- Email: [email protected]
CELL-MEDIATED IMMUNITY TO A HUMAN LIVER-SPECIFIC ANTIGEN IN PATIENTS WITH ACTIVE CHRONIC HEPATITIS AND PRIMARY BILIARY CIRRHOSIS
296
CELL-MEDIATED IMMUNITY TO A HUMAN LIVER-SPECIFIC ANTIGEN IN PATIENTS WITH ACTIVE CHRONIC HEPATITIS AND PRIMARY BILIARY CIRRHOSIS
JOANNA MILLER CHRISTINE G. MITCHELL A. L. W. F. EDDLESTON Liver Unit, King’s
M. G. M. SMITH W. D. REED ROGER WILLIAMS
College Hospital and Medical School, London S.E.5
An organ-specific lipoprotein has been Summary isolated from normal human liver and
cell-mediated immune responses
to
it have been
assayed in patients with chronic liver diseases using the leucocyte-migration test. Significant inhibition of migration was almost completely restricted to those patients with postulated autoimmune diseases of the liver, being found in 69% of patients with active chronic hepatitis and in 50% of those with primary biliary cirrhosis. The patients with active chronic hepatitis and normal migration indices had shown a satisfactory response to immunosuppressive therapy, with normal liver-function tests at the time of assay.
isolation of organ-specific antigens has proved difficult the use of such purified materials in the leucocytemigration test would represent a considerable advance. Meyer zum Buschenfelde has described the isolation of a human liver-specific lipoprotein,3and has induced histological lesions in rabbits similar to chronic aggressive hepatitis by repeated injections of this antigen.4 We have further purified this lipoprotein and used it as an antigen in the leucocyte-migration test, and we describe our findings in 23 healthy volunteers and 40 patients with various liver diseases. Patients and Methods Of the 40 patients, 12 had primary biliary cirrhosis, 16 active chronic hepatitis, 6 hxmochromatosis, and 6 alcoholic cirrhosis. The diagnoses were based on current clinical and biochemical criteria and in all cases were confirmed by histological examination of liver-biopsy specimens. The patients with active chronic hepatitis were participating in a double-blind controlled trial of
azathioprine or prednisone. Preparation of liver-specific protein-The supernatant of a homogenate of fresh human liver, freed of particulate matter by centrifugation at 150,000 g, was fractionated by sequential gel-filtration onSephadex’ G100 and G200.3 The
Introduction
BECAUSE are serum-autoantibodies commonly in active chronic and present hepatitis primary biliary cirrhosis, these diseases have been judged to be autoimmune, although antibodies directed specifically against the affected organ have rarely been detected. Smith et allusing the leucocyte-migration test, demonstrated cell-mediated immune responses to human fetal and adult liver homogenates in patients with active chronic hepatitis and primary biliary cirrhosis, and they suggested that these cell-mediated responses might be important in pathogenesis. The liver homogenates used by Smith et all2probably contained many antigens, only a few of which were involved in the immunological reaction. With suboptimal concentrations of antigen in the leucocytemigration test, stimulation rather than inhibition of migration may be observed in sensitised subjects, which makes interpretation difficult. Above a certain antigen concentration, non-specific inhibition of migration occurs, and with organ homogenates this toxic effect does not allow the optimum concentration of specific antigens to be achieved. Although the
lipoprotein, contaminated with traces of another liverspecific but cytoplasmic protein, is present in the first peak eluted from sephadex G200. Further separation was obtained by gel-filtration on’Sepharose’ 6B. The lipoprotein is present in the first peak eluted from the gel, the cytoplasmic protein being retarded. After each preparation the lipoprotein was assayed for contamination with other proteins by electrophoresis on 6% polyacrilamide gel using Poulik’s discontinuous buffer system, and for liver specificity by immunoelectrophoresis on agar-gel against specific antisera, supplied by Professor Meyer zum Buschenfelde. As the lipoprotein is only stable in solution for a few days, it was freshly prepared each week.
PROFESSOR YASE: REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9.
10.
Charcot, J-M., Joffroy, A. Arch. Physiol. norm. path. 1869, 2, 354 629, 744. Koerner, D. Ann. intern. Med. 1952, 37, 1204. Kurland, L. T., Mulder, D. W. Neurology, Minneap. 1954, 4, 355, 438 Kimura, K., et al. Proc. Japan Acad. 1961, 37, 417. Shiraki, H., Yase, Y. Handbook of Clinical Neurology (in the press) Handa, Y., Yase, Y. Igaku no Ayumi, 1970, 73, 478. Yase, Y., Matsumoto, N., Yoshimasu, F., Handa, Y. Proceeding 3rd Asian and Oceanian Congress of Neurology, Bombay, 197 (in the press). Yoshimasu, F. J. Wakayama med. Ass. 1969, 20, 31. Yase, Y., Shinjo, Y., Yoshimasu, F., Kumamoto, T. Proceeding 2nd International Congress on Muscle Diseases, Perth, Australis 1971 (in the press). Forbes, R. M., Cooper, A. R., Mitchell, H. H. J. biol. Chem. 1953
203, 359. 11. Forbes, R. M., Mitchell, H. H., Cooper, A. R. ibid. 1956, 233, 969. 12. Swanback, T. R. Plant Physiol. 1939, 14, 423. 13. Dastur, D. K., Manghani, D. K., Rachavendren, K. V. J. clin Invest. 1971, 50, 9.
Results of
Purified human
leucocyte-migration
test.
liver-specific lipoprotein
was
used
as
the
antigen in patients with active chronic hepatitis, primary biliary cirrhosis, hxmochromatosis, and alcoholic cirrhosis.
297
Leucocyte-migration test-The technique is described in detail elsewhere.5 Peripheral-blood leucocytes are allowed migrate out of capillary tubes for twenty hours at 37 °C into tissue-culture medium, and the area of migration in the presence of the antigen compared to that obtained in control medium. Preliminary experiments showed that the highest concentration of lipoprotein which could be used in the test system without obtaining non-specific inhibition of migration was 50 g. per ml., and this concentration was therefore used throughout the study. Inhibition of migration was measured without knowledge of the diagnosis or the results of liver-function tests. to
Results
The mean migration index in the normal subjects 0-90 (±0-075 s.D., normal range 0-75 — 1 -05 [mean ±2 S.D.]). Values in the patients with alcoholic cirrhosis or hxmochromatosis usually fell within the normal range, significant inhibition of migration being observed in 1 case and stimulation in 2 of the 12 patients in these two groups (see accompanying figure). In contrast, inhibition of migration was observed in 11(69%) of the 16 patients with active chronic hepatitis and stimulation of migration in 1. The 4 patients with normal migration indices had demonstrated a satiswas
factory response to prednisone
or
azathioprine therapy
and had normal liver-function tests at the time of assay. The mean serum-aspartate-aminotransferase level for these patients (41’3;5’5 m.U. per ml.) was significantly lower than that for the patients with abnormal migration indices (75-4:31-1 m.U. per ml., p < 0-01), although there was no difference in the mean plasma-bilirubin levels. If the 12 patients with abnormal migration indices alone are considered, no correlation could be detected between the extent of inhibition of migration and changes in liver function reflected in the plasma bilirubin and aminotransferase levels. There was inhibition of leucocyte migration in 6 (50%) of the 12 patients with primary biliary cirrhosis. All the patients had abnormal liver-function tests and, on histological criteria, the disease had progressed to stage three in 2 of the cases and to stage four in the remainder (stages according to Scheuer 6). There was no correlation between the extent of inhibition of migration and the serum bilirubin, aminotransferase, or alkaline-phosphatase levels. Discussion
We used
immunoelectrophoresis to demonstrate the a liver-specific lipoprotein in homogenates of liver similar to those used by Smith et al .2 In their study, 53% of 58 patients with active chronic hepatitis had abnormal migration indices, inhibition of migration being found in 40 % and stimulation in 13%. Although Soborgregards stimulation of migration as an in-vitro correlate of weak in-vivo sensitisation, this is not widely accepted and transition values are difficult to interpret. We found that inhibition of migration was more commonly recorded (69%) and stimulation was rarely encountered, although the concentration of antigen used was, in terms of protein content, eight times less than that of the homogenate. This suggests that the lipoprotein is an important antigenic constituent of liver homogenates, and, as it is organ specific, our results provide strong support for the postulated presence of
role of autoimmune processes in the pathogenesis of active chronic hepatitis. Further evidence for this hypothesis is the production in rabbits of chronic hepatitis by immunisation with human liver-specific
proteins.4 Treatment with prednisone and azathioprine has no consistent effect on the levels of serum-autoantibodies in patients with active chronic hepatitis, but our patients with active chronic hepatitis who had normal migration indices had responded satisfactorily to such therapy. This agrees with the view that cell-mediated immune responses, rather than humoral antibodies, may be more directly involved in the production of liver damage. However, an exact correlation between the degree of migration inhibition and the liverfunction tests would be unexpected as we have demonstrated, in a study of Mantoux positive and
negative subjects, that, although the leucocyte-migration test was capable of accurately detecting sensitisation, there was no correlation between the extent of inhibition with purified protein derivative of tuberculin and the intensity of an intradermal skin-test using the same antigen.5 Why some patients with primary biliary cirrhosis demonstrated inhibition of migration and others gave normal results is uncertain. It was clearly not related to disease activity as reflected by the serum bilirubin In primary biliary and aminotransferase levels. lesion is damage to the cirrhosis, early histological interlobular bileducts which are surrounded by mononuclear-cell infiltrates. Piecemeal necrosis of the hepatocytes, characteristic of active chronic hepatitis, is not usually seen until the later stages. One possibility is that bileduct antigens may be more important immunologically in the early stages of primary biliary cirrhosis, and that sensitisation to hepatocellular antigens does not occur until later in the disease. All our patients had stage three or four disease with histological evidence of hepatocellular necrosis and might, therefore, have been expected to be sensitised to the lipoprotein antigen. Although only half the patients demonstrated significant inhibition of migration, the migration indices in 4 of the other 6 cases were very close to the lower limit of the normal range. When purified antigens can be obtained from bileduct epithelial cells, it should be possible to define the immunological abnormalities more completely and to study those reported cases of active chronic hepatitis with biliary features 9 for sensitisation to bileduct antigens, as well as to those on the surface membrane of the hepatocyte. Miss A. Kemp gave excellent technical assistance and we are grateful to the Wellcome Trust for continued support. Requests for reprints should be addressed to R. W. REFERENCES
Doniach, D., Walker, J. G. Lancet, 1969, i, 813. Smith, M. G. M., Golding, P. L., Eddleston, A. L. W. F., Mitchell, C. G., Kemp, A., Williams, R. Br. med. J. 1972, i, 527. 3. Meyer zum Buschenfelde, K. H., Miescher, P. A. Clin. exp. Immun. 1972, 10, 89. 4. Meyer zum Buschenfelde, K. H. in Immunology of the Liver (edited by M. Smith and R. Williams). London, 1972. 5. Mitchell, C. G., Smith, M. G. M., Golding, P. L., Eddleston, A. L. W. F., Williams, R. Clin. exp. Immun. (in the press). 6. Scheuer, P. J. Liver Biopsy Interpretation. London, 1968. 7. Søborg, M. Acta med. scand. 1968, 184, 135. 8. Doniach, D., Murray-Lyon, I. M. Unpublished. 9. Datta, D. A., Sherlock, S., Scheuer, P. J. Gut, 1963, 4, 223.
1. 2.
CELL-MEDIATED IMMUNITY TO A HUMAN LIVER-SPECIFIC ANTIGEN IN PATIENTS WITH ACTIVE CHRONIC HEPATITIS AND PRIMARY BILIARY CIRRHOSIS
JOANNA MILLER CHRISTINE G. MITCHELL A. L. W. F. EDDLESTON Liver Unit, King’s
M. G. M. SMITH W. D. REED ROGER WILLIAMS
College Hospital and Medical School, London S.E.5
An organ-specific lipoprotein has been Summary isolated from normal human liver and
cell-mediated immune responses
to
it have been
assayed in patients with chronic liver diseases using the leucocyte-migration test. Significant inhibition of migration was almost completely restricted to those patients with postulated autoimmune diseases of the liver, being found in 69% of patients with active chronic hepatitis and in 50% of those with primary biliary cirrhosis. The patients with active chronic hepatitis and normal migration indices had shown a satisfactory response to immunosuppressive therapy, with normal liver-function tests at the time of assay.
isolation of organ-specific antigens has proved difficult the use of such purified materials in the leucocytemigration test would represent a considerable advance. Meyer zum Buschenfelde has described the isolation of a human liver-specific lipoprotein,3and has induced histological lesions in rabbits similar to chronic aggressive hepatitis by repeated injections of this antigen.4 We have further purified this lipoprotein and used it as an antigen in the leucocyte-migration test, and we describe our findings in 23 healthy volunteers and 40 patients with various liver diseases. Patients and Methods Of the 40 patients, 12 had primary biliary cirrhosis, 16 active chronic hepatitis, 6 hxmochromatosis, and 6 alcoholic cirrhosis. The diagnoses were based on current clinical and biochemical criteria and in all cases were confirmed by histological examination of liver-biopsy specimens. The patients with active chronic hepatitis were participating in a double-blind controlled trial of
azathioprine or prednisone. Preparation of liver-specific protein-The supernatant of a homogenate of fresh human liver, freed of particulate matter by centrifugation at 150,000 g, was fractionated by sequential gel-filtration onSephadex’ G100 and G200.3 The
Introduction
BECAUSE are serum-autoantibodies commonly in active chronic and present hepatitis primary biliary cirrhosis, these diseases have been judged to be autoimmune, although antibodies directed specifically against the affected organ have rarely been detected. Smith et allusing the leucocyte-migration test, demonstrated cell-mediated immune responses to human fetal and adult liver homogenates in patients with active chronic hepatitis and primary biliary cirrhosis, and they suggested that these cell-mediated responses might be important in pathogenesis. The liver homogenates used by Smith et all2probably contained many antigens, only a few of which were involved in the immunological reaction. With suboptimal concentrations of antigen in the leucocytemigration test, stimulation rather than inhibition of migration may be observed in sensitised subjects, which makes interpretation difficult. Above a certain antigen concentration, non-specific inhibition of migration occurs, and with organ homogenates this toxic effect does not allow the optimum concentration of specific antigens to be achieved. Although the
lipoprotein, contaminated with traces of another liverspecific but cytoplasmic protein, is present in the first peak eluted from sephadex G200. Further separation was obtained by gel-filtration on’Sepharose’ 6B. The lipoprotein is present in the first peak eluted from the gel, the cytoplasmic protein being retarded. After each preparation the lipoprotein was assayed for contamination with other proteins by electrophoresis on 6% polyacrilamide gel using Poulik’s discontinuous buffer system, and for liver specificity by immunoelectrophoresis on agar-gel against specific antisera, supplied by Professor Meyer zum Buschenfelde. As the lipoprotein is only stable in solution for a few days, it was freshly prepared each week.
PROFESSOR YASE: REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9.
10.
Charcot, J-M., Joffroy, A. Arch. Physiol. norm. path. 1869, 2, 354 629, 744. Koerner, D. Ann. intern. Med. 1952, 37, 1204. Kurland, L. T., Mulder, D. W. Neurology, Minneap. 1954, 4, 355, 438 Kimura, K., et al. Proc. Japan Acad. 1961, 37, 417. Shiraki, H., Yase, Y. Handbook of Clinical Neurology (in the press) Handa, Y., Yase, Y. Igaku no Ayumi, 1970, 73, 478. Yase, Y., Matsumoto, N., Yoshimasu, F., Handa, Y. Proceeding 3rd Asian and Oceanian Congress of Neurology, Bombay, 197 (in the press). Yoshimasu, F. J. Wakayama med. Ass. 1969, 20, 31. Yase, Y., Shinjo, Y., Yoshimasu, F., Kumamoto, T. Proceeding 2nd International Congress on Muscle Diseases, Perth, Australis 1971 (in the press). Forbes, R. M., Cooper, A. R., Mitchell, H. H. J. biol. Chem. 1953
203, 359. 11. Forbes, R. M., Mitchell, H. H., Cooper, A. R. ibid. 1956, 233, 969. 12. Swanback, T. R. Plant Physiol. 1939, 14, 423. 13. Dastur, D. K., Manghani, D. K., Rachavendren, K. V. J. clin Invest. 1971, 50, 9.
Results of
Purified human
leucocyte-migration
test.
liver-specific lipoprotein
was
used
as
the
antigen in patients with active chronic hepatitis, primary biliary cirrhosis, hxmochromatosis, and alcoholic cirrhosis.
297
Leucocyte-migration test-The technique is described in detail elsewhere.5 Peripheral-blood leucocytes are allowed migrate out of capillary tubes for twenty hours at 37 °C into tissue-culture medium, and the area of migration in the presence of the antigen compared to that obtained in control medium. Preliminary experiments showed that the highest concentration of lipoprotein which could be used in the test system without obtaining non-specific inhibition of migration was 50 g. per ml., and this concentration was therefore used throughout the study. Inhibition of migration was measured without knowledge of the diagnosis or the results of liver-function tests. to
Results
The mean migration index in the normal subjects 0-90 (±0-075 s.D., normal range 0-75 — 1 -05 [mean ±2 S.D.]). Values in the patients with alcoholic cirrhosis or hxmochromatosis usually fell within the normal range, significant inhibition of migration being observed in 1 case and stimulation in 2 of the 12 patients in these two groups (see accompanying figure). In contrast, inhibition of migration was observed in 11(69%) of the 16 patients with active chronic hepatitis and stimulation of migration in 1. The 4 patients with normal migration indices had demonstrated a satiswas
factory response to prednisone
or
azathioprine therapy
and had normal liver-function tests at the time of assay. The mean serum-aspartate-aminotransferase level for these patients (41’3;5’5 m.U. per ml.) was significantly lower than that for the patients with abnormal migration indices (75-4:31-1 m.U. per ml., p < 0-01), although there was no difference in the mean plasma-bilirubin levels. If the 12 patients with abnormal migration indices alone are considered, no correlation could be detected between the extent of inhibition of migration and changes in liver function reflected in the plasma bilirubin and aminotransferase levels. There was inhibition of leucocyte migration in 6 (50%) of the 12 patients with primary biliary cirrhosis. All the patients had abnormal liver-function tests and, on histological criteria, the disease had progressed to stage three in 2 of the cases and to stage four in the remainder (stages according to Scheuer 6). There was no correlation between the extent of inhibition of migration and the serum bilirubin, aminotransferase, or alkaline-phosphatase levels. Discussion
We used
immunoelectrophoresis to demonstrate the a liver-specific lipoprotein in homogenates of liver similar to those used by Smith et al .2 In their study, 53% of 58 patients with active chronic hepatitis had abnormal migration indices, inhibition of migration being found in 40 % and stimulation in 13%. Although Soborgregards stimulation of migration as an in-vitro correlate of weak in-vivo sensitisation, this is not widely accepted and transition values are difficult to interpret. We found that inhibition of migration was more commonly recorded (69%) and stimulation was rarely encountered, although the concentration of antigen used was, in terms of protein content, eight times less than that of the homogenate. This suggests that the lipoprotein is an important antigenic constituent of liver homogenates, and, as it is organ specific, our results provide strong support for the postulated presence of
role of autoimmune processes in the pathogenesis of active chronic hepatitis. Further evidence for this hypothesis is the production in rabbits of chronic hepatitis by immunisation with human liver-specific
proteins.4 Treatment with prednisone and azathioprine has no consistent effect on the levels of serum-autoantibodies in patients with active chronic hepatitis, but our patients with active chronic hepatitis who had normal migration indices had responded satisfactorily to such therapy. This agrees with the view that cell-mediated immune responses, rather than humoral antibodies, may be more directly involved in the production of liver damage. However, an exact correlation between the degree of migration inhibition and the liverfunction tests would be unexpected as we have demonstrated, in a study of Mantoux positive and
negative subjects, that, although the leucocyte-migration test was capable of accurately detecting sensitisation, there was no correlation between the extent of inhibition with purified protein derivative of tuberculin and the intensity of an intradermal skin-test using the same antigen.5 Why some patients with primary biliary cirrhosis demonstrated inhibition of migration and others gave normal results is uncertain. It was clearly not related to disease activity as reflected by the serum bilirubin In primary biliary and aminotransferase levels. lesion is damage to the cirrhosis, early histological interlobular bileducts which are surrounded by mononuclear-cell infiltrates. Piecemeal necrosis of the hepatocytes, characteristic of active chronic hepatitis, is not usually seen until the later stages. One possibility is that bileduct antigens may be more important immunologically in the early stages of primary biliary cirrhosis, and that sensitisation to hepatocellular antigens does not occur until later in the disease. All our patients had stage three or four disease with histological evidence of hepatocellular necrosis and might, therefore, have been expected to be sensitised to the lipoprotein antigen. Although only half the patients demonstrated significant inhibition of migration, the migration indices in 4 of the other 6 cases were very close to the lower limit of the normal range. When purified antigens can be obtained from bileduct epithelial cells, it should be possible to define the immunological abnormalities more completely and to study those reported cases of active chronic hepatitis with biliary features 9 for sensitisation to bileduct antigens, as well as to those on the surface membrane of the hepatocyte. Miss A. Kemp gave excellent technical assistance and we are grateful to the Wellcome Trust for continued support. Requests for reprints should be addressed to R. W. REFERENCES
Doniach, D., Walker, J. G. Lancet, 1969, i, 813. Smith, M. G. M., Golding, P. L., Eddleston, A. L. W. F., Mitchell, C. G., Kemp, A., Williams, R. Br. med. J. 1972, i, 527. 3. Meyer zum Buschenfelde, K. H., Miescher, P. A. Clin. exp. Immun. 1972, 10, 89. 4. Meyer zum Buschenfelde, K. H. in Immunology of the Liver (edited by M. Smith and R. Williams). London, 1972. 5. Mitchell, C. G., Smith, M. G. M., Golding, P. L., Eddleston, A. L. W. F., Williams, R. Clin. exp. Immun. (in the press). 6. Scheuer, P. J. Liver Biopsy Interpretation. London, 1968. 7. Søborg, M. Acta med. scand. 1968, 184, 135. 8. Doniach, D., Murray-Lyon, I. M. Unpublished. 9. Datta, D. A., Sherlock, S., Scheuer, P. J. Gut, 1963, 4, 223.
1. 2.