The Adaptive Immune System and Liver Toxicity

9.12 The Adaptive Immune System and Liver Toxicity M P Holt and C Ju, University of Colorado Denver, Denver, CO, USA ª 2010 Elsevier Ltd. All rights reserved.

9.12.1 9.12.2 9.12.2.1 9.12.2.2 9.12.3 9.12.3.1 9.12.3.1.1 9.12.3.1.2 9.12.3.2 9.12.3.3 9.12.4 9.12.4.1 9.12.4.2 9.12.4.3 9.12.4.4 9.12.5 9.12.5.1 9.12.5.2 9.12.5.3 9.12.6 9.12.6.1 9.12.6.2 9.12.7 9.12.7.1 References

Introduction The Immune System Components of the Immune System Development of an Adaptive Immune Response The Liver as an Immunological Organ Cellular Components of the Hepatic Immune System Antigen-presenting cells in the liver Hepatic lymphocytes Lymphocyte Recruitment to the Liver Clearance of Activated T Cells Primary Biliary Cirrhosis Apoptosis of BECs Autoimmune Responses Loss of Immune Tolerance and Induction of Autoimmunity Autoimmune Paradoxes of PBC Viral Hepatitis Innate Immune Response to Viral Infection Adaptive Immune Responses in HBV and HCV Infection Development of Chronic Hepatitis Alcoholic Liver Disease Adduct Formation and Initiation of an Immune Response Genetic Factors and Immune Response in ALD Nonalcoholic Fatty Liver Disease The Role of Adipocytokines and Cytokines in NAFLD

Abbreviations mm ADAR ALD AMA APC BCOADC-E2 BCR BEC BSA CD CREST

micrometer RNA-specific adenosine deaminase alcoholic liver disease antimitochondrial antibody antigen-presenting cell branched chain 2-oxoacid dehydrogenase complex B-cell receptor biliary epithelial cell bovine serum albumin cluster of designation calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia

CTL CTLA DC E3BP

HBV HCV HEL HER HLA HNE ICAM IFN Ig IL IP

276 276 276 277 279 279 279 279 280 281 281 281 282 282 283 283 284 284 285 286 286 288 288 288 290

cytotoxic T lymphocyte cytotoxic T-lymphocyte antigen dendritic cell dihydrolipoamide dehydrogenase (E3)-binding protein hepatitis B virus hepatitis C virus hen egg lysozyme hydroxyethyl radical human leukocyte antigen 4-hydroxy-2-nonenal intracellular adhesion molecule interferon immunoglobulin interleukin interferon-inducible protein

275

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KC LFA LSEC LSP MAA MCP MDA MHC MIG MIP NAFLD NASH NK OAS

Kupffer cell lymphocyte function associated antigen liver sinusoidal endothelial cell liver-specific protein MDA-acetaldehyde monocyte chemotactic protein malondialdehyde major histocompatibility complex monokine induced by gammainterferon macrophage inflammatory protein nonalcoholic fatty liver disease nonalcoholic steatohepatitis natural killer oligoadenylate synthetase

9.12.1 Introduction The liver is continuously exposed to a large number of substances, including pathogens, xenobiotics, tumor cells, and harmless dietary antigens. Infectious agents from systemic circulation need to be efficiently removed while the large number of antigens derived from the gastrointestinal tract must be tolerized. To respond to these challenges, the adaptive immune responses in the liver favor tolerance rather than immunity. However, when regulation of the immune system goes awry, the delicate balance of immunity and tolerance in the liver is compromised, which culminates in immune-mediated liver injury. In this chapter, we focus on the many hepatic disorders that are at least in part driven by immune mechanisms, specifically the adaptive immune system. We begin with a general summary of the adaptive immune system followed by an overview of the unique hepatic immune system. This is followed by a review of the current literature in which the pathogenesis of liver injury is advanced by adaptive immunity, specifically primary biliary cirrhosis (PBC), viral hepatitis, alcoholic liver disease (ALD), and nonalcoholic fatty liver disease (NAFLD). It is our hope that this chapter will shed light on the molecular and cellular mechanisms of adaptive immune responses in the development and progression of hepatic injury.

OGDC PBC PD PDC PKR RAG RANTES

RNA TCR TGF TH TNF Treg VAP VCAM

oxoglutarate dehydrogenase complex primary biliary cirrhosis programmed cell-death pyruvate dehydrogenase complex protein kinase R recombination-activating gene regulated upon activation, normal T cell expressed and secreted ribonucleic acid T-cell receptor transforming growth factor T helper tumor necrosis factor regulatory T cell vascular adhesion protein vascular cell adhesion molecule

9.12.2 The Immune System 9.12.2.1 System

Components of the Immune

The immune response to infectious or damaging agents is carried out by the innate or adaptive immune systems. Cellular mediators of innate immunity include tissue macrophages, polymorphonuclear leukocytes (neutrophils, eosinophils, and basophils), and natural killer (NK) cells. These cells provide an array of nonspecific responses to infection, such as recognition of microbial molecular patterns and generation of antimicrobial peptides, cytokine elaboration, activation of complement and mediation of opsonization, phagocytosis of infected cells and microbes, and direct killing of virus-infected cells (Janeway and Medzhitov 2002). A characteristic feature of innate immunity is that repeated exposure to the same infectious agent does not significantly alter the nature of the response. The innate immune system makes a further contribution by keeping the infectious agent in check, pending activation of adaptive immunity. T and B lymphocytes (T and B cells) are regarded as the central orchestrators of adaptive immunity. Unlike cells of the innate immune system, which use a fixed repertoire of inherited receptors, lymphocytes are able to mount a specific immune response against virtually any foreign antigen, due to their huge repertoire of highly diverse antigen

The Adaptive Immune System and Liver Toxicity

receptors. This diversity, acquired during lymphocyte development, is the result of the random recombination of variable, diversity, and joining gene segments, which are initiated by the products of recombination-activating genes (RAG)1 and RAG2 (Bassing et al. 2002). Lymphocytes that have encountered antigen persist over a long term within the host and provide for a more rapid response to a previously encountered challenge, a concept known as immunological memory. An additional feature of the adaptive immune system is tolerance, which is managed by the continuous deletion and removal of lymphocytes that react to self-protein, or autoantigens. Adaptive immunity is further classified into two classes of responses – humoral immunity and cellular immunity. Humoral immunity is mediated by B cells against extracellular pathogens. Upon activation of B cells, they secrete the soluble form of B-cell receptor (BCR), also known as antibody or immunoglobulin (Ig), which is classified by the isotype of the heavy chains (IgM, IgG, IgE, IgA, and IgD). Binding of these antibodies to viruses and microbial toxins neutralizes the pathogen by blocking its ability to bind to receptors on host cells. Antibody binding further marks invading pathogens, which enables phagocytic cells of the immune system to recognize and eliminate them. The cellular arm of the adaptive immune system is mediated by T cells that eliminate infected cells as well as assist in the activation and proliferation of other immune cells. The T-cell receptor (TCR) differs from BCRs in several ways. First, the TCR is not secreted, existing instead on the cell surface as a heterodimer of either  and or and  subunits. Second, TCRs recognize peptides produced by the proteolytic cleavage of antigen protein as opposed to the native protein recognized by BCRs.

9.12.2.2 Development of an Adaptive Immune Response The adaptive immune response mediated by T cells is considered to proceed in four stages: initiation/expansion, effector cell differentiation, contraction, and memory generation (Farber 2005). Antigen-presenting cells (APCs) reside in most tissues where their role is to survey the local environment for antigen. Upon internalization, the antigen is degraded and processed through several cytoplasmic compartments of the APC. A piece of the antigen, as a peptide fragment, is then loaded onto

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membrane glycoproteins, encoded in a cluster of genes known as the major histocompatibility complex (MHC) (Babbitt et al. 1985), in man the human leukocyte antigen (HLA). The peptide bound within the MHC is then transported to the cell surface through the Golgi system, a process known as antigen presentation. During antigen presentation, the APC matures into a professional APC and travels from the site of infection through the afferent lymphatic vessels into the peripheral lymphoid organs – the lymph nodes, spleen, and lymphoid tissues associated with mucosa (gut-associated tonsils, Peyer’s patches, and appendix). It is within the secondary lymphoid organs where the encounter between T cells and MHC-peptide complexes on the surface of professional APCs occurs (Forster et al. 1999). There are two types of MHC molecules: MHC class I and MHC class II molecules. MHC class I molecules present peptides derived from endogenous and viral antigens, such as those of viral proteins. Peptides consisting of 8–11 amino acids bound to MHC class I molecules are recognized via the TCR of T cells expressing the CD8 receptor (CD8 T cells). CD8 T cells, also known as cytotoxic T lymphocytes (CTLs), exert their cytolytic activity via Fas/FasL and perforin/granzyme pathways (Berke 1995). The Fas/FasL pathway entails binding of the ligand (FasL) to the Fas receptor on target cells leading to subsequent activation of the caspase cascade and apoptosis. The perforin/granzyme pathway involves perforin-mediated introduction of pores in the cell membrane and release of granzymes into the cytosol that activates caspases. CD8 T cells also produce inflammatory cytokines such as interferon (IFN)- and tumor necrosis factor (TNF)- (Guidotti and Chisari 1996). MHC class II molecules present peptides derived from proteins within intracellular macrophage vesicles or internalized by phagocytic cells and B cells. Peptides consisting of 12–18 amino acids bound to MHC class II molecules are recognized by the TCR of two distinct subsets of T cells expressing the CD4 receptor (CD4 T cells). These subsets are distinguished as TH1 and TH2 cells (Mosmann and Sad 1996). TH (T helper) 1 cells secrete TH1 cytokines, including IFN- and TNF , as well as stimulate CTLs and activate macrophages to control intracellular bacterial infections. TH2 cells, or helper T cells, secrete TH2 cytokines, such as interleukin (IL)-4, IL-5, and IL-13 and play a central role in the elimination of extracellular pathogens by activating B cells for a humoral response (Mosmann and Coffman 1989). The activation of an

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Macrophage

Hepatocyte

IFN-γ

TNF-α IL-1β Nitric oxide

TH1 cell

Inflammatory cytokines cytolytic granules

IL-2

Cytotoxic CD8 T cell

Y

Y Y

Y

B-cell activation

TH2 cell

B cell IL-4, IL-5, IL-13, etc.

Antibody production

APC

B7.1 B7.2

CD4 T cell

CD28

Antigen

T-cell activation Naive T cell

MHC Antigen processing and presentation

TCR

Peptide fragment

Figure 1 Activation of an adaptive immune response within secondary lymphoid organs.

adaptive immune response within secondary lymphoid organs is illustrated in Figure 1. The activation or priming of naive T cells to antigen-specific, effector T cells requires at least two signals. TCR recognition of a specific MHCantigenic peptide complex produces signal 1. Signal 2 is provided by the binding of costimulatory molecules expressed on APCs to their ligands on T cells. For example, CD40 and B7 molecules (B7.1 and B7.2) on APCs interact with CD40 ligand and CD28 on T cells, respectively (Paul 1999). Antigen presentation in the context of infection, which leads to upregulation of costimulatory molecules on APCs, allows for the generation of effector T cells, immunity, and memory. On the other hand, antigen presentation under steady-state conditions, in which the expression of costimulatory molecules is minimal, is likely to render the T cell anergic or tolerogenic.

The second stage of the adaptive immune response involves differentiation into effector T cells and proliferation (clonal expansion). Effector T-cell differentiation is mediated via changes in gene expression, resulting in the upregulation of specific factors such as IL-2. Effector T cells also lose expression of the lymph node homing receptor, L-selectin (CD62L), thereby facilitating their efficient homing to infected, nonlymphoid tissues such as the liver. Functionally, effector T cells further acquire the ability to secrete cytokines such as IFN and IL-4, and to become cytotoxic in the case of CD8 T cells. The third stage of adaptive immunity is a contraction phase in which a large proportion of the expanded effector T-cell population dies, with a small fraction surviving on as memory T cells. The final stage of the adaptive immune response consists of the development of immunological memory that persists throughout the lifetime of the host. These

The Adaptive Immune System and Liver Toxicity

memory T cells are specialized for migration into target tissue and neutralization of antigen upon reexposure (Sallusto et al. 2004).

9.12.3 The Liver as an Immunological Organ Due to the anatomical position of the liver between the gastrointestinal tract and the systemic circulation, and tremendous blood supply, the liver has a high rate of interaction with foreign agents, toxins, and dietary antigens. Despite the liver’s continuous exposure to various antigens, the adaptive immune response of the liver is known to favor induction of immunological tolerance rather than immunity. This is supported by numerous studies that have demonstrated that: (1) dietary antigens derived from the gastrointestinal tract are tolerized in the liver; (2) allogenic liver organ transplants are accepted across MHC barriers (Calne et al. 1969); (3) preexposure to donor cells through the portal vein of recipient animals increases their acceptance of solid tissue allografts (Gorczynski et al. 1994; Rao et al. 1988); and (4) preexposure of soluble antigens via the portal vein leads to systemic immune tolerance (Cantor and Dumont 1967; Chen et al. 2001). 9.12.3.1 Cellular Components of the Hepatic Immune System 9.12.3.1.1 Antigen-presenting cells in the liver

Blood flow within the liver slows down due to the narrow sinusoids (7–12 mm) and the temporary obstruction by resident macrophages (Kupffer cells, KCs). Due to this reduction in blood flow, circulating T cells are forced into contact with endothelium and are presented with opportunities to interact with various APCs within the liver, including liver sinusoidal endothelial cells (LSECs), hepatic dendritic cells (DCs), and KCs. It has been demonstrated that the LSECs are capable of presenting antigen to naive CD4 and CD8 T cells and inducing tolerance, as LSEC-activated CD8 T cells fail to differentiate into cytotoxic effector cells, and LSEC-activated CD4 T cells produce immune regulatory cytokines, such as IL-4 and IL-9 (Knolle et al. 1999; Limmer et al. 2000). The tolerogenicity of hepatic DCs has also been investigated, and several studies have demonstrated that, compared with spleen DC, those in the liver exhibit immature phenotypes and are

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ineffective stimulators of antigen-specific T-cell responses (Jomantaite et al. 2004; Lian et al. 2003; Pillarisetty et al. 2004). KCs represent the largest population of tissue-resident macrophages and are capable of antigen presentation following phagocytosis of pathogens. Furthermore, KCs are ideally situated to encounter and present antigen to circulating T cells in the sinusoidal lumen of the liver (MacPhee et al. 1995; McCuskey and Reilly 1993). It has been shown that KCs play a role in livermediated tolerance to allografts (Callery et al. 1989; Kamei et al. 1990; Sato et al. 1996) and in the induction of tolerance against soluble antigens (Ju et al. 2003). This composition of tolerogenic APCs could explain the tolerogenic nature of the liver. 9.12.3.1.2

Hepatic lymphocytes Based on analysis of hepatic lymphocytes it is estimated that the normal human liver possesses approximately 1  1010 lymphocytes (Norris et al. 1998). Figure 2 illustrates the composition of intrahepatic lymphocytes. Detailed analysis of normal human intrahepatic lymphocytes reveals that the CD4:CD8 T cell ratio is 1:3.5, compared with 1.8:1 in the circulation, lymph nodes, or spleen. In addition to conventional lymphocytes, the liver is populated by additional lymphoid cells that use alternative and more primitive mechanisms for target recognition. Such cells consist of T cells with similar properties as NK cells (NKT cells) and T cells expressing the

 TCR, which constitute approximately 65% of all lymphocytes present in the normal liver (Norris et al. 1998, 1999). In contrast to the very low frequency in the circulation and most lymphoid organs, NKT cells account for approximately half of hepatic T cells (Nakatani et al. 2004; Vicari and Zlotnik 1996). NKT cells express the NK cell marker (NK-1.1) γδ TCR (15%)

B cells (5%)

αβ TCR (20%) NKT cells (30%)

NK cells (30%) Figure 2 Composition of intrahepatic lymphocytes.

280 The Adaptive Immune System and Liver Toxicity

and the CD8  chain in the absence of the chain rather than the CD8  heterodimer found on conventional T cells. NKT cells represent a unique set of T cells that express a highly restricted TCR that recognizes glycolipid antigens presented in the groove of the nonclassical MHC class I-like CD1 molecule (Godfrey and Kronenberg 2004; Kronenberg 2005). NKT cells have been shown to suppress the generation of TH1 cells (Miyamoto et al. 2001) and CTLs (Smyth and Godfrey 2000). NKT cells also secrete a range of cytokines including both IFN- and IL-4, which facilitate their regulatory role in immune activation. Another type of unconventional T cells within the liver are  T cells, representing roughly 15% of hepatic lymphocytes, compared with only 2.7% in the blood (Norris et al. 1998). Unlike conventional  T cells,  T cells possess invariant TCRs, which recognize an array of antigens including soluble nonpeptide antigens and stress-inducible proteins without the need for antigen presentation in the context of MHC molecules (Wen et al. 1999). The recognition of nonpeptide components of pathogens allows NKT and  T cells to recognize antigens that are relatively fixed in structure, as they do not require antigen processing, including antigens derived from mycobacteria, plants, and viruses (Bukowski et al. 1999). A distinct population of regulatory T cells (Tregs), when activated, suppress effector T-cell responses (Sakaguchi 2004), thereby facilitating tolerance to harmless self or nonpathogenic environmental antigens. Tregs are CD4 T cells that express the IL-2 receptor  chain (CD25). Tregs are further characterized by their expression of the transcription factor FOXP3, which is a repressor of transcription and critical for Treg function (Hori et al. 2003). Tregs recognize antigens within the context of MHC class II molecules and mediate tolerance through depletion of IL-2 (de la et al. 2004; Shevach 2002) and the secretion of IL-10 and transforming growth factor (TGF)- (Sakaguchi 2004), both of which are potent immunosuppressive mediators of inflammation capable of suppressing effector cell proliferation (Maloy et al. 2003). The inhibition or deletion of Tregs is associated with autoimmunity in both mice and humans (Sakaguchi 2004). Furthermore, Treg infiltration into the liver during chronic viral hepatitis and liver cancer has been observed to lead to persistence of viral infection and cancer progression (Rushbrook et al. 2005; Unitt et al. 2005).

9.12.3.2 Liver

Lymphocyte Recruitment to the

Following antigen presentation by APCs to naive T cells in secondary lymphoid tissue, the activated lymphocytes migrate to nonlymphoid, inflammatory sites. The ability of lymphocytes to adhere to the endothelium and migrate into a particular tissue is dictated by various chemokines and adhesion molecules (Butcher and Picker 1996). This lymphocyte infiltration into the liver is critical to the development of inflammation during an adaptive immune response. However, in some circumstances, immune-mediated liver injury may develop due to prolonged inflammation and lead to damage by persistent recruitment, retention, and survival of effector lymphocytes within the inflamed liver. The recruitment of lymphocytes to the liver is mediated by chemokines, such as CC (macrophage inflammatory protein (MIP)-1, MIP-1 , monocyte chemotactic protein (MCP)-1, and regulated upon activation, normal T cell expressed and secreted (RANTES)), CXC (CXCL16, interferon-inducible protein (IP)-10, and monokine induced by gammainterferon (MIG)) chemokines (Afford et al. 1998; Fisher et al. 1996) derived from hepatocytes, KCs, and stellate cells, which bind to chemokine receptors such as CCR2, CCR5, and CXCR3 on the surface of lymphocytes (Yoong et al. 1999). LSEC express several adhesion molecules, including the intracellular adhesion molecule (ICAM)-1 and ICAM-2 (Iigo et al. 1997), as well as vascular adhesion protein (VAP)-1 (Salmi and Jalkanen 1996). There is a marked increase in the expression of vascular cell adhesion molecule (VCAM)-1 and ICAM-1 during inflammatory episodes (Adams et al. 1994; Steinhoff et al. 1993). The ICAMs engage lymphocyte function associated antigen (LFA)-1 on activated T cells (Morita et al. 1994) and are involved in the trapping of lymphocytes in tissue during pathological episodes, including ischemia/reperfusion (Marubayashi et al. 1997) and allograft rejection (el Wahsh et al. 1997). Once captured and adherent, lymphocytes must traverse the endothelium in order to enter the inflamed tissue. In most tissues, this requires migration through inter-endothelial tight junctions mediated by molecules such as junctional adhesion molecule 2 (ligand for LFA-1) and CD31 (ligand for v 3) (Ostermann et al. 2002). However, in the liver, the fenestrated LSEC do not form tight junctions, lack a basement membrane, and express low levels of CD31 (Scoazec and Feldmann 1991). These unique features

The Adaptive Immune System and Liver Toxicity

suggest that once trapped, lymphocytes transmigrate across the endothelium into the liver following unique signals, including chemotactic signals toward the site of inflammation. 9.12.3.3

Clearance of Activated T Cells

The liver has been dubbed the ‘elephant’s graveyard’ for activated T cells (Crispe et al. 2000). A number of studies using CD8 antigen-specific TCR-transgenic T cells demonstrated that, following a series of injections of the specific peptide, the activated T cells undergo apoptosis after a transient accumulation in the liver. This phenomenon coincides with the disappearance of these cells from the peripheral lymphoid organs (Bertolino et al. 1995; Huang et al. 1994). These findings support an important role of the liver in the induction of apoptosis and clearance of activated T cells. However, this hypothesis has been recently challenged by the findings that viralspecific CD8þ T cells can accumulate within the liver after an extrahepatic infection, and that these T cells are viable and retain effector functions (Keating et al. 2007; Polakos et al. 2007).

9.12.4 Primary Biliary Cirrhosis PBC is characterized by chronic and progressive destruction of biliary epithelial cells (BECs) lining the small intrahepatic bile ducts (Kaplan and Gershwin 2005). PBC is also identified as the first autoimmune liver disease. The histopathologic advancement of PBC involves ductopenia, progressive cholestasis, interface hepatitis, septal fibrosis, and ultimately biliary cirrhosis (Nakanuma et al. 1995). Additional symptoms include hyperlipidemia, osteopenia, and coexisting autoimmune diseases such as Sjo¨gren’s syndrome and a form of scleroderma known as CREST (calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia) syndrome (Pares and Rodes 2003). PBC predominately affects females (female to male ratio 10:1), with the majority of cases diagnosed in patients between 40 and 60 years of age (Talwalkar and Lindor 2003). Current evidence suggests that PBC susceptibility and pathogenesis is complex, including interactions between an individual’s genetic makeup (Invernizzi et al. 2005; Selmi et al. 2004) and environmental exposure. Approximately 10% of patients who exhibit symptoms of PBC

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present with additional features of an autoimmune disease directed predominately against the liver (autoimmune hepatitis). The established serological hallmark of PBC is the presence of antimitochondrial antibodies (AMAs) (Berg and Klein 1995; Mackay 2000), which are present in approximately 95% of patients (Kaplan and Gershwin 2005). It has been suggested that the presence of AMA in serum prior to clinical presentation of PBC may reflect the failure of the immune system to establish and maintain selftolerance and therefore act as an early disease marker. 9.12.4.1

Apoptosis of BECs

An early factor in the development of PBC involves upregulation of a member of the 2-oxoacid dehydrogenase complex, in particular the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), on the cell surface of BECs (Gershwin et al. 1992; Tsuneyama et al. 1995). PDC is normally restricted to the inner surface of the inner mitochondrial membrane and is therefore hidden from the immune system. However, expression of the PDC epitope on the cell surface enables recognition by immune cells and this process appears to facilitate the autoimmune-mediated destruction of BECs, presumably through T cell-mediated apoptosis, and pathogenesis of PBC (He et al. 2006). There is strong evidence to support this notion, as markers of apoptosis, including DNA fragmentation and expression of Fas and Bcl-xL have been observed within damaged intrahepatic portal tracts (Koga et al. 1997; Tinmouth et al. 2002) along with down-regulation of the antiapoptotic protein Bcl-2 (Iwata et al. 2000b). The process of BEC apoptosis appears to be primarily mediated via cytotoxic CD8 T cells due to the presence of granzyme and FasL-expressing cells within BEC damaged areas (Iwata et al. 2000a). Cytotoxic CD8 T cells isolated from patients with PBC have also been observed in vitro to induce BEC apoptosis (Kita et al. 2002). Although evidence suggests BEC apoptosis is the result of cytotoxic CD8 T cell activity, this relationship has recently been called into question (Jones 2007). It has been suggested that although the intensity of CD8 T-cell effector function is greatest during the early stages of liver injury, the actual peak of BEC apoptosis occurs later in disease progression (Ballardini et al. 2001). In addition, it is known that cholestatic liver injury leads to retention of bile salts within the liver that are themselves proapoptotic (Paumgartner and Beuers 2002; Rodrigues et al. 1998). Based on these occurrences,

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it has been proposed that BEC apoptosis may therefore represent a secondary process or consequence of autoimmunity and not the initiating event in the pathogenesis of PBC. 9.12.4.2

Autoimmune Responses

The autoantibodies that are characterisitic of PBC are directed against oxoacid dehydrogenase complexes on the inner mitochondrial membrane, including PDC-E2, the dihydrolipoamide dehydrogenase (E3)-binding protein (E3BP), the E2 subunit of the branched chain 2-oxoacid dehydrogenase complex (BCOADC-E2), and the E2 subunit of oxoglutarate dehydrogenase complex (OGDC-E2) (Yeaman et al. 2000). Approximately 95, 90, and 50% of PBC patients possess serum autoantibodies reactive against PDC-E2, OGDC-E2, and BCOADC-E2, respectively (Fussey et al. 1988; Mutimer et al. 1989). The widespread presence of autoantibodies and B cells in the liver and blood of patients with PBC (Bjorkland et al. 1994) supports the notion that PBC is an autoimmune disease. However, at present, there are limited data demonstrating a causal link between autoantibodies and the development of PBC (Neuberger and Thomson 1999). Although the level of serum IgG anti-PDC autoantibody has been demonstrated to escalate during disease progression (Heseltine et al. 1990; Kisand et al. 1998), another study has demonstrated the lack of bile duct damage following sensitization of experimental animals with mitochondrial autoantigens, such as PDC (Krams et al. 1989). While the function of autoantibodies remains elusive, the presence of infiltrating T cells within the portal tracts, the expression of MHC molecules on the surface of BECs, and the specificity of bile duct damage suggest that autoreactive T cells are responsible for the autoimmune-mediated responses directed against BECs in the pathogenesis of PBC. The lymphocyte infiltrate surrounding biliary ducts in PBC consists predominately of CD4 and CD8 T cells (Bjorkland et al. 1991; Leon et al. 1995). Furthermore, CD4 and CD8 T cells appear to recognize the same immunodominant target epitope of PDC-E2. Amino acid residues 163–176 presented within the context of MHC class II molecules have been identified as the major epitope of PDC-E2 specifically targeted by HLA DR40101-restricted CD4 T cells (Shimoda et al. 1995), while the dominant target epitope of HLA-A2-restricted CD8 T cells spans amino acid residues 159–167 in the context of MHC class I

molecules (Kita et al. 2002). CD8 T cells isolated from the livers of PBC patients have demonstrated specific cytotoxicity against target cells pulsed with the amino acid residue 159–167 of PDC-E2 (PDCE2159–167), while those from healthy patients did not (Kita et al. 2002). This cytotoxic activity of PDCE2159–167-specific CD8 T cells was further associated with production of IFN- , suggesting a potential role for this cytokine in the pathogenesis of BEC damage in PBC (Harada et al. 1997). 9.12.4.3 Loss of Immune Tolerance and Induction of Autoimmunity Although evidence supports a relationship between the presence of autoreactive immune responses and PBC, the mechanism(s) involving the breakdown of self-tolerance remains elusive. One potential explanation is the molecular mimicry model, which is based on cross-reactivity against epitopes on proteins from unrelated species, such as between microbial agents and self-antigens (Oldstone 1987; von Herrath and Oldstone 1996). For example, bacteria, particularly Escherichia coli, have been implicated in molecular mimicry during the pathogenesis of PBC, as the bacterial PDC-E2 epitope shares homology with that of the human PDC-E2 (Yeaman et al. 1988). Current theory maintains that molecular mimicry at the level of MHC class I and/or class II-restricted epitopes may participate in the generation of autoreactive T-cell responses. In this instance, T cells activated against mimicry peptides from an infectious agent would also be capable of cross-reacting with self-PDC-E2 peptides presented by MHC class I or class II molecules on the surface of BECs. The cross-recognition of the autoantigen could perpetuate the immune response, thereby leading to chronic autoimmunity. This view is supported by the finding that the MHC class II-restricted epitope of PDC-E2163–176 recognized by CD4 T cells in patients with PBC was also identified in homologous proteins of E. coli (Shimoda et al. 2000). An alternative explanation for the loss of tolerance to PDC autoantigens is based on the ineffective clearance of apoptotic cells, which upon secondary necrosis release potential intracellular antigens (Rovere et al. 1998). The apoptosis of BECs has been observed in the liver from patients during both the early phase (Tinmouth et al. 2002) and the later stages of PBC (Fox et al. 2001). Furthermore, it has been argued that unlike other cell types, which in effect ‘delete’ cytoplasmic PDC by glutathiolation (Odin et al. 2001),

The Adaptive Immune System and Liver Toxicity

thereby effectively removing the autoantigen, BECs undergoing apoptosis maintain an intact expression of PDC on the cell surface (Macdonald et al. 2004). In this model, defects in the clearance of apoptotic cells may facilitate uptake and presentation of the autoantigens by APCs to T cells, thereby contributing to the onset of autoimmunity (Hanayama et al. 2002; O’Brien et al. 2006). Inhibition of T-cell activation is one method by which the immune system regulates self-tolerance. Loss of this inhibition may provide another potential mechanism in the autoimmune response evident in PBC. Cytotoxic T-lymphocyte antigen (CTLA)-4 and programmed cell-death (PD)-1 are two molecules expressed by activated T cells that function to down-regulate and inhibit previously primed T-cell proliferation thereby promoting tolerance. A recent genotype analysis has demonstrated that putative functional variants of these two genes may influence the risk of PBC, despite the lack of any detectable association from the individual polymorphisms (Juran et al. 2008). The synergistic effect of these variants has been observed to reduce the amount of cell surface expression of CTLA-4 and PD-1, resulting in enhanced T-cell activation and proliferation that may facilitate autoimmunity (Juran et al. 2008). The initiation of an autoimmune response leading to PBC has also been proposed to be triggered by environmental factors, including chemical xenobiotics (Triger 1980; Uibo and Salupere 1999; Watson et al. 1995). The potential modification of selfproteins by xenobiotics may generate neoantigens and culminate in the loss of tolerance. Immunization of rabbits and guinea pigs with a laboratory synthesized organic chemical, 6-bromohexanoate, conjugated to bovine serum albumin (BSA) led to the development of AMA with the same antigenic specificity and enzyme inhibitory activity to PDC-E2 that is displayed by sera of patients with PBC (Amano et al. 2004; Leung et al. 2003, 2007). Although the autoimmune response of the immunized rabbits was limited to the generation of these AMA, immunized guinea pigs did eventually develop PBC-like liver lesions and autoimmune cholangitis (Leung et al. 2003, 2007). Such xenobioticimmunized guinea pigs represent the first inducible animal model of PBC and support the claim that exposure to xenobiotic-modified PDC-E2 may initiate an autoimmune response to PDC-E2 and trigger PBC. Further elucidation into the role of inflammation and autoimmunity associated with the pathogenesis

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of PBC has been aided by reports of three spontaneous, autoimmune biliary disease models. The NOD.c3c4 mouse is the first model animal to spontaneously develop AMA and PBC-like liver disease. Such mice develop similar clinical and autoimmune phenotypes as patients with PBC, including biliary lymphocytic infiltrates, AMA directed against PDCE2, and progressive biliary obstruction (Irie et al. 2006; Koarada et al. 2004). The dominant negative TGF- receptor II and IL-2 receptor / mouse models represent two additional spontaneous mouse models of autoimmune biliary disease. These mice also develop features characteristic of human PBC, including the generation of AMA against PDC-E2, lymphocytic infiltration with concurrent portal inflammation, and biliary ductal damage (Oertelt et al. 2006; Wakabayashi et al. 2006). 9.12.4.4

Autoimmune Paradoxes of PBC

Although there exists strong evidence favoring an autoimmune-mediated mechanism in the pathogenesis of PBC, there are also unresolved paradoxes and unique features of classic autoimmunity that are absent in this disease. First, although the E2 subunit of PDC is ubiquitously expressed (He et al. 2006), autoimmune-mediated destruction in PBC is predominately restricted to the BECs lining the intrahepatic bile ducts. The exclusive hepatic liver damage associated with PBC may be due to the liver’s unique exposure to environmental triggers, such as potential pathogens from the gastrointestinal tract that may initiate molecular mimicry. Second, a specific association with HLA phenotype may be expected for an autoimmune, T cell-mediated disease. However, this remains to be seen, as PBC has been observed following liver transplantation, which is across HLA boundaries (Robertson et al. 2007). Additional factors of PBC that do not correlate with autoimmunity include the lack of an animal model with identical antigen specificity and the absence of disease occurrence in children.

9.12.5 Viral Hepatitis Hepatitis B virus (HBV), a member of the Hepadnaviridae family (Ganem and Schneider 2001), and hepatitis C virus (HCV), of the family Flaviviridae (Lindenbach and Rice 2001), are the most common causes of liver disease. Worldwide, it

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is estimated that the number of people chronically infected with HBV (Ganem and Prince 2004) and HCV (Pawlotsky 2004) exceeds 400 million and 170 million, respectively. Although both viruses induce immune-mediated acute and chronic necroinflammatory liver disease, the history and outcome of HBV and HCV infection differ profoundly. HBV infection during adulthood results in transient liver disease and viral clearance in more than 95% of adults, while vertical transmission of HBV from mother to offspring results in approximately 90% chronic hepatitis (Fattovich 2003). In contrast, HCV readily establishes chronic hepatitis in 60–80% of otherwise immunocompetent adults (Hoofnagle 2002; Seeff 2002). Insight into the immunopathogenic mechanisms associated with HBV and HCV infection has been based on data from experimental studies in chimpanzees and infected human patients, which are the only species known to be susceptible to HBV and HCV (Grakoui et al. 2003; Guidotti et al. 1999; Nascimbeni et al. 2003; Shoukry et al. 2003; Thimme et al. 2003; Yanagi et al. 1997). Although infection from viral hepatitis is limited to primates, there has been investigation using transgenic mice with replication-competent copies of HBV and HCV genomes in their hepatocytes (Guidotti et al. 1995; Kawamura et al. 1997). Evidence suggests that while neither HBV nor HCV are directly cytopathic, the viral-specific liver injury associated with infection is determined by immune-mediated host–virus interactions. Successful clearance of a viral infection requires the coordinated response of both the innate and the adaptive immune system. 9.12.5.1 Innate Immune Response to Viral Infection It is widely accepted that hepatocytes are the primary site of both HBV and HCV infection. In response to infection, the innate immune system is activated to limit the extent of viral spread. NK and NKT cells are rapidly recruited and attempt to control and eliminate viral infected cells by direct killing as well as the secretion of large amounts of antiviral proteins, most notably type 1 interferon (IFN)- and IFN- (Tosi 2005), which in turn activate expression of numerous IFN-stimulated genes possessing antiviral, antiproliferative, and immunomodulatory functions (Der et al. 1998). Several IFN-induced proteins demonstrated to have anti-HCV activity include protein kinase R (PKR) (Pflugheber et al. 2002), the RNA-specific adenosine deaminase 1

(ADAR 1) (Taylor et al. 2005), the 29–59 oligoadenylate synthetases (2–5 OAS)/RNaseL system (Guo et al. 2004), and P56 (Wang et al. 2003). The antiviral effects of IFN- and IFN- have been observed in transgenic mice containing HBV-expressing hepatocytes, as they inhibited formation of new HBV capsids, destabilized existing capsids, and degraded preformed HBV-RNA (McClary et al. 2000; Wieland et al. 2000). These early defense mechanisms by the innate immune system, although beneficial, are not believed to significantly contribute to the control of infection or the pathogenesis of hepatic injury. 9.12.5.2 Adaptive Immune Responses in HBV and HCV Infection Virus-specific CD4 and CD8 T-cell responses are predominately responsible for the control and clearance of viral infection, as depletion of either of these cells prior to challenge of chimpanzees with HBV (Thimme et al. 2003) or HCV (Grakoui et al. 2003) results in chronic infection. Hepatic DCs play a critical role in the generation and maintenance of T-cell responses upon viral infection. During surveillance of the liver, DCs phagocytoze virus-infected cells and present antigen within the context of MHC molecules to T cells. Activated CTLs elicit specific cytolytic effects on virus-infected hepatocytes via Fas/FasL and perforin/granzyme pathways (Wakita et al. 2000). CTLs also secrete cytokines, such as IL-2, IFN- , and TNF-, which inhibit viral replication without adversely affecting the infected cell (Greenberg et al. 1997). Multispecific CTL responses have been associated with spontaneous clearance of HCV in both chimpanzee (Cooper et al. 1999; Thimme et al. 2002) and human infection (Gruner et al. 2000; Lechner et al. 2000). The protection provided by memory CTLs against viral reinfection has been revealed following depletion of these cells prior to rechallenge in chimpanzees that had previously recovered from HCV infection, as these animals subsequently developed reinfection (Shoukry et al. 2003). These studies suggest that virus-specific CTLs provide a protective role against viral infection. While CTLs are traditionally thought to be the main effector cells that eliminate virus-infected cells (Gremion and Cerny 2005), considerable evidence has emerged suggesting an important role for CD4 T cells in the elimination of both HBV and HCV. CD4 T-cell responses play a key role in the outcome of infection, since without this population, induction of new immune responses is impaired and memory

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CTLs cannot be sustained (Grakoui et al. 2003; Shedlock and Shen 2003). The importance of CD4 T cells during viral infection has been illustrated in both experimental chimpanzee and natural human infection as clearance of HCV is associated with a robust and sustained HCV-specific CD4 T-cell response (Missale et al. 1996; Takaki et al. 2000; Tsai et al. 1997) compared to the persistent infection of patients lacking this response (Missale et al. 1996; Tsai et al. 1997). Indications for the important role of CD4 T cells in HBV infection is due to the finding that transfer of HBV-specific TH1 cells into HBV transgenic mice assisted in clearance of the virus (Franco et al. 1997). Collectively, these data indicate that CTLs are the primary effector cells mediating protective immunity against HBV and HCV infection, with assistance from CD4 T cells. 9.12.5.3

Development of Chronic Hepatitis

In the event that CD4 T cells and CTLs are unable to keep the virus in check, the persistent necroinflammatory liver injury can progress to chronic hepatitis, liver fibrosis, and cirrhosis, and an increased risk for hepatocellular carcinoma. Numerous mechanisms of T-cell failure and dysfunction have been proposed to lead to the advancement of chronic viral hepatitis (Rehermann 2007). The impairment in antigen presentation to T cells has been proposed as one means by which a flawed immune response enables the progression of chronic infection. The ability of DCs to initiate adaptive immune responses is dependent on their maturation state. Along with a role in innate immune responses, NK cells further contribute to adaptive immunity via regulation of DC activation and maturation (Moretta 2005). It has been demonstrated that the capacity of NK cells, derived from chronic HCV-infected patients (HCV-NK), to activate DCs is severely compromised compared to NK cells from healthy patients (Jinushi et al. 2004). HCV-NK-stimulated DCs preferentially generated CD4 T cells comprising a TH2 phenotype. This evidence suggests that a shift in the cytokine profile of CD4 T cells, away from that of a TH1 phenotype (IFN- production and stimulation of CTLs), may enable HCV to evade antiviral responses (Tsai et al. 1997). Furthermore, it has been found that NK cells are responsible for modulating DCs to induce and maintain regulatory phenotypes and functions of Tregs (Jinushi et al. 2007), which may facilitate viral escape through T-cell inhibition. Recent data have

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supported a role for virus-specific Tregs in mediating defective immune responses via suppression of Tcell effector function and thereby contributing to the progression of chronic hepatitis. Elevated levels of Tregs have been observed in the livers of patients with chronic HBV (Stoop et al. 2005) and HCV (Accapezzato et al. 2004) infection compared to patients who resolved infection and healthy individuals. The efficacy of the adaptive immune response to eliminate viral infection is further influenced by the diversity of viral epitopes recognized by T cells and the magnitude of activated effector cells. Successful clearance of HCV infection has been demonstrated to involve CTL responses directed against multiple MHC class I-restricted epitopes in HCV proteins (Cooper et al. 1999) with a high frequency against each individual epitope (Lechner et al. 2000). This robust CTL activity during resolution of acute HCV infection coincides with a persistent CD4 T-cell response against multiple structural and nonstructural HCV proteins (Missale et al. 1996). On the other hand, chronic HCV infection is usually characterized by low CTL numbers that target few antigenic epitopes (Rehermann et al. 1996; Wedemeyer et al. 2002). Viral persistence may also be due to mutations within the viral antigen epitopes, particularly of HCV (Erickson et al. 2001), which contribute to CTL impairment and viral escape. Viral mutations have been demonstrated to impede T-cell binding to MHC molecules (Chang et al. 1997) and regulation of TCR expression (Tsai et al. 1998) leading to chronic hepatitis. Viral escape and the development of chronic viral infection may also be the result of liver tolerance via T-cell anergy (Bertoletti et al. 1994). Inactivation of T cells may occur as a result of exposure to antigen under suboptimal conditions, including antigen presentation by nonantigen presenting cells such as other T cells (Lamb et al. 1983) and lack of costimulation (Appleman and Boussiotis 2003). Chronic infection may further be the result of an inadequately mounted CD4 T-cell response, which influences the functional potential of CTLs (Aberle et al. 2006). Based on studies of acute HCV infection, it has been suggested that clearance of the virus is more likely to occur when patients display an HCV-specific TH1 profile (characteristic of IFN- and IL-2 production), while those patients with a more pronounced TH2 profile (IL-4 and IL-10) were more likely to develop chronic infection (Tsai et al. 1997). T-cell inhibition was observed using transgenic mice

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containing HBV-expressing hepatocytes, as HBVspecific CTLs transferred into the transgenic mice quickly lost their ability to produce IFN- while concurrently upregulating PD-1 (Isogawa et al. 2005), suggesting that this pathway may contribute to the progression of chronic hepatitis. Furthermore, chronic HCV infection has been shown to develop with persistent high levels of PD-1 expression on T cells (Urbani et al. 2006). In a further attempt to explain chronic viral infection, it is possible that the virus has developed the ability to evade and subvert the host’s immune system, via interference with the antiviral IFN system (Foy et al. 2003). Overall, an inefficient and/or dysfunctional adaptive immune response that fails to eradicate infection from HBV and HCV facilitates a chronic necroinflammatory environment, the end result of which is hepatic fibrosis, cirrhosis, and hepatocellular carcinoma.

9.12.6 Alcoholic Liver Disease The chronic consumption of alcohol results in a variety of liver abnormalities ranging from steatosis to steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. The mechanisms underlying ALD are complex and remain poorly understood. It is clear that there is individual variation in susceptibility to ALD, as only a minor proportion of chronic heavy drinkers present with clinically advanced forms of ALD, including hepatitis, fibrosis, and cirrhosis (Lieber 2000). Therefore, additional factors other than direct toxicity from ethanol and its metabolites need to be addressed as potential mediators of ALD. Numerous clinical and experimental studies have revealed the involvement of the immune system in the onset of this disease (Messingham et al. 2002; Nelson and Kolls 2002). Immune dysfunction has been demonstrated from the increase in mortality of alcoholics due to pneumonia (Cortese et al. 1992; Esposito 1984) and the increased immune deficiency of patients chronically consuming alcohol to infectious diseases such as HBV, HCV, and HIV (Cook 1998). Furthermore, the detection of circulating autoantibodies (Klassen et al. 1995; Paronetto 1993) and the presence of CD4 and CD8 T cells in areas of hepatic damage during excessive alcohol consumption indicate that the immune system, particularly the adaptive immune system, may be involved in the evolution of ALD (Bailey et al. 1976; Zetterman and Sorrell 1981).

9.12.6.1 Adduct Formation and Initiation of an Immune Response Ethanol metabolism via alcohol dehydrogenase and the cytochrome P450 pathway results in the generation of highly reactive metabolites, which can covalently modify hepatic cellular proteins. Protein adduction by alcohol metabolites is known to disrupt protein function, especially if there is a lysine residue in a functionally significant location, such as in tubulin- and lysine-dependent enzymes (Sorrell and Tuma 1985; Tuma et al. 1987). Acetaldehyde, the initial metabolite of ethanol, covalently binds to reactive lysine residues forming protein adducts (Lin et al. 1988; Niemela et al. 1995) that have been found in the serum of both rats (Worrall et al. 1989) and human patients (Lin et al. 1990) following consumption of alcohol. Adduct formation may contribute to the functional impairment of hepatocytes leading to cellular damage as well as initiate an immune response when the adduct is taken up and processed by APCs and presented to lymphocytes. Activation of the adaptive immune response is considered to be a critical determinant in the initiation, propagation, and/or severity of ALD. Acetaldehyde adducts are immunogenic as they induce specific antibody production by B cells in rats chronically exposed to alcohol (Israel et al. 1986) and in alcoholic patients (Niemela et al. 1987), particularly in those with severe hepatic damage (Viitala et al. 1997). Furthermore, immunization of guinea pigs with acetaldehyde adduct-modified proteins with concurrent chronic alcohol feeding resulted in hepatic fibrosis (Yokoyama et al. 1995). Aldehydic products of lipid peroxidation, including malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) are also capable of protein modification and adduct formation (Esterbauer et al. 1991). Both MDA and HNE adducts have been observed in patients with ALD (Worrall and Thiele 2001) with a potential correlation between the severity of disease and the extent of adduct formation (Stewart et al. 2004; Viitala et al. 2000). Studies have also shown that MDA and acetaldehyde are further capable of reacting to proteins in a synergistic manner resulting in the formation of hybrid adducts to a larger extent than either aldehyde alone (Tuma et al. 1996). The presence of MDA-acetaldehyde (MAA) adducts has been witnessed in both ethanol-fed rats (Xu et al. 1998) and alcoholic patients (Rolla et al. 2000) and similar to MDA and HNE adducts, the amount of antibody production was associated with the degree

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of liver disease (Rolla et al. 2000). In addition, the oxidation of ethanol to hydroxyethyl radicals (HER) by CYP2E1 (Albano et al. 1991) is able to generate stable protein adducts (Albano et al. 1993), and antibodies directed against these HER adducts have been detected in the serum of patients with ALD but not nonalcoholic liver disease (Clot et al. 1995). Altogether, this evidence suggests that the initiation of an adaptive immune response against various adducts formed during chronic alcohol consumption participates in the development and progression of ALD. Although ALD has been demonstrated in guinea pigs following immunization with acetaldehyde adduct-modified proteins and ethanol consumption, in this experiment, the acetaldehyde adducts were generated using foreign (human) protein. This raises the potential that adduction to foreign protein may significantly enhance the immunogenicity of the protein, culminating in an amplified immune response and liver injury (Klassen et al. 1995). To address this issue, mice were immunized with homologous (mouse) albumin adducted with acetaldehyde followed by ethanol feeding (Shimada et al. 2002). Although this study resulted in induction of T-cell activation, histological examination of the liver yielded neither significant hepatic necrosis nor inflammatory cell infiltrate, along with no significant elevation in either AST or ALT levels. It was suggested that the acetaldehyde adduct-mouse albumin may not participate in liver injury or simply induce diminished immune response. Previous studies to address the immune-stimulating properties of acetaldehyde adducts have used adjuvants (Niemela 1993; Worrall et al. 1990), which enhance the immune response. The use of adjuvants confounds the notion that this response occurs in vivo during chronic ethanol consumption. On the other hand, studies have observed unique biological properties of MAA adducts that may be relevant to the induction of liver injury under physiological conditions (Klassen and Thiele 1998; Thiele et al. 1998). Immunization with MAA adduct-self-proteins, in the absence of adjuvants, resulted in antibody production not only against the specific MAA epitope, but also epitopes on the unmodified self-protein (Klassen and Thiele 1998). MAA adducts were further able to elicit specific CTL response to the self-protein but not the MAA epitope itself. The ability of MAA adducts to induce an immune response in the absence of adjuvants makes it enticing to speculate that MAA modification of protein may contribute to immune

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reactions that stimulate the production of antibodies and/or T cells directed against MAA adducts and self-liver antigens, potentially resulting in specific hepatic damage. While there is a mixed inflammatory infiltrate in the liver during ALD, histological evaluation of the liver indicates that a significant percentage of the infiltrate consists of CD4 and CD8 T cells (Chedid et al. 1993). Evidence to support a role for T cells in the initiation of ALD was demonstrated by the adoptive transfer of hepatic T cells from ethanol-fed rats into normal rats followed by injection with concanavalin A, which resulted in severe hepatitis and was associated with elevated serum levels of TNF- and IL-6 (Cao et al. 1999). Furthermore, experimental studies of lymphocytes obtained from alcoholinduced cirrhotic patients have demonstrated that these cells may propagate the inflammatory process due to their increase in TNF- expression following coculture with ethanol compared to lymphocytes from control patients (Santos-Perez et al. 1996). The production of autoantibodies and cytotoxic responses directed at unmodified self-proteins (autoantigens), which are characteristics of an autoimmune reaction, have been reported in ALD and may represent another possible mechanism in the pathogenesis of ALD. As discussed above, MAA adducts have not only been demonstrated to elicit strong antibody response against the adduct, but against the self-protein as well, supporting the role of autoimmunity (Thiele et al. 1998; Tuma et al. 1996). The ability of MAA adducts to induce an autoimmune response was further demonstrated using a model carrier protein, hen egg lysozyme (HEL), for which the antibody response was directed against the carrier protein and not the adduct itself (Thiele et al. 1998; Willis et al. 2002). Autoantibodies against liver-specific protein (LSP), a lipoprotein associated with the plasma membrane of hepatocytes, have been detected in a large percentage of patients with untreated chronic active hepatitis and in approximately 29% of patients with ALD (Manns et al. 1980; Perperas et al. 1981). Furthermore, the relationship between the presence of anti-CYP2E1 antibodies during ALD and advanced liver disease (Vidali et al. 2003) suggests that CYP2E1 is an additional target of the immune system involved in the generation of an autoimmune response. Although there exists multiple clinical and experimental evidence suggesting a direct relationship between adduct formation, antigen-specific T- and B-cell immune and autoimmune responses, and liver injury,

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the data appear circumstantial, and a direct causation remains to be established. 9.12.6.2 Genetic Factors and Immune Response in ALD The potential role for genetic factors in mediating susceptibility to ALD has been demonstrated from a twin study in which the rate for alcoholic cirrhosis was threefold higher in monozygotic than dizygotic twin pairs (Hrubec and Omenn 1981). Genetic factors in combination with immune responses may further play a role in patient susceptibility to ALD. IL-10 is an immunoregulatory, anti-inflammatory cytokine that inhibits the activation of CD4 T cells and function of CD8 T cells and macrophages, as well as inhibits MHC class II expression on APCs. A polymorphism at position –627 in the IL-10 promoter has been associated with reduction in IL-10 production. Based on a clinical study of over 500 alcoholics presenting with and without advanced ALD, it has been reported that there exists a strong correlation between possession of this variant gene and ALD (Grove et al. 2000). Polymorphisms in the IL-1 gene have also been related to inherited susceptibility to ALD (Takamatsu et al. 2000). Further evidence to suggest a relationship between genetics and immune mechanisms is based on the increased frequency of ALD in patients with an exon 1 polymorphism in the T-cell inhibitory gene, CTLA-4 (Valenti et al. 2004).

9.12.7 Nonalcoholic Fatty Liver Disease In the absence of chronic alcohol consumption, fatty liver disease is one of the most common triggers of chronic liver disease (Clark et al. 2002) and is the most prevalent cause of asymptomatic abnormal liver function tests within the United States (Browning et al. 2004; Clark et al. 2003). The histopathology of NAFLD is markedly similar to ALD and constitutes a spectrum of liver disease ranging from steatosis alone, nonalcoholic steatohepatitis (NASH), inflammation, hepatocyte ballooning, fibrosis (Matteoni et al. 1999), and ultimate progression to cirrhosis (Brunt 2004). NAFLD further parallels ALD in terms of the small percentage of patients with nonalcoholic steatosis who develop clinically significant liver disease (Browning et al. 2004). Although the pathogenesis of NAFLD remains

elusive, it is strongly associated with obesity and type 2 diabetes mellitus (Matteoni et al. 1999). This relationship suggests that NAFLD may result from a dysfunction in hepatic and visceral adipose tissue signaling, thereby contributing to liver damage by enhancing insulin resistance, steatosis, and progression to steatohepatitis (Bugianesi et al. 2005; Marchesini et al. 2003). The initiation and propagation of NAFLD is most widely regarded to be attributed to a two-hit process (Day and James 1998). The first hit constitutes the development of insulin resistance and increase of free fatty acids in the liver resulting in hepatic steatosis. Hepatocytes counter this buildup of excess fat via metabolism and oxidation of the fatty acids, thereby facilitating the release of reactive oxygen species and rendering the hepatocytes susceptible to a second hit. This second hit involves oxidative stress, lipid peroxidation, and activation of the immune system, which elicits the lobular inflammatory component of steatohepatitis and hepatocyte damage. Expansion of visceral adipose tissue leads to an increase in the production of adipocytokines and cytokines, including leptin, adiponectin, and TNF as well as free fatty acids that may alter energy metabolism and lipid homeostasis and further contribute to liver injury. Although the triggers for the initiation of ALD and NAFLD are distinct, the presence of an inflammatory infiltrate and immune responses during the progression of steatohepatitis in both diseases are analogous. Furthermore, since investigation into the immunological mechanisms regulating the pathogenesis of NAFLD remains in its infancy, we instead focus on the potential role of adipocytokines and cytokines in mediating the immune response during NAFLD. 9.12.7.1 The Role of Adipocytokines and Cytokines in NAFLD Adipocytokines are classified as cytokines that are mainly, but not exclusively, produced by adipose tissue. Leptin and adiponectin constitute adipocytokines that are believed to mediate insulin resistance and related inflammatory disorders (La Cava and Matarese 2004; Wellen and Hotamisligil 2005). Known for its regulation of appetite and obesity (La Cava and Matarese 2004), leptin is also considered a proinflammatory cytokine that participates in the modulation of T-cell function. Leptin has been shown to increase the TH1 phenotype of CD4 T cells while suppressing the TH2 phenocyte, as well

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as inducing the proliferation of naive CD4 T cells and inhibiting the proliferation of memory CD4 T cells (Lord et al. 1998). Leptin may modulate the immune response in fatty liver disease as a recent study has shown that leptin suppresses proliferation of Tregs (De et al. 2007). As previously described, Tregs play a critical role in the regulation of hepatic inflammation and disease via suppression of T-cell effector function. Patients with PBC have demonstrated a relative reduction of Tregs (Lan et al. 2006), which contributes to autoimmunity while elevated levels of Tregs have been observed to play a part in the development of chronic HBV and HCV infection (Cabrera et al. 2004; Stoop et al. 2005). The potential role of Tregs in the pathogenesis of NAFLD has recently been investigated. Using an experimental model of mice fed a high fat diet to induce steatosis, it has recently been shown that oxidative stress, the second hit in the progression of NAFLD, triggered Treg apoptosis and depletion from the steatotic liver (Ma et al. 2007). This condition led to an exacerbation of hepatic inflammation upon exposure to secondary injury. These results identify a mechanism by which increased oxidative stress in the fatty liver induces Treg apoptosis and reduces the number of hepatic Tregs, which in turn facilitates increased inflammation and susceptibility to further injury. Therefore, hepatic Treg depletion in the fatty liver, whether due to oxidative stress or elevated leptin levels, may be a crucial factor in the progression from simple steatosis to NAFLD. Adiponectin is an adipocyte-specific plasma protein that acts as an endogenous regulator of endothelial cells. The binding of adiponectin to its receptor ADIPOR2, expressed mainly in the liver (Yamauchi et al. 2003) by the endothelial cells, suppresses the expression of TNF-induced adhesion molecules (Ouchi et al. 1999). Adiponectin has been shown to inhibit IFN- synthesis, induce the production of anti-inflammatory cytokines including IL-10, and its presence in a T-cell proliferation assay resulted in a diminished ability to evoke an allogenic T-cell response (Wolf et al. 2004). During states of insulin resistance, such as NAFLD, the serum levels of adiponectin have been observed to be reduced (Arita et al. 1999). Furthermore, a protective role for adiponectin against fatty liver diseases was confirmed in a ob/ob mouse model, as adiponectin replacement decreased hepatomegaly, steatosis, and ALT levels (Xu et al. 2003). This evidence suggests the following model in an adiponectin-deficient environment, such as NAFLD: the uninhibited expression of adhesion

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molecules by LSECs would facilitate the recruitment and retention of lymphocytes in the liver, which through an unimpeded process of activation, proliferation, and production of TH1 cytokines, such as IFN- , would mediate a chronic inflammatory environment, steatohepatitis, and progression of NAFLD. In addition to leptin and adiponectin, TNF-, of which serum levels have been found to be elevated in patients with NASH (Hui et al. 2004) has also been suggested to play a central role in the chronic inflammatory response during NAFLD. Evidence to support a pathogenic role for TNF- is based on the reduction in steatosis in TNF receptor knockout mice compared to wild-type mice (Tomita et al. 2006) and the improvement of hepatic histology, serum ALT levels, and several biochemical measures of hepatic insulin resistance following inhibition of TNF- with anti-TNF antibodies (Li et al. 2003). TNF- also induces the expression of IL-6 in the liver that can further increase insulin resistance and inflammatory responses (Abiru et al. 2006). It has further been suggested that high levels of TNF- may promote the recruitment of lymphocytes to the liver that may mediate steatohepatitis (FoxRobichaud and Kubes 2000). Although the exact mechanism of immune-mediated NAFLD remains undefined, adipocytokines and cytokines may be involved in a complex network that regulates the adaptive immune response in obesity and NAFLD. In response to a large number of substances, including pathogens, xenobiotics, tumor cells, and harmless dietary antigens, the liver provides an intricate balance between the initiation of an appropriate immune response and the generation of tolerance. However, when regulation of the immune system goes awry, the delicate interplay of immunity and tolerance in the liver is compromised, which culminates in immune-mediated liver injury. Further understanding of the activation of innate and adaptive immunity will lead to the identification and development of therapeutic targets against potentially infectious and harmful agents.

Acknowledgment This work was supported by U.S. National Institutes of Health Grant ROI ES012914 (to C. J.) and predoctoral fellowship in pharmacology/toxicology from the PhRMA Foundation (to M. P. H.).

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