Chapter 31Autoimmune hepatitis

Introduction

Due to its location and function, the liver is continually being antigenically challenged by pathogens, toxins, tumor cells, and less harmful antigens such as those acquired with food intake. The bloodstream travels along the hepatic sinusoids which are covered by an endothelial layer devoid of basal membrane and presenting “pores.” This sinusoid structure allows the transmigration of cells towards the hepatic parenchyma. Soon after differentiation and activation, circulating T cells are capable of entering non-lymphoid peripheral organs. Differentiation and activation of T cells within the peripheral lymphoid organs are known to take roughly 48 h. The liver is an exception to the general time course lymphoid trafficking due to its ability to retain mature naïve T cells, mainly CD8+ T cells which, after a 24 h period, are delivered into the peripheral circulation as activated cells (1).

Chronic inflammation of the liver occurs as a consequence of the recruitment and retention of lymphocytes in the tissue. The recruitment of effector and regulatory T cells (Tregs) from the circulation is dependent on interactions between lymphocytes and specific cell surface molecules expressed on endothelial cells (2). Once captured, the retention and positioning of leukocytes within tissue requires certain signals to locate and retain them at sites of target cell damage. These receptors and chemokine signals follow the accepted multistep paradigm of leukocyte adhesion to vascular endothelium, which is relevant to most organ systems although the specific signals involved differ among tissues. Chemokines are critical components of this adhesion cascade and are believed to play two crucial roles: triggering integrin-mediated stable adhesion and directing migration. Chemokines can bind to endothelial glycosaminoglycans and thus allow them to be presented to flowing leukocytes and also provide a mechanism for the paracrine presentation of chemokines secreted by other cells within the microenvironment. Similar mechanisms are believed to be involved in both normal immune surveillance and in inflammatory disease although the chemokines involved differ with constitutive chemokines playing the dominant role in physiological trafficking and inducible inflammatory cytokines involved in inflammation. Chemokines can be classified into four groups based on their amino acid sequence. The two largest groups are the CC chemokines, where conserved cysteine residues lie adjacent to each other, and CXC chemokines, where an amino acid separates the first two cysteine residues. Within the group of CXC chemokines, CXCL10 or interferon (IFN)-inducible protein (IP-10), CXCL9 or monokine induced by IFN-γ (MIG), CXCL11 or IFN-γ - inducible T-cell -chemoattractant (ITAC) display potent lymphocyte chemotactic activity and bind a common receptor and CXCR3, the expression of which is increased on tissue-infiltrating regulatory and Th1-polarized T cells. T cells infiltrating the inflamed liver express high levels of CXCR3. Moreover, CXCR3 ligands are up-regulated on hepatic endothelium at sites of T-cell infiltration in chronic hepatitis, and their presence has been correlated with outcome of inflammatory liver disease (3).

The liver is a residence organ for dendritic cells (DC), whose precursors derive from peripheral blood. The antigenic uptake that occurs within the liver by immature DC promotes their maturation and migration to peripheral lymph nodes, where they act as efficient antigen presenting cells. This is a key process in the initiation of the immune response. Tregs may be attracted to DC residing within the liver to modulate their local function and survival. In secondary lymphoid tissues, Tregs use multiple mechanisms to inhibit DC function and block initiation of autoimmunity. Tregs can suppress effector cell proliferation and function within the liver by secreting immunosuppressive cytokines (IL-10, TGFβ) or through contact-dependent mechanisms to restore immune homeostasis and promote resolution of hepatitis. Effector T cells cause damage to hepatocytes resulting in interface and lobular hepatitis. Infiltrating T cells, macrophages, and stromal cells secrete IFNγ, TNFα, and other proinflammatory cytokines that result in a microenvironment that promotes the persistence of chronic liver inflammation.

Apart from DC, hepatocytes, kupffer cells, and sinusoidal endothelial cells can present antigens to CD8+ T cells. The local presentation of antigens within the liver leads to activation-triggered T cell apoptosis. The balance between the different activation processes that take place within the liver leads to immunological tolerance, which explains the long survival of hepatic grafts and the chronic persistence of hepatotropic viruses (4). The development of intrahepatic tolerance would be due to the low expression of co-stimulatory molecules by hepatocytes (5).

Another feature that makes the liver a specialized immune organ is the type of resident T cells in the parenchyma (6). Fifty percent of intrahepatic lymphocytes (IHL) are non-conventional cells expressing membrane proteins that are typical of T cells and proteins belonging to natural killer cells (CD56, CD161, and/or KIRs, which are receptors that inhibit the cytotoxic response). These cells are termed NKT cells or “natural T cells.” IHLs represent 20% of the total NK cell population, 25% of the T cells, and 5% of the B cells. Classical NKT cells (type I) express only one α chain of the TCR called the invariant chain (iNKT) by which the recognition of antigenic epitopes presented by the non-classical histocompatibility molecule CD1 (7) is accomplished. These cells also express CD45RO, an isoform of the CD45 which features activated lymphocytes and CD161 (NKR-P1A), a co-stimulatory molecule that contributes to the antigenic presentation mediated by CD1.

The interaction between antigen presenting cells and NKT cells results in the rapid expression of regulatory/Th2 and proinflammatory (IFN-γ and TNF) cytokines by NKT cells. Activated NKT cells are a source of IFN-γ and IL-4, two cytokines with hepatocytotoxic properties (Figure 1).

Figure 1

Myeloid dendritic cells (DC) present the antigen to NKT cells within the context of CD1 HLA complex through the TCR Vα24. During antigen presentation, DC release IL-12 which 1- promotes NKT cytotoxicity and regulates the number of DC (negative (more...)

Autoimmune hepatitis (AIH)

Autoimmune hepatitis (AIH) is a chronic and progressive hepatitis of moderate to severe activity. AIH does not cause granulomatous biliary lesions, siderosis, or copper depots (8). The classification of AIH is based on the specificity of the autoantibodies found in the patient’s serum (Table 1). In this chapter, special reference will be made to type I AIH.

Table 1

Abbreviations. ANA: Antinuclear antibodies, SMA: anti-smooth muscle antibodies, p-ANCA: perinuclear anti-neutrophil cytoplasm antibodies, anti-ASGPR: anti-a sialoglycoprotein receptor antibodies, antiSLA/LP: anti-soluble liver antigens/liver-pancreas (more...)

Clinical manifestations and diagnosis

In individuals with unresolved hepatitis, it is common to find symptoms that are similar to those of acute viral hepatitis. In these patients, a combination of signs and symptoms can be found, e.g. jaundice, acolia, coluria, hepatomegaly, splenomegaly, spider veins, acne, malaise, fatigue, evidence of cirrhosis, or fulminant liver failure. In the first five years after the onset of the disease, 50% of untreated patients die while spontaneous remission is a less frequent phenomenon. Diagnosis of AIH-I is difficult, since clinical, biochemical, or histological findings are not conclusive if taken in isolation. The criteria for the diagnosis of this disease have been established and revised by the International Autoimmune Hepatitis Group (20, 21). More recently, a simplified score intended to be used in clinical practice has been proposed by this group (22). The diagnostic criteria that make it possible to distinguish probable from definite AIH are in Table 2.

Table 2

Diagnostic criteria for autoimmune hepatitis.

The current guidelines for the diagnosis of AIH are the following:

1. Diagnosis of AIH requires the assessment of aminotransferase activity, the levels of gammaglobulins, detection of ANA and/or SMA, or the presence of type 1 anti-liver and kidney microsome (anti-LKM-1) antibodies as well as the histological evaluation of a liver biopsy.
2. Diagnostic criteria of AIH must be applied to all patients.
3. In those cases where the diagnosis of AIH is not conclusive or when the clinical features of hepatitis are atypical, the scoring method must be used (Table 3).
4. Even though AIH-I presents histological and serological features that are common to pediatric and adult patients, previous work undertaken in our laboratory have shown that the clinical features and the genetic susceptibility for AIH-I differ between children and adults (Table 4).

Table 3

Differential characteristics between pediatric and adult autoimmune hepatitis.

Table 4

Scoring system for the diagnosis of atypical autoinmune hepatits in adults.

Treatment of AIH-I

AIH-I has been proven to respond favorably to the immunosuppressant treatment provided that there are no clinical signs of acute liver failure, a circumstance under which liver transplantation is mandatory.

The treatment of AIH-I comprises two phases. In newly diagnosed AIH, induction of remission is the main goal. The second phase of therapy is maintenance of remission with the lowest possible dose in order to maintain it while preventing significant side effects. If the diagnosis is correct, and the appropriate therapy is chosen, liver transplantation should be avoidable in patients with AIH.

The standard treatment consists of the administration of prednisolone with or without azathioprine. With this schedule, remission (normal liver tests) is achieved in more than 90% of the patients (26). In 20% of patients who have undergone remission, drugs can be withdrawn successfully after a few years of treatment, whereas in those patients suffering from AIH-II, the treatment must be maintained for the lifetime. A sustained remission of AIH has been reported in patients undergoing long-term treatment with azathioprine alone (27). Other effective drugs that are employed in the treatment of AIH-I are cyclosporine A and mophetil mycophenolate (28, 29)

Immunopathogenesis of AIH-I

The establishment of this disease may be considered a consequence of a breakdown of tolerogenic mechanisms which govern the liver milieu. Although the autoantigen/s that trigger AIH-I have not been identified so far, the family of asialoglycoprotein receptors, xenobiotic substances, bacteria, and viruses have been postulated as candidates. Infection with hepatitis A, B, C virus (HAV, HBV, HCV) or measles virus, in particular, can precede the onset of AIH-I.

In Argentina there is a high prevalence of HAV infection. In children, the allele HLA II DRB1*1301 is linked to the development of both chronic HAV hepatitis and AIH-I. Due to this fact, it has been suggested that in children, the development of AIH-I requires the expression of yet unknown genes. The latter hypothesis is in line with the polygenic nature of autoimmune diseases (32). The treatment of chronic B and C hepatitis with IFN-α promotes the synthesis of serum autoantibodies and in some patients, these autoantibodies have been shown to be pathogenic. The neoantigen formation, the molecular mimicry or the induction of proinflammatory cytokines could be considered factors modifying the tolerogenic liver microenvironment that would lead to the development of AIH-I.

It is known that 10% of chronic HCV patients have autoantibodies typical of AIH-II (Table 1). The molecular mimicry between viral proteins and the host’s antigens has been postulated to be the mechanism by which anti-LKM antibodies are generated (33).

Genetic expression studies done on liver biopsies obtained from pediatric patients with AIH-I have demonstrated the over-expression of primary transcripts of IFN-γ, IL-12p40 (a 40 kDa polypeptide), IL-12Rβ2 (the β2 subunit of the IL-12 receptor), IL-18, TGFβ1, IL-4, and Vα24 (34). The increase in the expression of these molecules, some of which were non-detectable in normal livers, has made it possible to infer their role in the mechanisms of liver injury. It is known that IL-2 is capable of inducing the synthesis of the IL-12 receptor chains in naïve T cells. Nevertheless, the rise in the expression of this receptor is dependent on the presence of IL-12, which is released by several antigen-presenting cells. Furthermore, the IL-12-dependent production of IFN-γ is responsible for the maintenance of high levels of IL-12Rβ2 (35, 36), a molecule controlling the differentiation of the Th1 phenotype of T cells (37). One of the effects of the IL-12 is to enhance the expression of the IL-18R present in IFN-γ-producing cells thus attaining a synergistic response between IL-12 and IL-18 to produce IFN-γ (38). The high expression levels of IFN-γ, IL-12p40, and IL-12Rβ2 found in liver biopsies of AIH-I patients is evidence of the role of a Th1-skewed response in this disease.

Apart from the classical Th1 and Th2 subsets, an effector T cell subset named Th17 has also been described. Th17 cells are characterized by the production of interleukin-17A (IL- 17A), IL-17F, IL-22, IL-21, IL-6, and tumor necrosis factor α (TNF-α) (39). Although the differentiation and maturation of human Th17 cells is still a matter of controversy, transforming growth factor β (TGF-β), IL-6, IL-21, IL-1β, and IL-23 have been postulated to induce the human Th17 cell lineage (40). IL-23, a heterodimer of the IL-12 family, composed of the p19 and p40 subunits, appears to be essential to maintain and expand Th17 effectors. Meanwhile, IL-27, which is composed of the p28 and EBI3 subunits, suppresses the Th17 differentiation and IL-17 production and promotes Th1 differentiation (41). The presence of Th17 cells has also been reported in inflamed human tissues from patients suffering from a variety of inflammatory and autoimmune disorders (42-44). Only circumstantial signs of Th17 effector functions were found in liver biopsies of patients with AIH-I as deduced from the limited production of IL-17A and IL-17F transcripts (45).

Regulatory T cells in AIH-IN

In addition to the Th1, Th2, and Th17 subsets, the functional quartet of CD4+ effectors also includes Tregs constitutively expressing the IL-2 receptor α-chain CD25 and the forkhead/winged helix (FoxP3) master regulatory transcription factor which is highly specific to the CD4+CD25high Treg lineage (46-48). Some studies carried out in mainly pediatric patients have addressed the possible impairment of Treg functions in AIH. These studies have shown that CD4+CD25+ Treg cells in peripheral blood seemed to feature both reduced frequencies and impaired suppressive functions (49, 50). Moreover, earlier studies have suggested that peripheral lymphocytes in autoimmune hepatitis exhibit increased immunosuppressive capability (51, 52). In these studies, however, Treg cells were defined only by staining for CD4 and CD25 since, at that time, it was not possible to distinguish human Tregs from activated effector T cells. In humans, a clear distinction of Tregs from activated conventional effector T cells is difficult since the Treg markers CD25 and Foxp3 can also be expressed transiently by activated effector T cells (53). More recently, a combination of more accurate methods has been developed. Those studies include the methylation status of the FOXP3 locus to search for demethylated FOXP3 TSDR (Treg-specific demethylated region) (54); the analysis of the expression of CD127, which is low in Treg cells and high in activated effector T cells (55); and the analysis of CD39, which seems to be one of the molecules that mediates the suppressive function of Treg cells by producing inhibitory adenosine (56). The results of these studies clearly established that Treg cells belonging to AIH patients are fully functional; Treg frequency is not decreased in AIH patients but rather seems to correlate with disease activity. Another important aspect of this study was the finding that the Treg frequency in the blood of AIH patients is associated with pharmacological immunosuppression, and therefore, not with the remission per se (30).

The complex cell and cytokine environment of the inflamed liver may have profound effects on Treg differentiation, stability, and function as well as alter the susceptibility of effector T cells to suppression. In addition to the above mentioned interaction between Tregs and dendritic cells, local cytokines could reprogram Tregs to express either Tbet which would lead to a Th1 phenotype (57) or RORc and a Th-17 phenotype (58). We have described a higher Vα24, IFN-γ, FoxP3, p28, IL-12p40, and IL-21 expression at the moment of diagnosis as well as a positive correlation between IL-21 and aminotransferase levels. It is interesting that only IFN-γ and FoxP3 decreased during biochemical remission following immunosuppressive treatment (AIH-Ir). An “AIH-I phenotype” described as “high Vα24, IFN-γ, and FoxP3 expression” was observed in a low percentage of AIH-Ir children but not in healthy, age-matched controls. Overall, the presence of a local deregulation of the innate (i.e., NKT cells) and adaptive arms of the immune responses (i.e., Th1, Treg cells) was described (45). In addition, IL-21 was highlighted as a mediator of liver injury. Together with Th17 cells, iNKT cells are a source of IL-21. Since a seemingly limited presence of a Th17 subpopulation and an amplified expression of Vα24+ transcripts were detected, it has been suggested that iNKT cells may be the main source for IL-21 in the livers of patients with AIH-I.

IL-21, together with IL-2 and IL-15 are known to induce the proliferation of NKT cells (59). Apart from its role as a differentiation factor for Th17 cells from naïve cells, the inhibitory role of IL-21 on the differentiation of Treg FoxP3+ cells from the same precursor cells as those of Th17 cells is also noteworthy. The same is true of the ability of this cytokine to confer CD4+ cell resistance to the suppressing effect of Treg cells (60). The high levels of IL-21 found in the AIH-I inflammatory scenario but not in other inflammatory liver diseases (e.g. non-alcoholic fatty liver disease and HCV infection) make it possible to suggest that the immunoregulation of Treg and NKT cell subsets would constitute one of the most probable roles of IL-21 in the pathogenesis of AIH-I. The latter hypothesis implies that the high levels of FoxP3 found in the livers of pediatric patients reflects an increase in the number of Treg cells and that the prevailing function of IL-21 would be to confer resistance to the suppressant effector of Treg cells. Thus, and in spite of their local increase, FoxP3+ cells would not be able to suppress the autoimmune response. An increase in the recruitment of Treg cells from the peripheral compartment to the liver would constitute an attempt to suppress the local immune response. Treatment of AIH will fail if adoptively transferred Treg cells are inhibited or pushed into an effector differentiation pathway by local factors in the liver. Consequently, the need to shift the balance from damage towards resolution of inflammation emphasizes the importance of understanding the effects of the local inflammatory microenvironment (61). The fact that Treg cells are detected at high frequencies in the liver of patients with AIH suggests that suppression of function locally rather than a basic defect in Treg generation or function underlies the persistence of chronic autoimmune hepatitis.

Figure 2 depicts the interaction between genetic susceptibility, an antigenic peptide, and the production of different cytokines favoring the autoimmune attack (62).

Figure 2

Autoimmune attack to hepatocytes. A specific autoantigenic peptide is presented to helper T cells (Th0 cells) within the context of class II HLA molecules together with co-stimulatory molecules present in the antigen-presenting cell (APC). Th0 cells become (more...)

Conclusions

Due to the high incidence in many geographical areas as well as to the high percentage of liver transplantations due to autoimmune hepatitis, it is important to encourage more insightful studies to be done. Experimental evidence allows us to conclude that the liver damage in AIH-I would be a consequence of not only a Th-1-biased response but also the deregulation of hepatic Treg cell function. The molecular species responsible for the development of the different types of this disease in both children and adults remains to be determined. Knowing the autoantigen that triggers these processes is of paramount importance for it would broaden the frontiers for the development of more accurate methodologies of diagnosis, prognosis, and treatment of this pathology.

Follow-up studies are necessary to elucidate the mechanisms of the immune response that remain active despite the treatment and whether those pathways would be amenable to further therapeutic intervention.

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