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Service de Gastroentérologie, Departement de Pédiatrie, Hôpital Sainte-Justine, Montréal, Québec, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several transgenic murine models of immune related liver diseases have
been designed to understand the mechanisms involved in the development
of the tissue injury and of the breakdown of tolerance, but these
models have many drawbacks. A case in point is the transgenic mouse
expressing a short peptide (aa 1–60) of the lymphocytic
choriomeningitis virus
(LCMV)3 glycoprotein
under the control of the albumin promoter (5). These
animals need to be bred with mice that exhibit increased susceptibility
to liver disease and require injection of specific T cells before
developing a transient elevation of serum transaminase levels
(5). An important determinant of the organ-specific immune
response is the promoter use for neo-self Ag expression. The albumin
promoter is responsible for high levels of neo-self Ag expression in
the liver, but also for low levels of expression in the thymus
(5). The expression of a neo-self Ag in this central
immune organ led to negative selection of most high affinity/avidity
self-reactive T cells. The fact that hepatitis can be induced by
neonatal thymectomy in mice confirms the importance of T cell selection
in this organ (6). Mouse models expressing IFN-
in the
liver or deficient in TGF-
1 have been reported to develop liver
tissue injury, thereby showing the significance of regulatory cytokines
in pathogenesis of cytolysis (7, 8). TGF-1-deficient
mice developed multiorgan inflammation, including the liver, but only
presented necrosis of hepatocytes in a BALB/c genetic background,
establishing the importance of genetic susceptibility
(8).
Autoimmune diseases have been generated after infection by LCMV in
transgenic mice expressing LCMV-nucleoprotein (NP) or glycoprotein in
cells of the islets of Langerhans or their oligodendrocytes
(9, 10, 11, 12). These transgenic mice did not develop any
immunopathology in the absence of the LCMV challenge
(9, 10, 11, 12). Such experiments showed that molecular mimicry
between self peptides and viral proteins can be responsible for
initiating and maintaining the autoimmune process. Diabetes and CNS
autoimmune disease in transgenic mice were mediated by
CD8+ cells, and a critical number of activated
CTLs were necessary to induce the disease (9, 10, 11, 12).
In view of the limitations of the models studied until now, a new transgenic murine model is presented in this study. A B and T lymphocyte-mediated immune response against a liver neo-self Ag is generated by DNA vaccination. To avoid local modifications produced in the liver by LCMV, a plasmid expressing LCMV-NP was delivered by i.m. injection to transgenic mice. Taking previous experience into consideration, it was elected to produce transgenic mice expressing LCMV-NP in their liver under the control of a liver-specific promoter. In this situation, the transthyretin (TTR) liver-specific minimal enhancer promoter sequence appeared to be the appropriate choice (13, 14). LCMV infection produces CD8+-mediated acute hepatitis, showing that it is an hepatotropic virus (15). DNA vaccination protects against LCMV infection, and even more interesting, generates a B and T lymphocyte response similar to that induced by live virus infection. The CTLA-4 extramembrane region was fused to NP in the expression vector, to improve the delivery of the neo-self Ag to APCs and to enhance the immune response. Several months after the vaccination, liver inflammation causing an increase of alanine aminotransferase (ALT) levels was recorded. In the conception of the LCMV transgenic mouse model of autoimmune hepatitis (AIH), at least two factors relevant to the pathogenesis of the human disease were considered: 1) the importance of a particular genetic background (it is known that mice with different background respond to distinct epitopes of LCMV proteins), and 2) the presence of an environmental trigger factor (such as immunization against LCMV proteins or the LCMV infection).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Full-length LCMV-NP cDNA (a gift from R. Zinkernagel, Zurich,
Switzerland) was subcloned in the BamHI cloning site of
pBluescript-KS II (Stratagene, La Jolla, CA), designated as pBS-NP.
SV40 t intron and poly(A) sequences were isolated from the
pPAP-GLVP (obtained from B. O’Malley, Houston, TX), and subcloned as a
850-bp BamHI-SpeI fragment into pBS-NP. The TTR
minimal enhancer/promoter region was isolated from pTTR-CAT (kindly
offered by R. Costa, Chicago, IL); this 330-bp HindIII
fragment was subcloned in the pBS-NP to create pBS-TTR-NP-SV40.
Subsequently, the fragment containing TTR-NP-SV40 was inserted between
two copies of the 5' element of the chicken -globin domain (HS4;
kindly offered by G. Felsenfeld, Bethesda, MD) to generate the
construct pHS4-TTR-NP-SV40-HS4. The capability of this construct to
allow NP expression in hepatocytes was tested by transient transfection
of HepG2 cells (see below).
Transgenic mice generation
The DNA transgene fragment originated from NtoI
endonuclease restriction of pBS-HS4-TTR-NP-SV40-HS4. The 7.8-kb DNA
obtained was isolated by gel electrophoresis and purified with a
QIAquick gel electrophoresis kit (Qiagen, Santa Clarita, CA). The
transgene was injected in fertilized eggs from C3H
(H-2k) x C57BL/6
(H-2b) mice. Genomic DNA was prepared from
founder mice and their offspring tail, and screened for the presence of
NP DNA by PCR amplification, using specific primers. The 5' primer
sequence was 5'-GGT GTA GTA AGG GTT TGG G-3', and the 3' primer
sequence was: 5'-CTT GGC CCC AAG GAC TGC-3'. Founder mice showing
integration of the transgene by PCR amplification and confirmed by
Southern blotting were bred with C57BL/6 mice to establish transgenic
lines with a H-2b background. Tissue-specific NP
expression was proved by RT-PCR amplification of total RNA and indirect
immunofluorescence of cryostat sections (see below). -Actin mRNA was
amplified with specific primers and used as control for RT-PCR. Two
lines, designated LCMV-NP-S1 and LCMV-NP-S2, were kept for and used in
future experiments.
DNA vaccination
Plasmids for vaccination were constructed in pRc/CMV (Invitrogen, Carlsbad, CA), with the human CMV promoter for expression in eukaryotic cells. Plasmid pCMV-NP was obtained by introducing NP cDNA in the NotI-ApaI cloning site of pRc/CMV. The capability of this construct to express NP was tested by transient transfection of HepG2 cells.
cDNA coding for the mouse CTLA-4 extracellular region was obtained by PCR amplification of a fragment of full-length cDNA in pUC9 American Type Culture Collection (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'. pCMV-CTLA-4-NP was constructed by subcloning CTLA-4 cDNA coding for the extracellular region of the protein into the restriction sites XbaI-BsmI of pBs-NP. The CTLA-4-NP fragment was then subcloned in the ApaI-NotI sites of pCMV. The insertion of CTLA-4 cDNA created a single open reading frame encoding the fusion protein CTLA-4-NP.
Plasmids were propagated in Escherichia coli by standard techniques and purified with a Qiagen Endofree Plasmid Maxi kit (Qiagen), according to the manufacturer’s instructions. Plasmid preparation purity was confirmed by spectrophotometric analysis and electrophoresis. Six- to 8-wk-old transgenic mice (TTR-NP-S1) were injected under general anesthesia in the internal tibial muscle with 100 µg (50 µl) of plasmid DNA dissolved in saline buffer. The plasmids pRc/CMV, pCMV-NP, or pVR-IL-12 (kindly provided by G. Prud’homme, Montreal, Quebec, Canada) were injected alone, respectively. In addition, two groups each containing six mice were vaccinated with pCMV-NP and pVR-IL-12 or pCMV-CTLA-4-NP and pVR-IL-12.
Transient transfection
HepG2 cells were transfected with pHS4-TTR-NP-SV40-HS4 or pCMV-NP, using Fugen reagent, according to the manufacturer’s instructions (Roche, Laval, Quebec, Canada). Forty-eight hours posttransfection, the cells were fixed with acetone, and NP was detected by indirect immunofluorescence. The first Ab was rabbit anti-LCMV serum (kindly provided by M. Burns, Hamburg, Germany) at a dilution of 1/200; and the second Ab was FITC-conjugated anti-rabbit IgG (Biosource, Camarillo, CA) at a dilution of 1/1000.
In vitro transcription-translation and immunoprecipitation
In vitro transcription-translation of pCMV-CTLA-4-NP was undertaken using the RiboMAX large scale RNA production system and the rabbit reticulocytes lysate system (Promega, Madison, WI), according to the manufacturer’s instructions. In vitro synthesized and [35S]methionine-labeled CTLA-4-NP fusion protein was immunoprecipitated with rabbit anti-LCMV serum or anti-CTLA-4 polyclonal Abs (Santa Cruz Biotechnology, Santa Cruz, CA), according to a previously published protocol (16). The immunoprecipitate was analyzed by 10% SDS-PAGE, followed by autoradiography.
Immunohistochemistry and histopathology
Liver samples from transgenic mice were embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA) and frozen in liquid nitrogen. Cryostat sections of these livers were fixed in acetone at 4°C for 10 min, and then treated by indirect immunofluorescence, as described above.
Transgenic LCMV-NP-S1-vaccinated mice were sacrificed at the fifth month after the last plasmid injection, and their livers were dehydrated, embedded in paraffin, sectioned, and stained with H&E. Semiquantitative grading of liver biopsies was assessed according to previously described numerical score (17, 18).
ELISA
NP cDNA was inserted in the pGEX-4T3 (Pharmacia Biotech,
Montreal, Quebec, Canada) cloning site to prepare GST-NP fusion protein
in isopropyl -D-thiogalactoside-induced bacterial
cultures. The fusion protein was purified using a glutathione Sepharose
column (Pharmacia Biotech). The purity of the preparation was assessed
by SDS-PAGE, and the protein was quantified by UV spectroscopy with BSA
as standard. Microwell plates were coated with 0.2 µg/well of GST-NP
fusion protein in 0.1 M NaHCO3, pH 8.6, overnight at 4°C.
After blocking with 2% BSA in PBS for 1 h at 37°C, 200 µl of
1/100 dilution of mice sera or standard dilution of mice anti-NP
serum with highest titer was loaded. Mouse sera were obtained before
and 1, 2, and 3 mo after vaccination. The presence of anti-NP Abs
was revealed by incubation with anti-mouse IgG alkaline
phosphatase-conjugated Ab at a dilution of 1/2000 (Biosource). Alkaline
phosphatase was developed by incubation with p-nitrophenyl
phosphate, and the result read at 405 nm. ODs were compared for sera
from different groups of vaccinated TTR-NP S1 mice. An antiserum was
considered positive if its specific OD was at least 2.5 times higher
than the mean OD of the preimmune sera from transgenic and
nontransgenic mice. The same technique was applied to establish the IgG
subclass. Briefly, mice sera were diluted to 1/50, and second Abs,
conjugated anti-IgG1, anti-IgG2a, anti-IgG2b, and IgM
alkaline phosphatase (Santa Cruz Biotechnology), were used at a
dilution of 1/1000.
CTLs against NP-dominant epitope
EL4 cells, an H-2b lymphoma T cell line (ATCC), served as targets. Briefly, 1 x 104 target cells coated with NP396–404 peptide (FQPQNGQFI) were incubated with serial dilutions of between 1 x 104 and 5 x 105 splenocytes (effector cells) in a final volume of 200 µl. After 5 h 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 instructions. Percent lysis was calculated by the formula: 100 x (A - B - C)/(D - C), in which A is experimental value (test release), B is spontaneous background signal value from effector cells, C is spontaneous background signal value from target cells, and D is the target maximum signal value. Maximum release and spontaneous release were determined by incubating the cells with lysis solution and culture medium, respectively.
Serum ALT activity
Serum ALT levels were measured in a Beckman-Synchron CX9 apparatus, from blood taken each month after the last plasmid injection.
Statistical analysis
Statistical significance was evaluated by the Student’s t test, and p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A murine-specific liver promoter (liver-specific TTR promoter) was
fused to cDNA coding for LCMV-NP (Fig. 1
A) and injected into
fertilized mouse embryos. Before injection, the construct was
transiently transfected into HepG2 cells, and NP expression was
demonstrated by indirect immunofluorescence (Fig. 1B). In 4
of 21 mice tested, the presence of the transgene was detected by
Southern blot analysis (data not shown) and PCR amplification (Fig. 1C). All such mice passed the TTR-NP transgene to their
offspring. Specific expression of LCMV-NP in the liver was evaluated by
RT-PCR amplification of liver-extracted total RNA and by indirect
immunofluorescence of liver cryostat sections (Fig. 2). Transcription of the NP transgene was
not detectable by RT-PCR in spleen, thymus, muscle, or kidney (Fig. 2A). Two founder lines, designated TTR-NP-S1 and TTR-NP-S2,
showed NP expression in their livers. TTR-NP-S1 was used for further
studies described in this work. The NP-dominant epitope differs
according to the mouse H-2 haplotype. In the H-2b
background, the epitope was located in the C-terminal region of the
protein and was constituted of 9 aa. To work on a homozygous
H-2b background, TTR-NP-S1 were bred with C57BL/6
to the fifth generation. The transgenic offspring were genotyped for
H-2b by PCR amplification, followed by dot blot
hybridization using sequence-specific oligonucleotide probes for
H-2b and H-2k. Observation
of TTR-NP-S1 for >1 year showed that none of these mice had any sign
of spontaneous liver or extrahepatic disease.
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Similar immune responses to Ags produced by the host were induced
by viral infection and DNA vaccines. For DNA vaccination, two vectors
were constructed, using the human CMV promoter and a SV40 poly(A)
sequence. One vector coded for NP, and the other for CTLA-4-NP (Fig. 3A). pCMV-NP coded for an
intracellular, mainly cytosolic, protein, and was tested by transient
transfection of HepG2 cells, followed by indirect immunofluorescence
(Fig. 3B). pCMV-CTLA-4-NP coded for a secretory protein and
was tested by in vitro transcription and translation, followed by
immunoprecipitation, using anti-NP and anti-CTLA-4 Abs as well
as gel electrophoresis (data not shown). TTR-NP-S1 mice were injected
between the ages of 6 and 8 wk to obtain an optimal response (see
protocol in Fig. 3C). Intracellular and secretory forms of
NP encoded by pCMV-NP or pCMV-CTLA-4-NP, respectively, were expressed
after i.m. injections, as shown by the production of
anti-NP-specific Abs (see below).
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Every month after the last injection, serum samples were obtained
for quantification of anti-NP Abs against purified NP Ag and
compared with preimmune sera. Three groups of mice sera injected with
different DNA preparations were compared; two of them were coinjected
with a plasmid encoding for IL-12 to enhance IgG production. Sera drawn
at different postinjection times were matched. Ab response was
significantly higher in mice injected with pCMV-NP compared with
pCMV-CTLA-4-NP (mean ± SD; 650 ± 150 vs 300 ± 200,
p = 0.0002). In mice immunized with pCMV-CTLA-4-NP,
anti-NP Abs were detected at similar levels at 1 and 3 mo after the
last injection. In contrast, a maximum level was found at the second
month, but did not reach statistical significance. The levels of these
Abs were similar during the 3 mo following vaccination with pCMV-NP
(Fig. 4A). Immunization did
not induce Abs against NP in the sera of transgenic mice vaccinated
with pCMV (blank vector) alone or IL-12-expressing plasmid alone (data
not shown).
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B). It is noteworthy that in sera from
pCMV-CTLA-4-NP-injected mice, IgG1 was absent, leading to the
conclusion that in these mice the immune response was of the Th1 type.
In addition, the absence of IgG1 can explain the lower level of IgG
observed with sera from mice injected with pCMV-CTLA-4-NP, when
compared with those injected with pCMV-NP (Fig. 4A). Detection of the CTL response against the NP-dominant epitope
CTLs play an important role in immune responses against virally
infected or tumoral cell as well as in tissue attack in autoimmune
diseases. CTL activation is mediated by specific cytokines, and in our
protocol is influenced by coinjection of DNA encoding for IL-12.
Specific cell lysis in the presence of the dominant epitope was
provoked by T cells isolated from the spleen of immunized mice (Fig. 5). Splenocytes from vaccinated TTR-NP-S1
mice were harvested at the second or fifth month after the last plasmid
injection and cultured in the presence of NP aa 396–404 synthetic
peptide for 5 days. Clearly, a linear rise in target cell lyses was
observed after incubation with an increasing number of effector
splenocytes, keeping stable the concentration of target cells coated
with the peptide representing the NP-dominant epitope (Fig. 5). T
lymphocytes analyzed in this cytotoxicity assay were from mice
coinjected with pVR-IL-12 and either pCMV-CTLA-4-NP or pCMV-NP and
showing the stronger Th1 immune response. pCMV-CTLA-4-NP and pCMV-NP
induced comparable levels of CTL response at the second or fifth month
after the last plasmid injection. In contrast, no CTL activity was
detected in splenocytes from naive TTR-NP-S1 mice or immunized with
pVR-IL-12 alone (Fig. 5).
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The presence of immune-mediated liver disease in the mouse model was detected by repeated measurements of ALT levels and microscopic assessment of liver injury.
In TTR-NP-S1 mice immunized with pCMV-NP and IL-12, an increase of
serum ALT was observed at the fifth month after the last injection.
However, in the pCMV-CTLA-4-NP- and the IL-12-injected mice, ALT
elevation was noted at the fourth month after the third DNA injection
(Fig. 6). It is therefore clear that
cytolysis appeared to be an earlier event in mice producing a secretory
protein targeted to APCs. In contrast, ALT levels were not increased in
TTR-NP-S1 injected with pCMV alone or pVR-IL-12 alone plasmids
(Fig. 6).
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). Semiquantitative grading score of
liver biopsies from pCMV-CTLA-4-NP- and the IL-12-injected mice was
between 9 and 12, according to Knodell et al. (17). Scarce
images of bridging necrosis were observed, but no fibrosis was found.
Therefore, biopsies of these mice were considered as showing moderate
chronic hepatitis (18). Histology of the liver from all
control groups did show neither inflammatory infiltrate nor
fibrosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A transgenic mouse expressing a neo-self Ag in the liver is an attractive model that has already been applied successfully to other autoimmune diseases (9, 10, 11, 12). The first problem to solve is the level of expression of any Ag in the liver, and to do so specifically in this organ, by avoiding the simultaneous expression of Ag in immune system central organs. To solve part of the problem, cDNA encoding for the neo-self Ag LCMV-NP was inserted between insulators (HS4). Frequently in the liver, transgenes are located in heterochromatin regions or at sites influenced by neighboring negative regulatory elements, and fail to be expressed. Insulator sequences have been shown to block the suppressive effects of the surrounding chromosome and direct integration site-independent expression of the transgene in mice (22, 23). To target transgene expression in mice specifically to the liver, a 300-bp enhancer/promoter region of TTR was a preferential choice for the expression of a neo-self Ag (13, 14). The TTR enhancer and promoter proximal DNA sequence direct transcription of minigenes in the liver, even in low copy number (13). Taking all these facts into consideration, it is possible that the success of the transgenic mouse model presented in this study is at least partially due to transgene construction.
DNA vaccination induces specific humoral and cellular mediated immune responses in animal models and humans (24, 25). The type of immune response obtained by DNA vaccination is qualitatively the same as that observed after a viral infection (24, 25). This immune response is protective against a persistent or acute, even otherwise lethal infection (26, 27). The strong B and T cell response can be optimized by i.m. injection. In muscle, proteins are rapidly released in the circulation, and their expression is longer than in other tissues. At least three injections of plasmids are necessary to generate CTL responses (28). This methodology allowed a vigorous and durable level of anti-NP Abs and CTL activity to be induced against the NP main epitope in TTR-NP-S1 mice. In TTR-NP-S1 mice injected with DNA coding for CTLA-4-NP fusion protein, liver inflammation was detected earlier than in mice injected with cDNA coding only for NP. When plasmid-coded proteins were secreted and especially when they were targeted to APCs, an increase in the Ab and cellular response was observed (29, 30). This was the case of the fusion protein in which the extracellular part of the CTLA-4 sequence was included, because CTLA-4 is a ligand of B7-1 and B7-2 receptors expressed on the surface of APCs.
Immune responses are influenced quantitatively and qualitatively by cytokines. IL-12 is a 70-kDa protein produced by APCs that boost the B and T cell-mediated response, even in neonates, driving differentiation of T cells toward a Th1 phenotype (28, 31, 32, 33). IL-12 acted locally and not systemically through an adjuvant effect to induce Th1 activity (28). Plasmid-producing IL-12 coinjected with DNAs modified the immune response to a Th1 phenotype and enhanced CTL activity (28). This known property of IL-12 led to the coinjection of a plasmid producing this cytokine with NP or CTLA-4-NP DNAs. The high levels of anti-NP Abs of the IgG2 subclass observed after vaccination with both plasmids in this model were interpreted as proof of the induction of Th1 cells. This particular phenotype was predominant in TTR-NP-S1 mice injected with CTLA-4-NP, showing that targeting of the neo-self Ag to IL-12-activated APCs was the best combination to ensure activation of the Th1 response. Protective immune responses during viral infections as well as pathological responses against self Ags in several autoimmune diseases are of the Th1 type (34, 35, 36, 37, 38). Therefore, the activity of this CD4+ pathway could be essential to induce AIH in the TTR-NP-S1 mouse model.
DNA vaccines are relatively easy to prepare, heat stable, and inexpensive to produce. In addition, one vaccine can be produced against multiple pathogens. However, occasionally DNA vaccination in some animal models has produced unexpected effects (39, 40). Clearly, an autoimmune process is triggered by molecular mimicry between vaccine DNA code Ags and mammalian proteins, as was shown in the case of TTR-NP-S1 mice. Moreover, because cross-reactivity between Ags does not always imply sequence similarity, there remains a potential for the development of unexpected immune responses directed toward endogenous protein (41, 42).
Previously described models of autoimmunity have shown similar delays between the induction of a specific immune response and consistent necrosis of the targeted cells (9, 10, 11, 12). Following serum ALT levels as a marker of hepatocyte lysis, it was apparent that this process started several weeks after the third plasmid injection, that it was only observed in mice vaccinated with plasmids containing NP cDNA, and that it was found earlier in mice injected with the CTLA-4-NP construct than in mice expressing an intracellular NP form. Altogether these results showed that hepatitis in TTR-NP-S1 vaccinated only with plasmids expressing NP was a specific event. The delay between the last injection and the increase in serum transaminases was probably due to the time necessary for activated T cells to migrate, divide, and follow avidity maturation in the liver to reach a critical number, as was described in the progression of autoimmune diabetes (43). Moreover, it is not known how many hepatocytes should be lysed to elevate serum ALT above normal limits.
The liver plays a particular role in T cell differentiation in humans and mice. This pathway is activated during infections, malignancies, and autoimmune diseases, acquiring a predominant place in athymic mice (44, 45). Naive and memory T lymphocytes migrate through the liver, showing a preference for the periportal field (2). Activated T lymphocytes proliferate in the periportal tract (2, 46), in which predominance of inflammation is found in chronic hepatitis. In addition, hepatocyte Ags in a HLA class I context are readily accessible to CTLs, mainly because of the lack of basement membranes in hepatic sinusoids and the discontinuity of the endothelial layer (2, 3). Altogether, these previously published results explained why in our model, CTLs specific for NP, activated in the peripheral lymphatic organs by DNA vaccination, could be responsible for tissue injury. Inflammation and cell necrosis were observed in the portal tracts and in the periportal areas, as well as intralobularly, findings consistent with those observed in patients with AIH. It will be particularly interesting in any future work to follow, for a longer time, the evolution of this chronic hepatitis in a TTR-NP-S1 mouse model, and to document the chronicity of the disease and the progressive development of liver fibrosis.
Break of tolerance and ignorance to a self liver Ag are absolutely necessary to induce AIH. The fate of specific T cells in the liver depends on the level of Ag expression (4) and on the simultaneous presence of inflammatory signals (47). High levels of Ag expression would lead to tolerance, and low levels would result in ignorance (4, 5). In the TTR-NP-S1 mouse model, the level of liver NP expression, even if not quantified, was not very high, as shown by a mediocre signal in immunofluorescence screening, and by its light signal detection in liver homogenates tested by Western blotting (data not reported). This level of NP expression probably prevented peripheral anergy or tolerance from developing in TTR-NP-S1 mice. Recently, it was shown that break of tolerance in the liver needs the transfer and strong activation of specific T cells to mice expressing a neo-self Ag under the control of the albumin promoter (5). However, in this model, high affinity T cells were mostly deleted in the thymus, in which low expression of the neo-self Ag was detected (5). In the transgenic mice described in this work, NP expression in the thymus from TTR-NP-S1 mice was not detected. In addition, taking into consideration the specificity of the TTR enhancer/promoter sequence used (48, 49), it could be expected that no major central deletion of specific T cells occurred in these animals. CTL sp. act. against cells presenting the NP main epitope in an H-2b background was found in vaccinated TTR-NP-S1 mice, concurrently with the development of hepatitis. These specific T cells were probably activated in the peripheral lymph nodes by Th1-induced lymphocytes, and migrated through the liver, in which the neo-self Ag peptide H-2Db on the surface of the hepatocytes became the target. Recently, it was shown that T lymphocytes, stimulated in distant lymphoid sites, migrated to the liver, in which they provoke hepatocyte injury (50).
In conclusion, in TTR-NP-S1 mice expressing LCMV-NP in their liver, tolerance to the neo-self Ag was broken by DNA vaccination. The immune response appears to be Th1 mediated, and resulted in high levels of IgG autoantibodies and activated CTLs specific for the neo-self Ag-dominant epitope. This model showed several of the characteristics of AIH, namely elevation of serum ALT, hepatitis characterized by lymphocytic infiltration, and a predominance of Th1 phenotype leading to the presence of autoantibodies and the activation of specific CTLs.
Analysis of the breakdown of tolerance to a liver self Ag and specifically the role of CD4+ (Th1/Th2) and CD8+ lymphocytes into the liver should help to elucidate pathogenic mechanisms leading to tissue destruction. Furthermore, study of different triggering mechanisms (LCMV infection) and the role of cytokines on the chronic tissue injury will orientate the exploration of factors involved in human AIH.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Fernando Alvarez, Division of Gastroenterology and Nutrition, Sainte-Justine Hospital, 3175, Cote-Sainte-Catherine Road, Montréal (Québec) H3T 1C5, Canada. E-mail address: fernando.alvarez{at}umontreal.ca
3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; AIH, autoimmune hepatitis; ALT, alanine aminotransferase; NP, nucleoprotein; TTR, transthyretin.
Received for publication March 8, 2002. Accepted for publication August 22, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the liver. Proc. Natl. Acad. Sci. USA 91:614.1-deficient mice develop necroinflammatory IFN--dependent hepatitis. J. Immunol. 166:6413.-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila. Cell 74:505.[Medline]
effector pathway by CD4+ T cells selected by I-Ag7 on a nonobese diabetic versus C57BL/6 genetic background. J. Immunol. 167:1693.
chain expression by adult human hepatic T cells: evidence for extrathymic T cell maturation. Eur. J. Immunol. 26:3114.[Medline]
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