Humoral and Cell-mediated Immunity to Hepatitis B Virus Antigens in Acute and Chronic Liver Disease
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J. ALDERSHVILE, 0. DIETRICHSON, F. HARDT, J. 0. NIELSEN & P. SKINHdJ 2nd Dept. of Medicine, Kommunehospitalet, Dept. of Medicine, Division of Hepatology, Hvidovre Hospital, Dept. of Infectious Diseases M, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
Aldershvile, J., Dietrichson, 0.. Hardt, F., Nielsen, J. 0. & Skinhoj, P. Humoral and cell-mediated immunity to hepatitis B virus antigens in acute and chronic liver disease. Scand. J. Gastroent. 1977, 12, 917-922 The humoral immune response to hepatitis B virus antigens and the cell-mediated immunity to hepatitis B surface antigen (HB,Ag) were investigated in 12 healthy persons and 5 6 patients with various liver diseases. In patients with acute viral hepatitis type B, anti-hepatitis B core antigen was present constantly in serum in all phases, and after clinical recovery simultaneouslywith anti-HB,. A transitory cellular immune response to HB,Ag was demonstrated at the time the antigen was cleared, while a patient with persisting HB, antigenaemia and another with transient hepatitis Be antigen showed no response during the course of the disease. Cellular immune response to HB,Ag was present only infrequently in patients with chronic liver disease type B and non-B. thus suggesting that a cellular immunity to HB,Ag is not a prerequisitefor the development of these conditions. Key-words: Acute hepatitis; cellular immunity; hepatitis antigen; liver disease; virus
J . Aldershvile, 2nd Depr. of Medicine, Kommunehospiralet, DK-1399 Copenhagen K , Denmark
Parallel with other virus infections, the elimination sient cell-mediated immunity to purified HB,Ag has of hepatitis B virus (HBV) is dependent on both been demonstrated during the acute phase of type B humoral and cellular immune mechanisms. The im- hepatitis and, in contrast, no migration inhibition mune response seems to be directed both against the was found in patients with chronic hepatitis (3, 9). virus antigens and against liver antigens exposed or However, in other studies, cell-mediated immunity made immunogenic by hepatitis B virus infection. A to HB,Ag positive serum or partially purified direct cytopathogenic effect of the HBV has not HB,Ag has been demonstrated in both acute and been demonstrated. The liver cell damage observed chronic hepatitis (4, 13). in acute and chronic hepatitis B infections may thus In the present study, the specific humoral immune be due to the immune reaction. Whereas uniform response to HBV antigens (HB,Ag, hepatitis B core results have been reported regarding the occurrence antigen (HB,Ag) and hepatitis Be antigen (HBeAg)) of anti-hepatitis B surface antigen (anti-HB,) and was correlated with the cellular immunity to HB,Ag anti-hepatitis B core antigen (anti-HB,), the results and the clinical course of the hepatitis B infection. of studies of the specific cell-mediated immune response to hepatitis B surface antigen (HB,Ag) are PATIENTS AND METHODS conflicting (3, 4, 9, 13). The total material consisted of 56 patients and 12 This might be due to methodological differences, healthy staff members. A liver biopsy was performed in all patients, and since the methods and antigen solutions used to estimate the cell-mediated immune response in vitro based on the histological and serological findings, are far from standardized. In some studies, a tran- the patients were classified as follows:
918
J . Aldershvile, 0 . Dietrichson, F. Hardr, J . 0. Nietsen & P. Skinhoj
Scand J Gastroenterol Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
Acute viral hepatitis type B
.
i
described previously (16). The final preparation contained 0.25 mg/ml of protein in phosphatebuffered saline, and no serum protein contamination could be detected by immunoelectrophoresis.
Twenty-five patients had acute viral hepatitis and circulating HB,Ag at the time of admission to hospital. Thirteen of the patients were examined during the acute phase, and seven of these were followed-up Agarose leucocyte migration inhibition test ( 2 ) by repeated examinations during the course of the The fresh agarose medium was a 2 per cent disease. Twelve patients were investigated after clinsolution of agarose (Litex, Glostrup, Denmark) ical and biochemical recovery. mixed at 47OC with horse serum (Statens Seruminstitut, Copenhagen, Denmark), sterilized water, Chronic active liver disease type B The group comprised 13 patients with chronic and a 10-fold concentration of tissue culture active hepatitis with or without cirrhosis. All these medium 199 (TC-medium 199 - l o x from Difco patients showed serological evidence of present or Laboratories, Detroit, Michigan, USA) to a final previous HBV infection, since seven patients had solution containing 1 per cent agarose and 10 per cent horse serum in single strength TC-medium HB,Ag, four had anti-HB,, and two anti-HB,. 199. Penicillin and streptomycin (TC-penicillinstreptomycin, Difco) was added to a final concentraChronic active liver disease, non-B tion of 66 units penicillin per ml and 66 pg streptoEight patients had chronic active hepatitis with or mycin per ml. Sodium bicarbonate solution (TC without cirrhosis. None of these patients had sodium bicarbonate solution 10 per cent, Difco) was HB,Ag, anti-HB, or anti-HB, in serum. added to adjust the pH in the agarose medium. Five Ten of the patients with chronic active hepatitis ml agarose medium was poured into each disposable with or without cirrhosis were being treated with plastic petri dish (millipore, Mass., USA). After corticosteroid and four with azathioprine at the time incubation in a 2 per cent COz, 98 per cent atmosof the investigation. pheric air saturated with water vapour, the pH was 7.2-7.4. After the gel had formed, eight holes Miscellaneous liver diseases (2.5mm in diameter) were cut in each agarose plate. This group comprised 10 patients, five with alco20 per cent dextran solution ( 5 per cent dextran holic cirrhosis, four with steatosis, and one with 250, Pharmacia, Sweden, in saline solution) was primary biliary cirrhosis. None of the patients had added to a heparinized venous blood sample. The serological evidence of present or previous HBV mixture was allowed to form sediment at 37 "C for infections. one hour. The supernatant leucocyte-rich plasma H B J g and anti-HB, was demonstrated by a solid was removed and centrifuged at 220g for 5 minutes. phase radioimmunoassay (Austria 11-125 and Au- The cell pellet was washed three times in Hanks sab, Abbott Diagnostic Division). balanced salt solution. The leucocytes were then Anti-HB, was determined by the indirect resuspended in Hepes-buffered TC-medium 199 immunofluorescence technique, using frozen sec- with 10 per cent horse serum added to give a final tions of liver tissue containing a high amount of concentration of 2.2 x lo8 Ieucocytes per ml. HG,Ag (kindly supplied by Professor Nowos- 2 x lo8 leucocytes were incubated with lop1 purilawski, National Institute of Hygiene, Warsaw, fied HB,Ag (protein concentration 0.05 mg/ml) for Poland), and an FITC-labelled monospecific rabbit half an hour. This protein concentration was the anti-human IgG. highest concentration with no inhibition of leucoH B d g and anti-hepatitis Be antigen (anti-HB,) cytes from healthy controls. As control, a similar number of cells was incuwas demonstrated by immunodiffusion, as debated with phosphate buffer without HB,Ag. Seven scribed previously (4). Preparation ofHBJg. HB,Ag was purified from pI of the leucocyte-antigen mixture was placed in the serum of an apparently healthy blood donor with three wells, and the control mixture was applied in a high titre of circulating HB,Ag by a method two other wells on the same plate. The plates were
Immunity to Hepatitis B Virus Antigens
919
Table 1. Cellular and humoral immune response to HBV antigens in 25 patients with acute viral hepatitis type B ~
Type B hepatitis
Scand J Gastroenterol Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
Acute phase After clinical recovery?
No.
HB,Ag in serum
13
13
12
I
~~~~~~~
Anti-HB, in serum
HB,Ag in serum
Anti-HB, in serum
CMI * to HB,Ag
0
13
1
0
4
12
12
0
0
7
Anti-HB, in serum
*CMI: cell-mediated immunity to HB,Ag is defined as migration index t 0 . 9 3 . t6- 12 months after the onset of hepatitis. None of the patients had clinical or biochemical signs of liver disease at that time. incubated for 24 hours at 3 7 "C in a 2 per cent CO, in air saturated with water vapour. T h e migration area was studied under a projection microscope. T h e migration index (MI) was measured as R p 2- r2 MI R c 2- r2
In the present study, significant inhibition of leucocyte migration was defined as a migration index less than 0.93, corresponding t o the mean minus two standard deviation of 12 healthy controls.
where R p is the radius of the migration with antigen, Rc is the migration radius in the control, and r is the radius of the wells.
RESULTS
~
Table I gives a summary of the presence of HBV antigens and corresponding immune response in the
MIGRATION INDEX A
1.2
1
2
]:l1.1
3
1.0 1.0 -
L 5 I -
0.9-
oa 6
0.706 -
7 01 7
017
I
Anti -HB, Anti -HB,
017 717
ACUTE PHASE
I/ 7
117
I1 7 617
31 7 6/7
CONVALESCENCE
Fig. 1. Cellular immune response to HB,Ag during the course of acute viral hepatitis type B in 7 patients. Migration index < 0.93 in the leucocyte migration test is regarded as a significant response. (Patient 2 had transient HB,Ag and patient 3 developed persistent HB, antigenaemia).
J. Aldershvile, 0. Dietrichson. F. Hardt, J. 0. Nielsen & P. Skinhoj
Scand J Gastroenterol Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
920
acute phase and after clinical recovery in the patients with acute viral hepatitis. At the time of the follow-up examinations, one of the 12 patients had developed persistent HB, antigenaemia. None of the patients was positive for anti-HB, in the acute phase, but all 12 convalescent patients had anti-HB,. All patients had anti-HB, at the time of admission to hospital and remained positive during the observation period. A transient presence of HB,Ag was found in one patient only. A cellular immune response to HB,Ag (MI < 0.93) was found in four out of 13 patients during the acute phase of type B hepatitis and in seven out of the 12 patients in the convalescent phase. Seven of the patients were studied longitudinally for cellular immune response to HB,Ag (Fig. 1). During the acute phase, migration inhibition was found in two patients only. In contrast, at the time the HB,Ag was cleared in six of the patients, five had a cellular immune response to HB,Ag. One of the
non-responding patients developed persistent HB, antigenaemia, and the other patient .had been HB,Ag-positive for some weeks. After the HB,Ag was cleared, the MI returned to normal values for all five patients with previous inhibition. Only one out of 13 patients with chronic type B hepatitis showed a cellular immune response to HB,Ag (Fig. 2), which is similar to the finding of two patients with MI(0.93 in each of the two remaining groups of patients. DISCUSSION In all the patients with acute hepatitis B virus infection in this study, circulating antibody to HB,Ag was the first immunological response detected. This finding is in agreement with other reports (1, 12) where anti-HB, was detected early in the antigenic phase, at the time of elevated serum transaminases. In the present study, the development of anti-HB, was found within a range of from 0 to 40 weeks after
MIGRATION INDEX
1.4
1.2
1.0
.. -
?. -
a8
.. ..
1.
i'
.. ..
!.
06 0.4. 0.2
CONTROLS
ACUTE VIRAL HEPATITIS
TYPE 0 TYPE NONE CHRONIC ACTIVE LIVER DISEASE WITH AND WITHOUT CIRRHOSIS
MISCELLANEOUS LIVER DISEASE
Fig. 2. Cellular immune response in the leucocyte migration test in 12 controls and 56 patients with acute and chronic liver disease. Migration index < 0.93 indicates a significant response.
Scand J Gastroenterol Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
Immunity to Hepatitis B Virus Antigens
clearing of HB,Ag. During this period, anti-HB, may be the only serological evidence of a recent HBV infection (8). There is considerable evidence to suggest that the humoral response, whether mediated through a direct antigen-antibody interaction, activation of complement, or antibody-directed cellular cytotoxicity, is not the primary cause of the liver cell damage (7, 1 1 , 12). Therefore, cellular immunity would appear to be a more likely cause of the pathological changes in hepatitis type B (6, 1 1 , 15). This theory is in agreement with the finding of a less severe clinical course of hepatitis B in patients with congenital or acquired T-cell defect. In the present follow-up study, a transitory cellular immune response to HB,Ag was demonstrated at the time the HB,Ag was cleared. This is parallel with the finding of de Moura et al. (3) and Ibrahim et al. (9), who also used the leucocyte migration inhibition (agarose technique) as a measure of cellmediated immunity to HB,Ag.
In earlier reports by Dudley et al. ( 4 ) and Irwin et al. (lo), using the same methods, the maximum cellular immune response was obtained early in the disease. However, the in vitro studies of cellular immunity to HB,Ag are difficultto compare, owing to differences in the techniques and in the level of purification of the antigen. In our follow-up study, and also in that of Ibrahim et al. (9), the transitory nature of the T-cell response was a marked feature. This observation is parallel to numerous observations in various in vitro systems, thus indicating that the appearance of cellular immunity is a transient phenomenon in acute viral infections. This phenomenon may be a result of the occurrence of circulating antibodies (anti-HB,, anti-HB,) which either block the viral antigens or the infected cells or, in complexes with the HB,Ag, interfere with the function of the sensitized Tlymphocytes. It has been postulated that an altered immune response to the HBV antigens is a prerequisite for the development of chronic active liver disease both in patients with and without persistent HB,Ag ( 5 ) . The same group (13) found a cellular immune response to HB,Ag in 2 4 out of 39 HB,Ag negative patients with chronic active liver disease, and only
921
six of these patients had anti-HB,. This indicated that at least in some patients, the cellular immune response to HB,Ag was more persistent than the humoral immune response. However, in the present study, using highly purified HB,Ag, only one of the patients with type B chronic active hepatitis showed a migration inhibition. This finding was parallel with the findings in the groups of patients without serological evidence of previous HBV infection. Thus a specific cellular immunity to HB,Ag seems to be important for the termination of the acute HBV infection, but is not a prerequisite for the development of chronic active hepatitis type B or non-B. The only patient in the present study who did not show migration inhibition to HB,Ag during the actute phase of type B hepatitis subsequently developed persistent HB, antigenaemia indicative of later progression to chronic liver disease. AC KN 0WLEDGEMENTS This work was supported by grants from the Ebba Celinder Foundation, the P. Carl Petersen Foundation, the Danish State Medical Research Council (5 12-6504), the Medical Research Foundation for Copenhagen, Faeroe Islands and Greenland. Thanks are expressed to Anne Marie Moller for skilful technical assistance. REFERENCES 1. Bradley, D. W., Maynard, J. E., Berquist, K. R. & Krushak, D. H. Nature (Lond.) 1974,251,356-357 2. Clausen, J. E. Dan. Med. Bull. 1975, 22, 181-194 3. De Moura. M. C., Vernace, S. J. & Paronetto, F. Gastroenterology 1975, 69, 3 10-3 I 7 4. Dudley, F. J., Giustino, V. & Sherlock, S. Brit. Med. J. 1912, IV, 154-756 5. Eddleston, A. L. W. F. & Williams, R. Lancet 1974, 11, 1543-1545 6. Galbraith, R. M., Sheik, E., Portmann, N., Eddleston, A. L. W. F., Williams, R., Parsons,V., Bewick, M. & Ogg, C. S. Brit. Med. J. 1976, I , 1495-1497 7. Hoofnagle, J. H., Gerety, R. J. & Barker, L. F. Lancet 1973,II, 869-873 8 . Hoofnagle, J. H., Gerety, R. J. & Barker, L. F. Amer. J . Med. Sci. 1975, 270, 179-182 9. Ibrahim, A. B., Vyas, G. N. & Perkins, H. A. Infection and Immunity 1975, 1 1 , 137-141 10. Irwin, G. R., Hierholzer, W. J., Cimis R. & McCollum, R. W. J. Infect. Dis. 1974, 130, 580-587 1 1. Kohler, P. F., Tranbath, J., Mevnill, D. A., Singleton, J. W. & Dubois, R. S. Clin.Imrnunol. Immunopathol. 1974, 2; 465-47 1
922
J . Aldershvile, 0. Dietrichson, F. Hardt, J . 0. Nielsen & P. Skinhoj
12. Krugman, S., Hoofnagle, J. H.,Gerety, R. J.,Kaplan, P. M. & Gerlin, T. L. New Engl. J. Med. 1974, 290, 133 1-1335 13. Lee. W. M., Reed, W. D., Mitchell,C. G., Galbraith, R. M., Eddleston, A. L. W. F., Zuckerman, A. J. & Williams, R. Brit. Med. J . 1975, I , 705-708
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Received 23 May 1977 Accepted 10 June 1977
14. Nielsen, J . O., Dietrichson, 0. & Juhl, E. Lancet 1974. II, 913-915 IS. Sherlock, S . Amer. J. Med. 1970, 49, 693-706 16. Skinhoj, P. & Hansen, J. F. Scand. J. Lab. Clin. Invest. 1974, 33, 199-203
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J. ALDERSHVILE, 0. DIETRICHSON, F. HARDT, J. 0. NIELSEN & P. SKINHdJ 2nd Dept. of Medicine, Kommunehospitalet, Dept. of Medicine, Division of Hepatology, Hvidovre Hospital, Dept. of Infectious Diseases M, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
Aldershvile, J., Dietrichson, 0.. Hardt, F., Nielsen, J. 0. & Skinhoj, P. Humoral and cell-mediated immunity to hepatitis B virus antigens in acute and chronic liver disease. Scand. J. Gastroent. 1977, 12, 917-922 The humoral immune response to hepatitis B virus antigens and the cell-mediated immunity to hepatitis B surface antigen (HB,Ag) were investigated in 12 healthy persons and 5 6 patients with various liver diseases. In patients with acute viral hepatitis type B, anti-hepatitis B core antigen was present constantly in serum in all phases, and after clinical recovery simultaneouslywith anti-HB,. A transitory cellular immune response to HB,Ag was demonstrated at the time the antigen was cleared, while a patient with persisting HB, antigenaemia and another with transient hepatitis Be antigen showed no response during the course of the disease. Cellular immune response to HB,Ag was present only infrequently in patients with chronic liver disease type B and non-B. thus suggesting that a cellular immunity to HB,Ag is not a prerequisitefor the development of these conditions. Key-words: Acute hepatitis; cellular immunity; hepatitis antigen; liver disease; virus
J . Aldershvile, 2nd Depr. of Medicine, Kommunehospiralet, DK-1399 Copenhagen K , Denmark
Parallel with other virus infections, the elimination sient cell-mediated immunity to purified HB,Ag has of hepatitis B virus (HBV) is dependent on both been demonstrated during the acute phase of type B humoral and cellular immune mechanisms. The im- hepatitis and, in contrast, no migration inhibition mune response seems to be directed both against the was found in patients with chronic hepatitis (3, 9). virus antigens and against liver antigens exposed or However, in other studies, cell-mediated immunity made immunogenic by hepatitis B virus infection. A to HB,Ag positive serum or partially purified direct cytopathogenic effect of the HBV has not HB,Ag has been demonstrated in both acute and been demonstrated. The liver cell damage observed chronic hepatitis (4, 13). in acute and chronic hepatitis B infections may thus In the present study, the specific humoral immune be due to the immune reaction. Whereas uniform response to HBV antigens (HB,Ag, hepatitis B core results have been reported regarding the occurrence antigen (HB,Ag) and hepatitis Be antigen (HBeAg)) of anti-hepatitis B surface antigen (anti-HB,) and was correlated with the cellular immunity to HB,Ag anti-hepatitis B core antigen (anti-HB,), the results and the clinical course of the hepatitis B infection. of studies of the specific cell-mediated immune response to hepatitis B surface antigen (HB,Ag) are PATIENTS AND METHODS conflicting (3, 4, 9, 13). The total material consisted of 56 patients and 12 This might be due to methodological differences, healthy staff members. A liver biopsy was performed in all patients, and since the methods and antigen solutions used to estimate the cell-mediated immune response in vitro based on the histological and serological findings, are far from standardized. In some studies, a tran- the patients were classified as follows:
918
J . Aldershvile, 0 . Dietrichson, F. Hardr, J . 0. Nietsen & P. Skinhoj
Scand J Gastroenterol Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
Acute viral hepatitis type B
.
i
described previously (16). The final preparation contained 0.25 mg/ml of protein in phosphatebuffered saline, and no serum protein contamination could be detected by immunoelectrophoresis.
Twenty-five patients had acute viral hepatitis and circulating HB,Ag at the time of admission to hospital. Thirteen of the patients were examined during the acute phase, and seven of these were followed-up Agarose leucocyte migration inhibition test ( 2 ) by repeated examinations during the course of the The fresh agarose medium was a 2 per cent disease. Twelve patients were investigated after clinsolution of agarose (Litex, Glostrup, Denmark) ical and biochemical recovery. mixed at 47OC with horse serum (Statens Seruminstitut, Copenhagen, Denmark), sterilized water, Chronic active liver disease type B The group comprised 13 patients with chronic and a 10-fold concentration of tissue culture active hepatitis with or without cirrhosis. All these medium 199 (TC-medium 199 - l o x from Difco patients showed serological evidence of present or Laboratories, Detroit, Michigan, USA) to a final previous HBV infection, since seven patients had solution containing 1 per cent agarose and 10 per cent horse serum in single strength TC-medium HB,Ag, four had anti-HB,, and two anti-HB,. 199. Penicillin and streptomycin (TC-penicillinstreptomycin, Difco) was added to a final concentraChronic active liver disease, non-B tion of 66 units penicillin per ml and 66 pg streptoEight patients had chronic active hepatitis with or mycin per ml. Sodium bicarbonate solution (TC without cirrhosis. None of these patients had sodium bicarbonate solution 10 per cent, Difco) was HB,Ag, anti-HB, or anti-HB, in serum. added to adjust the pH in the agarose medium. Five Ten of the patients with chronic active hepatitis ml agarose medium was poured into each disposable with or without cirrhosis were being treated with plastic petri dish (millipore, Mass., USA). After corticosteroid and four with azathioprine at the time incubation in a 2 per cent COz, 98 per cent atmosof the investigation. pheric air saturated with water vapour, the pH was 7.2-7.4. After the gel had formed, eight holes Miscellaneous liver diseases (2.5mm in diameter) were cut in each agarose plate. This group comprised 10 patients, five with alco20 per cent dextran solution ( 5 per cent dextran holic cirrhosis, four with steatosis, and one with 250, Pharmacia, Sweden, in saline solution) was primary biliary cirrhosis. None of the patients had added to a heparinized venous blood sample. The serological evidence of present or previous HBV mixture was allowed to form sediment at 37 "C for infections. one hour. The supernatant leucocyte-rich plasma H B J g and anti-HB, was demonstrated by a solid was removed and centrifuged at 220g for 5 minutes. phase radioimmunoassay (Austria 11-125 and Au- The cell pellet was washed three times in Hanks sab, Abbott Diagnostic Division). balanced salt solution. The leucocytes were then Anti-HB, was determined by the indirect resuspended in Hepes-buffered TC-medium 199 immunofluorescence technique, using frozen sec- with 10 per cent horse serum added to give a final tions of liver tissue containing a high amount of concentration of 2.2 x lo8 Ieucocytes per ml. HG,Ag (kindly supplied by Professor Nowos- 2 x lo8 leucocytes were incubated with lop1 purilawski, National Institute of Hygiene, Warsaw, fied HB,Ag (protein concentration 0.05 mg/ml) for Poland), and an FITC-labelled monospecific rabbit half an hour. This protein concentration was the anti-human IgG. highest concentration with no inhibition of leucoH B d g and anti-hepatitis Be antigen (anti-HB,) cytes from healthy controls. As control, a similar number of cells was incuwas demonstrated by immunodiffusion, as debated with phosphate buffer without HB,Ag. Seven scribed previously (4). Preparation ofHBJg. HB,Ag was purified from pI of the leucocyte-antigen mixture was placed in the serum of an apparently healthy blood donor with three wells, and the control mixture was applied in a high titre of circulating HB,Ag by a method two other wells on the same plate. The plates were
Immunity to Hepatitis B Virus Antigens
919
Table 1. Cellular and humoral immune response to HBV antigens in 25 patients with acute viral hepatitis type B ~
Type B hepatitis
Scand J Gastroenterol Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
Acute phase After clinical recovery?
No.
HB,Ag in serum
13
13
12
I
~~~~~~~
Anti-HB, in serum
HB,Ag in serum
Anti-HB, in serum
CMI * to HB,Ag
0
13
1
0
4
12
12
0
0
7
Anti-HB, in serum
*CMI: cell-mediated immunity to HB,Ag is defined as migration index t 0 . 9 3 . t6- 12 months after the onset of hepatitis. None of the patients had clinical or biochemical signs of liver disease at that time. incubated for 24 hours at 3 7 "C in a 2 per cent CO, in air saturated with water vapour. T h e migration area was studied under a projection microscope. T h e migration index (MI) was measured as R p 2- r2 MI R c 2- r2
In the present study, significant inhibition of leucocyte migration was defined as a migration index less than 0.93, corresponding t o the mean minus two standard deviation of 12 healthy controls.
where R p is the radius of the migration with antigen, Rc is the migration radius in the control, and r is the radius of the wells.
RESULTS
~
Table I gives a summary of the presence of HBV antigens and corresponding immune response in the
MIGRATION INDEX A
1.2
1
2
]:l1.1
3
1.0 1.0 -
L 5 I -
0.9-
oa 6
0.706 -
7 01 7
017
I
Anti -HB, Anti -HB,
017 717
ACUTE PHASE
I/ 7
117
I1 7 617
31 7 6/7
CONVALESCENCE
Fig. 1. Cellular immune response to HB,Ag during the course of acute viral hepatitis type B in 7 patients. Migration index < 0.93 in the leucocyte migration test is regarded as a significant response. (Patient 2 had transient HB,Ag and patient 3 developed persistent HB, antigenaemia).
J. Aldershvile, 0. Dietrichson. F. Hardt, J. 0. Nielsen & P. Skinhoj
Scand J Gastroenterol Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
920
acute phase and after clinical recovery in the patients with acute viral hepatitis. At the time of the follow-up examinations, one of the 12 patients had developed persistent HB, antigenaemia. None of the patients was positive for anti-HB, in the acute phase, but all 12 convalescent patients had anti-HB,. All patients had anti-HB, at the time of admission to hospital and remained positive during the observation period. A transient presence of HB,Ag was found in one patient only. A cellular immune response to HB,Ag (MI < 0.93) was found in four out of 13 patients during the acute phase of type B hepatitis and in seven out of the 12 patients in the convalescent phase. Seven of the patients were studied longitudinally for cellular immune response to HB,Ag (Fig. 1). During the acute phase, migration inhibition was found in two patients only. In contrast, at the time the HB,Ag was cleared in six of the patients, five had a cellular immune response to HB,Ag. One of the
non-responding patients developed persistent HB, antigenaemia, and the other patient .had been HB,Ag-positive for some weeks. After the HB,Ag was cleared, the MI returned to normal values for all five patients with previous inhibition. Only one out of 13 patients with chronic type B hepatitis showed a cellular immune response to HB,Ag (Fig. 2), which is similar to the finding of two patients with MI(0.93 in each of the two remaining groups of patients. DISCUSSION In all the patients with acute hepatitis B virus infection in this study, circulating antibody to HB,Ag was the first immunological response detected. This finding is in agreement with other reports (1, 12) where anti-HB, was detected early in the antigenic phase, at the time of elevated serum transaminases. In the present study, the development of anti-HB, was found within a range of from 0 to 40 weeks after
MIGRATION INDEX
1.4
1.2
1.0
.. -
?. -
a8
.. ..
1.
i'
.. ..
!.
06 0.4. 0.2
CONTROLS
ACUTE VIRAL HEPATITIS
TYPE 0 TYPE NONE CHRONIC ACTIVE LIVER DISEASE WITH AND WITHOUT CIRRHOSIS
MISCELLANEOUS LIVER DISEASE
Fig. 2. Cellular immune response in the leucocyte migration test in 12 controls and 56 patients with acute and chronic liver disease. Migration index < 0.93 indicates a significant response.
Scand J Gastroenterol Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
Immunity to Hepatitis B Virus Antigens
clearing of HB,Ag. During this period, anti-HB, may be the only serological evidence of a recent HBV infection (8). There is considerable evidence to suggest that the humoral response, whether mediated through a direct antigen-antibody interaction, activation of complement, or antibody-directed cellular cytotoxicity, is not the primary cause of the liver cell damage (7, 1 1 , 12). Therefore, cellular immunity would appear to be a more likely cause of the pathological changes in hepatitis type B (6, 1 1 , 15). This theory is in agreement with the finding of a less severe clinical course of hepatitis B in patients with congenital or acquired T-cell defect. In the present follow-up study, a transitory cellular immune response to HB,Ag was demonstrated at the time the HB,Ag was cleared. This is parallel with the finding of de Moura et al. (3) and Ibrahim et al. (9), who also used the leucocyte migration inhibition (agarose technique) as a measure of cellmediated immunity to HB,Ag.
In earlier reports by Dudley et al. ( 4 ) and Irwin et al. (lo), using the same methods, the maximum cellular immune response was obtained early in the disease. However, the in vitro studies of cellular immunity to HB,Ag are difficultto compare, owing to differences in the techniques and in the level of purification of the antigen. In our follow-up study, and also in that of Ibrahim et al. (9), the transitory nature of the T-cell response was a marked feature. This observation is parallel to numerous observations in various in vitro systems, thus indicating that the appearance of cellular immunity is a transient phenomenon in acute viral infections. This phenomenon may be a result of the occurrence of circulating antibodies (anti-HB,, anti-HB,) which either block the viral antigens or the infected cells or, in complexes with the HB,Ag, interfere with the function of the sensitized Tlymphocytes. It has been postulated that an altered immune response to the HBV antigens is a prerequisite for the development of chronic active liver disease both in patients with and without persistent HB,Ag ( 5 ) . The same group (13) found a cellular immune response to HB,Ag in 2 4 out of 39 HB,Ag negative patients with chronic active liver disease, and only
921
six of these patients had anti-HB,. This indicated that at least in some patients, the cellular immune response to HB,Ag was more persistent than the humoral immune response. However, in the present study, using highly purified HB,Ag, only one of the patients with type B chronic active hepatitis showed a migration inhibition. This finding was parallel with the findings in the groups of patients without serological evidence of previous HBV infection. Thus a specific cellular immunity to HB,Ag seems to be important for the termination of the acute HBV infection, but is not a prerequisite for the development of chronic active hepatitis type B or non-B. The only patient in the present study who did not show migration inhibition to HB,Ag during the actute phase of type B hepatitis subsequently developed persistent HB, antigenaemia indicative of later progression to chronic liver disease. AC KN 0WLEDGEMENTS This work was supported by grants from the Ebba Celinder Foundation, the P. Carl Petersen Foundation, the Danish State Medical Research Council (5 12-6504), the Medical Research Foundation for Copenhagen, Faeroe Islands and Greenland. Thanks are expressed to Anne Marie Moller for skilful technical assistance. REFERENCES 1. Bradley, D. W., Maynard, J. E., Berquist, K. R. & Krushak, D. H. Nature (Lond.) 1974,251,356-357 2. Clausen, J. E. Dan. Med. Bull. 1975, 22, 181-194 3. De Moura. M. C., Vernace, S. J. & Paronetto, F. Gastroenterology 1975, 69, 3 10-3 I 7 4. Dudley, F. J., Giustino, V. & Sherlock, S. Brit. Med. J. 1912, IV, 154-756 5. Eddleston, A. L. W. F. & Williams, R. Lancet 1974, 11, 1543-1545 6. Galbraith, R. M., Sheik, E., Portmann, N., Eddleston, A. L. W. F., Williams, R., Parsons,V., Bewick, M. & Ogg, C. S. Brit. Med. J. 1976, I , 1495-1497 7. Hoofnagle, J. H., Gerety, R. J. & Barker, L. F. Lancet 1973,II, 869-873 8 . Hoofnagle, J. H., Gerety, R. J. & Barker, L. F. Amer. J . Med. Sci. 1975, 270, 179-182 9. Ibrahim, A. B., Vyas, G. N. & Perkins, H. A. Infection and Immunity 1975, 1 1 , 137-141 10. Irwin, G. R., Hierholzer, W. J., Cimis R. & McCollum, R. W. J. Infect. Dis. 1974, 130, 580-587 1 1. Kohler, P. F., Tranbath, J., Mevnill, D. A., Singleton, J. W. & Dubois, R. S. Clin.Imrnunol. Immunopathol. 1974, 2; 465-47 1
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J . Aldershvile, 0. Dietrichson, F. Hardt, J . 0. Nielsen & P. Skinhoj
12. Krugman, S., Hoofnagle, J. H.,Gerety, R. J.,Kaplan, P. M. & Gerlin, T. L. New Engl. J. Med. 1974, 290, 133 1-1335 13. Lee. W. M., Reed, W. D., Mitchell,C. G., Galbraith, R. M., Eddleston, A. L. W. F., Zuckerman, A. J. & Williams, R. Brit. Med. J . 1975, I , 705-708
Scand J Gastroenterol Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
Received 23 May 1977 Accepted 10 June 1977
14. Nielsen, J . O., Dietrichson, 0. & Juhl, E. Lancet 1974. II, 913-915 IS. Sherlock, S . Amer. J. Med. 1970, 49, 693-706 16. Skinhoj, P. & Hansen, J. F. Scand. J. Lab. Clin. Invest. 1974, 33, 199-203
