be explained by these structures. In the eight liver tissue specimens, in which intranuclear spherical particles probably representing virus-core material were found, nuclear fluorescence with anti-IgG sera was present. Since the RaHBAg and the three different rabbit anti-human IgG sera did not produce reactions of identity with human HBAgpositive serum in the Ouchterlony technique, it is highly unlikely that these anti-IgG sera prepared against IgG isolated from HBAg-negative serum pools, contain antibodies against core material of Dane particles. The finding of ultrastructural particles in hepatocyte nuclei of those liver tissue specimens, in which nuclear IgG and sometimes a small amount of Clq and C3c was demonstrated, suggest the presence of virus antigen/IgG complexes in these nuclei. In the passive haemagglutination technique using only the " virus-coat system " no antibody against HBAg could be found in our HBAg-positive patients. The absence of this antibody can be explained by the excess of virus-coat antigen in persistent HB antigenaemia. In the study of Hoofnagle et al., however, none of the chronic HBAg carriers had antibodies against virus-coat material, but in
6
C. M. ROOS, THEA M. FELTKAMP-VROOM AND A. W. HELDER
10 per cent. of these patients antibodies against virus-core material were demonstrated. Therefore, anti-core antibody fixation in vivo might occur in the nuclei of hepatocytes containing core material, similarly to the in-vivo nuclear anti-nuclear antibody fixation in skin, liver and renal tissue from systemic lupus erythematosus patients (Tan and Kunkel, 1966; FeltkampVroom, unpublished observations). We have no explanation for the finding, that in only eight out of the 15 HBAg-positive patients with chronic persistent or active hepatitis, intranuclear particles of c. 23 nm were present. From this observation it is clear that electron microscopy of liver biopsy specimens is by itself insufficient for the diagnosis of hepatitis B. The strong cytoplasmic fluorescence in a focal pattern and the peripheral fluorescence of all hepatocytes in a diffuse pattern suggest an uninhibited production of virus-coat protein in chronic hepatitis B. This uninhibited production of antigenic material is in agreement with the great amount of HBAg in the serum of patients with persistent antigenaemia and chronic liver disease. According to Dudley et al. (1972) and to Sutnick (1974) this uninhibited HBAg production might be due to a decrease of the cellular immune defence of these patients. The small number of cells with cytoplasmic fluorescence in HBAgpositive patients with acute liver necrosis in contrast to the large number of fluorescent cells in patients with chronic hepatitis might then be the result of different immune reactions of the individual patient on the hepatitis B antigen. When specific antisera directed against virus-core, virus-coat and coat-subspecificity antigens become available, more insight will be gained about the localisation of the distinct HB antigens in liver tissue from HBAg-positive patients. SUhlhlARY
Liver tissue specimens from 43 HBAg seropositive patients and 22 HBAg seronegative individuals were investigated for the presence of HBAg with the immunofluorescence technique. Histological diagnoses were established with light microscopy on routinely stained frozen liver sections. The liver specimens of 42 out of the 43 HBAg-positive patients showed positive fluorescence. Three distinct fluorescence patterns could be observed : diffuse, focal and solitary. The diffuse pattern appeared to occur particularly in chronic aggressive or persistent hepatitis, the focal pattern was seen only in the group of patients without obvious alterations and the solitary pattern especially in acute hepatic necrosis. Fifteen out of the 42 HBAg-positive liver specimens were investigated with the electron microscope. Eight out of these 15 specimens revealed c. 23 nm nuclear particles. In these eight cases nuclear IgG was observed with the immunofluorescence technique as well as in 14 other specimens, which could not be studied with the electron microscope. The presence of immune complexes consisting of virus-core antigen and IgG in the nuclei of hepatocytes is suggested. Dr B. Houwen, Academic Hospital, Groningen, typed the anti-hepatitis B antigen serum for subspecificity. The authors thank Ir P. Duimel for the isolation of hepatitis B antigen
ANTIGEN AND ANTIBODY IN LIVER
7
and Miss M. van der Giessen for the preparation of the anti-immunoglobulin and anticomplement component sera. The technical assistance of Mr H. J. Ruis and Mrs c. de Graaff-Reitsma as well as the secretarial assistance of Miss H. R. Hertroijs is gratefully acknowledged.
REFERENCES AHMED,M. N., HUANG,S. N., AND SPENCE, L. 1971. Australia antigen in hepatitis. Arch. Path., 92, 66. J. D., RUBENSTEIN, D., AND SCOTT,E. J. 1971. New antigen-antibody system in ALMEIDA, Australia-antigen positive hepatitis. Lancet, 2, 1225. B. S.,AND WERNER, N. 1968. Particles associated with Australia BAYER, M. E., BLUMBERG, antigen in the sera of patients with leukaemai, Down’s syndrome and hepatitis. Nature (Lond.),218, 1957. BRZOSKO, W. J., MADALINSKI, K., KRAWCZYNSKI, K., AND NOWOSLAWSKI, A. 1973. Duality of hepatitis B antigen and its antibody. I. Immunofluorescence studies. J. Infect. Dis.,127, 424. COYNE,V. E., MILLMAN, I., GERDA,J., GERSTLY, B. J. S., LONDON, TH., SUTNICK, A., AND BLUMBERG, B. S. 1970. The localization of Australia antigen by immunofluorescence. J. Exp. Med., 131, 307. C. H., AND BRIGGS, M. 1970. Virus-like particles in serum of DANE,D. S., CAMERON, patients with Australia-antigen-associated hepatitis. Lancet 1, 695. S. 1972. Cellular immunity and hepatitisDUDLEY,F. J., Fox, R. A., AND SHERLOCK, associated, Australia-antigen liver disease. Lancet, 1, 723. H. G. J., AND KRIJNEN,H. W. 1972. The purification of DUIMEL, W. J., BRUMMELHUIS, hepatitis-associated antigen (HAA/SH/AU) from human serum and preparation of a sheep anti-HAA serum. Vox Sang.,23, 249. , J. 1971. Intrahepatic expression of serum hepatitis virus EDGINGTON, T. S., AND R I T ~D. associated antigens. J. Exp. Med., 134, 871. J. H. M. 1970. An embedding and sectioning FELTKAMP-VROOM, THEAM., AND BOODE, technique for immunohistochemical studies of minute specimens of tissue. J. Clin. Path., 23, 188. FELTKAMP, T. E. W. 1970. Titration of conjugates. In Standardization in immunofluorescence, ed. by E. J. Holborow, Blackwell Sci. Publ., Oxford, p. 189. M. A., SCHAFFNER, F., AND PARONETTO. F. 1972. Immuno-electron microscopy of GERBER, hepatitis. Proc. SOC.Exp. Biol.,140, 1334. DE GROOTE, J., DESMET,V. J., GEDIGK,P., KORB,G., POPPER,H., POTJLSEN, H., SCHEWR, P. J., SCHMID,M., THALER, H., AND UEHLINGER, E. 1968. A classification of chronic hepatitis. Lancet, 2, 626. HADZIYANNIS, ST., VISSOULIS,CH., Moussomos, A., AND AFROUDAKIS, A. 1972. Cytoplasmic localization of Australia antigen in the liver. Lancet, 1, 976. J. H., GERETY, R. J., AND BARKER, L. F. 1973. Antibody to hepatitis B-virus HOOFNAGLE, core in man. Lancet, 2, 869. S. N. 1971. Hepatitis associated antigen hepatitis. Amer J. Path., 64, 483. HUANG, HUANG, S. N., MILLMAN, I., O’CONNELL, A., ARONOFF, A., GAULT,H., AND BLUMBERG, B. S. 1972. Virus-like particles in Australia antigen associated hepatitis. An immunoelectron microscopic study of human liver. Amer. J. Path., 67, 453. H. R. E., BANGSMA, A. P. M., AND PLOEM, J. S . 1970. Performance HIJMANS, W., SCHUIT, testing of fluorescent antisera against human immunoglobulins. In Standardization in immunofluorescence, ed. by E. J. Holborow, Blackwell Sci. Publ., Oxford, p. 193. M. J. 1965. A formaldehyde-glutaraldehydefixative of high osmolality for use KARNOVSKY, in electron microscopy. J. Cell Biol., 27, 137a. KATER, L., BORST-EILERS, E., AND GORP,L. H. M. V. 1972. Immunopathological studies on liver biopsies. Folia Med. N e d , 15, 214.
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MILLMAN,I., ZAVATONE, V., GERSTLY,B. J.S., AND BLUMBERG, B. S. 1969. Australia antigen detected in the nuclei of liver cells of patients with viral hepatitis by the fluorescent antibody technique. Nature, 222, 181. MILLONIG, G. 1961. Advantages of phosphate buffer for solutions in fixation. J. appl. Physiol., 32, 16. NIELSEN, J. O., AND ELLING,P. 1971. Investigations of liver biopsies for Australia antigen by immunofluorescence technique. Clin.Exp. Immunol.,9, 699. NOWOSLAWSKI, A., BROZOSKO, W. J., MADALINSKI, K., AND KRAWCZYNSKI, K. 1970. Cellular localization of Australia antigen in the liver of patients with lymphoproliferative disorders. Lancet, 1, 494. NOWOSLAWSKI, A., KRAWCZYNSKI, K., BRZOSKO, W., AND MALADINSKI, K. 1972. Tissue localization of Australia antigen immune complexes in acute and chronic hepatitis and liver cirrhosis. Amer. J . Path., 68, 31. PLOEM, J. S. 1967. The use of a vertical illuminator with interchangeable dichroic mirrors for fluorescence microscopy with incident light. Z. Win. Mikr., 68, 129. STEIN,O., FAIRNARU, M., AND STEIN,Y. 1972. Visualization of virus-like particles in endoplasmic reticulum of hepatocytes of Australia antigen carriers. Lab. Invest., 26, 262. SUTNICK,A. 1. 1974. Australia antigen and the immune response in human diseases. J. Allergy Clin.Immunol., 53, 42. TAN,E. M., AND KUNKEL,H. G. 1966. An immunofluorescent study of skin lesions in systemic lupus erythematosus. Arthr. and Rheum., 9, 37. THE,T. H., AND FELTKAMP, T. E. W. 1970. Conjugation of fluorescein isothiocyanate to antibodies. 11. A reproducible method. Immunology, 18, 875.
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PLATE
1
ANTIGEN AND ANTIBODY IN LIVER
FIG.1.---Fluorescence of a liver section incubated with RaHBAg, showing a diffuse pattern. x 250.
FIG.2.--Fluorescence of a liver section incubated with RaHBAg, showing a focal pattern. x 250.
Roos, FELTKAMP-VROOM AND HELDER
PLATEI1
ANTIGEN AND ANTIBODY IN LIVER
FIG.3.--FIuorescence of a liver section incubated with RaHBAg, showing a solitary pattern. x 500.
FIG.4.--Fluorescence of a liver section incubated with rabbit anti-human IgG, showing a perisinusoidal pattern and fluorescence of several nuclei. x 250.
Roos, FELTKAMP-VROOM AND HELDER
PLATE111
ANTIGEN AND ANTIBODY IN LIVER
FIG. 5.-Liver
section incubated with rabbit anti-human IgG, showing bright granular nuclear fluorescence. x 500.
FIG.6.-Electron micrograph of nucleus of hepatocyte, showing small c. 23 nm particles. x 90,000.
