IL-10, regulatory T cells, and Kupffer cells mediate tolerance in concanavalin A–induced liver injury in mice

Authors

  • Annette Erhardt,

    1. Institute of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen-Nuremberg, Erlangen, Germany
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    • These authors contributed equally to this work.

  • Markus Biburger,

    1. Institute of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen-Nuremberg, Erlangen, Germany
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    • These authors contributed equally to this work.

  • Thomas Papadopoulos,

    1. Institute of Pathology, University of Erlangen-Nuremberg, Erlangen, Germany
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  • Gisa Tiegs

    Corresponding author
    1. Institute of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen-Nuremberg, Erlangen, Germany
    • Institute of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen-Nuremberg, Fahrstrasse 17, D-91054 Erlangen, Germany
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    • fax: (49) 9131-85-22-774


Abstract

The liver appears to play an important role in immunological tolerance, for example, during allo-transplantation. We investigated tolerance mechanisms in the model of concanavalin A (ConA)-induced immune-mediated liver injury in mice. We found that a single injection of a sublethal ConA dose to C57BL/6 mice induced tolerance toward ConA-induced liver damage within 8 days. This tolerogenic state was characterized by suppression of the typical Th1 response in this model and increased IL-10 production. Tolerance induction was fully reversible in IL-10−/− mice and after blockade of IL-10 responses by anti-IL10R antibody. Co-cultures of CD4+CD25+ regulatory T cells (Tregs) and CD4+CD25 responder cells revealed Treg from ConA-tolerant mice being more effective in suppressing polyclonal T cell responses than Treg from control mice. Moreover, Treg from tolerant but not from control mice were able to augment in vitro IL-10 expression. Depletion by anti-CD25 monoclonal antibody (MAb) indicated a functional role of Tregs in ConA tolerance in vivo. Cell depletion studies revealed Tregs and Kupffer cells (KC) to be crucial for IL-10 expression in ConA tolerance. Studies with CD1d−/− mice lacking natural killer T (NKT) cells disclosed these cells as irrelevant for the tolerogenic effect. Finally, cellular immune therapy with CD4+CD25+ cells prevented ConA-induced liver injury, with higher protection by Treg from ConA-tolerized mice. Conclusion: The immunosuppressive cytokine IL-10 is crucial for tolerance induction in ConA hepatitis and is mainly expressed by CD4+CD25+ Treg and KC. Moreover, Tregs exhibit therapeutic potential against immune-mediated liver injury. (HEPATOLOGY 2007;45:475–485.)

In the liver, local presentation of antigen causes T cell inactivation, tolerance, and apoptosis, possibly resulting from the need to maintain immunological silence to gut-derived food antigens.1 The overall predisposition of intrahepatic T cell responses toward tolerance might account for survival of liver allografts without immunosuppression and persistence of liver pathogens such as hepatitis B or C viruses.1, 2 In the past, tolerogenic mechanisms of the liver have been studied mainly with respect to CD8+ T cells, whereas less attention has been addressed to CD4+ T cells in this scenario,3 although they occur at highest frequency within the liver.4

To study cellular and molecular mechanisms of immune-mediated hepatitis, we have developed a T-cell–dependent liver injury model induced by concanavalin A (ConA) in immunocompetent mice. Liver damage in this model depends on CD4+ T cells,5 natural killer T (NKT) cells,6, 7 and Kupffer cells (KC).8 The Th1 cytokines tumor necrosis factor (TNF-α),9 interferon gamma (IFN-γ),10 IL-12,11 and IL-18,12 are important for disease development, whereas IL-1013, 14 is protective. ConA hepatitis has been referred to as a model for certain mechanisms of autoimmune hepatitis, because of responsiveness to immunosuppressive drugs,5 genetic prevalence of certain mouse strains with respect to susceptibility,15 and immunosuppression in state of remission. The mechanisms of this immunosuppression and potential involvement of regulatory cell types have not been elucidated so far. Two types of T cells particularly appear as potential candidates: CD4+CD25+ FoxP3+ Tregs or NKT cells.

Tregs control autoreactive T cells in vivo and their reduction or dysfunction causes certain autoimmune diseases in animals and humans.16, 17 Moreover, Tregs can control allograft rejection18 and inflammatory diseases, for example, in colon and kidney.19, 20 According to their capacity to suppress T-cell responses Tregs may worsen tumor immunity21 or microbial defense, for example, against hepatitis viruses.22 Tregs are characterized by co-expression of CD4 and CD25.16 The best marker with respect to their regulatory function is Foxp3.23 Other established but not necessarily specific markers are the glucocorticoid-induced TNF receptor-related protein,24 CD62L,25 and CD103.26 CD4+CD25+ T cells express cytotoxic T lymphocyte–associated protein 4, which mediates inhibition of effector T cell proliferation via cell–cell contact and inhibition of IL-2 expression.27 Interference with cytotoxic T-lymphocyte–associated protein 4 is sufficient to cause autoimmune disease in otherwise normal animals.28 Tregs have also been described to suppress T cell effector functions and inflammatory disease via IL-1019 or transforming growth factor beta production.29 Depending on the cytokine milieu, CD4+ T cells can differentiate into regulatory IL-10–producing Tr1 or transforming growth factor beta–secreting Th3 cells,30 thereby representing adaptive Treg populations. The differentiation to Tr1 or Th3 cells probably depends on natural CD4+CD25+ Tregs.31

NKT cells also have been described as suppressing certain autoimmune diseases, and their absence has been associated with autoimmune pathology.32–34 NKT cells are most frequent in the liver of mice but are also present in thymus, bone marrow, lymph nodes, and spleen. Their thymic development depends on the glycolipid-presenting MHC class I–like protein CD1d.32 Besides a restricted T cell receptor repertoire with an invariant Vα14-Jα18 chain in mice and homologous Vα24-Jα18 in man, most NKT cells express CD4 or are double negative and reveal a phenotype of activated T cells together with NK cell determinants, for example, NK1.1+ in C57BL/6 mice.32 On activation, NKT cells rapidly secrete high amounts of cytokines, mainly IL-4 and IFN-γ. NKT cell activation in vivo by their surrogate antigen α-galactosylceramide has been shown to prevent autoimmune disease, probably via an IL-10–dependent mechanism.35–37

Here we show that in C57BL6 mice, a single injection of a sublethal dose of ConA induces tolerance toward ConA-induced liver damage within 8 days, accompanied by an anti-inflammatory cytokine profile with prominent induction of IL-10. Indeed, this cytokine was identified to be crucial for ConA tolerance. However, the accompanying inhibition of IL-2 production, which was observed in ConA tolerance, appears to be IL-10 independent. Moreover, Tregs but not NKT cells were identified to be necessary for induction of the tolerogenic state. Tregs together with KC appear essential for pronounced IL-10 expression during tolerance. Studies using FACS-purified CD4+CD25+ Tregs suggest a therapeutic potential of these cells in immune-mediated liver injury.

Abbreviations

ConA, concanavalin A; CFSE, carboxyfluorescein-diacetate-succinimidyl-ester; IFN-γ, interferon gamma; KC, Kupffer cells; MAb, monoclonal antibody; NKT, natural killer T; RT, reverse transcription; Tregs, regulatory T cells.

Materials and Methods

Mice.

Male C57BL/6 wild-type, IL10−/−38 or Rag−/−39 mice (8-10 weeks old) were obtained from Janvier, Le Genest-St-Isle, France; Jackson Laboratory, Bar Habor, ME; or from animal facilities of the Institute of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen-Nuremberg, Germany. CD1d−/− mice40 (8-10 weeks old, C57BL/6 background) were a gift from Luc van Kaer, Nashville, TE. Animals were maintained under controlled conditions (22°C, 55% humidity, 12-hour day/night rhythm) and fed standard laboratory chow. All mice received humane care according to the guidelines of the National Institutes of Health and legal requirements in Germany.

Animal Treatments.

Concanavalin A type IV (ConA) was purchased from Sigma Chemical Co. (St. Louis, MO) and 10 to 15 mg/kg was administered intravenously in pyrogen-free saline. Control mice were injected with saline. Eight days later animals were restimulated with ConA. For KC-depletion, 100 μl liposome-encapsulated dichloromethylene-biphosphonate (Clodronate liposomes; provided by Dr. van Rooijen, Vrije Universiteit, Amsterdam, The Netherlands) was injected intravenously 48 hours before ConA-re-challenge. Dichloromethylene-biphosphonate for their preparation was a gift of Roche Diagnostics, (Mannheim, Germany). To block IL-10 responses, 500 μg anti–IL-10-receptor MAb (DNAX/ Schering-Plough Biopharma) were injected intravenously per mouse 1 hour before ConA pretreatment. In vivo depletion of CD4+CD25+ Tregs was achieved by intraperitoneal injection of 250 μg anti-CD25 MAb (clone PC-61.5) or isotype-control rat IgG 24 hours before ConA-restimulation. In an immune-therapeutic approach, 2 groups of wild-type mice received 1 × 106 splenic CD4+CD25+ Tregs intravenously from ConA-pretreated or saline-pretreated mice. Control mice received 1 × 106 CD4+CD25− cells. All groups received ConA challenge 24 hours after cell injection.

Sampling of Material.

Mice were anesthetized lethally (150 mg/kg intravenous methohexital + 15 mg/kg heparin) 8 hours after ConA injection. Cardiac blood was withdrawn for plasma cytokine determination and analysis of plasma transaminases. After excision, small liver samples were frozen in liquid nitrogen for RNA isolation/reverse transcription (RT)-PCR, a second part was embedded in GSV-1 tissue-embedding medium (Slee Technik GmbH, Mainz, Germany) and frozen at −75°C for immunofluorescent analysis.

Analysis of Plasma Aminotransferases.

Liver injury was quantified by automated measurement of plasma activities of ALT and AST 8 hours after ConA administration using a COBAS Mira System (Roche).

Cytokine Quantification by ELISA.

Sandwich ELISAs for murine plasma TNF-α, IFN-γ, IL-2, IL-6, and IL-10 were performed using Nunc-Immuno 96-well flat-bottom high-binding Maxisorb-polystyrene microtiter plates (Nalge-Nunc International). Antibodies were purchased from BD Pharmingen for IL-2, IL-6, and IL-10. Streptavidin-peroxidase was purchased from Roche Diagnostics. IFN-γ and TNF-α were quantified using DuoSet ELISA-Development Systems (R&D Systems) and the TMB-Substrate Reagent Set (BD Pharmingen) according to manufacturers' instructions.

Isolation of RNA and Real-Time PCR for Cytokine mRNAs.

Total RNA was isolated from liver tissue using the NucleoSpin RNA II Isolation Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's protocol. One microgram total RNA was transcribed using SuperScript II RnaseH reverse transcriptase, oligonucleotides, and oligo(dT) primers from Invitrogen (Karlsruhe, Germany). Real-time RT-PCR was performed using a LightCycler system and LightCycler-FastStart DNA-Master SYBR-Green-1 mix (Roche). Primer pairs were used as described previously.41 To confirm amplification specificity, melting curves of PCR products were analyzed. Relative mRNA levels were calculated by means of 2ΔCP (ΔCP = difference of crossing points of test samples and respective control samples as extracted from amplification curves by the LightCycler software) after normalization to reference β-actin levels.

Isolation of Splenocytes, Lymph Node Cells, and Nonparenchymal Liver Cells.

Tissues were passed through 100 μm nylon meshes. If applicable, hepatocytes were removed by centrifugation (800g, 20 minutes) in isotonic 37% Percoll solution (Amersham-Biosciences, Freiburg, Germany) containing 100 U/mL heparin. Erythrocytes were removed using lysis solution (139 mM NH4Cl, 19 mM Tris).

Flow-Cytometric Analysis.

Typically 4 × 105 leukocytes were stained using a standard protocol including pre-blocking of Fc receptors. The following MAbs were used: anti-CD16/32 (“Fc-block”; clone 93; eBioscience), FITC-labeled anti-mouse-NK1.1 (clone PK136), anti-mouse-CD103-FITC (clone M290), anti-mouse-CD45RB-biotin (clone 16A), CyChrome-labeled anti-mouse-CD3σ (clone 145-2C11; all BD Pharmingen), anti-mouse CD4-Tricolor (clone RM4-5; Caltag-Laboratories, Hamburg, Germany), anti-mouse-CD62L-FITC (clone MEL-14), anti-mouse-CD4-FITC (clone YTS.1.2) (Immunotools, Friesoythe, Germany), and anti-mouse-CD25-PE (clone 7D4; Miltenyi).

For intracellular FoxP3 staining, biotinylated anti-FoxP3 (clone FJK-16s) (eBioscience/NatuTec, Frankfurt, Germany) and streptavidin-CyChrome (BD-Pharmingen) were used together with “FoxP3-Staining Buffer Set” (eBioscience/NatuTec) according to manufacturer's instructions. Intracellular IL-10 staining was carried out using anti-mouse IL-10-FITC (clone JES5-2A5, Caltag) concomitant with FoxP3 detection. Data were recorded and analyzed using a FACScan Flow Cytometer (BD Biosciences) and Cellquest software.

Isolation of CD4+CD25+ Tregs and Responder T Cells.

A combined sorting procedure was carried out using magnetic bead separation (CD4+CD25+ Regulatory T-Cell-Isolation Kit, mouse; Miltenyi Biotec, Bergisch Gladbach, Germany) and FACS-sorting. Briefly, CD4+ T-cell were enriched using a biotinylated antibody cocktail, depleting all other blood cell types and anti-biotin microbeads. CD4+CD25+ T cells were isolated by positive selection using PE-labeled anti-CD25 MAb and anti-PE microbeads. Purity was controlled by flow cytometry and reached approximately 85%. Subsequently, CD4+CD25+ Tregs, and splenic CD4+CD25 responders or liver-derived CD4+CD25-NK1.1 responders were purified to approximately 98% by FACS-sorting using a MoFlo Cellsorter (Dako Cytomation; Freiburg, Germany).

Culture of Sorted Splenocytes and Hepatic Lymphocytes.

1 × 105 splenic responder cells (CD4+CD25) or CD25/NKT-cell–depleted hepatic lymphocytes were cultured alone or with 1 × 105 CD4+CD25+ Tregs for 72 hours in 96-well round-bottom plates in the presence of either ConA (5 μg/ml) or immobilized anti-CD3 MAb (5 μg/mL; clone 145-2C11, Immunotools). Cytokine concentrations in supernatant were measured by ELISA.

Carboxyfluorescein-Diacetate-Succinimidyl-Ester Cell-Proliferation Assay.

CD4+CD25 responder cells were labeled with carboxyfluorescein-diacetate-succinimidyl-ester (CFSE) using “Molecular Probes Vybrant CFDA-SE Cell-Tracer Kit” (Invitrogen) and cultured alone or together with Tregs in 96-well round-bottom plates for 3 days under different stimulation conditions as indicated. Proliferation (reflected by successive diminution of fluorescence intensities by dye distribution to daughter cells) was measured by flow cytometry.

Histology.

For histological analysis of tissue structure, livers were fixed in 4% formalin and subsequently embedded in paraffin. Sections were stained with hematoxilin-eosin using a standard procedure and analyzed by light microscopy.

Statistical Analysis.

Data are presented as mean ± SEM. Results were analyzed using Student t test if 2 groups were compared, the Tukey's test if more groups were compared. P values less than 0.05 were considered significant.

Results and Discussion

ConA Pretreatment Protects Mice From Liver Injury on Rechallenge.

To analyze the potential of ConA-mediated immune activation to induce a tolerogenic/immune-suppressive milieu, C57BL/6 mice were pretreated with a sublethal ConA dose or saline as a negative control, 3, 8, or 14 days before ConA rechallenge. In fact, in comparison with saline-treated control mice, ConA-pretreated mice were partially protected from liver injury mediated by ConA rechallenge 8 or 14 days after pretreatment, as reflected by histological analysis (Fig. 1). In contrast, liver damage on ConA rechallenge as early as 3 days after ConA pretreatment was even more pronounced than in control mice. Earlier timepoints were not analyzed in this assay, because within a few hours after ConA injection NKT cells, which are essential for ConA-induced hepatitis, are well known to transiently disappear (or at least become undetectable and incapable of being stimulated) for a few days as discussed below. This time course experiment prompted us to analyze characteristics of ConA tolerance at 8 days after ConA pretreatment, when a tolerogenic state was already reproducibly reached as reflected by significantly lower activities of plasma ALT and AST (Fig. 2).

Figure 1.

ConA tolerance develops within 8 days after a single ConA pretreatment. To assess the effects of ConA pretreatment on ConA-induced damage of liver tissue, mice were rechallenged at indicated timepoints after pretreatment, and liver samples taken 8 hours after ConA rechallenge were analyzed using hematoxylin-eosin staining. Samples from mice without ConA challenge or saline-pretreated, ConA-challenged mice were used as negative or positive controls, respectively. On ConA rechallenge at 3 days pretreatment, necrotic liver injury (indicated by black arrows) was even more pronounced than in saline-pretreated control mice and resulted in extended bridging necroses. Whereas at later timepoints of restimulation tolerance was established, resulting in diminished necrotic liver damage, ConA could still provoke infiltration of leukocytes into liver tissue (white arrows).

Figure 2.

Protection from ConA-induced liver injury by ConA pretreatment. ConA or saline as control were injected intravenously into mice 8 days before ConA restimulation. Plasma transaminase activities were determined 8 hours after ConA rechallenge. The experiment was repeated independently more than 10 times (mean ± SEM; n ≥ 3; *P ≤ 0.05 vs. saline-pretreated control).

Passive mechanisms like depletion of KC or CD4+ T or NKT cells (which all are crucial for ConA liver injury) might cause the same effects. However, we could clearly demonstrate that ConA tolerance is not caused by depletion of KC or effector lymphocytes (supplement). Also, using CD1d-knockout mice, we were able to exclude NKT cells, which are known to be capable of mediating immunosuppression, to be relevant for ConA tolerance (see Supplementary Figures).

ConA Tolerance Is Associated With an Anti-inflammatory Cytokine Profile.

ConA-induced liver injury is accompanied by production of a broad range of cytokines, including TNF-α and IFN-γ, whose cooperative signaling is indispensable for the onset of ConA hepatitis. Protection from liver injury in mice pretreated with ConA at day −8 was associated with significantly lower IFN-γ, IL-2, and IL-6 responses on ConA rechallenge, as measured by ELISA in plasma 8 hours after rechallenge, that is, at the timepoint of ALT quantification (Fig. 3A, for other timepoints of rechallenge see Supplementary Figures). On quantitative real-time RT-PCR-analysis, the significantly diminished IL-2 and IFN-γ plasma levels were partially reflected in the liver, with their intrahepatic mRNA levels being lower in ConA-pretreated than saline-control mice at 8 hours after rechallenge (Fig. 3B). IFN-γ expression is significantly elevated shortly (1.5 hours, data not shown) after rechallenge in ConA-pretreated mice compared with saline controls, indicating that lymphocytes in pretreated mice still responded to ConA rechallenge and, thus, induction of broad-range anergy is probably not responsible for ConA-induced tolerogenic effects. Tolerization did not significantly affect plasma TNF-α concentrations on ConA challenge (Fig. 3A). However, TNF-α expression—and also IL-6 expression—in liver tissue 8 hours after ConA rechallenge was significantly lower in ConA-tolerized than in saline-pretreated mice (Fig. 3B). Relevant changes in transforming growth factor beta expression were found neither in plasma nor intrahepatically (not shown). Aggravated liver damage on ConA rechallenge observed 3 days after ConA pretreatment corresponded well to accompanying exacerbated IFN-γ and TNF-α responses (Supplementary Figures).

Figure 3.

Induction of an anti-inflammatory cytokine profile on ConA restimulation. Cytokine expression in ConA- or saline-pretreated mice 8 hours after ConA rechallenge was measured (A) in plasma by ELISA, or (B) with respect to intrahepatic cytokine mRNA expression by quantitative real-time RT-PCR. For RT-PCR analysis, β-actin mRNA from each sample was used as an internal standard to normalize for equal levels of total mRNA. X-fold induction was calculated referring to mRNA levels of the respective cytokines in saline-pretreated mice. The graphs depict the summary of two independent experiments (mean ± SEM; n ≥ 5; *P ≤ 0.05 vs. saline control), and this pattern of anti-inflammatory cytokine modulation by ConA pretreatment also was found in several additional experiments.

In contrast to all other cytokines tested, the anti-inflammatory cytokine IL-10 showed significantly higher expression in ConA-pretreated than in control mice, reflected on both its plasma concentrations as well as intrahepatic IL-10 mRNA expression levels (Fig. 3A, B). These observations point to a potential role of IL-10 in the development of ConA tolerance.

To test whether the in vivo effect of reduced proinflammatory cytokine responses was reproducible in vitro, splenocytes from ConA- or saline-pretreated mice were isolated 8 days after pretreatment and restimulated ex vivo. In fact, on both restimulation with either ConA or anti-CD3, splenocytes from ConA-pretreated mice responded by significantly reduced IL-2, IFN-γ, and TNF-α expression and increased IL-10 production (Fig. 4).

Figure 4.

Modified cytokine responses of splenocytes from ConA-tolerized mice to ConA- or anti-CD3 stimulation in vitro. Splenocytes from both tolerized and non-tolerized mice were isolated 8 days after pre-treatment, cultivated (2 × 105/well), and restimulated with either ConA (5 μg/ml) or immobilized anti-CD3 MAb (5 μg/ml). After 72 hours, cytokine concentrations in supernatant were measured by ELISA, revealing a pronounced bias to anti-inflammatory cytokine responses in cells from ConA-tolerized mice. (mean ± SEM; n = 4; *P < 0.05 vs. respective saline controls).

IL-10 Is Important for the Antipathogenic Effect of ConA Tolerance.

A critical role of IL-10 in the onset of ConA tolerance was demonstrated by reversal of ConA tolerance regarding suppression of hepatocyte damage, IFN-γ, and IL-6 production in IL-10−/− mice (Fig. 5A) as well as by injection of anti-IL-10R MAb (Fig. 5B). In contrast, IL-2 production on ConA challenge was largely suppressed also in ConA-tolerized IL-10−/− and anti-IL-10R–treated mice, indicating that IL-2 impairment is IL-10 independent in this model. This clearly shows that IL-2 diminution, which is used in many reports as a main—and sometimes the only—indicator for immunoregulation, is actually not related to pathophysiology. In ConA tolerance, IL-10 might act directly via its immunosuppressive properties—corresponding to protection from ConA hepatitis by exogenous IL-1014—or might be important for development or differentiation of immunoregulatory cells such as regulatory T cells or Dendritic cells (reviewed in Mocellin et al.42).

Figure 5.

Loss of ConA tolerance in IL-10 knockout mice and anti–IL-10R–treated mice regarding pathophysiology but not IL-2 suppression. ConA or saline were injected intravenously to C57BL/6 wild type and (weight- and age-matched) IL10−/− mice (A), or mice that had been injected intravenously 24 hours before with 500 μg anti–IL-10R antibody (B). Eight days after ConA pretreatment, animals were rechallenged with ConA, and after an additional 8 hours, ALT levels and cytokine concentrations were determined. Both in IL-10−/− mice and anti–IL-10R–treated mice tolerization-induced suppression of liver injury (ALT), IFN-γ, and IL-6 expression was fully reversed, whereas IL-2 suppression remained unaffected. The results of the experiment depicted here (mean ± SEM; n ≥ 3; *P ≤ 0.05 vs. respective saline controls) were confirmed by two additional independent experiments for IL-10−/− mice and one additional anti–IL-10R experiment with intraperitoneal injection of the antibody.

CD4+CD25+ Regulatory T Cells In Vitro.

IL-10 has been shown to mediate the tolerogenic effect of CD4+CD25+ Tregs in models of autoimmune disease and inflammation.17 Therefore, we compared the features of CD4+CD25+ Tregs from ConA-tolerized to non-tolerized mice in co-cultures with equal numbers of responder cells of splenic or hepatic origin. In fact, purified CD4+CD25+ T cells from non-tolerized mice were able to suppress IL-2 production of CD4+CD25 splenic responder cells and also to significantly suppress their IFN-γ production. However, Tregs from ConA-tolerized mice showed significantly higher suppression than those of nontolerized mice (Fig. 6A).

Figure 6.

Tregs from ConA-pretreated mice reveal increased suppressive capacity and IL-10 induction in vitro. CD4+CD25+ Tregs were isolated from either ConA-pretreated or saline-pretreated mice, and 1 × 105 Tregs/well were cocultivated with (A) splenic responder T cells (CD4+CD25; 1 × 105/well) or (B) hepatic T cells, depleted from both CD25+ regulatory T cells and NKT cells by FACS sorting (CD3+NK1.1CD25; 1 × 105/well). Cocultures were stimulated with anti-CD3 MAb (plate-bound, 5 μg/mL), and cytokine concentrations in supernatant was measured after 72 hours of cultivation by ELISA (mean ± SEM; *P ≤ 0.05). (C) To detect IL-10–producing cell populations, CD4+CD25 responder cells from non-tolerized mice or CD4+CD25+ regulatory T cells from saline- or ConA-pretreated mice were cultivated for 14 hours in the presence of ConA (5 μg/mL) for stimulation and BD GolgiStop™ containing Monesin, to achieve intracellular cytokine accumulation. Finally, IL-10 production of CD4+CD25 FoxP3 responders or CD4+CD25+FoxP3+ Tregs was assessed by combined intracellular staining for FoxP3 and IL-10 and subsequent FACS analysis. (D) To test whether isolated Tregs were in principle capable of suppressing proliferation of responder cells, CD4+CD25 responders were labeled with CFSE and cultivated alone or—at a responder/Treg ratio of 10:1—together with CD4+CD25+ Tregs derived from ConA-pretreated mice for 3 days in the presence of 25 ng/mL TPA and 1 μmol/L Ionomycin. Proliferation is shown by means of the proliferative index, which represents a mathematical approximation to the median number of cell divisions the entirety of responder cells has passed through since the timepoint of labeling. Proliferative index was calculated using the algorithm proliferative index = Log[FInd/MFIall]/Log[2], with MFIall = median fluorescence intensity of all responder cells and FInd = peak-fluorescence of non-proliferating cells (mean ± SEM; *P ≤ 0.05 vs. unstimulated control; #P ≤ 0.05 vs. stimulated responder cells without Tregs).

We also analyzed the effect of Tregs on hepatic CD3+NK1.1CD25 T cells (from which Tregs and NKT cells had been removed by FACS-sorting) as responder cells. In this assay we also compared cytokine responses of responder cells from tolerized and nontolerized animals. Cocultivation of responder cells from control mice with Tregs of either ConA-tolerized or non-tolerized mice almost completely abrogated the measurable IL-2 response. Interestingly, IL-2 concentrations in culture supernatants of responder cells from ConA-pretreated mice was largely diminished compared with those from mock-treated mice, even in the absence of Tregs (Fig. 6B), indicating that IL-2 impairment is not only independent from IL-10 (see above) but ex vivo also largely independent from Tregs. (A discussion of tolerization-induced IL-2 suppression is provided as Supplementary information.) In contrast to IL-2, a significant reduction of IFN-γ production by hepatic T cells was achieved only on cocultivation with Tregs. Here again, Tregs from ConA-tolerized mice revealed significantly stronger suppression than those from saline-pretreated animals.

Single cultures of Tregs from tolerized mice showed significantly higher IL-10 concentrations than those from control mice. Also, cocultures of CD25 responder T cells with Tregs from ConA-tolerized mice but not with Tregs from control mice revealed IL-10 concentrations even higher than the sum of those of corresponding single cultures. Intracellular cytokine staining showed pronounced IL-10 expression being not conferred by CD4+CD25 FoxP3 responders cultured alone or with Tregs with or without ConA restimulation, but rather by CD4+CD25+FoxP3+ Tregs from saline-pretreated or—even more pronounced—from ConA-pretreated animals. The percentage of Tregs showing pronounced IL-10 expression increased on ConA restimulation and for Tregs from ConA mice significantly on co-culture with responder cells (Fig. 6C). Future experiments will show whether enhanced IL-10 production is caused by increased expression by original Tregs or—in a manner of infectious tolerance43—Treg-mediated engagement of originally FoxP3-CD25–negative cells in analogy to recent publications.44, 45

The observed effects of Tregs on responder cells in vitro were not caused by inhibition of proliferation of cytokine-producing responders, because under these culture conditions responder cell proliferation even without Tregs was only marginal as measured using CFSE-labeled responder cells in corresponding parallel cultures (not shown). However, to test the principle capability of MACS + FACS isolated Tregs used in our assays, to suppress proliferation of responder cells, CD4+CD25 cells were labeled with CFSE and cultivated alone or with Tregs under TPA/Ionomycin stimulation. Even at the low Treg:responder ratio of 1:10 analyzed here, Tregs significantly suppressed responder-cell proliferation (Fig. 6D).

The Role of CD4+CD25+ Regulatory T Cells In Vivo.

To investigate the potential role of CD4+CD25+ Tregs in ConA tolerance in vivo, these cells were depleted by injection of monoclonal anti-CD25 PC61.5 MAb 24 hours before ConA rechallenge. The efficiency of this depletion (>95%) was verified by FACS analysis of the splenic Treg population using anti-CD25 MAb 7D4 recognizing a different epitope than PC61.5 (Fig. 7A). Anti-CD25-treatment did not affect effector T cells that had been activated by the first ConA stimulus, because the transient activation-induced CD25 upregulation expires within approximately 3 to 5 days (own observations and demonstrated for rats in Cao et al.46). CD25-positive Treg depletion caused lower IL-2 suppression factors (Fig. 7B), suggesting that CD25+ Treg cells were involved in ConA tolerance.

Figure 7.

Involvement of Tregs in IL-2 suppression in ConA-tolerized mice. ConA or saline were injected intravenously into mice 8 days before ConA restimulation. Twenty-four hours before ConA rechallenge, half of the ConA- or saline-pretreated mice were injected with anti-CD25 MAb PC61.5 to deplete CD25-positive Tregs. (A) Efficient depletion of CD25+ Tregs was verified by FACS analysis of splenocytes. Cells were gated on viable lymphocytes by their light-scatter characteristics and on CD4-positive cells. Mean ± SEM of percentages of CD4+CD25+ Tregs within the CD4+ T-cell population is depicted. (B) Measurement of IL-2 concentrations in plasma 8 hours after ConA rechallenge showed a significant IL-2 suppression in both groups of ConA-tolerized mice (black square) in comparison with saline-pretreated mice (white square) (mean ± SEM; n ≥ 4; *P ≤ 0.05). However, the extent of suppression was significantly reduced among the Treg-depleted mice in comparison with mock-treated mice (P < 0.005, Welch t test), as indicated by the suppression factor, that is, IL-2 cytokine values of saline-pretreated mice divided by those of the ConA-pretreated mice.

To address the question of whether ConA tolerance might be associated with either an expansion of local Treg populations or modulation of their function, we analyzed their frequency in liver, spleen, and liver draining lymph nodes. We found a transient increase of CD4+CD25+FoxP3+ frequencies in all three organs (mainly at day 1); however, tolerance establishment (days 8/14) was not associated with increased Treg frequencies (Fig. 8A). This suggests that ConA pretreatment induces predominantly qualitative rather then quantitative changes in the Treg population, thereby correlating with the higher immune-modulatory potential of Tregs from ConA-tolerized than saline-pretreated mice as found in the in vitro experiments described above. We tried to assess whether such qualitative changes might be reflected in altered distributions of naïve/effector phenotypes among Tregs by measuring CD62L and CD103 expression, respectively. Again, pronounced but only transient alterations were found after ConA treatment, but on establishment of the tolerogenic state (day 8), frequencies of both the CD62L+ and CD103+ Treg populations had reached their base levels again (Fig. 8B). Future experiments will aim at the identification of other markers that might be associated with the qualitative changes among Tregs on ConA tolerization.

Figure 8.

Occurrence of FoxP3-positive Treg populations in liver, spleen, and liver-draining portal lymph nodes after ConA treatment. The corresponding organs were excised at the indicated timepoints after ConA injection or saline injection as a negative control, lymphocytes were isolated, stained for CD4 and FoxP3, and the frequencies of CD4+FoxP3+ Tregs among the CD4+ T cells was calculated (A). For identification of Treg subpopulations, cells were additionally stained for CD103 or CD62L, respectively (B). To enable direct comparisons of population frequencies at the different timepoints in spite of normal experimental day-to-day variations, the relative frequency of the respective populations in ConA-treated samples was normalized for their rate in saline controls of the same timepoint, with the latter frequency being defined as “1.” The percentages depicted in each panel represent the actual frequencies of the respective population in saline controls (calculated as mean ± SEM of the measured frequencies in saline controls from all timepoints).

Tregs and Kupffer Cells Are the Main Sources of IL-10.

To test whether Tregs might be involved in the ConA-induced IL-10 response, plasma IL-10 levels were measured in Treg-depleted ConA- or saline-pretreated mice after ConA rechallenge. Depletion of CD4+CD25+ Tregs caused reduced plasma IL-10 levels in saline-pretreated mice and a partial but significant reduction in ConA-pretreated mice, suggesting that Treg cells were involved in the IL-10 response (Fig. 9A). KC represent an intrahepatic cell population able to produce significant amounts of IL-10 and IL-6, with the IL-6-production by KC and liver-endothelial cells being suppressed by high IL-10 concentrations47 (an effect markedly resembling the cytokine profile found here in ConA tolerance). We depleted KC by clodronate-liposomes before ConA rechallenge. Successful KC depletion was verified by staining of cryostat liver sections with the BM8 MAb as described before8 (data not shown). In KC-depleted mice the relative tolerization-induced IL-10 augmentation was reduced in plasma (not shown) and especially pronounced regarding intrahepatic IL-10 mRNA levels (Fig. 9B). This indicates that KC contribute to IL-10 production in ConA tolerance. Double depletion of both Tregs and KC before ConA restimulation caused a largely diminished IL-10 response in both saline- and ConA-pretreated mice (Fig. 9C), suggesting that CD4+CD25+ Tregs and KC together are crucial for both primary IL-10 production and IL-10 augmentation in tolerized mice. We cannot exclude the possibility that additional cell types may contribute to the augmented IL-10 response, such as CD25-negative Tr1 cells, being well known for their production of IL-10 (reviewed in Veldman et al.48) or non–T cells. However, within the T-cell population only CD25+ Tregs but not Treg- (and NKT-) depleted T cells from ConA-tolerized mice had shown higher in vitro IL-10 expression compared with control mice.

Figure 9.

Critical role of Tregs and Kupffer cells for IL-10 production on ConA tolerization. Before ConA rechallenge, both saline-pretreated mice and ConA-pretreated mice were either mock-treated or subjected to (A) Treg depletion by injection of anti-CD25 MAb, thereby partially affecting the tolerization-induced IL-10 boost in plasma, or (B), KC depletion by clodronate liposomes that caused a significant reduction of the tolerization-induced IL-10 boost, especially in the liver, as detected by IL-10 mRNA quantification via real-time RT-PCR. (C) Double depletion of Tregs and KC significantly reduced ConA-induced IL-10 production in saline controls and largely abolished the tolerization-induced IL-10 augmentation on ConA rechallenge (mean ± SEM; n ≥ 3; *P ≤ 0.05; NS, not significant). All experiments were repeated once with corresponding results.

Injection of CD4+CD25+ Tregs Alleviates ConA Hepatitis.

To assess the immune-therapeutic potential of Tregs, 1 × 106 FACS-purified CD4+CD25+ Tregs or CD4+CD25 control lymphocytes were injected into C57BL/6 mice 24 hours before ConA treatment (Fig. 10). Mice injected with Tregs showed considerably lower liver injury than control mice. Tregs from ConA-pretreated mice appeared more efficient, with statistically significant suppression of liver injury. These results indicate that Tregs in fact have a therapeutic potential in this model of immune-mediated hepatitis. Most models of induction/encouragement of regulatory T-cell populations in vivo use defined antigens for initial activation. However, besides several reports showing polyclonal activation of pre-existing natural Tregs with superagonistic anti-CD28 MAb, repeated superantigen exposure was recently reported to induce “new” regulatory T cells more potent than natural CD4+CD25+ Tregs.49 The mechanism of improved Treg function without specific antigen in ConA tolerance is not yet elucidated; however, both encouragement of preexisting Tregs as well as induction of adaptive Tregs, for example, by the cytokine milieu or activated CD4+CD25+ Tregs on ConA pretreatment might be involved.

Figure 10.

Significant suppression of liver injury by adoptively transferred Tregss from ConA-pretreated mice. Mice were injected with 1 × 106 FACS-sorted Tregs from either ConA-tolerizedb) or those from saline-pretreated micea) or with 1 × 106 CD4+CD25 cells as control 24 hours before ConA treatment. After 8 hours, plasma ALT activities were measured.

In summary, our results disclose a ConA-induced differentiation of Kupffer cells and Tregs that instigates them to increased IL-10 production on restimulation, with IL-10 playing an important role in ConA tolerance. Interestingly ConA-induced protection from ConA liver damage was not restricted to C57BL/6 mice, but we also found protection in male Balb/c mice in a single pilot experiment (supplement). Also, in Balb/c mice (female), IL-10 had been identified as an essential mediator of protection in a work on chronic ConA hepatitis after several weeks of repeated restimulation, thereby supporting our interpretation of IL-10 as an important tolerance factor.50 These results fit into the emerging pattern that, whereas CD4+CD25+ Tregs were initially believed to exert regulatory functions by cell contact, there is increasing evidence for cytokine-dependent suppression by Tregs in vivo.16 Thus, IL-10, autologous patients' Tregs, or still unidentified differentiation factors may be promising tools for therapeutic intervention against immune-mediated liver injury.

Acknowledgements

The technical assistance of Sonja Heinlein and Andrea Agli is gratefully acknowledged. Anti–IL-10R antibody was kindly provided by Schering-Plough Biopharma, Palo Alto, CA.

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