SEMINARS IN LIVER DISEASE-VOL.
1 1 , NO. 3, 1991
Experimental Models of Autoimmune Hepatitis ANSGAR W. LOHSE, M.D.
From the I . M~riizinischeKlinik und Poliklitlik Johantlr.~Gutrnbrrg U ~ ~ i v e r s i t Muin-, iit Mainz, German!. Reprint requests: Dr. Lohse. I. Medizinische Klinik und Poliklinik, Johanncs Gutenberg Universitlt Mainz, Langenbeckstrcssc 1 , D-6500 Mainz, Germany.
CHRONIC ACTIVE HEPATITIS IN RABBITS In 1968 Kossling and Meyer zum Biischenfelde8 presented a model of chronic hepatitis in rabbits induced by repeated immunization with heterologous liver antigen. This model of chronic hepatitis in rabbits was described in more detail in subsequent studies."' Characteristically, chronic inflammatory lesions could be induced in rabbits by repeated immunization with soluble human liver antigen, but not by immunization with rabbit antigen. Nonetheless, the rabbits did develop antibodies and positive skin tests against rabbit liver antigen in the course of immunization. Although the animals did develop antibodies against liver antigen and membrane-bound immunoglobulin could be detected on isolated hepatocytes, these antibodies did not correlate with histologic disease activity.".i2 These studies suggested that cellular immune mechanisms rather than autoantibodies were likely to be responsible for the histologic injury. Subsequent studies by other groups in a similar rabbit model found marked responses to the immunizing liver antigen preparation in lymphocyte proliferation assays.13 This was true both for the whole preparation called liver-specific protein (LSP) and its subfraction LP2.'"IJ However, these preparations consisted of a multitude of liver proteins.15 The antigen or antigens responsible for this form of experimental hepatitis have thus never really been identified. The major limitations of this model for further immunologic studies was the problem that rabbits are not available as inbred, genetically homogeneous strains. Furthermore, the need for immunization with heterologous antigen and the lack of hepatitis following immunization with alloantigen as well as the nonspecific hepatitis found by some in animals immunized with irrelevant control antigensi3was undesirable for an animal model of autoimmune disease.
EXPERIMENTAL AUTOIMMUNE HEPATITIS IN MICE Scheiffarth et allh were the first to succeed in inducing an experimental hepatitis in inbred mice by immunization with allogeneic liver antigens in adjuvant. ~ 5 7 ~ mice ~ 1 were 6 immunized intramuscularly and intraperitoneally six times in weekly intervals with liver animals dehomOgenate from AKR mice in CFA. veloped mild inflammatory lesions only, but 12 of the 89
Copyright 0 1991 by Thieme Medical Publ~ahers.Inc., 38 1 Park Avenue South, New York, NY 100 16. All rights reserved.
241
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The study of autoimmune diseases in animal models can help in the understanding of the pathogenesis and regulation of human autoimmune diseases. At the same time, it may open new avenues in the prevention and therapy of autoimmune diseases. Various approaches have been taken to establish animal models for the study of autoimmune hepatitis. The establishment of reliable, reproducible, and useful models has been more difficult than in other autoimmune diseases and thus far no model has been recognized as the standard modcl for autoimmune hepatitis. Nonetheless, several lessons have already been learned from the work done to date and continuing work in this field should lead to further progress in the near future. The first attempts to induce an experimental hepatitis in animals by the injection of liver antigen date back to the beginning of the century.' Fiessinger' described cirrhotic changes in rabbits injected intraperitoneally with a nuclear antigen preparation, no histologic changes were seen in guinea pigs injected with liver antigen.' Forty years later, Casals and Olitsky' found inflammatory lesions and focal necroses in four of ten mice injected repeatedly with a mouse liver homogenate. Although these first two studies used antigen without adjuvant, all groups since have used adjuvant for immunization, mostly complete Freund's adjuvant (CFA). The late 1950s and the 1960s saw the publication of a number of reports on the induction of hepatitis by immunization with heterologous, allogeneic, or syngeneic antigen, as summarized in Table 1 . The several unsuccessful attempts of induction of experimental hepatitis by many groups are not cited, since little can be learned from these negative reports. Because immunology was in its infancy at the time of early reports of experimental hepatitis, immunologic information on these early models is missing. This review will therefore concentrate on the two types of experimental hepatitis that have been most thoroughly characterized, namely chronic active hepatitis (CAH) in rabbits and experimental autoimmune hepatitis (EAH) in mice.
SEMINARS IN LIVER DISEASE-VOLUME
I I , NUMBER 3. 1991
TABLE 1. Animal Models of Autoimmune Hepatitis*
Fiess~nger'
Specic,s
Rabbit
Antigc>rr?
Rr.s~i1r.s Biliary cirrhosis in 3 of 7 animals; negative results in dogs and gulnea pigs Periportal inflammation and necroses in 4 of 10 ~ n i c c Early focal necroses in I I of 31 animals (days 3-4) Focal necroses in only 3 of 16 hamsters Periportal infiltrations after I week Hepatocellular necroses
Nuclear protein\
Casals and Olitsky? Behar and Tal'
Mouse
--
Guinca pig hamster
Homo. LH
CFA
Dodd et al-' lliesco et ali Coppo and Tedeschih Scheiffarth et al' Scheiffarth ct al'"
Rabbit Rabbit
Rat LH Horno. LH
CFA CFA
Kossling et a1 (8-1 2)
Rabbll
Kuriki et al"
Moue
Mori ct ?? Watdnabe et a]'O '7
Moube
Lohse et alL"'J
Mouse
Rat
CFA
Rabbit Mouse
Mou\e
CFA
Sy ng . S- I00 Syng. S-100
CFA CFA
Syng. S-100
At least 12 x sc 9 x sc
Focal ~nflammatorychanges and necroses Periportal infiltrations and necroses (in sonic animals) Periportal infiltrations and necroses, no lesions with kidney antigen; ~uccessfulpasslve transfer Chronic hepatitis: no hepatitis following irnrnunization with rabbit LSP Frequent i m m u n i ~ a t i o n smore effective. passivc transfer ot discasc possible Passive transfer possible, T-cell mediated disease, specific cytvtoxity Thymcctomy fac~litateddi\ea\e induction and prolonged d ~ s e a s e duration Subacute '1'-cell mediated hepat~tis. sptcific T-cell reactivity. autoantibodies follow disease
*Only those experiments in which experimental animals showed different and niore severe changes than the control unirnals are mentioned. Some early studies are included for historical reasons despite the lack of suitable controls. TLH: liver hornogenate; S-100: 100.000 g m supernatant of liver hornogenate: LSP: liver specific protein: CFA: complete Frcund'h adjuvanl; ip: intrapcritoncally; 1111:intra~nuscularly;sc: subcutaneously; homo: homologous: hum: human: syng: synpeneic.
mice studied developed moderate to severe periportal inf i l t r a t i o n ~ .CFA ' ~ alone or immunization with kidney homogenate in CFA did only rarely lead to mild or noder rate inflammatory responses. The most important finding of this pioneering work was the adoptive transfer of the hepatitis with spleen cells from diseased animals. '' This finding strongly suggested that cell-mediated immunity was responsible for this model of experimental hepatitis. No reasons were given why liver from AKR mice rather than from syngeneic mice was used. It is thus not possible to assess whether this was a truly autoimmune process or whether allogencic immune responses may have played a role in the pathogenesis of the observed lesions. It was only in the 1980s that the importance of studying inbred animals in immunologic research was fully appreciated by researchers in the field of immuncmediated liver disease. In 1983 Kuriki et all7 presented a mouse model of autoimmune hepatitis following immunization with syngeneic liver antigen. Hepatitis in this model was induced in SMA mice with LSP-enriched liver homogenate. Because of their good experience with the application of the polysaccharide of Kleb.siellu pt~euinoniue as adjuvant, these authors used this adjuvant instead of CFA.IXImmunization was done intramuscularly at monthly intervals up to a total of 1 I injections. Mild to moderate inflammatory lesions could be observed in
all mice immunized with liver antigen in adjuvant: no lesions or minimal lesions only were observed in the groups receiving liver antigen only, adjuvant only, or kidney antigen in adjuvant." More severe lesions were observed in those animals immunized 8 to 1 1 times compared with those immunized three to six times. Minimal lesions were also observed in the kidneys, whereas other organs appeared to be unaffected. Spleen cells from diseased animals could transfer hepatitis to irradiated recipient mice." The results of transfer into nonirradiated naive recipients were not reported. Kuriki et all7 found that their animals did develop antibodies against liver antigens, but did not describe whether antibody development and titers correlated with hepatitis. They also observed a very interesting phenomenon only in those mice that received liver antigen in adjuvant: splenocytes from these animals markedly suppressed the mitogen-stimulated proliferation of normal spleen cells (17). The interpretation of this finding is difficult. It appears that the animals' immune system noticed the aberrant autoimmune response to liver self-antigen and may have initiated a counterregulatory immune response leading to a generalized suppression. Mori et al1"-" in a series of publications have studied EAH in mice in considerable detail. This group of investigators used syngeneic 100,000 gm supernatant of freshly prepared liver homogenate as immunogen. Pro-
~
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Autl~or
tein, 10 mg, was injected intramuscularly in CFA. Immunization was repeated at weekly intervals. The initial article describes six consecutive injection^;'^ later on only four injections were used.".'2 Male C57BLl6, C3HI He, and BALBiC mice were studied. Hepatitis could best be induced in C57BLl6 mice, the same strain that was used in the original study by Scheiffarth et a1.16 The ability of the antigen preparation to induce hepatitis was quickly lost by freezing and thawing the material. '' The mice developed antibodies against the antigen preparation, which did not react with the material that had been frozen and thawed more than three times. No further information on the character of the antigen or antigens responsible for the experimental hepatitis has become available as yet. Subsequent passive transfer experiments have given contradictory results." Unseparated spleen cells and nylon wool column nonadherent, T-cell enriched spleen cells could transfer mild hepatitis to naive recipients to a similar degree, whereas the nylon wool adherent fraction was able to transfer disease more effectively. However, when this nylon wool column adherent (with supposedly few T cells to start with) spleen cell population was further depleted of T cells by treatment with the pan-T-cell antibody anti-Thy 1.2 and guinea-pig complement, the ability to transfer disease was almost completely lost. Thus, although the first finding suggests that cells other than T cells were responsible for the disease transfer, the latter finding shows T cells to be of critical importance. It is difficult to interpret these findings. Two explanations seem possible. The most likely possibility is that several cell populations are involved-ii the pathogenesis of this experimental model. T cells are a prerequisite for the disease, but may require other cells, predominantly found in the nylon wool adherent compartment, for disease induction. This possibility is supported by earlier findings demonstrating that T cells are required in the mouse for the production of anti-LSP antib~dies.'~ The other possible interpretation is that the T-cell enriched fraction included, like the unseparated spleen cells, a counterregulatory suppressor cell population that was less predominant in the nylon wool adherent cell population. In view of the findings by Kuriki et all7 demonstrating active suppression in animals with EAH, this possibility deserves further study. Support is given to this interpretation by the subsequent finding in the model of Mori et that passive transfer of hepatitis was facilitated by prior irradiation of donor mice with 300 rad,'2 a procedure known preferentially to inactivate suppressor lymphocytes. Mori et al. also tested the proliferative response of splenocytes from their animals against LSP. No proliferative response against the ethylene diaminetetraacetic acid (EDTA)-containing LSP preparation (isolated as described by McFarlane et could be measured, but EDTA-free LSP was able to stimulate spleen cells in a standard T-cell proliferation assay.19 However, spleen cells from normal, untreated C57BLl6 mice and immunized animals showed similar proliferative responses." The role of antigen specific T-cell responses is thus not entirely clear. It should be remembered that Mori et al immunized with crude liver proteins, the 100,000 gm supernatant of liver homogenate. This antigen prepara-
tion will have included several antigens not present or present in only very small amounts in the LSP preparation. T cells from the experimental hepatitis animals may have been directed against other antigens not tested in their proliferation assay. Cytotoxicity studies by the same group21 suggests that specific cytotoxic T cells preferentially found in the nylon wool adherent spleen cell population of mice with experimental hepatitis were of pathogenetic importance. Specific lysis of syngeneic hepatocytes, but not renal cells, was found to be present 1 and 2 weeks after the last immunization.'' Taken together these studies present an apparently T-cell mediated model of EAH. Araki et al" presented a further murine model of Tcell mediated EAH in BlO.A(SR) mice. However, in their model low-dose irradiation to decrease the suppressor cell activity in the experimental animals was required to induce hepatitis. In addition, for moderate hepatitis to develop up to eight immunizations spread over a period of 6 months were required, making this model rather unattractive for further pathogenetic studies. Nonetheless, the adoptive transfer experiments give further support for the importance of T cells in the pathogenesis of autoimmune hepatitis.'" Watanabe and colleagues'"~" examined the effect of neonatal thymcctomy on the induction and persistence of EAH. Hepatitis was induced by a similar protocol to that used by Mori et al, except that A/J mice were used for their studies. It was found that neonatal thymectomy both facilitated the induction of hepatitis2' and led to persistence of the d i ~ e a s e . 'Transfer ~ of normal spleen cells, but not the transfer of spleen cells from thymectomized mice, abolished the effect of t h y m e ~ t o m y These . ~ ~ studies suggest that normal spleen cells have a suppressive ability against EAH and that this suppressor activity is lost by neonatal thymectomy. We have recently presented a simplified model of EAH in mice.28 Hepatitis was likewise induced by immunization with the 100,000 gm supernatant (called S100) of freshly prepared syngeneic liver homogenate in CFA. C57BLl6 mice were more susceptible than C3H or BALBIC mice, and no hepatitis could be induced in Lewis rats. The study of various modes and schedules of immunizaticln showed that intraperitoneal immunization was more effective than intramuscular or subcutaneous immunization. Three weekly injections led to moderate hepatitis in almost all animals, but a similar degree of inflammatory damage could be achieved with just a single intraperitoneal immunization with syngeneic S- 100 antigen (Fig. 1). Severity of EAH induced by a single immunization peaked around 3 to 4 weeks after the injection, but mild to moderate hepatitis was still detectable 6 months after disease i n d u ~ t i o nBiochemical .~~ evidence (raised transaminases) of liver cell damage was detectable mainly during the first 6 weeks of EAH (Fig. 2). EAH could be transferred by activated T cells from diseased animals. As in the other studies, adoptively transferred EAH was milder and more transient than the antigen-induced active disease.28Studying T-cell reactivity further, it became clear that the S-100 antigen preparation was toxic in cell culture studies. The S-100 antigen was therefore separated into three peaks over a CL-6B Sepharose column. The second peak seemed to retain
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MODELS OF AUTOIMMUNE HEPATITIS-LOHSE
SEMINARS IN LIVER DISEASE-VOLUME
11, NUMBER 3, 1991
at the peak of inflammatory lesions, no or almost no autoantibodies were detectable (Fig. 2). Yet, when, histologically, hepatitis was improving and when there was no more biochemical evidence of ongoing hepatocyte destruction, autoantibodies continued to develop and titers continued to rise.29These results present strong evidence for T cells being solely responsible for EAH.
FIG. 1. Liver histology at week 3 of experimental autoimmune hepatitis induced in a male C57BLl6 mouse by a single immunization with syngeneic soluble liver antigen (S100) in complete Freund's adjuvant.
the toxic component of the S-100 preparation. T-cell reactivity against the first and third peaks was very marked in animals with EAH (Fig. 3), and no response was observed in control animals. BALBIC mice, which were less susceptible to EAH, showed a weaker T-cell response to peaks 1 and 3 than the more susceptible C57BL!6 mice." Future studies will have to show where to look further for the target antigens of T cells in EAH. In our model of EAH mice developed characteristic a~toantibodies.~" Rather than producing a multitude of antibodies against the crude liver immunogen. S-100, only a few specific autoantibodies developed. The autoantibody pattern differed from that seen in most cases of human autoimmune hepatiti~.'~."The target structures recognized by these autoantibodies are possible target antigens for T-cell autoreactivity in the EAH mice. Interestingly, autoantibodies developed only weeks after histologic and biochemical evidence of hepatitis. Indccd
Numerous studies of toxic, infectious, and genetically induced hepatitis have been described. Those with pohsible relevance to the study of autoimmune liver disease will be reviewed briefly. Poralla et a13?were able to induce hepatitis in rabbits by injecting monoclonal antibody to rabbit liver antigen.'? The antibodies were raised by immunization with LSP. Several antibodies with various reactivity patterns could be isolated, thus confirming the heterogeneity of the preparation called LSP.'? One of these antibodies showing membrane reactivity was able to induce transient liver cell damage in rabbits. These experiments show that antibody-mediated cytotoxicity is a possible mechanism of hepatitis in immune-mediated liver disease (both viral and autoimmune). Two rat strains developing spontaneous hepatitis have been d e ~ c r i b e d . ' " ~These ~ arc substrains of the Long-Evans strain, named LEA and LEC, bred at Hokkaido University in Japan. Hepatitis develops around the age of 3 to 4 months. The lack of inflammatory response, however, suggests that this genetically determined disease is not in any way immune mediated. However, it is precisely this lack of inflammatory response that should deserve further study. In view of the abundant liver cell necroses with the subsequent release of a multitude of intracellular antigens into the circulation and thus into the arms of the immune system, it would be interesting to know which systemic immune reactions
T i m e c o u r s e of EAH i n d u c e d by a s i n g l e i m m u n i z a t i o n
= ---t
Histology
GPT
-
120
- 100 -80
$
- 60
- 40 L 20
0
Autoantibodies:
weeks
10
-
(+I
+
++
20
+++
FIG. 2. Time course of histologic and biochemical changes and occurrence of autoantibodies in experimental autoimmune hepatitis (EAH) in C57BL16 mice induced by a single intraperitoneal immunization with 2.5 mg of S-100 antigen in complete Freund's adjuvant (CFA).
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OTHER FORMS OF EXPERIMENTAL HEPATITIS
PROLIFERATIVE RESPONSE OF EAH SPLENOCYTES
K?
peak 1
peak 2
peak 3
MT
BP
Antigen (1 0 rnicrograrnWrnl)
FIG. 3. T-cell responses to fractions of the immunizing S100 antigen preparation (peaks 1-3), M. tuberculosis (MT) as part of the complete Freund's adjuvant (CFA) and control antigen myelin basic protein 1 week after induction of experimental autoimmune hepatitis (EAH).
to liver antigen are or are not induced. This would help us to differentiate to what extent autoantibodies and Tcell autoreactivity can develop simply in response to antigen release following necrosis. Intraperitoneal injection of group A streptococcal cell walls leads to hepatic granulomas with subsequent fibrosis. Study of this model showed fibrogenesis to be T-cell dependent, apparently mcdiated by a 40 kd fibroblast-activating factor secreted by the inflammatory T ~ e l l s . ~ ~model h i s may help in the elucidation of thc link between an (auto-)immune response and liver fibrosis and cirrhosis. Of the various models for virus-induced liver disease, mention should be made of the model of lymphocytic choriomeningitis virus (LCMV) induced hepatitis in the mouse.37 Hepatitis in this model is mediated by class I restricted cytotoxic T cclls. Various strains are susceptible to LCMV hepatitis to a differing degree. Among the strains that are quite susceptible are the C57BLi6 mice, which may allow studies in the same strain as in EAH. It would be particularly interesting to know whether LCMV hepatitis is associated with autoimmune phenomena.
LESSONS FROM OTHER MODELS OF AUTOIMMUNE DISEASES A multitude of animal models for various human autoimmune diseases have been described, such as experimental arthritis, encephalomyelitis, thyroiditis, diabetes mellitus, systemic lupus erythematosus, or uveitis. Several of the observations made in these models are of relevance to all autoimmune diseases in general and autoimmune hepatitis in particular.
Most of these models are like EAH, based on immunization of healthy animals with autoantigcn in adjuvant. Others, however, can be induced by immunization with a microbial antigen, such as adjuvant arthritis following the immunization with CFA (that is, Mycobacterium ruberculosis in oil) only38." and streptococcal cell wall-induced a r t h r i t i ~The . ~ ~ study of adjuvant arthritis in particular has shown that cross-reactivity between a foreign antigen and self-antigen may lead to autoimmune disease. T-cell clones reactive with a defined cpitope on thc 65 kd heat shock protein of M. tuberculosis were isolated from animals with adjuvant arthritis, and these cells were able to mediate a r t h r i t i ~ . ~ ' . ~ T h experiese ments demonstrate that foreign antigen by presenting the immunc system with cross-reactive epitopes in an immunogenic environment may indeed induce autoimmune disease. Mercury induces in Brown-Norway rats an autoimmune disease that is in particular characterized by a glomerulonephritis, autoantibodies, and autoreactive Tc c l l S , ~ ~This . ~ ~ model thus provides evidence of yet another possible mechanism in the induction of autoimmune disease, namely, through environmental agents such as drugs and toxins. This mechanism may be of particular relevance to liver-kidney microsound (LKM)positive autoimmunc hepatitis, which is associated with autoantibodics against isoenzymes of the drug-metabolizing cytochrome P450 systemJkJhand in the case of ticrynafen-induced hepatitis is clearly drug induced.j7 Experimental models cannot only help to illucidate the pathogenesis of autoimmune diseases, but also open the way for the study of new therapeutic approaches. Experimental protocols include the therapy with monoclonal antibodies specific for T-cell receptor rearrangements of those T-cells primarily responsible for autoimmune disease" and vaccination with attenuated autoreactive Tcell clone^.'^^^^ These studies open the prospects of specific, nontoxic therapies for autoimmune diseases. None of the models of autoimmune hepatitis have so far been used for therapeutic studies. It will be important to know whether EAH is amenable to these new therapeutic interventions.
QUESTIONS FOR THE FUTURE The experimental models of autoimmune hepatitis have been developed sufficiently to allow detailed studies into the pathogenesis, regulation, and therapy of autoimmune hepatitis. The evidence to date is very strong that T cells are responsible, and probably solely responsible for EAH. The subtypes and characteristics of the T-cell subpopulations involved have to be studied. Immunohistochemical studies will have to characterize the inflammatory infiltrates, and these studies may tell us most about the T-cell subpopulations responsible for the hepatitis. Ideally the T cells should be grown as lines and clones. This would also allow the identification of the target antigen or antigens responsible for the disease process. Furthermore, the T-cell regulatory disturbances in EAH have received hardly any attention as yet. As mentioned in this review, there are observations sug-
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MODELS OF AUTOIMMUNE HEPATITIS-LOHSE
gesting the presence of counterregulatory suppressive mechanisms in EAH. Since autoimmune disease probably represents a disequilibrium between autoreactive and suppressive mechanisms,Y it is just as important to study the regulatory suppressive mechanisms as the autoreactive ones. New therapeutic approaches should be of the tested in EAH' Last' but not least, the studies in experimental animals has to be checked in human autoimmune hepatitis. Patients should be tested for T-cell responses against the antigen preparation5 recognized to be relevant in the animals, and T-cell regulatory circuits should be studied in an analogous fashion to the animals. Animal models will never exactly reflect human disease, but animal models can help us to understand the underlying basic mechanisms, help to ask the right questions for the study of human disease. and allow the development of new therapeutic strategies. Acknowletlgtne~~rs. I arn grateful to Prof. Meyer rum Buschenfelde
for helpful discussions. Financial help by the Deutsche Forschungsgemeinschaft is gratefully acknowledged.
REFERENCES Fiessinger N: Histogenise des processus de cirrhose hipatique. Lesion parenchymateuse el cirrhose. In: Maloine A (Ed): Etude histologique experimentale et pathologique. Parls, Thesis. 1908. Casals I . Olitsky PK: Tests for hcpatic dysfunction in ~iiicc. Proc Soc Exp Hiol 63:383-389. 1946. Hehar AJ. Tal C: Expcri~ncntallivcr necrosis produccd by the injection of homologous whole liver with adjuvant. J Pathol Bacterial 77:591-596, 1959. Dodd MC, Bigley NJ, Geyer VB, et al: Autoirnrnune response in rabbits injected with rat liver ribosomes. Science 137:688689, 1962. lliesco M, Berceanu S. Radu I. Hergot L: Recherches sur I'hipatite toxique expCrimentale. 11. ConsidKrations sur les IKsions dans I'hkpatite toxique et dans I'hepatite par isoirnmunisation avec des antigenes hepatiques. Arch Roum Pathol Exp Microbiol 22:41-47. 1963. Coppo M. Tedeschi G: Essais de provocation experimentale (rat) d'une hipatite par autoagression. Gastroenterologia 7:3954. 1964. Scheiffarth F. warn at^ H, Niederer W: Tierexperimentelle Studien zur Pathogenese der chronischen Hepatitis. I. hlitteilung. Morphologische Studien an der Leber nach Sensibilislerung mi1 homologen Leberzellfraktionen. Virchous Arch Pathol Anat 339:358-365, 1965. Kiissling FK, Meyer zum Buschcnfclde KH: Zur lnduktion ciner aktiven chronischen Hepatitis durch heterologe Leberproteine. Virchous Arch Pathol Anat 345:365-376. 1968. Meyer zum Buschenfelde KH, Kiissling FK. Miescher PA: Experimental chronic active hepatitis in rabbits following immunization with human livcr protcins. Clin Exp lmmunol 10:99-108, 1972. Meyer zum Biischenfelde KH, Hopf U: Studies on the pathogenesis of experimental chronic active hepatitis in rabbits. I. Induction of the disease and protective effect of allogeneic liver specific proteins. Br J Exp Pathol 55:498-508, 1974. Hopf U , Meyer zum Buschenfelde KH: Studies on the pathogenesis of experimental chronic activc hepatitis in rabbits. 11. Demonstration of immunoglobulin on isolated hepatocytes. Br J Exp Pathol 55509-5 13, 1974. Hopf U , Meyer zum Buschenfelde KH, Hutteroth TH: Hu-
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morale Im~nunrcaktioncnan der hepatocelluliren Plasmamembran bei dcr cxpcrimentellen chronisch-aktiven Hepatitis in1 Kaninchcn. Klin Worhenschr 54591-598, 1976. 13. Feighery C , McDonald GSA, Greally JF. Weir DG: Histological and immunological investigation of liver-specific protein (LSP) immunized rabbits compared with patients with liver disease. Clin Exp Immunol 45: 143-1 5 1. 1981. 14. Uibo RM. Helin HJ, Krohn KJE: Imlnunological reactions to liver-specific membrane lipoprotein (LSP) in experimental autoimmune liver disease in rabbits. Clin Exu Imrnunol 48:505512. 1982. Feighery C, Weir DG: How specific is liver specific protein'? Gastroenterology 79: 179-1 82, 1980. Scheiffarth F, Warnatz H, Maycr K: Studies concerning the importance of mononuclear cells in the dcvclopment of experimental hepatitis. J lmrnunol 98:396-401, 1967. Kuriki J . Murakami H, Kakumu S , et al: Expcrimcntal autoimrnune hepatitis in mice after irn~uunizationwith syngenelc liver proteins together with the polysaccharide of Klcbsiclla pneumoniae. Gastroenterology 84:596-603, 1983. Nakashima I: Adjuvant action of capsular polysaccharidc of Klehsiella pneumoniae on antibody response. I. Intensity of its action. J lmmunol 108:1009-1016. 1972. Mori Y. Mori T. Yoshida H, el al: Study of cellular immunity in cxperimental autoimmune hepatitis in mice. Clin Exp Inlmunol 57:85-92, 1984. Mori Y. Mori T, Ueda S , et al: Study of cellular immunity in experimental autoirnmune hepatitis in mice: l'ransfer of spleen cells sensitized with liver proteins. Clin Exp lmmunol 61 :577584, 1985. Mori T, Mori Y, Yoshida H. et al: Cell-mediated cytotoxicity of sensiti~edspleen cells against target liver cells-in vivo and in vitro \tudy with a mouse model of experimental autoimmune hepatitis. Hepatology 5:770-777. 1985. Ogawa M. Mori Y, Mori T. et al: Adoptive transfer of experimental autoimmune hepatiti\ in mice
1 1 , NO. 3, 1991
Experimental Models of Autoimmune Hepatitis ANSGAR W. LOHSE, M.D.
From the I . M~riizinischeKlinik und Poliklitlik Johantlr.~Gutrnbrrg U ~ ~ i v e r s i t Muin-, iit Mainz, German!. Reprint requests: Dr. Lohse. I. Medizinische Klinik und Poliklinik, Johanncs Gutenberg Universitlt Mainz, Langenbeckstrcssc 1 , D-6500 Mainz, Germany.
CHRONIC ACTIVE HEPATITIS IN RABBITS In 1968 Kossling and Meyer zum Biischenfelde8 presented a model of chronic hepatitis in rabbits induced by repeated immunization with heterologous liver antigen. This model of chronic hepatitis in rabbits was described in more detail in subsequent studies."' Characteristically, chronic inflammatory lesions could be induced in rabbits by repeated immunization with soluble human liver antigen, but not by immunization with rabbit antigen. Nonetheless, the rabbits did develop antibodies and positive skin tests against rabbit liver antigen in the course of immunization. Although the animals did develop antibodies against liver antigen and membrane-bound immunoglobulin could be detected on isolated hepatocytes, these antibodies did not correlate with histologic disease activity.".i2 These studies suggested that cellular immune mechanisms rather than autoantibodies were likely to be responsible for the histologic injury. Subsequent studies by other groups in a similar rabbit model found marked responses to the immunizing liver antigen preparation in lymphocyte proliferation assays.13 This was true both for the whole preparation called liver-specific protein (LSP) and its subfraction LP2.'"IJ However, these preparations consisted of a multitude of liver proteins.15 The antigen or antigens responsible for this form of experimental hepatitis have thus never really been identified. The major limitations of this model for further immunologic studies was the problem that rabbits are not available as inbred, genetically homogeneous strains. Furthermore, the need for immunization with heterologous antigen and the lack of hepatitis following immunization with alloantigen as well as the nonspecific hepatitis found by some in animals immunized with irrelevant control antigensi3was undesirable for an animal model of autoimmune disease.
EXPERIMENTAL AUTOIMMUNE HEPATITIS IN MICE Scheiffarth et allh were the first to succeed in inducing an experimental hepatitis in inbred mice by immunization with allogeneic liver antigens in adjuvant. ~ 5 7 ~ mice ~ 1 were 6 immunized intramuscularly and intraperitoneally six times in weekly intervals with liver animals dehomOgenate from AKR mice in CFA. veloped mild inflammatory lesions only, but 12 of the 89
Copyright 0 1991 by Thieme Medical Publ~ahers.Inc., 38 1 Park Avenue South, New York, NY 100 16. All rights reserved.
241
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The study of autoimmune diseases in animal models can help in the understanding of the pathogenesis and regulation of human autoimmune diseases. At the same time, it may open new avenues in the prevention and therapy of autoimmune diseases. Various approaches have been taken to establish animal models for the study of autoimmune hepatitis. The establishment of reliable, reproducible, and useful models has been more difficult than in other autoimmune diseases and thus far no model has been recognized as the standard modcl for autoimmune hepatitis. Nonetheless, several lessons have already been learned from the work done to date and continuing work in this field should lead to further progress in the near future. The first attempts to induce an experimental hepatitis in animals by the injection of liver antigen date back to the beginning of the century.' Fiessinger' described cirrhotic changes in rabbits injected intraperitoneally with a nuclear antigen preparation, no histologic changes were seen in guinea pigs injected with liver antigen.' Forty years later, Casals and Olitsky' found inflammatory lesions and focal necroses in four of ten mice injected repeatedly with a mouse liver homogenate. Although these first two studies used antigen without adjuvant, all groups since have used adjuvant for immunization, mostly complete Freund's adjuvant (CFA). The late 1950s and the 1960s saw the publication of a number of reports on the induction of hepatitis by immunization with heterologous, allogeneic, or syngeneic antigen, as summarized in Table 1 . The several unsuccessful attempts of induction of experimental hepatitis by many groups are not cited, since little can be learned from these negative reports. Because immunology was in its infancy at the time of early reports of experimental hepatitis, immunologic information on these early models is missing. This review will therefore concentrate on the two types of experimental hepatitis that have been most thoroughly characterized, namely chronic active hepatitis (CAH) in rabbits and experimental autoimmune hepatitis (EAH) in mice.
SEMINARS IN LIVER DISEASE-VOLUME
I I , NUMBER 3. 1991
TABLE 1. Animal Models of Autoimmune Hepatitis*
Fiess~nger'
Specic,s
Rabbit
Antigc>rr?
Rr.s~i1r.s Biliary cirrhosis in 3 of 7 animals; negative results in dogs and gulnea pigs Periportal inflammation and necroses in 4 of 10 ~ n i c c Early focal necroses in I I of 31 animals (days 3-4) Focal necroses in only 3 of 16 hamsters Periportal infiltrations after I week Hepatocellular necroses
Nuclear protein\
Casals and Olitsky? Behar and Tal'
Mouse
--
Guinca pig hamster
Homo. LH
CFA
Dodd et al-' lliesco et ali Coppo and Tedeschih Scheiffarth et al' Scheiffarth ct al'"
Rabbit Rabbit
Rat LH Horno. LH
CFA CFA
Kossling et a1 (8-1 2)
Rabbll
Kuriki et al"
Moue
Mori ct ?? Watdnabe et a]'O '7
Moube
Lohse et alL"'J
Mouse
Rat
CFA
Rabbit Mouse
Mou\e
CFA
Sy ng . S- I00 Syng. S-100
CFA CFA
Syng. S-100
At least 12 x sc 9 x sc
Focal ~nflammatorychanges and necroses Periportal infiltrations and necroses (in sonic animals) Periportal infiltrations and necroses, no lesions with kidney antigen; ~uccessfulpasslve transfer Chronic hepatitis: no hepatitis following irnrnunization with rabbit LSP Frequent i m m u n i ~ a t i o n smore effective. passivc transfer ot discasc possible Passive transfer possible, T-cell mediated disease, specific cytvtoxity Thymcctomy fac~litateddi\ea\e induction and prolonged d ~ s e a s e duration Subacute '1'-cell mediated hepat~tis. sptcific T-cell reactivity. autoantibodies follow disease
*Only those experiments in which experimental animals showed different and niore severe changes than the control unirnals are mentioned. Some early studies are included for historical reasons despite the lack of suitable controls. TLH: liver hornogenate; S-100: 100.000 g m supernatant of liver hornogenate: LSP: liver specific protein: CFA: complete Frcund'h adjuvanl; ip: intrapcritoncally; 1111:intra~nuscularly;sc: subcutaneously; homo: homologous: hum: human: syng: synpeneic.
mice studied developed moderate to severe periportal inf i l t r a t i o n ~ .CFA ' ~ alone or immunization with kidney homogenate in CFA did only rarely lead to mild or noder rate inflammatory responses. The most important finding of this pioneering work was the adoptive transfer of the hepatitis with spleen cells from diseased animals. '' This finding strongly suggested that cell-mediated immunity was responsible for this model of experimental hepatitis. No reasons were given why liver from AKR mice rather than from syngeneic mice was used. It is thus not possible to assess whether this was a truly autoimmune process or whether allogencic immune responses may have played a role in the pathogenesis of the observed lesions. It was only in the 1980s that the importance of studying inbred animals in immunologic research was fully appreciated by researchers in the field of immuncmediated liver disease. In 1983 Kuriki et all7 presented a mouse model of autoimmune hepatitis following immunization with syngeneic liver antigen. Hepatitis in this model was induced in SMA mice with LSP-enriched liver homogenate. Because of their good experience with the application of the polysaccharide of Kleb.siellu pt~euinoniue as adjuvant, these authors used this adjuvant instead of CFA.IXImmunization was done intramuscularly at monthly intervals up to a total of 1 I injections. Mild to moderate inflammatory lesions could be observed in
all mice immunized with liver antigen in adjuvant: no lesions or minimal lesions only were observed in the groups receiving liver antigen only, adjuvant only, or kidney antigen in adjuvant." More severe lesions were observed in those animals immunized 8 to 1 1 times compared with those immunized three to six times. Minimal lesions were also observed in the kidneys, whereas other organs appeared to be unaffected. Spleen cells from diseased animals could transfer hepatitis to irradiated recipient mice." The results of transfer into nonirradiated naive recipients were not reported. Kuriki et all7 found that their animals did develop antibodies against liver antigens, but did not describe whether antibody development and titers correlated with hepatitis. They also observed a very interesting phenomenon only in those mice that received liver antigen in adjuvant: splenocytes from these animals markedly suppressed the mitogen-stimulated proliferation of normal spleen cells (17). The interpretation of this finding is difficult. It appears that the animals' immune system noticed the aberrant autoimmune response to liver self-antigen and may have initiated a counterregulatory immune response leading to a generalized suppression. Mori et al1"-" in a series of publications have studied EAH in mice in considerable detail. This group of investigators used syngeneic 100,000 gm supernatant of freshly prepared liver homogenate as immunogen. Pro-
~
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Autl~or
tein, 10 mg, was injected intramuscularly in CFA. Immunization was repeated at weekly intervals. The initial article describes six consecutive injection^;'^ later on only four injections were used.".'2 Male C57BLl6, C3HI He, and BALBiC mice were studied. Hepatitis could best be induced in C57BLl6 mice, the same strain that was used in the original study by Scheiffarth et a1.16 The ability of the antigen preparation to induce hepatitis was quickly lost by freezing and thawing the material. '' The mice developed antibodies against the antigen preparation, which did not react with the material that had been frozen and thawed more than three times. No further information on the character of the antigen or antigens responsible for the experimental hepatitis has become available as yet. Subsequent passive transfer experiments have given contradictory results." Unseparated spleen cells and nylon wool column nonadherent, T-cell enriched spleen cells could transfer mild hepatitis to naive recipients to a similar degree, whereas the nylon wool adherent fraction was able to transfer disease more effectively. However, when this nylon wool column adherent (with supposedly few T cells to start with) spleen cell population was further depleted of T cells by treatment with the pan-T-cell antibody anti-Thy 1.2 and guinea-pig complement, the ability to transfer disease was almost completely lost. Thus, although the first finding suggests that cells other than T cells were responsible for the disease transfer, the latter finding shows T cells to be of critical importance. It is difficult to interpret these findings. Two explanations seem possible. The most likely possibility is that several cell populations are involved-ii the pathogenesis of this experimental model. T cells are a prerequisite for the disease, but may require other cells, predominantly found in the nylon wool adherent compartment, for disease induction. This possibility is supported by earlier findings demonstrating that T cells are required in the mouse for the production of anti-LSP antib~dies.'~ The other possible interpretation is that the T-cell enriched fraction included, like the unseparated spleen cells, a counterregulatory suppressor cell population that was less predominant in the nylon wool adherent cell population. In view of the findings by Kuriki et all7 demonstrating active suppression in animals with EAH, this possibility deserves further study. Support is given to this interpretation by the subsequent finding in the model of Mori et that passive transfer of hepatitis was facilitated by prior irradiation of donor mice with 300 rad,'2 a procedure known preferentially to inactivate suppressor lymphocytes. Mori et al. also tested the proliferative response of splenocytes from their animals against LSP. No proliferative response against the ethylene diaminetetraacetic acid (EDTA)-containing LSP preparation (isolated as described by McFarlane et could be measured, but EDTA-free LSP was able to stimulate spleen cells in a standard T-cell proliferation assay.19 However, spleen cells from normal, untreated C57BLl6 mice and immunized animals showed similar proliferative responses." The role of antigen specific T-cell responses is thus not entirely clear. It should be remembered that Mori et al immunized with crude liver proteins, the 100,000 gm supernatant of liver homogenate. This antigen prepara-
tion will have included several antigens not present or present in only very small amounts in the LSP preparation. T cells from the experimental hepatitis animals may have been directed against other antigens not tested in their proliferation assay. Cytotoxicity studies by the same group21 suggests that specific cytotoxic T cells preferentially found in the nylon wool adherent spleen cell population of mice with experimental hepatitis were of pathogenetic importance. Specific lysis of syngeneic hepatocytes, but not renal cells, was found to be present 1 and 2 weeks after the last immunization.'' Taken together these studies present an apparently T-cell mediated model of EAH. Araki et al" presented a further murine model of Tcell mediated EAH in BlO.A(SR) mice. However, in their model low-dose irradiation to decrease the suppressor cell activity in the experimental animals was required to induce hepatitis. In addition, for moderate hepatitis to develop up to eight immunizations spread over a period of 6 months were required, making this model rather unattractive for further pathogenetic studies. Nonetheless, the adoptive transfer experiments give further support for the importance of T cells in the pathogenesis of autoimmune hepatitis.'" Watanabe and colleagues'"~" examined the effect of neonatal thymcctomy on the induction and persistence of EAH. Hepatitis was induced by a similar protocol to that used by Mori et al, except that A/J mice were used for their studies. It was found that neonatal thymectomy both facilitated the induction of hepatitis2' and led to persistence of the d i ~ e a s e . 'Transfer ~ of normal spleen cells, but not the transfer of spleen cells from thymectomized mice, abolished the effect of t h y m e ~ t o m y These . ~ ~ studies suggest that normal spleen cells have a suppressive ability against EAH and that this suppressor activity is lost by neonatal thymectomy. We have recently presented a simplified model of EAH in mice.28 Hepatitis was likewise induced by immunization with the 100,000 gm supernatant (called S100) of freshly prepared syngeneic liver homogenate in CFA. C57BLl6 mice were more susceptible than C3H or BALBIC mice, and no hepatitis could be induced in Lewis rats. The study of various modes and schedules of immunizaticln showed that intraperitoneal immunization was more effective than intramuscular or subcutaneous immunization. Three weekly injections led to moderate hepatitis in almost all animals, but a similar degree of inflammatory damage could be achieved with just a single intraperitoneal immunization with syngeneic S- 100 antigen (Fig. 1). Severity of EAH induced by a single immunization peaked around 3 to 4 weeks after the injection, but mild to moderate hepatitis was still detectable 6 months after disease i n d u ~ t i o nBiochemical .~~ evidence (raised transaminases) of liver cell damage was detectable mainly during the first 6 weeks of EAH (Fig. 2). EAH could be transferred by activated T cells from diseased animals. As in the other studies, adoptively transferred EAH was milder and more transient than the antigen-induced active disease.28Studying T-cell reactivity further, it became clear that the S-100 antigen preparation was toxic in cell culture studies. The S-100 antigen was therefore separated into three peaks over a CL-6B Sepharose column. The second peak seemed to retain
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MODELS OF AUTOIMMUNE HEPATITIS-LOHSE
SEMINARS IN LIVER DISEASE-VOLUME
11, NUMBER 3, 1991
at the peak of inflammatory lesions, no or almost no autoantibodies were detectable (Fig. 2). Yet, when, histologically, hepatitis was improving and when there was no more biochemical evidence of ongoing hepatocyte destruction, autoantibodies continued to develop and titers continued to rise.29These results present strong evidence for T cells being solely responsible for EAH.
FIG. 1. Liver histology at week 3 of experimental autoimmune hepatitis induced in a male C57BLl6 mouse by a single immunization with syngeneic soluble liver antigen (S100) in complete Freund's adjuvant.
the toxic component of the S-100 preparation. T-cell reactivity against the first and third peaks was very marked in animals with EAH (Fig. 3), and no response was observed in control animals. BALBIC mice, which were less susceptible to EAH, showed a weaker T-cell response to peaks 1 and 3 than the more susceptible C57BL!6 mice." Future studies will have to show where to look further for the target antigens of T cells in EAH. In our model of EAH mice developed characteristic a~toantibodies.~" Rather than producing a multitude of antibodies against the crude liver immunogen. S-100, only a few specific autoantibodies developed. The autoantibody pattern differed from that seen in most cases of human autoimmune hepatiti~.'~."The target structures recognized by these autoantibodies are possible target antigens for T-cell autoreactivity in the EAH mice. Interestingly, autoantibodies developed only weeks after histologic and biochemical evidence of hepatitis. Indccd
Numerous studies of toxic, infectious, and genetically induced hepatitis have been described. Those with pohsible relevance to the study of autoimmune liver disease will be reviewed briefly. Poralla et a13?were able to induce hepatitis in rabbits by injecting monoclonal antibody to rabbit liver antigen.'? The antibodies were raised by immunization with LSP. Several antibodies with various reactivity patterns could be isolated, thus confirming the heterogeneity of the preparation called LSP.'? One of these antibodies showing membrane reactivity was able to induce transient liver cell damage in rabbits. These experiments show that antibody-mediated cytotoxicity is a possible mechanism of hepatitis in immune-mediated liver disease (both viral and autoimmune). Two rat strains developing spontaneous hepatitis have been d e ~ c r i b e d . ' " ~These ~ arc substrains of the Long-Evans strain, named LEA and LEC, bred at Hokkaido University in Japan. Hepatitis develops around the age of 3 to 4 months. The lack of inflammatory response, however, suggests that this genetically determined disease is not in any way immune mediated. However, it is precisely this lack of inflammatory response that should deserve further study. In view of the abundant liver cell necroses with the subsequent release of a multitude of intracellular antigens into the circulation and thus into the arms of the immune system, it would be interesting to know which systemic immune reactions
T i m e c o u r s e of EAH i n d u c e d by a s i n g l e i m m u n i z a t i o n
= ---t
Histology
GPT
-
120
- 100 -80
$
- 60
- 40 L 20
0
Autoantibodies:
weeks
10
-
(+I
+
++
20
+++
FIG. 2. Time course of histologic and biochemical changes and occurrence of autoantibodies in experimental autoimmune hepatitis (EAH) in C57BL16 mice induced by a single intraperitoneal immunization with 2.5 mg of S-100 antigen in complete Freund's adjuvant (CFA).
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OTHER FORMS OF EXPERIMENTAL HEPATITIS
PROLIFERATIVE RESPONSE OF EAH SPLENOCYTES
K?
peak 1
peak 2
peak 3
MT
BP
Antigen (1 0 rnicrograrnWrnl)
FIG. 3. T-cell responses to fractions of the immunizing S100 antigen preparation (peaks 1-3), M. tuberculosis (MT) as part of the complete Freund's adjuvant (CFA) and control antigen myelin basic protein 1 week after induction of experimental autoimmune hepatitis (EAH).
to liver antigen are or are not induced. This would help us to differentiate to what extent autoantibodies and Tcell autoreactivity can develop simply in response to antigen release following necrosis. Intraperitoneal injection of group A streptococcal cell walls leads to hepatic granulomas with subsequent fibrosis. Study of this model showed fibrogenesis to be T-cell dependent, apparently mcdiated by a 40 kd fibroblast-activating factor secreted by the inflammatory T ~ e l l s . ~ ~model h i s may help in the elucidation of thc link between an (auto-)immune response and liver fibrosis and cirrhosis. Of the various models for virus-induced liver disease, mention should be made of the model of lymphocytic choriomeningitis virus (LCMV) induced hepatitis in the mouse.37 Hepatitis in this model is mediated by class I restricted cytotoxic T cclls. Various strains are susceptible to LCMV hepatitis to a differing degree. Among the strains that are quite susceptible are the C57BLi6 mice, which may allow studies in the same strain as in EAH. It would be particularly interesting to know whether LCMV hepatitis is associated with autoimmune phenomena.
LESSONS FROM OTHER MODELS OF AUTOIMMUNE DISEASES A multitude of animal models for various human autoimmune diseases have been described, such as experimental arthritis, encephalomyelitis, thyroiditis, diabetes mellitus, systemic lupus erythematosus, or uveitis. Several of the observations made in these models are of relevance to all autoimmune diseases in general and autoimmune hepatitis in particular.
Most of these models are like EAH, based on immunization of healthy animals with autoantigcn in adjuvant. Others, however, can be induced by immunization with a microbial antigen, such as adjuvant arthritis following the immunization with CFA (that is, Mycobacterium ruberculosis in oil) only38." and streptococcal cell wall-induced a r t h r i t i ~The . ~ ~ study of adjuvant arthritis in particular has shown that cross-reactivity between a foreign antigen and self-antigen may lead to autoimmune disease. T-cell clones reactive with a defined cpitope on thc 65 kd heat shock protein of M. tuberculosis were isolated from animals with adjuvant arthritis, and these cells were able to mediate a r t h r i t i ~ . ~ ' . ~ T h experiese ments demonstrate that foreign antigen by presenting the immunc system with cross-reactive epitopes in an immunogenic environment may indeed induce autoimmune disease. Mercury induces in Brown-Norway rats an autoimmune disease that is in particular characterized by a glomerulonephritis, autoantibodies, and autoreactive Tc c l l S , ~ ~This . ~ ~ model thus provides evidence of yet another possible mechanism in the induction of autoimmune disease, namely, through environmental agents such as drugs and toxins. This mechanism may be of particular relevance to liver-kidney microsound (LKM)positive autoimmunc hepatitis, which is associated with autoantibodics against isoenzymes of the drug-metabolizing cytochrome P450 systemJkJhand in the case of ticrynafen-induced hepatitis is clearly drug induced.j7 Experimental models cannot only help to illucidate the pathogenesis of autoimmune diseases, but also open the way for the study of new therapeutic approaches. Experimental protocols include the therapy with monoclonal antibodies specific for T-cell receptor rearrangements of those T-cells primarily responsible for autoimmune disease" and vaccination with attenuated autoreactive Tcell clone^.'^^^^ These studies open the prospects of specific, nontoxic therapies for autoimmune diseases. None of the models of autoimmune hepatitis have so far been used for therapeutic studies. It will be important to know whether EAH is amenable to these new therapeutic interventions.
QUESTIONS FOR THE FUTURE The experimental models of autoimmune hepatitis have been developed sufficiently to allow detailed studies into the pathogenesis, regulation, and therapy of autoimmune hepatitis. The evidence to date is very strong that T cells are responsible, and probably solely responsible for EAH. The subtypes and characteristics of the T-cell subpopulations involved have to be studied. Immunohistochemical studies will have to characterize the inflammatory infiltrates, and these studies may tell us most about the T-cell subpopulations responsible for the hepatitis. Ideally the T cells should be grown as lines and clones. This would also allow the identification of the target antigen or antigens responsible for the disease process. Furthermore, the T-cell regulatory disturbances in EAH have received hardly any attention as yet. As mentioned in this review, there are observations sug-
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MODELS OF AUTOIMMUNE HEPATITIS-LOHSE
gesting the presence of counterregulatory suppressive mechanisms in EAH. Since autoimmune disease probably represents a disequilibrium between autoreactive and suppressive mechanisms,Y it is just as important to study the regulatory suppressive mechanisms as the autoreactive ones. New therapeutic approaches should be of the tested in EAH' Last' but not least, the studies in experimental animals has to be checked in human autoimmune hepatitis. Patients should be tested for T-cell responses against the antigen preparation5 recognized to be relevant in the animals, and T-cell regulatory circuits should be studied in an analogous fashion to the animals. Animal models will never exactly reflect human disease, but animal models can help us to understand the underlying basic mechanisms, help to ask the right questions for the study of human disease. and allow the development of new therapeutic strategies. Acknowletlgtne~~rs. I arn grateful to Prof. Meyer rum Buschenfelde
for helpful discussions. Financial help by the Deutsche Forschungsgemeinschaft is gratefully acknowledged.
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morale Im~nunrcaktioncnan der hepatocelluliren Plasmamembran bei dcr cxpcrimentellen chronisch-aktiven Hepatitis in1 Kaninchcn. Klin Worhenschr 54591-598, 1976. 13. Feighery C , McDonald GSA, Greally JF. Weir DG: Histological and immunological investigation of liver-specific protein (LSP) immunized rabbits compared with patients with liver disease. Clin Exp Immunol 45: 143-1 5 1. 1981. 14. Uibo RM. Helin HJ, Krohn KJE: Imlnunological reactions to liver-specific membrane lipoprotein (LSP) in experimental autoimmune liver disease in rabbits. Clin Exu Imrnunol 48:505512. 1982. Feighery C, Weir DG: How specific is liver specific protein'? Gastroenterology 79: 179-1 82, 1980. Scheiffarth F, Warnatz H, Maycr K: Studies concerning the importance of mononuclear cells in the dcvclopment of experimental hepatitis. J lmrnunol 98:396-401, 1967. Kuriki J . Murakami H, Kakumu S , et al: Expcrimcntal autoimrnune hepatitis in mice after irn~uunizationwith syngenelc liver proteins together with the polysaccharide of Klcbsiclla pneumoniae. Gastroenterology 84:596-603, 1983. Nakashima I: Adjuvant action of capsular polysaccharidc of Klehsiella pneumoniae on antibody response. I. Intensity of its action. J lmmunol 108:1009-1016. 1972. Mori Y. Mori T. Yoshida H, el al: Study of cellular immunity in cxperimental autoimmune hepatitis in mice. Clin Exp Inlmunol 57:85-92, 1984. Mori Y. Mori T, Ueda S , et al: Study of cellular immunity in experimental autoirnmune hepatitis in mice: l'ransfer of spleen cells sensitized with liver proteins. Clin Exp lmmunol 61 :577584, 1985. Mori T, Mori Y, Yoshida H. et al: Cell-mediated cytotoxicity of sensiti~edspleen cells against target liver cells-in vivo and in vitro \tudy with a mouse model of experimental autoimmune hepatitis. Hepatology 5:770-777. 1985. Ogawa M. Mori Y, Mori T. et al: Adoptive transfer of experimental autoimmune hepatiti\ in mice
