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Immunology and Liver Disease

Dig Dis 2010;28:86–92 DOI: 10.1159/000282069

Tolerance Induction in Response to Liver Inflammation

Annette Erhardt Gisa Tiegs

Institute of Experimental Immunology and Hepatology, Center of Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg , Germany

Moreover, regulatory T cells from ConA-tolerant mice dis-played a higher immunosuppressive potential in vitro andin vivo compared to those from non-tolerant animals. Inter-estingly, ConA hepatitis was aggravated in CCR5 –/– and CXCR3 –/– mice. Conclusion: These results suggest that ConA tolerance is mediated by induced IL10+ regulatory T cells, probably trafficking into the liver depending on the IFN- � -inducible chemokine receptors CCR5 and CXCR3.

Copyright © 2010 S. Karger AG, Basel

Introduction

The liver appears to be a privileged organ regarding immune regulation and tolerance as it occupies a par-ticular position within the human body linking the gas-trointestinal tract and the systemic venous circulation [1, 2] . Hence, the liver is permanently exposed to intestinal antigens including pathogens, toxins and harmless di-etary antigens. As the liver assumes the role of a ‘scaven-ger’ organ and is involved in the clearance of foreign an-tigens as well as bacterial and toxic products from the gut, it is compulsory to circumvent any dispensable and inad-equate immune activation to prevent liver damage.

However, gut-derived antigens are not ignored by the immune system; rather the liver has been considered to

Key Words Immunological tolerance � Murine model � Concanavalin A

Abstract Background/Aims: The liver plays an important role in im-munological tolerance due to its anatomical location, as it links the gastrointestinal tract and the systemic venous cir-culation. Therefore, immune reactions against dietary or bacterial antigens from the gut have to be avoided. However, immune responses resulting in elimination of harmful hepa-totrophic pathogens have to be induced. We investigated mechanisms of tolerance induction in response to liver in-flammation in a murine model of immune-mediated liver in-jury. Methods: Liver damage was induced by injection of the plant lectin concanavalin A (ConA). Cytokine levels were measured in plasma and liver tissue. The frequencies of in-trahepatic and splenic cell subsets were measured by FACS analyses. Results: ConA hepatitis was mediated by acti-vation of CD4+ T cells, NKT cells and Kupffer cells releasing IFN- � and TNF- � . Tolerance developed towards ConArechallenge within 8 days, lasted for several weeks andwas characterized by significantly reduced plasma trans-aminase activities, decreased Th1/Th17 responses and anincreased IL-10 release, the latter being produced by CD4+CD25+FoxP3+ regulatory T cells and Kupffer cells.

Gisa Tiegs Institute of Experimental Immunology and HepatologyUniversity Medical Center Hamburg-Eppendorf, Martinistrasse 52 DE–20246 Hamburg (Germany) Tel. +49 40 7410 58732, Fax +49 40 7410 57150, E-Mail g.tiegs   @   uke.de

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Immune Tolerance in the Liver Dig Dis 2010;28:86–92 87

favor the induction of peripheral tolerance. Furthermore, the overall predisposition of the intrahepatic immune re-sponse might also account for long-term survival of al-logeneic liver transplants despite MHC mismatches and even in the absence of immunosuppression [3] . Addition-ally, the presence of a liver allograft can suppress the re-jection of other solid tissue grafts from the same donor, whereas further organ transplants from another donor can lead to graft rejection, indicating antigen-specific in-duction of tolerance by the transplanted liver [4, 5] . More-over, oral application of autoantigens induces immune tolerance [6] , also depending on liver-specific mecha-nisms.

This clearly indicates that active immune regulation occurs in the liver, promoting the development of periph-eral tolerance. In contrast, pathogens settling the liver might exploit this benefit of local tolerance. Therefore, infections of the liver by pathogens (e.g. hepatitis B or hepatitis C viruses) require induction of an effective im-mune response to break down the infection and to pre-vent progression of persistence [7] since chronic infec-tions might result in liver fibrosis, liver cirrhosis or (in the worst case) hepatocellular carcinoma.

Therefore, the liver is an organ with paradoxical im-munological properties. On the one hand, immune reac-tions against harmless antigens have to be avoided, buton the other hand, immune responses against harmful hepatotrophic pathogens have to be induced effectively. Therefore, liver lymphocytes have to switch rapidly from a tolerant to a responsive state. To cope with these differ-ent challenges, the liver produces cytokines, chemokines, or acute-phase proteins, and harbors large amounts of immune cells, comprising conventional and unconven-tional subpopulations of the innate [natural killer (NK) and natural killer T (NKT) cells] and adaptive immune system (T and B cells) [8, 9] . Conventional T cells include CD4+ and CD8+ T cells. Unconventional T cells com-prise NK cell-marker-positive T cells, namely classical and nonclassical NKT cells, and NK cell-marker-negative � � T cells. NKT cells are more abundant in the liver than in other organs (20–30% of the intrahepatic lymphocyte population) [8] . NK cells are also enriched up to 30% among liver-resident lymphocytes. Hence, the liver is a pool of an unusual and unique composition of lympho-cytes in comparison to peripheral blood.

Besides liver-associated lymphocytes, the liver con-tains parenchymal hepatocytes (60–80%) and nonparen-chymal cells (20–40%) [8] composed of sinusoidal endo-thelial cells (LSECs); intrahepatic macrophages, namely Kupffer cells (KCs); and hepatic stellate cells [7] . LSECs

are lining sinusoids and form a fenestrated endothelium; therefore, they are in direct contact with blood cells of the immune system. Kupffer cells are derived from circulat-ing monocytes and constitute the largest population of resident macrophages in the body. They are well posi-tioned in the sinusoidal space, thus fulfilling their func-tions, e.g. phagocytosis of apoptotic cells and micro-or-ganisms, antigen presentation and involvement in toler-ance [2] .

Besides antigen trapping mediated by KCs and LSECs, antigen presentation to T cells is also maintained by ‘pro-fessional’ antigen-presenting cells in the form of dendrit-ic cells. In the healthy liver, dendritic cells are mostly im-mature and reside around portal areas. Since interleukin (IL)-10 and transforming growth factor (TGF)- � are con-stitutively expressed by KCs and LSECs, the liver offers a cytokine milieu that might render resident dendritic cells tolerogenic [8, 10] . A small proportion of the nonparen-chymal cells is allocated to hepatic stellate cells/Ito cells (5%) found in the perisinusoidal space. After liver dam-age, hepatic stellate cells are activated, characterized by adoption of a myofibroblast-like phenotype, prolifera-tion, contractility, expression of interstitial collagen I and III, and chemotaxis. In chronic injury, activated hepatic stellate cells are the major source of the collagens that comprise fibrosis and cirrhosis [11] .

The Immune-Mediated Model of Concanavalin A-Induced Hepatitis in Mice

In the past, tolerogenic mechanisms of the liver were studied mainly with respect to CD8+ T cells [12, 13] , whereas less attention has been addressed to CD4+ T cells in this scenario [1] , although they occur at the highest frequency within the liver [9] . Therefore, we established a model of immune-mediated liver injury induced by in-jection of concanavalin A (ConA) [14] .

ConA is a mitogenic plant lectin isolated from jack bean and often used in vitro to activate T cells. It binds mannose residues of different glycoproteins and thereby activates lymphocytes. Upon a single intravenous injec-tion to mice [14] or rats [15] , ConA induces acute liver damage within 8 h in a dose range from 10 to 25 mg/kg. Immune-mediated liver injury in this model depends on activation of CD4+ T cells, NKT cells and KCs [16, 17] .

Liver damage is mediated by T cell-derived interferon (IFN)- � [18] and KC-derived tumor necrosis factor (TNF)- � [19, 20] , followed by necrotic cell death of hepa-tocytes and release of the transaminases ALT and AST

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from the cytoplasm of hepatocytes into the blood. Addi-tionally, IL-12 [21] and IL-18 [22] are important for dis-ease development.

CD4+ T cells are indispensable for the development of liver injury in vivo confirmed by the usage of SCID (se-vere combined immunodeficiency disorder) and RAG (recombinase activating gene) knockout mice, both lack-ing T and B cells, athymic nude mice, lacking only Tcells, and by experiments using depletion of CD4+ T cells [14] . However, depletion of CD8+ T cells did not pre-vent ConA-induced liver injury [14] . NKT cell-deficient CD1dKO mice were also highly resistant to ConA-in-duced liver damage, indicating CD4+ T cells might large-ly refer to CD4+ NKT cells [17, 23] .

In contrast to the pro-inflammatory cytokines TNF- � and IFN- � , the immunosuppressive and anti-inflamma-tory cytokine IL-10 plays a protective role in this model [24, 25] .

ConA hepatitis has been referred to as a model for cer-tain mechanisms of autoimmune hepatitis due to the prevalence of CD4+ T cells, responsiveness to immuno-suppressive drugs [14] , genetic prevalence regarding hap-lotype specificity and susceptibility of certain mouse strains [26, 27] , gender-related differences [28, 29] and immunosuppression in the state of remission [30, 31] .

Autoimmune Hepatitis

Autoimmune hepatitis (AIH) with an incidence of 1–2: 10,000 is characterized by a misdirected immune re-action against autoantigens leading to the high titers of a wide range of circulating autoantibodies, hypergamma-globulinemia and endocrine abnormalities [28] . Com-mon autoantibodies measured during AIH include anti-nuclear, smooth muscle, type 1 liver-kidney microsomal, soluble liver antigen and perinuclear staining antineutro-phil cytoplasmic antibodies.

At present, the treatment of choice is the corticosteroid prednisone alone or a combination with prednisone and azathioprine aiming at a downmodulation of an overac-tive immune system. Both treatment protocols show high survival rates and work best when AIH is diagnosed ear-ly. However, a treatment failure rate of 13% and the failure to induce permanent remission in most patients under-lines the urgent need to develop additional treatment reg-imens [32, 33] . Furthermore, management of side effects such as weight gain, high blood pressure, anxiety, osteo-porosis and diabetes is very important.

Since AIH generally shows a marked female predomi-nance (70–80% of affected individuals are women) and is especially induced in peri-and postmenopausal women, it is possible that changes in hormonal regulation of the immune system might contribute to AIH development in addition to environmental factors and genetic predispo-sition regarding certain haplotypes of HLA antigens. Moreover, in patients with AIH: (i) peripheral numbers and function of CD4+CD25+FoxP3+ regulatory T cells (T reg ) are depressed compared with controls, (ii) the per-centage of T reg inversely correlates with autoantibody ti-ters and (iii) T reg numbers are lower in patients at the time of diagnosis than during remission [34, 35] . The mecha-nisms of this immunosuppression and potential involve-ment of regulatory cell types have not yet been elucidated in autoimmune-mediated liver damage. The pool of reg-ulatory T cells contains both naturally arising T cells (nT reg ) generated in the thymus and adaptive or induced regulatory T cells (iT reg ) generated in the periphery from naïve T cells. Both regulatory cell types have already been described as IL-10 producers in vivo [36–39] . Until now, the most specific marker of nT reg is the transcription fac-tor FoxP3, which is relevant for maintaining self-toler-ance [40] . In this context, nT reg have been shown to be less frequent or functional in patients with autoimmune dis-eases other than AIH, e.g. multiple sclerosis or type 1 di-abetes [41] . This is supported by the fact that mutations of the foxp3 gene lead to depletion of T reg , resulting in se-vere autoimmunity and lymphoproliferative disorders in mouse and man [42] .

ConA Hepatitis: A Model for Induction of Peripheral Tolerance?

To investigate immunosuppressive mechanisms in the liver, we used the model of ConA-induced immune-me-diated liver injury in mice. As previously mentioned, a single intravenous injection of ConA triggered acute liver damage in wild-type mice. Interestingly, injection of a sublethal dose of ConA induced tolerance towards re-challenge with the same agent within 8 days. This tolero-genic state lasted for several weeks and was characterized by suppression of the typical T helper 1 (Th1) and Th17 response, improvement of liver histopathology and an anti-inflammatory cytokine profile, i.e. downmodula-tion of IFN- � , TNF- � , IL-6, IL-17 and IL-2 production and a concomitant increase of IL-10 release both in plas-ma and liver tissue [31] . CD4+CD25+ T reg , as well as KCs, contributed to IL-10 release in tolerant mice as demon-

Immune Tolerance in the Liver Dig Dis 2010;28:86–92 89

strated by depletion experiments with an anti-CD25 mAb (clone PC61.5) or clodronate liposomes, respec-tively.

Normally, KCs fulfill diverse functions in the liver, in-cluding clearance of endotoxin from the portal circula-tion, antigen-presentation and the release of soluble me-diators such as cytokines [43] . In this context, KCs could be differentiated from TNF- � - and IL-6-producing type I macrophages into IL-10-producing type II or alterna-tively activated macrophages [44] due to ConA-induced immune-activation followed by T cell tolerance in the liv-er [45] .

Moreover, tolerization-induced IL-10 production and reduction of plasma transaminase activity were not de-tectable upon in vivo depletion of CD4+CD25+FoxP3+ T reg in DEREG mice (depletion of regulatory T cell) [46] prior to ConA restimulation [unpubl. data], confirming the results of T reg depletion by injection of an anti-CD25 mAb (clone PC61.5) and indicating a functional role of T reg in ConA tolerance in vivo. DEREG mice express a diphtheria toxin (DT) receptor-enhanced green fluores-cent protein fusion protein under the control of the foxp3 gene locus, allowing selective and efficient depletion of FoxP3+ T reg cells by DT, whereas anti-CD25 mAb treat-ment might also affect activated CD4+CD25+ effector T cells.

Double depletion of both T reg and KCs prior to ConA restimulation caused a largely diminished IL-10 response in both saline- and ConA-pretreated mice [31] , verifying that CD4+CD25+ T reg and KCs together are crucial for primary IL-10 production and especially for IL-10 aug-mentation in tolerized mice provoked by ConA-induced differentiation to IL-10-producing CD4+CD25+FoxP3+ T reg and to type II macrophages, respectively. Experi-ments using IL-10 –/– mice or blocking anti-IL-10R mAbs argued for a critical role of IL-10 regarding inductionof tolerance since tolerance was totally reversed both in IL-10 –/– mice and upon injection of anti-IL-10R mAbprior to the first or second ConA stimulation, demon-strated by enhanced plasma transaminase activitiesand pronounced pro-inflammatory cytokine responses in ConA-pretreated IL10 –/– mice or ConA-pretreatedanti-IL-10R mAb-treated mice.

NKT cells, which are abundant in the murine liver and essential for ConA-induced hepatitis due to IFN- � pro-duction, did not mediate tolerance against ConA, al-though the frequency of NKT cells is nearly doubled in the liver of ConA-treated mice on day 8 in comparison to saline-treated mice. Nevertheless, NKT cell-deficient, ConA-pretreated CD1d –/– mice displayed the character-

istic cytokine profile with reduced Th1/Th17 response and concomitant increased IL-10 release upon ConA re-stimulation [31] .

In vitro analysis of T reg from ConA-tolerant mice re-vealed a higher immunosuppressive capacity compared to T reg from non-tolerant mice, since T reg from ConA-tolerant mice suppressed polyclonal T cell responses and IL-2/IFN- � release of responder cells more effectively in co-culture systems compared to T reg from control mice. Moreover, T reg from tolerant mice were able to augment in vitro IL-10 expression in contrast to nT reg from naïve control mice [31] . Furthermore, T reg from ConA-tolerant mice also exhibited a higher immunomodulatory activity in vivo, since adoptive transfer of T reg from ConA-toler-ant mice 24 h prior to ConA challenge resulted in sig-nificantly reduced plasma transaminase activity. In con-trast, T reg from IL10 –/– mice failed to exhibit therapeutic potential against immune-mediated liver injury in the cellular immune-therapy approach, confirming the in-dispensability of IL-10 in vivo. Interestingly, the adop-tively transferred CD4+CD25+ T reg from ConA-tolerant mice were 1 93% FoxP3-positive.

To conclude, ConA tolerance is mediated by an active process in vivo, namely by tolerogenic T reg and the im-munosuppressive cytokine IL-10, whereas passive mech-anisms such as nonresponsiveness/anergy or depletion of disease-mediating T cells, NKT cells or KCs due to the first ConA stimulus are not relevant in this model.

The Role of Chemokines and Chemokine Receptors in ConA Hepatitis

To mediate protection from ConA-induced liver dam-age, T reg have to migrate to the site of inflammation. In this context, chemokines play an important role since they mediate migration of immune cells into infected and inflamed tissue in order to initiate an effective immune response [47, 48] . CD4+ Th1 cells are characterized by expression of CCR5, CXCR3, and CXCR6. CD4+ Th2 cells preferentially express CCR4 and CCR8, whereas CD4+ Th17 cells express CCR6. However, specific che-mokine receptor expression by T reg is still unclear.

The chemoattractant function of resident liver cells such as hepatocytes or LSECs has already been identified [49, 50] . Recently, it has been demonstrated that CCR5-binding chemokines CCL3, CCL4 and CCL5 were ex-pressed in the liver upon ConA challenge. Moreover, two independent studies showed that CCR5-deficient mice developed more severe liver damage than wild-type mice

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[51–53] . Due to the loss of CCR5, high levels of the cor-responding ligands, especially CCL5, were expressed in the liver. Hence, CD4+ T cells and NKT cells expressing the alternate CCL5-binding receptor CCR1 were recruit-ed into the liver of CCR5-deficient mice, worsening liver damage [53] . Consequently, in vivo neutralization of CCL5 resulted in protection from ConA-induced hepati-tis in CCR5-deficient mice. Moreover, it has been dem-onstrated that CCR5-deficient NKT cells produced more IL-4 and resisted apoptosis after ConA administration [51] . Subsequently, IL-4 release transactivated NK cells resulting in higher IFN- � release and more severe liver damage.

Recent studies have also identified CCR5 expression not only on Th1 cells, but also on T reg [54, 55] . Hence, an alternative explanation might be the failure of recruit-ment of CCR5+ T reg from portal-draining lymph nodes into the inflamed liver in CCR5-deficient mice [54] .

We have also analyzed the involvement of the chemo-kine receptor CXCR3, which is preferentially expressed on Th1 cells. CXCR3 and its ligands are involved in the pathogenesis of various autoimmune diseases such as rheumatoid arthritis, type 1 diabetes mellitus, or system-ic lupus erythematosus [56] , but also in acute allograft rejection [57] . However, CXCR3-deficient mice have in-dicated controversial results regarding tissue infiltration and disease severity in experimental models of multiple sclerosis [56] . Therefore, CXCR3-expressing T cells might exert either protective or deleterious functions regarding the affected organ.

During ConA-induced hepatitis, CXCR3-binding chemokines CXCL9, CXCL10, and CXCL11 were strong-ly upregulated in the liver. Surprisingly, CXCR3-deficient mice developed more severe liver damage upon ConA challenge compared to wild-type mice indicated by high-er lethality of CXCR3-deficient mice even upon treat-ment with a low dose of ConA (unpubl. data). Interest-ingly, CXCR3 is not only expressed on Th1 cells, but also on non-lymphoid tissue homing CD4+CD25+ T reg be-sides high levels of CD103, CCR5, CCR4 and CCR8 [58] , suggesting that T reg from CXCR3-deficient mice were not able to migrate into the liver and suppress ConA-induced inflammation. Accordingly, CXCR3 high CCR7 low T reg , representing an activated tissue-infiltrating phenotype, were identified in the liver of patients with autoimmune liver diseases, whereas CXCR3 low CCR7 high T reg were pre-dominant in the blood [59] . In this context, it has recent-ly been shown that CXCR3 expression was induced by an acute systemic Th1 response depending on the Th1-spe-cific transcription factor T-bet, since T-bet-deficient mice

completely lack CXCR3+FoxP3+ T reg [60] . De novo T-bet expression in these T reg was induced under IFN- � -depen-dent Th1 conditions.

It is noteworthy that T reg from both CCR5- [55] and CXCR3-deficient mice (unpubl. data) retained their sup-pressive capacity in in vitro suppression assays, suggest-ing a defective T reg migration capacity from secondary lymphoid organs to the inflamed liver rather than anintrinsic impairment of T reg -mediated suppression in CCR5- and CXCR3-deficient mice.

Conclusion

In summary, these results suggest that ConA tolerance is mediated by CD4+CD25+FoxP3+ IL-10 producing Treg trafficking into the liver due to IFN- � -inducible chemo-kine receptors CCR5 and CXCR3. CCR5+CXCR3+IL-10+ T reg specifically recruited to the liver might represent a novel therapeutic option for improvement of autoim-mune liver disease. In the past, clinical trials using low-molecular-weight chemokine receptor antagonists for treatment of autoimmune and inflammatory disease failed, although beneficial effects have been observed in animal models [61] . Our data clearly demonstrated that this might have been due to loss of T reg accumulation at the inflamed site. Hence, blockade of receptors by low-molecular-weight antagonists might have detrimental consequences with respect to regulation of an inflamma-tory response in the liver and has to be precisely investi-gated.

Disclosure Statement

The authors disclose no conflicts of interest. This work was funded by research grants from the Deutsche Forschungsgemein-schaft (Ti 169/8-1 and SFB 841-B1) to G.T.

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