Animal experiments cannot predict the probability of idiosyncratic drug toxicity; consequently, an important goal of the pharmaceutical industry is to develop a new methodology for preventing this form of drug reaction. Although the mechanism remains unclear, immune reactions are likely involved in the toxic processes underlying idiosyncratic drug toxicity: the drug is first activated into a chemically reactive metabolite that binds covalently to proteins and then acts as an immunogen. Therefore, screening tests to detect chemically reactive metabolites are conducted early during drug development and typically involve trapping with glutathione. More quantitative methods are then used in a later stage of drug development and frequently employ (14)Cor (3)H-labeled compounds. It has recently been demonstrated that a zone classification system can be used to separate risky drugs from likely safe drugs: by plotting the amount of each protein-bound reactive metabolite in vitro against the dose levels in vivo, the risk associated with each drug candidate can be assessed. A mechanism for idiosyncratic drug-induced hepatotoxicity was proposed by analogy to virus-induced hepatitis, in which cytotoxic T lymphocytes play an important role. This mechanism suggests that polymorphism in human leukocyte antigens is involved in idiosyncrasy, and a strong correlation with a specific genotype of human leukocyte antigens has been found in many cases of idiosyncratic drug toxicity. Therefore, gene biomarkers hold promise for reducing the clinical risk and prolonging the life cycle of otherwise useful drugs.

Increasing evidence in recent years has suggested that B cells act as a crucial regulator in autoimmune diseases. However, little is known about their role in autoimmune hepatitis (AIH) and the underlying regulatory mechanisms. In this study, we show that B cells ameliorated experimental AIH (EAH) by suppressing CD4+ T-cell responses and that CD11b expression on B cells was required for the regulatory function of B cells. In vitro studies reveal that the suppressive function of CD11b was mediated by the impairment of T-cell antigen receptor (TCR) signaling transduction and the promotion of TCR down-regulation. Moreover, we show that the increased CD11b expression on B cells was interleukin (IL)-10 dependent and that additional IL-10 stimulation promoted CD11b expression on B cells, thereby enhancing B-cell regulatory effects.

These findings reveal a previously unrecognized role for CD11b in B-cell regulatory function and its protective effect on EAH.

Autoimmune hepatitis (AIH) is a chronic liver disease which is normally recognized during late stage of the disease. Due to limited knowledge about the onset and course of disease and need for chronic immunosuppression with significant side-effects there is a requirement for a good preclinical animal model, mirroring main characteristics of AIH. In addition to the exclusion of other liver diseases, AIH is characterized by elevated serum transaminases, specific autoantibodies and elevated gammaglobulins as well as a specific liver histopathology. A good preclinical model should mirror most of these criteria. In the last decades several models have been published using different approaches to break hepatic tolerance and induce liver damage. The induction of a chronic hepatitis similar to the human disease remained a difficult challenge. Nevertheless, these models helped to get more information about the aspects of AIH induction and liver immunology.

Drug-induced liver injury (DILI) represents a major impediment to the development of new drugs and is a leading cause of drug withdrawal. The occurrence of hepatotoxicity has been closely associated with the formation of chemically reactive metabolites. Huge investment has focused on the screening of chemically reactive metabolites to offer a pragmatic approach to produce safer drugs and also reduce drug attrition and prevent market place withdrawal. However, questions surrounding the importance of chemically reactive metabolites still remain. Increasing evidence now exists for the multi-factorial nature of DILI, in particular the role played by the host immune system or disease state in the pathogenesis of DILI. This review aims to evaluate the current measures for the prediction and diagnosis of DILI and to highlight investigations being made to understand the multidimensional nature. Some of the steps being made to generate improved physiological systems to identify more sensitive, reflective mechanism-based biomarkers to aid the earlier identification of DILI and develop safer medicines are also discussed.

Adverse drug reactions, such as hepatotoxicity, blood dyscrasias and hypersensitivity are a major obstacle for the use and the development of new medicines. Many forms of organ-directed toxicity can arise from the bioactivation of drugs to so-called chemically reactive metabolites, which can modify tissue macromolecules. It is well established that the toxicities of model hepatotoxins, such as acetaminophen, furosemide, bromobenzene and methapyrilene can be correlated with the generation of chemically reactive metabolites, which can be detected by measurement of the irreversible binding of radiolabelled material to hepatic protein and/or the detection of stable phase II metabolites such as glutathione conjugates. The basic chemistry of the reaction of such metabolites with model nucleophiles is relatively well understood. A major challenge is to define how certain reactive intermediates may chemically modify critical proteins and how modification of specific amino acids may alter protein function which in turn may affect cell signalling, regulation, defence, function and viability. This in turn will determine whether or not bioactivation will result in a particular form of drug-induced injury. It is now clear that even relatively simple reactive intermediates can react in a discriminative manner with particular cellular proteins and even with specific amino acids within those proteins. Therefore both non-covalent, as well as covalent bonds will be important determinants of the target protein for a particular reactive metabolite. Mammalian cells have evolved numerous defence systems against reactive intermediates. Sensitive redox proteins such as Nrf-2 recognize oxidative stress and electrophilic agents. This is achieved by chemical modification of cysteine groups within keap-1, which normally forms an inactive heterodimer with Nrf-2. Modification of keap-1 releases Nrf-2 that translocates to the nucleus and effects gene transcription of a number of genes involved in the detoxication of chemically reactive metabolites. Diminution of protein function can occur by either covalent modification of nucleophilic amino acids (e.g. cysteine, lysine, histidine etc.) or oxidation of thiols, which can be reversible or irreversible. In the case of acetaminophen, more than 30 target proteins have been identified and for several of them, corresponding alterations in protein function have been defined in the context of tissue necrosis. Alternatively, protein modification may induce signalling systems which initiate cell death, an immune response or to an altered tissue genotype.

NKT cells expressing phenotypic markers of both T and NK cells seem to be pivotal in murine models of immune-mediated liver injury, e.g., in Con A-induced hepatitis. Also alpha-galactosylceramide (alpha-GalCer), a specific ligand for invariant Valpha14 NKT cells, induces hepatic injury. To improve the comprehension of NKT-cell mediated liver injury, we investigated concomitants and prerequisites of alpha-GalCer-induced hepatitis in mice. Liver injury induced by alpha-GalCer injection into C57BL/6 mice was accompanied by intrahepatic caspase-3 activity but appeared independent thereof. alpha-GalCer injection also induces pronounced cytokine responses, including TNF-alpha, IFN-gamma, IL-2, IL-4, and IL-6. We provide a detailed time course for the expression of these cytokines, both in liver and plasma. Cytokine neutralization revealed that, unlike Con A-induced hepatitis, IFN-gamma is not only dispensable for alpha-GalCer-induced hepatotoxicity but even appears to exert protective effects. In contrast, TNF-alpha was clearly identified as an important mediator for hepatic injury in this model that increased Fas ligand expression on NKT cells. Whereas intrahepatic Kupffer cells are known as a pivotal source for TNF-alpha in Con A-induced hepatitis, they were nonessential for alpha-GalCer-mediated hepatotoxicity. In alpha-GalCer-treated mice, TNF-alpha was produced by intrahepatic lymphocytes, in particular NKT cells. BALB/c mice were significantly less susceptible to alpha-GalCer-induced liver injury than C57BL/6 mice, in particular upon pretreatment with d-galactosamine, a hepatocyte-specific sensitizer to TNF-alpha-mediated injury. Finally, we demonstrate resemblance of murine alpha-GalCer-induced hepatitis to human autoimmune-like liver disorders. The particular features of this model compared with other immune-mediated hepatitis models may enhance comprehension of basic mechanisms in the etiopathogenesis of NKT cell-comprising liver disorders.

The importance of reactive metabolites in the pathogenesis of drug-induced toxicity has been a focus of research interest since pioneering investigations in the 1950s revealed the link between toxic metabolites and chemical carcinogenesis. There is now a great deal of evidence that shows that reactive metabolites are formed from drugs known to cause hepatotoxicity, but how these toxic species initiate and propagate tissue damage is still poorly understood. This review summarizes the evidence for reactive metabolite formation from hepatotoxic drugs, such as acetaminophen, tamoxifen, diclofenac, and troglitazone, and the current hypotheses of how this leads to liver injury. Several hepatic proteins can be modified by reactive metabolites, but this in general equates poorly with the extent of toxicity. Much more important may be the identification of the critical proteins modified by these toxic species and how this alters their function. It is also important to note that the toxicity of reactive metabolites may be mediated by noncovalent binding mechanisms, which may also have profound effects on normal liver physiology. Technological developments in the wake of the genomic revolution now provide unprecedented power to characterize and quantify covalent modification of individual target proteins and their functional consequences; such information should dramatically improve our understanding of drug-induced hepatotoxic reactions.

In autoimmune hepatitis, strong TGF-beta1 expression is found in the inflamed liver. TGF-beta overexpression may be part of a regulatory immune response attempting to suppress autoreactive T cells. To test this hypothesis, we determined whether impairment of TGF-beta signaling in T cells leads to increased susceptibility to experimental autoimmune hepatitis (EAH). Transgenic mice of strain FVB/N were generated expressing a dominant-negative TGF-beta type II receptor in T cells under the control of the human CD2 promoter/locus control region. On induction of EAH, transgenic mice showed markedly increased portal and periportal leukocytic infiltrations with hepatocellular necroses compared with wild-type mice (median histological score = 1.8 +/- 0.26 vs. 0.75 +/- 0.09 in wild-type mice; P < 0.01). Increased IFN-gamma production (118 vs. 45 ng/ml) and less IL-4 production (341 vs. 1,256 pg/ml) by mononuclear cells isolated from transgenic livers was seen. Impairment of TGF-beta signaling in T cells therefore leads to increased susceptibility to EAH in mice. This suggests an important role for TGF-beta in immune homeostasis in the liver and may teleologically explain TGF-beta upregulation in response to T cell-mediated liver injury.

Although several mouse models of AIH have been described, no model is ideal. Indeed, the disease is self-limited in each model, and none is associated with significant liver fibrosis or progression to cirrhosis. Nevertheless, these models should be useful for testing different hypotheses regarding the initiation of AIH. Still, each model poses unique limitations. The EAIH model initiated by immunization with crude liver antigens in CFA has been plagued by a high prevalence of hepatitis lesions in CFA controls and inconsistencies in results. The TGF beta-1 and IL-2 deficient models lead to high in utero mortality and short median life spans in the surviving animals. Lastly, all models require more detailed characterization of the antigen specificity of infiltrating liver T cells and the pathogenesis of liver cell injury.

The histopathological features of liver biopsies from 20 cats with progressive lymphocytic cholangitis/cholangiohepatitis are reported. These biopsies were subject to immunohistochemical investigation for expression of CD3, CD79. Major Histocompatibility Complex (MHC) Class II molecules, and feline IgG, IgM and IgA. Livers from five normal cats, which were also examined showed constitutive expression of MHC Class II by sinusoidal Kupffer cells and bile duct epithelium, in addition to a population of portal, and bile duct inter-epithelial, CD3+ T lymphocytes. In liver biopsies from cats with the active phase of lymphocytic cholangitis/cholangiohepatitis (n = 11), the portal lymphocytes were predominantly CD3+ T cells that infiltrated bile duct epithelium and periportal hepatic parenchyma, CD79+ B lymphocytes formed distinct aggregates or follicles within the regions of T-cell infiltration. Low numbers of plasma cells were present, and these predominantly expressed IgA. MHC Class II was expressed by Kupffer cells, infiltrating T and B lymphocytes and macrophages. There was membrane and cytoplasmic Class II expression by bile duct epithelium, some vascular endothelium, and fibroblasts within areas of fibrosis. In liver biopsies from cats with chronic lymphocytic cholangitis/cholangiohepatitis (n = 9), there was less in flammation, but the composition of the infiltrates was similar to that in the active phase of disease. The findings provide further evidence for an immune mediated pathogenesis in progressive lymphocytic cholangitis/cholangiohepatitis.

The aim of this study was to investigate liver histology in mice after immunization with the conserved self molecule dihydrolipoamide dehydrogenase, E3, a subunit of the mitochondrial 2-OADC enzyme family identified as the M2 autoantigen in the liver disease, primary biliary cirrhosis. Mice were immunized by a novel procedure. The autoantigen E3 was introduced by pinocytosis into hypertonically treated syngeneic lymphoid cells to facilitate intracellular antigen processing and presentation and the generation of a cytolytic T cell response. Liver sections were examined and scored for evidence of an inflammatory response by two independent procedures: standard microscopy with visual scoring, and automated scanning with computerized scoring. There was a close correlation between read-outs of liver histology by standard microscopy and automated scanning, using the index of mononuclear cellular infiltrations in hepatic portal tracts. Such infiltrates were prominent in the immunized mice, but, unexpectedly, the degree of infiltration was similar in mice injected with autoantigen (E3)-loaded syngeneic cells, or syngeneic cells treated only with hypertonic medium. The equivalent changes in the liver with the experimental and control protocol is indicative of the reactivity of the liver to any provocative immune stimulus, and is cautionary for protocols designed for the induction of autoimmune liver disease in experimental animals.

Experimental autoimmune hepatitis (EAH) is an animal model for autoimmune hepatitis. The disease is T cell-mediated and runs a subacute course, with maximal disease activity around week four after disease induction and a slow ensuing recovery. The aim of the present study was to investigate the immunoregulatory mechanisms that may account for recovery in EAH. It was found that T cell reactivity to liver antigens preceded histological disease, but at the peak of disease activity this T cell response was already suppressed. Active and antigen-specific suppression could be demonstrated, as irradiated splenocytes from animals at the beginning of recovery from EAH were able to suppress in vitro the T cell response to liver antigens by 42%. The response to an irrelevant antigen was suppressed by 16%, showing additional antigen non-specific suppression in vitro. The response to an in vivo immunization with an unrelated microbial antigen (tetanus toxoid) at the peak of disease was markedly reduced (by 90%). These data demonstrate in vitro and in vivo immunosuppression associated with the overcoming of and recovery from EAH. They stress the importance of immunoregulatory cycles in the control of autoimmune hepatitis.