Normal morphology of biliary tree and peribiliary glands 516
Biliary epithelial injury 520
Cholangiopathy and cholangitis 524
Pathological features secondary to impaired bile flow 529
Biliary interface activity 530
Biliary fibrosis/cirrhosis 533
Vanishing bile duct syndrome 534
Diseases primarily affecting intrahepatic bile ducts 535
Primary biliary cholangitis 535
Idiopathic adulthood ductopenia 547
Bile duct injury in liver allograft and graft-versus-host disease 547
Other disorders associated with intrahepatic bile duct injury 547
Isolated idiopathic bile ductular hyperplasia 550
Diseases affecting extrahepatic and intrahepatic biliary tree 550
Primary sclerosing cholangitis 550
Other forms of sclerosing cholangitis 559
Chronic cholangitis and malignancy 568
Pathology of the peribiliary glands 568
Necroinflammatory and degenerative changes 568
Peribiliary cysts (multiple hilar cysts) 568
Hyperplasia of peribiliary glands 568
Atypical hyperplasia and neoplasia of peribiliary glands 569
Role of peribiliary glands in biliary disease 569
Diseases primarily affecting extrahepatic bile ducts 570
This chapter covers the different diseases in which there is injury to the intrahepatic and/or extrahepatic bile ducts ( Table 9.1 ). The disorders are varied; some, although selectively affecting the bile ducts, are discussed in more detail elsewhere in this volume, and appropriate chapter cross-references are given. In this chapter we emphasize the histological features that characterize the early stages of the disease in which distinctive bile duct lesions may be identified in liver biopsies. In some of these conditions, especially primary biliary cholangitis/cirrhosis (PBC), chronic liver allograft rejection and graft-versus-host disease (GVHD), the interlobular bile ducts up to a diameter of 100 µm are selectively damaged and progressively disappear from the liver, the so-called vanishing bile duct or ductopenic disorders. These are characterized histologically by ductopenia, which has an increasing number of recognized aetiologies and associations. In other diseases, these structures may be affected together with larger intrahepatic and extrahepatic bile ducts, especially in both primary sclerosing cholangitis (PSC) and secondary sclerosing cholangitis. Progressive destruction and loss of the intrahepatic bile ducts typically results in other changes that lead to portal-portal bridging fibrosis and eventually biliary cirrhosis. Thus in their later stages, the various diseases share many pathological features, which in turn closely resemble the changes observed with secondary biliary cirrhosis (reviewed at the end of this chapter).
|Neonates and children|
The term ‘cholangiopathy’ is collectively applied to the diseases in which cholangiocytes are the primary target of the disease process. In contrast to extrahepatic biliary obstruction, chronic intrahepatic bile duct lesions may evolve over many years without obvious morphological cholestasis or bilirubinostasis. This period of evolution is characterized by profound and characteristic changes at the interface between portal tracts and parenchyma: cholate stasis or biliary interface activity, previously known as ‘biliary piecemeal necrosis’. We review these diseases in depth because in the absence of characteristic bile duct lesions, their recognition is essential to distinguish biliary from other forms of chronic liver disease. The role of liver biopsies in patients with PBC or PSC has changed over the last decade, as discussed in relevant sections. Biliary disorders selectively affect different segments of the biliary tree, so the first section outlines the anatomical features relevant to the classification and nomenclature of the biliary disorders (see Chapter 1 ). We then review basic pathogenetic mechanisms common to many of the distinct entities discussed later in the chapter.
Normal morphology of biliary tree and peribiliary glands
The right and left hepatic ducts, their confluence and both the common hepatic duct and the common bile duct are classified as the extrahepatic bile ducts, while the bile ducts proximal to the right or left hepatic duct are referred to as intrahepatic bile ducts. The bile ducts are lined by biliary epithelial cells (BECs) or cholangiocytes, which play a number of roles, in particular contributing to about 30–40% of total bile secretion, participating in bile acid reabsorption and drug metabolism and mediating immune responses. They constitute approximately 4–5% of the liver mass. The intrahepatic branching of the bile ducts is best visualized on a biliary injection cast ( Fig. 9.1 ). There is no sharp delineation of the various segments, but the intrahepatic bile ducts can be practically classified into two main categories:
Large intrahepatic bile ducts. These consist of the right and left hepatic bile ducts and their first to third branches (segmental and area bile ducts), all of which are grossly visible and are also referred to as the ‘perihilar’ bile ducts. They are lined by tall columnar epithelium and are surrounded by a layer of hypocellular collagen (duct wall).
Small intrahepatic bile ducts. These are the branches of the large intrahepatic bile duct and are classified into the ‘septal’ and ‘interlobular’ bile ducts, which are visible only under the microscope, the former being inconsistently sampled in needle biopsy specimens.
As with the large intrahepatic ducts, the septal ducts (>100 µm in diameter) are lined by tall columnar cells with basal nuclei and are surrounded by a fibrous duct wall. In contrast, the interlobular bile ducts are lined by cuboidal cells resting on a basement membrane. The interlobular bile ducts are connected to the bile canalicular network by bile ductules (<20 µm diameter) lined by cuboidal cells and the canals of Hering, which are lined partly by biliary epithelium and partly by hepatocytes. Bile ductules and canals of Hering become obvious in various pathological conditions and are more easily identifiable by immunostaining for biliary keratins.
Peribiliary glands are present within the fibromuscular walls of extrahepatic bile ducts ( Fig. 9.2 A and B ) and also along the large intrahepatic bile ducts. Glandular acini along the large intrahepatic bile duct arise from ductal plates, and their density decreases progressively through infancy and childhood. Similar glandular elements are also found at the neck of gallbladder and the ampulla of Vater. Peribiliary glands around the large intrahepatic bile ducts are subdivided into intramural, nonbranching tubular glands and extramural ramified glands. The latter lie in the periductal connective tissue and have a linear distribution along two opposite sides of the bile ducts in a three-dimensional (3D) model ( Fig. 9.2 C ); they indirectly drain into the bile duct lumen via their own conduit ( Fig. 9.3 A ). The extramural glands show anastomosing bridges and consist of serous and mucous acini ( Fig. 9.3 B and C ). Pancreatic acini without Langerhans islets are found intermingled with peribiliary glandular acini in about 4% of autopsy livers and are probably an intrinsic component of these glands ( Fig. 9.3 D), most likely reflecting a common embryological origin. In addition to their absorptive and secretory activities, the peribiliary glands contain multipotent stem/progenitor cells that can differentiate into hepatocytes, cholangiocytes and pancreatic cells. These cells supposedly contribute to epithelial regeneration, and the defects of the repair process may be involved in the progression of biliary diseases, including the development of periductal fibrosis and bile duct dysplasia in PSC.
The bile duct shares embryological and histological features with the pancreas, since the two organs develop from the endoderm foregut and have nearly identical ductal epithelium, with analogous morphology and immunohistochemical phenotypes. Similar neoplastic and non-neoplastic diseases can also occur in the two organ systems ( Table 9.2 ).
Self-defence system and innate immunity
The human biliary tree is sterile under normal conditions, yet bacterial components such as pathogen-associated molecular patterns (PAMPs) including lipopolysaccharide (LPS) and bacterial DNA from intestinal flora, are detectable in bile and BECs in chronic inflammatory biliary diseases, and even in normal bile. BECs are also consistently exposed to bile contents, including toxic hydrophobic bile acids. To prevent unnecessary inflammatory responses, the biliary tract is equipped with tightly regulated defence mechanisms, which are physical (bile flow, biliary mucus), chemical (bile salts) and immunological (secretory IgA).
In the biliary tree from the intrahepatic septal to extrahepatic bile ducts, BECs contain mucin droplets in the supranuclear cytoplasm, and the mucin secreted into the duct lumen serves as a barrier against unfriendly bile constituents. Another protective mechanism against toxic hydrophobic bile acids is called the ‘bicarbonate (HCO 3 − ) umbrella’. BECs secrete HCO 3 − through anion exchanger 2 (AE2), which contributes to the maintenance of a high pH on the apical surface of the biliary epithelium. The alkaline environment inhibits bile acid uptake and toxicity in BECs ( Fig. 9.4 A ).
Nonspecific bactericidal enzymes such as lactoferrin and lysozyme are demonstrated in the intrahepatic biliary tree and bile. Human β-defensins (hBDs) and cathelicidin, antimicrobial peptides contributing to innate immunity at mucosal surfaces, are expressed in the biliary tract ; hBD1 is constitutively expressed in biliary epithelium, while hBD2 is upregulated in large intrahepatic bile ducts in extrahepatic biliary obstruction, hepatolithiasis and to a lesser degree in PBC and PSC ( Fig. 9.4 B ). Cathelicidin is expressed by normal BECs in addition to hepatocytes. Moreover, bile salts including chenodeoxycholic acid and ursodeoxycholic acid enhance cathelicidin expression through the farnesoid X receptor (FXR) and the vitamin D receptor. Trefoil factor family (TFF) peptides expressed at the apical surface of the epithelium play a major role in mucosal repair.
Although human bile contains several PAMPs in normal as well as diseased livers, PAMPs physiologically do not elicit an inflammatory response in the biliary tree, suggesting that tolerance against commensal PAMPs is important in maintaining the homeostasis of biliary innate immunity. Toll-like receptors (TLRs), which are involved in the recognition of PAMPs, are expressed in normal and damaged BECs ( Fig. 9.4 C ). BECs express TLRs 2, 3, 4 and 5, which bind to ligands, such as PAMPs, double-stranded RNA, LPS and flagellin, respectively. Evidence for the maintenance of tolerance by BECs was demonstrated by the upregulation of interleukin-1 receptor-associated kinase M (IRAK-M), a negative regulator of TLR signalling, in isolated human BECs on stimulation with TLR2 and TLR4. Peroxisome proliferator activating receptor γ (PPARγ), another negative regulator of intracellular TLR signalling, is also associated with the endotoxin tolerance in BEC.
Immunoglobulin A is known to be secreted into bile by binding with the secretory component. Secretory IgA (sIgA) functions in a number of ways to protect the biliary tract; it can directly bind and neutralize bacterial toxins and can bind to bacteria and prevent their adhesion to the mucosal membrane. Also, IgA has been demonstrated to neutralize intracellular microbes and their products. Biliary intraepithelial lymphocytes (bIELs), which are greatly increased in immune-mediated cholangitis, are occasionally encountered in normal intrahepatic bile ducts. Most bIELs are positive for CD8, and some are CD57 positive; these cells may participate in biliary innate immunity.
Disruption of the defence system may contribute to the development and progression of biliary diseases. The distribution of the relevant molecules along the intrahepatic biliary tree is schematically shown in Fig. 9.4 D.
Biliary epithelial injury
Biliary epithelium may undergo a range of histopathological changes that often lack specificity and differ according to the size of the affected ducts; bile duct damage can be classified into several categories, as summarized in this section. More characteristic histopathological alterations are considered and illustrated later in relation to individual disorders.
Basic changes in response to injury
In the extrahepatic and large intrahepatic bile ducts, biliary epithelial swelling and atrophy, disordered nuclear polarity and sloughing are encountered in both inflammatory and neoplastic conditions. This may lead to ulceration with a granulomatous or xanthomatous reaction induced by extravasated bile products. In the interlobular bile ducts, cytoplasmic vacuolation, eosinophilic change, nuclear pleomorphism and hyperchromasia are common degenerative changes. Nuclear ‘piling up’ suggests regeneration, which may be substantiated by immunostaining for proliferative markers, although mitoses are rarely seen. Proliferation of biliary epithelium is associated with subsequent luminal enlargement. Nuclear pyknosis, apoptosis, fading and uneven spacing anticipate duct loss. Injured ducts can be variably distorted with an irregularly shaped lumen. Rupture of the duct basement membrane is followed by dilation which, as in the case of early PBC, may lead to an overestimation of the actual size of the affected duct; small diverticula may rarely form.
Other epithelial changes
Haemobilia and related lesions
Impacted erythrocytes are encountered in bile duct lumens in cases of haemobilia ( Fig. 9.5 A ) or within the epithelium of the small bile ducts in liver biopsy specimens taken after recent endoscopic or surgical biliary manipulation ( Fig. 9.5 B ) or in association with primary or secondary malignancy.
Mucobilia and related lesions
Mucin may be impacted in the duct lumen, and this is occasionally marked, leading to leakage and extravasation with the formation of mucus lakes. This feature is occasionally seen in ‘intraductal papillary neoplasm of the bile duct’ (IPNB, formerly known as ‘biliary papillomatosis’) or in other mucin-producing bile duct tumours. Similar changes are also encountered in non-neoplastic biliary diseases such as PSC and hepatolithiasis, and this lesion is occasionally lined by pleomorphic epithelium, which may be misinterpreted as carcinomatous transformation ( Fig. 9.5 C and D ). Accumulation of foamy macrophages around the damaged bile ducts (xanthogranulomatous cholangitis) should be differentiated from mucin extravasation.
Mucin may become detectable histochemically in the supranuclear cytoplasm and on the luminal surface of interlobular bile ducts as a nonspecific response to various injuries. Metaplasia of biliary epithelium can be observed as follows. Gastrointestinal metaplasia (e.g. pyloric gland metaplasia or intestinal metaplasia) is seen in chronically inflamed large bile ducts and peribiliary glands ( Fig. 9.6 A ). The change is associated with increased expressions of gastric-type mucin core protein (MUC) 5AC and MUC6 and intestinal type MUC2. This MUC expression pattern differs from the normal constitutive MUC3. Goblet cells are frequently seen among the lining epithelial cells, but infrequently in pathological peribiliary glands, where intestinal metaplasia may include Paneth cells ( Fig. 9.6 B ). Intestinal-type epithelium with goblet cells is physiologically present in otherwise normal gallbladders of children, particularly those younger than 6 years. Thus intestinal metaplasia in the bile ducts may represent a reacquisition of the immature phenotype rather than a real metaplastic process. Expression of other molecules in the lining of large intrahepatic bile ducts, such as REG I and trefoil factors, appears related to intestinal or gastric metaplasia. Pancreatic acinar metaplasia is also found infrequently in PSC. Hepatocytic metaplasia occurs in interlobular bile ducts and bile ductules in various pathological situations but remains of uncertain significance ( Fig. 9.6 C ). The cells have a clear or weakly eosinophilic, glycogen-rich cytoplasm with a distinct border and rest on a basement membrane alongside normal biliary cells. They are morphologically almost identical to hepatocytes, although some may express a mixed hepatocytic and biliary phenotype. Squamous metaplasia is rarely encountered in longstanding inflammation of large bile ducts ( Fig. 9.6 D) or in the lining of biliary cysts.
Hyperplasia of lining epithelia of the septal and large bile ducts manifests as micropapillary projections or as a stratification of the epithelium with or without dilation of the duct lumen. In choledochal cysts with pancreatobiliary malunion, the surface epithelium of the dilated ducts often shows high papillary hyperplasia. Peribiliary glands, intramural or extramural, also show hyperplasia and proliferation and participate in the secretion of neutral, carboxylated and sulphated mucins into the bile duct lumen. When prominent, in particular with Clonorchis sinensis infection or hepatolithiasis, the term ‘adenomatous hyperplasia’ or ‘chronic proliferative cholangitis’ has been used ( Fig. 9.6 A ). Hyperplasia of small interlobular bile ducts and ductules leads to an increased number of apparently tortuous structures within the portal tracts.
Biliary intraepithelial neoplasia (bile duct dysplasia)
Biliary intraepithelial neoplasia (BilIN), previously referred to as ‘bile duct dysplasia’, is characterized by atypical, enlarged and hyperchromatic nuclei, an increased nuclear/cytoplasmic ratio and a loss of polarity ( Fig. 9.7 ). It is either micropapillary or flat, with a portion of the duct circumference replaced by a single-layer or multilayered dysplastic epithelium. Three grades are applied according to the degree of cytological and architectural atypia. BilIN-1 demonstrates the mildest changes. BilIN-2 corresponds to intermediate grades of atypia, with more nuclear atypia and focal disturbance of cellular polarity than BilIN-1. BilIN-3 includes so-called carcinoma in situ of the biliary tract. BilIN grades1, 2 and 3 are seen in association with chronic inflammation such as PSC, hepatolithiasis or clonorchiasis, within both large intrahepatic and extrahepatic bile ducts and also in peribiliary glands, and are considered to reflect multistep neoplastic transformation of the biliary epithelium. Metaplastic changes along with expression of gastrointestinal MUCs are frequently observed during the process of malignant transformation.
Necrosis, apoptosis, senescence and autophagy
In response to diverse stresses, cholangiocytes exhibit various cellular changes, such as swelling and shrinkage. This process is followed by either regeneration of the epithelium or cell death leading to bile duct loss. Important cytopathic processes of biliary epithelium include necrosis, apoptosis and cellular senescence. Coagulative or lytic necrosis is occasionally encountered in toxic cholangiopathy.
The role of apoptosis in bile duct injury may be underestimated because apoptotic cells are rapidly eliminated from the biliary tree and are probably shed into bile. Apoptosis is difficult to identify and quantify in H&E-stained sections, but shrunken slender cells with pyknotic nuclei and fragmented and condensed nuclei in the biliary epithelial layer and bile duct lumen are regarded as apoptotic bodies ( Fig. 9.8 A ). This can be confirmed using in situ nick-end labelling and immunostaining of single-strand DNA, both of which detect DNA fragmentation ( Fig. 9.8 B ). Ultrastructurally, there is a reduction of cell volume with aggregated cytoplasmic organelles.
Apoptosis of cholangiocytes has been considered as an important pathogenetic process in several cholangiopathies. Excessive apoptotic activity beyond the proliferative response of bile duct cells will result in ductopenia. In contrast, inhibition of the apoptotic process may cause hyperplasia of bile duct epithelium, with an increased risk of neoplastic transformation. In addition, the induction of apoptosis in cholangiocytes by leukocytes is itself important for the clearance of pathogens from the bile ducts.
BECs of the damaged bile duct in PBC and also chronic allograft rejection frequently show characteristic features such as eosinophilic cytoplasm, cellular and nuclear enlargement, nuclear pleomorphism, multinucleation and irregular arrangement with uneven nuclear spacing ( Fig. 9.9 A ). The affected bile ducts may consequently acquire an atrophic or ‘dysplastic-like’ appearance. These changes are used for the diagnosis of early chronic rejection and may represent ongoing cellular senescence. In support of this, cells with these appearances present cellular senescence markers such as cell cycle regulator genes, p16 INK4 and p21 WAF1/CIP , and increased activity of senescence-associated β-galactosidase (SA-β-gal) ( Fig. 9.9 B ). Cellular senescence has also been observed in damaged bile ducts in PSC and biliary atresia. Cellular senescence is a condition in which the cell no longer has the ability to proliferate, and senescent cells are irreversibly arrested at the G1 phase of the cell cycle. The findings that the expression of senescence-related p21 WAF1/CIP protein is increased in BECs during early chronic rejection and decreased with successful recovery suggest that in chronic liver allograft rejection and PBC, a cellular senescence of BECs is responsible for impaired regeneration of bile ducts. Also, a relatively insufficient proliferative response caused by cellular senescence is also responsible for the progressive loss of bile ducts from apoptosis. Quantitative fluorescence and in situ hybridization showed a significant decrease in telomere length in BECs of the damaged small bile ducts in PBC, compared with normal-looking bile ducts in PBC, chronic viral hepatitis and normal livers. Telomere shortening could be a trigger of cellular senescence.
Senescent cells are also known to remain metabolically active and then contribute to a persistent inflammation. As with cholangiocytes activated by TLRs, senescent BECs participate in biliary proinflammatory responses in various conditions because they are known to aberrantly express chemokines, cytokines, growth factors and matrix metalloproteinases (MMPs), leading to alterations of peribiliary microenvironments ( Fig. 9.9 C ). This process is called a senescence-associated secretory phenotype. Cytokines and chemokines produced by senescent BECs may also facilitate cellular senescence in adjacent BECs and other types of cells in the periductal tissue. These processes are presumably involved in both tissue repair and disease progression of the biliary tract.
Another cellular function closely related to senescence is autophagy, an intracytoplasmic process of self-degradation to remove damaged proteins, organelles and external pathogens (e.g. bacteria) that are engulfed. This process occurring inside lysosomes contributes to cellular homeostasis. Autophagy is activated during cellular senescence, in which it inhibits apoptotic signals. This process also appears to be involved with autoimmunity by presenting degraded intracytoplasmic ‘self’ antigens with major histocompatibility complex (MHC) class II molecules. In the bile duct, molecules involved in autophagy (e.g. microtubule-associated protein-light chain 3b [LC3], p62/sequestosome-1) are expressed in cholangiocytes, particularly of PBC and dysplastic biliary epithelium. Although autophagy may play a role in the development or progression of biliary diseases, further studies are required to conclude whether activated autophagy of cholangiocytes is a primary event or simply secondary to increased cellular stress in damaged bile ducts.
Biliary epithelial cell renewal
Homeostasis of the biliary epithelium operates through a balance between apoptotic cell death or senescence and cell renewal. This is mainly regulated by the Bcl-2 family of proteins. Bcl-2, which prolongs cell survival by counteracting apoptosis, is diffusely expressed by cells of the interlobular bile ducts and ductules, while bax , a promoter of apoptosis, is expressed throughout the biliary tree. Renewal of BECs of small bile ducts and ductules may be secured by progenitor or stem cells located in the canal of Hering, a process unlikely to operate in the septal and large intrahepatic bile ducts. As already mentioned, the conduits of peribiliary glands may be a renewal site for the lining epithelium of large bile ducts and for the regeneration and proliferation of peribiliary glands. Ductular reaction, a common process associated with intra- and extrahepatic cholestasis, has been re-evaluated recently (see later discussion, page 531).
Bile duct loss or ductopenia
Ductopenia is defined as the absence of an appropriately sized bile duct in the portal tract. When severe inflammation is present, the duct may be obscured by numerous inflammatory cells, and immunostaining for keratins 19 or 7 may assist with their identification. Within portal tracts, hepatic arterial branches and interlobular bile ducts of similar size run parallel. This parallelism and the proportion of arteries without accompanying bile ducts in the portal tracts can be used in clinical practice to appreciate the size of affected ducts and also the degree of bile duct loss. Several well-formed portal tracts in a liver needle or wedge biopsy specimen are usually considered enough for the evaluation of bile duct loss, with ductopenia usually defined as the absence of interlobular bile ducts in at least 50% of the portal tracts. Anomalous arteries without portal venous or biliary elements are occasionally encountered in the diseased or even normal livers, and such arteries should not be counted in the evaluation. Alternatively, examination of serial sections can also show actual disappearance of affected bile duct.
Cholangiopathy and cholangitis
Cholangiopathy can be divided into several categories based on morphological and aetiological factors. Cholangitis is morphologically defined as cholangiopathy with inflammatory reactions.
Cholangitis is histologically classified into a suppurative and a nonsuppurative form. These terms are descriptive and not necessarily related to a specific aetiology.
Suppurative cholangitis implies the presence of numerous polymorphonuclear cells around and within the wall as well as within the lumen of bile ducts. This may involve ducts of any size and is occasionally associated with abscess formation—cholangitic abscess ( Fig. 9.10 A ). Microbial infection is often responsible, but the change also occurs in the presence of sterile bile, particularly after bile extravasation, where the detergent effect of the bile salts may produce a chemical cholangitis, and in the acutely rejected liver graft (rejection cholangitis) and cholangitis lenta, where the release of chemokines or cytokines is the likely cause.
Nonsuppurative cholangitis includes a spectrum of bile duct inflammation ( Fig. 9.10 B ), which may be granulomatous, lymphoid or sclerosing, according to the predominant type of inflammatory reaction present.
Granulomatous cholangitis of the interlobular bile ducts constitutes the hallmark of PBC. It is also found in drug-induced liver disease and in sarcoidosis. Nonspecific biliary damage with a granulomatous or epithelioid reaction and lymphoid follicle formation occasionally occurs in livers with other longstanding biliary diseases, in particular PSC. PBC-type granulomatous cholangitis with epithelioid cells forming a mantle around the injured bile duct must be distinguished from the giant cell reaction to extravasated bile seen in nearby ruptured ducts, as in PSC and biliary obstruction.
Lymphoid cholangitis refers to a close association between duct branches, usually interlobular bile ducts, and lymphocytic aggregates, which may show a follicular arrangement. Occasionally, bile ducts are embedded in such aggregates and show epithelial injury. This is found in PBC and PSC with concomitant bile duct destruction, or in nonbiliary disorders, in particular autoimmune hepatitis and viral hepatitis C, where progressive bile duct destruction is generally not a feature. An extreme example of large-duct lymphocytic cholangitis characterized by many lymphoid follicles is referred to as ‘follicular cholangitis’ (see later).
Sclerosing cholangitis with evident bile duct fibrosis develops as a result of longstanding inflammatory, obstructive or ischaemic injury of the bile ducts; it can be obliterative or nonobliterative. The former is characteristic of PSC, although in our experience, it may be seen in secondary forms of sclerosing cholangitis as well. Progressive loss of BECs leads to replacement of ducts by fibrous cords (fibro-obliterative duct lesion). The bile duct wall in longstanding sclerosing cholangitis shows a marked increase in the number of c-kit receptor-expressing mast cells which secrete fibrogenic factors such as histamine, basic fibroblast growth factor (bFGF) and tumour necrosis factor α (TNFα). The biliary epithelium itself produces and secretes fibrogenic substances such as bFGF, transforming growth factor β (TGFβ) and platelet-derived growth factor (PDGF), as well as basement membrane proteins and extracellular matrix proteins. BECs are variably expressing vimentin and might acquire phenotypes of mesenchymal cells, although definite epithelial mesenchymal transition (EMT) of biliary epithelium has not been convincingly demonstrated. The biliary epithelium also has receptors for epidermal growth factor (EGF), leading to autocrine growth stimulation, and c-met for hepatocyte growth factor (HGF), which is released from the activated portal fibroblasts or adjacent hepatic stellate cells. In all forms of sclerosing cholangitis, a marked attenuation of the peribiliary vascular plexus is frequently seen within the sclerotic duct wall, but it remains uncertain whether these changes are secondary to, or responsible for, the bile duct fibrosis.
Aetiological and pathogenetic classification
PBC, PSC, GVHD and hepatic allograft rejection are examples of autoimmune- or alloimmune-mediated cholangitis. Recent studies showed that biliary atresia, particularly the acquired type, and IgG4-related sclerosing cholangitis are also associated with altered immunity. The level and extent of the biliary tree affected differ across and within individual diseases. The peribiliary glands are involved in PSC, GVHD and IgG4-related sclerosing cholangitis. Immune-mediated events are evidenced by an accumulation of activated auto- or alloreactive T lymphocytes at the site of bile duct destruction. There is a mixture of immunocompetent cells, particularly CD3+, CD4+ and CD8+ T cells that bear the T-cell receptor α/β. This supports T-cell cytotoxicity and cytokine release in the pathogenesis of the bile duct lesion. The proportion of CD4+ and CD8+ T cells does, however, differ according to individual diseases or stage of development. Regulatory T cells (Tregs) seem to play an important role in the initiation of the process.
Altered innate immunity
At the initiation and during progression of immune-mediated diseases, there is evidence that innate immunity is involved and molecules related to innate immunity are expressed in biliary epithelium and infiltrating inflammatory cells. An innate immune response to bacterial components is speculated in the pathogenesis of PBC and PSC. CD4+ T helper type 17 (Th17) cells, characterized by the secretion of interleukin (IL)-17, are generated from Th0 cells by IL-6 and IL-1β and maintained by IL-23. IL-17-positive cells are identified in the chronic inflammation of bile ducts in PBC, which is causally related to innate immune responses to PAMPs. PSC is also triggered by bacteria or PAMPs (e.g. LPS, lipoteichoic acid) that enter the portal circulation through inflamed intestinal mucosa. PAMPs activate inflammatory cells in the liver through pattern recognition receptors, including TLRs and CD14, leading to the secretion of cytokines, which in turn activate natural killer (NK) cells (IL-12) and promote recruitment and activation of lymphocytes (TNFα, IL-1β, CXCL8).
Immunopathological environments of portal tracts and bile ducts
Target antigen(s) in bile ducts
The immune process is targeted against allo- or autoantigens that may be abnormally or aberrantly expressed in the BECs or absorbed from the bile and deposited in the BECs. The alloimmune response preferentially affects the interlobular bile ducts. In liver allograft rejection the recipient’s immune response is directed against allogeneic antigens (peptide antigens presented by allogeneic MHC or allogeneic MHC antigens themselves) on the donor bile ducts. In GVHD, MHC-related antigens induced on the BECs by locally released cytokines or viral infections are targeted by grafted lymphoid cells. Antimitochondrial antibodies (AMAs) are detected against pyruvate dehydrogenase complex E2 subunit (PDC-E2) in PBC. They may cross-react with exogenous microbial components such as LPS, which bear a molecular mimicry for PDC-E2. In PSC, cross-reactive peptides shared by biliary and colonic epithelium and bacterial antigens have been proposed as possible targets. Increased expression of both heat shock protein 60 (HSP60) and lipid A, a constituent of endotoxin, has been demonstrated in the cholangiocytes in PBC and PSC. In addition, the BECs contain antigenic substances such as carbonic anhydrase II, against which autoantibodies have been detected in sera of patients with immune-mediated cholangitis.
Antigen presentation and recognition
Two types of antigen-presenting cells (APCs), professional and nonprofessional, of which BECs are a possible example, may be involved in immune-mediated cholangitis. Professional APCs, especially dendritic cells and to a lesser degree macrophages, are the most important regulators of initial T-cell activation. Dendritic cells that express B7, HLA-DR and S100 molecules are observed around and occasionally within the epithelial layer of damaged bile ducts ( Figs 9.11 and 9.12 A and B ). At least two molecules, B7-1 and B7-2, work as co-stimulatory ligands for CD28 expressed on surrounding T cells. Focal staining for B7-2 on BECs expressing aberrantly HLA-DR suggests that the BECs may present self antigens, although this issue is still controversial.
Bile ducts and the portal microenvironment
In addition to being the main target of immune-mediated cholangitis, bile ducts participate actively and passively in accumulation, activation and proliferation of immunocompetent cells. Expression and secretion of cytokines, chemokines and their receptors by multiple cell types around the damaged bile ducts as well as by BECs themselves are central to the progression of bile duct injury. However, a triggering event that damages BECs or changes the inflammatory microenvironments of the portal tract is required to start the process in genetically susceptible individuals.
BECs interact with members of the immune system in a number of ways. BECs, particularly in small bile ducts, express constitutively low levels of surface immune molecules such as MHC class I; very late antigen (VLA) 2, 3 and 6; intercellular adhesion molecule 1 (ICAM1) and lymphocyte-associated antigen 3 (LFA3). Importantly, during an inflammatory response, BECs upregulate their expression of these molecules. This is shown by the addition of inflammatory cytokines (e.g. TNFα, IL-1, IFN-γ) to culture systems. Local release of cytokines, particularly IFN-γ, TNFα and chemokines, also induces and upregulates the bile duct expression of MHC class II ( Fig. 9.12 A and B ), ICAM1, LFA3 and vascular cell adhesion molecule 1 (VCAM1) ( Fig. 9.12 C and D ). LFA3 on BECs can interact with CD2 molecules on killer T cells, and VCAM1 binds to VLA4 on leukocytes. Overall, the increase in leukocyte adhesion molecules facilitates tissue-specific migration by slowing down leukocyte circulation around the damaged epithelium, promoting trafficking to the target site. Human BECs also constitutively express IL-6, IL-8 and monocyte chemotactic protein 1 (MCP1), which are important chemotactic agents for neutrophils, monocytes and T cells.
Recruitment of inflammatory cells and epitheliotropism
Induction and upregulation of immune molecules also occur on the adjacent microvasculature and lead to recruitment of more inflammatory cells. The leukocytes, which accumulate around the bile ducts, express integrins, LFA1 and VLA4. They migrate from the peribiliary vascular plexus, whose constituent capillaries strongly express chemokine-induced adhesion molecules (ICAM1, VCAM1 and ELAM1/E-selectin), which secures extravasation and localization of inflammatory cells. The process facilitates cell positioning or adhesion around the bile ducts, antigen presentation, co-stimulatory signalling and activation of antigen-specific CD4+ T cells. After migration, the immunocompetent cells must penetrate into the bile duct epithelium, epitheliotropism ( Fig. 9.13 A ). The presence on duct basement membranes of binding sites for lymphocyte receptors and the upregulation of integrins and other adhesion molecules facilitate the process. Surface expression of ICAM1, LFA3 and VCAM1 on bile duct cells ( Fig. 9.12 C and D ) suggests linkages between damaged bile ducts and lymphocytes which secure antigen presentation to periductal lymphocytes and trigger effector mechanisms. Point and broad contacts between infiltrating lymphocytes and both BECs and extracellular matrix are demonstrated ultrastructurally, and such interactions appear to involve binding of CX3CR1 to fractalkine, which is upregulated in injured bile duct epithelium.
Th1/Th2 balance and regulation of immune reactions
Along with the breakdown of self-tolerance, the relative strength of helper T cell type 1 (Th1) and type 2 (Th2) responses and the resultant cytokine milieu are determinants for the characterization and continuation of bile duct damage. In PBC, PSC and liver allograft rejection, there is a predominance of Th1 subsets, which produce IL-2 and IFN-γ and stimulate both the proliferation of cytotoxic T lymphocytes (CTLs) and the local production of IFN-γ and TNFα. The latter are known to induce and upregulate expression of MHC class I and II and adhesion molecules on BECs. Expressions of IL-5, IL-6 and TGFβ are also noted in the majority of cases of immune-mediated cholangitis. These alterations in the cytokine milieu are supposed to contribute to antibody production by B lymphocytes. Unlike classic autoimmune biliary diseases, Th2 predominance is a characteristic feature in IgG4-related sclerosing cholangitis, where the combined overproduction of IL-4 and IL-10 is thought to play a central role in IgG4 induction.
Tregs are important for the regulation of immune-mediated cholangitis and play a critical role in self-tolerance. One study showed that FoxP3-expressing Tregs were lower in PBC portal tracts than in chronic hepatitis C and autoimmune hepatitis; a deficiency in Tregs was also found in daughters and sisters of PBC patients when compared with controls. However, another study showed that the amounts of infiltrating FoxP3+ Tregs in portal tracts were similar in PBC and chronic viral hepatitis and correlated with the degree of portal inflammation regardless of the disease. Interestingly, the Treg population is increased in the bile duct lesions of IgG4-related sclerosing cholangitis.
Several effector mechanisms have been proposed for immune-mediated bile duct damage, but auto- or alloreactive T cells, particularly CTLs, probably play the major role in causing epithelial cell death.
T cell-mediated cytotoxicity
The initiation of apoptosis, a major form of cell death in immune-mediated cholangiopathy, may occur through different yet interrelated mechanisms, as follows:
Direct cytotoxicity by CD4+ and CD8+ T cells; the former are mainly dependent on the Fas/Fas ligand (FasL) interaction, whereas the latter rely on the perforin–granzyme exocytosis pathway. Most CTLs in allograft rejection are MHC class I-restricted CD8+ T cells, while CD4+ cells belonging mainly to the Th1 subset predominate in PBC and PSC. These T cells may recognize PDC-E2.
Activation of the Fas receptor (FasR)/FasL system is the best-studied model of apoptosis. In PBC, PSC and allograft rejection, FasR is strongly expressed on the damaged bile duct cells ( Fig. 9.13 B ), which are surrounded or infiltrated by FasL-expressing cytotoxic T cells of Th1 subset. The exact stimuli for FasR expression on the bile duct epithelium may vary with individual diseases.
Induction of cytokines, TNFα and IFN-γ
The occurrence of apoptotic cell death through TNFα is supported by expression of both mRNA and protein for the TNFα and TNFα receptor on damaged bile ducts. The expression of TNF-related apoptosis-inducing ligand (TRAIL) by diseased BECs in PBC and PSC may be an attempt by BECs to control the inflammatory responses by targeting death receptor (DR)-expressing leukocytes.
Serum autoantibodies are detected in PBC and PSC, but it remains debatable whether AMA or centromere-type antinuclear antibodies (ANAs) in PBC and ANA or perinuclear antineutrophil cytoplasmic antibodies (pANCAs) in PSC have a pathogenetic role. A majority of the plasma cells infiltrating the portal tracts in PBC secrete AMAs of IgG, IgA and IgM classes, and these antibodies may be involved in antibody-dependent cytotoxicity. CD20+ B cells and immunoglobulin-positive cells detected in the portal areas may result from a proliferation of autoreactive B cells stimulated by CD4+ T cells of Th2 subset. The demonstration of selective caspase activation by dimeric IgA with specificity for the PDC-E2 component of AMA in an in vitro cell line transfected with human polymeric immunoglobulin receptor may support a pathogenetic role of AMA in bile duct injury of PBC.
In liver allograft rejection and GVHD, preformed antidonor MHC class I antibodies and antibodies directed against major ABO blood group antigens, which are normally expressed on the BECs, may be responsible for antibody binding and complement-fixing damage.
Oxidative stress and other mechanisms
Reactive oxygen species (ROS) generated in BECs themselves and also by inflammatory cells around and within the bile duct epithelium may be responsible for apoptosis and cellular senescence of BECs in PBC and allograft rejection.
In acute liver allograft rejection and to a lesser degree in PBC and PSC at early stages, there is a marked eosinophil infiltration in portal tracts with widespread deposition of eosinophil cationic protein granules known to be cytotoxic for bile ducts. Blood eosinophilia may be associated with this infiltrate. Eosinophils are correlated with an upregulation of eosinophil chemotactic factor IL-5. Eosinophils are also prominent in IgG4-related sclerosing cholangitis.
Predisposing and augmenting factors
There is good evidence that genetically determined abnormalities of immunoregulation play a role in the development of bile duct damage in PBC and PSC. Several HLA haplotypes are known to increase the risk of PBC and PSC. Deficiency in Tregs found in daughters and sisters of PBC patients suggest that genetic predisposition exists before disease development. Although recent genome-wide association studies (GWAS) have identified non-HLA susceptibility loci for biliary disorders, many of them have previously been detected in other conditions, suggesting that risk loci detected by GWAS are likely to represent broad phenotypic features such as inflammation rather than disease-specific affections. Genetic factors in addition to HLA disparity are also likely determinants of whether individuals will be strong or weak rejectors after liver grafting, but none has been specifically identified, except for non-Caucasian recipients being at increased risk for graft rejection.
Bacterial or viral infections may play a primary or secondary role in the progression of immune-mediated bile duct injury. Bacteria, particularly enterobacteria, have been implicated in the progression of PBC, PSC and hepatolithiasis. Bacterial or viral infections may aggravate or initiate the alloimmune-mediated bile duct lesions in GVHD and hepatic allograft rejection. Cytomegalovirus (CMV) infection has been shown to increase the risk of ductopenic rejection, although the pathogenetic role of CMV in this regard remains in doubt. Hepatitis C infection and treatment with IFN-α may predispose to the development of ductopenic rejection after orthotopic liver transplantation (OLT).
Hydrophobic bile acids
Normally, the biliary epithelium is protected against the detergent effects of bile acids by phospholipids, resulting from the incorporation of the bile acids into mixed micelles with phospholipids and cholesterol. The detergent power of bile acids augments with increasing relative hydrophobicity of bile acids. Endogenous hydrophobic bile acids may enhance the bile duct lesion by dissolving membrane lipids or stripping key hydrophobic proteins off the outer layer of the apical membrane of BECs and by promoting apoptosis.
Bacterial, fungal, parasitic and viral cholangitis may affect normal and immunocompromised hosts. In particular, the biliary tract and the liver are prone to infestation by liver flukes such as Clonorchis sinensis and Opisthorchis viverrini, which are endemic in East Asia, especially northern Thailand and some parts of Korea (see Chapter 7 ).
Bile ducts are affected in Alagille syndrome, cystic fibrosis, fibropolycystic diseases (e.g. congenital hepatic fibrosis, Caroli disease) and MDR3 deficiency (see Chapter 3 ).
Drug- or toxin-induced cholangiopathy
Although cholangiocytes have a low metabolic activity compared with hepatocytes, bile duct injury, cholangitis and ductopenia are occasionally reported in drug-induced reactions. Drug-induced cholangiopathies (see also Chapter 12 ) often result in severe cholestasis. Variable types of cholangitis may be associated and at times lead to progressive loss of bile ducts and prolonged cholestasis. Pathogenically, some may be related to immune-mediated processes such as hypersensitivity reaction or to ischaemic damage such as floxuridine (FUDR)-induced cholangiopathy (see later). Toxic cholangiopathy has also been reported experimentally and clinically. Similar but more cytotoxic or cytopathic bile duct injury has been produced experimentally or accidentally by α-naphthylisothiocyanate, 4,4′-diaminodiphenyl methane or paraquat.
The peribiliary vascular plexus is essentially supplied by branches of the hepatic artery, which makes the bile ducts, unlike the parenchyma, particularly vulnerable to any interference with arterial flow. The small constituent vessels are well demonstrated by immunostaining for CD34 or factor VIII-related antigen ( Fig. 9.14 A ). They form a three-layer plexus around the extrahepatic, large intrahepatic and septal bile ducts, with the small portal vein branches and small arteries around the bile ducts (peribiliary arteries) being included in the outer layer. This organization becomes less evident around the interlobular bile duct and ductules, which nevertheless remain entirely dependent on arterial blood. Primary and secondary damage to the hepatic arterial branches or to the peribiliary vascular plexus itself, whether from thrombotic occlusion, foam cell arteriopathy or vasculitis, may lead to ischaemic cholangitis. This may take the form of BEC damage, cholangitis without necrosis and bile duct fibrosis. Erosion, focal necrosis or infarction of bile ducts and extravasation of bile (biloma) may ensue. Constituent vessels of the perivascular plexus may be affected variably ( Fig. 9.14 A and B ). Ductopenia, biliary stricture and cholangiectasis are common complications, with infection often developing in the devascularized tissue. The biliary tree in allografts is particularly susceptible to ischaemia because of a lack of vascular collaterals in the early post-transplant period (see Chapter 14 ). Necrotizing arteritis (polyarteritis nodosa) of hepatic arterial branches and microvascular occlusion from sickle cell disease are rare causes of ischaemic bile duct injury.
Pathological features secondary to impaired bile flow
The complex mechanisms of bile secretion are described in Chapter 1 . Cholestasis denotes a defect in bile secretory mechanisms leading to an accumulation in the blood of substances normally excreted in the bile— biochemical cholestasis . This is often but not invariably associated with deposition of bile in the liver, which can be visualized microscopically— morphological cholestasis . Mechanical or obstructive cholestasis usually refers to lesions of the extrahepatic biliary tract (extrahepatic cholestasis) but is occasionally produced by intrahepatic processes that cause jaundice only when the flow is compromised in the majority of the intrahepatic biliary passages, as in the late stage of PBC. Although mechanical obstruction thus clearly contributes to cholestasis originating within the anatomical confines of the liver, most use the term ‘intrahepatic cholestasis’ to indicate cholestatic syndromes without demonstrable mechanical obstruction. Such functional intrahepatic cholestasis is common to a number of parenchymal injuries of various aetiologies, when complex metabolic alterations at the level of the hepatocyte produce an abnormally thick bile that becomes inspissated in the canaliculi, first and predominantly in the perivenular region.
There may be a striking discrepancy between morphological and clinical/biochemical cholestasis. Conditions that are clinically cholestatic and considered as primarily biliary diseases may progress without evident bilirubinostasis on biopsy. This is mainly the case in partial obstruction or destruction of the biliary passages, as in the early stages of PBC and PSC. In this situation the patient experiences pruritus and shows biochemical signs of cholestasis, such as elevated serum alkaline phosphatase (ALP), γ-glutamyltransferase (GGT), cholesterol and bile acids, whereas the level of conjugated bilirubin remains normal or is only marginally raised and remains so until the disease is well advanced. Morphologically, this preicteric phase is characterized by profound changes at the portal or portoseptal interface (biliary interface activity).
Microscopically, cholestasis of any aetiology is characterized by an exclusively or predominantly perivenular localization of bilirubin deposition in hepatocytes, inspissated bile in dilated canaliculi and variable degree of bile regurgitation into the perisinusoidal space with phagocytosis by Kupffer cells ( Fig. 9.15 ). This may be more correctly described as ‘bilirubinostasis’, although products other than the bilirubin are concomitantly retained but not readily visualized microscopically. In severe cholestasis or in cholestasis of longer duration, such pigment deposition may extend to the periportal region, and cholestatic or biliary liver cell rosettes may develop. These are particularly common in children and consist of dilated canaliculi surrounded by more than two hepatocytes in a pseudotubular arrangement (cholestatic rosettes) ( Fig. 9.16 A ). The rosettes express some biliary keratins ( Fig. 9.16 B ) and on 3D reconstruction are found to communicate with bile ducts and ductules. The predominantly perivenular localization of bilirubinostasis may be explained by both the lower oxygen tension in the perivenular region and the fact that bile acid secretion and corresponding bile water flow are regulated by mechanisms different from those responsible for bilirubin secretion. Since the periportal zone is the main contributor to ‘bile acid-dependent’ bile flow, the canaliculi of the perivenular zones are perfused with a smaller amount of fluid generated by mechanisms progressively independent of bile acids as one approaches the hepatic venule. Such zonal differences predispose to a primarily perivenular distribution of canalicular precipitates during cholestasis of any cause.
In addition to bilirubinostasis, cholestasis is associated with changes in staining patterns of various enzymes. In particular, the basolateral membrane of the hepatocyte acquires microvilli that show a positive reaction for Mg 2+ -ATPase, whereas this has disappeared in the canalicular membrane. The pattern suggests a reverse secretory polarity, as confirmed by the demonstration of the canalicular bile salt export carrier on the basolateral membrane. The latter also shows a striking increase in histochemically demonstrable serum ALP and GGT, possibly from a high local concentration in detergent bile acids causing release of these enzymes from the liver cell membrane.
On electron microscopy, cholestasis is recognized by a typical pattern of changes in and around the canaliculi ( Figs 9.17 and 9.18 ), even in the absence of any visible bile pigment accumulation on light microscopy. The canaliculi are variably dilated with a loss of microvilli, and their lumen may be filled with electron-dense material representing bile concretions. However, the tight junctions are preserved; the pericanalicular ectoplasm is thickened, and the hepatocytes show dilated endoplasmic reticulum, with the presence of vacuoles containing whorled membranous material and lipofuscin inclusions. Mitochondria exhibit curled or circular cristae.
Biliary interface activity
Biliary interface activity, previously known as ‘biliary piecemeal necrosis’, refers to a disruption of the parenchymal limiting plates by a complex process which includes cholate stasis, ductular reaction and fibroplasia in variable combinations ( Figs 9.19 and 9.20 A–C ). The recognition of biliary interface activity is important because in the absence of cholestasis and of characteristic bile duct injury, it is the main clue to diagnosing a primarily biliary disorder. The two main components, cholate stasis and ductular reaction, are discussed separately because either may dominate the picture and evolve distinctively, which may reflect the severity and duration of interference with bile flow, or at times for no obvious reason. Fibrosis invariably develops when cholate stasis and ductular reaction persist.
The term ‘cholate stasis’ acknowledges that the cytological alterations are thought to be caused by the intracellular detergent action of retained bile acids (see Chapter 1 ). Periportal hepatocytes are preferentially affected because they are the site of maximal bile acid transport in the normal liver. The hepatocytes are swollen and rounded with a distinct border and a clear cytoplasm that may contain web-like membranous and perinuclear granular remnants ( Fig. 9.19 A ). They often contain copper and its binding protein, a polymerized form of metallothionein, which accumulates in lysosomes where it is readily demonstrated as black/brown granules by orcein stain (see Chapter 2 ). This indirect method is a useful diagnostic tool since, at an early stage, in the absence of bilirubinostasis, the demonstration of orcein-positive granules in the periportal regions is a good indicator of a chronic biliary process ( Fig. 9.19 B ). As the lesion progresses, the thread-like reticular cytoplasm, feathery degeneration, acquires a greenish brown tinge from impregnation with bilirubin, and bile pigment may later accumulate ( Fig. 9.19 C ). During the process there is cytoskeletal injury, which often results in the formation of Mallory–Denk bodies (MDBs) ( Fig. 9.19 D). This is thought to result from a toxic effect of bile acids, and possibly copper, on the microtubules with consequent aggregation of intermediate microfilament proteins. MDBs in chronic cholestasis are identical morphologically and chemically to those observed in alcoholic liver disease, but differ in their periportal rather than centrilobular location.
The small intralobular bile ducts, ductules and canals of Hering have the property of increasing in number in many forms of liver disease, especially in cholestasis. The ductules are accompanied by an inflammatory infiltrate and by fibrosis, the composite picture referred to as ‘ductular reaction’ ( Fig. 9.20 B and C ). Their origin is still a matter of debate. The neoductules, lined by cuboidal or flattened cells, seem to arise from ‘ductular metaplasia’ of periportal hepatocytes in longstanding cholestasis, and to a lesser extent from a proliferation of pre-existing ductules, although the relative role of these two mechanisms may vary depending on degree of biliary obstruction or duration of cholestasis. A shift from the hepatocellular to the biliary cell phenotype can be demonstrated for keratins ( Fig. 9.20 D), blood group antigens, chromogranin and ‘bile duct-type’ integrins VLA 2, 3 and 6. The mechanism inducing ductular metaplasia of periportal hepatocytes appears to be complex, involving pericellular matrix components, possibly bile salts, hepatic stellate cells (HSCs) and various humoral and neural factors. A contribution of bone marrow-derived progenitor or stem cells to the ductular reaction remains speculative in the human liver.
Reactive ductular cells contain dense-core neuroendocrine-type granules and express chromogranin, neural cell adhesion molecule (NCAM or CD56) and parathyroid hormone-like peptide, a factor which modulates cellular growth and differentiation. These findings led to speculation that reactive ductular cells may produce a substance that exerts an autocrine or paracrine regulatory role on the growth of ductules or in the ductular metaplasia of periportal hepatocytes. In the setting of primary cholangiopathies, reactive ductules coexpress NCAM and Bcl-2, suggesting an histogenesis that recapitulates the early stages of biliary ontogenesis.
The role of the ductular reaction is incompletely understood. The ductules may provide abortive bypass mechanisms for the drainage of bile in diseases associated with destruction of interlobular bile ducts, such as PBC. The ductular cells reabsorb bile acids and may thus protect the hepatocytes from the deleterious effect of bile acid overload. Reabsorption with leakage of bile components leads to a marked inflammatory reaction, which consists mainly of polymorphonuclear leukocytes closely associated with the reactive ductules in the oedematous fibrous matrix at the portal periphery ( Fig. 9.20 C ). These morphological features of acute cholangiolitis represent a tissue reaction to irritant chemical stimuli rather than a true bacterial infection.
Ductular reaction and the accompanying inflammatory reaction play an important role in portal and periportal fibrosis. The ductules produce and secrete a variety of biologically active materials such as cytokines and chemokines, including TNFα, IL-6, IL-8, monocyte chemotactic protein 1 (MCP1) and nitric oxide (NO), and also express aberrant NCAM, underpinning their potential role in the inflammatory reaction and concomitant fibrogenesis. Surrounded by a periodic acid-Schiff (PAS)-positive basement membrane, the neoductules are invariably set in a loose connective tissue matrix deposition which contains type IV collagen and laminin, in part synthesized by the ductular cells themselves. Fibrosis also results from stimulation of mesenchymal cells. Reactive ductules express growth factors, such as connective tissue growth factor (CTGF), TGFβ2 and platelet-derived growth factor (PDGF), which activate mesenchymal cells and matrix production. Intimately associated with the ductules are myofibroblasts and transitional cells between HSCs and myofibroblasts expressing smooth muscle actin and desmin. They are responsible for the production of tenascin in the early stage of ductular reaction, followed later on by deposition of type VI collagen, matrix components and interstitial collagen types I, III and V.
In this way, the ductular reaction is a pacemaker for the development of progressive fibrosis in chronic cholestatic liver disease, eventually resulting in biliary cirrhosis and the associated haemodynamic alterations. In the two chronic cholestatic disorders, paucity of the intrahepatic bile ducts (Alagille syndrome) and chronic liver allograft rejection, in which ductular reaction is essentially absent, periportal fibrosis is similarly inconspicuous. The ductular reaction and periductular fibrosis are reversible in the early stage. After removal of the proliferative stimulus (e.g. after relief of bile duct obstruction), the excess ductular cells are deleted by apoptosis. Regression of the ductular reaction is accompanied by regression of the periductular fibrosis.
Ductular metaplasia of hepatocytes
Periportal hepatocytes facing the portal connective tissue show ductular features with coexpression of biliary markers such keratin 7 (K7) or 19 (K19) while still bearing hepatocellular phenotypes, such as deposition of orcein-positive granules. Small-cell changes of periportal and occasionally midzonal hepatocytes are a reflection of chronic biliary disease, and such changes could suggest diagnosis of PBC. Expression by periportal hepatocytes of biliary K7 and K19 is also an early marker of chronic cholestasis, with K7 usually becoming positive earlier than K19.
In chronic large-duct obstruction and intrahepatic bile duct loss, biliary interface activity and fibroplasia steadily progress. The limiting plates become irregular with separation of hepatocytes by deposition of loose fibrous tissue, which contrasts sharply with the compact collagen of the original portal tract or later of the central core of the fibrous septa ( Fig. 9.21 A ). Hepatocytes manifesting cholate stasis become intermingled with smaller hepatocytes having a darker-staining cytoplasm that acquire phenotypic characteristics of duct cells, in particular the expression of K7 and K19 ; these merge imperceptibly with the reactive neoductules. The associated inflammatory infiltrate is usually less dense and of different cellular composition than that observed at the interface in autoimmune hepatitis (see Chapter 8 ). Neutrophils predominate, and there is variable accumulation of cholesterol/lipid-laden macrophages with a foamy or microvesicular appearance—xanthomatous cells (see Fig. 9.19 C ). These may be difficult to distinguish from hepatocytes showing cholate stasis on haematoxylin and eosin (H&E)-stained sections.
Progressive fibrosis in the periportal zone produces gradual enlargement of the portal tracts, followed by the formation of fibrous septa bridging adjacent portal tracts, and eventually the development of a micronodular cirrhosis with parenchymal regeneration and distortion ( Fig. 9.21 B ). Until well advanced, the basic architecture is preserved, with hepatic venules and portal tracts maintaining an almost normal anatomical relationship, thus the term monolobular cirrhosis . This provides a hint as to the biliary aetiology of the lesion but may lead to difficulty in deciding morphologically when a true cirrhosis has been established ( Fig. 9.21 B ). The features of cholate stasis and the deposition of oedematous collagen matrix produce a striking ‘halo’ at the interface between hepatocytes and septa, further indicating the biliary nature of the lesion ( Fig. 9.21 B–D ).
Much of the cellular and tissue damage is caused by retention of hydrophobic, cytotoxic bile acids. Ursodeoxycholic acid (UDCA) appears to protect rat hepatocytes in vitro and in vivo against such cytotoxic damage. UDCA inhibits, at least in part, the biological and toxic effects of the endogenous bile acids by reducing their concentration within and around the liver cells, while UDCA itself is devoid of toxicity. This explains the wide use of this drug in the treatment of chronic cholestatic disorders in both adults and children.
Vanishing bile duct syndrome
Vanishing bile duct syndrome (VBDS) defines a clinicopathological complex in which cholestasis is associated with loss of the intrahepatic bile ducts or ductopenia. A heterogeneous group of conditions have been variably associated with VBDS ( Table 9.3 ). Immune processes, infections, drugs and ischaemia have been incriminated as causes of the bile duct destruction which may occur within months or develop over many years. In this respect the clinician can distinguish an acute or subacute form evolving over months, in which ductular reaction and portal fibrosis are inconspicuous and features of acute rather than chronic cholestasis dominate the picture; this is exemplified by the ductopenia observed in allograft rejection, severe GVHD, with some drugs and in treated Hodgkin lymphoma. In contrast, progressive disappearance of the bile ducts may evolve over one or two decades accompanied by the changes of chronic cholestasis, portoseptal fibrosis and eventually a biliary cirrhosis; this is the pattern characteristic of PBC, sclerosing cholangitides and occasionally sarcoidosis and drugs. The interlobular bile ducts and bile ductules are mainly affected in PBC, GVHD, hepatic allograft rejection and drug-induced ductopenia, whereas ductopenia generally follows the involvement of septal and large intrahepatic bile ducts in PSC and in extrahepatic biliary atresia.
|Infantile and childhood diseases ( Chapter 3 )|
|Infectious diseases ( Chapter 7 )|
|Drug- or toxin-induced biliary injury ( Chapter 12 )|
|Neoplastic diseases ( Chapter 13 )|
Recovery from VBDS has been observed, especially in the liver allograft or after drug toxicity. Regrowth of the bile ducts is preceded by marginal ductular reaction. HSCs or progenitor cells, presumably migrating from the periportal areas into the biliary channels, may be involved in the process.
Diseases primarily affecting intrahepatic bile ducts
Primary biliary cholangitis
PBC is an autoimmune liver disease of unknown aetiology that selectively affects the small intrahepatic bile ducts and variably the hepatocytes ( Fig. 9.22 ). Along with the progressive bile duct damage and chronic cholestasis, biliary fibrosis/cirrhosis then develops, leading eventually to hepatic failure. The term ‘primary biliary cirrhosis’ was established by Ahrens et al. in 1950 and has been widely used for more than six decades. However, the term is inaccurate for many patients in whom a true cirrhosis only develops in the later stages of the disease, or sometimes not at all. Because of these limitations, an alternative term ‘primary biliary cholangitis’ was recently proposed by an international panel of experts to describe this condition. The move in terminology from ‘cirrhosis’ to ‘cholangitis’ was intended to remove the misconceptions and possible stigmata associated with having a diagnosis of cirrhosis while still allowing continued use of the acronym ‘PBC’. Although there are also criticisms of the newly proposed terminology, notably the tautologous use of the words ‘biliary’ and ‘cholangitis’, it is now being used widely. ‘Chronic nonsuppurative destructive cholangitis’ (CNSDC) accurately describes the morphological changes involving bile ducts in PBC, but this term has not been widely accepted for routine clinical practice.
PBC usually affects middle-aged to elderly women, with a peak incidence in the 40–60 age-group. Unlike PSC, it is rare before age 30. There is a female preponderance of 9 : 1–10 : 1. This could be related to the increased expression of oestrogen receptor found in bile duct cells and hepatocytes in PBC, because expression of oestrogen receptor and relevant molecules in cholangiocytes increases susceptibility to Bcl-2 family-mediated apoptosis. The disease is generally similar in both genders, except that males were found to have less pruritus and skin pigmentation, and a higher incidence of hepatocellular carcinoma. Although PBC occurs worldwide and has been found in all races, there is some geographical variation in incidence, with few cases reported from the Indian and African continents. In a combined European study, overall prevalence was 23 per 1 million, but with regional variations ranging from <10 to >60 per 1 million. An increased awareness of the condition and the diagnosis of asymptomatic patients seem to account for the apparent rise in incidence and prevalence of PBC.
Insidious in onset, the presenting features are usually intense pruritus and lethargy with increasing skin pigmentation and eventually cholestatic jaundice, although icterus may not develop for years after the initial symptoms. The number of asymptomatic PBC patients is relatively increased, and some remain so, whereas others gradually progress to symptomatic PBC. Some patients first present with jaundice, particularly during pregnancy or after drug intake, and hepatic decompensation occasionally may bring the patient to clinical notice. Portal hypertension may become manifest before a true cirrhosis is established. Xanthomas/xanthelasma occurs in 30% of patients. The incidental finding of hepatomegaly, increased serum ALP or serum AMA may lead to the recognition of patients who are asymptomatic. They may remain asymptomatic for years and experience a normal life expectancy, although an increased mortality is shown for those in whom symptoms subsequently develop.
PBC eventually progresses in most cases. Its natural history is presumed to be about 20 years, and the onset of jaundice, usually in the last 5–7 years, heralds clinical deterioration. Death is usually caused by hepatocellular failure, with bleeding from oesophageal varices in approximately 30% of patients ; nonhepatic causes of death are found in <20%. Predicting the prognosis is a major issue, with the use of liver transplantation as the only treatment option in patients with advanced disease. Several models based on multiple regression analysis of clinical, biochemical and histopathological variables have been proposed, and serum bilirubin levels, presence of bridging fibrosis/cirrhosis and tissue deposition of abundant copper-associated proteins are the most significant risk factors. Ductopenia has also been shown to predict disease progression in PBC.
The coexistence of PBC with other autoimmune diseases is found in about 55% of patients. Features of Sjögren syndrome or the ‘sicca complex’ of dry eyes and dry mouth may be found in more than half the patients. PBC may be associated with all or some of the features of the CREST syndrome—calcinosis, Raynaud phenomenon, oesophageal dysfunction, sclerodactyly and telangiectasis —to which the presence of anticentromere antibodies and keratoconjunctivitis sicca were later added. Other potential associations include rheumatoid arthritis, autoimmune thyroiditis, renal tubular acidosis, coeliac disease, systemic lupus erythematosus and vasculitis. Many other conditions have also been reported, but most of these rare associations are probably coincidental.
Liver enzymes show a normal or mildly elevated bilirubin level (34–68 µmol/L; 2–4 mg/dL) at early stages, accompanied by a striking and disproportionate elevation of serum ALP to levels three to five times normal, although a normal value does not preclude the diagnosis. Increased bilirubin raises a concern of advanced disease. There is a moderate elevation of serum aminotransferase (transaminase) levels (100–150 IU/L). Serum immunoglobulins of all three major classes may be increased, but greatly elevated IgM in excess of 150% of normal is most consistent and is a distinctive feature of PBC. The most helpful diagnostic test is the demonstration of AMAs, which are found in the sera of more than 95% of patients. Following antigen cloning and better definition, the major autoantigens which AMAs recognize are now identified as components of the 2-oxo-acid dehydrogenase complex (2-OADC), an enzyme complex located on the mammalian inner mitochondrial membranes. AMAs in more than 95% of PBC patients are strongly reactive with epitopes in PDC-E2 components and also with components of other 2-OADCs. In about half of PBC patients, AMAs are known to react with BCOAD-E2 (E2 component of branched-chain 2-oxo-acid dehydrogenase complex, 52 kD) and in 40–88% of PBC patients with OGDC-E2 (E2 component of 2-oxoglutarate dehydrogenase complex, 48 kD). Occurrence of ANA of centromere type is also characteristic for PBC. In addition, autoantibodies against a 210-kD glycoprotein of the nuclear membrane (gp210) and Sp100 nucleoprotein are highly specific for PBC. Although these ANAs are detectable less often than AMAs, they are useful for the diagnosis in AMA-negative cases.
Profile of autoantibodies and clinical course
The significance of AMAs and other autoantibodies in the long-term outcome of PBC remains elusive. IgM levels are lower, and high-titre ANA positivity is more common in AMA-negative than AMA-positive PBC cases. The two groups are similar in all other aspects. There is immunophenotypic similarity of the infiltrating inflammatory cells and of the ANA reactivity with gp210 and Sp100 in both groups. In addition, the majority of AMA-negative PBC cases are positive for recombinant elements of either of the two OADC-E2. Thus the general view is that ‘autoimmune cholangitis’, the term applied initially to AMA-negative cases, can be considered as synonymous with AMA-negative PBC. Thus far, AMAs do not seem to correlate with disease activity, particularly with the degree of bile duct injury. Recently, positive anti-gp210 antibodies have been reported to be associated with severe interface hepatitis, lobular inflammation, ductopenia and a more frequent progression to cholestatic hepatic failure. In contrast, positive anticentromere antibodies are significantly associated with severe ductular reaction, less severe ductopenia and a more frequent progression to cirrhosis with portal hypertension. Antiprothrombin IgM is reportedly associated with a worse prognosis, as demonstrated by a higher Mayo score.
Aetiology and pathogenesis
PBC is a multifactorial disease. Its aetiology remains enigmatic but most likely is related to a combination of genetic and environmental factors.
Genetic factors are suggested by a familial predisposition and an increased prevalence of autoantibodies in relatives of PBC patients. Also, a high concordance rate of up to 60% in monozygotic twins has been described. However, the identification of three discordant pairs of eight monozygotic pairs emphasizes that epigenetic factors and/or environment play a critical role. The finding that PBC recurs in an allogeneic transplanted liver suggests genetic rather than host PBC-specific susceptibility of bile duct or liver. With regard to genetic risk biomarkers, there are reports of weak associations between IL-1 and endothelial nitric oxide synthase polymorphisms and early stage of PBC, and the c2 allele in CYP2E1 is associated with advanced disease. Genetic associations HLA DRBI*08 haplotypes, CTLA-4, TNFα, vitamin D receptor and CTLA-4/ICOS gene variants remain controversial. Zhong et al. reported that K8/K18/K19 variants are overrepresented in PBC patients and associated with disease severity, suggesting that these may serve as genetic modifiers.
Recent GWAS and subsequent meta-analysis in people of European descent have identified HLA and over 20 non-HLA susceptibility loci ( IL12A , IL12RB2 , STAT4 , IRF5 , IKZF3 , MMEL1 , SPIB , DENND1B , CD80 , IL7R , CXCR5 , TNFRSF1A , CLEC16A , NFKB , RAD51L1 , MAP3K7IP1 , PLCL2 , RPS6KA4 , TNFAIP2 , 7p14 and 16q24) as the genes associated with susceptibility to PBC. Japanese studies found two other foci ( TNFSF15 and POU2AF1 ), suggesting different disease susceptibility among ethnic groups. A pathway analysis indicated that signalling cascades related to IL-12, IL-27 and JAK-STAT are relevant to the development of PBC. Although the mechanisms whereby these genetic alterations may be involved in mediating liver damage in PBC remain to be elucidated, these global approaches have provided new insights into the pathogenesis and potential drug targets in PBC.
Geographical clustering of PBC cases favours an environmental factor for development of PBC in genetically predisposed hosts. The association of PBC with environmental factors such as microbes, drugs and chemicals has been suggested, and AMA formation as an early step in the pathogenesis of PBC is presumably triggered by these environmental factors. Bacterial infections or mycobacteria have long been incriminated. A report of symptomatic PBC diagnosed after sequential immunization with a lactobacillus vaccine for recurrent vaginitis over years showed cross-reactivity between serum antibody against microbial PDC-E2 and β-galactosidase of Lactobacillus species administered by the vaccine, suggesting that lactobacillus vaccination therapy may be responsible for the development of PBC in genetically susceptible women.
Granuloma formation, a well-known product of mycobacterial infection, is part of the early bile duct injury in PBC. To date, positive bacterial culture from liver tissue with PBC has not been obtained, but CD1, a family of four distinct nonpolymorphic HLA class I-like molecules that can present microbial antigens to T cells, is expressed in epithelioid granulomas and epithelial cells of the small bile ducts in the early stage of PBC and may be involved in presenting microbial lipid antigen(s) to surrounding T cells. DNA of several indigenous bacteria (e.g. Propionibacterium acnes ) were identified in granulomas of PBC, suggesting that P. acnes and other enteric bacteria are involved in the pathogenesis of granuloma. In addition, Chlamydia pneumoniae antigens have been universally found in all liver tissue samples of patients with PBC, and the cases tested for C. pneumoniae 16S RNA by in situ hybridization were positive.
Reduced expression of anion exchanger 2
Chloride-bicarbonate AE2 is important in maintaining intracellular pH and creating ‘HCO 3 − umbrella’ by bicarbonate transport. AE2 is present mainly on bile canaliculi and the luminal surface of bile ductules and interlobular bile ducts in normal livers. Its expression is reduced in interlobular bile ducts of PBC, in which process overexpression of microRNA-506 is supposedly involved. Interestingly, a combination of UDCA and dexamethasone restores expression of AE2 in cholangiocytes. In a genetic study, variations in AE2 gene were found to be a significant prognostic factor in patients with PBC.
In experimental models, most AE2-deficient mice had AMA and increased serum levels of IgG, IgM and ALP. About one-third of AE2-deficient mice also had extensive portal inflammation, with CD4+ or CD8+ T lymphocytes surrounding damaged bile ducts. Cholangiocytes isolated from these mice showed gene expression profiles suggesting oxidative stress and increased antigen presentation. This model may indicate that the reduced expression of the anion exchanger may be a primary event, followed by the development of serological autoimmune abnormalities and histological cholangitis in PBC.
Cellular senescence and autophagy
Damaged bile ducts in PBC, particularly those undergoing CNSDC, express molecular markers of cellular senescence such as p16 and p21. The senescent biliary epithelium is known to secrete proinflammatory cytokines and chemokines such as TNFα, CCL2 and CX3CL1, as well as enzymes such as MMPs that may trigger fibroinflammatory responses around the bile ducts. Autophagy is also presumed to occur in cholangiocytes of PBC, along with increased senescence. Autophagy markers such as microtubule-associated protein-light chain 3b (LC3) and p62/sequestosome-1 (p62) are aggregated in the cytoplasm of senescent cholangiocytes in PBC.
Molecular mimicry and autoantibodies
PBC is characterized by a breakdown in self-tolerance of T and B cells to the conserved mitochondrial ‘self’ antigen E2 component of 2-OADC, particularly the major self antigen PDC-E2, and then by occurrence of autoreactive T and B cells against PDC-E2. The events provoking initial activation of AMAs and PDC-E2-recognizing T cells remain unknown. The hypothesis of ‘molecular mimicry’ implies that foreign pathogens with homology to self protein or modified self protein can break tolerance. PDC-E2 are intracellular enzymes highly conserved during evolution, and there is cross-reactivity between AMAs and subcellular constituents of microbes, in particular Escherichia coli and mycobacteria. AMA seropositivity is reported in women with recurrent urinary tract infections, with or without evidence of PBC, further supporting an involvement of E. coli infection in the induction of PBC-specific autoimmunity. Also, the large number of E. coli immunogenic mimics may account for the dominance of the major PDC-E2 autoepitope. IgG3 antibodies that react to both PDC-E2 and β-galactosidase of Lactobacillus delbrueckii and enhanced immune reactivity to the mycoplasma PDC-E2 antigen were identified in the serum of PBC patients. Another possible mechanism is that liver autoantigens exposed to chemicals (e.g. metabolized xenobiotics) are modified and become immunogenic. Thus exposure to the environmental xenobiotics may be one of the initiating factors that break the tolerance to self proteins in genetically susceptible hosts.
The major AMA-related antigen, PDC-E2, is aberrantly expressed on the small intrahepatic bile ducts in PBC, a staining pattern that does not parallel the distribution of mitochondria ( Fig. 9.23 ). BECs can uniquely preserve the PDC-E2 epitope after apoptosis, and PDC-E2 was found to localize unmodified within apoptotic blebs of intrahepatic BECs, but not within blebs of various other cell lineages studied. AMA-containing sera reacting with PDC-E2 on apoptotic BECs suggests that the autoantigen is accessible to the immune system during apoptosis. In another study, PDC-E2 was found to co-localize with autophagy-related molecules such as LC3 and p62 in damaged cholangiocytes of PBC. In cultured cholangiocytes, various cellular stresses increased the number of granules positive for both LC3 and PDC-E2 and also enhanced the expression of PDC-E2 on the cellular membrane. Dysregulated autophagy of mitochondria may cause dislocation of mitochondrial antigens, which may lead to an autoimmune reaction against cholangiocytes. PBC patients exhibit aberrant HLA class II expression in addition to increased HLA class I expressions on injured BECs (see Fig. 9.12 A ). The periductal presence of proinflammatory cytokines such as IFN-γ may induce such HLA I and II expression in BECs, which probably facilitates antigen presentation such as PDC-E2 to T cells.
Antimitochondrial antibodies and bile duct lesions
AMAs may be pathogenetic, but AMA titres are not correlated with disease activity; thus their precise role in mediating liver damage in PBC remains uncertain. It should be noted that the 2-OADC, which is recognized by AMAs, is not restricted to the small intrahepatic bile ducts but is distributed throughout the body, whereas the destruction in PBC is limited to small intrahepatic bile ducts. IgA, which is both produced by portal plasma cells and derived from the portal blood and then bound to secretory component, is transported to the luminal surface of BECs. Bile from PBC patients has been shown to contain IgG and IgA anti-PDC-E2 antibodies. IgA PDC-E2 antibody may bind to PDC-E2 within the cytoplasm, preventing uptake of PDC-E2 into the inner mitochondrial membrane and leading to deficient PDC-E2 transport into the mitochondria. This in turn could cause mitochondrial dysfunction and damage or induction of apoptosis, both followed by cell death. Bile duct lesions in PBC are sometimes surrounded by plasma cells coronally. Given that this histological finding is associated with higher AMA titres, periductal plasma cells may be a major source of AMAs.
Autoreactive T cells
Autoreactive T cells (both helper and cytotoxic) may be involved in the pathogenesis of bile duct injuries. Epitopes recognized by AMAs and T cells are located within overlapping areas of mitochondrial antigens. In patients with autoimmune diseases, the organ damage is caused by CD4+ CD28− T cells, which express high levels of IFN-γ and possess cytolytic activity. In PBC, CD4+ T cells have been shown to recognize amino acids 163–176 of PDC-E2, and there is a corresponding marked increase of CD4+ CD28− T cells in PBC livers. Furthermore, CD8+ CTLs that recognize components of amino acids 159–167 of PDC-E2 have an effector role in the bile duct injury.
Participation of innate immunity
In addition to the adaptive immune response, innate immunity is also important in PBC, in that a hypersensitive innate immune system to pathogen-associated stimuli may facilitate loss of tolerance. Negative regulators of intracellular TLR signalling, PPARγ and IRAK-M, are known to be associated with the endotoxin tolerance of BECs, and both molecules are ubiquitously expressed in intrahepatic biliary epithelium, whereas expression of PPARγ is significantly reduced in damaged bile ducts of PBC. A Th1-type-cytokine IFN-γ downregulates PPARγ and upregulates TLR, consequently increasing the susceptibility of biliary innate immunity. Cytokine Th1-dominant milieu around the ducts increases susceptibility to PAMPs, which may be associated with the development of cholangitis. Increased serum IgM is thought to be a result of chronic B-cell activation through TLR signalling pathway activated by CpG-B (a ligand of TLR). Interestingly, monocytes from patients with PBC are more sensitive to the activation process by TLRs, resulting in secretion of selective proinflammatory cytokines that may be critical in the breakdown of self-tolerance.
Th17 cells, characterized by the secretion of IL-17, have been implicated in the pathogenesis of PBC, as in other autoimmune diseases, because Th17 cells are accumulated around bile ducts undergoing CNSDC. Th17 cells are part of the mucosal host defence system, and their major role seems to be protection against infections sustained by extracellular bacteria. This subset of T cells is induced by IL-6 and IL-1β, which are thought to be produced by cholangiocytes in PBC. The biliary epithelium stimulated by innate immunity increases expressions of chemokines (e.g. CCL2 and CX3CL1), which may recruit lymphocytes and APCs into peripheral portal tracts. NK cells, another player in innate immunity, are also known to be present around injured bile ducts of PBC.
The early stages of PBC are characterized by portal inflammation with destructive injury to the small intrahepatic bile ducts, which eventually undergo destruction ( Fig. 9.24 ). Simultaneously, parenchymal necroinflammatory changes involving lobular and periportal areas frequently develop, but these changes are generally mild in severity. Gradually, cholestatic and fibrotic changes are superimposed and lead to extensive biliary fibrosis and cirrhosis.
Bile duct injury
The initial injury affects the small interlobular bile ducts 40–80 µm in diameter, with the smaller branches being the first to disappear, and to a lesser degree the septal bile ducts. Epithelial cells of the bile ducts are variably swollen, with a vacuolated cytoplasm and an irregular luminal border, or show an eosinophilic shrunken appearance with pyknotic nuclei. The epithelium also shows proliferative changes with stratification. This, together with some duct ectasia after rupture of its basement membrane, leads to an overestimation of the actual size of the duct affected. The damage to the interlobular bile ducts occurs through apoptotic loss and senescence of BECs. The majority of lymphocytes in the portal tracts are CD4+ and CD8+ T cells (Th1 subset), although B cells are occasionally dense around the bile ducts and may form lymphoid follicles. Plasma cells and eosinophils can be conspicuous in the early stages, and a coronal arrangement of plasma cells around the bile ducts is unique for PBC.
Bile duct damage and portal inflammation are distributed heterogeneously within the liver and might not be accurately sampled in a percutaneous liver biopsy. Nonspecific portal inflammation can therefore be found in early liver biopsies from patients with a clinical diagnosis of PBC. Aggregates of epithelioid cells are common, and well-defined, noncaseating granulomas are characteristically seen in the early stages. Epithelioid cell granulomas, particularly when intimately associated with damaged bile ducts, are a characteristic finding of PBC ( Figs 9.22 C and 9.24 C ). A few epithelioid cells may be loosely arranged in the vicinity of the bile ducts. There may be an admixture of foamy macrophages, suggesting phagocytosis of phospholipid substances, probably leaked from the injured duct. Hyaline deposits may also be seen in the portal areas, at times within lymphocytic aggregates. Around some ducts, oedema may be prominent. These bile duct lesions have been called ‘chronic nonsuppurative destructive cholangitis’ by Rubin et al., or florid duct lesions.
Serial sections disclose the segmental disappearance of interlobular bile ducts associated with a variable degree of granulomatous and lymphocytic reaction ( Fig. 9.25 ). Necrotic or ruptured bile ducts are variably surrounded by lymphocytes, plasma cells, epithelioid cells ( Fig. 9.26 A ) and foamy macrophages ( Fig. 9.26 B ). Later, only remnants of duct epithelium will be identifiable as isolated K7-positive cells or small, amorphous deposits of PAS-positive material among the inflammatory cell infiltrate. Finally, even such vestigial remnants are absent, and only some focal condensation of the portal fibrous tissue or a lymphocytic aggregate remains to identify the site from which a bile duct has disappeared ( Fig. 9.27 A ). The presence of arteries unaccompanied by ducts is used as a useful but rough marker of bile duct loss or ductopenia ( Fig. 9.27 B ). Foci of concentric periductal fibrosis, first described by Scheuer in 1967, have more recently been observed in approximately 20% of patients with PBC and may cause problems in the distinction between PBC and PSC. In contrast, the septal and large intrahepatic ducts, although they may show some inflammation in their wall, are preserved even at an advanced stage of the disease.
The canals of Hering (CoH) connecting bile canaliculi to the interlobular bile ducts have been found to be decreased in number in all stages of PBC, suggesting that they are destroyed in concert with the destruction of small bile ducts. One study found that loss of CoH, identified by K19 immunostaining, may be a very early manifestation of PBC that precedes bile duct loss and can be seen in biopsies that otherwise appear normal/near normal (‘minimal change’ PBC). Another study found that CoH loss was associated with histological and biochemical features of more advanced disease, suggesting that lack of CoH may impair regeneration of cholangiocytes after bile duct injury, which in turn promotes disease progression in PBC. An association between CoH loss and hepatitis activity was also observed, suggesting that CoH, like bile ducts, may be targets of immune-mediated injury.
Parenchymal and interface changes
The parenchymal and interface changes consist of necroinflammatory and chronic cholestatic alterations. Necroinflammatory changes include single-cell necrosis, acidophilic bodies, Kupffer cell hyperplasia and sinusoidal infiltration with lymphocytes and pigment-laden macrophages and are generally mild. Rarely, more intense perivenular inflammation may occur with hepatocellular loss ( Fig. 9.28 A ). Well-formed epithelioid granulomas may also be found outside the portal areas ( Fig. 9.28 B ). The activity at the interface (see below) is predominantly biliary, but may at times simulate autoimmune hepatitis (AIH) ( Fig. 9.28 C ).
In the early stages of PBC, bile duct injury and associated inflammation generally remain confined within the portal tract boundaries. The subsequent progression of the disease is characterized by an extension of the necroinflammatory process into the periportal parenchyma–interface activity. This may take two different forms, which often occur in combination.
Lymphocytic interface activity resembles the lesion seen in AIH and suggests an extension to adjacent hepatocytes of the same immunological process as that affecting the duct system ( Fig. 9.28 C ). Lymphocytic interface activity is present in a substantial number of patients, generally early in conjunction with florid bile duct damage and inflammation. In less than 10%, it may even dominate the picture and may be associated with clinical features of AIH, so-called PBC-AIH overlap syndrome. IgM elevation and predominantly IgM+ plasma cells in portal tracts are typical of PBC, whereas IgG elevation and predominantly IgG+ plasma cells favour AIH. The extent to which immunohistochemical staining for IgM and IgG may be helpful as an adjunct to the conventional histological diagnosis of PBC and AIH is uncertain.
Biliary interface activity is superimposed or, as the duct loss advances, becomes the main feature. In common with other chronic cholestatic disorders, it results from the ‘toxic’ effect of hydrophobic bile acids and other retained bile products. There is cholate stasis and deposition of copper or copper-associated protein granules ( Fig. 9.29 A and B ; see also Fig. 9.19 A and B ). The degree of copper deposition generally correlates with biochemical markers of cholestasis and stages of PBC. Xanthoma cells, often in aggregates, may be present ( Fig. 9.29 C ). Ductular reaction may be a striking feature, although the ductular elements often disappear in the advanced stages of the disease when pericellular fibrosis ( Fig. 9.30 ), MDBs and intracellular or canalicular bile pigment dominate the picture at the interface (see Fig. 9.19 D).
Small-cell change of periportal hepatocytes and nodular hyperplasia of hepatocytes occur in noncirrhotic stages of PBC. Twin-cell and pseudoglandular plates consisting of small hepatocytes may be prominent from an early stage ( Fig. 9.31 A and B ); this has been confirmed by increased uptake of bromodeoxyuridine and enhanced proliferating cell nuclear antigen (PCNA) immunostaining. In contrast to that in other chronic liver diseases such as viral hepatitis, the small-cell change seen in PBC appears to be a regenerative phenomenon rather than a premalignant lesion. Nodular regenerative hyperplasia (NRH) is also recognized to occur in noncirrhotic PBC patients ( Fig. 9.31 C ). It is probably an important factor both in the hepatomegaly of PBC and in the development of portal hypertension, which may be clinically significant before cirrhosis has developed. The underlying mechanism of NRH in precirrhotic PBC is uncertain; it may reflect collateral damage to small portal veins lying in the vicinity of inflammatory bile duct lesions.
Progressive fibrosis leading to cirrhosis
With time, the necroinflammatory and cholestatic process accompanied by fibrosis extends along the terminal distribution of the portal tracts, leading to portal-portal bridging septa. Dilated lymphatics and venules forming microcavernous structures are frequently seen in the enlarged portal tracts (see Fig. 9.27 A ). Periseptal changes are usually continuous with and similar to the periportal changes. The biliary-type fibrosis is dense, scar-like in the deeper portions of the septa, but oedematous at the periphery, where it gives rise to a halo effect (see Figs 9.21 B, 9.29 A and 9.30 ).
Two mechanisms appear to be involved in the progression of PBC. On one hand, extensive bile duct destruction, chronic cholestasis and biliary interface activity lead to biliary-type fibrosis or cirrhosis, the most common pattern. On the other hand, lymphocytic interface activities may lead to cirrhosis resembling that observed with AIH. The latter pattern occurs concomitantly or variably alternates with the biliary pattern during the course of the disease and in individual cases.
Macroscopically, the cirrhotic liver is generally larger than that of posthepatitic cirrhosis after viral or autoimmune hepatitis. It is predominantly micronodular, of a regular appearance, and there is variable bile staining ( Fig. 9.32 ). Rarely a shrunken, macronodular liver is observed. The histological changes may be difficult to distinguish from those of cirrhosis of other aetiology. PBC should be suspected, however, with the following features:
Virtual absence of medium-sized and small bile ducts
Focal lymphocytic aggregates in portal areas
Periportal cholate stasis or cholestasis, with MDBs and copper deposition regularly highlighting the parenchymal limiting plates
Biliary or monolobular pattern of cirrhosis
Partial or focal preservation of the normal architecture
In a few cases the diagnostic bile duct lesions may persist at the cirrhotic stage, and granulomas may also be seen. The histological distinction among PBC, PSC and IgG4-related sclerosing cholangitis is discussed later. Table 9.4 compares the main clinicopathological features of PBC and PSC.
|Gender, M/F||1 : 9||2 : 1|
|Alkaline phosphatase (ALP)||++++||++++|
|Bilirubin||+ to +++||+/++|
|Antimitochondrial antibodies (AMA)||>95%||Absent or low titre|
|Other autoantibodies||<30%||Children > adults|
|HLA association||Not certain||B8; DR3, DR2, DRw52a|
|Inflammatory bowel disease||<4%||>65%|
|Histological features (needle biopsy)|
|Granulomatous cholangitis||30–50%||Very rarely|
|Fibro-obliterative cholangitis||Minor degrees of periductal fibrosis rarely seen||10-20%|
Role of liver biopsy
According to the guidelines proposed by the European Association for the Study of Liver (EASL) and the American Association of the Study of Liver Diseases (AASLD), liver biopsy is no longer regarded as mandatory to make a diagnosis of PBC in patients with a cholestatic serum enzyme pattern and serum AMA, in view of the high specificity of AMA for the diagnosis of PBC. However, liver biopsy is required in AMA-negative patients and to exclude other concomitant diseases such as AIH and nonalcoholic steatohepatitis. Steatosis may be present in up to 50% of liver biopsies from PBC patients, but its relevance in disease progression is uncertain.
Histological assessment of disease severity
Classic histological staging systems
Many pathologists worldwide have used the systems described by Ludwig et al. and Scheuer and Lefkowitch, which divide the histological changes of PBC into four successive stages. In Scheuer’s system, stage 1 is characterized by florid duct lesions, stage 2 by proliferation of bile ductules, stage 3 by fibrosis or scarring (see Fig. 9.29 ) and stage 4 by cirrhosis ( Fig. 9.32 ). In Ludwig’s system the histological features used for staging chronic hepatitis are applied: stage 1 is portal hepatitis, stage 2 interface hepatitis, stage 3 bridging necrosis or bridging fibrosis and stage 4 cirrhosis. Although these classic systems are simple and readily applicable to PBC and other cholangiopathies, heterogeneity in the distribution of the changes within PBC liver and sampling errors with needle biopsy specimens have long been recognized. In addition, these classic staging systems incorporate features of disease activity (histological ‘grade’), such as bile duct inflammation and interface hepatitis, and do not consider other histological features, such as bile duct loss and accumulation of copper-associated protein, which worsen as the disease progresses. Such information may be desirable to evaluate disease activity and progression, now that UDCA therapy is universally used in PBC patients.
Novel systems for histological staging and grading
To overcome limits of the classic schemes, a novel system applicable to needle liver biopsy specimens has been proposed by Nakanuma and other Japanese researchers. Three features—fibrosis, bile duct loss and chronic cholestasis—are used for staging PBC ( Table 9.5 ). Chronic cholestasis is evaluated by the deposition of orcein-positive granules, which are detectable in the relatively early stages of PBC and increase with disease progression (see Figs 9.19 B and 9.29 B ). A final stage (0–3) is obtained by adding the scores for the three items, where 0 is stage 1 (no or minimum progression), 1–3 is stage 2 (mild progression), 4–6 is stage 3 (moderate progression), and 7–9 is stage 4 (advanced disease) ( Table 9.6 ). If orcein staining is not available, the sum of scores for fibrosis and bile duct loss is converted to an overall severity score ( Table 9.7 ).
|0||No fibrosis or fibrosis limited to portal tracts|
|1||Fibrosis extends beyond portal areas with occasional incomplete septa|
|2||Bridging fibrosis with variable lobular distortion|
|3||Cirrhosis (extensive fibrosis and regenerative nodules)|
|Bile duct loss|
|0||Bile ducts are present in all portal tracts|
|1||Bile ducts are missing in <1/3 of portal tracts|
|2||Bile ducts are missing in 1/3–2/3 of portal tracts|
|3||Bile ducts are missing in >2/3 portal tracts|
|Chronic cholestasis (deposition of orcein-positive granules)|
|1||Deposition in periportal hepatocytes in <1/3 of portal tracts|
|2||Deposition in variable numbers of periportal hepatocytes in 1/3–2/3 of portal tracts|
|3||Deposition in many hepatocytes in >2/3 of portal tracts|
|1 (no progression)||0|
|2 (mild progression)||1–3|
|3 (moderate progression)||4–6|
|4 (advanced progression)||7–9|
|Stages||Scores fibrosis + bile duct loss|
|1 (no progression)||0|
|2 (mild progression)||1–2|
|3 (moderate progression)||3–4|
|4 (advanced progression)||5–6|
Both chronic cholangitis and hepatitis features are used to grade necroinflammatory activity ( Table 9.8 ). Chronic cholangitis activity (CA) is categorized into four grades. Grade 0 represents absent or ambiguous bile duct damage, which may include mild biliary epithelial damage. In grade 3, at least one damaged bile duct shows features of CNSDA, including bile ducts partly or entirely surrounded by epithelioid cell granuloma (granulomatous cholangitis). In grade 1, one damaged bile duct entirely surrounded by mild to moderate lymphoplasmacytic infiltrate is found, a type of cholangitis occasionally encountered in chronic viral hepatitis. Interlobular bile ducts surrounded by a small amount of lymphoplasmacytic infiltrate or adjacent to lymphoid cells are not regarded as evident chronic cholangitis. In grade 2, two or more foci of evident chronic cholangitis are present.
|Grade||Activity of cholangitis (CA)|
|CA 0 (no activity)||No cholangitis ± mild duct epithelial damage|
|CA 1 (mild activity)||At least one focus of evident chronic cholangitis|
|CA 2 (moderate activity)||Two or more foci of evident chronic cholangitis|
|CA 3 (marked activity)||At least one focus of chronic nonsuppurative destructive cholangitis|
|Activity of hepatitis (HA)|
|HA 0 (no activity)||No interface hepatitis and no or minimal lobular hepatitis|
|HA 1 (mild activity)||Focal interface hepatitis affecting 10 continuous hepatocytes in one portal tract and mild to moderate lobular hepatitis|
|HA 2 (moderate activity)||Focal interface hepatitis affecting 10 continuous hepatocytes in ≥2 portal tracts and mild to moderate lobular hepatitis|
|HA 3 (marked activity)||Interface hepatitis affecting 20 continuous hepatocytes in ≥50% of portal tracts, moderate to marked lobular hepatitis or bridging /zonal necrosis|
Hepatitis activity (HA) is evaluated by both interface hepatitis and lobular hepatitis and is categorized into four grades. Grade 0 represents no interface hepatitis. Grades 1 and 2 show interface hepatitis affecting about 10 continuous hepatocytes at the interface of one portal tract or fibrous septum, as well as two or more portal tracts or fibrous septa, respectively. Grade 3 requires the presence of interface hepatitis affecting >20 continuous hepatocytes at the limiting plate of many portal tracts or fibrous septa. Entrapment of hepatocytes in the widened portal tract is also found in grade 3 HA. Absent or minimal lobular hepatitis is found in grade 0, mild to moderate lobular hepatitis in grade 1 or 2 and moderate lobular hepatitis in grade 3. Occasional zonal necrosis and bridging necrosis are regarded as grade 3. Interobserver agreement with this system has been fair for the staging but poor for necroinflammatory scores.
There are merits and drawbacks with all the scoring systems proposed for PBC. Interobserver agreement is a common issue; the use of precise definitions and prior discussion between pathologists should help to increase interobserver reproducibility. Sampling variability is another important problem and especially applies to the variable severity of fibrosis that occurs in PBC and other chronic cholestatic biliary diseases. A number of studies have suggested that the Nakanuma system for staging PBC is better at predicting outcomes in PBC than the systems proposed by Ludwig and Scheuer and may also be helpful in predicting responses to treatment. The improved predictive value of the Nakanuma system may relate to its assessment of three separate features of disease progression rather than relying predominantly on the fibrosis stage. Of the three features assessed, deposition of orcein-positive granules has been found to be the strongest predictor of adverse outcomes. Another scoring system recently proposed by a French group incorporated assessment of lymphocytic interface hepatitis, fibrosis and bile duct loss, all of which are thought to be important in disease progression. This system showed better interobserver agreement and correlation with biochemical abnormalities than the Ludwig or Scheuer system, but predictive value for adverse outcomes could not be assessed.
Noninvasive methods have increasingly been used to assess disease severity in many chronic liver diseases, notably chronic viral hepatitis and nonalcoholic fatty liver disease. These include serum markers and radiological assessments (e.g. transient elastography) which are used as surrogate markers of liver fibrosis. Although studies have documented the utility of noninvasive markers of liver fibrosis in PBC, their role in the routine assessment of patients with PBC (and other chronic cholestatic liver diseases such as PSC) has not been clearly established. Liver biopsy may thus continue to play an important role in assessing disease severity and progression, particularly in the context of clinical trials.
Variants of primary biliary cholangitis
Antimitochondrial antibodies are undetectable in about 5% of patients with PBC. A number of studies have identified differences in clinical, biochemical and immunological features of AMA-negative compared with AMA-positive PBC. Cases of AMA-negative PBC typically have other autoantibodies (ANA and SMA). Other changes reported to occur more frequently in AMA-negative PBC patients include less severe pruritus, higher aspartate transaminase (AST) levels, lower IgM levels and differences in the composition of inflammatory infiltrates in the liver (e.g. more plasma, T, CD5+ and CD20+ cells). However, these differences are mostly subtle, and the overall clinical features, natural history, histopathological findings and response to UDCA are similar to those of AMA-positive patients.
PBC with features of autoimmune hepatitis (‘PBC-AIH overlap syndrome’)
PBC and AIH (hepatitic form of PBC; see Chapter 8 ), the two main autoimmune liver diseases, differ in their clinical, biochemical, serological and histological features and, in most patients, a diagnosis of PBC or AIH can be achieved using accepted criteria. However, in about 10% of PBC cases, problems arise because features of both conditions in various combinations coexist in a single patient. The term ‘PBC-AIH overlap syndrome’ has been used to describe such patients. Most cases are best regarded as having PBC with unusually prominent inflammatory activity (PBC with ‘hepatitic features’) rather than representing a distinct entity.
Chazouilleres et al. have proposed diagnostic criteria for the PBC-AIH overlap syndrome. These include the presence of at least two of three diagnostic features of PBC—(1) GGT ≥5× upper limit of normal (ULN) or ALP ≥2× ULN, (2) positive AMA and (3) florid bile duct lesion on histology—and at least two of three diagnostic features of AIH: (1) increased alanine transaminase (ALT) levels ≥5× ULN, (2) serum IgG levels ≥2× ULN or positive anti-smooth muscle antibody (SMA) and (3) moderate or severe lymphocytic interface activity on histology. Using these criteria, 9.2% of PBC patients had an overlap syndrome. Similar diagnostic criteria have been recommended in guidelines produced by EASL and the International Autoimmune Hepatitis Group. The reported frequency of PBC-AIH overlap syndrome in other studies is 5–15% of PBC patients. This variation is probably a result of broader or narrower definitions; long-term follow-up with frequent histological examinations may reduce the number of cases initially considered as PBC-AIH overlap.
Although a diagnosis of ‘overlap syndrome’ could theoretically be made without a liver biopsy, uncertainty about establishing the diagnosis means that liver biopsy is still recommended in this situation. A recent study suggested that classic histological features of AIH such as hepatocyte rosettes, emperipolesis and lobular hepatitis are less frequent and severe in PBC patients with interface hepatitis than in those with classic AIH. It must be stressed that the histological finding of PBC-like bile duct injury in otherwise classic AIH or lymphocytic interface hepatitis in otherwise classic PBC is insufficient for a diagnosis of overlap syndrome.
Patients typically present with simultaneous features supporting a diagnosis of PBC and AIH. Some patients may have a ‘sequential syndrome’, presenting initially with features of one disease (usually PBC), then with features of the other disease several months or years later. In two patients, clinicopathological features of AIH occurred after liver transplantation (LT) for PBC. In another patient with PBC who underwent auxiliary LT, AIH developed in the allograft liver while PBC persisted in the native liver. Classic AIH evolving into typical PBC has also been reported. Interestingly, a few patients with overt AIH who test positive for AMA at initial presentation and are treated with corticosteroid therapy have shown no clinical or histologic evidence of PBC despite the continued detection of AMAs over up to 27 years of follow-up.
A worse outcome is observed in PBC patients with overlap features. The frequency of cirrhosis, portal hypertension, gastrointestinal bleeding, ascites and oesophageal varices are significantly higher and there are more frequent liver-related deaths and LTs in the overlap group at the time of follow-up than in PBC patients without AIH features. There is some evidence to suggest that PBC patients with prominent hepatitic features may benefit from treatment with immunosuppressive therapy, although the criteria for using immunosuppression in this setting are not clearly defined.
Other ‘overlap syndromes’ involving PBC
The rare case reports of patients with overlapping features of PBC and PSC most likely reflect the lack of genetic overlap between these two conditions. In most of the eight cases recently reviewed by Floreani et al., the diagnosis was based on a combination of AMA positivity and abnormal cholangiography. Histological findings in the seven patients who underwent liver biopsy were variable: two had inflammatory bile duct lesions supporting a diagnosis of PBC, one showed periductal fibrosis in keeping with PSC, and the remainder simply had features of chronic cholestasis with ductopenia.
A single case report has described an ‘overlap syndrome’ involving PBC and IgG4-related sclerosing cholangitis. Studies have described PBC patients with mass-forming lymphoid lesions, thought to be reactive in nature (‘reactive lymphoid hyperplasia’ or ‘pseudolymphoma’). The pathogenesis of these lesions and their relationship to PBC is uncertain; two cases were associated with large numbers of IgG4+ plasma cells but were not thought to be a manifestation of IgG4-related disease.
Portal hypertension is frequently observed in patients with cirrhotic PBC, and it may appear even at the early stages. A possible contributing factor to the earlier presentation is associated nodular regenerative hyperplasia of the liver parenchyma, as previously discussed.
Hepatocellular carcinoma (HCC) typically develops several years after the onset of cirrhosis and carries a poor prognosis, similar to that in cirrhosis of other aetiologies. In a study of 667 patients, the overall incidence of HCC was 5.9% in patients with stage 3–4 disease, with a male incidence of 20% versus 4.1% in female patients. A Japanese nationwide study also indicated that an advanced stage of liver fibrosis is an independent risk factor for the development of HCC in female but not male patients, suggesting that men are at risk of developing HCC at any stage. A recent multicentre study of 4565 patients, 123 of whom developed HCC, showed an overall incidence of 3.4 cases per 1000 patient-years; in addition to confirming male gender and disease stage as independent risk factors, biochemical nonresponse to treatment was the most significant factor predictive of future HCC risk. An autopsy series suggested that the tumour cells in PBC are more likely to accumulate fat, copper and its binding protein as well as MDBs, particularly at an early stage. Other studies report an increased incidence of extrahepatic malignancies, notably breast cancer, which was found to be 4.4 times that predicted for the general population.
The treatment of choice for PBC is UDCA, a hydrophilic bile acid that reduces the intrahepatic, more hydrophobic bile salt concentration and competitively inhibits its reabsorption in the gut, thereby reducing the overall bile acid pool size. UDCA may also exert immunological effects ; in particular, UDCA therapy is associated with a reduction of HLA molecule expression within the liver. UDCA may also induce biliary HCO 3 − secretion, leading to the reinforcement of the HCO 3 − umbrella to protect cholangiocytes against toxic bile acids. Clinically, UDCA often improves or even normalizes serum levels of cholestatic enzymes and delays disease progression. However, results of studies assessing the benefit of UDCA on liver histology diverge, which can be expected given the known sampling variation and the small number of paired biopsies reviewed. Bezafibrate, a commonly used medication for hyperlipidaemia which increases phospholipid output into bile and reduces the cytotoxicity of hydrophobic bile acids, was found to lower liver enzyme levels, apparently through its action on MDR3 and PPARα. Combination therapy with bezafibrate and UDCA improved the biochemical profile and Mayo risk score of patients with PBC who respond only partially to UDCA. However, the survival rate was not significantly improved, whereas long-term combination therapy significantly increased the serum creatinine levels. Immunosuppressive therapy has been disappointing in PBC, although as discussed earlier, the combination of corticosteroids and UDCA, and in some cases prednisone and/or azathioprine, may be required to achieve biochemical remission in patients with PBC-AIH overlap syndromes. Discovery of the interaction between bile acids and nuclear hormone receptor farnesoid X receptor (FXR) has led to the identification of a novel drug target in cholestatic disorders. Obeticholic acid, an agonist of FXR, has been shown to improve biochemical profiles in patients with PBC who had an inadequate response to UDCA.
Idiopathic adulthood ductopenia
Some authors have identified patients who present in young adulthood with a chronic cholestatic syndrome. Their liver showed a loss of interlobular bile ducts in more than 50% of portal tracts, leading to biliary fibrosis/cirrhosis, but their clinical, radiological and immunological features did not satisfy the criteria for any of the recognized causes of vanishing bile duct syndrome. Serum biochemical tests indicated severe and progressive cholestasis. Ludwig first used the term ‘idiopathic adulthood ductopenia’ for this condition. Patients show a male preponderance (2 : 1), are AMA negative and have an essentially normal cholangiogram, a negative drug history and no evidence of chronic inflammatory bowel disease or sarcoidosis. Viral hepatitis markers and circulating autoantibodies are absent. Most patients were younger than the lower age limit encountered in PBC, making a diagnosis of AMA-negative PBC unlikely. Some cases may represent small-duct PSC without associated ulcerative colitis, whereas others may be late onset of infantile nonsyndromic paucity of interlobular bile ducts. The diagnosis is one of exclusion. The prognosis is variable, but most patients reported present with pruritus or jaundice and show a progressive course resulting in LT or death. LT is followed by prompt and complete resolution of the jaundice and pruritus. UDCA and immunosuppression have been successful in some patients.
Idiopathic adulthood ductopenia may constitute a heterogeneous patient group. Although most cases are sporadic, familial cases do occur. A series of five members of an extended family spanning three generations suggests that genetics may play a role. Hereditary or acquired dysfunction of the canalicular membrane transporters bile salt export pump (BSEP, ABCB11 ) and multidrug-resistance protein type 3 (MDR3, ABCB4 ), which do not seem to play a role in PBC or PSC, remains to be evaluated in patients with idiopathic adulthood ductopenia. A missense mutation in the canalicular phospholipid transporter gene ABCB4 was reported in 11 siblings with a range of cholestatic disorders, three of whom died of cirrhosis (ages 5, 7 and 43 years), and three had adult-onset disease with small-duct cholangiopathy, including ductopenia. Clinicopathologically, two types of idiopathic adulthood ductopenia associated with different prognoses are recognized. Patients with type 1 or mild idiopathic adulthood ductopenia (MIAD) are asymptomatic or have symptoms of cholestatic liver disease. They tend to have less destruction of the intrahepatic bile ducts on liver biopsy specimens (bile duct loss in <50% of portal tracts). Their clinical course ranges from spontaneous improvement to chronic progressive cholestasis. MIAD cases may be the result of a partial genetic defect, as suggested by a higher frequency of multiple HLA-DRB1 alleles. In contrast, patients with type 2 idiopathic adulthood ductopenia generally manifest initial symptoms of decompensated biliary cirrhosis, have extensive destruction of the intrahepatic bile ducts on liver biopsy and frequently require OLT.
Bile duct injury in liver allograft and graft-versus-host disease
The interlobular bile ducts are main targets of the immune attack during liver allograft rejection and GVHD, and thus these lesions are of significant relevance to the discussion of biliary injury in the liver. These changes are discussed in detail in Chapter 14 and so are not considered further here.
Other disorders associated with intrahepatic bile duct injury
Acute and chronic hepatitis (viral and autoimmune)
A hepatitis-associated bile duct lesion was first reported by Poulsen and Christoffersen, and subsequent studies by these and other investigators have shown that this lesion occurs most often in chronic hepatitis C and also in AIH. The approximate frequency is 10–15% in acute viral hepatitis, 20–35% in chronic viral hepatitis overall and 25–30% in AIH. The lesion is particularly common in chronic hepatitis C, with a reported incidence ranging from 35% to 90%. Lymphocytic cholangitis has also been described in hepatitis E. The prevalence of bile duct lesions varies greatly in different series, probably reflecting uncertainty about the aetiology of the underlying acute or chronic hepatitis in early series and variations in the criteria used to diagnose bile duct lesions.
The distinctive lesions are characterized by prominent swelling and vacuolation of the small bile duct epithelium with piling up of irregularly spaced and hyperchromatic nuclei ( Fig. 9.33 A ). The surrounding inflammatory cell infiltrate often includes lymphocytic aggregates with or without a follicular arrangement. The lumen may be considerably narrowed ( Fig. 9.33 B ). The lesions involve only a segment of the duct circumference. Although histological differentiation of hepatitis-associated bile duct lesions from PBC is possible in most cases, bile duct injury indistinguishable from the PBC lesion can be encountered. The bile duct injury of chronic hepatitis C differs from that seen in immune-mediated cholangitis such as PBC in that ectopic expression of HLA-DR and enhanced expression of HLA-A,B,C are generally absent or mild (see Fig. 9.12 A and B ). Furthermore, ‘hepatitic bile duct lesions’ rarely, if ever, result in bile duct loss and therefore do not lead to development of chronic cholestasis.
Other viral infection
Reversible injury to biliary epithelium is not unusual in CMV hepatitis. In the neonate, viral inclusions of biliary epithelium are seen as a feature of CMV hepatitis ( Fig. 9.34 ). It has been suggested that the paucity of interlobular bile ducts of neonates may be a sequela of CMV infection, but this is not supported by hard data. In adults, mostly in immunocompromised hosts, CMV infection can occasionally be a cause of cholangitis, severe bile duct necrosis or haemobilia.
Drug- and toxin-induced injury of bile ducts
Drug- and toxin-induced cholangiopathy is reviewed in Chapter 12 . Some injuries are associated with progressive duct loss, whereas others show cholangitis, cholangiocyte degeneration or necrosis. In many patients, severe cholestatic jaundice develops and persists. Although severe and prolonged jaundice eventually disappears, a biochemical cholestasis may persist for months.
The most commonly implicated drugs are neuroleptics (e.g. chlorpromazine), tricyclic antidepressants, anticonvulsants (e.g. phenytoin), ajmaline, antibiotics (e.g. clindamycin, flucloxacillin, erythromycin, thiabendazole, amoxicillin/clavulanic acid) and nonsteroidal anti-inflammatory drugs (e.g. ibuprofen). Although drug-induced liver injury caused by TNFα antagonists usually exhibits a hepatitis picture, cholestasis with severe bile duct injury or loss can also rarely occur.
Morphologically, perivenular bilirubinostasis is a constant finding irrespective of duct loss. The epithelium of the small bile ducts shows variable vacuolation or eosinophilic degeneration with nuclear pleomorphism, pyknosis and signs of regeneration with nuclear crowding and mitotic activity ( Fig. 9.35 A ). The portal tracts are oedematous with a mild to moderate inflammatory cell infiltrate. This may show a periductal reinforcement with a predominance of eosinophils and neutrophils ( Fig. 9.35 A ), but granulomatous destructive cholangitis indistinguishable from PBC is recorded exceptionally. A granulomatous reaction with eosinophils may be seen in patients presenting signs of hypersensitivity ( Fig. 9.35 B and C ). There may be extensive duct loss with or without significant portal inflammation ( Fig. 9.35 D). In longstanding cases, progressive ductopenia leads to portal fibrosis and signs of chronic cholestasis with copper accumulation in periportal hepatocytes. In this form, extensive ductular reaction and some fibrosis precede restoration of the interlobular bile ducts, whose numbers, however, may remain reduced after clinical recovery. The prognosis has been reported as favourable, with ultimate resolution in most patients. However, a few cases show a rapid progression, even in children, and development of cirrhosis is recorded. Some patients present with a syndrome that closely resembles PBC; when AMA is found in the serum, it seems reasonable to conclude that the drug has unmasked or triggered genuine PBC.
Similar bile duct injury has been produced experimentally or accidentally by toxic substances such as α-naphthylisothiocyanate and 4,4′-diaminodiphenylmethane. The latter, which is an aromatic amine contained in bread, was the cause of so-called Epping jaundice in the United Kingdom; liver biopsy specimens from the patients showed cholangitic inflammation with many eosinophils, bile duct necrosis and bilirubinostasis. In paraquat poisoning, eosinophilic shrinkage of duct epithelium, nuclear pyknosis and epithelial detachment from the basement membrane have been noted (see Fig. 12.13 ) but with a scanty inflammatory reaction. Biliary toxicity of chlorinated organic solvents, such as dichloromethane and 1.2-dichloropropane, was recently identified. This association was proved by investigations of workers in Japanese printing companies diagnosed with cholangiocarcinomas in their 30s or 40s. In addition to bile duct cancers, multiple foci of epithelial dysplasia and features of sclerosing cholangitis were found throughout the biliary tree.
The Stevens–Johnson syndrome , a drug hypersensitivity reaction with severe mucocutaneous manifestations, has been associated with the vanishing bile duct syndrome (VBDS). The association occurs more often in patients with an abnormal immune status. Stevens–Johnson syndrome is accompanied by immune complex deposition, followed by cytokine release and/or cell-mediated response. Several drugs have been linked with both VBDS and Stevens–Johnson syndrome, which provides evidence for immunopathogenetic mechanisms being common to both syndromes. Chamuleau et al. reported a case of toxic epidermal necrolysis of unknown origin and VBDS; both eventually improved after 1 year, leaving a mild elevation of ALP and GGT as the only sequela.
Liver involvement in sarcoidosis is discussed in detail in Chapter 15 . However, some cases of sarcoidosis may be associated with granulomatous cholangitis and progressive ductopenia, leading to a chronic cholestatic syndrome that resembles PBC. In most patients, bile ducts are probably damaged as a ‘bystander effect’ in portal tracts containing sarcoid granulomas, rather being specific targets of immune-mediated injury, as seen in PBC. Furthermore, the large multinodular granulomas with fibrous scarring characteristic of sarcoidosis are different than the smaller portal granulomas typically seen in PBC. A few patients will have convincing features of both sarcoidosis and PBC, and sarcoidosis may also mimic features of PSC.
Sepsis and toxic shock syndrome
Cholestasis is a common complication of severe extrahepatic bacterial infection, particularly gram-negative strains, and septicaemia. Translocation of endotoxin through the intestinal mucosa may cause intrahepatic cholestasis in patients with no detectable bacteria in the blood. Liver pathology will be modified by additional ischaemic injury in patients with severe septic shock.
The mechanisms of cholestasis in severe septic/endotoxic shock are complex, with altered bile production in hepatocytes and impaired bile flow in the biliary tree. Biopsy specimens show a marked cholangiolitis with bile plugging of cholangioles, so-called cholangitis lenta, in addition to canalicular bile plugs. The portal tracts are surrounded by dilated ductules and canals of Hering which extend into the periportal zone; their lumens contain PAS-positive, bilirubin-stained casts with neutrophil polymorphs present within and around the ductules ( Fig. 9.36 A ). This is an important lesion to recognize, in that this pattern of acute cholangiolitis without an associated suppurative cholangitis suggests septicaemia rather than duct obstruction. In longstanding cases of septicaemia or at autopsy, the portal tracts are ringed by the bile-containing dilated cholangioles ( Fig. 9.36 B ). In experimental models, increased bacterial components (e.g. LPS) in the circulation cause production of inflammatory cytokines (e.g. TNFα, IL-1b, IL-6, IL-8) in bile ducts and ductules, which eventually leads to the generation of nitric oxide (NO) in cholangiocytes. NO inhibits cyclic adenosine monophosphate (cAMP)-dependent fluid secretion and HCO 3 − transport mediated by cystic fibrosis transmembrane regulator (CFTR) and AE2 in cholangiocytes, resulting in impaired bile secretion, increased susceptibility to hydrophobic toxic bile acids and disruption of intercellular tight junction. The production of proinflammatory cytokines also contributes to the recruitment of inflammatory cells, particularly neutrophils, toward bile ducts and reactive ductules.
Similar, striking cholangiolar bile retention may often be seen at autopsy or in liver explants after submassive liver cell necrosis of viral or drug aetiology, as well as in decompensated cirrhosis. In these situations the changes seen may reflect septic complications. Similar changes with absence of duct and ductular inflammation and structurally normal interlobular bile ducts were an uncommon yet important histological finding in liver biopsies from a paediatric liver transplant series. They differed from biliary obstruction and other causes of post-transplant cholestasis. Compared with a control group of large-duct obstruction, the rate of proven bacterial or fungal infection in the study group was 100% versus 54.5% in the control group. Graft and patient survival were similarly poor.
The toxic shock syndrome is thought to be caused by an exotoxin produced by Staphylococcus aureus and in some cases was associated with the use of vaginal tampons; skin infection was present in other patients, but no source of infection could be identified in many cases. Patients present with multisystem involvement; vomiting and diarrhoea, impaired renal function, thrombocytopenia, mental deterioration and jaundice with elevated serum transaminases. Jaundice was a feature in 50% of patients. Histologically, there is a severe cholangitis and cholangiolitis with intense mural, luminal and periductal infiltration of neutrophil polymorphs ( Fig. 9.37 ). The lesions were attributed to chemical irritation, probably resulting from exotoxin excretion in the bile, combined with liver hypoperfusion.