Structure, Function and Responses to Injury

Portal circulation

The portal tract derives its name from the ramifications of the portal venous system through the liver. The portal system also provides the tracts along which the hepatic artery system and biliary tree travel, giving rise to the alternate term ‘portal triad’. However, as the terminal portal tracts reach their own terminus, the portal vein system ends, ironically leaving a final portal ‘dyad’ lacking a terminal portal vein but containing hepatic artery–bile duct pairs before these also end after a short distance. In the periphery of the normal adult liver sampled by percutaneous liver biopsy, portal ‘triads’ containing portal vein, hepatic artery and bile duct profiles constitute only 70% of portal tracts. Careful histological examination also reveals an additional 30% of diminutive ‘portal dyads’ containing only hepatic artery and bile duct profiles. Lastly, the portal system ramifies through approximately 17–20 orders of branches to supply the entire corpus of the liver. The subdivisions are not strictly dichotomous, however, in that one branch may ultimately have fewer subbranches than another. Thus the liver ultimately has an irregular lobular organization at the microscopic level, consistent with the observations from 3D reconstruction.

Beginning at the porta hepatis, the portal vein divides into successive generations of distributing or conducting veins, so called because they do not directly give off inlet venules. The vein diameters remain in the macroscopic range, introducing negligible vascular resistance. According to their diminishing position in the hierarchy of branching, they may be classified as interlobar , segmental and interlobular . The portal venous system branches in a nondichotomous manner, such that smaller portal tracts may branch off conducting portal tracts without the latter losing their conducting capability.

These further branches of the portal system are those that produce the smallest portal veins, which do distribute their blood into the sinusoids. These succeeding branches are (1) the preterminal portal venules, microscopically found in portal tracts of triangular cross-section, and (2) the terminal portal venules, which taper to about 20–30 µm in diameter and are surrounded by scanty connective tissue in portal tracts of circular rather than triangular cross section. As denoted in Fig. 1.6 , from both the preterminal and terminal portal venules arise side branches, designated inlet venules , which have an endothelial lining with a basement membrane and scanty adventitial fibrous connective tissue but no smooth muscle in their walls. They pass through the periportal connective tissue sheath and enter into the parenchyma to open into the sinusoids. Although these inlets were reported to be guarded by sphincters composed of sinusoidal lining cells—the afferent or inlet ­sphincters, this concept has not been supported in more recent years. Some early branching of the smallest conducting veins may produce more than one portal vein within some portal tracts, and these supply only those sinusoids which abut the portal tract.

According to the anatomical studies of Matsumoto et al. ^, describing the primary hepatic lobule, the conducting portal system and the terminal portal branches can both give rise directly to penetrating inlet venules. The inflow-front for portal venous blood thus has a sickle shape. This is at variance with the concept of the acinus, in which portal blood flow exhibits radial symmetry only around the inlet venule arising from terminal portal vein branches. Moreover, the inlet venules do not run the entire length of the distance between two portal tracts, but rather taper off into a sinusoidal configuration near the middle of the interportal distance. In other words, the vascular watershed areas are not located at the nodal points of Mall (see Fig. 1.6 ), but at the midpoint of the interportal distance (midseptum M of Zou et al. ). Zonal gradients of metabolic activities and of enzyme histochemical staining patterns also conform to the sickle-zone pattern, not with the acinus diagram. The sickle-zone pattern has been confirmed by others. It is also important to note that the septal venules are not orthogonal to the portal tract, but in three dimensions follow a somewhat arcuate course into the parenchyma.

The importance of keeping this more sophisticated anatomical perspective becomes clear when examining the damaged liver. For example, according to the acinar concept, with severe hepatic congestion involving destruction of hepatocytes in acinar zone 3 and potentially in zone 2, the surviving parenchyma of acinar zone 1 would appear clover shaped, with the portal tract at its centre, but still damaged. However, the primary hepatic lobular concept predicts that immediate periportal damage does not actually occur in vascular insufficiency or chronic venous congestion. Instead, the ischaemic hepatic damage occurs in sickle-shaped zones standing in relief with preserved viable parenchyma.

Arterial circulation

The hepatic artery supplies branches throughout the portal tract system. In the largest interlobar portal tracts, there are typically four hepatic arteries accompanying the interlobar bile ducts. The hepatic artery/bile duct ratio drops until, in the most distal portal tracts, hepatic arteries are paired in close proximity with interlobular bile ducts in a 1:1 ratio. Even at this level, there are typically two hepatic artery–bile duct pairs per portal vein in the peripheral portal tracts sampled by percutaneous liver biopsy.

The terminal blood flow of the arteries is by three routes: into a plexus around the portal vein, into a plexus around a bile duct, and into terminal hepatic arterioles. The perivenous plexus is characteristically distributed around interlobar, segmental and interlobular portal vein branches within the portal area; it drains into hepatic sinusoids. Arterioportal anastomoses between perivenous arterioles and terminal portal venules can be observed in mammalian liver, but the frequency of these in normal human livers is uncertain. ^, By the level of the terminal portal veins, the arteriolar plexus is absent.

A peribiliary arterial-derived plexus supplies all the intrahepatic bile ducts. Around the larger ducts the peribiliary plexus is two layered, with a rich inner, subepithelial layer of fine capillaries and an outer, periductular venous network which receives blood from the inner layer. The smallest, terminal bile ducts have only a single layer of fine capillaries. Ultrastructural studies have shown that the capillaries are lined by fenestrated endothelium. The peribiliary plexus drains principally into hepatic sinusoids. The peribiliary plexus develops in parallel with the development of the intrahepatic bile ducts, spreading from the hepatic hilum to the peripheral area of the liver and becoming fully developed with the full maturation of the biliary system. In addition to providing the vascular supply to bile ducts, the peribiliary vascular plexus is thought to be involved in the reabsorption of bile constituents (including bile acids) taken up apically by bile duct epithelial cells and secreted across their basal plasma membrane into the portal tract interstitium, constituting a ‘cholehepatic’ circulation. The cholehepatic reuptake of biliary substances may play a key adaptive role during times of downstream bile duct obstruction, because these solutes may be ‘dumped’ into the systemic circulation for disposal by the kidneys. Conversely, cholehepatic circulation of medications such as sulindac may compete with normal biliary secretion of bile acids, thereby inducing functional cholestasis.

Terminal hepatic arterioles have an internal elastic lamina and a layer of smooth muscle cells, and they open into periportal sinusoids via arteriosinus twigs. Although some mammalian species exhibit hepatic arterioles that penetrate deeply into the parenchyma before entering sinusoids near to the hepatic veins, reports of such penetrating arterioles in humans have been disputed. Regardless, isolated parenchymal arterioles may sometimes be seen in liver biopsies. Ekataksin has suggested that these vessels supply isolated vascular beds in the parenchyma. Perhaps more importantly, isolated arteries that traverse the hepatic parenchyma to feed the hepatic capsule and hepatic veins may play a principal role in the development of subcapsular haemorrhage and in arterial collateral formation following transcatheter arterial chemoembolization for liver cancers.

Venous drainage

Having perfused the parenchyma through the sinusoids, blood enters the terminal hepatic venules (‘central veins’ of classic lobules). Scanning electron microscopy (SEM) has clearly demonstrated in the walls of terminal hepatic veins the fenestrations through which the sinusoids open, and the astute observer can see these sinusoidal openings into terminal hepatic veins in histological sections. The terminal vein branches drain into sublobular veins, which in turn form larger hepatic vein branches, whose macroanatomy is described earlier. The venous anatomy does not strictly parallel the distribution of the portal system, because hepatic veins traverse between portal system-defined lobules as the venous system exits the liver. This is understandable; ultimately the hepatic veins need to exit through the dorsum of the liver, whereas the portal system enters the liver ventrally. The hepatic venous system also does not ramify as extensively as the portal system, so there is a slight preponderance of terminal portal tracts to terminal hepatic veins throughout the liver.

Plasma membrane

As with other epithelial cells, the hepatocyte is highly polarized ( Fig. 1.8 ). Within its plasma membrane, three specialized regions, or domains, are recognized: sinusoidal , which faces the sinusoid and the perisinusoidal space; lateral , facing the intercellular space between hepatocytes; and canalicular , bounding that specific part of the intercellular space constituting the bile canaliculus. ^, Using a more generic terminology, the sinusoidal and lateral domains constitute the basolateral plasma membrane of the hepatocyte, and the canalicular domain is the apical domain. This polarity is largely maintained by the tight junctions formed between adjacent hepatocytes, which delineate the basolateral domain from the canalicular domain and create a barrier between fluid in the intercellular space and bile in the canaliculus. ^, In addition to tight junctions, there are also gap junctions and desmosomes in the lateral domain, and it is across lateral-domain gap junctions that intercellular communication takes place. Stereological studies in the rat have shown that the basolateral, canalicular and lateral domains constitute approximately 70%, 15% and 15%, respectively, of the total cell surface area.

The domains of the hepatocyte plasma membrane are not simply topographical entities. They are specialized to serve different basolateral and apical functions of the hepatocyte. Molecular differences include composition of the lipids in the plasma membrane, the complement of membrane proteins, function of endocytic and exocytic compartments and relationship to the cytoskeleton. Beyond direct regulation of membrane protein function, hepatocytes can dynamically regulate actual membrane lipid content and the concentration of specific proteins in each domain, providing a powerful additional mechanism for functional control of plasma membrane function. ^,

Canalicular domain

The ‘bile canaliculus’ is defined as an intercellular space formed by the apposition of the edges of gutter-like hemicanals delimited by tight junctions, on the facing surfaces of adjacent hepatocytes ( Fig. 1.9A, B ). Canalicular diameter varies from 0.5 to 1.0 µm in the perivenular area and from 1 to 2.5 µm in the periportal zone, in accordance with flow of bile from the centrilobular region of the lobule toward the portal tract. The canalicular surface is unevenly covered by microvilli, which are more abundant along a ‘marginal ridge’ at each edge of the hemicanaliculus. In experimental biliary obstruction the canaliculi become dilated and the microvilli disappear, except along the marginal ridges. Intracellular subapical microfilaments are concentrated around the canaliculi, forming distinct, organelle-free pericanalicular sheaths and extending into the microvilli. This pericanalicular microfilament web is contractile, enabling hepatocytes to propel secreted bile along the canalicular channel. The presence of contractile elements in the pericanalicular zone can be demonstrated by indirect immunofluorescence ( Fig. 1.9C ). The presence of adenosine triphosphatase (ATPase) can be demonstrated histochemically; CD10 is a reliable immunostain for identifying the bile canaliculus ( Fig. 1.9D ). Brown-tinted accumulations of lipofuscin may also outline the canalicular pole of the hepatocyte, reflecting the presence of pericanalicular lysosomes, more evident in the adult liver because of the aging process. When present, lysosomal lipofuscin deposits are more prominent in perivenular hepatocytes.

The canalicular surface is isolated from the rest of the intercellular surface by junctional complexes (see Fig. 1.9A ): desmosomes, intermediate junctions, tight junctions and gap junctions. The ‘tight junctions’ constitute a permeability barrier to macromolecules between the bile canaliculus and the rest of the intercellular space. ‘Tightness’, however, is a relative term; there seems to be a positive correlation between degrees of tightness and the number of strands forming the junction. On this basis, the canalicular tight junctions are comparable to those elsewhere in the body (e.g. rete testis, vasa efferentia) regarded as only ‘moderately tight’. Intriguingly, tight junction proteins are also involved in key regulatory functions at nonjunctional locations, such as hepatocyte differentiation, proliferation and migration.

Nucleus

The hepatocyte nucleus shows characteristics expected in the nucleus of a cell actively engaged in protein synthesis: large, occupying 5–10% of the volume of the cell; spherical, with one or more prominent nucleoli; and with scattered chromatin. The nuclear membrane is double layered and contains many pores ( Fig. 1.10 ). At birth, all but a few hepatocytes are mononuclear and of uniform size. In the adult liver there is considerable variation in both number and size of nuclei. About 25% of the adult hepatocytes are binucleate; the two nuclei are similar in size and staining properties. Hepatocyte nuclei fall into various sizes, with volumes in the ratio 1:2:4:8. This variation reflects polyploidy, with the DNA content increasing correspondingly. At birth in humans, almost all hepatocytes are diploid (and mononucleate). From the eighth year, when more than 90% of hepatocytes are diploid, the number of tetraploid nuclei (i.e. those with twice the normal DNA content) increases, to reach about 15% in children of 15 years and 40% by middle adulthood. ^, Tetraploid cells are thought to arise by mitosis of cells with two diploid nuclei. The DNA content of each nucleus doubles, but the chromosomes are then arranged on a single mitotic spindle, so that division produces two daughter cells, each with a single tetraploid nucleus. The significance of polyploidy in hepatocytes is unknown. Since cell size is proportional to cell ploidy, polyploidy does not provide an increased amount of genetic material per unit volume of cytoplasm.

Hepatocyte mitotic division provides for intrauterine and postnatal growth of the liver, which continues well into childhood. By adulthood the liver has a very low mitotic index, with estimates ranging from 1 mitosis per 10,000–20,000 cells to up to 2.2 mitoses per 1000 cells. Nevertheless, a high percentage of hepatocyte nuclei are euchromatic, indicating that transcription of most of the genome is occurring continuously. Almost all the DNA is in the extended configuration, and minimal heterochromatin is observed.

Conversely, hepatocytes engaged in protein biosynthesis have a large nucleolus (sometimes several) that can be recognized on light microscopy. The nucleolus is where ribosomal genes are located and where ribosome biogenesis occurs. Electron microscopy (EM) reveals the nucleolus to contain three main components: rounded fibrillar centres composed of thin, loosely distributed fibrils; a dense fibrillar component containing tightly packed fibrils that surround the fibrillar centres; and the granular component, constituted by granules which embed both fibrillar components. Ribosomal genes exist in an extended, ready-to-be-transcribed configuration within the fibrillar centres and partly in the dense fibrillar component. Although the precise location of ribosomal gene transcription remains unclear, newly transcribed RNA molecules undergo early processing and maturation in the dense fibrillar component and are assembled into preribosomes in the granular component. Protein-rich ribosomal subunits then exit the nucleus through pores in the double-membrane nuclear envelope.

Endoplasmic reticulum

The endoplasmic reticulum (ER) is a network of parallel, flattened sacs or cisternae on whose cytoplasmic surfaces may be attached polyribosomes to constitute the rough ER (RER) ( Fig. 1.11 ). Clusters of RER are scattered randomly throughout the hepatocyte cytoplasm and constitute approximately 60% of the ER. The remaining 40% is the smooth ER (SER), lacking a ribosomal coating. The SER also forms anastomosing networks of tubules and vesicles of varying diameters which are continuous with the cisternae of the RER. The outer nuclear membrane also has attached ribosomes and is continuous with the membrane of the RER. The SER is often found in the region of the Golgi apparatus and communicates with it. The SER frequently has a close topographical relationship with glycogen ( Fig. 1.12 ). The ER occupies 15% of the total cell volume, and its surface area—approximately 60,000 µm ² per hepatocyte—is more than 35 times the area of the plasma membrane. There is also zonality in the distribution of the ER; the surface area of SER in the centrilobular area is twice that in the periportal zone. ^,

The cell functions associated with the ER include (1) protein synthesis, both of secretory proteins and some of the protein constituents of the cell and organelle membranes; (2) metabolism of fatty acids, phospholipids and triglycerides; (3) production and metabolism of cholesterol and possibly production of bile acids; (4) xenobiotic metabolism; (5) ascorbic acid synthesis; and (6) haem degradation. In the case of protein synthesis, polypeptides synthesized by RER ribosomes are retained in the membrane or are ejected into the lumen of the RER for folding and post-translational modification. The protein products pass through the SER to the Golgi apparatus for packaging and insertion into membranes throughout the cell (in the case of membrane proteins) or for exocytotic secretion across the basolateral or apical membranes of the hepatocyte.

The cytochrome P-450 system is localized in the ER membrane; this is the system whereby the liver cell metabolizes and detoxifies xenobiotics. This enzyme system can be reversibly induced by certain xenobiotics, such as phenobarbital, and this is accompanied by the synthesis and hypertrophy of the ER; the mechanisms involved in new membrane production are not clear. The preponderance of SER in the centrilobular region of the lobule and the presence of haem in cytochrome P-450 enzymes explain the darker hue of the centrilobular region, which can be observed by visual inspection of cut-liver sections.

Glucose 6-phosphatase is localized on the ER, playing a key role in dephosphorylation of intracellular glucose 6-phosphate before release of glucose into the circulation by hepatocytes. As a correlate, the SER proliferates during synthesis of glycogen and thus is available for hepatocyte glycogenolysis when hepatocellular release of glucose is required for metabolic needs elsewhere.

Golgi complex

Each hepatocyte contains as many as 50 Golgi zones (which may not be separate but rather form a tridimensional continuity) situated most frequently beside the nucleus or in the vicinity of the bile canaliculus. Each complex appears as a stack of four to six curved, flattened parallel sacs, often with dilated bulbous ends containing electron-dense material. The convex or cis surface is directed toward the ER, and small vesicles in the cis -Golgi transfer synthesized proteins from the ER. The concave or trans surface is the origin of secretory vesicles. Vesicles break off from the ends of the sacs and carry the contained secretory proteins, including lipoproteins, for discharge at the sinusoidal surface or less often at the canalicular surface. Membrane proteins destined for insertion into any of the plasma membrane domains also are routed through the Golgi complex. The complex and its associated cytoplasm constitute approximately 2–4% of the cell volume. In addition to its role in the secretion of proteins, the Golgi complex has a large complement of glycosylating enzymes, important in the glycosylation of secretory proteins and in the synthesis and recycling of membrane glycoprotein receptors. The Golgi complex is capable of rapid and reversible structural reorganization into a tubuloglomerular network while maintaining its biosynthetic capabilities. With the SER, RER, lysosomes, other intermediate organelle compartments and even the nuclear and mitochondrial envelope membranes, the Golgi is an integral part of the complex intracellular organelle network involving vesicular trafficking that enables uptake, sorting, degradation, biosynthesis, trafficking and secretion of cellular proteins and lipids. ^{, ,}

Lysosomes

The existence of lysosomes was first predicted by De Duve on the basis of biochemical studies on liver homogenates. He subsequently identified them as the ‘peribiliary dense bodies’ found in electron micrographs of liver and established them as a new species of cell organelle. Their functions in health and disease have been reviewed ^, and are of particular importance to pathologists because of their involvement in several storage diseases (see Chapter 3 ).

Lysosomes present a variety of appearances in electron micrographs of liver and may be part of the intracellular membranous network known as the ‘GERL’ (Golgi-SER-lysosome). The GERL is involved with endocytosis and exocytosis, serving as a site for sorting of secretory proteins for secretion and for trafficking of endocytosed proteins to lysosomes for degradation. Indeed, the GERL is the site where the lysosomal enzyme acid phosphatase makes its first appearance, most likely playing a role in formation of lysosomes.

Several classes of lysosomes can be identified within the hepatocyte cytoplasm: (1) primary lysosomes , small in size, are considered to be functionally in a resting phase; (2) secondary lysosomes are functionally activated and delimited by a single membrane (see Fig. 1.11 ); (3) autophagic vacuoles contain parts of the degrading cytoplasmic organelles and are often delimited by a double membrane; and (4) residual bodies are larger than primary and secondary lysosomes and are usually more numerous in older individuals. The residual bodies contain the residues of nondigested material or pigments such as lipofuscins (considered undigestible permanent residues). Lipofuscin granules are the most numerous lysosomal bodies present in human hepatocytes.

Lysosomes are frequently found near the plasma membrane proximal to the bile canaliculus and can discharge their contents into the biliary space. The lysosomes in periportal hepatocytes are often larger and more positive for acid phosphatase than those in centrilobular hepatocytes. ^,

Lysosomal pleomorphism reflects a variety of functions. First, although the liver cell is long-lived, there is evidence for turnover of its cytoplasm and organelles. Cytoplasmic constituents may be incorporated within and digested by the primary lysosome, forming an autophagic vacuole, then forming a secondary lysosome. Autophagic vacuoles therefore show fragments of organelles or cell inclusions in various stages of digestion. Second, lysosomes also incorporate lipofuscin pigment, which may accumulate undigested over long periods, forming residual bodies; material of exogenous origin, including iron, stored as ferritin, which accumulates in large quantities in iron overload states; and copper, which accumulates in copper-overload conditions and cholestasis. Third, coated vesicles and multivesicular bodies result from receptor-mediated endocytosis. In a complicated sequence of intracellular events, ligand–receptor complexes in clathrin-coated pits on the basolateral cell plasma membrane are internalized to form endocytic vesicles, or endosomes . Soluble ligands which are internalized in this way include insulin, low-density lipoproteins, transferrin, immunoglobulin A (IgA) and asialoglycoproteins. Fusion of endosomes occurs to form multivesicular bodies. Some of these vesicles are responsible for transcytosis or intracellular transport from the basolateral domain to the canalicular domain. Others fuse with primary lysosomes, and their contents undergo partial degradation before being exocytosed at the canalicular or basolateral domain. Still other vesicles undergo complete degradation and become increasingly electron dense with the formation of dense bodies. ^, Microtubules appear to have an important role in sorting the pathways along which endocytic vesicles move within the hepatocyte.

Peroxisomes

Peroxisomes are single membrane-bound ovoid granules 0.2–1.0 µm in diameter (see Fig. 1.12 ). They were first described as ‘microbodies’ by Rouiller and Bernhard in 1956. The properties of peroxisomes in liver have been reviewed elsewhere. Each hepatocyte may contain 300–600 peroxisomes, and they comprise 1.5–2% of cell volume. Peroxisomes may be more numerous in perivenular hepatocytes, but they are generally homogeneously distributed within the hepatic lobule. ^, There is morphological heterogeneity between species; in the rat, peroxisomes contain a paracrystalline striated core or nucleoid in which urate oxidase is concentrated; human peroxisomes lack a core. Peroxisomes contain oxidases that use molecular oxygen to oxidize a number of substrates with the production of hydrogen peroxide (thus the name of the organelle), which in turn is hydrolysed by peroxisomal catalase. Approximately 20% of the oxygen consumption of the liver is used in peroxisomal activity. The energy produced by this oxidation is dissipated as heat. An alcohol overload may be metabolized in the liver by peroxisomal catalase. Drugs, such as clofibrate, which lower blood lipids cause a proliferation of peroxisomes, an increase that has been causally linked to the hypolipidaemic action. Alterations in hepatocyte perixosomes have been reported in bacterial infections, viral hepatitis, Wilson disease and alcoholic liver diseases (ALDs). ^, Various metabolic disorders have been described in which there is either an absence of peroxisomes or a deficiency of peroxisomal enzymes ^, (see Chapter 3 ). Peroxisomes have become of particular interest owing to their critical role in hepatic lipid metabolism and their contribution to hepatocellular function or dysfunction in nonalcoholic fatty liver disease (NAFLD).

Mitochondria

Mitochondria are large organelles (1.5 µm in diameter and up to 4 µm long) that number approximately 1000–2000 per hepatocyte and constitute about 20% of the cytoplasmic volume of hepatocytes. Mitochondria are the site of oxidative phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP), constituting the source of cellular aerobic metabolism. Longer (up to 4 µm) and larger (up to 1.5 µm in diameter) mitochondria are more numerous in periportal hepatocytes. Mitochondria are bounded by an outer and an inner membrane, each 5–7 nm thick (see Fig. 1.11 ). The outer membrane possesses special pores that allow the passage of molecules smaller than approximately 2000 daltons. The inner membrane’s surface area is greatly increased by the presence of numerous cristae, which fold within the mitochondrial matrix. The space between inner and outer membranes presents a low-density matrix and ranges from about 7 to 10 nm in thickness. Mitochondria have a relatively low-density matrix in which lamellar or tubular cristae and a variable amount of small, dense granules can be observed; these granules may represent a concentration of calcium ions (Ca ²⁺ ), a small circular DNA and ribosomes. The dense granules have a diameter of 20 to 50 nm. In addition, filaments of circular mitochondrial DNA about 3–5 nm in width and granules approximately 12 nm in diameter containing mitochondrial RNA are also present. The DNA codes for some of the mitochondrial proteins are synthesized in ribosomes within the organelle, but most of the mitochondrial protein is encoded by nuclear DNA. Mitochondria are self-replicating and have a half-life of approximately 10 days. Mitochondria may fuse and are remarkably mobile organelles, moving about in the cytoplasm and closely associated with microtubules.

Mutations in the mitochondrial genome account for various mitochondrial myopathies and may also play a role in development of NAFLD. Indeed, it has become apparent that specific biochemical abnormalities of mitochondria may play an important role in the pathogenesis of certain liver diseases and that genetic defects in mitochondrial proteins and enzyme systems may be the underlying cause of other liver and metabolic diseases. Because mitochondria possess a distinct and unique extranuclear genome, a new class of maternally, or mitochondrially, inherited diseases have emerged (see Chapter 3 ).

The structural compartmentation of mitochondria provides for topographical localization of various enzyme systems, the details of which form almost a science in themselves. It need only be said here that the outer membrane is relatively unimportant as a locus for enzymes; it keeps the inner membrane together and contains porin , a transport protein that forms channels permeable to molecules of less than 2000 daltons. The inner membrane and crystal lamellae support the respiratory chain enzymes involved in oxidative phosphorylation, which generates ATP. The matrix contains most of the components of the citric acid cycle and the enzymes involved in β-oxidation of fatty acids and in the urea cycle. Mitochondria are randomly distributed within individual hepatocytes but are larger and more numerous in periportal cells, corresponding with the greater levels of oxidative respiration and metabolism in the periportal region.

Cytoplasm

The cytoplasm of any cell is a highly concentrated matrix of proteins and microfilaments, within which organic and inorganic solutes diffuse. ^, Movement of larger components through the matrix, especially membrane-bound vesicles, involves directed transport along the cytoskeletal fibres, the composition of which is described in the next section. A hepatocyte is extremely rich in non-membrane-bound cytoplasmic inclusions, including glycogen granules, lipid droplets and various pigments. Glycogen granules are the most abundant and on EM may occur in the monoparticulate form (β particles, 15–30 nm in size) or more frequently as aggregates of smaller particles arranged to form ‘rosettes’ (α particles; see Fig. 1.12 ). Glycogen granules are dispersed in the cytoplasm but are often associated with the SER. Intranuclear glycogen usually appears as β particles. Glycogen is depleted during fasting, disappearing first from periportal hepatocytes and then from perivenular cells. On refeeding the sequence reverses. In this manner, hepatocytes constitute a major metabolic energy reserve during fasting, thus supporting systemic glucose homeostasis.

Lipid inclusions appear as empty vacuoles in histological sections or as osmiophilic droplets on TEM and usually are not surrounded by membranes. Lipid droplets consist of triglycerides in their interior and are coated with a monolayer of phospholipids. Small lipid droplets have a high surface/volume ratio and are accessible to cytoplasmic lipases, which may degrade the retained triglyceride quickly. Large lipid droplets have a low surface/volume ratio and may reside in hepatocytes long after the metabolic processes in their deposition have subsided.

A variable number of iron-containing granules is often present within the hepatocyte cytoplasm, depending heavily on the iron status of the host. These are usually in the form of ferritin particles. With an approximately spherical shape, the iron-containing protein ferritin consists of a protein shell (apoferritin) 11 nm in diameter and an iron-containing central core approximately 5 nm in diameter. Hepatocyte iron deposits may also occur as single membrane-bound lysosomal bodies (residual bodies), forming aggregates of iron-containing electron-dense particles (siderosomes, haemosiderin granules). In addition to hepatocytes, liver endothelial cells and Kupffer cells also accumulate intracellular iron under conditions of iron overload.

Cytoskeleton

The major components of the cytoskeleton of most eukaryotic cells comprise 6-nm-diameter microfilaments , 8–10-nm-diameter intermediate filaments (IFs) and 20-nm-diameter microtubules ; the definitive review of their structure and function in the hepatocyte remains that of Feldmann. These are structurally, chemically and functionally distinct, linear macromolecules coursing through the cytoplasm. IFs are relatively stable macromolecules capable of modulation measured in minutes and longer intervals. Microfilaments and microtubules are highly dynamic structures capable of rapid polymerization and depolymerization on a second-to-second basis and thus rapid adaptation in response to functional demands. For all these macromolecules, polymerization and depolymerization of their constituent molecules are under the influence of various intracellular factors, including free Ca ²⁺ ions, high-energy compounds and associated proteins. In addition, various accessory proteins modulate these components and link them to one another, to cell organelles and to the cell membrane; these are part of a microtrabecular lattice, or cytomatrix. These structures interact to regulate internal organization, cell shape, movement, division, secretion, metabolism and intercellular communication.

Liver sinusoidal endothelial cells

Liver sinusoidal endothelial cells (LSECs) originate from bone marrow and form a highly dynamic barrier between the vascular space and underlying hepatocytes. There is no underlying basal lamina of the sinusoidal endothelium; instead, LSECs rest on a delicate mesh of ECM that supports the LSECs but does not block fluid or solute movement. The main characteristics of LSECs are (1) their smooth, thin, attenuated cytoplasmic sheet, about 150–170 nm in maximum thickness ; (2) the numerous holes (fenestrae) that perforate the cytoplasmic sheet, facilitating exchange between blood and hepatocytes; and (3) their numerous intracytoplasmic pinocytotic vesicles, indicating a high capacity for endocytosis (see Figs 1.13B and 1.15A ). The cytoplasmic extensions of LSECs may overlap, but there are no intercellular junctions (e.g. endothelial adherence, tight) otherwise found in vascular endothelial cells. Under appropriate stimuli, inflammatory cells can undergo ready diapedesis (migration) from the vascular compartment into the space of Disse.

Metabolism and molecular phenotype

The LSECs have a predominantly anaerobic metabolism, reflected by a low number of mitochondria and production of high levels of lactate. They have limited but profound biosynthetic activity, producing nitrous oxide (NO), endothelins, prostaglandins and possibly cytokines such as interleukin-1 (IL-1) and IL-6, all of which have potent effects on vascular tone and the functions of nearby cells. Through both the generation of NO and the influence of their secreted cytokines on the contractile function of underlying stellate cells (see later), LSECs play a key role in the regulation of sinusoidal blood flow. LSECs are subject to damage by some hepatotoxins. For example, LSECs are the initial target of some hepatotoxic drugs (e.g. acetaminophen) and toxins, which occurs before hepatocyte injury. Direct toxic damage to LSECs also is the initiating event in sinusoidal obstruction syndrome (SOS), in which endothelial cell debris occludes the vascular space, with life-threatening consequences.

LSECs show a number of phenotypic differences from other vascular endothelium as well as other types of liver cells, some of which can be used as LSEC markers. Normal LSECs do not bind the lectin Ulex europaeus and, in most species, do not express factor VIII-related antigen (von Willebrand factor [VWF]). Furthermore, LSECs contain absent to low levels of other molecules characteristically found in vascular endothelium, such as E-selectin, CD31 (PECAM) and CD34, but do express Fc IgG receptors (CD16 and CDw32), CD4, CD14 and aminopeptidase N. CD34-positive LSECs are restricted to a few periportal sinusoids, staining the cell body and processes ( Fig. 1.16A ). The cell bodies of CD34-negative LSEC are immunoreactive for stabilin-2 (stab-2), a HA-binding protein ( Fig. 1.16B, C ). Therefore, CD34 and stab-2 are mutually exclusive markers of LSEC in the normal liver. Under conditions promoting sinusoidal capillarization, a greater proportion of LSECs become CD34-positive.

LSECs exhibit membrane immunoreactivity for intercellular adhesion molecule 1 (ICAM1). The natural ligand for this adhesion molecule, lymphocyte-associated antigen 1 (LFA1), is present on Kupffer cells; this LSEC receptor may therefore be involved in the adhesion of Kupffer cells to the endothelial lining. Upregulation of ICAM1 expression in LSECs may also be important in ‘trapping’ LFA1-positive lymphocytes in inflammatory liver diseases.

There is heterogeneity of LSECs along the acinar axis. In addition to the increased porosity in the perivenular zone previously mentioned, variation in cell size, heterogeneous lectin binding and expression of various receptors, cytoplasmic density, endocytic capacity and surface glycosylation have also been demonstrated. ^{, , ,}

In summary, as a fenestrated barrier, LSECs substantially influence the trafficking of molecules and cells between liver parenchyma and blood. They have major functions in hepatic homeostasis, including plasma ultrafiltration, regulation of hepatic microcirculation and vascular tone, inflammation and thrombosis. In addition, they are essential for control of the hepatic immune response. ^,

Kupffer cells

Kupffer cells, present in the lumen of hepatic sinusoids (see Fig. 1.15C ), can be identified with monoclonal antibodies such as CD68. Kupffer cells are specialized liver macrophages, representing the largest population of resident tissue macrophages. They belong to the mononuclear phagocytic system but manifest phenotypic differences which distinguish them from other macrophages. Kupffer cells are of considerable importance in host defense mechanisms; their inflammatory response can be considered as a main mediator in liver injury and repair. Indeed, all of the liver macrophages (resident Kupffer cells and exogenous monocyte-derived macrophages) have an important role in both liver homeostasis and the pathogenesis of various liver diseases, giving opportunity for therapeutic targeting of these cells under appropriate circumstances.

On SEM, Kupffer cells have an irregular stellate shape, and their luminal surface shows many of the structural features associated with macrophages, such as small microvilli, microplicae and sinuous invaginations of the plasma membrane. Kupffer cells bulge into the sinusoidal lumen. Their cell body rests in contact over a more or less large area on the endothelial lining ( Fig. 1.17 ), but they never form junctional complexes with endothelial cells. However, Kupffer cells may be found in gaps between adjacent endothelial cells, and their protoplasmic processes may extend through the larger endothelial fenestrae into the perisinusoidal space of Disse.

Kupffer cells are more numerous in the periportal sinusoids, and as noted earlier, there is some evidence that, as with hepatocytes and LSECs, Kupffer cells also manifest functional heterogeneity in the lobule, with different subsets distributed through lobular zones. Kupffer cells have been considered to be fixed tissue macrophages, but they appear capable of actively migrating along the sinusoids, both with and against the blood flow. They can migrate into areas of liver injury and into regional lymph nodes. Because of their location and shape, Kupffer cells can interact with blood and cells as they transit.

Kupffer cells contain numerous lysosomes (almost one-quarter of all lysosomes of the liver) and phagosomes, and the cisternae of their ER are rich in peroxidase. Their primary functions include the removal, by ingestion and degradation, of particulate and soluble material from the portal blood, in which they discriminate between ‘self’ and ‘non-self’ particles. They act as scavengers of microorganisms and degenerate normal cells (e.g. effete erythrocytes), circulating tumour cells and various macromolecules. These functions are in part carried out nonspecifically, but Kupffer cells are also involved in the initiation of immunological responses and the induction of tolerance to antigens absorbed from the gastrointestinal tract. The efficiency of this clearance function is shown by the fact that removal of particulate material is limited only by the magnitude of hepatic blood flow; removal of particles may approach single-pass efficiency.

Kupffer cells play a major role in the clearance of gut-derived endotoxin from the portal blood, achieved without the induction of a local inflammatory response. The precise mechanisms involved are not fully understood, but there appears to be finely balanced autoregulation between the release of proinflammatory and inflammatory mediators such as IL-1, IL-6, TNF-α and interferon-γ (IFN-γ) and mediators such as IL-10 that suppress macrophage activation and inhibit their cytokine secretion. The role of Toll-like receptors (TLRs) in this response is considered in other chapters.

Several cytokines, chemokines and reactive nitrogen and oxygen species released by activated Kupffer cells are thought to have local effects, modulating microvascular responses and the functions of hepatocytes and stellate cells. Although Kupffer cells can express class II histocompatibility antigens and can function in vitro as antigen-presenting cells (APCs), they appear to be considerably less efficient at this than macrophages at other sites. Their principal roles in the immune response therefore appear to be antigen sequestration by phagocytosis and clearance of immune complexes.

Kupffer cells exhibit an important plasticity, expressing different phenotypes depending on the local metabolic and immune environment. Therefore, they can express the proinflammatory M1 phenotype or several M2 phenotypes involved in the resolution of inflammation and wound healing. Kupffer cells can play a protective role through their tolerogenic phenotype, as in some drug- and toxin-induced liver injury but can also shift to a pathologically activated state and contribute to chronic inflammation in various liver diseases. Impairment of their activity as scavengers of hepatic blood can contribute to pathogens entering the systemic circulation and thus systemic infection.

There is firm evidence from bone marrow and liver transplant studies showing that Kupffer cells are derived at least in part from circulating monocytes. ^, However, Kupffer cells are capable of replication, and their local proliferation accounts for a substantial part of the expansion of this cell population in response to liver injury. ^, Kupffer cells can proliferate in response to T-helper type 2 (Th2) inflammatory signals. Furthermore, Kupffer cells appear in the fetal liver of the mouse before there are circulating monocytes, and evidence indicates that they are derived from primitive macrophages which first appear in the yolk sac. These data suggest that Kupffer cells may have a dual origin.

Liver-associated lymphocytes

Liver lymphocytes include cells of the adaptive immune system (T and B lymphocytes) and innate immune system: natural killer (NK) and natural killer T (NKT) cells. NK cells constitute 31% of hepatic lymphocytes and NKT cells, 26%. The liver is thus particularly enriched with cells of the innate immune system compared with other parenchymal organs. While this has immediate value for the liver dealing with foreign antigens released from the gut into the splanchnic circulation, it also means that the liver is well equipped for an immune response to neoantigens expressed within its substance. ^,

It has been estimated that an average normal human liver contains approximately 1 × 10 ¹⁰ lymphoid cells. These lymphocytes are predominantly located within portal tracts but are also found scattered throughout the parenchyma, where they are found in loose luminal contact with Kupffer cells or LSECs. In the peripheral blood, 85% of the lymphocytes are T and B cells, which possess clonotypic antigen-specific receptors. In contrast, up to 65% of lymphocytes within the liver are NK cells, T cells expressing γδ T-cell receptors (TCRs) and T cells expressing NK molecules (NKT cells); clonotypic T and B cells are only a minority of intrahepatic lymphocytes.

Because of their particular location in the space of Disse in the intercellular recess between adjacent hepatocytes, hepatic NK cells were first described as ‘pit cells’ in the rat liver in the 1970s. Recent studies have shown that two distinct NK-cell subsets are present in mouse and human livers: conventional NK cells and liver-resident NK cells. The latter, which are those appearing morphologically as the pit cells, are large granular lymphocytes, usually also in contact with Kupffer and endothelial cells; some lymphocyte microvilli can protrude into the space of Disse. ^, They present characteristic dense granules, visible on EM, as well as a few other organelles usually concentrated on one side of the cell.

The liver contains the largest number of γδ T cells in the body. ^, γδ T cells are also found in the skin, gut and respiratory mucosa and in the pregnant uterus and accumulate at sites of infection. They secrete various cytokines and can lyse antigen-bearing target cells. Their precise function in the liver is not yet clear.

Hepatic NKT cells coexpress TCR and NK activating and inhibitory receptors. They can be further subclassified on the basis of their expressing various types of TCRs and various NK receptors. Functional studies have demonstrated that hepatic NKT cells have numerous cytotoxic activities and produce multiple cytokines. Their major role therefore may be to effect local immunological reactions through the production of cytokines.

The relative frequency of the various subpopulations of liver-associated lymphocytes varies between individuals, probably reflecting each individual’s immunological status; this in turn is affected by genetic background and both previous and current antigen exposure. The hepatic environment itself also may influence the distribution of the various subsets. The rich array of subtypes of liver-resident lymphocytes points towards extensive immunosurveillance mechanisms in response to infectious, inflammatory and tumoural liver disease, with direct implications for novel therapies.

Hepatic stellate cells

Originally identified by Boll and von Kupffer in the 1870s, HSCs were largely ignored until 1951, when Ito described their morphological features on light microscopy. They were subsequently referred to under a variety of terms—Ito cells, hepatic lipocytes, fat-storing cells, stellate cells and para- or perisinusoidal cells. ^, The now-accepted nomenclature for them is ‘hepatic stellate cells’ (HSCs). Their pathobiology and their role in regulating microcirculation, vitamin A storage and synthesis of ECM components, as well as their contribution to liver inflammation and immunology, have been extensively reviewed.

During embryogenesis, HSCs originate from the mesenchyme of the septum transversum, along with portal tract fibrocytes and perivascular mesenchymal cells. In the normal adult liver, HSCs account for about 5–8% of cells and are regularly spaced along the sinusoids (∼40 µm from HSC nucleus to HSC nucleus). There are about 5–20 HSCs per 100 hepatocytes. Despite their relatively sparse distribution, HSCs have long, thin cytoplasmic processes which have a perisinusoidal distribution in the space of Disse, organized into a sheath surrounding the sinusoid network. Indeed, the close proximity of HSCs with LSECs resembles that of pericytes in capillaries. The cell body of HSCs is often located in the interhepatocytic recess.

Quiescent state

HSCs in the normal liver are ‘quiescent’, having received no stimuli to transform to their ‘active’ myofibroblastic state. The relationship of HSCs with other cells in the normal sinusoidal environment is closely related to their normal morphological and functional features, as follows:

• The subendothelial processes of HSCs terminate as very thin, thorn-like microprojections which contact the plasma membrane of the hepatocyte microvilli and also the LSECs. Thus the HSCs adhere to the LSECs and hepatocytes, each strongly influencing the behaviour of the other cell types in response to hepatic injury and stimulation of hepatic regeneration.

• Autonomic nerve endings in the space of Disse come into contact with HSCs, ^, and they respond to α-adrenergic stimulation and contract in response to several vasoactive substances, such as prostaglandin, thromboxane A2, ET-1, substance P and angiotensin II. These morphological and functional features support the role of HSCs in the haemodynamic regulation of sinusoidal blood flow.

• In their quiescent phenotype, HSCs store vitamin A (80% of the body’s vitamin A) in intracellular lipid droplets. Dietary retinyl esters reach hepatocytes in chylomicron remnants, which pass from the sinusoidal lumen through the endothelial fenestrae and are taken up by hepatocytes. Most of the endocytosed retinol is rapidly transferred to the HSCs for storage. About 75% of HSCs in the normal liver contain lipid droplets, which are intermingled with the usual cytoplasmic organelles and generally abundant cytoplasm. Similar stellate cells, which also share the property of vitamin A storage, exist in the human pancreas, and evidence also indicates a more widespread distribution involving lung, kidney and gut. ^,

• In accordance with the needs of the host organism, retinol (vitamin A) is released to peripheral target tissues in the form of RBP complexes.

By EM, HSCs are present in the space of Disse, subjacent to LSECs and adjacent to hepatocytes ( Fig. 1.18 ). In normal conditions, HSCs are not readily visualized on light microscopy ( Fig. 1.19A ), but the lipid droplets rich in vitamin A can be demonstrated by fluorescence microscopy of frozen tissue when excitation light of 328 nm is used, or by gold chloride impregnation of tissue during fixation. HSCs can be visualized by GFAP in their quiescent state ^, ( Fig. 1.19B ), but this staining is not always reproducible; cellular RBP 1 (CRBP1) immunostaining is the best marker to visualize HSCs in their quiescent as well as activated state. When prominent in some pathological conditions such as hypervitaminosis A, HSCs can be visualized in routine histological sections without special stains.