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 tract ‘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.

Beginning at the porta hepatis, the portal vein divides into successive generations of distributing or conducting veins, so called because they do not directly feed the sinusoidal circulation. 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. From both the preterminal and terminal portal venules arise very short 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. These inlets are reported to be guarded by sphincters composed of sinusoidal lining cells—the afferent or inlet sphincters. 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 canal.

Drawing on the anatomical studies of Matsumoto et al., the conducting portal system and the terminal portal branches 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 with 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, Matsumoto et al. claim that immediate periportal damage actually never occurs in vascular insufficiency or chronic venous congestion, and that typically the liver biopsy always shows sickle zones standing in relief.

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 a peripheral portal ‘triad’.

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. Occasional arterioportal anastomoses between perivenous arterioles and terminal portal venules have been observed, 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 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.

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.

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 unite to form intercalated 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. 1.9 ). 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.

Rat liver preparations. A, Light microscopy of liver cell plates cut longitudinally showing a centrally placed nucleus and occasional binucleate cells, the sinusoidal surface against which Kupffer cell nuclei are abutting, the canalicular pole and the intercellular surface. B, Scanning electron micrograph showing several cell types. Hepatocytes (H) contain a nucleus (N), and at the junction between cells the bile canaliculus (bc) and the intercellular surface are clearly defined. The sinusoidal surface is seen, and in the sinusoidal area a Kupffer cell (Kc), endothelial cell (Ec) and two perisinusoidal cells (psc) are present.

(Courtesy of Professor E. Wisse, Brussels.)

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. 1.10 A and 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. 1.10 C ). The presence of adenosine triphosphatase (ATPase) can be demonstrated histochemically; CD10 is a reliable immunostain for identifying the bile canaliculus ( Fig. 1.10 D ). 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.

A, Transmission electron micrograph of bile canaliculus (bc), human liver . Note microvilli projecting into the lumen and the organelle-free pericanalicular cytoplasm (asterisk). Tight junctions and desmosomes (arrows) are present between the two adjacent hepatocytes (H). B, Scanning electron micrograph. On the surface of a number of neighbouring hepatocytes, an interconnected network of bile hemicanaliculi show bifurcations (arrows) together with blunt ends (asterisks). C, Bile canaliculi in rabbit liver. Immunofluorescence preparation in which the section was reacted with a human serum containing smooth muscle antibody. The pericanalicular microfilaments have produced strong positive immunofluorescence. D, CD10 immunoreactivity of canalicular network in normal human liver.

( A courtesy of Professor P. Bioulac-Sage.)

The canalicular surface is isolated from the rest of the intercellular surface by junctional complexes (see Fig. 1.10 A ): 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’.

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. 1.11 ). 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.

Freeze-fracture replica illustrating the inner leaflet (P face) and the outer leaflet (E face) of the nuclear membrane of a hepatocyte. Note the numerous nuclear pores. (Human liver, magnification ×44,800.)

(Courtesy of Professor R. De Vos.)

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 one 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 centers composed of thin, loosely distributed fibrils; a dense fibrillar component containing tightly packed fibrils that surround the fibrillar centers; 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 centers 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 endoplasmic reticulum (RER) ( Fig. 1.12 ). Clusters of RER are scattered randomly throughout the hepatocyte cytoplasm and constitute approximately 60% of the ER. The remaining 40% is the smooth endoplasmic reticulum (SER), lacking a ribosomal coating. The SER also forms anastomosing networks of tubules and vesicles of varying diameter 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. 1.13 ). 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.

A, Transmission electron micrograph (TEM) illustrating cytoplasmic organelles, nucleus and bile canaliculus (C) between two adjacent hepatocytes. L, Lysosomes; M, mitochondria. (Human liver; ×23,000.) B, TEM illustrating rough endoplasmic reticulum (RER) and mitochondria (M) with matrix granules. Part of the nucleus (N) with inner and outer membrane also shown. (Human liver, ×36,800.)

(Courtesy of Professor R. De Vos.)

Transmission electron micrograph illustrating networks of smooth endoplasmic reticulum (SER) ; Golgi apparatus (G) and peroxisomes (P). Note also glycogen rosettes (GL). (Human liver, ×28,800.)

(Courtesy of Professor R. De Vos.)

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 a number of 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. 1.12 ); (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 organisms. 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 are capable of discharging 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. 1.13 ). 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 lobular. 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. Various metabolic disorders have been described in which there is either an absence of peroxisomes or a deficiency of peroxisomal enzymes (see Chapter 3 ).

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. 1.12 ). 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 that 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. More recently 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 has 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 2 kilodaltons (kD). The inner membrane and cristal 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 smaller and less numerous in centrilobular than in periportal cells, the zonal implications of which are beyond the scope of this chapter.

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. 1.13 ). 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 amount 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 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. Intermediate filaments 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 extracellular matrix 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 1.14 B and 1.16 A ). 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.

Kupffer cells

Kupffer cells, present in the lumen of hepatic sinusoids (see Fig. 1.16 C ), 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 defence mechanisms and the innate immune response and can be considered as a main mediator in liver injury and repair. As such, they have an important role in the pathogenesis of various liver diseases.

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.

Scanning electron microscopy. Part of Kupffer cell lying in a sinusoid and showing characteristic microvilli projecting at the cell surface (arrows). Small rims of fenestrated endothelium can be observed at both sides of the Kupffer cell (f). A small bundle of collagen fibres (c) is situated in the space of Disse (SD) on the right.

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 endoplasmic reticulum 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 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, tumour necrosis factor alpha (TNFα) 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, 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.

Strong evidence from bone marrow and liver transplant studies show 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 presence of these cells in such large numbers indicates that they must serve important roles in normal hepatic immune responses and immune homeostasis.

Hepatic stellate cells

Originally identified by Boll and von Kupffer in the 1870s, hepatic stellate cells 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 extracellular matrix components, as well as their contribution to liver inflammation and immunology, have been extensively reviewed.

HSCs account for about 5–8 % of cells in the normal liver 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:

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  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 behavior of the other cell types in response to hepatic injury and stimulation of hepatic regeneration.

  
  
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  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, endothelin-1, substance P and angiotensin II. These morphological and functional features support the role of HSCs in the haemodynamic regulation of sinusoidal blood flow.

  
  
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  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.

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

In normal conditions, HSCs are not readily visualized on light microscopy ( Fig. 1.18 A ), 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 glial fibrillary acidic protein (GFAP) in their quiescent state ( Fig. 1.18 B ), but this staining is not always reproducible; cellular retinol-binding protein 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.

Hepatic stellate cells (HSCs). A, HSCs may occasionally be seen on optical microscopy; the fat globules are phloxinophilic in this section stained by Masson trichrome, and the perisinusoidal location of the cell is readily appreciated. B, GFAP (glial fibrillary acidic protein) immunoreactivity of HSCs in normal rat liver.

(Courtesy of Dr. Liena Zhao.)

Space of Disse

The space of Disse lies subjacent to LSECs and is the fluid space in direct contact with the basal plasma membrane surface of hepatocytes (see Fig. 1.16 A ). Abundant hepatocyte microvilli project into the space and may be important in keeping the space open. This space is not normally discernible in biopsy material, but in autopsied liver the hepatocytes shrink from the sinusoids, and the space of Disse is then characteristically evident. Nevertheless, this fluid space represents 2–4% of the volume of the hepatic parenchyma.

Studies with TEM show continuity of the space of Disse with the lateral intercellular space between adjacent hepatocytes, considerably expanding the perisinusoidal fluid compartment (see Fig. 1.16 B ). The space of Disse thus forms an extensive and essentially unique extravascular space. As noted earlier, the sinusoidal endothelium not only is discontinuous and extensively fenestrated, but in many species (including humans) lacks a basement membrane. It is thus freely permeable to blood plasma, which enters the space of Disse and comes into direct contact with the hepatocytes and the HSCs. This extravascular plasma constitutes the immediate medium of exchange between blood and hepatocytes, whose surface area of contact is increased by the abundant microvilli. The plasma is then presumed to flow toward the hepatic veins. However, there is evidence that fluid within the space of Disse may also be taken up by lymphatic spaces in the periportal zone in a retrograde fashion, either re-entering the sinusoidal blood or continuing within lymphatic channels to exit via the hilar lymphatics. The nature of the anatomical link between the periportal ends of the space of Disse and the lymphatics is discussed later.

As mentioned, the extracellular matrix (ECM) within the space of Disse is produced predominantly by the HSCs and constitutes the structural ‘reticulin’ framework of the liver. Hepatocytes may produce some collagen and some proteoglycans. Kupffer cells and LSECs influence the parenchymal ECM primarily through the production of cytokines which modulate the synthetic activity of the HSCs, but may themselves produce small amounts of proteoglycans. There is a gradient in the ECM in the space of Disse, with variation in amount and composition with increasing distance from the portal tracts. Thus, laminin, collagen type IV and heparan sulphate predominate in the periportal zone, whereas in the perivenular zones, fibronectin, collagen type III and dermatan sulphate are more abundant. In addition, it has been shown that fibrillin-1 is present in high amounts in the space of Disse in the absence of elastin, contrary to portal zones, where fibrillin-1 is co-localized with elastin to form elastic fibres, as in other sites (e.g. dermis or vessel walls). Fibrillin-1 forms a continuous network of slender fibres in the space of Disse between HSC processes and the sinusoidal membrane of hepatocytes.

The ECM plays a major role in the normal biology of the liver, influencing hepatocyte, LSEC and HSC function. Interaction between the ECM and these cells is of fundamental importance in maintaining their differentiation, growth and function. In contrast to activities in other organs, the ECM does not act as a diffusion barrier between the plasma and the hepatocytes.

The ECM components in the space of Disse interact with the hepatocyte and endothelial cell membranes through various surface integrins and other receptors. Hepatocytes have receptors for fibronectin, laminin and types I and IV collagen, and they can also bind proteoglycans. The proteoglycans can also act as adhesion and receptor molecules and are an important reservoir for cytokines and growth factors.

Unmyelinated nerve fibres are distributed throughout the hepatic lobules, localized mainly in the space of Disse. These nerve endings are oriented toward LSECs, hepatocytes and mainly HSC processes. Various neurotransmitters (e.g. substance P, noradrenaline, acetylcholine) are present in vesicles of these nerve endings, participating with vasoactive agents (e.g. endothelin-1, prostaglandin, nitric oxide) as well as adrenergic receptors present on HSCs in the haemodynamic regulation of sinusoidal microcirculation.

Architecture

The intrahepatic biliary system, discussed earlier with regard to hepatic embryology, is now examined in detail regarding the architecture of the biliary tree. First, the bile canaliculi that run between adjacent hepatocytes form a complicated intraparenchymal polygonal network. Based on rat studies, canalicular diameter increases gradually from the perivenular region to the periportal region; the diameter enlarges physiologically during periods of high bile flow.

Near the portal tract interface, canaliculi drain into the canals of Hering, partly lined by hepatocytes and partly by cholangiocytes ( Fig. 1.19 ). The canals of Hering drain into bile ductules (also termed cholangioles ), defined as having a basement membrane and lined entirely by cholangiocytes, usually three to six in circumference. The ductules may run their course for a short distance within the periportal parenchyma or, more often, begin at the limiting plate of the portal/parenchymal interface. Ductules therefore have an obligate portal tract segment and a variable intralobular segment.

Transmission electron microscopy of a section through a canal of Hering (CH), whose wall shows cholangiocytes, (ductal cells, D ) and a hepatocyte (H). (Human liver, ×9200.)

(Courtesy of Professor R. De Vos.)

In the portal tracts the ductules join the interlobular bile ducts ( Fig. 1.20 ), the smallest branches of which are 15–20 µm in diameter. These interlobular bile ducts are lined by a single layer of flattened cuboidal epithelium, have a basement membrane and are in turn ensheathed in the fibrous tissue of the portal tracts. As the interlobular bile ducts merge downstream, they increase in size and form larger septal ducts more than 100 µm in diameter and lined by a simple, tall, columnar epithelium with basally situated nuclei. Portal tract fibrous tissue shows some condensation around these septal ducts, but there is no well-marked concentric orientation. Table 1.1 summarizes the terminology and heterogeneity of the intrahepatic bile ducts as proposed by Desmet et al. A definitive quantitative 3D study of the human biliary system has been published. The larger ducts further anastomose to form intrahepatic bile ducts, 1–1.5 mm in diameter, which give rise to the main hepatic ducts. As described earlier, the intrahepatic bile ducts are supplied by an anastomosing peribiliary vascular plexus derived from the hepatic artery and draining into periportal sinusoids.

A, Transmission electron microscopy (TEM) of a cross section of a bile duct. L, Lumen; basement membrane (arrow). (Human liver, ×5750.) B, TEM of epithelium of a bile duct. Note contorted intercellular space (IC) ; apical microvilli (MV) ; junctional complexes (J). (Human liver, ×36,800.) C, Cross section of a portal tract containing a normal, medium-sized interlobular bile duct similar in size to A. (Haematoxylin and eosin [H&E] stain.)

( B courtesy of Professor R. De Vos.)

Peribiliary glands

The presence of glandular elements has been demonstrated around the extrahepatic and larger intrahepatic bile ducts ( Fig. 1.21 ). These peribiliary glands are of two types: (1) intramural mucous glands, which communicate directly with the bile-duct lumen, and (2) extramural mixed seromucinous glands, which form branching tubuloalveolar lobules and secretory ducts that drain into the bile duct lumen. These glands are relatively dense in the hilar bile ducts, cystic duct and periampullary region. SEM has shown that hilar ducts may also have irregular side branches and pouches in which bile may be stored and probably modified. The peribiliary glands secrete several substances, such as lactoferrin and lysozyme. They also are stem cell niches of the biliary tree, capable of differentiating into hepatobiliary and pancreatic endocrine or exocrine cells. These glands have been implicated in pathogenesis of both intrahepatic and extrahepatic cholangiocarcinoma.

Large intrahepatic bile duct near the hilum. Note the surrounding mucous and seromucinous peribiliary glands; one mucous gland opens into the duct lumen. (H&E stain.)

Cholangiocytes

Cholangiocytes account for 3–5% of the endogenous liver cell population, lining the intrahepatic and extrahepatic bile duct system. More than cells lining inert conduits, cholangiocytes modify the composition of bile during its transit through bile ducts, involving secretion and absorption of water, electrolytes and other organic solutes. Ultrastructurally, the cholangiocyte has a prominent Golgi complex, numerous cytoplasmic vesicles and short luminal microvilli. Studies in the rat suggest that 10–15% of basal bile flow is produced by ductal epithelium, and the corresponding contribution in humans is estimated at 40%. Secretion is under hormonal control (secretin and somatostatin); secretin is released from the duodenum after vagal stimulation and with the presence of acid in the duodenum and stimulates the secretion of bicarbonate-rich bile. The duct epithelium also secretes IgA and IgM (but not IgG), as shown immunohistochemically on human duct cells. Reabsorption involves water, glucose, glutamate and urate. Bile acids are reabsorbed through biliary epithelium and are recirculated by a cholehepatic shunt pathway through the peribiliary plexus; this recycling promotes bile acid-dependent bile flow in the ducts.

Extensive studies of cholangiocytes over the past 25 years have increased our understanding of both the normal formation of bile and how bile duct biology is altered during nonobstructive or obstructive cholestasis. The following concepts merit emphasis:

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  Cholangiocytes exhibit morphological and functional heterogeneity along the length of the intrahepatic biliary tree. Bile ductules are lined by four to five small cuboidal cholangiocytes. In interlobular bile ducts ranging from 20 to 100 µm in diameter, cholangiocytes become progressively larger and more columnar in shape and ultimately display a primary cilium.

  
  
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  Cholangiocytes are capable of proliferating in response to liver injury or surgical reduction, a process enhanced by secretin receptor signalling and secretin-stimulated choleresis. Small cholangiocytes are more resistant to liver injury than large cholangiocytes, and replicate during damage.

  
  
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  In turn, because of their larger cytoplasmic size, large cholangiocytes exhibit more membrane receptors, transporters and channels and are more responsive to physiologic agonists such as secretin and somatostatin for the modification of secreted bile.

  
  
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  Cholangiocytes are highly dynamic cells with regard to inter- and intracellular signalling, involving such mechanisms as transmembrane G protein-coupled receptors (TGR5), Farnesoid X receptor (FXR) and sphingosine 1–phosphate receptor 2 (S1PR2). Bile acids are strong modulators of cholangiocyte function. The secretory activity of cholangiocytes is regulated by both cyclic adenosine monophosphate (cAMP) and calcium signalling pathways.

  
  
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  Cholangiocytes are responsive to innervation, as discussed later.

Accumulation of bile acids during cholestasis (obstructive or nonobstructive) may have profound effects on cholangiocytes, including stimulation of proliferation and increasing secretin responsiveness. However, since cholangiocytes contain an apical sodium-dependent bile acid transporter (ASBT; official symbol NTCP2; gene symbol Slc10a2 ), they are capable of taking up bile salts from the bile duct lumen and returning them to the peribiliary circulatory plexus. While the resultant cholehepatic shunt can further stimulate bile secretion by hepatocytes, in the setting of obstructive cholestasis, bile salts can enter the systemic circulation for elimination in urine.

Cholangiocytes express receptors for epidermal growth factor (EGF), secretin and somatostatin. Normally they express class I major histocompatibility complex (MHC) antigens but not class II; cytokine-induced expression of class II antigen is seen in graft-versus-host disease, allograft rejection, primary biliary cirrhosis and primary sclerosing cholangitis and may be important in the pathogenesis of the bile duct injury in these diseases. Indeed, cholangiocytes may themselves secrete proinflammatory cytokines and contribute to the destructive events that occur in autoimmune or secondary inflammatory biliary diseases. Cholangiocytes express more cell–matrix adhesion molecules or integrins than hepatocytes, including α ₂ β ₁ , α ₃ β ₁ , α ₅ β ₁ , α ₆ β ₁ and α ₆ β ₄ , concurring with those expressed by most simple epithelial cells. Bile duct epithelium also expresses glutamyl transpeptidase, carcinoembryonic antigen (CEA) and epithelial membrane antigen, representing other phenotypic differences from hepatocytes, the precise significance of which is as yet uncertain. Thus the cells lining the biliary tree clearly have an extremely dynamic biology.

Extracellular matrix

The liver normally has only a small amount of connective tissue in relation to its size. Whereas in the human body, collagen constitutes about 30% of the total protein, the corresponding figure for the liver is 5–10%. Underlying the visceral peritoneum of the liver, or serosa, there is a layer of dense connective tissue admixed with elastic fibres which varies in thickness from 40 to 70 µm. This constitutes the Glisson capsule , irregular extensions of which extend as delicate fibrous septa up to 0.5 cm into the superficial parenchyma. These anatomically normal septa produce some architectural distortion and the appearance of subcapsular ‘fibrosis’, which must not be misinterpreted when wedge liver biopsies are examined. Condensation of the Glisson capsule occurs at the porta hepatis, and the fibrous tissue then extends into the liver, supporting and investing the portal vein, hepatic artery and bile-duct branches, thereby constituting the portal tracts. Some extension of the capsular tissue also accompanies the large hepatic vein branches, but no fibrous sheath surrounds terminal hepatic venules, which are in direct contact with perivenular hepatocytes.

The components of the ECM are collagens, glycoproteins and proteoglycans ( Table 1.2 ). In the normal liver parenchyma, interstitial collagens (types I and III) are concentrated in portal tracts and around terminal hepatic veins, with occasional bundles in the space of Disse. Delicate strands of type IV collagen (reticulin) course alongside hepatocytes in the space of Disse. This so-called reticulin network or framework is usually visualized by silver impregnation staining methods ( Fig. 1.22 ). Far from being inert scaffolding, the intrahepatic ECM is a dynamic tissue compartment that has profound impact on cellular function and the hepatic response to injury.

A, Liver biopsy from a child of 17 months. Note the twin-cell liver plates; these are better shown on a reticulin preparation at the same magnification in B, in which the nuclei are seen in a perisinusoidal position. C, Adult liver showing normal single-cell liver plates with centrally placed nuclei. D, Adult liver at the same magnification as C, showing a regenerative response with twin-cell liver plates. As in B, the nuclei tend to be in a perisinusoidal position; there is also rosette formation. ( A, H&E stain. B, C, D, Gordon-Sweet reticulin stain.)

Hepatic nerves

The liver is innervated by sympathetic and parasympathetic nerve fibres, with innervation of the ductal plate, bile ducts, peribiliary glands, portal veins and hepatic arteries early in human fetal life ( Fig. 1.24 ). The role of hepatic innervation was a source of puzzlement because the orthotopically transplanted liver, which has no innervation, functions well in the recipient. Recent studies have now made clear that hepatic innervation has considerable significance under specific conditions.

Schematic representation of hepatic afferent and efferent innervation. BD, Bile duct; PV, portal vein; HA, hepatic artery; CoH, canal of Hering; Hc, hepatocyte; GJ, gap junction; HSC, hepatic stellate cell; HV, hepatic vein.

Autonomic nerve fibres reach the liver in two separate but intercommunicating plexuses around the hepatic artery and portal vein and are distributed with branching vasculature. The nerve fibres include preganglionic parasympathetic fibres derived from the anterior and posterior vagi and sympathetic fibres which are mostly postganglionic with cell bodies in the coeliac ganglia, and which receive their preganglionic sympathetic connections from thoracic spinal segments T7–T10. The hilar plexuses also include visceral afferent fibres and some phrenic nerve fibres, probably afferent in character. Immunohistochemistry (IHC) studies of human liver, using antibodies to common neural proteins such as protein gene product (PGP) 9.5 and N-CAM, have shown that nerve fibres not only are present around vascular structures in portal tracts but extend into the parenchyma, running along the sinusoids. Fluorescence histochemistry and IHC using antibodies to dopamine–β-hydroxylase and tyrosine hydroxylase have shown that the majority of intrasinusoidal fibres are sympathetic; many contain neuropeptide tyrosine (NPY), a regulatory peptide commonly found in adrenergic nerves. Unmyelinated nerve fibres can be seen in the space of Disse using TEM. They are frequently surrounded by Schwann cell processes, but a few bare nerve endings or varicosities are found in close apposition to hepatocytes or HSCs. Synaptic clefts have been identified at points of contact, suggesting direct innervation of these cells, but true synapses are not found.

An extensive cholinergic (parasympathetic) network is found in rat liver. In human liver, Amenta et al. described parasympathetic cholinergic innervation of portal tract vessels with only limited innervation of the parenchyma. IHC studies have also identified intrahepatic fibres containing two neuropeptides—substance P and calcitonin gene-related peptide (CGRP)—which are commonly found in afferent nerves. Such afferent fibres may be involved in chemo- and osmoreception as well as in vasomotor regulation. It seems likely that the liver would have sensory receptors since it is exposed to the nutrient and solute load delivered via the portal circulation from the gut.

Within the parenchyma, release of neurotransmitters from the intrasinusoidal sympathetic fibres may modulate hepatocyte, LSEC and HCS function. Adrenergic nerves play a role in the control of hepatocyte carbohydrate and lipid metabolism and in intrahepatic haemodynamics and response to metabolic stress, including helping to maintain hepatic glucose homeostasis even when insulin regulation of hepatic glucose uptake is disrupted. Beyond that, stimulation of hepatic sympathetic nerves in vivo produces hyperglycaemia. This effect is caused by enhanced glycogenolysis in hepatocytes and appears to be under α-adrenergic control. The regulation of hepatic carbohydrate metabolism by sympathetic nerve fibres may be enhanced further by gap junctional communication between hepatocytes.

Bioulac-Sage et al. have speculated that adrenergic nerves may induce contraction in HSCs, thereby regulating intrasinusoidal blood flow. This may have relevance to evidence from experimental animals and humans, that the liver’s normal response to hypovolaemic shock is impaired by denervation, and this may result in hepatic ischaemic injury. Loss of intrasinusoidal nerve fibres in the cirrhotic liver may contribute to impaired metabolic function. Whether this loss accounts for some of the abnormalities of portal blood flow is not clear.

Within portal tracts, the neurobiology of cholangiocytes also has garnered considerable interest. Cholangiocytes express the M3 acetylcholine (ACh) receptor. Acetylcholine, acting on M3 receptor subtypes, potentiates secretin-induced choleresis and can stimulate cholangiocyte proliferation. Pharmacological or surgical denervation can induce cholangiocyte apoptosis, suggesting that adrenergic innervation has a key role in regulating cholangiocyte proliferation during regeneration. Cholangiocytes may also express receptors for the neurotransmitter histamine (the G-protein-coupled H3R receptor), for serotonin, and for the α-type calcitonin gene-related peptide 1 (α-CGRP1), all of which may influence cholangiocyte proliferation. Recognizing that the cholangiocyte compartment of bile ductules and canals of Hering contains putative stem cells with evidence for neuroresponsiveness.

Kupffer cells and dendritic cells

Antigen-presenting cells (APCs), including liver sinusoidal endothelial cells (LSECs), Kupffer cells and circulating blood dendritic cells (DCs), take up antigens, process them and present them to other immune cells. Because blood flow in hepatic sinusoids is slow, interaction of liver sinusoidal cell populations with circulating immune cells, both naive and with memory, is possible. LSECs exhibit particular competence for antigen cross-presentation to CD8+ T cells. Kupffer cells play a role in all forms of hepatitis, as activated Kupffer cells release TNFα, IL-2, IL-12 and leukotriene B3. In addition to the local effects of such cytokines, these mediators also are involved in the hepatic recruitment of circulating neutrophils and cytotoxic T lymphocytes (CTLs). DCs are highly immunogenic in response to hepatotropic viral infection and serve as major APCs to support the local CD8+ T-cell response. DCs express Toll-like receptors (TLRs) that recognize particular components of infectious organisms. The activation of TLR signalling pathways results in production of proinflammatory cytokines and end-maturation of DCs. The activated DCs and Kupffer cells stimulate NK cells, NKT cells, T cells and B cells through secretion of cytokines such as IL-12, TNFα, IFN-α and IL-10. The DC subsets that produce IL-12 and TNFα support generation of cytotoxic Th1 T lymphocytes. DCs producing IL-10 promote generation of Th2 T cells, which on further secretion of IL-4, IL-5, IL-10 and IL-13, stimulate B-cell antibody production.

Hepatocellular death of any sort is rapidly followed by Kupffer cell phagocytosis of the residual debris. For example, when an isolated hepatocyte undergoes apoptosis, it is routinely engulfed by a nearby Kupffer cell within 2–4 hours. With smouldering hepatocellular apoptosis, clumps of macrophages can accumulate in the parenchyma ( Fig. 1.26 ). Such macrophages can persist in the parenchyma for an extended period, probably weeks to months, serving as sentinels of prior hepatocellular injury and death.

Lobular inflammation in chronic hepatitis, in which hepatocyte debris has been phagocytosed by resident macrophages, leaving clumps of parenchymal macrophages; apoptotic bodies are also seen. (H&E stain.)

Hepatic damage more extensive than apoptosis of isolated hepatocytes engenders recruitment of circulating macrophages. The most dramatic example is massive hepatic necrosis, in which the vast expanse of the hepatocellular parenchyma undergoes cell death. With survival of the patient over the ensuing hours and days, the hepatic parenchyma becomes a sea of macrophages amidst the cellular debris. Their phagocytic and migratory action facilitates removal of the nonviable material, clearing the way for regeneration and recovery of the liver tissue.

Natural killer cells and natural killer T cells

Liver-specific natural killer (NK) cells are the major lymphocyte population in the human liver, making up 30–50% of the lymphocyte population. As noted earlier, NK cells reside in interstices of the space of Disse as ‘pit cells’ with a granular cytoplasm and are cytotoxic lymphocytes that can also produce cytokines. NK cells also express DC markers and share properties with DCs. Liver NK cells play an important role in first-line, innate defense against viral infection. During inflammatory liver disease, circulating NK cells can be recruited to the liver. Circulating NK cells exhibit a more dynamic maturation process, and there is evidence that their phenotype and function are modified on arrival in the hepatic microenvironment. Both locally resident and recruited NK cells can lyse target cells through multiple, nonredundant mechanisms and amplify the local inflammatory response.

Intrahepatic natural killer T (NKT) cells are a heterogeneous group of T lymphocytes. NKT cells are found less frequently in the liver, residing within the sinusoids adherent to endothelial cells, capable of crawling rapidly along these vessel walls. Compared to NK cells, NKT cells exhibit a lower nuclear/cytoplasmic ratio and have dense granules containing perforin. Activated NKT cells are capable of inducing liver injury. NK cells and NKT cells can produce high levels of proinflammatory (Th1) and anti-inflammatory (Th2) cytokines. They can kill virus-infected hepatocytes by secretion of perforin and granzyme and, in the case of NK cells, by surface expression of TNF-related apoptosis-inducing ligand.

Unlike T and B lymphocytes, which require clonotypic antigen receptors to recognize antigens, NK and NKT cells can participate in the immune response without prior antigenic stimulation. Stimulated populations of NKT cells can expand locally and undergo phenotypic maturation. In so doing, NKT precursors convert from a stage which is Th2 and IL-4 secreting to a stage which is Th1 and thus IFN-γ secreting. IFN-γ enhances the DC expression of proteins involved in cellular antigen processing and presentation, including proteosome subunits and MHC molecules. In addition, IFN-γ induces additional chemokines that enhance and direct the adaptive immunity of T and B cells. The expanded NKT cell populations are thus a very effective resident proinflammatory effector arm of the host immune system.

Along with Kupffer cells and LSECs, the constitutive activation of NK and NKT cells in the liver and their ability to rapidly produce copious amounts of cytokines enable the liver to respond quickly to remove pathogens, toxins and noxious food antigens that enter the liver through splanchnic blood. This innate immune system also is key to eliminating hepatocytes expressing aberrant antigens. Specifically, after hepatitis B virus (HBV) or hepatitis C virus (HCV) infects the liver, viral replication within hepatocytes occurs, and viral particles are continuously released into the circulation. Through the action of APCs (especially DCs), activation of NK and NKT cells occurs. In addition to direct cytolytic action of NK and NKT cells on hepatocytes expressing viral antigens, the secretion of cytokines such as IFN-γ by NK/NKT cells and CTLs can inhibit replication of HBV and HCV in other hepatocytes through a noncytolytic mechanism (see Fig. 1.25 ). Expression of IFN-α also occurs (by both NK and NKT cells and by infected hepatocytes); this interferon initiates intracellular signalling involving the signal transducer and activator of transcription (STAT) pathway to increase expression of genes that block viral replication within infected hepatocytes. Thus, during HBV infection the innate immune cells contribute significantly to the suppression of viral replication. Unfortunately, the same is not true for HCV infection because HCV is capable of blocking the antiviral IFN-α effect, through inhibition of STAT signalling within hepatocytes.

Regulatory T cells

A population of lymphocytes garnering particular attention are CD4+ T cells constitutively expressing the IL-2-receptor α-chain (CD25) and the forkhead family transcriptional regulator box P3 (FoxP3): CD4+ CD25+ FoxP3+ T cells, or ‘Tregs’. Tregs represent about 5–10% of peripheral CD4+ T cells. They are highly differentiated T cells that have limited proliferative ability and are prone to apoptosis. Tregs regulate the activation of CD4+ and CD8+ T cells by suppressing their proliferation and effector function. This suppressive action is critical in preventing the activation of autoreactive T cells, thus limiting collateral autoimmune damage during an immune response. Suppression occurs both through cell–cell contact and possibly through release of inhibitory cytokines. Once stimulated, Tregs act in an antigen-nonspecific fashion. Patients with autoimmune hepatitis have a reduced number of circulating Tregs at diagnosis. The numbers are replenished during remission on immunosuppression but never reach normal values. The presence of Tregs in the active phases of hepatitis makes intuitive sense, but their persistence during chronic viral hepatitis and in autoimmune hepatitis raises key questions about their contribution to these chronic inflammatory conditions. Tregs can inhibit virus-specific T-cell immunity in the pathogenesis of chronic HBV or chronic HCV infection. Tregs also have been shown to play a role in causing a persistent fibrotic state, inhibiting resolution of fibrosis.

T-follicular helper cells

The CXCR5+ CD4+ T-follicular helper (Tfh) cells are important for the formation of germinal centre and humoral responses. A subset of Foxp3+ Bcl6+ Tfh cells, defined as ‘follicular regulatory T (TFR) cells’, inhibit immune responses and share many characteristics with Tregs. TFRs are capable of downregulating antigen-specific immune responses to viral infection and are associated with poor virus eradication. In particular, there is a positive correlation between the numbers of circulating TFR cells and the levels of serum hepatitis B surface antigen (HBsAg) and HBV DNA in patients with chronic HBV infection, and with increased serum HCV RNA in patients with chronic HCV infection.

Neutrophils

Neutrophils have long been known to be part of the parenchymal inflammatory infiltrate of alcoholic hepatitis and the portal tract inflammation of obstructive cholestasis. One common theme is gut leakage of lipopolysaccharide (endotoxin), which is a potent inducer of intrahepatic proinflammatory cytokine production, including TNFα, IL-1β and IL-6. Hepatocytes, Kupffer cells, LSECs and HSCs all express CD14 and TLR4, the co-receptors for lipopolysaccharide, and are capable to sending homing signals to neutrophils for recruitment to the liver. The generation of reactive oxygen species (ROS) during alcoholic hepatitis and also hepatic ischemia-reperfusion injury activates a complex system of intrahepatic signalling cascades and inflammatory responses which promote neutrophil margination, adhesion and migration into the liver tissue. Toxic injury to the liver, from direct hepatotoxins such as acetaminophen and in many other forms of drug-induced liver injury, is abetted by neutrophilic inflammation. Also, neutrophils are a key feature of nonalcoholic steatohepatitis (NASH). The metabolic derangements of steatotic liver disease activate AP-1, a hallmark of lipotoxicity. AP-1 is a transcriptional regulator of IL-8, and it is hypothesized that this may be a mechanism for how metabolic derangement may stimulate neutrophil recruitment to the liver in NASH; activation of NFκβ in steatotic livers alone is not sufficient to generate NASH.

Most neutrophils involved in hepatic injury are extravasated from sinusoids and not from postsinusoidal venules (unlike elsewhere in the body). Neutrophils are readily activated by cytokines and other inflammatory mediators. Although neutrophil-generated proteases such as elastase and cathepsin G play a role in hepatic injury, a major destructive mechanism is neutrophil generation of ROS. Superoxide created by NADPH oxidase (NOX2) leads to hydrogen peroxide (H ₂ O ₂ ) generation, which can diffuse directly into hepatocytes. Alternatively, myeloperoxidase uses H ₂ O ₂ to created hypochlorous acid (HClO), which also diffuses into target cells. Collectively, it is clear that neutrophils can be potent inflammatory cells once they arrive on the scene.