Impairment of TGF-beta signaling in T cells increases susceptibility to experimental autoimmune hepatitis in mice

Christoph Schramm1, Martina Protschka2, Heinz H. Köhler3, Jürgen Podlech4, Matthias J. Reddehase4, Peter Schirmacher5, Peter R. Galle1, Ansgar W. Lohse1, and Manfred Blessing2

1 First Department of Medicine, 3 Department of Pathology, 2 First Department of Medicine Pathophysiology Section, and 4 Department of Virology, Johannes Gutenberg-University, 55101 Mainz; and 5 Department of Pathology, University of Cologne, 50931 Cologne, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In autoimmune hepatitis, strong TGF-beta 1 expression is found in the inflamed liver. TGF-beta overexpression may be part of a regulatory immune response attempting to suppress autoreactive T cells. To test this hypothesis, we determined whether impairment of TGF-beta signaling in T cells leads to increased susceptibility to experimental autoimmune hepatitis (EAH). Transgenic mice of strain FVB/N were generated expressing a dominant-negative TGF-beta type II receptor in T cells under the control of the human CD2 promoter/locus control region. On induction of EAH, transgenic mice showed markedly increased portal and periportal leukocytic infiltrations with hepatocellular necroses compared with wild-type mice (median histological score = 1.8 ± 0.26 vs. 0.75 ± 0.09 in wild-type mice; P < 0.01). Increased IFN-gamma production (118 vs. 45 ng/ml) and less IL-4 production (341 vs. 1,256 pg/ml) by mononuclear cells isolated from transgenic livers was seen. Impairment of TGF-beta signaling in T cells therefore leads to increased susceptibility to EAH in mice. This suggests an important role for TGF-beta in immune homeostasis in the liver and may teleologically explain TGF-beta upregulation in response to T cell-mediated liver injury.

transgenic mice; dominant-negative transforming growth factor-beta type II receptor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AUTOIMMUNE HEPATITIS (AIH) is thought to be a T cell-mediated disease (28). Little is known about the factors influencing susceptibility to AIH (11). If left untreated, the disease may rapidly lead to liver fibrosis, and cirrhosis quite often is the first presentation of a patient with AIH (10). The fibrogenic potential of TGF-beta 1 is well known and has been shown in experimental models overexpressing TGF-beta 1 in the liver (5, 24). In patients with AIH, strong TGF-beta 1 expression in the liver has been detected, which seems to correlate with disease activity (4).

In addition to its fibrogenic and mitoinhibitory potential, TGF-beta 1 is a key regulator of immune homeostasis (34). TGF-beta 1 knockout mice show a multifocal inflammatory disorder and die within 4 wk of birth (29, 49). Crossing TGF-beta 1 knockout mice with SCID mice results in mice with no inflammatory lesions, suggesting a role for lymphocytes in the observed immune dysregulation (13). However, because of its multiple effects on different cell types, it has been difficult to delineate the actions of TGF-beta as specific to T cells in the knockout model. In vitro studies have shown that TGF-beta 1 exerts profound and partly contradictory regulatory effects on T cells that depend on the context of its production (34). Inhibitory effects on proliferation, IL-2 production, and various effector functions such as cytokine production and signaling (1, 15, 16, 18, 21, 25) as well as cytolytic activity (42, 48) have been described.

All three isoforms of TGF-beta (TGF-beta 1, 2, and 3) bind to a single receptor consisting of two subunits and mediate their highly pleiotropic effects depending on their expression pattern and microenvironment (41). On binding of the ligand to the TGF-beta type II receptor, the type I receptor associates with this complex and is subsequently phosphorylated by the intracellular serine-threonine kinase domain of the type II receptor (2, 3, 14). The TGF-beta type I receptor then phosphorylates and activates Smad proteins, which mediate expression of TGF-beta response genes (54, 12). The mutated dominant-negative TGF-beta type II receptor (Delta kTbeta RII) lacks the intracellular kinase domain. It titrates the type I receptor into inactive complexes, resulting in the inhibition of the signaling cascade (7).

In AIH, TGF-beta might be produced to suppress autoreactive T cells invading the liver. This local immunoregulatory response may help to prevent fulminant exacerbations of AIH and enable induction of spontaneous remissions (37), but at the same time it may promote fibrosis. To test this hypothesis of an important immunoregulatory role of TGF-beta in AIH, we generated transgenic mice overexpressing a dominant-negative TGF-beta type II receptor in T cells, thereby overcoming the restrictions of the TGF-beta 1 knockout model. The truncated receptor was expressed under the control of the human CD2 promoter/enhancer, which is expressed in a copy-dependent manner in T cells (56). The hCD2-Delta kTbeta RII transgenic mice were characterized and subsequently used to study the possible effects of TGF-beta on T cells in the induction of experimental autoimmune hepatitis (EAH), an animal model for AIH. EAH shares several features with AIH and is induced by immunizing mice with syngenic liver homogenate in adjuvant. The disease is most likely T cell mediated because lymphocytic infiltrates and T cell reactivity to liver antigens can be demonstrated (27, 38, 52) and the disease can adoptively be transferred by using activated splenocytes (38, 44).

The hCD2-Delta kTbeta RII mice demonstrated increased susceptibility to induction of EAH, as demonstrated histologically and biochemically. This suggests that indeed the regulatory effects of TGF-beta on T cells are important in maintaining immune homeostasis within the liver. TGF-beta seems to be required for suppression of autoreactive T cells infiltrating the liver.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation and maintenance of hCD2-Delta kTbeta RII mice. The cDNA of the type II receptor lacking the cytoplasmic kinase domain (Delta kTbeta RII) (7) was inserted in the SmaI site of the human CD2 (hCD2) minilocus expression vector (56). The expression cassette was excised by NotI/SalI digestion, gel purified, and used for pronuclear microinjection of fertilized eggs of strain FVB/N, essentially as described (22). Offspring were biopsied at ears or tails and analyzed for genotype by PCR using an hCD2-specific primer [5'-TTTGTAGCCAGCTTCCTTCTG-3', corresponding to positions 243-263; GenBank accession no. X07871 (32)] and a human TGF-beta type II receptor-specific oligonucleotide [5'-TGCACTCATCAGAGCTACAGG-3', corresponding to positions 272-252; GenBank accession no. U52242 (50)].

All transgenic lines were established and maintained as heterozygotes on a FVB/N background. For all experimental procedures, age-matched nontransgenic littermates were used as controls. Animal care was in accordance with all governmental and institutional guidelines.

Northern blot analysis. Total RNAs from various tissues were isolated with TRI Reagent (Sigma, Deisenhofen, Germany) according to the manufacturer's instructions. Aliquots (~20 µg) of RNA were separated by electrophoresis using 1% agarose formaldehyde gels and were subsequently blotted onto nylon membranes (Hybond N; Amersham, Braunschweig, Germany). For the analysis of RNA expression of the transgenic mouse line designated line 2 (see RESULTS for descriptions of each line), 30 µg of RNA were loaded for nonlymphoid organs to better demonstrate the lack of transgene expression in these organs. Filters were processed at high stringency as described (9) and were hybridized with the 32P-labeled human TGF-beta type II receptor cDNA (920 bp). Before electrophoresis, ethidium bromide was added to the samples to assess similar loading of RNA and the transfer efficiency of the RNA samples.

Separation of lymphoid cells. For the separation of B and T lymphocytes, splenocytes from 6-wk-old mice were marked with anti-mouse CD3-FITC or anti-mouse CD45R/B220-FITC MAb (BD Pharmingen, Heidelberg, Germany). MACS beads conjugated with anti-FITC antibodies were added, and cells were separated by using LS+ cell separation columns from Miltenyi Biotech (Bergisch Gladbach, Germany). B cells were removed in a first separation step, and T cells were further purified in a second separation step. Purity of T cells was assessed by FACS analysis and ranged between 90 and 95%.

FACS analysis. For assessment of the purity of cell separation, the FITC-marked cells were counterstained with anti-mouse CD19-PE or anti-mouse CD3-PE (BD Pharmingen). Spleen, thymic, and lymph node cells of transgenic mice and nontransgenic littermates were analyzed by using anti-mouse CD4-FITC, anti-mouse CD8-PE/FITC, anti-mouse CD62L-PE, and anti-mouse CD44-PE MAb (all from BD Pharmingen). FACS analysis was performed with a FACScan using CELL-quest software (Becton Dickinson, Heidelberg, Germany).

Cell culture and inhibition of IL-2 secretion. Spleen cells were cultivated in RPMI 1640 medium (Biochrom, Berlin, Germany) containing 5% FCS supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml; Life Technologies, Eggenstein, Germany). Cells (2 × 106 cells/ml) were plated and incubated with different concentrations of human TGF-beta 1 as indicated (Strathmann Biotech, Hannover, Germany). After 12 h of cultivation, T cells were separated and plated onto precoated 24-well plates (Greiner, Frickenhausen, Germany). Antibody (10 µg/ml anti-mouse CD28 MAb; BD Pharmingen) was added to the medium. For precoating, plates were incubated at 4°C with 10 µg/ml anti-mouse CD3 MAb in 0.1 M sodium phosphate buffer (pH 8.5) overnight. Cells were grown at 37°C in a water-saturated atmosphere with 5% CO2 in air. Supernatants were collected after 24 h and frozen in liquid nitrogen.

ELISA. Concentrations of IL-2, IFN-gamma , and IL-4 in cell culture supernatants were measured by using mouse OptEIA ELISA sets (BD Pharmingen) according to the manufacturer's instructions. Immunoglobulin subclasses in sera of mice were assayed by using the SBA clonotyping system (Southern Biotechnology, Birmingham, AL).

Cell proliferation assay. Splenocytes were seeded at 5 × 105 cells/well in 96-well flat-bottom plates (Greiner) in RPMI medium supplemented with 5% FCS and antibiotics (as described in Cell culture and inhibition of IL-2 secretion) or in serum-free medium (PAN Biotech, Aidenbach, Germany). For cell stimulation, plates were precoated with anti-mouse CD3 MAb overnight, and anti-mouse CD28 MAb was added to the medium at a concentration of 10 µg/ml. Cells were incubated at 37°C in 5% CO2 for 72 h and pulsed with 2.5 µCi/well [3H]thymidine for the last 24 h of culture. Samples were harvested and counted in a Betaplate liquid scintillation counter (Wallac, Freiburg, Germany).

Immunization of mice. EAH was induced by immunizing mice with the 100,000-g supernatant from livers of wild-type syngenic mice in adjuvant basically as described (39). Briefly, for the preparation of liver antigen, the portal vein was cannulated and flushed with cold PBS. The liver was removed and homogenized in PBS containing aprotinin (Sigma) and trypsin inhibitor (Serva, Heidelberg, Germany) and centrifuged at 150 g for 10 min. The supernatant was subsequently centrifuged for 1 h at 100,000 g, resulting in the S-100 supernatant. The protein concentration was determined, and mice were immunized intraperitoneally with 2.5 mg of fresh S-100 protein emulsified in the same volume of complete Freund's adjuvant (Difco, Detroit, MI). Immunizations were done twice or three times in weekly intervals, and mice were bled and killed 2 wk after the last immunization. Mice of line 2 were used for induction of hepatitis.

Isolation of inflammatory cells from livers. Cell suspensions were generated from livers by using 40-µm cell strainers (Becton Dickinson). Cells from several livers were pooled, washed in Hanks' balanced salt solution, and resuspended in 100% Percoll (Amersham, Uppsala, Sweden). The suspension was overlaid with 40% Percoll and centrifuged, and the cells were recovered from the interphase. After being washed twice, cells were either used for flow cytometric analysis or cultured for 48 h in RPMI medium supplemented with 5% FCS and antibiotics for the analysis of the cytokine secretion profile. Tissue culture plates were precoated overnight with anti-CD3 MAb, and anti-CD28 MAb was added to the medium as described.

Histology. Selected tissues were fixed in 4% formalin and embedded in paraffin. Sections (2 µm) were stained with hematoxylin and eosin and analyzed. Elastica van Gieson staining was used for assessment of fibrosis. Cryostat sections (5 µm) were fixed in cold acetone and stained with hematoxylin and eosin.

Immunohistochemical staining was performed after endogenous peroxidase was blocked with methanol and hydrogen peroxide. For the staining of T cells, anti-CD3 MAb (Novocastra, Newcastle, UK), biotinylated anti-rat antibody (BD Pharmingen), and the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) were used. Staining was enhanced with ammonium nickel sulfate. For the staining of B cells, anti CD45R/B220 MAb (BD Pharmingen) was used as primary antibody. Granulocytes were stained by using the specific Naphtole-AS-D-Chloracetate-Esterase kit (Sigma) according to the manufacturer's instructions.

All visible portal tracts of a liver section were evaluated by a pathologist in a blinded fashion and graded as follows: grade 0, normal histomorphology; grade 1, minor inflammatory infiltrates with occasional liver cell necrosis; grade 2, moderate liver damage with inflammatory infiltrates and focal necroses; and grade 3, extensive infiltrates in portal tracts and lobules accompanied by diffusely distributed liver cell necroses. At least two separate sections were assessed per liver. For single animals, multiple cryostat sections were assessed from different parts of the liver.

Statistical analysis. Means ± SE are given. For comparison of groups, the Wilcoxon two-sample test or the Mann-Whitney rank sum test was applied. P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of hCD2-Delta kTbeta RII mice. To interrupt TGF-beta signaling in T cells, we inserted the cDNA of a truncated TGF-beta type II receptor (Delta kTbeta RII), which lacks the intracellular kinase domain, into the human CD2 minilocus expression vector (Fig. 1A). The hCD2 promoter/enhancer drives the copy number-dependent expression in T cells (56). We obtained three founder animals that were fertile and gave rise to three transgenic lines, which were designated according to the Institute for Laboratory Animal Research guidelines as TgN(CD2Delta kTbeta RII)1Mbl, TgN(CD2Delta kTbeta RII)2Mbl, and TgN(CD2Delta kTbeta RII)4Mbl. These transgenic lines are abbreviated from here on as lines 1, 2, and 4. Heterozygous animals of all three lines showed no macroscopic abnormalities. Mice were observed until the age of 9 mo. Examination of hematoxylin- and eosin-stained sections of various organs and tissues including the liver, lung, heart, spleen, kidneys, and intestine revealed normal histology (data not shown).


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Fig. 1.   Generation and characterization of transgenic mice. A: schematic presentation of the hCD2-Delta kTbeta RII construct. The open box represents the cDNA for the human dominant-negative TGF-beta receptor II mutant (Delta kTbeta RII) that was inserted into the second exon of the hCD2 minilocus expression vector. Localization of the primers used for transgene detection is shown. B and C: cell type-specific RNA expression of the transgene (tg) is shown by Northern blot analysis using total RNAs extracted from purified wild-type (wt) T cells, transgenic T and B cells, and various tissues of line 1 (B) and line 2 (C). The length of the transgene-derived RNA was 1.5 kb, which is consistent with the size of the truncated TGF-beta type II receptor and the adhering vector sequences. The signal of the wild-type RNA is found at 4.2 kb. Ethidium bromide staining of 18S rRNA was used as a RNA loading control. C: increased amount of RNA (30 µg) was loaded from nonlymphoid organs to underline the lack of transgene expression in these organs. D: comparison of transgene-derived RNA expression in T cells between the three transgenic lines by Northern blotting. Strongest transgene expression was found in line 2. E: TGF-beta induced IL-2 suppression is impaired in transgenic mice. Splenocytes from lines 1, 2, and 4 were cultured for 12 h at various concentrations of TGF-beta 1. T cells were purified and stimulated with anti-CD3/CD28 MAbs for 24 h, and IL-2 levels were measured in the supernatants. Values are given as %baseline IL-2 concentration (baselines: line 2 = 625 pg/ml; line 1 = 433 pg/ml; line 4 = 335 pg/ml; wild-type T cells = 219 pg/ml).

Tissue-specific transgene expression was assessed by Northern blot analysis. Total RNA was isolated from various tissues and cell types. RNA expression in tissues of lines 1 and 2 is shown in Fig. 1, B and C. Strong expression of the transgene is seen in T cells. Weak transgene expression was observed in the B cell fraction, probably due to contaminating T cells. However, weak transgene expression in B cells cannot be excluded. Transgene expression was detectable in T cells of all three transgenic lines and was strongest in line 2 (Fig. 1D).

Next, the ability of the truncated receptor to compete with the endogenous TGF-beta type II receptor was assessed. TGF-beta 1 was added at various concentrations to splenocyte cultures. After purification and stimulation of T cells with anti-CD3 and anti-CD28 MAb, a marked TGF-beta -mediated inhibition of IL-2 production by wild-type T cells was noted [17% of baseline value; baseline = 216 (range 166-275) pg/ml; Fig. 1E]. In contrast, IL-2 production of transgenic T cells from line 2 could not be inhibited even at a concentration of 10 ng/ml TGF-beta 1 [100% of baseline value; baseline = 625 (range 558-688) pg/ml]. T cells from lines 1 and 4 were less resistant to inhibition of IL-2 production (Fig. 1E). The degree of resistance to TGF-beta correlated with the RNA expression pattern of the transgene, as demonstrated by Northern blot analysis.

Phenotypic analysis of hCD2-Delta kTbeta RII mice. The cell surface phenotypes of thymus, spleen (Fig. 2; Table 1). and lymph node cells (data not shown) were analyzed in mice up to the age of 15 wk and revealed no major differences between transgenic and nontransgenic mice. Specifically, there was no difference in total splenic cell numbers or in the CD4/CD8 ratio in spleen or lymph nodes. Staining of CD4- and CD8-positive splenic and lymph node T cells for CD62L and CD44 suggested normal numbers of naive and memory T cells.


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Fig. 2.   Thymic development in hCD2-Delta kTbeta RII mice of line 2. Thymocytes from 8- to 14-wk-old transgenic (TG) and nontransgenic (WT) mice were stained with anti-CD4 and anti-CD8 MAbs and were analyzed by flow cytometry. At least 10,000 events were collected.


                              
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Table 1.   Phenotypic analysis of mononuclear cells from spleens of 8- to 12-week-old hCD2-Delta kTbeta RII transgenic and nontransgenic mice

Due to the reciprocal relationship between TGF-beta and IFN-gamma , we examined whether the impairment of TGF-beta signaling leads to differences in IFN-gamma secretion of splenocytes. Transgenic splenocytes that were stimulated with anti-CD3 and anti-CD28 MAbs secreted more IFN-gamma than wild-type splenocytes [51 (range 42-79) ng/ml vs. 33 (range 21-42) ng/ml for culture with serum-free medium; Fig. 3]. Differences observed in the proliferation rates of stimulated [91,620 (range 86,620-94,762) counts per minute (cpm) vs. 98,518 (range 97,995-101,595) cpm for culture with serum-free medium, Fig. 3] or unstimulated (data not shown) splenocytes between wild-type and transgenic mice were not significant.


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Fig. 3.   IFN-gamma production and cell proliferation by transgenic splenocytes. Total spleen cells from transgenic (tg; line 2) and nontransgenic (wt) mice were stimulated for 72 h with anti-CD3/CD28 MAbs. IFN-gamma levels in culture supernatants were measured by using ELISA (left). For cell proliferation assays (right), cells were pulsed with 2.5 µCi/well [3H]thymidine for the last 24 h of culture. Data are presented as %wild-type concentrations/counts per minute. Two separate experiments with six animals per group are included. *P < 0.005.

Since TGF-beta 1 influences immunoglobulin class switching, we compared the levels of T cell-dependent IgA, IgG1, and IgG2a in transgenic and nontransgenic mouse sera. Significantly increased levels of TGF-beta /IL-5-dependent IgA were found in transgenic mice of line 2 (P < 0.05 for all dilutions tested; Fig. 4). Similar results were obtained for line 1, although with smaller differences between wild-type and transgenic animals. No differences were detected in Th1-dependent IgG2a or in Th2-dependent IgG1-levels.


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Fig. 4.   Increased IgA levels in sera of transgenic mice. Four fourfold dilutions of sera from 8- to 10-wk-old hCD2-Delta kTbeta RII mice of line 2 (tg) and nontransgenic littermates (wt) were assayed for the levels of IgA (A), IgG1 (B), and IgG2a (C) by using ELISA. The results are presented as means of 6 mice/group for assessment of IgA and of 3 mice/group for the assessment of IgG1 and IgG2a. The differences in IgA levels were statistically significant for all dilutions tested (**P < 0.005, *P < 0.05).

Increased susceptibility to EAH in hCD2-Delta kTbeta RII mice and characterization of hepatic infiltrates. To test whether loss of TGF-beta signaling might influence the susceptibility to EAH, we induced EAH by repeated injection with syngenic liver homogenate in complete Freund's adjuvant. Liver histology was analyzed 2 wk after the last immunization. Wild-type mice showed only minor granulocytic infiltrates restricted to the portal tracts (Fig. 5A). In contrast, transgenic mice showed portal and periportal leukocytic infiltrates with hepatocellular necroses (Fig. 5, B and C). Immunohistochemical staining was performed to further characterize the phenotype of cells infiltrating the liver. A significant amount of CD3-positive T cells (Fig. 5D) and some B cells (Fig. 5F) were seen within the hepatic infiltrate. Scattered T cells were also seen within the hepatic lobule. The majority of cells at this stage of hepatitis induction were granulocytes (Fig. 5E). The severity of hepatitis was graded in a blinded fashion, and at least two separate sections were analyzed per animal.


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Fig. 5.   Histology of experimental autoimmune hepatitis (EAH) in hCD2-Delta kTbeta RII mice. Hepatitis was induced by repeated intraperitoneal injection of 2.5 mg of protein from 100,000-g supernatants of syngenic liver homogenate in complete Freund's adjuvant. Two weeks after the last injection, mice were bled and killed. Results are representative of 4 separate experiments with 5-10 mice/group. Liver sections were stained with hematoxylin and eosin and assessed for severity of hepatitis by a pathologist in a blinded fashion. A: wild-type mice show very little signs of hepatitis with occasional granulocytes around bile ducts. B and C: transgenic mice show portal and periportal leukocytic infiltrates with hepatocellular necroses. D-F: infiltrates were characterized by immunohistochemical staining for CD3 (T cells; D) and CD45R/B220 (B cells; F) and were characterized histochemically by using chloracetate esterase assay for granulocytes (E). Bars = 50 µm.

In addition, in selected animals cryosections were assessed from different parts of the liver. In case of inflammation, hepatic infiltrates and hepatocellular necroses were quite evenly distributed throughout the liver, making a sampling error unlikely. A significantly higher histological hepatitis score (median score = 1.8 ± 0.26 vs. 0.75 ± 0.09; P < 0.01; Fig. 6A) was found in transgenic mice. Serum transaminases were measured as a marker of hepatocellular damage. At baseline, no difference could be detected between wild-type and transgenic mice (aspartate aminotransferase = 59 ± 2 vs. 55 ± 2 U/l; Fig. 6B). A gradual rise in transaminases was observed after the start of immunizations, which had already reached significance for transgenic mice at day 10 after immunization compared with baseline values (P < 0.001; Fig. 6B). The difference in aspartate aminotransferase levels in wild-type and transgenic mice on day 10 after immunization (74 + 8 vs. 116 + 22 U/l; P = 0.09) increased until 2 wk after the last immunization, when the mice were killed (144 + 32 vs. 322 + 73 U/l; P = 0.02; Fig. 6B). As a control, transgenic mice were also immunized with complete Freund's adjuvant alone containing only mycobacterial antigens. In two separate experiments, severity of hepatitis was found to be less than in transgenic animals immunized with syngenic liver homogenate emulsified in complete Freund's adjuvant but more pronounced than in wild-type animals, indicating that inflammation in the liver can be induced in transgenic animals by using adjuvant alone (data not shown).


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Fig. 6.   Severity of EAH in hCD2-Delta kTbeta RII mice. Liver sections were graded by a pathologist in a blinded fashion. Results are representative of 5 separate experiments including 5-10 mice/group. A: mean histological score was significantly higher in transgenic mice (*P < 0.01). B: aspartate aminotransferase levels as a marker of hepatocellular damage did not differ at baseline between wild-type and transgenic mice. After immunizations with the 100,000-g supernatant were initiated, transgenic mice developed significantly elevated serum aminotransferase levels shortly after the first immunization (P = 0.001 compared with baseline values), which further increased until 2 wk after the last immunization (**P = 0.02 for comparison between wild-type and transgenic mice).

Various other organs were examined histologically. Examination of the hearts showed normal histology. Little peribronchial inflammation was found in lungs. Occasionally, interstitial nephritis was observed but with no significant difference between transgenic and wild-type mice (data not shown).

Inflammatory cells were isolated from the livers by using Percoll gradient centrifugation. Flow cytometric analysis revealed that one-third of mononuclear cells isolated from transgenic livers were CD3-positive T cells, whereas in wild-type animals only 14% T cells were found (Fig. 7A). These cells were then analyzed for their cytokine secretion profile after anti-CD3/28 stimulation in vitro. Liver-infiltrating cells in transgenic animals were found to secrete more IFN-gamma (118 vs. 45 ng/ml; Fig. 7B) and less IL-4 (341 vs. 1,256 pg/ml; Fig. 7C) than their wild-type counterparts. The experiments shown in Fig. 7 were performed twice, yielding similar results each time.


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Fig. 7.   Characterization of hepatic mononuclear infiltrate. Cells were isolated from inflamed livers by using Percoll gradient centrifugation 2 wk after the last immunization. A: cells from wild-type (WT) and transgenic (TG) mice were stained with anti-CD3-FITC and analyzed by flow cytometry. Cells were stimulated with anti-CD3/28 for 48 h, and cytokines were analyzed in culture supernatants by using ELISA. B: ELISA for IFN-gamma . C: ELISA for IL-4. Median values are given.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results presented demonstrate that impairment of the suppressive effects exerted by TGF-beta on T cells can facilitate or augment autoimmune inflammatory liver disease. The important role of TGF-beta in immune homeostasis has been previously shown in TGF-beta 1 knockout mice that develop a multifocal inflammatory disorder (29, 49). In these mice, lack of TGF-beta 1 leads to a number of abnormalities, including upregulation of major histocompatability complex (MHC) class I and II (17), dysregulation of hematopoiesis (55), and abnormal development as demonstrated by a 50% embryonic lethality (30). Furthermore, TGF-beta 1 knockout mice still express TGF-beta 2 and 3, which might, in part, compensate for the lack of TGF-beta 1.

The importance of T and B lymphocytes in the development of the autoimmune disorder observed in TGF-beta 1 knockout mice has been suggested by studies using TGF-beta 1 knockout SCID mice (13) and TGF-beta 1/MHC class II knockout mice (33). Yet the multifocal inflammatory disorder spontaneously developing in TGF-beta 1 knockout mice also affects the liver and leads to early death. Together with the above-mentioned restrictions of delineating the effects of TGF-beta to a certain cell type, TGF-beta 1 knockout mice seemed unsuitable for the study of the role of TGF-beta signaling in T cells in the maintenance of immune homeostasis within the liver.

We therefore generated transgenic mice that overexpress a dominant-negative TGF-beta type II receptor in T cells. The impairment of TGF-beta signaling in T cells did not lead to spontaneous autoimmunity, as observed in the TGF-beta 1 knockout model and as recently described for another model of T cell-specific loss of TGF-beta signaling (19). This might be due to the FVB/N mouse strain we used in our experiments, because C57BL/6 mice seem to be more prone to immune dysregulation (38). Indeed, the importance of the genetic background in the development of autoimmune disease has been demonstrated by the strain-dependent phenotypes of IL-2 knockout mice (46, 47). For example, Fcgamma RIIB knockout mice on the C57BL/6 background develop a lethal, lupus-like syndrome, whereas mice on the BALB/c background present with a normal phenotype (6). Very recently, strain specificity could be demonstrated for the development of hepatic inflammation in TGF-beta 1 knockout mice bred to the BALB/c or to a hybrid 129/CF-1 background (20).

Conflicting results have emerged from the impairment of TGF-beta signaling in T cells in three different models recently described (19, 40, 43). Expression of a dominant-negative TGF-beta type II receptor under the control of the CD4 promoter lacking the CD8 silencer has resulted in spontaneous autoimmunity with lymphocytic infiltrates in various organs, including the liver (19). In another model using the hCD2 promoter, mice developed a CD8+ T cell lymphoproliferative disorder without any inflammatory component (40). In contrast, overexpression of Smad7, which is inhibitory for several members of the TGF-beta superfamily, did not produce a spontaneous phenotype (43). The reason for the differences observed remain to be elucidated but might relate to different promoter specificity and expression during thymic development as well as differences in the degree of impairment of TGF-beta signaling and differences in the genetic background. Although we were able to uncouple IL-2 production in T cells of line 2 from TGF-beta 1-mediated suppression in our model, it was not designed for complete abrogation of TGF-beta signaling to analyze the role of TGF-beta in the initiation phase of EAH.

Since our transgenic mice did not show spontaneous histological abnormalities, they provide a suitable model to study the regulatory role of TGF-beta on T cells in the induction phase of autoimmune liver injury. We induced EAH by immunizing mice with syngenic liver homogenate in complete adjuvant. This has been shown to result in autoimmune liver injury with varying intensity in different mouse strains (38). Only minimal biochemical or histological signs of hepatitis were detected in nontransgenic FVB/N mice after three immunizations with liver homogenate in adjuvant, demonstrating the relative resistance of this mouse strain to EAH. In contrast, transgenic mice developed portal and periportal leukocytic infiltrates with hepatocellular necroses and significantly elevated aminotransferase levels, indicating that TGF-beta signaling in T cells is required for maintenance of immune regulation in autoimmune liver injury.

A significant proportion of liver-infiltrating cells were T cells, the proportion in transgenic mice being about twice as high as in wild-type mice. Stimulated in vitro by anti-CD3/28 cross-linking, the inflammatory cells isolated from transgenic livers produced more IFN-gamma and less IL-4 than their wild-type counterparts. IFN-gamma production by liver-infiltrating T cells has been made responsible for the phenotype of necroinflammatory hepatitis recently described for TGF-beta 1 knockout mice on the BALB/c background (20). Impaired TGF-beta signaling in T cells might be partially responsible for the increased IFN-gamma production observed in this model. The importance of IFN-gamma in the pathogenesis of liver injury has been demonstrated in transgenic mice with increased IFN-gamma production in the liver (51) and in the T cell-dependent hepatitis models induced by injection of concanavalin A (31) or hepatitis B virus surface antigen (45). However, there is no direct proof of IFN-gamma being directly involved in the pathogenesis of EAH and the more severe reaction observed in our transgenic model.

In addition, IFN-gamma has been detected within the inflammatory lesions of patients with AIH (23). EAH shares several other features with human AIH periportal hepatitis, such as lymphocytic infiltrates (27, 38, 52), T cell reactivity to liver antigens (38), autoantibody production (35), and response to immunosuppressive therapy (36). In patients with AIH, strong TGF-beta 1 expression can be found in inflamed livers, which seems to correlate with disease activity (4). It could thus be speculated that TGF-beta in the inflamed liver is at least in part produced to suppress infiltrating autoreactive T cells.

In addition to the increased susceptibility to induction of EAH, we demonstrated that in hCD2-Delta kTbeta RII mice lymphocytes produce higher amounts of Th1-like cytokines. Baseline IL-2 production of line 2 T cells was nearly three times the amount of wild-type T cells. Transgenic splenocytes secreted more IFN-gamma on CD3/CD28 cross-linking, which was also shown by using serum-free medium, thus eliminating the possible inhibitory effect of serum-derived TGF-beta on wild-type splenocytes. These findings are in accordance with the study by Gorelik and Flavell (19), who demonstrated that, in the absence of TGF-beta signaling, most of the T cells differentiated into effector cells.

Although our study and others demonstrate the importance of T cells in EAH, a possible contribution of B cells cannot be excluded. In our transgenic animals, increased levels of serum IgA were found. TGF-beta is an important immunoglobulin class switch factor for IgA (26), and decreased serum IgA-levels can be found in TGF-beta knockout mice (53). TGF-beta signaling in B cells seems to be essential for the secretion of IgA, since a recently published conditional mutagenesis of the TGF-beta type II receptor in B cells was associated with a virtually complete absence of serum IgA (8). In this context it is important to note that weak expression of the transgene in the B cell fraction of our transgenic mice cannot be excluded. However, the fact that serum IgA levels are increased in our transgenic animals shows that TGF-beta signaling in B cells is functional. This is consistent with the finding that only strong overexpression of a dominant-negative type II receptor over the endogenous receptor impairs signal transduction. It was suggested that T cells with loss of TGF-beta signaling might stimulate IgA secretion of B cells by increased TGF-beta production (19).

The results of this study demonstrate the important role of TGF-beta in counteracting T lymphocyte-mediated liver injury. TGF-beta signaling in T cells is necessary to suppress infiltration of the liver on EAH induction. Overexpression of TGF-beta occurring in active AIH is thus likely to be an important regulatory mechanism toward reestablishing homeostasis, albeit at the expense of increased fibrogenesis, eventually leading to cirrhosis.


    ACKNOWLEDGEMENTS

We thank Karin Nicol and Marina Snetkova for excellent technical assistance.


    FOOTNOTES

This work was supported by the Deutsche Forschungsgemeinschaft, project C3, Sonderforschungs Bereich 548, and MAIFOR, Faculty of Medicine, University of Mainz and the Boehringer-Ingelheim Foundation.

Address for reprint requests and other correspondence: M. Blessing, I. Dept. of Medicine, Johannes-Gutenberg Univ., Obere Zahlbacherstr. 63, 55131 Mainz, Germany (E-mail:blessing{at}mail.uni-mainz.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published December 4, 2002;10.1152/ajpgi.00286.2002

Received 16 July 2002; accepted in final form 20 November 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 284(3):G525-G535
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