Journal of A utoimmunity (1992) 5 ( Supplement A), 293-300
I m m u n o i n t e r v e n t i o n in P r i m a r y B i l i a r y Cirrhosis: an O v e r v i e w
Gary P. Bray and Roger Williams
Institute of Liver Studies, King's College Hospital School of Medicine and Dentistry, Denmark Hill, London SE5 8RX, UK Since a u t o i m m u n e processes are p r o b a b l y involved in the early stages of p r i m a r y biliary cirrhosis (PBC), i m m u n o m o d u l a t o r y drugs have been investigated with the a i m of prolonging survival, delaying t r a n s p l a n t a t i o n , slowing histological progression and relieving s y m p t o m s . Corticosteroids, azathioprine, c h l o r a m b u c i l and, m o r e recently, cyclosporin A a n d m e t h o trexate have all be subjected to clinical investigation. In the latest of these, a E u r o p e a n m u l t i c e n t r e trial, cyclosporin A has been shown to delay death or t r a n s p l a n t a t i o n with a reduction in liver related deaths a n d slowing of the rise of s e r u m bilirubin. The incidence of nephrotoxicity and hypertension are low at the doses used.
Introduction Primary biliary cirrhosis (PBC) is a cholestatic liver disease which predominantly affects middle aged females. T h e diagnosis is made after presentation with pruritus or jaundice or after the incidental finding of abnormal liver function tests. Less c o m m o n l y it m a y present with variceal bleeding. Considerable geographical variation is reported and in one recent study, the prevalence was as high as 151 per million [1]. Since the first descriptions of P B C [2, 3], the discovery of effective treatment has been a major goal of research into the condition although the exact aetiology of the disease remains uncertain.
Autoimmunity and PBC T h e r e is considerable evidence that a u t o i m m u n e processes are involved in the aetiology of PBC. Initiation of a u t o i m m u n i t y may result in granulomata and inflammation around small intrahepatic bile ducts, leading to fibrosis, increasing cholestasis and liver failure. 293 0896-8411/92/0A0293 + 08 $03.00/0
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Non-organ specific antibodies are common with anti-mitochondrial antibodies ( A M A ) being found in over 95% of patients with P B C T h e four different subtypes of these antibodies have been identified by Berg and his colleagues as M2, M4, M8 and M9 [4]. A n t i - M 2 antibodies are those most frequently found and recognize antigens on the inner mitochondrial m e m b r a n e which are now known to be enzymes involved in intermediary metabolism. T h e s e are the E2 components of the pyruvate dehydrogenase (PDC), 2-oxoglutarate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes as well as protein X (part of P D C - E 2 ) , and the a and 13 subunits of P D C - E 1 [5]. Although A M A could have a direct role in pathogenesis, the occurrence of AMA-negative PBC argues against this as does the failure to produce the disease by immunization of animals with a recombinant form of the E2 component of P D C [6]. H o w an humoral antibody response to intracellular proteins can result in T-cell mediated damage localized to the biliary tree [5] is difficult to understand as it is unknown whether mitochondrial antigens can be expressed by the cell m e m b r a n e of hepatocytes [7, 8].
Lymphocytic infiltrates are found around damaged bile ducts and lymphocytes from patients with P B C may be able to transmit the disease to experimental animals T h e lymphocytic infiltration around bile ducts consists of predominantly C D 3 +, C D 4 +, C D 8 - T cells expressing the T-cell 13chain [9]. Injection of peripheral blood lymphocytes from patients with P B C into female mice with severe combined i m m u nodeficiency disease has been found to result in A M A appearing in the serum with a portal infiltrate with bile duct damage in the liver of the mice [10]. Although the histological changes induced in these mice were not identical to those found in h u m a n PBC, this work and the characteristic lymphocytic infiltrate found in the portal tracts of patients with the disease suggest that T cells have an important role in the pathogenesis of the disorder.
P B C is associated with H L A - D R w 8 and C4B2 E11, 12] Association of a particular H L A allele with a disease suggests that this may complex with an antigen in a way that may lead to an a u t o i m m u n e response [13]. In this way, H L A - D R w 8 and C4 B2 may confer susceptibility to the disease.
M H C Class II antigens are expressed by bile duct epithelium In PBC, bile duct epithelial cells have been shown to express H L A - D R , - D P and - D Q M H C class I I antigens [14, 15]. M H C class I I antigens are known to control the induction of C D 3 +, C D 4 + T-cell mediated i m m u n e responses. Disease progression and prognosis With progression of the disease and deepening cholestasis, liver decompensation, as evidenced by jaundice, ascites, coagulopathy, severe infection, renal failure and encephalopathy, may occur. Several prognostic models have been developed in order
Immunointervention in primary biliary cirrhosis 295 Table 1. Comparison of Cox multiple regression
models in PBC, showing independent variables identified in five studies Prognostic models European Serum bilirubin Serum albumin Age Prothrombin time Oedema Therapy Fibrosis/cirrhosis Cholestasis
Yes Yes Yes
Mayo Yes
Yes Yes Yes Yes
Yes Yes Yes
to help to predict outcome and life expectancy in individual patients. T h e y have also been used in assessing the value of treatments such as liver transplantation and the optimal timing for this procedure [16-21] (Table 1). T h e European model, which was developed after Cox multiple regression analysis of the results of the multicentre trial of azathioprine [17], and the M a y o clinic model [18], which does not involve histology, are the two most frequently used. In most cases, the progression of PBC is relatively slow with a reported median survival of 11.9 years from the onset of s y m p t o m s [16] but it is now recognized that the median survival of patients who are asymptomatic at the time of diagnosis is also worse than for a normal age matched population. T h e M a y o clinic found asymptomatic patients to have a four-fold increase in mortality compared to an age, race and sex matched population in the United States, with 89 % of these patients developing s y m p t o m s during the study period [22].
Rationale for i m m u n o m o d u l a t o r y therapy and design o f clinical trials Despite the evidence in favour of an a u t o i m m u n e aetiology for PBC, the response to i m m u n o s u p p r e s s i o n is, as we shall discuss later, not as p r o m p t as in the a u t o i m m u n e variety of chronic active hepatitis. T h e i m m u n o m o d u l a t o r y drugs which have been used in P B C are listed in T a b l e 2. T h e r e are several difficulties which are encountered in the design of any clinical trial and, in particular, those conducted in PBC [23]. In order that a trial has sufficient statistical power to detect an effect exerted by the drug on the course of the disease, there m u s t be an adequate n u m b e r of patients recruited and no unplanned interim analyses should be allowed. I f an interim analysis is p e r f o r m e d then the clinical investigators and patients should not be unblinded and, unless significant toxicity is encountered, the results should not determine whether the trial is continued. T h e length of trial should be decided prior to starting and the trial should be double blind and placebo controlled with patients both randomized and their outcome analysed on an 'intent to treat' basis. Small, non-randomized, unblinded and
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T a b l e 2. Immunomodulatory
drugs which have been used in p r i m a r y biliary cirrhosis and major trials
Drug used Prednisolone Azathioprine Chlorambucil Cyclosporin
Reference Mitchison et al. 1989 [25] Heathcote et al. 1976 [28] Christensen et al. 1985 [17] Hoofnagle et al. 1986 [29] Wiesner et al. 1988 [23] Lombard et al. 1990 [34]
Methotrexate Penicillamine and colchicine may also modulate the immune system but have been principally used for their anticupretic and antifibrotic activity, respectively, uncontrolled studies cannot give certain answers as to whether a drug affects survival or progression of histology and are not included in this review. In view of the long and variable time course of the illness, follow-up of patients needs to be protracted in order to show a beneficial effect on survival (or the need for liver transplantation) of any intervention. As the clinical course of PBC prior to development of s y m p t o m s is unpredictable, analysis of trials recruiting both symptomatic and asymptomatic patients may need to be stratified. In addition to survival, other endpoints of treatment can be used, e.g. amelioration of s y m p t o m s such as pruritus, reduction in histological progression or an i m p r o v e m e n t in biochemical parameters. Corticosteroids
Corticosteroids inhibit cell division and cytokine production by all elements of the i m m u n e system and prednisolone specifically has been shown to be able to correct defective regulation of immunoglobulin production by lymphocytes in vitro [24]. Surprisingly, the first and only controlled trial of corticosteroids in PBC, from Newcastle, was not published until 1990 [25] despite their intermittent use in this disease for over 35 years [26]. In the Newcastle 1-year double-blinded study, 36 patients were randomized to prednisolone or placebo after matching for age, menopausal status, histological stage and serum bilirubin. While cholestatic s y m p t o m s and biochemical parameters, such as serum alkaline phosphatase, improved on prednisolone, the trial was too short and had too few patients to assess any change in histology or mortality. S e r u m bilirubin and albumin, which are the best two biochemical prognostic predictors in PBC, were unchanged. O f considerable importance was the finding that the rate of bone density loss, as measured by photon absorptiometry and trabecular bone volume, was doubled by the use of prednisolone. Such an effect would suggest a considerable hazard, as osteoporosis is not only c o m m o n in patients with advanced P B C but is often clinically manifest by pain and vertebral collapse [27].
Immunointervention in primary biliary cirrhosis 297 Azathioprine Azathioprine, in a similar way to corticosteroids, has widespread inhibitory effects on the immune system. A prospective, although unblinded, trial of a 2 mg/kg dose of azathioprine or placebo, given to 45 patients for up to 6 years and reported by the Royal Free Hospital in 1976, failed to show a significant difference in biochemistry, histology or survival [28]. As this had insufficient statistical power to detect an effect on the latter two parameters, a much larger European multicentre study involving 248 patients was carried out, including patients from King's College Hospital and with doses of 0.5 to 1.5 mg/kg [17]. Using Cox multiple regression analysis and adjusting for the more advanced disease in the patients randomized to azathioprine, an improvement in survival of 20 months was shown for the average patient taking azathioprine with a 41% reduction in the risk of dying during the treatment period ( P = 0 . 0 1 ) . Despite these results and only minor side effects encountered in this study, there have been continued concerns about the safety of azathioprine and in particular of the development of malignancies. It is also disappointing that an improvement in biochemistry or histology was not apparent in the trial.
Chlorambucil This alkylating agent has been used successfully in other autoimmune diseases such as rheumatoid arthritis and glomerulonephritis. In a study performed by the National Institute of Health, 24 patients were randomized, unblinded, to 0.5 to 4 mg/kg chlorambucil or placebo and followed for 2-6 years [29]. An improvement in biochemical parameters such as serum bilirubin and albumin and a reduction in inflammatory infiltrate on liver biopsy was shown in the treated group but in association with a high incidence of bone marrow suppression, herpes infection and early onset of the menopause.
Cyclosporin Cyclosporin A (CsA) is a cyclic endecapeptide of fungal origin which, by reducing cytokine production, inhibits both helper/inducer a n d cytotoxic T-cell activities while promoting suppressor cell function, leading to a fall in peripheral blood helper/ inducer:suppressor/cytotoxic cell ratio (CD4:CD8) [30] and inhibition of B-cell function [31 ], including the response to T-cell-independent antigens. It also disrupts T-cell-macrophage interactions and intrathymic maturation of T cells [32]. CsA has now been assessed in one large multicentre trial and two smaller studies. In 1988, a pilot study of only 12 patients, of whom six were treated with CsA maintaining blood levels between 60 and 200 ng/ml, showed a reduction in serum alkaline phosphatase and 7-glutamyltransferase but no change in serum bilirubin or albumin. Serum creatinine increased by 51% in those given CsA [33]. T w e n t y - n i n e patients were enrolled in a placebo controlled, double-blind study by the Mayo Clinic using an initial dose of CsA of 4 mg/kg per day and aiming for a blood level of between 80 and 120 ng/ml. T h e y found an improvement in fatigue, pruritus, and the biochemical markers of serum bilirubin, alkaline phosphatase and titre of antimitochondrial antibody. Although there was a reduction in histological progression
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over 2 years of follow up, nephrotoxicity was observed in 12 and hypertension in nine of the 19 patients treated with CsA. T h e results of the European multicentre trial of CsA have recently been analysed; this was carried out by the same group as performed the azathioprine trial already referred to and enrolled 349 patients [34]. This double-blind, placebo controlled trial is the largest study so far reported with patients followed for up to 6 years. Sixty-one patients died during the study and 29 were given a transplant. Cox multiple regression analysis was used to eliminate imbalance in prognostic features between the treated and placebo groups at presentation. This showed that time from entry into the trial to death or transplantation was prolonged by treatment with CsA (50-60% increase, P < 0.05) and that there were significantly fewer liver related deaths in the group given CsA. T h e rise in serum bilirubin was also slower in those patients given CsA and histology tended to change less. Nephrotoxicity was seen less frequently than in the Mayo Clinic study. A serum creatinine greater than 150 ~M occurred in 9% of the CsA-treated patients and nephrotoxicity resulted in discontinuation of the drug in 5%. Hypertension was observed in 11%. In all, 77% CsA-treated patients completed the study. This trial suggests that CsA may delay the need for liver grafting and that the incidence of significant nephrotoxicity is less. In view of this toxicity and since absorption in PBC may be poor [35], monitoring of blood CsA levels is mandatory. CsA, like other immunomodulatory agents is likely to be most effective early in the natural history of PBC, when autoimmune processes are likely to be occurring, and of least benefit once cirrhosis or liver decompensation have developed. CsA is probably the most successful irnmunomodulatory drug so far used in this puzzling disease. Once serum bilirubin has risen to above 100 I~M, liver transplantation is likely to be needed within the next 1-2 years. Because of the better results, surgery should not be delayed once the serum bilirubin rises above 150 ~tM. Other agents with some immuno-modulatory
activity
Methotrexate, a cytotoxic agent used in malignancies and in inflammatory disorders such as rheumatoid arthritis and asthma, has recently been reported to be beneficial in primary biliary cirrhosis and primary sclerosing cholangitis. Apart from being a folic acid antagonist, it may also inhibit the biological activity of interleukin-1 on T-cell proliferation [36]. Although initial reports have been encouraging [37, 38], methotrexate has not yet been subjected to a randomized, controlled trial. Worries about drug-induced hepatotoxicity may have been overestimated [39] but will only be resolved by a properly controlled clinical trial. References
1. Danielsson, A., L. Boqvist, and P. Uddenfeldt. 1990. Epidemiology of primary biliary cirrhosis in a defined rural population in the northern part of Sweden. Hepatology 11: 458-464 2. Sherlock, S. 1959. Primary biliary cirrhosis (chronic intrahepatic obstructive jaundice). Gastroenterology 37:574-586 3. Ahrens, E. H., M. A. Pater, H. G. Kunkel, W. J. Eisenmenger, and S. H. Blondhem. 1950. Primary biliary cirrhosis. Medicine 29:299-364
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4. Berg, P. A. and R. Klein. 1989. Heterogeneity of antimitochondrial antibodies. Semin. Liver. Dis. 9" 103-116 5. James, O. F., S. J. Yeaman, and M. F. Bassendine. 1989. Molecular aspects of the M2 autoantigens in primary biliary cirrhosis: what a difference a year makes. Hepatology 10: 247-251 6. Krams, S. M., C. D. Surh, R. t . Coppel, A. Ansari, B. Ruebner, and M. E. Gershwin. 1989. Immunization of experimental animals with dihydrolipoamide acetyltransferase, as a purified recombinant polypeptide, generates mitochondrial antibodies but not primary biliary cirrhosis. Hepatology 9" 411-416 7. Baum, H. 1989. Nature of the mitochondrial antigens of primary biliary cirrhosis and their possible relationships to the etiology of the disease. Semin. Liver Dis. 9:117-123 8. Sundin, U. and K. G. Sundqvist. 1991. Plasma membrane association of primary biliary cirrhosis mitochondrial marker antigen M2. Clin. Exp. Immunol. 83:407-412 9. Gershwin, M. E. and I. R. Mackay. 1991. Primary biliary cirrhosis: paradigm or paradox for autoimmunity. Gastroenterology 100:822-833 10. Krams, S. M., K. Dorshkind, and M. E. Gershwin. 1989. Generation of biliary lesions after transfer of human lymphocytes into severe combined immunodeficient (SCID) mice. J. Exp. Med. 170:1919-1930 11. Underhill, J., P. Donaldson, D. Doherty, G. Bray, M. Lombard, and R. Williams. 1990. R F L P analysis of H L A class I I associations in primary cirrhosis (PBC). Gut 31:A1208 (abstract) 12. Briggs, D., P. T. Donaldson, P. Hayes, K. I. Welsh, R. Williams, and J. M. Neuberger. 1987. A major histocompatibility complex class I I I allotype (C4B2) associated with primary biliary cirrhosis (PBC). Tissue Antigens 29:141-145 13. Segall M. and F. H. Bach. 1990. H L A and disease. N. Engl.J. Med. 322:1879-1881 14. Spengler, U., G. R. Pape, R. M. Hoffmann, J. P. Johnson, J. Eisenburg, G. Paumgartner, and G. Riethmuller. 1988. Differential expression of M H C class I I subregion products on bile duct epithelial cells and hepatocytes in patients with primary biliary cirrhosis. Hepatology 8:459-462 15. Ballardini, G., F. B. Bianchi, R. Mirakian, M. Fallani, E. Pisi, a n d G. F. Bottazzo. 1987 H L A - A , B, C, H L A - D / D R and H L A - D / D Q expression on unfixed liver biopsy sections from patients with chronic liver disease. Clin. Exp. Immunol. 70:35-46 16. Roll, J., J. L. Boyer, D. Barry, and G. Klatskin. 1983. T h e prognostic importance of clinical and histologic features in assymptomatic and symptomatic primary biliary cirrhosis. N. Engl. J. Med. 308:1-7 17. Christensen, E., J. Neuberger, J. Crowe, D. G. Altman, H. Popper, B. Portmann, D. Doniach, L. Ranek, N. Tygstrup, and R. Williams. 1985. Beneficial effect of azathioprine and prediction of prognosis in primary biliary cirrhosis. Final results of an international trial. Gastroenterology 89:1084-1091 18. Dickson, E. R., P. M. Grambsch, T. R. Fleming, L. D. Fisher, and A. Langworthy. 1989. Prognosis in primary biliary cirrhosis: model for decision making. Hepatology 10:1-7 19. Goudie, B. M., A. D. Burt, G. J. Macfarlane, P. Boyle, C. R. Gillis, R. N. MacSween, and G. Watkinson. 1989. Risk factors and prognosis in primary biliary cirrhosis. Am. J. Gastroenterol. 84:713-716 20. Bonsel, G. J., I. J. Klompmaker, F. van't Veer, J. D. Habbema, and M. J. Slooff. 1990. Use of prognostic models for assessment of value of liver transplantation in primary biliary cirrhosis. Lancet 335:493-497 21. Grambsch, P. M., E. R. Dickson, R. H. Wiesner, and A. Langworthy. 1989. Application of the Mayo primary biliary cirrhosis survival model to Mayo liver transplant patients. Mayo Clin. Proc. 64:699-704 22. Balasubramaniam, K., P. M. Grambsch, R. H. Wiesner, K. D. Lindor, and E. R. Dickson. 1990. Diminished survival in asymptomatic primary biliary cirrhosis. Gastroenterology 98:1567-1571 23. Wiesner, R. H., P. M. Grambsch, K. D. Lindor, J. Ludwig, and E. R. Dickson. 1988. Clinical and statistical analyses of new and evolving therapies for primary biliary cirrhosis. Hepatology 8:668-676
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24. A1Aghbar, M. N., G. J. Alexander, J. Neuberger, K. T. Nouri-Aria, A. L. Eddleston, and R. Williams. 1986. The effect of prednisolone in vitro on immunoglobulin production in primary biliary cirrhosis. Clin. Exp. Immunol. 63:663-670 25. Mitchison, H. C., M. F. Bassendine, A. J. Malcolm, A. J. Watson, C. O. Record, and O. E. James. 1989. A pilot, double-blind, controlled 1-year trial of prednisolone treatment in primary biliary cirrhosis: hepatic improvement but greater bone loss. Hepatology 10: 420-429 26. Carman, C. T. and J. E. Giansiracusa. 1955. Effect of steroid therapy on the clinical and laboratory features of primary biliary cirrhosis. Gastroenterology 28:193-207 27. Maddrey, W . C . 1990. Bone disease in patients with primary biliary cirrhosis. Prog. Liver. Dis. 9:537-554 28. Heathcote, J., A. Ross, and S. Sherlock. 1976. A prospective controlled trial of azathioprine in primary biliary cirrhosis. Gastroenterology 70:656-660 29. Hoofnagle, 3. H., G. L. Davis, D. F. Schafer, M. Peters M, M. I. Avigan, S. C. Pappas, R. G. Hanson, G. Y. Minuk, G. M. Dusheiko, G. Campbell, R. N. M. MacSween, and E. A. Jones. 1986. Randomized trial of chlorambucil for primary biliary cirrhosis. Gastroenterology 91:1327-1334 30. Routhier, G., O. Epstein, G. Janossy, H. C. Thomas, S. Sherlock, P. C. Kung, G. Goldstein. 1980. Effects of cyclosporin A on suppressor and inducer T lymphocytes in primary biliary cirrhosis. Lancet ii: 1223-1226 31. A1Aghbar, M. N., G. J. Alexander, K. T. Nouri-Aria, J. Neuberger, A. L. Eddleston, and R. Williams. 1986. In vitro effect of cyclosporin A on immunoglobulin production and concanavalin A induced suppression in primary biliary cirrhosis. Gut 27" 317-323 32. Kahan, B. D. 1990. Cyclosporine. N. Engl.J. Med. 321:1725-1738 33. Minuk, G. Y., C. E. Bohme, E. Burgess, N. B. Hershfield, J. K. Kelly, E. A. Shaffer, L. R. Sutherland, and G. Van Rosendaal. 1988. Pilot study of cyclosporin A in patients with symptomatic primary biliary cirrhosis. Gastroenterology 95:1356-1363 34. Lombard, M., B. Portmann, J. Neuberger, R. Williams, N. Tygstrup, L. Ranek, H. R. Larsen, J. Rodes, M. Navasa, C. Trepo, G. Pape, G. Schou, J. H. Badsberg, and P. K. Andersen. 1990. Cyclosporin A treatment in primary biliary cirrhosis. Hepatology 12:872 (Abstract) 35. Robson, S., J. Neuberger, H. P. Keller, E. Abisch, W. Neiderberger, B. yon Graffenried, and R. Williams. 1984. Pharmacokinetic study of cyclosporin A (Sandimmun) in patients with primary biliary cirrhosis. Br. J. Clin. Pharmaeol. 18:627-631 36. Kaplan, M. M. 1989. Methotrexate treatment of chronic liver diseases: friend or foe? Quart. J. Med. 72:757-761 37. Kaplan, M. M., T. A. Knox, and S. A. Arora. 1988. Primary biliary cirrhosis treated with low-dose oral pulse methotrexate. Ann. Intern. Med. 109:429-431 38. Kaplan, M . M . a n d T . A. Knox. 1989. Effective treatment ofpre-cirrhotic primary biliary cirrhosis (PBC) with methotrexate (MTX). Hepatology 10:585 39. Rau, R., T. Karger, G. Herborn, and H. Frenzel. 1989. Liver biopsy findings in patients with rheumatoid arthritis undergoing longterm treatment with methotrexate. J. Rheumatol. 16" 489-493
I m m u n o i n t e r v e n t i o n in P r i m a r y B i l i a r y Cirrhosis: an O v e r v i e w
Gary P. Bray and Roger Williams
Institute of Liver Studies, King's College Hospital School of Medicine and Dentistry, Denmark Hill, London SE5 8RX, UK Since a u t o i m m u n e processes are p r o b a b l y involved in the early stages of p r i m a r y biliary cirrhosis (PBC), i m m u n o m o d u l a t o r y drugs have been investigated with the a i m of prolonging survival, delaying t r a n s p l a n t a t i o n , slowing histological progression and relieving s y m p t o m s . Corticosteroids, azathioprine, c h l o r a m b u c i l and, m o r e recently, cyclosporin A a n d m e t h o trexate have all be subjected to clinical investigation. In the latest of these, a E u r o p e a n m u l t i c e n t r e trial, cyclosporin A has been shown to delay death or t r a n s p l a n t a t i o n with a reduction in liver related deaths a n d slowing of the rise of s e r u m bilirubin. The incidence of nephrotoxicity and hypertension are low at the doses used.
Introduction Primary biliary cirrhosis (PBC) is a cholestatic liver disease which predominantly affects middle aged females. T h e diagnosis is made after presentation with pruritus or jaundice or after the incidental finding of abnormal liver function tests. Less c o m m o n l y it m a y present with variceal bleeding. Considerable geographical variation is reported and in one recent study, the prevalence was as high as 151 per million [1]. Since the first descriptions of P B C [2, 3], the discovery of effective treatment has been a major goal of research into the condition although the exact aetiology of the disease remains uncertain.
Autoimmunity and PBC T h e r e is considerable evidence that a u t o i m m u n e processes are involved in the aetiology of PBC. Initiation of a u t o i m m u n i t y may result in granulomata and inflammation around small intrahepatic bile ducts, leading to fibrosis, increasing cholestasis and liver failure. 293 0896-8411/92/0A0293 + 08 $03.00/0
© 1992 Academic Press Limited
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G.P. Bray and R. Williams
Non-organ specific antibodies are common with anti-mitochondrial antibodies ( A M A ) being found in over 95% of patients with P B C T h e four different subtypes of these antibodies have been identified by Berg and his colleagues as M2, M4, M8 and M9 [4]. A n t i - M 2 antibodies are those most frequently found and recognize antigens on the inner mitochondrial m e m b r a n e which are now known to be enzymes involved in intermediary metabolism. T h e s e are the E2 components of the pyruvate dehydrogenase (PDC), 2-oxoglutarate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes as well as protein X (part of P D C - E 2 ) , and the a and 13 subunits of P D C - E 1 [5]. Although A M A could have a direct role in pathogenesis, the occurrence of AMA-negative PBC argues against this as does the failure to produce the disease by immunization of animals with a recombinant form of the E2 component of P D C [6]. H o w an humoral antibody response to intracellular proteins can result in T-cell mediated damage localized to the biliary tree [5] is difficult to understand as it is unknown whether mitochondrial antigens can be expressed by the cell m e m b r a n e of hepatocytes [7, 8].
Lymphocytic infiltrates are found around damaged bile ducts and lymphocytes from patients with P B C may be able to transmit the disease to experimental animals T h e lymphocytic infiltration around bile ducts consists of predominantly C D 3 +, C D 4 +, C D 8 - T cells expressing the T-cell 13chain [9]. Injection of peripheral blood lymphocytes from patients with P B C into female mice with severe combined i m m u nodeficiency disease has been found to result in A M A appearing in the serum with a portal infiltrate with bile duct damage in the liver of the mice [10]. Although the histological changes induced in these mice were not identical to those found in h u m a n PBC, this work and the characteristic lymphocytic infiltrate found in the portal tracts of patients with the disease suggest that T cells have an important role in the pathogenesis of the disorder.
P B C is associated with H L A - D R w 8 and C4B2 E11, 12] Association of a particular H L A allele with a disease suggests that this may complex with an antigen in a way that may lead to an a u t o i m m u n e response [13]. In this way, H L A - D R w 8 and C4 B2 may confer susceptibility to the disease.
M H C Class II antigens are expressed by bile duct epithelium In PBC, bile duct epithelial cells have been shown to express H L A - D R , - D P and - D Q M H C class I I antigens [14, 15]. M H C class I I antigens are known to control the induction of C D 3 +, C D 4 + T-cell mediated i m m u n e responses. Disease progression and prognosis With progression of the disease and deepening cholestasis, liver decompensation, as evidenced by jaundice, ascites, coagulopathy, severe infection, renal failure and encephalopathy, may occur. Several prognostic models have been developed in order
Immunointervention in primary biliary cirrhosis 295 Table 1. Comparison of Cox multiple regression
models in PBC, showing independent variables identified in five studies Prognostic models European Serum bilirubin Serum albumin Age Prothrombin time Oedema Therapy Fibrosis/cirrhosis Cholestasis
Yes Yes Yes
Mayo Yes
Yes Yes Yes Yes
Yes Yes Yes
to help to predict outcome and life expectancy in individual patients. T h e y have also been used in assessing the value of treatments such as liver transplantation and the optimal timing for this procedure [16-21] (Table 1). T h e European model, which was developed after Cox multiple regression analysis of the results of the multicentre trial of azathioprine [17], and the M a y o clinic model [18], which does not involve histology, are the two most frequently used. In most cases, the progression of PBC is relatively slow with a reported median survival of 11.9 years from the onset of s y m p t o m s [16] but it is now recognized that the median survival of patients who are asymptomatic at the time of diagnosis is also worse than for a normal age matched population. T h e M a y o clinic found asymptomatic patients to have a four-fold increase in mortality compared to an age, race and sex matched population in the United States, with 89 % of these patients developing s y m p t o m s during the study period [22].
Rationale for i m m u n o m o d u l a t o r y therapy and design o f clinical trials Despite the evidence in favour of an a u t o i m m u n e aetiology for PBC, the response to i m m u n o s u p p r e s s i o n is, as we shall discuss later, not as p r o m p t as in the a u t o i m m u n e variety of chronic active hepatitis. T h e i m m u n o m o d u l a t o r y drugs which have been used in P B C are listed in T a b l e 2. T h e r e are several difficulties which are encountered in the design of any clinical trial and, in particular, those conducted in PBC [23]. In order that a trial has sufficient statistical power to detect an effect exerted by the drug on the course of the disease, there m u s t be an adequate n u m b e r of patients recruited and no unplanned interim analyses should be allowed. I f an interim analysis is p e r f o r m e d then the clinical investigators and patients should not be unblinded and, unless significant toxicity is encountered, the results should not determine whether the trial is continued. T h e length of trial should be decided prior to starting and the trial should be double blind and placebo controlled with patients both randomized and their outcome analysed on an 'intent to treat' basis. Small, non-randomized, unblinded and
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T a b l e 2. Immunomodulatory
drugs which have been used in p r i m a r y biliary cirrhosis and major trials
Drug used Prednisolone Azathioprine Chlorambucil Cyclosporin
Reference Mitchison et al. 1989 [25] Heathcote et al. 1976 [28] Christensen et al. 1985 [17] Hoofnagle et al. 1986 [29] Wiesner et al. 1988 [23] Lombard et al. 1990 [34]
Methotrexate Penicillamine and colchicine may also modulate the immune system but have been principally used for their anticupretic and antifibrotic activity, respectively, uncontrolled studies cannot give certain answers as to whether a drug affects survival or progression of histology and are not included in this review. In view of the long and variable time course of the illness, follow-up of patients needs to be protracted in order to show a beneficial effect on survival (or the need for liver transplantation) of any intervention. As the clinical course of PBC prior to development of s y m p t o m s is unpredictable, analysis of trials recruiting both symptomatic and asymptomatic patients may need to be stratified. In addition to survival, other endpoints of treatment can be used, e.g. amelioration of s y m p t o m s such as pruritus, reduction in histological progression or an i m p r o v e m e n t in biochemical parameters. Corticosteroids
Corticosteroids inhibit cell division and cytokine production by all elements of the i m m u n e system and prednisolone specifically has been shown to be able to correct defective regulation of immunoglobulin production by lymphocytes in vitro [24]. Surprisingly, the first and only controlled trial of corticosteroids in PBC, from Newcastle, was not published until 1990 [25] despite their intermittent use in this disease for over 35 years [26]. In the Newcastle 1-year double-blinded study, 36 patients were randomized to prednisolone or placebo after matching for age, menopausal status, histological stage and serum bilirubin. While cholestatic s y m p t o m s and biochemical parameters, such as serum alkaline phosphatase, improved on prednisolone, the trial was too short and had too few patients to assess any change in histology or mortality. S e r u m bilirubin and albumin, which are the best two biochemical prognostic predictors in PBC, were unchanged. O f considerable importance was the finding that the rate of bone density loss, as measured by photon absorptiometry and trabecular bone volume, was doubled by the use of prednisolone. Such an effect would suggest a considerable hazard, as osteoporosis is not only c o m m o n in patients with advanced P B C but is often clinically manifest by pain and vertebral collapse [27].
Immunointervention in primary biliary cirrhosis 297 Azathioprine Azathioprine, in a similar way to corticosteroids, has widespread inhibitory effects on the immune system. A prospective, although unblinded, trial of a 2 mg/kg dose of azathioprine or placebo, given to 45 patients for up to 6 years and reported by the Royal Free Hospital in 1976, failed to show a significant difference in biochemistry, histology or survival [28]. As this had insufficient statistical power to detect an effect on the latter two parameters, a much larger European multicentre study involving 248 patients was carried out, including patients from King's College Hospital and with doses of 0.5 to 1.5 mg/kg [17]. Using Cox multiple regression analysis and adjusting for the more advanced disease in the patients randomized to azathioprine, an improvement in survival of 20 months was shown for the average patient taking azathioprine with a 41% reduction in the risk of dying during the treatment period ( P = 0 . 0 1 ) . Despite these results and only minor side effects encountered in this study, there have been continued concerns about the safety of azathioprine and in particular of the development of malignancies. It is also disappointing that an improvement in biochemistry or histology was not apparent in the trial.
Chlorambucil This alkylating agent has been used successfully in other autoimmune diseases such as rheumatoid arthritis and glomerulonephritis. In a study performed by the National Institute of Health, 24 patients were randomized, unblinded, to 0.5 to 4 mg/kg chlorambucil or placebo and followed for 2-6 years [29]. An improvement in biochemical parameters such as serum bilirubin and albumin and a reduction in inflammatory infiltrate on liver biopsy was shown in the treated group but in association with a high incidence of bone marrow suppression, herpes infection and early onset of the menopause.
Cyclosporin Cyclosporin A (CsA) is a cyclic endecapeptide of fungal origin which, by reducing cytokine production, inhibits both helper/inducer a n d cytotoxic T-cell activities while promoting suppressor cell function, leading to a fall in peripheral blood helper/ inducer:suppressor/cytotoxic cell ratio (CD4:CD8) [30] and inhibition of B-cell function [31 ], including the response to T-cell-independent antigens. It also disrupts T-cell-macrophage interactions and intrathymic maturation of T cells [32]. CsA has now been assessed in one large multicentre trial and two smaller studies. In 1988, a pilot study of only 12 patients, of whom six were treated with CsA maintaining blood levels between 60 and 200 ng/ml, showed a reduction in serum alkaline phosphatase and 7-glutamyltransferase but no change in serum bilirubin or albumin. Serum creatinine increased by 51% in those given CsA [33]. T w e n t y - n i n e patients were enrolled in a placebo controlled, double-blind study by the Mayo Clinic using an initial dose of CsA of 4 mg/kg per day and aiming for a blood level of between 80 and 120 ng/ml. T h e y found an improvement in fatigue, pruritus, and the biochemical markers of serum bilirubin, alkaline phosphatase and titre of antimitochondrial antibody. Although there was a reduction in histological progression
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over 2 years of follow up, nephrotoxicity was observed in 12 and hypertension in nine of the 19 patients treated with CsA. T h e results of the European multicentre trial of CsA have recently been analysed; this was carried out by the same group as performed the azathioprine trial already referred to and enrolled 349 patients [34]. This double-blind, placebo controlled trial is the largest study so far reported with patients followed for up to 6 years. Sixty-one patients died during the study and 29 were given a transplant. Cox multiple regression analysis was used to eliminate imbalance in prognostic features between the treated and placebo groups at presentation. This showed that time from entry into the trial to death or transplantation was prolonged by treatment with CsA (50-60% increase, P < 0.05) and that there were significantly fewer liver related deaths in the group given CsA. T h e rise in serum bilirubin was also slower in those patients given CsA and histology tended to change less. Nephrotoxicity was seen less frequently than in the Mayo Clinic study. A serum creatinine greater than 150 ~M occurred in 9% of the CsA-treated patients and nephrotoxicity resulted in discontinuation of the drug in 5%. Hypertension was observed in 11%. In all, 77% CsA-treated patients completed the study. This trial suggests that CsA may delay the need for liver grafting and that the incidence of significant nephrotoxicity is less. In view of this toxicity and since absorption in PBC may be poor [35], monitoring of blood CsA levels is mandatory. CsA, like other immunomodulatory agents is likely to be most effective early in the natural history of PBC, when autoimmune processes are likely to be occurring, and of least benefit once cirrhosis or liver decompensation have developed. CsA is probably the most successful irnmunomodulatory drug so far used in this puzzling disease. Once serum bilirubin has risen to above 100 I~M, liver transplantation is likely to be needed within the next 1-2 years. Because of the better results, surgery should not be delayed once the serum bilirubin rises above 150 ~tM. Other agents with some immuno-modulatory
activity
Methotrexate, a cytotoxic agent used in malignancies and in inflammatory disorders such as rheumatoid arthritis and asthma, has recently been reported to be beneficial in primary biliary cirrhosis and primary sclerosing cholangitis. Apart from being a folic acid antagonist, it may also inhibit the biological activity of interleukin-1 on T-cell proliferation [36]. Although initial reports have been encouraging [37, 38], methotrexate has not yet been subjected to a randomized, controlled trial. Worries about drug-induced hepatotoxicity may have been overestimated [39] but will only be resolved by a properly controlled clinical trial. References
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