This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.
In 2007, a review on the “current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies” was published (Hewitt et al. 2007). This review was the result of a symposium dedicated to the characterization and use of hepatocytes (organized by the “Medicon Valley Hepatocyte User Forum”) and written by presenters at the meeting. Since this time, there have been a number of advances in the understanding of hepatocyte functions, cell signaling, and mechanisms in liver toxicity, as well as culture techniques such as 3D cultures and co-culture with non-parenchymal cells (NPCs). More recently, great advances have also been made in the generation of hepatocyte alternative models from iPS cells, embryonic stem cells, etc. This prompted a number of original authors to collaborate with other experts in the fields of hepatocytes, NPCs, toxicology and drug metabolism to compile an update of research since 2007—there have been many developments, reflected quite magnificently in the size of this tome! Most sections include a list of key questions and “take home messages” so that the reader can select topics accordingly. The result is a comprehensive overview of “all that is hepatic,” from the structure of the liver to cell isolation tips (including a supplementary section with detailed protocols for the isolation and culture of human and rodent hepatocytes) and to mechanisms involved in hepatocyte differentiation and function, metabolism, disease and drug-related liver injury.
2 Structure and cellular components of the liver
2.1 Cell composition and organization
The cellular composition of the liver is summarized in Fig. 1a, b and Table S1 (see ESM). The liver lobule is the histologically well-defined structural unit of the liver (Fig. 2). A lobule has a hexagonal shape, a diameter of approximately 1 mm and the thickness is about 2 mm. In adults, the lobule consists of hepatocyte plates (aka cords), which radiate from a central vein in the center of the hexagon. Adjacent hepatocytes are joined by tight junctions. The tight junctions delimit the bile canaliculi with a diameter of about 1 μm. The hepatocytes in a plate are exposed on both sides to capillaries (sinusoids). The human liver contains about one million lobules. At each vertex of the hexagonal, lobule is a portal triad. A portal triad comprises an artery, a vein and a bile duct bundled by connective tissue. Liver metabolism, oxygenation and extracellular matrix (ECM) distribution are best understood by assuming that the portal triad is the center of symmetry instead of the central vein. Then, the hepatic acinus becomes the smallest liver functional unit and is defined as the population of hepatocytes supplied by one portal triad, i.e. a microcirculatory functional unit. The acinus extends over a roughly elliptical region comprising the hepatocytes from two adjacent lobules. The short axis of the ellipse is the line connecting two portal triads, the long axis connects two central veins (Fig. 2). The length of the long axis is approximately 1 mm. In the acinus area, the hepatocytes are exposed to a spatial biochemical gradient that influences metabolism and gene expression. The gradient is established by the changes in plasma composition and oxygenation occurring downstream to the blood flow in the space between the periportal and the perivenous areas (Jungermann and Kietzmann 1996, 1997; Kietzmann and Jungermann 1997). The blood from the portal vein supplies 80 % of the liver’s blood and contains nutrients that are absorbed from the digestive tract. From the periportal to the perivenous zone, the oxygen concentration drops from about 13 % v/v (equivalent to partial pressure of 60–65 mmHg and to a free concentration of 84–91 μmol/l, periportal) to 9 % v/v (mixed periportal) and finally to 4 % v/v (equivalent to a partial pressure of 30–35 mmHg and to a free concentration of 42–49 μmol/l, perivenous) (Allen and Bhatia 2003; Kietzmann et al. 2006). The oxygen gradient in the acinus regulates the spatial expression of genes encoding carbohydrate-metabolizing enzymes, including pyruvate carboxykinase 1 (mostly expressed in the periportal region), glucokinase and liver pyruvate kinase (both mostly expressed in the perivenous region), through oxygen-responsive transcription factors, such as NRE and HIFs (Kietzmann et al. 2006). This is the so-called metabolic zonation of the liver.
Compared to other organs, the liver is not particularly rich in ECM. Nevertheless, the ECM plays an important role in maintaining the differentiated phenotype of hepatocytes and NPCs (Martinez-Hernandez and Amenta 1993; Schuppan et al. 2001). Significant ECM alterations are observed in liver cirrhosis and fibrosis (Schuppan et al. 2001; Wells 2008a). The phenotypic changes induced by increasing the ECM stiffness are summarized in Table 1. As expected, isolated hepatocytes de-differentiate when cultured on hard 2D substrates that increase the ECM stiffness to favor a proliferative rather than differentiated cellular phenotype (Wells 2008a, b). The ECM composition roughly follows a gradient in the region comprised between the periportal and the perivenous areas (Table S2; see ESM). Basement membrane proteins (consisting of laminin, collagen type IV and perlecan) are mostly concentrated around the portal blood vessels and the larger venes. Here, the ECM composition is similar to that of other epithelial organs. By contrast, the basement membrane is absent in the parenchyma. The ECM in the parenchyma is located in the space of Dissé between the hepatocyte plates and the sinusoids (Fig. 3). Fibronectin and collagen I dominate in the parenchyma, with smaller amounts of collagen type III. The effect of the matrix components is striking in hepatic progenitor cells. Collagen I favors the differentiation of hepatic stem cells, while laminin maintains stemness (McClelland et al. 2008).
Hepatocytes take up substances destined for the bile, e.g. bile salts, via the basolateral membrane and secrete or excrete them across the canalicular membrane into the canaliculi, where they enter the biliary tree (Hofmann 2009). This functional polarity requires a strict partition of protein and lipid components in the different plasma membrane domains (Evans 1980; Coleman 1987; Wang and Boyer 2004). As a consequence, transport functions and transport proteins are expressed in a highly polar manner in hepatocytes (Meier 1988) (Fig. 4). To yield a domain-specific polar distribution of membrane proteins in the hepatocyte plasma membrane, the distribution of newly synthesized membrane proteins requires sorting processes. Hepatocyte basolateral proteins, as well as many of the canalicular proteins, after their biosynthesis in the endoplasmic reticulum, are targeted directly from the trans-Golgi network to the basolateral membrane, where canalicular proteins are subsequently endocytosed and transported to the apical domain by transcytosis (Bartles et al. 1987; Schell et al. 1992). This is different in columnar epithelial cells, where sorting occurs at the level of the trans-Golgi (Hubbard et al. 1989). By contrast, newly synthesized canalicular ABC transporters are directly targeted to the canalicular membrane (Sai et al. 1999; Kipp and Arias 2002). For Ntcp, the basolateral uptake transporter for conjugated bile salts, sorting to the basolateral membrane relies on (a) cytoplasmic sorting signal(s), as site-directed mutagenesis of Ntcp identified two tyrosine residues located in the cytoplasmic tail of Ntcp to be crucial for basolateral sorting of Ntcp (Sun et al. 2001). Studies investigating sorting of apical proteins identified a multiplicity of signals and mechanisms (Delacour and Jacob 2006; Weisz and Rodriguez-Boulan 1992), which are cell-type-specific. Interestingly, in hepatocyte cell lines, lipid rafts were shown to be involved in the transcytosis and direct apical trafficking of canalicular proteins (Nyasae et al. 2003; Slimane et al. 2003).
All the different functions of the liver are tightly linked to the complex assembly of highly specialized cell types organized in the sinusoidal unit embedding hepatocytes into a structural–functional organization, with the different NPCs of the liver, such as sinusoidal endothelial cells, hepatic stellate cells and liver macrophages (also termed as Kupffer cells). Hepatocytes are the major parenchymal cells carrying out most of the metabolic functions and account for the majority of the total liver cell population. The majority of circulating plasma proteins such as albumin, transporters, protease inhibitors, blood coagulation factors and modulators of immune complexes and inflammation is expressed by hepatocytes. They control the homeostasis of molecules such as glucose/glycogen, triglycerides, cholesterol, bile acids, and vitamins A and D and metabolize amino acids, metals and endogenous compounds such as heme and bilirubin. Ammonia detoxication and pH regulation need urea synthesis, that is performed by hepatocytes, so ammonia metabolism is often used as a functional marker of hepatic phenotype (Lippincott 1993; Saunders 1996; Michalopoulos 2007; Tanaka et al. 2011). Classic columnar epithelial cells are “leaning” with their basal membrane on the ECM and are facing with their apical or brush border membrane the external space. They are in addition connected to neighboring cells at their lateral membrane by tight junctions and desmosomes. In contrast, hepatocytes bear a unique topology: their apical domain (canalicular plasma membrane) is forming a tubular system by the connection of two adjacent hepatocytes by tight junctions. These tubuli form an anastomosing network, are called canaliculi and represent the smallest branches of the biliary tree (Jansen 2000). The basolateral domain of hepatocytes is formed by the sinusoidal and lateral plasma membrane. At the sinusoidal side, hepatocytes are directly in contact with blood plasma since the sinusoidal capillaries are fenestrated and surrounded by a discontinuous basal lamina. At the lateral membrane, hepatocytes are in contact with neighboring hepatocytes via desmosomes and gap junctions. This unique architecture allows the basolateral plasma membrane to mediate an intense solute exchange with blood plasma. Bile salts are mild detergents (Hofmann and Small 1967), and therefore, the canalicular membrane needs special biophysical properties and/or protective mechanisms to prevent it from being solubilized by the high concentrations of bile salts present in the canaliculus. Lipid composition of hepatocyte plasma membrane is specific for each domain. The rat canalicular liver plasma membrane contains about two times more cholesterol and total phospholipids and has about a two times higher sphingomyelin content than the basolateral rat liver plasma membrane (Meier et al. 1984). This enrichment of the canalicular plasma membrane in cholesterol and sphingomyelin is crucial for keeping its membrane integrity. For example, in vitro experiments showed that an increase in cholesterol content in phospholipid liposomes reduces bile salt induced membrane solubilization (Zhou et al. 2009). In addition, membrane microdomains or lipid rafts contain sphingomyelin and cholesterol in tightly packed, liquid-ordered state (Rajendran and Simons 2005). Indeed, recent studies demonstrated the presence of detergent and bile salt inducible microdomains in the canalicular membrane (Ismair et al. 2009; Guyot and Stieger 2011).
The non-parenchymal areas of the liver are mainly formed by endothelial cells (19 % of the total liver cell mass) (Kmiec 2001). The liver endothelial cells lining the sinusoids are uniquely specialized. They line the sinusoids and have large pores (fenestrae) with a diameter between 0.1 µm and 0.3 µm that allow a free flow of molecules (toxicants, nutrients, hormones, proteins and further plasma soluble components) from the plasma to the hepatocytes (Fig. 2). Since, in contrast to other organs, the liver endothelial cell sinusoids lack a basal lamina; the liver has no continuous barrier between epithelial cell surface and the plasma. The remaining major cell types populating the liver are stellate cells (6 %) (Kmiec 2001) and Kupffer cells (15 %). Liver sinusoidal endothelial cells (LSCEs) are not simply barrier cells that restrict access of blood-borne compounds to the parenchyma, they are functionally specialized cells that have complex roles and display some similarities to lymphatic endothelial cells, underscoring the view that the liver also displays features of a lymphatic organ. This includes not only receptor-mediated clearance of endotoxins, bacteria and other compounds, but also the regulation of inflammation, leukocyte recruitment and host immune responses to pathogens. Apart from being equipped with scavenger receptors that facilitate efficient uptake of potential antigens, sinusoidal endothelial cells also have the unique ability to function as antigen-presenting cells for T cells, which is considered to play a role for generating immunological tolerance (Limmer and Knolle 2001). Hepatic stellate cells in turn reside within the perisinusoidal space of Dissé that is lined by parenchymal cells and sinusoidal endothelial cells. Under physiological conditions, these cells are characterized as vitamin A–storing cells, displaying neuronal and neuroendocrine markers but also a variety of markers that characterize stem cells (Kordes et al. 2007; Kordes et al. 2008, 2009). Their recognition as the cellular source of myofibroblasts characterizing hepatic fibrosis has launched an era of astonishing progress in understanding the mechanistic basis of hepatic fibrosis progression and regression during chronic inflammatory diseases of the liver (Reeves and Friedman 2002; Atzori et al. 2009). This rather simple view of hepatic stellate cells as the major source of proliferative, contractile and fibrogenic cells has meanwhile been replaced by a remarkably broad spectrum of functions including stem cell–like features not only in liver injury, but also in regeneration (Kordes et al. 2009), intermediary metabolism and immunoregulation (Crispe 2009; Atzori et al. 2009). Liver macrophages are present in the microvessels of the sinusoids and under homeostatic conditions represent about 15 % of total liver cell population. The fact that the liver harbors almost 80–90 % of all tissue macrophages in the body (Bouwens et al. 1986), located in a strategic position for screening of pathogens, which enter the liver via the portal venous blood underscores the important role of the liver for systemic acute phase response and innate immunity. Apart from having vital homeostatic functions as a kind of “janitorial” cell responsible for the removal of cellular debris and clearance of exogenous material, macrophages are central to innate immunity with key functions in host defense against invading pathogens. Macrophages have a remarkable plasticity, enabling them to efficiently respond to environmental signals and modify their phenotype. They rapidly recognize potential danger from exogenous and endogenous sources and undergo activation, enabling them to launch biochemical attack and to involve hepatocytes and the other NPCs of the liver into the inflammatory process by releasing a variety of mediators including cytokines, chemokines, eicosanoids, proteolytic enzymes, reactive oxygen species (ROS) and nitric oxide; as they induce the expression of adhesion molecules and secrete chemotactic signals, liver macrophages are also involved in recruiting and retaining non-resident cellular players to the liver such as neutrophils, natural killer cells, and may further enlarge their own population by recruiting monocytes from circulation that subsequently differentiate into macrophages (Kolios et al. 2006). Thereby macrophages are not only important constituents of innate immunity but also relevant for regulation of liver regeneration and are critical regulators of hepatocyte function. Hence, they are considered to be the major source of mediators that control acute phase protein production in hepatocytes but also influence the metabolic and detoxifying capacity of hepatocytes. An in-depth understanding of the intercellular communication between hepatocytes and macrophages and the integration of macrophage-derived signals into hepatocyte function therefore is of high clinical and scientific relevance. A more detailed description of NPCs and their role in drug-induced liver injury (DILI) is reviewed in section “Non-parenchymal cells and their role in hepatotoxicity,” and in vitro models using macrophages are detailed in section “Co-cultures of hepatocytes and macrophages.”
2.2 Zonal heterogeneity of hepatocytes
Liver metabolism comprises an immense spectrum of interrelated anabolic and catabolic functions which are performed simultaneously without affecting each other or leading to futile cycles and other forms of wasting energy. In order to cope with this challenge, liver parenchyma shows a considerable heterogeneity and functional plasticity, known as “metabolic zonation” (Jungermann and Katz 1982; Gebhardt 1992). Different metabolic pathways are carried out in different zones and sometimes even single cell of the liver lobules, the smallest structural and functional units that can be discerned in liver sections. They appear mainly as hexagonal entities, but may also comprise pentagons as well as heptagons that are clearly defined by vascular elements (for comprehensive review of lobule structures in murine liver see Lamers et al. (1989). At their periphery, three adjacent lobules are grouped around a triangular structure of dense connective tissue, the Glisson trias, which contains the blood supply for conical sectors of all three adjacent lobules. Each Glisson trias contains two afferent vessels (the portal venule and the hepatic arteriole) as well as the bile ductule. In the center of the lobules, one efferent vessel, the hepatic venule or so-called central vein, is located that drains the blood from the lobule, i.e. from at least three different portal venules. According to their localization along the porto-central axis, hepatocytes are grouped into three different zones, the zone 1 (periportal), zone 2 (midzonal) and zone 3 (pericentral). This distinction is only semantic and does not reflect the real localization of any (marker) protein.
2.2.1 Key methods for investigating metabolic zonation
In the past, parenchymal heterogeneity has been extensively characterized with respect to the major metabolic pathways, namely carbohydrate, lipid, amino acid and drug metabolism. The most frequently used techniques for investigating the microdiversity of hepatocytes in liver parenchyma were immunocytochemistry or immunofluorescence and in situ hybridization which all provided a comprehensive overview about the exact lobular expression and localization of many enzymes of these metabolic pathways (reviewed by Meijer et al. 1990; Gebhardt 1992; Jungermann and Kietzmann 1996). For example, studies suggest that gluconeogenesis is present in all hepatocytes, but predominates in the periportal zone (Fig. 5). By contrast, glycolysis is most active in part of the pericentral zone, but generally shows a relatively low activity in hepatocytes. This distribution is dynamic and varies with feeding conditions. The zonation of other major metabolic pathways is schematically illustrated in Fig. 5. The immunochemical approach was also used for localizing hepatocytes involved in the synthesis of major serum proteins usually revealing shallow gradients in protein expression (Racine et al. 1995).
Studies on the functional consequences of this heterogeneity required other techniques. For example, strongly zonated hepatic ammonia metabolism was studied using isolated perfused livers performed in the antegrade (portal to central) and retrograde (central to portal) direction. This method also revealed other functions, such as intercellular glutamine cycling (Häussinger 1983) and bile salt transport (Groothuis et al. 1982). This technique was improved by including separate perfusion of both afferent vessels instead of only the portal vein (Comar et al. 2010) and provided new insight into to the influence of arterial blood in the regulation of ammonia elimination. A distinct and more versatile approach was the isolation of hepatocytes from different locations of the liver lobules. Various techniques were applied to achieve this goal. For instance, hepatocytes from different lobular zones were isolated according to their different size and density by centrifugal elutriation (Wilton et al. 1993; Botham et al. 1998; Romero et al. 1999). The most suitable separation technique leading to consistent results, the so-called digitonin–collagenase perfusion method developed independently by Quistorff (1985) and Lindros and Penttilä (1985), allows isolation of two distinct subpopulations of hepatocytes, one enriched in periportal and the other one enriched in pericentral cells. The major drawback of this ingenious isolation procedure is that only one of these subpopulations of hepatocytes can be obtained from a given liver. A general and reliable protocol of this technique was published by Gebhardt (1998). When the subpopulations are isolated from different livers (from either mice or rats), the periportal fraction amounted to 60–70 % of the hepatocytes and the pericentral to 30–40 %. Because of the inter-individual differences between the mice, the high yield for both subpopulations achieved with this technique is obtained at the expense of low comparability of the subpopulations. Therefore, another technique aiming at isolating periportal and pericentral hepatocytes from one and the same liver was developed (Tordjmann et al. 1997). However; the method is more demanding, it results in a lower cell yield, and has been successful only in rats so far. More recently, laser microdissection has proven an elegant technique for isolating cellular material from few hepatocytes located anywhere in the parenchyma including RNA samples from pericentral glutamine synthetase (GS)-expressing hepatocytes [Gebhardt, unpublished observation], but this technique does not allow the isolation of viable cells. The enrichment of periportal and pericentral hepatocytes in the isolated subpopulations is usually estimated by measuring the activities of several highly zonated enzymes such as glutamine synthetase, alanine aminotransferase and pyruvate kinase (Gebhardt and Mecke 1983; Burger et al. 1989). Since E-cadherin in the liver is present only in the periportal zone (~50 % of hepatocytes) (Ueberham et al. 2010), it can be used as a suitable marker for revealing the enrichment of periportal cells by immunocytochemical staining.
The extensive use of the digitonin–collagenase perfusion technique has provided a detailed picture of metabolic zonation. In particular, a microarray study based on the comparison of periportal and pericentral hepatocytes considerably improved our knowledge of zonation (Braeuning et al. 2006). Thus, this study provided for the first time a many-facetted picture of the subtle differences in zonation of enzymes involved in xenobiotic metabolism. While most enzymes show pericentral dominance, a small number of these enzymes exhibit preferentially periportal expression. Another remarkable paper shows a very detailed zonal distribution of enzymes involved in heme synthesis (Braeuning and Schwarz 2010a). Further contributions concern the refinement of knowledge on zonation of amino acid metabolism (Braeuning et al. 2006) and iron metabolism (Troadec et al. 2008). Despite these advances in understanding metabolic zonation, it is important to note that separation of merely two subpopulations is not sufficient to elucidate all different aspects of hepatocyte heterogeneity. For instance, a recent proteomic study in adenomatous polyposis coli KO mice provided evidence that induced GS-expressing hepatocytes are characterized by an unexpectedly low amount of glycolytic enzymes and a downregulation of many components of mitochondrial oxidative phosphorylation (Vasilj et al. 2012). It is likely, though not yet proven, that the normal GS-expressing hepatocytes residing close to the central vein in up to three cell layers, exhibit the same features (Fig. 5).
2.2.2 Factors determining metabolic zonation
Since its discovery, parenchymal heterogeneity has raised the question of how it is determined by regulatory factors. Alhough they play an important role in the determination of local cell function, hormones and metabolic signals were found not to act as primary cues of metabolic zonation (Gebhardt and Gaunitz 1997; Jungermann and Kietzmann 2000). After several decades of intensive but slow-moving investigations, it became apparent that Wnt/β-catenin signaling is a master regulator of liver zonation (Loeppen et al. 2002; Benhamouche et al. 2006). Knockout studies of β-catenin, on the one hand, resulting in interruption of the pathway, and of APC, on the other hand, resulting in its over-activation revealed that Wnt/β-catenin signaling acts in a gradient-like manner with increasing activity from the periportal to the pericentral zone (reviewed in Gebhardt and Hovhannisyan 2010). Even though the origin of this gradient and other details of Wnt pathway function remain unknown, the mystery of liver zonation seems essentially solved. For the first time, it was shown that a morphogen may determine the function of a differentiated cell according to its spatial location within a specific tissue, termed “post-differentiation patterning” (Gebhardt and Hovhannisyan 2010).
In addition to Wnt/β-catenin signaling, it was speculated that Ha-ras-dependent signaling participates in determining zonal differences in gene expression (Hailfinger et al. 2006). However, this assumption is based mainly on comparisons of mRNA and protein expression patterns of periportal and pericentral hepatocytes with those of liver tumors containing different types of mutations in signaling pathways and, thus, is not completely convincing (Gebhardt and Ueberham 2006), since tumor signaling usually shows multiple deviations from the normal counterpart. Nonetheless, there is independent evidence that other morphogens cooperate with Wnt/β-catenin signaling in specifying liver zonation and that epidermal growth hormone (EGF)-induced Ha-ras-dependent signaling may be one of these (Braeuning et al. 2007a). Up until now, however, it remains to be elucidated how other morphogens aid in specifying the zonal heterogeneity of hepatocytes.
2.3 Non-parenchymal cells and their role in hepatotoxicity
The major cell type of the liver is the hepatocyte, a parenchymal cell, which makes up to 80 % of the entire liver mass and performs the majority of the liver functions (Kmiec 2001; Lippincott 1993; Saunders 1996; Michalopoulos 2007; Tanaka et al. 2011). The other 20 % of the liver mass are comprised of NPCs such as stellate cells of the connective tissue, endothelial cells of the sinusoids, Kupffer cells functioning as in situ macrophages and immune cells, such as lymphocytes (T cells, B cells, natural killer (NK) and especially NKt cells) and leukocytes (neutrophils, monocytes and dendritic cells) (Taub 2004; Tacke et al. 2009). Most of the current activities in developing in vitro test system for hepatotoxicity focus on the parenchymal cell, the hepatocyte itself. Great efforts are made to establish conditions to maintain in vivo activities of primary hepatocytes. Moreover, large research programs have been initiated to differentiate human hepatocyte-like cells from stem or precursor cells (Fletcher et al. 2008; Agarwal et al. 2008; Cai et al. 2007). However, recent studies have provided evidence that, upon an initial damage to hepatocytes, a secondary response occurs that involves several types of NPC or immune cells and may dramatically aggravate the initial damage (Liu et al. 2004, 2006a; Ochi et al. 2004), The main cell types involved in hepatotoxin-induced liver damage are shown in Table 2. It is thus questionable whether hepatotoxicity can be sufficiently predicted in vitro by analyzing only one cell type, i.e. the parenchymal cell. For example, many hepatotoxic compounds, e.g. methapyrilene, thioacetamide, piperonyl-butoxide (Ellinger-Ziegelbauer et al. 2008), do not, or only at extremely high concentrations, kill hepatocytes in vitro, which might be explained by the lack of a “second hit,” perhaps inflammatory cells, absent in in vitro systems that use hepatocytes alone. Although there is a wealth of information on the functional properties of NPCs in different pathological context, their precise contribution to hepatotoxicity has only recently been investigated. These seminal studies have generated great interest in the scientific community and in some cases also have raised important questions that challenge the validity of the experimental approaches. Evidently, a definite role for each NPC cannot be drawn based on a single methodology. Nevertheless, these studies have succeeded in setting up the stage for more refined investigations. It is critical to understand the communication between NPC cell types and hepatocytes and how this contributes to hepatotoxicity. These hepatocyte–NPC interactions would gain even further relevance if their degree depends on physicochemical properties of the compounds. Relatively little is known in this field (Rubbia-Brandt et al. 2004; DeLeve 1996; Wang et al. 2000); however, one example demonstrating a compound-specific effect is vinyl chloride. Like many other compounds, it is metabolically activated in hepatocytes and is hepatotoxic. However, a long-term effect of vinyl chloride is not only hepatocellular cancer but it also causes a very rare tumor of the liver, hemangiosarcoma, which arises from LSECs (Cohen et al. 2009). This “communication” between hepatocytes and LSECs is very specific for vinyl chloride and not observed for many other genotoxic compounds activated by hepatocytes (Cohen et al. 2009).
In the following section, the characteristics and transporter function of a number of NPCs and their contribution to hepatotoxicity with a particular focus on acetaminophen are reviewed. Acetaminophen-induced liver damage is perhaps the best understood model of drug-induced liver injury. Hence, it is not surprising that most studies on the role of NPCs in hepatotoxicity are based on acetaminophen intoxication. Acetaminophen induces direct cell death with features of apoptosis and necrosis (Cover et al. 2005). It is well established that necrotic cells release strong pro-inflammatory molecules such as DNA and high mobility group box protein-1 (HMGBP1) (Jaeschke et al. 2012b). Thus, it is very likely that this early necrotic cells trigger an inflammatory response (Kono and Rock 2008). Another indication for the involvement of NPCs in acetaminophen toxicity is the finding that precision-cut liver slices, that contain all the liver cell types, are more sensitive than isolated hepatocytes for acetaminophen cell death (Hadi et al. 2013). The role of NPCs in immune-mediated hepatotoxicity is described in detail in section “Immune-mediated iDILI.”
2.3.1 Liver sinusoidal endothelial cells
Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells characterized by fenestrations and the lack of a basement membrane (Wisse et al. 1996; Iwakiri and Groszmann 2007). This vascular endothelium provides more than just a physical barrier for blood circulation. It actively participates in inflammatory reactions by several mechanisms, including (1) detection of pathogen-associated molecular patterns (PAMPs, e.g. lipopolysaccharide, lipoteichoic acid (LTA), N-acetyl muramyl peptide (NAM)) or damage-associated molecular patterns (DAMPs, e.g. DNA), (2) secretion of cytokines and chemokines to recruit and activate leukocytes and (3) expressing adhesion molecules that favor the attachment and extravasation of leukocytes to the site of injury (Pober and Sessa 2007). LSECs have unique properties. They possess a strong scavenging capacity, which mediates the uptake of several waste macromolecules such as hyaluronic acid, collagen α-chains and modified low-density lipoproteins (LDL) (Li et al. 2011; McCourt et al. 1999; Malovic et al. 2007).
As described above, LSECs contain numerous fenestrae (Elvevold et al. 2008) that allow passage of proteins and large macromolecules (e.g. lipoproteins). In acute liver damage, LSEC suffer structural alterations that can promote inflammation. Electron microscopy revealed that within 2 h of acetaminophen intoxication in mice, LSECs exhibited many gaps throughout the cytoplasm that were formed by destruction and/or coalescence of fenestrae (McCuskey et al. 2005; Ito et al. 2003a). This effect was observed both in isolated LSECs and LSECs in liver tissue. Moreover, the gaps formed through LSECs permitted the passage of erythrocytes to the space of Dissé, indicative of hemorrhage and collapse of the sinusoidal wall (McCuskey et al. 2005).
LSECs express Toll-like receptors (TLRs) that detect bacteria or self-damage debris and trigger signal transduction pathways that promote inflammation (Wu et al. 2009). Upon acute intoxication, damaged hepatocytes release intracellular molecules which can activate TLRs in LSECs, including heat shock proteins (Hsp) and fragmented DNA rich in cytidinephosphate-guanosine (CpG-DNA) (Jaeschke et al. 2012a, b). LSECs express TLR9, and can efficiently recognize CpG-DNA in vitro and in vivo, as demonstrated by the uptake of FITC-labeled CpG-DNA either added to culture medium or injected intravenously in vivo (Martin-Armas et al. 2006). In addition to TLR9, LSECs express the adaptor molecule, MyD88, which mediates signal transduction pathways from activated TLR. Indeed, uptake of CpG-DNA into LSECs led to activation of the NF-kappaB signaling pathway, as indicated by nuclear localization of phosphorylated NF-kappaB (Martin-Armas et al. 2006). Furthermore, culture of LSECs in the presence of CpG-DNA enhanced the secretion of interleukin (IL)-1β (20 % over control) and IL-6 (40 % over control) into the medium (Martin-Armas et al. 2006). LSECs express adhesion molecules that are important for leukocyte attachment and further extravasation to the site of injury, namely intercellular adhesion molecule-1 (ICAM-1). Under conditions of liver damage by CCl4, the expression of ICAM-1 increases with a peak 24 h after injection (Neubauer et al. 2000). This may have important consequences in terms of tissue damage, since engagement of ICAM-1 by its receptor Mac-1 in neutrophils causes their degranulation and extensive oxidative stress (Jaeschke 2003; Shappell et al. 1990).
Recently, a study based on 3D tissue reconstruction and mathematical modeling has demonstrated that LSECs play a key role in the establishment of functional tissue structure (Hoehme et al. 2010). After cell division, hepatocytes orient themselves in the direction of the closest sinusoid, a process named “hepatocyte-sinusoid alignment” (HSA), which is essential for the restoration of liver microarchitecture. The importance of LSECs for liver regeneration is also illustrated by the fact that many hepatotoxic compounds that require metabolic activation by cytochrome P450 enzymes (CYPs) (e.g. acetaminophen and CCl4) kill almost all hepatocytes in the center of the liver lobules, because the relevant CYPs are mostly expressed in this pericentral region. While most hepatocytes are killed, a substantial fraction of LSECs survive and seem to be sufficient to serve as “guide rails” for regenerating hepatocytes that migrate from the outer surviving hepatocyte fraction into the inner dead cell mass (Hoehme et al. 2010). Besides their role in establishing functional liver microarchitecture, LSECs also coordinate hepatocyte proliferation during liver regeneration (Ding et al. 2010). Using a zoo of knockout mice, it has been demonstrated that LSEC-derived factors, particularly HGF and Wnt2, play a critical role in regenerative LSEC–hepatocyte communication.
2.3.2 Kupffer cells
Kupffer cells represent resting tissue macrophages which upon liver damage synthesize and secrete the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and IL-1β (Roberts et al. 2007). Both cytokines can potentially cause hepatocyte killing by activation of signal transduction pathways that lead to apoptosis, such as p38, JNK and generation of ROS (Wajant et al. 2003). In addition, TNFα from Kupffer cells was reported to activate LSECs leading to deposition of fibrin in liver tissue which may cause ischemia and hypoxia (Roberts et al. 2007). Furthermore, Kupffer cell–secreted cytokines may attract and activate immune cells which in certain cases can exacerbate the initial damage (Roberts et al. 2007). Hence, they can potentially have a damaging role in acetaminophen toxicity. In early studies, their contribution to hepatotoxicity was assessed by treating acetaminophen-intoxicated mice with gadolinium chloride (GdCl3), a compound that inhibits phagocytic activity and generation of ROS in macrophages (Lee et al. 2004), and depletes macrophages in the periportal zone of the liver (Hardonk et al. 1992). Mice and rats injected with GdCl3 showed reduced liver damage after acetaminophen intoxication (Michael et al. 1999), supporting the concept of a harmful role of Kupffer cells in acetaminophen-induced hepatotoxicity (Michael et al. 1999; Laskin et al. 1995). However, recent reports suggest that these results are questionable. In these studies, Kupffer cell depletion was achieved by clodronate liposomes injection, a technique that efficiently depletes all resident macrophages from liver and spleen (van Rooijen 1989; van Rooijen and Sanders 1997). As expected, Kupffer cell depletion abrogated both TNFα and IL-1β induction (Ju et al. 2002; Campion et al. 2008), which would be expected to reduce the extent of acetaminophen toxicity. However, contrary to the GdCl3 reports, clodronate Kupffer cell depletion enhanced hepatotoxicity induced by acetaminophen (Ju et al. 2002; Campion et al. 2008). The mechanism for the protective effect of Kupffer cells was explained in part by a Kupffer cell–dependent induction of hepatic transporters, particularly Mrp4, which may be interpreted as a defense against exposure to toxic compounds (Campion et al. 2008). An alternative (or complementary) mechanism for the protective role of Kupffer cells might depend on the expression of the anti-inflammatory cytokine, IL-10. Indeed, IL-10 is strongly induced 72 h after acetaminophen intoxication, and this was completely abrogated by depletion of Kupffer cells (Ju et al. 2002; Campion et al. 2008). Of note, IL-10 was shown to protect against acetaminophen toxicity by downregulation of iNOS and peroxynitrite formation (Bourdi et al. 2002). The rather surprising fact that enhanced toxicity occurred in spite of downregulation of TNFα can be explained by studies showing that TNFα per se is not strongly hepatotoxic, as demonstrated by the observed low degree of liver damage in mice injected with TNFα (Beraza et al. 2007). Subsequent studies using GdCl3 found very little or no protection at all to acetaminophen-induced hepatotoxicity (Ito et al. 2003b; Knight and Jaeschke 2004; Ju et al. 2002). Thus, it seems that Kupffer cells are responsible for hepatoprotective responses mediated in part by induction of export pumps in hepatocytes and by secretion of anti-inflammatory cytokines.
The role of infiltrating macrophages in liver toxicity is controversial, largely due to the heterogeneity and plasticity of macrophages and difficulties in establishing effective markers to identify and study different populations of macrophages in the liver. As for Kupffer cells, it has been described that macrophages can contribute to acetaminophen-induced hepatotoxicity by producing pro-inflammatory cytokines such as TNFα and IL-1β (Goldin et al. 1996). However, macrophages can also secrete IL-10-, TGF-β- and IL-18-binding proteins, which are anti-inflammatory cytokines (Gordon 2003). In addition, in recent years, enormous progress has been achieved in the identification of different macrophage subtypes by flow cytometry, a technique that allows quantitative detection of surface antigens that reflect different macrophage populations (Geissmann et al. 2003, 2010). A current phenotypic definition of macrophages is based on their pro- or anti-inflammatory properties, which depends on the cytokine cocktail they secrete: M1 or classically activated macrophages are induced by lipopolysaccharide and Th1 cytokines (e.g. IFNγ, IL-1); M2 or alternatively activated macrophages are induced by apoptotic bodies or Th2 cytokines (e.g. IL-4, IL-10 and IL-13) (Gordon 2003; Geissmann et al. 2003, 2010). M1 macrophages release TNFα, IL-8, RANTES and IL-1β that promote the inflammatory process, whereas M2 macrophages secrete IL-1ra, TGF-β, IL-10 and PGE2, cytokines that repress inflammation and contribute to the regeneration process (Geissmann et al. 2003; Fadok et al. 1998). An elegant study by Holt et al. (2008) identified two different macrophage populations in rodent liver that reflected resident macrophages (Kupffer cells) and infiltrating macrophages (IM) based on flow cytometric analysis with two widely used markers for these cells, that is CD11b and F4/80 (Holt et al. 2008). Resident macrophages were CD11blow-F4/80high while infiltrating macrophages were CD11bhigh-F4/80low. Under control conditions, the majority of liver non-parenchymal cells were Kupffer cells (75 %), while the IM population was almost absent. However, upon acetaminophen intoxication, there was a strong and transient increase in IM that peaked at 48 h after intoxication (35 % of total liver non-parenchymal cells), while Kupffer cells followed an opposite trend, with a transient decrease that peaked also at 48 h (16 % of total liver non-parenchymal cells) (Holt et al. 2008). PCR analysis indicated that IM expressed markers that are characteristic of M2–alternatively activated macrophages (e.g. Ym1, Fizz1, Arg-1). These markers were not expressed in isolated Kupffer cells. Furthermore, IM expressed CCR2, a receptor for the macrophage-specific chemokine Mcp-1. In agreement with a role of the Mcp-1/CCR2 axis in recruitment of circulating monocytes, the IM population was completely absent in acetaminophen-treated CCR2 knockout mice. The role of IM in acetaminophen hepatotoxicity was determined by specifically depleting circulating monocytes, the precursors of infiltrating macrophages, by bone marrow irradiation 3 days prior to acetaminophen intoxication (Holt et al. 2008). This procedure only depleted the IM population (CD11bhigh-F4/80low) without affecting the KC population (CD11blow-F4/80high). Ablation of IM had a similar effect as depletion of Kupffer cells, which is a delayed recovery after acetaminophen intoxication (Holt et al. 2008). The mechanism by which IM promote the wound healing process during hepatotoxicity might depend on the secretion of cytokines that repress inflammation and contribute to the regeneration (IL-1ra, TGF-β, IL-10) (Geissmann et al. 2003; Fadok et al. 1998), but also in their ability to promote apoptosis of neutrophils. Indeed, IM from acetaminophen-treated mice induced apoptosis of mouse neutrophils in direct co-cultures (Holt et al. 2008). This effect depended on direct contact between the two cells, since IM-induced neutrophil apoptosis was almost completely abrogated when these cells were in trans-well culture dishes (Holt et al. 2008). In conclusion, the role of IM seems to be in promoting the regenerative response that follows acute liver damage, by secreting anti-inflammatory cytokines and by killing and eliminating infiltrating neutrophils at the site of injury.
Macrophages phagocytic activity as well as cytokine production can be modulated by bile acids (Calmus et al. 1992; Funaoka et al. 1999; Minter et al. 2005; Scott-Conner and Grogan 1994; Sung and Go 1999; Graf and Bode 2012). The influence of bile acids on the immune response has been reviewed recently (Fiorucci et al. 2010; Graf and Bode 2012). FXR, PXR and VDR as well as the TGR5 are expressed in peripheral blood mononuclear cells, macrophages and Kupffer cells (Fiorucci et al. 2010; Graf and Bode 2012; Kawamata et al. 2003; Keitel et al. 2008b; Schote et al. 2007). Treatment of isolated human mononuclear cells with the FXR agonist obeticholic acid (INT-747) decreased TNFα secretion and prevented differentiation of CD14+ monocytes into dendritic cells (Gadaleta et al. 2011). Because PXR activation suppresses NF-kappaB transcriptional activity in hepatocytes, a similar mechanism may apply to Kupffer cells (Hu and Li 2010; Zhou et al. 2006; Fiorucci et al. 2010). TGR5 mRNA and protein expression has been detected in CD14+ monocytes of the peripheral blood as well as in macrophages of lung, liver (Kupffer cells) and intestine (Wang et al. 2011; Keitel et al. 2008a, b; Kawamata et al. 2003). Stimulation of TGR5 in Kupffer cells by bile acids or specific agonists suppressed the lipopolysaccharide-induced mRNA expression of inflammatory cytokines such as IL-1β, TNFα, IL-6 and monocyte chemoattractant protein-1 (MCP-1) (Pols et al. 2011; Wang et al. 2011; Keitel et al. 2008a, b). TGR5 activation reduced the phosphorylation of IκBα, thereby preventing nuclear translocation of p65 and inhibiting NF-kappaB transcriptional activity (Pols et al. 2011). Thus, bile acids suppress NF-kappaB target gene expression through activation of both NRs and TGR5. The differential expression of nuclear and membrane-bound bile acid receptors in parenchymal cells and liver NPCs enables a cell-type and bile-acid-specific bile acid signaling in this organ.
Neutrophils, the most abundant leukocytes in the blood, are professional phagocytes which are quickly recruited to sites of inflammation (e.g. bacterial infection) (Mantovani et al. 2011), where they release proteolytic enzymes stored in their granules and generate ROS (Mantovani et al. 2011). The signals triggering their recruitment are also present in sterile inflammation such as tissue injury by chemicals or trauma (McDonald et al. 2010; Rock et al. 2011). It is well established that acetaminophen toxicity induces a strong recruitment of neutrophils into the liver (Liu et al. 2004, 2006a; Williams et al. 2010; Cover et al. 2006). Yet, the role of these leukocytes in this context is highly controversial (Liu et al. 2004; Jaeschke et al. 2012b). Independent studies demonstrated that depletion of neutrophils in mice with antibodies directed against the epitope, Gr-1, dramatically decreased acetaminophen-induced liver damage (Liu et al. 2006a; Ishida et al. 2006). The increase in serum ALT was less than 50 % after neutrophil depletion (Liu et al. 2006a). Also the dead cell area in the lobules center was decreased by more than 50 %. Moreover, also survival of the mice was improved (Liu et al. 2006a). A proposed explanation for the role of neutrophils is the release of cytotoxic hypochlorous acid and chloramines from their granules. These cells can also release serine proteases that contribute to hepatocyte killing (Ramaiah and Jaeschke 2007). However, increasing evidence reveals highly controversial aspects of the neutropenia-inducing antibody approach, indicating that it induces a protective pre-conditioning in the liver. Kupffer cells actively removing antibody-tagged neutrophils become activated (Bautista et al. 1994; Jaeschke and Liu 2007). This also causes a stress response in hepatocytes inducing the expression of protective genes like metallothionein, heme oxygenase and others (Jaeschke and Liu 2007). Consistent with this, application of the neutropenia-inducing antibodies after acetaminophen injection but before the onset of injury was not protective (Cover et al. 2006). Furthermore, neutrophils recruited into the liver after acetaminophen intoxication are not activated, as indicated by their low ROS production upon phorbol ester (PMA) stimulation (Williams et al. 2010). Finally, mice deficient in ICAM-1 (Cover et al. 2006), CD18 (Williams et al. 2010) and NADPH oxidase (James et al. 2003) are not protected against acetaminophen toxicity. Altogether, in spite of initial reports on neutrophil-mediated acetaminophen hepatotoxicity, cumulative evidence strongly argues against this hypothesis. Nevertheless, it is important to consider that under certain conditions, neutrophils may induce an inflammation response that rather aggravates than repairs the injured tissue (Jungermann and Kietzmann 2000). Neutrophil-mediated toxicity has been extensively documented in ischemia/reperfusion, endotoxic shock and cholestasis-induced liver damage (Jaeschke 2003; Jaeschke and Hasegawa