Autoimmune hepatitis (AIH) is characterized by an immune-mediated injury of the hepatic parenchyma of unknown pathogenesis. Type 2 AIH is identified by the presence of anti-liver-kidney microsomes type 1 (anti-LKM1) and anti-liver cytosol type 1 (anti-LC1) autoantibodies. The current study shows that a murine model of AIH can be generated by DNA immunization against type 2 AIH self-antigens (P450 2D6 and formiminotransferase-cyclodeaminase). A pCMV plasmid containing the N-terminal region of mouse CTLA-4 and the antigenic region of human CYP2D6 (672-1,377 bp) and human formiminotransferase cyclodeaminase (FTCD; 1,232-1,668 bp) was used for DNA immunization of C57BL/6 female mice. Immunized mice showed elevated levels of alanine aminotransferase (ALT), with peaks at 4 and 7 months postinjection. Periportal, portal, and intralobular liver inflammatory infiltrates were observed at histology. Mainly CD4+ lymphocytes, but also CD8+ and B lymphocytes, were found in the liver. Cytotoxic-specific T cells were found in both the liver and spleen of these animals. Mice developed anti-LKM1 and anti-LC1 antibodies of immunoglobulin G2 (IgG2) subclass, against specific mouse autoantigens. The ALT levels correlated with both the presence of anti-LKM1/anti-LC1 antibodies and the presence of liver necroinflammation. In conclusion, in mice, DNA immunization against human autoantigens breaks tolerance and induces an autoimmune liver disease. Molecular mimicry between foreign and self-antigens explains the liver injury. This model of AIH resembles human type 2 AIH and will be helpful for the study of its pathogenesis. (HEPATOLOGY 2004;39:1066–1074.)
Autoimmune hepatitis (AIH) is a disorder of unknown etiology characterized by an immune-mediated injury that gradually destroys the hepatic parenchyma. Most patients have circulating autoantibodies, which are considered to be specific markers of the disease.1 Two types of AIH are recognized, according to the autoantibodies found in sera samples. Type 1 AIH is characterized by the presence of anti-smooth muscle and/or anti-nuclear antibodies, whereas type 2 AIH shows anti-liver-kidney microsomal type 1 (anti-LKM1) and/or anti-liver cytosol type 1 (anti-LC1) antibodies.2–4 Previous work has shown that the target of LC1 antibodies is formiminotransferase cyclodeaminase (FTCD)5 and that anti-LKM1 antibodies are directed against P450 2D6 (CYP2D6).6 These two enzymes are mainly expressed in hepatocytes. Titers of these autoantibodies show a good correlation with AIH activity and it has been speculated that they might play a role in the pathogenesis of this inflammatory disease.7 Information obtained from clinical data can be applied to develop an animal model that will give new and broader insights into the relative importance of different factors involved in the pathogenesis of AIH.
Several murine models of AIH have been described, but none of them are completely satisfactory. One of these models showed the development of a subacute hepatitis after immunization with a liver subcellular fraction.8 Other models are based on the breaking of immune homeostasis by overexpression of interferon gamma,9 a proinflammatory cytokine, in the liver or by the deficiency of transforming growth factor-β1, an immunomodulatory cytokine.10 A transgenic mouse model expressing the main epitope of the lymphocyte choriomeningitis virus (LCMV)-glycoprotein under the control of the albumin promoter was produced.11 This promoter supported the expression of the protein not only in the liver but also in the thymus, resulting in the deletion of most T-specific cells and the development of tolerance. Therefore, LCMV infection did not trigger chronic hepatitis. In this model, AIH was observed after transplant of specific cytotoxic lymphocytes, and only in double transgenic mice (LCMV-glycoprotein and hepatitis B surface antigen). Hepatitis B surface antigen expression renders hepatocytes sensitive to cell death induced by different cytokines.11
Recently, a transgenic mouse expressing LCMV-nucleoprotein (NP) under the control of the liver-specific transthyretin promoter was generated. This animal developed an immune-mediated liver injury after vaccination with plasmids coding for NP. This study showed that AIH can be triggered by molecular mimicry and that activated T lymphocytes in the periphery migrated and caused cytolysis of targeted cells.12 Based on these facts, a new murine model of AIH was developed by molecular mimicry against self-antigens in naive mice. Aiming to reproduce type 2 AIH, CYP2D6 and FTCD were used as antigens. Cross-species immunization13–16 has proved to be successful in producing animal models of autoimmune diseases14, 16, 17 or inducing immune response against cross-species viral18 or cancer antigens.19, 20 Mice vaccinated with a plasmid coding for human CYP2D6 and FTCD developed a specific immune reaction and, consequently, AIH. We describe the development of this experimental model and its similarities with human type 2 AIH.
Complementary DNA (cDNA) Construction and DNA Vaccination.
The DNA vaccination was done using the pRc/CMV vector (Invitrogen, Carlsbad, CA) for expression in eukaryotic cells. All the plasmids were propagated in Escherichia coli using standard techniques and purified using the Endofree Plasmid Giga kit (Qiagen, Santa Clarita, CA), according to the manufacturer's guidelines. The pVR-interleukin-12 (IL-12) plasmid was kindly provided by G. Prud'homme (Montreal, Canada).21
CTLA-4-CYP2D6-FTCD cDNA was constructed first in pCRII-TOPO and then transferred to pCMVand cDNA coding for the mouse CTLA-4 extracellular region was obtained by polymerase chain reaction amplification of a fragment of the full-length cDNA in pUC9 (American Type Culture [ATCC], Manassas, VA). The following primers were used: 5′-GCT CTA GAA CTA GTG AAT TCC CGG GG-3′ and 5′-CCA AGA ATG CAC AGT AGA ATC CGG GCA TGG-3′. This polymerase chain reaction fragment was then cloned using the pCRII-TOPO TA cloning Kit (Invitrogen). To receive the CYP2D6-FTCD fragment, the pCRII-TOPO-CTLA-4 vector was digested with Bsm1 and Xba1 restriction enzymes (New England Biolabs, Beverly, MA) and gel purified with the QIAQUICK gel extraction kit (Qiagen). The CYP2D6-FTCD cDNA cloned in pMAL-cR122 was digested with Bsm1 and Xba1 restriction enzymes. The DNA fragment containing the CYP2D6-FTCD was gel purified and cloned in the Bsm1 and Xba1 restriction sites in pCRII-TOPO-CTLA-4. The complete CTLA-4-CYP2D6-FTCD cDNA was removed from pCRII-TOPO with HindIII and Xba1 and cloned in the corresponding sites in pRc/CMV.
Female C57BL/6 mice, 6 to 8 weeks old, were injected under general anesthesia in the anterior tibialis muscle with 100 μg (50 μL) of plasmid DNA dissolved in saline buffer. The mice were separated in four groups according to the plasmid injected: (1) pCMV alone (n = 5); (2) pVR-interleukin-12 (IL-12) alone (n = 5); (3) pCMV-CTLA-4-CYP2D6-FTCD alone (n = 5); or (4) pCMV-CTLA-4-CYP2D6-FTCD and pVR-IL-12 (n = 46). Mice were injected three times, 2 weeks apart. Some animals were lost during anesthesia (n = 5) at different times. No animals were lost to organ failure or other manifestation of an extrahepatic condition attributable to inflammation.
In Vitro Transcription-Translation of pCMV-CTLA-4-CYP2D6-FTCD.
In vitro transcription-translation of pCMV-CTLA-4-CYP2D6-FTCD was performed using the RiboMAX large-scale RNA production system and the rabbit reticulocyte lysate system (Promega, Madison, WI), according to the manufacturer's instructions. In vitro synthesized and [35S] methionine-labeled CTLA-4-CYP2D6-FTCD fusion protein was immunoprecipitated with patient's serum (positive for anti-LKM1 and anti-LC1 autoantibodies), according to a previously published protocol.5 The immunoprecipitate was analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by autoradiography.
Preparation of Mouse Liver Homogenate.
C57BL/6 normal mouse liver samples were homogenized with a Potter-Elvehjem in lysis buffer (1% Nonidet P40, 1% deoxycholate (DOC), 0.1% SDS, 150 mmol/L NaCl, 10 mmol/L Tris-HCl, pH 7.4) at 4°C. The crude homogenate was centrifuged at 500g for 10 minutes and the supernatant was removed. The cleared homogenate was then analyzed by SDS-PAGE.
Western Blot Analysis.
Proteins (30 μg of mouse liver homogenate or 2 μg of recombinant fusion proteins) were separated by electrophoresis on 10% SDS-PAGE and transferred to nitrocellulose filters (Amersham Life Sciences, Oakville, Canada). The membrane was blocked with 5% powder milk in TBST (10 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 0.05% Tween 20). The nitrocellulose filter was then incubated for 2 hours at room temperature with the first antibody in TBST/1% powder milk. The secondary antibody used was conjugated to peroxidase (Biosource International, Camarillo, CA), diluted to 1:10,000 in TBST/1% powder milk. Bound peroxidase was detected with chemiluminescence blotting substrate (Boehringer Mannheim, Germany) according to the manufacturer's instructions.
Enzyme-Linked Immunosorbent Assay (ELISA).
ELISA was performed as described,22 using the fusion protein coded by pMAL-cR1-CYP2D6-FTCD and purified in a maltose-Sepharose column (New England Biolabs). The purity of the preparation was assessed by SDS-PAGE and the protein was quantified by ultraviolet spectroscopy with bovine serum albumin as the standard. Briefly, microwell plates were coated with 0.2 μg per well of CYP2D6-FTCD fusion protein in 0.1 mol/L NaHCO3, pH 8.6, overnight at 4°C. After blocking with 2% bovine serum albumin in phosphate-buffered saline for 1 hour at 37°C, 200 μL of mouse sera sample was loaded under different dilutions. The presence of anti-CYP2D6-FTCD antibodies was revealed by incubation with anti-mouse immunoglobulin G (IgG)/alkaline phosphatase-conjugated antibodies at a dilution of 1:2,000 (Biosource). Alkaline phosphatase was developed by incubation with p-nitrophenyl phosphate and the result read at 405 nm. Optical densities (ODs) were compared for serum samples from different groups of vaccinated mice. A serum sample was considered positive if its specific OD was at least two times higher than the mean OD of the preimmune mice serum samples. The same technique was applied to establish the IgG subclasses. Briefly, mice serum samples were tested at a dilution of 1:50, followed by alkaline phosphatase-conjugated anti-IgG1, anti-IgG2a, anti-IgG2b, and IgM (Santa Cruz Biotechnology, Santa Cruz, CA), at a dilution of 1:1,000.
Immunohistochemistry and Histopathology.
Vaccinated mice were sacrificed 8 to 10 months after the last plasmid injection. Their liver samples were dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin-eosin. The livers were also tested by immunohistochemistry experiments to characterize the lymphocyte infiltrate using antibodies specific for mouse CD3, CD4, CD8, and CD23 (Santa Cruz Biotechnology) using published protocols.23
Analysis of Liver and Spleen CTLs Against CYP2D6 and FTCD.
EL4, an H-2b lymphoma T-cell line (ATCC), was used as the target. Briefly, 1 × 104 target cells were left in contact with the CYP2D6-FTCD fusion protein for 24 hours at 37°C. These cells were then incubated with serial dilutions of 2.5 × 103 to 2.5 × 104 splenocytes and/or liver isolated lymphocytes (effector cells) in a final volume of 200 μL. After 5 hours of incubation at 37°C, the release of lactate dehydrogenase was measured at 490 nm using the CytoTox 96 assay kit (Promega), according to the manufacturer's guidelines. Maximum and spontaneous release values were determined by incubating the cells with lysis solution and culture medium, respectively.
Serum Alanine Aminotransferase (ALT) Activity.
Serum ALT levels were measured in a Beckman-Synchron CX9 apparatus 1 to 9 months after the last plasmid injection.
Statistical significance was evaluated by the Student's t test and P < .05 was considered significant.
A chimeric cDNA sample was constructed with the mouse CTLA-4 cDNA coding for the extracellular domain, the minimal antigenic region of human CYP2D6,22 and the antigenic region of human FTCD5 (Fig. 1A). The CTLA-4 portion of the chimeric cDNA sample was used to enable the secretion of the chimeric protein.12 This construction was verified by in vitro expression and the chimeric protein was immunoprecipitated with anti-LC1 and anti-LKM1 human sera (Fig. 1B). This plasmid, pVR-IL-12, and the pCMV vector were used to immunize 8-week-old C57BL/6 female mice at 2-week intervals (Fig. 1C).
Characterization of Liver Disease.
The ALT activity level was measured up to 12 months and was used to monitor the activity of the liver inflammation. Increase in ALT activity was not found in control mice (pCMV and pVR-IL-12). However, three of five (60%) mice in the pCMV-CTLA-4-CYP2D6-FTCD group and 37 of 46 (77%) mice in the pCMV-CTLA-4-CYP2D6-FTCD+pVR-IL-12 group showed ALT levels at more than 2 SD above normal in C57BL/6 mice. As described in Fig. 2, the vaccination with pVR-IL-12 and pCMV-CTLA-4-CYP2D6-FTCD gave the highest ALT levels. Two ALT peaks, at 4 and 7 months, were observed for mice injected with both plasmids and to a lesser extent with the mice injected with pCMV-CTLA-4-CYP2D6-FTCD alone. These findings are compatible with the physiological role of IL-12, which is to increase the B-cell and T-cell–mediated response.
Histopathological examination of mice liver samples with elevated levels of ALT (8-9 months after the last injection) showed inflammatory infiltrates in the portal tracts and interface hepatitis (Fig. 3, mice 1 and 2), small foci of interlobular inflammation, and necrosis. The grading score of liver biopsy samples was between 8 and 12.24, 25 ALT levels in serum correlate with the presence of inflammation (P = .001) as revealed by liver histology. No mouse in the control groups showed any inflammation (Figs. 2 and 3, control). No fibrosis was observed in any of the mice.
To characterize the lymphocyte infiltrate, specific antibodies were used (anti-CD3, anti-CD4, anti-CD8, and anti-CD23; Fig. 3). The majority of the T lymphocytes were CD4+. These cells were distributed throughout the infiltrate. CD8+ cells were found mainly in the interface and perivascular areas of the portal tract. B lymphocytes were also present sparsely in the liver inflammatory infiltrate. To confirm that these lymphocytes were specific for either the CYP2D6 and/or FTCD protein, lymphocytes were isolated from the spleen and/or the liver of five animals with elevated ALT levels and their cytotoxic T-cell (CTL) activity was measured. We found two animals with detectable CTL activity, at an effector/target ratio of 2.5:1. T cells isolated from the spleen of one mouse showed 8,3% specific lysis. Isolated liver lymphocytes from the other mouse showed 3% specific lysis. These preliminary results indicate that CYP2D6 and/or FTCD-specific lymphocytes can be activated and found in the liver of vaccinated C57BL/6 mice.
Characterization of the B-Lymphocyte Immune Response.
A specifically developed ELISA test allowed the simultaneous detection of anti-LKM1 and anti-LC1.22 These results are summarized in Fig. 4. Higher titers of combined anti-LKM1 and anti-LC1 were found in mice immunized with IL-12 compared with the group immunized without IL-12. IL-12 also affected the timing of the increase in antibodies titers, which appeared 1 month earlier than in the group without IL-12. A slight increase in titers at 7 months postinjection for both groups was observed. The levels of ALT correlate well with the presence or absence of anti-LKM1/anti-LC1 antibodies (P = .0067). To further characterize the B-cell response, IgG subclasses were determined and the results are summarized in Fig. 5A . The predominance of the IgG2 subclass is compatible with a Th1 immune response and seems to be independent of the adjunction of IL-12 which is known to orient the immune response toward a Th1 phenotype.
The reactivity of serum samples from each group of mice was evaluated by ELISA and Western blot with either the fusion protein (CYP2D6-FTCD), CYP2D6 alone, or FTCD alone. Results are summarized in Table 1. As expected, ELISA is more sensitive than Western blot analysis. In addition, ELISA and Western blot results showed that human FTCD is less antigenic than human CYP2D6 in C57BL/6 mice.
Table 1. Characterization of the B Cell Response for the Four Groups of Mice Used
Plasmids Used for Injection
Western Blot (n)
Fusion Protein Only
Fusion Protein + CYP2D6
Fusion Protein + CYP2D6 + FTCD
Fusion Protein Only
Fusion Protein + CYP2D6
Fusion Protein + CYP2D6 + FTCD
NOTE. This table describes the results obtained when mouse sera was tested by either western blot or ELISA, against CYP2D6-FTCD, CYP2D6 and FTCD. Mouse sera reacted mainly against the fusion protein (CYP2D6-FTCD) by ELISA. Reactivity against the denatured antigens as tested by western blot was infrequent.
CTLA4-CYP2D6-FTCD + IL12
Antibodies detected by ELISA recognized the human proteins used for the immunization. To evaluate autoantibodies, serum samples from vaccinated mice were tested against proteins from C57BL/6 mouse liver homogenates in a Western blot analysis (Fig. 5B). The mouse serum samples either recognize a 48-kd protein, a 58-kd protein, or both. These molecular weights correspond to the mouse cytochrome from the 2D subfamily, CYP2D9 (48 kd), and mouse FTCD (58 kd).
The development of a murine model of type 2 AIH and its analysis may shed light on an otherwise poorly understood autoimmune pathology. The reasons behind the development of specific autoantibodies in type 2 AIH, anti-LKM1 and anti-LC1, against proteins that are neither secreted nor expressed at the surface of the hepatocyte remain unknown.5, 26 The possible involvement of these autoantibodies in the pathogenicity of type 2 AIH is, at least, controversial.7 Previously described animal models of AIH have been of help in understanding some of the underlying processes that could give rise to a liver autoimmune disease.8, 9, 11, 27 Many identified shortcomings of those models led us to develop a new mouse model based on the hypothesis of molecular mimicry, between a foreign and a neo-self antigen, as the trigger of the autoimmune process.12
DNA vaccination has been proven to induce a specific humoral and cellular immune response in animals.28, 29 The plasmid used for the DNA vaccination was constructed to produce a 62-kd chimeric fusion protein containing the antigenic regions of both human CYP2D6 and FTCD. This chimeric protein also contains the extracellular region of mouse CTLA-4. This fragment acts as a secretory signal and as an immunological modulator.12 The extracellular part of CTLA-4 allows binding of the fusion protein to the B7-1 and B7-2 receptors at the surface of antigen-presenting cells. An increase in the cellular and humoral response to fusion proteins containing this CTLA-4 fragment has been previously described.30, 31 To increase the immunologic response to the product of this construct, pVR-IL-12 was coinjected. IL-12 is a strong B and T-cell stimulator naturally produced by antigen-presenting cells, driving T cells toward a Th1 phenotype differentiation and enhancing the CTL reactivity.12, 32–34 The protocol, three intramuscular injections of plasmid DNA at 2-week intervals, has been proven to maintain a prolonged level of protein expression. It continually stimulates the immune system (both the T and B-lymphocyte systems) and produces a strong CTL response to the antigen.32
Two peaks of ALT serum activity were found at 4 months and at a higher level at 7 months postinjection. These findings are similar to previous results obtained using a transgenic mouse model of AIH.12 An interesting observation is that the ALT serum activity peak found at 4 months is only the first event in a two-step process. The first event occurs after the development of autoantibodies and probably after the activation and migration of T cells to the liver. A second event occurs 7 months after the last immunization, when an increase in ALT serum activity and production of autoantibodies occurs simultaneously. If the initial increase in autoantibodies is dependent on the secretion of antigens from the muscle, the 7-month peak was most probably secondary to the liberation of the autoantigen from the hepatocytes. In type 2 AIH, it was already shown that inflammatory activity corresponds with the autoantibody titers of anti-LC1.7
A characteristic portal and periportal inflammatory infiltrate and an intralobular infiltrate were only observed in mice with elevated ALT levels. The characterization of this infiltrate shows that most lymphocytes are CD4+ T-helper (Th) cells, but CD8+ CTLs are also present, mainly in the periphery of portal areas, along with B lymphocytes (CD23+) which are sparsely distributed. A predominance of CD4+ lymphocytes was also observed in liver biopsy specimens of AIH patients.35 Autoreactive anti-LKM1 (anti-CYP2D6) lymphocytes in type 2 AIH patients were also CD4+, displaying a Th1 phenotype.36 As in the model described in the current study, the presence of B lymphocytes in the liver of AIH patients is sparse. Most cells in the infiltrate are T lymphocytes. CD4+ cells are widely distributed in septal inflammatory tissue, whereas CD8+ cells are predominantly located in areas of active hepatocellular necrosis.37 The analysis of T lymphocytes from the spleen and liver of vaccinated mice indicates that there are activated CYP2D6 and/or FTCD-specific CTL in both organs of animals with elevated levels of ALT. The levels of specific lysis observed are similar to those previously obtained in an NP transgenic mouse model of AIH.12 These preliminary data warrant further investigation to fully characterize the cellular autoimmune response in this model of type 2 AIH.
The finding that CYP2D9 and mouse FTCD are mainly expressed in the liver like CYP2D6 and FTCD in humans can explain the liver specificity of this inflammatory disease as observed in human type 2 AIH. In this human disease, the immune T-cell reactivity against CYP2D6 and FTCD is limited to the liver. Nonetheless, the presence of inflammation in other organs should be investigated in future experiments, even if the level of expression of CYP2D9 and mouse FTCD is very low (e.g., kidney, brain).38, 39
The production of autoantibodies against CYP2D9 and murine FTCD is an interesting aspect of this model of type 2 AIH. In the group coinjected with pVR-IL-12, the LKM1/LC1 antibodies were found earlier and at higher titers than in mice injected only with pCMV-CTLA4-CYP2D6-FTCD. These results showed that B-lymphocyte activation was induced by IL-12. The autoantibodies produced are mainly of the IgG2a and IgG2b subclasses and are representative of a Th1-type response. This type of immune response (Th1) was already found in other organ-specific autoimmune diseases.40 Autoantibodies detected in mice recognized both the human and the mouse antigens. They were directed mainly against conformational epitopes on cytochromes from the 2D subfamily and FTCD. It is noteworthy that CYP2D6 was more antigenic than FTCD, as it has been found in type 2 AIH patients.22
In conclusion, type 2 AIH can be induced in naive C57BL/6 mice using xenoimmunization with known human type 2 AIH antigens. A break of tolerance against mouse liver proteins occurs and an autoimmune process develops. Mice with elevated ALT levels have a characteristic liver inflammatory infiltrate, constituted mainly of CD4+ lymphocytes, but also CD8+ and B lymphocytes. Mice show anti-LKM1 and anti-LC1 autoantibodies, the hallmark of type 2 AIH and display a Th1 phenotype of immune response. This break of tolerance against self-proteins induced by molecular mimicry is proof that foreign antigens can use the same mechanism to trigger an autoimmune process.