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The pathobiology of biliary epithelia
GASTROENTEROLOGY 1997;112:269–279
VIEWPOINTS IN DIGESTIVE DISEASES The Pathobiology of Biliary Epithelia STUART K. ROBERTS,* JURGEN LUDWIG,* and NICHOLAS F. LARUSSO‡ Division of Gastroenterology and Internal Medicine, *Department of Biochemistry and Molecular Biology, and ‡Division of Anatomic Pathology, Mayo Medical School, Clinic and Foundation, Rochester, Minnesota
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ntrahepatic biliary epithelial cells, or cholangiocytes, account for 3%–5% of the hepatic cell population and line a complex three-dimensional network of interconnecting conduits in the liver, termed the intrahepatic biliary ductal system. Early in this century, studies suggested that cholangiocytes play an important role in water and electrolyte transport; however, subsequent attempts to further delineate the functions of cholangiocytes were hindered by the lack of suitable experimental models. Recently, the knowledge of cholangiocyte biology has advanced considerably, based primarily on new experimental models that have allowed investigators to isolate and culture individual cholangiocytes and intact intrahepatic bile ducts. These advances have shown that cholangiocytes make an important contribution to basal and agonist-induced ductal bile formation. Moreover, these cells are candidate targets in many immune- and nonimmune-mediated liver diseases; as a collective name for these conditions, we propose the term cholangiopathies. Because of their morbidity, mortality, and overall cost to society, cholangiopathies have assumed major importance among liver diseases and syndromes.
Embryology and Anatomy The intrahepatic biliary ductal system consists of a network of progressively larger tributaries that include ductules (synonymous with cholangioles) and ducts. Ductules (õ20 mm) normally connect the canals of Hering (the openings adjacent to the canalicular domain of hepatocytes) with the interlobular bile ducts (20–100 mm), which are the smallest ducts that are always accompanied by hepatic artery and portal vein radicles. Bile drains from interlobular bile ducts through progressively larger septal (100–400 mm) and segmental ducts into the hepatic ducts (400–800 mm).1 The histogenesis of biliary epithelium is controversial. The traditional concept has been that biliary epithelial cells derive from extrahepatic hilar ductal structures. Activation of stem cells late in fetal life also has been postulated.2 However, these concepts are now challenged by / 5E16$$0062
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several recent data suggesting that bile duct formation stems from the transformation of parenchymal cell precursors (i.e., hepatoblasts) into differentiated biliary epithelial cells during late fetal liver development.2 – 4 Under light microscopy (Figure 1A and B), the intrahepatic bile ductules and ducts are lined by a heterogeneous population of cholangiocytes that vary from cuboidal to columnar (Ç6–15 mm). Ultrastructurally, cholangiocytes lining a typical bile duct show apical and basolateral domains easily distinguished by features such as apically oriented vesicles and tight junctions and numerous apical microvilli (Figure 1C, inset). These microvilli are a feature of both intrahepatic and extrahepatic cholangiocytes (Figure 1D). Scanning electron micrographs confirm the uniform distribution of microvilli; they also show that cholangiocytes can secrete mucus (Figure 1D, inset). Intrahepatic bile ducts receive their blood supply from a periductal network of minute vessels, termed the peribiliary vascular plexus. This plexus originates from hepatic artery branches and drains mostly into the sinusoids (Figure 1E).5 Insufficient perfusion of this system can cause profound, albeit incompletely appreciated, duct damage.6 Structural alterations of the peribiliary plexus occur in various hepatobiliary diseases, but reliable pathophysiological correlations have not been made at this time.
Experimental Models Traditionally, radiolabeled derivatives of inert carbohydrates, such as [14C]mannitol, have been used as in vivo markers of canalicular bile flow. Unfortunately, the ability of these markers to discriminate canalicular from Abbreviations used in this paper: AIDS, acquired immunodeficiency syndrome; AMA, antimitochondrial antibody; ANA, antinuclear antibody; CFTR, cystic fibrosis transmembrane conductance regulator; GVHD, graft-vs.-host disease; HIV, human immunodeficiency virus; PBC, primary biliary cirrhosis; PDC, pyruvate dehydrogenase complex; PSC, primary sclerosing cholangitis. q 1997 by the American Gastroenterological Association 0016-5085/97/$3.00
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Figure 1. Normal human bile duct (BD) morphology. (A ) Interlobular bile duct lined by cuboidal cholangiocytes. The accompanying hepatic artery (A) and portal vein (V) radicles are not visible in this particular frame (H&E). (B ) Septal bile duct lined by columnar cholangiocytes (H&E). These ducts are fed by two or more interlobular bile ducts as shown in A. Note that in both frames the ducts appear empty. (C ) Transmission electron micrograph of interlobular bile duct. Note microvilli in the lumen of the duct (arrow ). (Inset ) Close-up view of microvilli. (D ) Scanning electron micrograph of cholangiocytes lining the common bile duct. Each cell is delineated by a groove. Note the uniformly distributed microvilli on the surface of each cholangiocyte. (Inset ) View into a choledochal lacuna showing mucus secretion (arrowheads ) by cholangiocytes. (E ) Schematic drawing of the peribiliary vascular plexus (PBVP). DV, drainage vein for PBVP; FA, feeder artery for peribiliary vascular plexus; PB, portal branch (original magnification: A, 4001; B, 1001; C, 14001; inset, 63001; D, 30001; and inset, 7001). (Reprinted with permission62.)
ductal bile secretion is quite limited, and new approaches are clearly needed. In rodents, selectively proliferated cholangiocytes can be studied after bile duct ligation or dietary manipulation with noncarcinogenic (e.g., a-naphthylisothiocyanate or lithocholic acid) or carcinogenic regimens, for example, / 5E16$$0062
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with choline-deprived diet containing ethionine. Ductal bile secretion, cholangiocyte proliferation, and the ontogeny of intrahepatic biliary epithelia have all been investigated with these models.1 Recently, intact intrahepatic bile duct units have been isolated from normal rats by enzymatic digestion and WBS-Gastro
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microdissection; these units retain their in situ morphology, polarity, and cholangiocyte-specific phenotypes (Figure 2A–C).7 This technique already has proved useful for the study of several ductal transport processes, such as agonist-stimulated HCO30 and H/ secretion, and tubulovesicular transcytosis and will likely be used to study other functions of intrahepatic bile ducts.1 Despite technical difficulties, many groups studied cholangiocyte biology with isolated cholangiocyte-enriched preparations (20%–95% pure) from normal rat liver using different separation methods, including isopycnic centrifugation, counterflow elutriation, and immunoaffinity separation.1 The last approach yields ¢95% pure cholangiocytes that retain their morphological polarity for a short time (Figure 2D and E).8 Others used cholestatic livers of bile duct–ligated or a-naphthylisothiocyanate–fed rats to isolate and study large numbers of purified hyperplastic cholangiocytes.9 For the study of normal human livers10 and cholestatic liver diseases,11 highly purified human cholangiocytes also have been isolated, again using immunoaffinity techniques. For the study of primary monolayer cultures of human10 – 13 and rodent14 – 16 cholangiocytes, researchers have used fibroblasts as a feeder layer, plated cells onto suitable extracellular matrix substitutes, or used isolated segments of bile ducts as starting material. This last technique (Figure 2F–G) yielded long-term confluent monolayer cultures of normal rat cholangiocytes that not only retained morphological polarity and cholangiocyte-specific phenotypes but also generated high transepithelial electrical resistances that recently helped to identify an apical glucose transporter in cholangiocytes.14,17 Using retroviral transduction with simian virus (SV40) large T antigen,13 continuous human cell lines were established from immortalized normal cholangiocytes; they remained positive for g-glutamyl-transferase and cytokeratin-19. Carcinoma cell lines of human ductal origin are also available. They are well differentiated, easy to culture, and often retain specific cholangiocyte and epithelial phenotypes. For example, they allowed the study of ion secretion,18 secretory products such as membrane glycoproteins,19 tumor-associated proteins, growth factors, and the regulation of cell growth and proliferation.20
Cholangiocyte Function Ductal Bile Formation Bile formation by cholangiocytes, which is in part hormone-induced, contributes approximately 40% to total bile flow21 by adding ions and water to canalicular bile. In vivo studies with rats with ductal hyperplasia confirmed that an increase in the number of cholangio/ 5E16$$0062
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Figure 2. Experimental in vitro models of biliary epithelia. (A ) Light micrographs of freshly isolated bile duct units in 1-mm cross sections stained with methylene blue. CT, connective tissue; L, lumen. (B ) Transmission electron microscopy. Note the apical microvilli (arrowheads ) projecting into the lumen (L), basally located nucleus (N), and numerous folds and interdigitations at the basal surface. (C ) Illustration showing the bile duct unit lumen containing oil (arrow ) after microinjection of an oil droplet (OD) with a micropipette (P). (Reprinted with permission.7). (D ) Scanning electron micrograph of a group of isolated cholangiocytes after immunomagnetic separation on magnetic beads (M). Note the prominent microvilli (arrowheads ) are limited to one side of the cell. (E ) Low-magnification view of isolated cholangiocytes. Note the microvilli (arrows ) limited to one side of the cell. (Reprinted with permission8.) (F ) Phase-contrast microscopy shows the epithelial character of normal rat cholangiocytes grown on thick collagen. (G ) Scanning electron microscopy of the apical surface of normal rat cholangiocytes. The ‘‘cobblestone-like’’ appearance of cholangiocytes can be seen at low magnification. (Reprinted with permission.14)
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We do not know how ductal ion transport regulates ductal water movement. However, water channels appear to be involved; indeed, several species, including humans, express at least one subtype of membrane water channels (i.e., aquaporin channel-forming integral membrane protein) at the apical and/or basolateral domains,32,33 and rodent cholangiocytes transport water transcellularly via a channel-mediated mechanism.33 This water movement may occur at the apical membrane of cholangiocytes in response to transient transmembrane osmotic gradients created by hormone-stimulated ion transport (see Figure 3). Figure 3. Plasma membrane protein topography in cholangiocytes. A working model of the cellular mechanisms regulating ductal ion and water secretion. Cx, connexin; CHIP 28, channel integral membrane protein of 28 kilodaltons; EGF, epidermal growth factor.
cytes is accompanied by a large increase in both basal and secretin-induced bile flow.22 Biliary bicarbonate secretion is also invariably increased during secretin-stimulated choleresis1,22 and decreased during somatostatininduced cholestasis,23 suggesting that ductal HCO30 transport is closely related to ductal water secretion. Recent in vitro studies showed that both secretin and somatostatin regulate ductal bile secretion by interacting with surface receptors on cholangiocytes; each hormone altered intracellular levels of adenosine 3*,5*-cyclic monophosphate (cAMP).24,25 Secretin also stimulated exocytosis in cholangiocytes via a cAMP mediated mechanism.24 Two important transport proteins, a Cl0/HCO30 exchanger and a cyclic adenosine monophosphate–activated Cl0 channel (i.e., cystic fibrosis transmembrane conductance regulator [CFTR]), are functionally26 – 29 expressed in the apical domain of cholangiocytes. Secretin stimulates the activity of the Cl0/HCO30 exchanger, likely via the activation of CFTR and possibly other cAMP-activated Cl0 channels.26 Thus, analogous to the observations in ductal cells of the pancreas,30 secretin appears to stimulate biliary ductal HCO30 secretion by initially binding to a receptor on the basolateral domain of cholangiocytes, thereby stimulating the activity of CFTR on the apical domain through a cAMP-mediated mechanism. This sequence results in an increased activity of an apical Cl0/HCO30 exchanger in which luminal Cl0 is exchanged for intracellular HCO30, resulting in cholangiocyte HCO30 secretion (see Figure 3). Exocytosis in ductal bile secretion may involve hormone-regulated insertion of vesicles containing ion channels into the apical plasma membrane or insertion of vacuolar H/ pumps into the basolateral membrane of cholangiocytes.24,31 / 5E16$$0062
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Cholangiocyte Proliferation and Growth Cholangiocytes proliferate in a variety of patterns, not only in rat experiments but in human biliary liver diseases such as primary biliary cirrhosis (PBC) and in alcoholic liver disease or viral and drug-induced hepatitis.1,34 Type I ductular hyperplasia in rats follows bile duct ligation or feeding either a-naphthylisothiocyanate, lithocholic acid, or 4*,4*-diaminodiphenylmethane. Cholangiocytes proliferate and form an organized network of well-defined tubular structures that remain confined to portal tracts.1,34 Although these hyperplastic cells resemble normal cholangiocytes, their origin and the mechanism(s) responsible for their proliferation under these circumstances remain obscure.1 In contrast, type II ductular hyperplasia is observed in conditions such as severe hepatitis B virus or PBC.1,34 Proliferating bile ductules form a three-dimensional network of tortuous, poorly defined conduits that extend into the parenchyma.1 Current data suggest that the cells lining these ductules represent retrodifferentiated hepatocytes and not replicated cholangiocytes.1 Type III ductular hyperplasia appears in the early stages of experimentally induced carcinogenesis in rat liver. Oval-shaped cells proliferate and form disorganized, ill-defined tubular structures extending into the parenchyma and distorting the lobular architecture.1 These oval cells resemble both cholangiocytes and hepatocytes and express cytokeratin-19, albumin, and a-fetoprotein typically observed with cells of ductular, parenchymal, or neoplastic origin. The origin of oval cells and their role in the development of hepatocellular carcinoma remain controversial. Recently identified proteins that may regulate cholangiocyte growth and proliferation include hepatocyte growth factor, epidermal growth factor, transforming growth factor a, proline, cholecystokinin, and insulinlike growth factors.1,35 Hepatocyte growth factor may be WBS-Gastro
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an important mitogen for human cholangiocytes in vivo because its receptors are expressed by cholangiocytes36 and because human hepatocyte growth factor stimulates DNA synthesis of cultured human cholangiocytes.10 Cholangiocyte Heterogeneity Epithelia such as hepatocytes, renal tubular cells, and intestinal epithelial cells are functionally heterogeneous. For example, renal tubular epithelial cells function to dilute or concentrate luminal contents depending on their location in the renal tubular system. Similarly, cholangiocytes appear functionally heterogeneous.1 Thus, agonist-induced ductal transport of ions and water occurs principally in cholangiocytes lining the medium and large bile ducts, not the smaller radicles. Further studies are required to completely map the predicted functional diversity of the intrahepatic bile ducts. Of particular interest are small cholangiocytes because they appear to be the prime target of immune attacks as observed in PBC or allograft rejection.
Cholangiocytes in Human Disease: The Cholangiopathies The cholangiopathies represent diseases and syndromes of the biliary system at any site between the canals of Hering and the ampulla of Vater. Depending on the purpose of study, pathophysiological, morphological, and etiologic classifications can be applied. In Table 1, we propose a classification based principally on known or purported etiologies. Herein we briefly review some of the conditions listed in Table 1, in particular, the immune-mediated and infectious cholangiopathies; they are the focus of our discussion because much progress has been made in our understanding of these disorders. Immune-Mediated Cholangiopathies Hepatic allograft rejection. Allograft rejection is a major cause of morbidity after liver transplantation. By convention, cellular (acute) rejection is distinguished from ductopenic (chronic) rejection.37 Cellular rejection is characterized by nonsuppurative cholangitis (Figure 4A) and endotheliitis caused by alloimmune-mediated injury to cholangiocytes and to portal and hepatic vein endothelia, respectively.38 Ductopenic rejection connotes progressive loss of interlobular bile ducts, often in association with foam cell arteriopathy (vascular rejection).38 Rejection cholangitis is characterized by the aberrant expression of HLA class II antigens in cholangiocytes38,39 and by the presence of recipient T cells infiltrating cholangiocytes. The resulting damage usually is reversible, but rejection also may lead to irreversible duct loss, i.e.,
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Table 1. Classification of the Cholangiopathies Immune-mediated (definite and putative) Hepatic allograft rejection (definite) GVHD (definite) PBC (putative) Autoimmune cholangitis (putative) PSC (putative) Infectious Viral (including HIV-associated cholangitis) Bacterial Fungal Parasitic Protozoan (HIV associated) Genetic and developmental Cystic fibrosis Allagille’s syndrome (syndromatic paucity of intrahepatic bile ducts) Fibropolycystic liver diseases (e.g., congenital hepatic fibrosis) Drug-induced (e.g., amoxicillin) Ischemic Idiopathic Biliary atresia Idiopathic childhood ductopenia (nonsyndromatic paucity of intrahepatic bile ducts) Idiopathic adulthood ductopenia Chronic cholestasis of sarcoidosis Neoplastic: cholangiocarcinoma (bile duct adenocarcinoma) NOTE. By convention, cholelithiasis and choledocholithiasis are excluded.
ductopenic rejection. The traditional two types of rejection are essentially features of the same process, with the prognosis depending primarily on the degree of duct loss. Vascular rejection may damage ducts indirectly by causing biliary ischemia. Cytomegalovirus infection, biliary ischemia unrelated to vascular rejection, and other complications may play a pathogenetic role, possibly by stimulating the expression of HLA antigens in cholangiocytes. Graft-vs.-host disease. Graft-vs.-host disease (GVHD) after bone marrow transplantation may cause cholangiocyte injury and duct loss; this phenomenon probably is mediated by allogeneic T cells because removal of graft T cells prevents experimental chronic GVHD.38 However, autoreactivity may also play an important role, particularly in cases with injury to interlobular and septal bile ducts, as shown by aberrant expression of HLA class II molecules by cholangiocytes, presence of hypergammaglobulinemia, and appearance of autoantibodies, including antimitochondrial antibodies (AMAs), antinuclear antibodies (ANAs), and rheumatoid factor.38 The triggering antigens of GVHD remain unknown, but the frequent occurrence of GVHD in recipients of bone marrow from HLA-identical sibling donors raises the possibility that a cholangiocyte-specific antiWBS-Gastro
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Figure 4. Pathology of cholangiopathies. (A ) Cholangitis in cellular rejection 5 days after liver transplantation. The interlobular bile duct (asterisk ) is surrounded by a dense mixed inflammatory infiltrate (H&E). (B ) Fibrous obliterative cholangitis in PSC. Note interlobular bile duct with periductal fibrosis, infiltration of bile duct epithelium by neutrophils, and degeneration of cholangiocytes (H&E). (Reprinted with permission54.) (C ) Ischemic cholangiopathy with bile duct necrosis and secondary inflammation (ischemic cholangitis) 11 months after liver transplantation (H&E). (Reprinted with permission.63) (D ) Cholangiocarcinoma complicating PSC with growth in a perineural space (H&E) (original magnification: A and D, 3201; B, 4001; and C, 72.51) N, nerve.
gen, acting as a minor histocompatibility antigen, is the target protein for the immunologic reaction.38 PBC. PBC affects adults and mostly women and is characterized by inflammatory and often granulomatous destruction of small bile ducts.38,40 PBC has become a common indication for orthotopic liver transplantation. Several observations suggest that PBC is an autoimmune disease, including the high frequency of disease-specific AMAs of the M2 type and other serum autoantibodies; the increased frequency of HLA C4A-Q0, which is a class / 5E16$$0062
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III allele associated with many autoimmune diseases; the presence of activated T (predominantly CD4/ and CD8/) and B lymphocytes in and around cholangiocytes aberrantly expressing MHC class II; and the frequent association of PBC with autoimmune diseases, such as Sjo¨gren’s disease, rheumatoid arthritis, and autoimmune thyroiditis.38,40 Despite this evidence, trials with immunomodulatory agents such as D-penicillamine, azathioprine, methotrexate, or cyclosporine A have not been successful, thus casting some doubt on the concept of autoimmuWBS-Gastro
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nity.41 Unfortunately, no reliable animal models of PBC exist. So far, experimental strategies have included immunizing animals with a number of different immunogens, such as gallbladder mucous membranes, mitochondrial antigens, cholangiocytes, and peripheral blood lymphocytes from patients with PBC. Murine chronic GVHD40 also has been studied in this context. The most promising recent reports suggest that immune-mediated cholangitis can be induced in neonatally thymectomized mice that were stimulated with cholangiocyte antigens42 or in adult rats that were immunized with highly purified cholangiocytes.43 The mitochondrial 2-oxo-acid dehydrogenase multienzyme complex is the M2 autoantigen specific for PBC serum, and the E2 component of the pyruvate dehydrogenase complex (PDC) is the major autoantigen. This may mean that M2 in cholangiocytes is the target autoantigen in PBC38,40 because cholangiocytes express higher levels of PDC-E2 than other cells in normal liver, cholangiocytes express high levels of PDC-E2 in PBC livers, cultured cholangiocytes from patients with PBC express PDC-E2 on their surface, and autoepitopes of E2 are preferentially expressed in the luminal region of cholangiocytes in PBC livers. However, M2-related T-cell cytotoxicity has not been shown in PBC, and the mechanism of autoantibody production or the putative action of antiM2 antibodies has not been elucidated.38,40 One hypothesis to explain the immunologic injury to bile ducts in PBC suggests that, initially, T cells recognize epitopes of microbial proteins that share cross-reactive epitopes with PDC-E2. This is followed by a T- and B-cell reaction against these self-antigens that become aberrantly expressed with MHC class II on cholangiocytes.44 This molecular mimicry between microbial proteins and self-peptides is supported by AMA cross-reactivity with subcellular constituents of gram-negative and gram-positive organisms, the presence of certain PBCspecific reactive proteins in several Enterobacteriaceae species, the observation that mutant forms of bacteria such as Escherichia coli in urinary tract infection may elicit PBC-specific M2 antibody positivity, and the existence of familial AMA.45 An alternative hypothesis proposes that the transport of PDC-E2 to the mitochondria for assembly is affected by a genetic mutation (e.g., alteration to the leader sequence) that leads to aberrant or modified expression of PDC-E2 on the surface of cholangiocytes. Given that cholangiocytes may function as antigen-presenting cells in PBC, altered processing of normal PDC-E2 could conceivably lead to the presentation of nontolerized peptides of PDC-E2 to immunocytes resulting in immune-mediated cholangitis. / 5E16$$0062
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Autoimmune cholangitis. Autoimmune cholangitis is a controversial cholestatic disease that shares features with both PBC and autoimmune hepatitis. The diagnosis requires clinical and/or biochemical cholestasis, a liver biopsy specimen with nonsuppurative cholangitis, laboratory evidence of AMA and anti-M2 antibody negativity, high titers of serum ANAs, and immunoglobulin (Ig) M concentrations that tend to be lower and IgG concentrations that tend to be higher than in PBC.38,46–52 The etiology of autoimmune cholangitis is unknown, and the pathogenesis is poorly understood. Responses to corticosteroid therapy may be dramatic,47 – 49 but these are not invariable.51,52 Although PBC and autoimmune hepatitis probably may occur together or in sequence,48 some cases appear unrelated; terms such as autoimmune cholangiopathy,47 immunocholangitis,49 overlap syndrome,50 and primary autoimmune cholangitis51 have been suggested to describe such instances. The classification of autoimmune cholangitis remains controversial particularly because many patients with AMA-negative PBC also have ANA (often in high titers) and/or anti–smooth muscle antibodies and comparatively low IgM levels.46,52,53 Indeed, autoimmune cholangitis is often indistinguishable from AMA-negative PBC46,51 – 53 and, in many instances, can only be distinguished from AMA-positive PBC by its immunoserological profile.52 Adding to the controversy is the fact that autoimmune cholangitis has been classified by some as part of the spectrum of autoimmune hepatitis47 and by others as a distinctive subgroup of AMA-negative adulthood ductopenic disorders.51 Thus, to bring this controversy into better focus, we like others52 suggest to use the name autoimmune cholangitis as a collective term, akin to autoimmune hepatitis, with multiple subgroups of the disease identified by different autoantibody profiles; these would include AMA-positive PBC, AMAnegative PBC, and ANA-positive, AMA-negative cholangitis, among other possible constellations. This would by no means preclude using the term PBC as a diagnosis, but it would define the situation much more clearly and would place the serological variations in a context that easily lends itself to further studies. Primary sclerosing cholangitis. Primary sclerosing cholangitis (PSC) is characterized cholangiographically by strictures, beading, and irregularities of the biliary tree and pathologically by inflammation and fibrosis of intrahepatic (Figure 4B) and extrahepatic bile ducts. The chronic disease typically affects young men and is associated with inflammatory bowel disease, especially chronic ulcerative colitis54,55 in approximately 75% of the cases. Current evidence suggests that, among a
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multitude of proposed mechanisms, genetic and immunologic factors likely play the most significant pathogenetic role in PSC.54,55 A few reports of familial PSC suggest a genetic predisposition and so do haplotype studies associating PSC with HLA B8, DR2, DR3, DR4, and DRw52a.54,55 After comparison with appropriate controls, DR52a and Dw2 emerge as the best candidate alleles for the HLA association with PSC. The association attains particular importance in patients who possess the HLA DR4 allele because it may herald rapid disease progression. Cellular immune abnormalities in PSC include decreased peripheral blood T lymphocytes with a relative increase in the CD4/CD8 ratio, T-lymphocyte predominant infiltrates in the vicinity of proliferating and damaged bile ducts, enhanced autoreactivity of portal tract T lymphocytes, and an increased number and proportion of circulating B cells.56 Humoral immune abnormalities in PSC include hypergammaglobulinemia, increased circulating immune complexes, and circulating autoantibodies such as perinuclear antineutrophil cytoplasmic antibodies, antineutrophil nuclear antibodies, anticolon epithelial autoantibodies, and sometimes the classic autoantibodies ANA, AMA, and anti– smooth muscle antibody.54– 56 Furthermore, HLA class II antigens are aberrantly expressed by cholangiocytes at an early stage of the disease, and peripheral blood lymphocytes from patients with PSC are sensitized to unidentified biliary antigens. Moreover, intracellular adhesion molecule 1, a ligand that facilitates lymphocyte attachment to target cells, appears in cholangiocytes in late-stage PSC, and PSC is associated with immune diseases such as type I diabetes and thyroiditis, among others. The morphological distribution of PSC with its involvement of large ducts differs profoundly from the findings in the other autoimmune or putative autoimmune cholangitis. This strongly suggests that very important pathogenetic differences exist. Although, evidence supports the view that PSC results from autoimmune-mediated injury to cholangiocytes, the data appear less convincing than in PBC. Autoimmunity may be linked to an antibody that recognizes a cross-reactive peptide in small and large duct cholangiocytes as well as in colonic epithelial cells from patients with ulcerative colitis.57 Indeed, two thirds of patients with PSC have circulating IgG autoantibodies against such an antigen.57 Such a mechanism could explain the frequent coexistence of PSC with ulcerative colitis. Infectious Cholangiopathies Bile ducts may be affected by viral, bacterial, fungal, protozoan, and parasitic infections. Recent experi/ 5E16$$0062
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ences have accumulated mostly in the acquired immunodeficiency syndrome (AIDS). AIDS cholangiopathy. Cholangiography in human immunodeficiency virus (HIV)-infected patients may show five distinct patterns: sclerosing cholangitis, polypoid cholangitis, papillary stenosis, combined papillary stenosis and sclerosing cholangitis, and long, extrahepatic bile duct strictures.58 Probable causative microorganisms are identified in about 60% of the AIDS cholangiopathies; they include cytomegalovirus, Cryptosporidium, Microsporidium, and Mycobacterium avium.58,59 Cytomegalovirus is very common in patients with AIDS and is readily recognizable in biopsy specimens, whereas Cryptosporidium, another commonly isolated agent, is easily overlooked although it may attach to the luminal surface of cholangiocytes. In patients with AIDS without evidence of an opportunistic biliary infection, the HIV itself has been implicated. However, HIV infection of cholangiocytes has not yet been shown. Like many other cholangiopathies, aberrant HLA class II expression on cholangiocytes is seen in AIDS; whether this is an important observation is unclear. Genetic and Developmental Cholangiopathies This group of cholangiopathies comprises a considerable number of conditions, including Alagille’s syndrome and the fibropolycystic liver diseases such as congenital hepatic fibrosis. Major new insights have been gained only in cystic fibrosis. Cystic fibrosis. This lethal autosomal recessive disorder of exocrine glands is caused by the abnormal CFTR, a chloride channel found on chloride-secreting epithelia.60 In adult patients with cystic fibrosis, mucus obstruction of intrahepatic bile ducts is often found, with or without biliary fibrosis. Mutations in CFTR, which is in the apical domain of cholangiocytes, lead to defective ductal Cl0 and water secretion, resulting in biliary disease. No specific therapy is available for cystic fibrosis liver disease; there is some hope for preventive treatment in the near future based on recombinant adenoviruses expressing human CFTR; infusion of these viruses into rat livers temporarily generated CFTR.61 Drug-Induced Cholangiopathies Little progress has been made in the understanding of these conditions although the number of drugs that may cause cholangitis and lead to ductopenia is steadily growing; close to 20 are now known (Table 2). Vascular Cholangiopathies Surgical trauma to hepatic arteries, anastomotic arterial strictures, hepatic artery thrombosis, and foam WBS-Gastro
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Table 2. Drugs That May Cause Nonsuppurative Cholangitis and Ductopenia Generic or chemical name Acetaminophen Amitryptilin Amoxicillin Ampicillin Carbamazepine Chlorpromazine Cromolyn sodium Cyproheptadine Diazepam Erythromycin Haloperidol Imipramine Methyltestosterone Phenylbutazone Prochlorperazine Thiabendazole Tolbutamide Trifluoperazine hydrochloride Troleandomycin
Product classification Analgesic and antipyretic Antidepressant Antibiotic Antibiotic Anticonvulsant Tranquilizer:phenothhiazine Mast cell stabilizer Antihistamine and antiserotonergic Sedative and hypnotic:benzodiazepine Antibiotic Antipsychotic Antidepressant Androgen Anti-inflammatory Tranquilizer:phenothiazine Anthelmintic Oral hypoglycemic Tranquilizer:phenothiazine Antibiotic
cell arteriopathy in allograft rejection are the main causes of vascular cholangiopathies. The low perfusion of the biliary plexus may lead to ductal inflammation with fibrosis and diffuse or segmental degeneration of cholangiocytes (Figure 4C).6 If inflammatory changes are prominent, the condition can be classified as ischemic cholangitis. Neoplasia: Cholangiocarcinoma Cholangiocarcinoma, a malignant tumor of biliary epithelia (Figure 4D), is the most important neoplasm of intrahepatic bile ducts because it is frequent and so far almost universally fatal. Early diagnosis is difficult, and effective therapy is lacking.20 Risk factors for cholangiocarcinoma include PSC, thorium dioxide (Thorotrast) and anabolic steroid administration, biliary cysts such as choledochal cysts, Caroli’s disease and other fibropolycystic liver diseases, and in the Far East, chronic infestation of the bile ducts with the parasitic flukes Opisthorchis viverrini and Clonorchis sinensis. The molecular mechanisms responsible for the malignant transformation of cholangiocytes are unknown, in part because little information is available on the factors regulating cholangiocyte proliferation. Still, several cholangiocarcinoma cell lines prominently express transcript for one subtype of somatostatin receptor SSTR2.20 Based on this observation, investigators have recently shown that a radiolabeled derivative of somatostatin can accurately detect the presence of cholangiocarcinoma in humans and that long-acting somatostatin analogues can inhibit the growth of cholangiocarcinoma cell lines in vitro and in vivo after injection in nude mice.20 Pilot / 5E16$$0062
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studies are now under way in humans to assess somatostatin analogues in the diagnosis and treatment of cholangiocarcinoma.
Summary Our understanding of the pathobiology of biliary epithelia is rapidly growing because of a surge of investigative activity. This became possible after suitable experimental models and techniques were developed with which to study cholangiocyte biology. Although the molecular mechanisms of bile formation by cholangiocytes and the role of these cells as a major cellular target in a variety of severe hepatobiliary diseases are currently being investigated, many questions remain unanswered, particularly regarding cholangiocellular functions, both in normal and abnormal conditions. As current experimental models become more refined, scientists with interests as diverse as cell biology and physiology, morphology, pharmacology, immunology, genetics, and oncology can be expected to further clarify the pathobiology of biliary epithelia.
References 1. Alpini G, Phillips JO, LaRusso NF. The biology of biliary epithelia. In: Arias IM, Boyer JL, Fausto N, Jakoby WB, Schachter DA, Shafritz DA, eds. The liver: biology and pathobiology. New York: Raven, 1994:623–653. 2. Desmet VJ, Roskams T, Van Eyken P. Ductular reaction in the liver. Pathol Res Pract 1995;191:513–524. 3. Shiojiri N. The origin of intrahepatic bile duct cells in the mouse. J Embryol Exp Morphol 1984;79:25–39. 4. Shah K, Gerber MA. Development of intrahepatic bile ducts in humans. Immunohistochemical study using monoclonal cytokeratin antibodies. Arch Pathol Lab Med 1989;113:1135–1138. 5. Terada T, Ishida F, Nakanuma Y. Vascular plexus around intrahepatic bile ducts in normal livers and portal hypertension. J Hepatol 1989;8:139–149. 6. Ludwig J, Batts KP, McCarty RL. Ischemic cholangitis in hepatic allografts. Mayo Clin Proc 1992;67:519–526. 7. Roberts SK, Kuntz SM, Gores GJ, LaRusso NF. Regulation of bicarbonate-dependent ductular bile secretion assessed by lumenal micropuncture of isolated rodent intrahepatic bile ducts. Proc Natl Acad Sci USA 1993;90:9080–9084. 8. Ishii M, Vroman B, LaRusso NF. Isolation and morphologic characterization of bile duct epithelial cells from normal rat liver. Gastroenterology 1989;97:1236–1247. 9. Sirica AE, Cihla HP. Isolation and partial characterizations of oval and hyperplastic bile ductular cell-enriched populations from the livers of carcinogen and noncarcinogen-treated rats. Cancer Res 1984;44:3454–3466. 10. Joplin R, Strain AJ, Neuberger JM. Immuno-isolation and culture of biliary epithelial cells from normal human liver. In Vitro Cell Dev Biol 1989;25:1189–1192. 11. Joplin R, Strain AJ, Neuberger JM. Biliary epithelial cells from the liver of patients with primary biliary cirrhosis: isolation, characterization, and short-term culture. J Pathol 1990;162:255–260. 12. Demetris AJ, Markus BH, Saidman S, Fung JJ, Makowka L, Graner S, Duquesnoy R, Starzl TE. Isolation and primary cultures of hu-
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man intrahepatic bile ductular epithelium. In Vitro Cell Dev Biol 1988;24:464–470. Grubman SA, Perrone RD, Lee DW, Murray SL, Rogers LC, Wolkoff LI, Mulberg AE, Cherington V, Jefferson DM. Regulation of intracellular pH by immortalized human intrahepatic biliary epithelial cell lines. Am J Physiol 1994;266:G1060–G1070. Vroman B, LaRusso NF. Development and characterization of polarized primary cultures of rat intrahepatic bile duct epithelial cells. Lab Invest 1996;74:303–313. Yang L, Faris RA, Hixson DC. Long-term culture and characteristics of normal rat liver bile duct epithelial cells. Gastroenterology 1993;104:840–852. Mathis GA, Walls SA, Sirica AE. Biochemical characteristics of hyperplastic rat bile ductular epithelial cells cultured ‘‘on top’’ and ‘‘inside’’ different extracellular matrix substitutes. Cancer Res 1988;48:6145–6153. Lazaridis K, Vroman B, LaRusso NF. Direct evidence that rat cholangiocytes can absorb and transport glucose. J Invest Med 1995;43:398A. Basavappa S, Middleton J, Mangel AW, McGill JM, Cohn JA, Fitz JG. Cl0 and K/ transport in human biliary cell lines. Gastroenterology 1993;104:1796–1805. Yamaguchi N, Morioka H, Okhura H, Hirohashi S, Kawai K. Establishment and characterization of the human cholangiocarcinoma cell line HChol-Y1 in a serum-free, chemically defined medium. J Natl Cancer Inst 1985;75:29–35. Tan CK, Podila PV, Taylor JE, Nagorney DM, Wiseman GA, Gores GJ, LaRusso NF. Human cholangiocarcinomas express somatostatin receptors and respond to somatostatin with growth inhibition. Gastroenterology 1995;108:1908–1916. Nathanson MH, Boyer JL. Mechanisms and regulation of bile secretion. Hepatology 1991;14:551–566. Alpini G, Ulrich CD, Pham LD, Phillips JO, Miller LJ, LaRusso NF. Upregulation of secretin receptor gene expression in rat cholangiocytes after bile duct ligation. Am J Physiol 1994;266:G922– G928. Tietz PS, Alpini G, Pham L, LaRusso NF. Somatostatin inhibits secretin-induced ductal hypercholeresis and exocytosis by cholangiocytes. Am J Physiol 1995;269:G110–G118. Kato A, Gores GJ, LaRusso N. Secretin stimulates exocytosis in isolated bile duct epithelial cells by a cyclic AMP-mediated mechanism. J Biol Chem 1992;267:15523–15529. Farouk M, Vigna SR, McVey DC, Meyers WC. Localization and characterization of secretin binding sites expressed by rat bile duct epithelium. Gastroenterology 1992;102:963–968. Alvaro D, Cho WK, Mennone A, Boyer JL. Effect of secretin on intracellular pH regulation in isolated rat bile duct epithelial cells. J Clin Invest 1993;92:1314–1325. Strazzabosco M, Mennone A, Boyer JL. Intracellular pH regulation in isolated rat bile duct epithelial cells. J Clin Invest 1991;87: 1503–1512. Fitz JG, Basavappa S, McGill J, Melhus O, Cohn JA. Regulation of membrane chloride currents in rat bile duct epithelial cells. J Clin Invest 1993;91:319–328. Martinez-Anso E, Castillo JE, Diez J, Medina JF, Prieto J. Immunohistochemical detection of chloride/bicarbonate anion exchangers in human liver. Hepatology 1994;19:1400–1406. Raeder MG. The origin of and subcellular mechanisms causing pancreatic bicarbonate secretion. Gastroenterology 1992;103: 1674–1684. Villanger O, Veel T, Raeder MG. Secretin causes H/ secretion from intrahepatic bile ductules by vacuolar-type H/-ATPase. Am J Physiol 1993;265:G719–G724. Nielson S, Smith BL, Christensen EI, Agre P. Distribution of aquaporin CHIP in secretory and resorptive epithelia and capillary endothelia. Proc Natl Acad Sci USA 1993;90:7275–7279.
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33. Roberts SK, Yano M, Ueno Y, Pham L, Alpini G, Agre P, LaRusso NF. Cholangiocytes express the aquaporin CHIP and transport water via a channel-mediated mechanism. Proc Natl Acad Sci USA 1994;91:13009–13013. 34. Desmet VJ. Current problems in the diagnosis of biliary disease and cholestasis. Semin Liver Dis 1986;6:233–245. 35. de Groen PC, Vroman B, LaRusso NF. Insulin is a key regulator of cholangiocyte proliferation in vitro. Hepatology 1995;22:323A. 36. Prat M, Narsimhan RP, Crepaldi T, Nicotra PG, Natali PG, Comoglio PM. The receptor encoded by the human c-Met oncogene is expressed in hepatocytes, in epithelial cells and in solid tumors. Int J Cancer 1991;49:323–328. 37. Weisner RH, Ludwig J, Krom RA, Hay JE, van Hoek B. Hepatic allograft rejection: new developments in terminology, diagnosis, prevention, and treatment. Mayo Clin Proc 1993;68:69–79. 38. Woolf GM, Vierling JM. Disappearing intrahepatic bile ducts: the syndromes and their mechanisms. Semin Liver Dis 1993;13: 261–275. 39. Wiesner RH, Ludwig J, van Hoek B, Krom RAF. Current concepts in cell-mediated hepatic allograft rejection leading to ductopenia and liver failure. Hepatology 1991;14:721–729. 40. Kaplan MM. Primary biliary cirrhosis. N Engl J Med 1987;316: 521–528. 41. Kaplan MM. New strategies needed for treatment of primary biliary cirrhosis. Gastroenterology 1993;104:654–662. 42. Kobashi H, Yamamoto K, Yoshioka T, Tomita M, Tsuji T. Nonsuppurative cholangitis is induced in neonatally thymectomized mice: a possible animal model for primary biliary cirrhosis. Hepatology 1994;19:1424–1430. 43. Ueno Y, Phillips JO, Ludwig J, Lichtman SN, LaRusso NF. Development and characterization of a rodent model of immune-mediated cholangitis. Proc Natl Acad Sci USA 1996;93:216–220. 44. Burroughs AK, Butler P, Sternberg MJE, Baum H. Molecular mimicry in liver disease. Nature 1992;358:377–378. 45. Sherlock S. Primary biliary cirrhosis: clarifying the issues. Am J Med 1994;96:27S–33S. 46. Michieletti P, Wanless IR, Katz A, Scheuer PJ, Yeaman SJ, Bassendine MF, Palmer JM, Heathcote EJ. Anti-mitochondrial antibody negative primary biliary cirrhosis: a distinct syndrome of autoimmune cholangitis. Gut 1994;35:260–265. 47. Ben-Ari Z, Dhillon AP, Sherlock S. Autoimmune cholangiopathy: part of the spectrum of autoimmune chronic active hepatitis. Hepatology 1993;18:10–15. 48. Colombato LA, Alvarez F, Cote J, Huet P-M. Autoimmune cholangiopathy: the result of consecutive primary biliary cirrhosis and autoimmune hepatitis? Gastroenterology 1994;107:1839– 1843. 49. Brunner G, Klinge O. A cholangitis with antinuclear antibodies (immunocholangitis) resembling chronic non-suppurative destructive cholangitis. Dtsch Med Wochenschr 1987;112:1454– 1458. 50. Lindor K, Wiesner RH, LaRusso NF, et al. Chronic active hepatitis: overlap with primary biliary cirrhosis and primary sclerosing cholangitis. In: Czaja AJ, Dickson ER, eds. Chronic active hepatitis. The Mayo Clinic experience. New York: Marcel Dekker, 1986: 171–187. 51. Taylor SL, Dean PJ, Riley CA. Primary autoimmune cholangitis. An alternative to antimitochondrial antibody-negative primary biliary cirrhosis. Am J Surg Pathol 1994;18:91–99. 52. Goodman ZD, McNally PR, Davis DR, Ishak KG. Autoimmune cholangitis: a variant of primary biliary cirrhosis. Clinicopathologic and serologic correlations in 200 cases. Dig Dis Sci 1995;40: 1232–1242. 53. Lacerda MA, Ludwig J, Dickson ER, Jorgenson R, Lindor K. Antimitochondrial antibody-negative primary biliary cirrhosis. Am J Gastroenterology 1995;90:247–249.
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54. LaRusso NF, Weisner RH, Ludwig J, MacCarty RL. Primary sclerosing cholangitis. N Engl J Med 1984;310:899–903. 55. Lee Y-M, Kaplan MM. Primary sclerosing cholangitis. N Engl J Med 1995;332:924–933. 56. Chapman RW. Role of immune factors in the pathogenesis of primary sclerosing cholangitis. Semin Liver Dis 1991;11:1–4. 57. Mandal A, Dasgupta A, Jeffers L, Squillante L, Hyder S, Reddy R, Schiff E, Das KM. Autoantibodies in sclerosing cholangitis aganist a shared peptide in biliary and colon epithelium. Gastroenterology 1994;106:185–192. 58. Cello JP. Human immunodeficiency virus–associated biliary tract disease. Semin Liver Dis 1992;12:213–218. 59. Pol S, Romana CA, Richard S, Amouyal P, Deportes-Livage I, Carnot F, Pays JF, Berthelot P. Microsporidia infection in patients with the human immunodeficiency virus and unexplained cholangitis. N Engl J Med 1993;328:95–99. 60. Denning GM, Ostedgaard LS, Cheng SH, Smith AE, Welsh MJ. Localization of cystic fibrosis transmembrane conductance regulator in chloride secretory epithelia. J Clin Invest 1992;89:339– 349.
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61. Yang Y, Raper S, Cohn J, Engelhardt J, Wilson J. An approach for treating the hepatobiliary disease of cystic fibrosis by somatic gene transfer. Proc Natl Acad Sci USA 1993;90:4601–4605. 62. Ludwig J, Kim CH, Wiesner RH, Krom RAF. Floxuridine-induced sclerosing cholangitis: an ischemic cholangiopathy? Hepatology 1989;9:215–218. 63. Ludwig J, Batts KP. Transplantation pathology. In: MacSween RNM, Anthony PP, Scheuer PJ, Burt AD, Portmann BC, eds. Pathology of the liver. 3rd ed. Edinburgh, Scotland: Churchill Livingstone, 1994.
Received May 10, 1996. Accepted August 20, 1996. Address requests for reprints to: Nicholas F. LaRusso, M.D., Center for Basic Research in Digestive Diseases, Mayo Medical School, Clinic and Foundation, 200 First Street Southwest, Rochester, Minnesota 55905. Fax: (507) 284-0762. Dr. Roberts’ current address is: Department of Gastroenterology, Alfred Hospital, Prahran, Victoria, Australia.
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VIEWPOINTS IN DIGESTIVE DISEASES The Pathobiology of Biliary Epithelia STUART K. ROBERTS,* JURGEN LUDWIG,* and NICHOLAS F. LARUSSO‡ Division of Gastroenterology and Internal Medicine, *Department of Biochemistry and Molecular Biology, and ‡Division of Anatomic Pathology, Mayo Medical School, Clinic and Foundation, Rochester, Minnesota
I
ntrahepatic biliary epithelial cells, or cholangiocytes, account for 3%–5% of the hepatic cell population and line a complex three-dimensional network of interconnecting conduits in the liver, termed the intrahepatic biliary ductal system. Early in this century, studies suggested that cholangiocytes play an important role in water and electrolyte transport; however, subsequent attempts to further delineate the functions of cholangiocytes were hindered by the lack of suitable experimental models. Recently, the knowledge of cholangiocyte biology has advanced considerably, based primarily on new experimental models that have allowed investigators to isolate and culture individual cholangiocytes and intact intrahepatic bile ducts. These advances have shown that cholangiocytes make an important contribution to basal and agonist-induced ductal bile formation. Moreover, these cells are candidate targets in many immune- and nonimmune-mediated liver diseases; as a collective name for these conditions, we propose the term cholangiopathies. Because of their morbidity, mortality, and overall cost to society, cholangiopathies have assumed major importance among liver diseases and syndromes.
Embryology and Anatomy The intrahepatic biliary ductal system consists of a network of progressively larger tributaries that include ductules (synonymous with cholangioles) and ducts. Ductules (õ20 mm) normally connect the canals of Hering (the openings adjacent to the canalicular domain of hepatocytes) with the interlobular bile ducts (20–100 mm), which are the smallest ducts that are always accompanied by hepatic artery and portal vein radicles. Bile drains from interlobular bile ducts through progressively larger septal (100–400 mm) and segmental ducts into the hepatic ducts (400–800 mm).1 The histogenesis of biliary epithelium is controversial. The traditional concept has been that biliary epithelial cells derive from extrahepatic hilar ductal structures. Activation of stem cells late in fetal life also has been postulated.2 However, these concepts are now challenged by / 5E16$$0062
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several recent data suggesting that bile duct formation stems from the transformation of parenchymal cell precursors (i.e., hepatoblasts) into differentiated biliary epithelial cells during late fetal liver development.2 – 4 Under light microscopy (Figure 1A and B), the intrahepatic bile ductules and ducts are lined by a heterogeneous population of cholangiocytes that vary from cuboidal to columnar (Ç6–15 mm). Ultrastructurally, cholangiocytes lining a typical bile duct show apical and basolateral domains easily distinguished by features such as apically oriented vesicles and tight junctions and numerous apical microvilli (Figure 1C, inset). These microvilli are a feature of both intrahepatic and extrahepatic cholangiocytes (Figure 1D). Scanning electron micrographs confirm the uniform distribution of microvilli; they also show that cholangiocytes can secrete mucus (Figure 1D, inset). Intrahepatic bile ducts receive their blood supply from a periductal network of minute vessels, termed the peribiliary vascular plexus. This plexus originates from hepatic artery branches and drains mostly into the sinusoids (Figure 1E).5 Insufficient perfusion of this system can cause profound, albeit incompletely appreciated, duct damage.6 Structural alterations of the peribiliary plexus occur in various hepatobiliary diseases, but reliable pathophysiological correlations have not been made at this time.
Experimental Models Traditionally, radiolabeled derivatives of inert carbohydrates, such as [14C]mannitol, have been used as in vivo markers of canalicular bile flow. Unfortunately, the ability of these markers to discriminate canalicular from Abbreviations used in this paper: AIDS, acquired immunodeficiency syndrome; AMA, antimitochondrial antibody; ANA, antinuclear antibody; CFTR, cystic fibrosis transmembrane conductance regulator; GVHD, graft-vs.-host disease; HIV, human immunodeficiency virus; PBC, primary biliary cirrhosis; PDC, pyruvate dehydrogenase complex; PSC, primary sclerosing cholangitis. q 1997 by the American Gastroenterological Association 0016-5085/97/$3.00
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Figure 1. Normal human bile duct (BD) morphology. (A ) Interlobular bile duct lined by cuboidal cholangiocytes. The accompanying hepatic artery (A) and portal vein (V) radicles are not visible in this particular frame (H&E). (B ) Septal bile duct lined by columnar cholangiocytes (H&E). These ducts are fed by two or more interlobular bile ducts as shown in A. Note that in both frames the ducts appear empty. (C ) Transmission electron micrograph of interlobular bile duct. Note microvilli in the lumen of the duct (arrow ). (Inset ) Close-up view of microvilli. (D ) Scanning electron micrograph of cholangiocytes lining the common bile duct. Each cell is delineated by a groove. Note the uniformly distributed microvilli on the surface of each cholangiocyte. (Inset ) View into a choledochal lacuna showing mucus secretion (arrowheads ) by cholangiocytes. (E ) Schematic drawing of the peribiliary vascular plexus (PBVP). DV, drainage vein for PBVP; FA, feeder artery for peribiliary vascular plexus; PB, portal branch (original magnification: A, 4001; B, 1001; C, 14001; inset, 63001; D, 30001; and inset, 7001). (Reprinted with permission62.)
ductal bile secretion is quite limited, and new approaches are clearly needed. In rodents, selectively proliferated cholangiocytes can be studied after bile duct ligation or dietary manipulation with noncarcinogenic (e.g., a-naphthylisothiocyanate or lithocholic acid) or carcinogenic regimens, for example, / 5E16$$0062
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with choline-deprived diet containing ethionine. Ductal bile secretion, cholangiocyte proliferation, and the ontogeny of intrahepatic biliary epithelia have all been investigated with these models.1 Recently, intact intrahepatic bile duct units have been isolated from normal rats by enzymatic digestion and WBS-Gastro
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microdissection; these units retain their in situ morphology, polarity, and cholangiocyte-specific phenotypes (Figure 2A–C).7 This technique already has proved useful for the study of several ductal transport processes, such as agonist-stimulated HCO30 and H/ secretion, and tubulovesicular transcytosis and will likely be used to study other functions of intrahepatic bile ducts.1 Despite technical difficulties, many groups studied cholangiocyte biology with isolated cholangiocyte-enriched preparations (20%–95% pure) from normal rat liver using different separation methods, including isopycnic centrifugation, counterflow elutriation, and immunoaffinity separation.1 The last approach yields ¢95% pure cholangiocytes that retain their morphological polarity for a short time (Figure 2D and E).8 Others used cholestatic livers of bile duct–ligated or a-naphthylisothiocyanate–fed rats to isolate and study large numbers of purified hyperplastic cholangiocytes.9 For the study of normal human livers10 and cholestatic liver diseases,11 highly purified human cholangiocytes also have been isolated, again using immunoaffinity techniques. For the study of primary monolayer cultures of human10 – 13 and rodent14 – 16 cholangiocytes, researchers have used fibroblasts as a feeder layer, plated cells onto suitable extracellular matrix substitutes, or used isolated segments of bile ducts as starting material. This last technique (Figure 2F–G) yielded long-term confluent monolayer cultures of normal rat cholangiocytes that not only retained morphological polarity and cholangiocyte-specific phenotypes but also generated high transepithelial electrical resistances that recently helped to identify an apical glucose transporter in cholangiocytes.14,17 Using retroviral transduction with simian virus (SV40) large T antigen,13 continuous human cell lines were established from immortalized normal cholangiocytes; they remained positive for g-glutamyl-transferase and cytokeratin-19. Carcinoma cell lines of human ductal origin are also available. They are well differentiated, easy to culture, and often retain specific cholangiocyte and epithelial phenotypes. For example, they allowed the study of ion secretion,18 secretory products such as membrane glycoproteins,19 tumor-associated proteins, growth factors, and the regulation of cell growth and proliferation.20
Cholangiocyte Function Ductal Bile Formation Bile formation by cholangiocytes, which is in part hormone-induced, contributes approximately 40% to total bile flow21 by adding ions and water to canalicular bile. In vivo studies with rats with ductal hyperplasia confirmed that an increase in the number of cholangio/ 5E16$$0062
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Figure 2. Experimental in vitro models of biliary epithelia. (A ) Light micrographs of freshly isolated bile duct units in 1-mm cross sections stained with methylene blue. CT, connective tissue; L, lumen. (B ) Transmission electron microscopy. Note the apical microvilli (arrowheads ) projecting into the lumen (L), basally located nucleus (N), and numerous folds and interdigitations at the basal surface. (C ) Illustration showing the bile duct unit lumen containing oil (arrow ) after microinjection of an oil droplet (OD) with a micropipette (P). (Reprinted with permission.7). (D ) Scanning electron micrograph of a group of isolated cholangiocytes after immunomagnetic separation on magnetic beads (M). Note the prominent microvilli (arrowheads ) are limited to one side of the cell. (E ) Low-magnification view of isolated cholangiocytes. Note the microvilli (arrows ) limited to one side of the cell. (Reprinted with permission8.) (F ) Phase-contrast microscopy shows the epithelial character of normal rat cholangiocytes grown on thick collagen. (G ) Scanning electron microscopy of the apical surface of normal rat cholangiocytes. The ‘‘cobblestone-like’’ appearance of cholangiocytes can be seen at low magnification. (Reprinted with permission.14)
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We do not know how ductal ion transport regulates ductal water movement. However, water channels appear to be involved; indeed, several species, including humans, express at least one subtype of membrane water channels (i.e., aquaporin channel-forming integral membrane protein) at the apical and/or basolateral domains,32,33 and rodent cholangiocytes transport water transcellularly via a channel-mediated mechanism.33 This water movement may occur at the apical membrane of cholangiocytes in response to transient transmembrane osmotic gradients created by hormone-stimulated ion transport (see Figure 3). Figure 3. Plasma membrane protein topography in cholangiocytes. A working model of the cellular mechanisms regulating ductal ion and water secretion. Cx, connexin; CHIP 28, channel integral membrane protein of 28 kilodaltons; EGF, epidermal growth factor.
cytes is accompanied by a large increase in both basal and secretin-induced bile flow.22 Biliary bicarbonate secretion is also invariably increased during secretin-stimulated choleresis1,22 and decreased during somatostatininduced cholestasis,23 suggesting that ductal HCO30 transport is closely related to ductal water secretion. Recent in vitro studies showed that both secretin and somatostatin regulate ductal bile secretion by interacting with surface receptors on cholangiocytes; each hormone altered intracellular levels of adenosine 3*,5*-cyclic monophosphate (cAMP).24,25 Secretin also stimulated exocytosis in cholangiocytes via a cAMP mediated mechanism.24 Two important transport proteins, a Cl0/HCO30 exchanger and a cyclic adenosine monophosphate–activated Cl0 channel (i.e., cystic fibrosis transmembrane conductance regulator [CFTR]), are functionally26 – 29 expressed in the apical domain of cholangiocytes. Secretin stimulates the activity of the Cl0/HCO30 exchanger, likely via the activation of CFTR and possibly other cAMP-activated Cl0 channels.26 Thus, analogous to the observations in ductal cells of the pancreas,30 secretin appears to stimulate biliary ductal HCO30 secretion by initially binding to a receptor on the basolateral domain of cholangiocytes, thereby stimulating the activity of CFTR on the apical domain through a cAMP-mediated mechanism. This sequence results in an increased activity of an apical Cl0/HCO30 exchanger in which luminal Cl0 is exchanged for intracellular HCO30, resulting in cholangiocyte HCO30 secretion (see Figure 3). Exocytosis in ductal bile secretion may involve hormone-regulated insertion of vesicles containing ion channels into the apical plasma membrane or insertion of vacuolar H/ pumps into the basolateral membrane of cholangiocytes.24,31 / 5E16$$0062
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Cholangiocyte Proliferation and Growth Cholangiocytes proliferate in a variety of patterns, not only in rat experiments but in human biliary liver diseases such as primary biliary cirrhosis (PBC) and in alcoholic liver disease or viral and drug-induced hepatitis.1,34 Type I ductular hyperplasia in rats follows bile duct ligation or feeding either a-naphthylisothiocyanate, lithocholic acid, or 4*,4*-diaminodiphenylmethane. Cholangiocytes proliferate and form an organized network of well-defined tubular structures that remain confined to portal tracts.1,34 Although these hyperplastic cells resemble normal cholangiocytes, their origin and the mechanism(s) responsible for their proliferation under these circumstances remain obscure.1 In contrast, type II ductular hyperplasia is observed in conditions such as severe hepatitis B virus or PBC.1,34 Proliferating bile ductules form a three-dimensional network of tortuous, poorly defined conduits that extend into the parenchyma.1 Current data suggest that the cells lining these ductules represent retrodifferentiated hepatocytes and not replicated cholangiocytes.1 Type III ductular hyperplasia appears in the early stages of experimentally induced carcinogenesis in rat liver. Oval-shaped cells proliferate and form disorganized, ill-defined tubular structures extending into the parenchyma and distorting the lobular architecture.1 These oval cells resemble both cholangiocytes and hepatocytes and express cytokeratin-19, albumin, and a-fetoprotein typically observed with cells of ductular, parenchymal, or neoplastic origin. The origin of oval cells and their role in the development of hepatocellular carcinoma remain controversial. Recently identified proteins that may regulate cholangiocyte growth and proliferation include hepatocyte growth factor, epidermal growth factor, transforming growth factor a, proline, cholecystokinin, and insulinlike growth factors.1,35 Hepatocyte growth factor may be WBS-Gastro
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an important mitogen for human cholangiocytes in vivo because its receptors are expressed by cholangiocytes36 and because human hepatocyte growth factor stimulates DNA synthesis of cultured human cholangiocytes.10 Cholangiocyte Heterogeneity Epithelia such as hepatocytes, renal tubular cells, and intestinal epithelial cells are functionally heterogeneous. For example, renal tubular epithelial cells function to dilute or concentrate luminal contents depending on their location in the renal tubular system. Similarly, cholangiocytes appear functionally heterogeneous.1 Thus, agonist-induced ductal transport of ions and water occurs principally in cholangiocytes lining the medium and large bile ducts, not the smaller radicles. Further studies are required to completely map the predicted functional diversity of the intrahepatic bile ducts. Of particular interest are small cholangiocytes because they appear to be the prime target of immune attacks as observed in PBC or allograft rejection.
Cholangiocytes in Human Disease: The Cholangiopathies The cholangiopathies represent diseases and syndromes of the biliary system at any site between the canals of Hering and the ampulla of Vater. Depending on the purpose of study, pathophysiological, morphological, and etiologic classifications can be applied. In Table 1, we propose a classification based principally on known or purported etiologies. Herein we briefly review some of the conditions listed in Table 1, in particular, the immune-mediated and infectious cholangiopathies; they are the focus of our discussion because much progress has been made in our understanding of these disorders. Immune-Mediated Cholangiopathies Hepatic allograft rejection. Allograft rejection is a major cause of morbidity after liver transplantation. By convention, cellular (acute) rejection is distinguished from ductopenic (chronic) rejection.37 Cellular rejection is characterized by nonsuppurative cholangitis (Figure 4A) and endotheliitis caused by alloimmune-mediated injury to cholangiocytes and to portal and hepatic vein endothelia, respectively.38 Ductopenic rejection connotes progressive loss of interlobular bile ducts, often in association with foam cell arteriopathy (vascular rejection).38 Rejection cholangitis is characterized by the aberrant expression of HLA class II antigens in cholangiocytes38,39 and by the presence of recipient T cells infiltrating cholangiocytes. The resulting damage usually is reversible, but rejection also may lead to irreversible duct loss, i.e.,
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Table 1. Classification of the Cholangiopathies Immune-mediated (definite and putative) Hepatic allograft rejection (definite) GVHD (definite) PBC (putative) Autoimmune cholangitis (putative) PSC (putative) Infectious Viral (including HIV-associated cholangitis) Bacterial Fungal Parasitic Protozoan (HIV associated) Genetic and developmental Cystic fibrosis Allagille’s syndrome (syndromatic paucity of intrahepatic bile ducts) Fibropolycystic liver diseases (e.g., congenital hepatic fibrosis) Drug-induced (e.g., amoxicillin) Ischemic Idiopathic Biliary atresia Idiopathic childhood ductopenia (nonsyndromatic paucity of intrahepatic bile ducts) Idiopathic adulthood ductopenia Chronic cholestasis of sarcoidosis Neoplastic: cholangiocarcinoma (bile duct adenocarcinoma) NOTE. By convention, cholelithiasis and choledocholithiasis are excluded.
ductopenic rejection. The traditional two types of rejection are essentially features of the same process, with the prognosis depending primarily on the degree of duct loss. Vascular rejection may damage ducts indirectly by causing biliary ischemia. Cytomegalovirus infection, biliary ischemia unrelated to vascular rejection, and other complications may play a pathogenetic role, possibly by stimulating the expression of HLA antigens in cholangiocytes. Graft-vs.-host disease. Graft-vs.-host disease (GVHD) after bone marrow transplantation may cause cholangiocyte injury and duct loss; this phenomenon probably is mediated by allogeneic T cells because removal of graft T cells prevents experimental chronic GVHD.38 However, autoreactivity may also play an important role, particularly in cases with injury to interlobular and septal bile ducts, as shown by aberrant expression of HLA class II molecules by cholangiocytes, presence of hypergammaglobulinemia, and appearance of autoantibodies, including antimitochondrial antibodies (AMAs), antinuclear antibodies (ANAs), and rheumatoid factor.38 The triggering antigens of GVHD remain unknown, but the frequent occurrence of GVHD in recipients of bone marrow from HLA-identical sibling donors raises the possibility that a cholangiocyte-specific antiWBS-Gastro
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Figure 4. Pathology of cholangiopathies. (A ) Cholangitis in cellular rejection 5 days after liver transplantation. The interlobular bile duct (asterisk ) is surrounded by a dense mixed inflammatory infiltrate (H&E). (B ) Fibrous obliterative cholangitis in PSC. Note interlobular bile duct with periductal fibrosis, infiltration of bile duct epithelium by neutrophils, and degeneration of cholangiocytes (H&E). (Reprinted with permission54.) (C ) Ischemic cholangiopathy with bile duct necrosis and secondary inflammation (ischemic cholangitis) 11 months after liver transplantation (H&E). (Reprinted with permission.63) (D ) Cholangiocarcinoma complicating PSC with growth in a perineural space (H&E) (original magnification: A and D, 3201; B, 4001; and C, 72.51) N, nerve.
gen, acting as a minor histocompatibility antigen, is the target protein for the immunologic reaction.38 PBC. PBC affects adults and mostly women and is characterized by inflammatory and often granulomatous destruction of small bile ducts.38,40 PBC has become a common indication for orthotopic liver transplantation. Several observations suggest that PBC is an autoimmune disease, including the high frequency of disease-specific AMAs of the M2 type and other serum autoantibodies; the increased frequency of HLA C4A-Q0, which is a class / 5E16$$0062
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III allele associated with many autoimmune diseases; the presence of activated T (predominantly CD4/ and CD8/) and B lymphocytes in and around cholangiocytes aberrantly expressing MHC class II; and the frequent association of PBC with autoimmune diseases, such as Sjo¨gren’s disease, rheumatoid arthritis, and autoimmune thyroiditis.38,40 Despite this evidence, trials with immunomodulatory agents such as D-penicillamine, azathioprine, methotrexate, or cyclosporine A have not been successful, thus casting some doubt on the concept of autoimmuWBS-Gastro
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nity.41 Unfortunately, no reliable animal models of PBC exist. So far, experimental strategies have included immunizing animals with a number of different immunogens, such as gallbladder mucous membranes, mitochondrial antigens, cholangiocytes, and peripheral blood lymphocytes from patients with PBC. Murine chronic GVHD40 also has been studied in this context. The most promising recent reports suggest that immune-mediated cholangitis can be induced in neonatally thymectomized mice that were stimulated with cholangiocyte antigens42 or in adult rats that were immunized with highly purified cholangiocytes.43 The mitochondrial 2-oxo-acid dehydrogenase multienzyme complex is the M2 autoantigen specific for PBC serum, and the E2 component of the pyruvate dehydrogenase complex (PDC) is the major autoantigen. This may mean that M2 in cholangiocytes is the target autoantigen in PBC38,40 because cholangiocytes express higher levels of PDC-E2 than other cells in normal liver, cholangiocytes express high levels of PDC-E2 in PBC livers, cultured cholangiocytes from patients with PBC express PDC-E2 on their surface, and autoepitopes of E2 are preferentially expressed in the luminal region of cholangiocytes in PBC livers. However, M2-related T-cell cytotoxicity has not been shown in PBC, and the mechanism of autoantibody production or the putative action of antiM2 antibodies has not been elucidated.38,40 One hypothesis to explain the immunologic injury to bile ducts in PBC suggests that, initially, T cells recognize epitopes of microbial proteins that share cross-reactive epitopes with PDC-E2. This is followed by a T- and B-cell reaction against these self-antigens that become aberrantly expressed with MHC class II on cholangiocytes.44 This molecular mimicry between microbial proteins and self-peptides is supported by AMA cross-reactivity with subcellular constituents of gram-negative and gram-positive organisms, the presence of certain PBCspecific reactive proteins in several Enterobacteriaceae species, the observation that mutant forms of bacteria such as Escherichia coli in urinary tract infection may elicit PBC-specific M2 antibody positivity, and the existence of familial AMA.45 An alternative hypothesis proposes that the transport of PDC-E2 to the mitochondria for assembly is affected by a genetic mutation (e.g., alteration to the leader sequence) that leads to aberrant or modified expression of PDC-E2 on the surface of cholangiocytes. Given that cholangiocytes may function as antigen-presenting cells in PBC, altered processing of normal PDC-E2 could conceivably lead to the presentation of nontolerized peptides of PDC-E2 to immunocytes resulting in immune-mediated cholangitis. / 5E16$$0062
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Autoimmune cholangitis. Autoimmune cholangitis is a controversial cholestatic disease that shares features with both PBC and autoimmune hepatitis. The diagnosis requires clinical and/or biochemical cholestasis, a liver biopsy specimen with nonsuppurative cholangitis, laboratory evidence of AMA and anti-M2 antibody negativity, high titers of serum ANAs, and immunoglobulin (Ig) M concentrations that tend to be lower and IgG concentrations that tend to be higher than in PBC.38,46–52 The etiology of autoimmune cholangitis is unknown, and the pathogenesis is poorly understood. Responses to corticosteroid therapy may be dramatic,47 – 49 but these are not invariable.51,52 Although PBC and autoimmune hepatitis probably may occur together or in sequence,48 some cases appear unrelated; terms such as autoimmune cholangiopathy,47 immunocholangitis,49 overlap syndrome,50 and primary autoimmune cholangitis51 have been suggested to describe such instances. The classification of autoimmune cholangitis remains controversial particularly because many patients with AMA-negative PBC also have ANA (often in high titers) and/or anti–smooth muscle antibodies and comparatively low IgM levels.46,52,53 Indeed, autoimmune cholangitis is often indistinguishable from AMA-negative PBC46,51 – 53 and, in many instances, can only be distinguished from AMA-positive PBC by its immunoserological profile.52 Adding to the controversy is the fact that autoimmune cholangitis has been classified by some as part of the spectrum of autoimmune hepatitis47 and by others as a distinctive subgroup of AMA-negative adulthood ductopenic disorders.51 Thus, to bring this controversy into better focus, we like others52 suggest to use the name autoimmune cholangitis as a collective term, akin to autoimmune hepatitis, with multiple subgroups of the disease identified by different autoantibody profiles; these would include AMA-positive PBC, AMAnegative PBC, and ANA-positive, AMA-negative cholangitis, among other possible constellations. This would by no means preclude using the term PBC as a diagnosis, but it would define the situation much more clearly and would place the serological variations in a context that easily lends itself to further studies. Primary sclerosing cholangitis. Primary sclerosing cholangitis (PSC) is characterized cholangiographically by strictures, beading, and irregularities of the biliary tree and pathologically by inflammation and fibrosis of intrahepatic (Figure 4B) and extrahepatic bile ducts. The chronic disease typically affects young men and is associated with inflammatory bowel disease, especially chronic ulcerative colitis54,55 in approximately 75% of the cases. Current evidence suggests that, among a
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multitude of proposed mechanisms, genetic and immunologic factors likely play the most significant pathogenetic role in PSC.54,55 A few reports of familial PSC suggest a genetic predisposition and so do haplotype studies associating PSC with HLA B8, DR2, DR3, DR4, and DRw52a.54,55 After comparison with appropriate controls, DR52a and Dw2 emerge as the best candidate alleles for the HLA association with PSC. The association attains particular importance in patients who possess the HLA DR4 allele because it may herald rapid disease progression. Cellular immune abnormalities in PSC include decreased peripheral blood T lymphocytes with a relative increase in the CD4/CD8 ratio, T-lymphocyte predominant infiltrates in the vicinity of proliferating and damaged bile ducts, enhanced autoreactivity of portal tract T lymphocytes, and an increased number and proportion of circulating B cells.56 Humoral immune abnormalities in PSC include hypergammaglobulinemia, increased circulating immune complexes, and circulating autoantibodies such as perinuclear antineutrophil cytoplasmic antibodies, antineutrophil nuclear antibodies, anticolon epithelial autoantibodies, and sometimes the classic autoantibodies ANA, AMA, and anti– smooth muscle antibody.54– 56 Furthermore, HLA class II antigens are aberrantly expressed by cholangiocytes at an early stage of the disease, and peripheral blood lymphocytes from patients with PSC are sensitized to unidentified biliary antigens. Moreover, intracellular adhesion molecule 1, a ligand that facilitates lymphocyte attachment to target cells, appears in cholangiocytes in late-stage PSC, and PSC is associated with immune diseases such as type I diabetes and thyroiditis, among others. The morphological distribution of PSC with its involvement of large ducts differs profoundly from the findings in the other autoimmune or putative autoimmune cholangitis. This strongly suggests that very important pathogenetic differences exist. Although, evidence supports the view that PSC results from autoimmune-mediated injury to cholangiocytes, the data appear less convincing than in PBC. Autoimmunity may be linked to an antibody that recognizes a cross-reactive peptide in small and large duct cholangiocytes as well as in colonic epithelial cells from patients with ulcerative colitis.57 Indeed, two thirds of patients with PSC have circulating IgG autoantibodies against such an antigen.57 Such a mechanism could explain the frequent coexistence of PSC with ulcerative colitis. Infectious Cholangiopathies Bile ducts may be affected by viral, bacterial, fungal, protozoan, and parasitic infections. Recent experi/ 5E16$$0062
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ences have accumulated mostly in the acquired immunodeficiency syndrome (AIDS). AIDS cholangiopathy. Cholangiography in human immunodeficiency virus (HIV)-infected patients may show five distinct patterns: sclerosing cholangitis, polypoid cholangitis, papillary stenosis, combined papillary stenosis and sclerosing cholangitis, and long, extrahepatic bile duct strictures.58 Probable causative microorganisms are identified in about 60% of the AIDS cholangiopathies; they include cytomegalovirus, Cryptosporidium, Microsporidium, and Mycobacterium avium.58,59 Cytomegalovirus is very common in patients with AIDS and is readily recognizable in biopsy specimens, whereas Cryptosporidium, another commonly isolated agent, is easily overlooked although it may attach to the luminal surface of cholangiocytes. In patients with AIDS without evidence of an opportunistic biliary infection, the HIV itself has been implicated. However, HIV infection of cholangiocytes has not yet been shown. Like many other cholangiopathies, aberrant HLA class II expression on cholangiocytes is seen in AIDS; whether this is an important observation is unclear. Genetic and Developmental Cholangiopathies This group of cholangiopathies comprises a considerable number of conditions, including Alagille’s syndrome and the fibropolycystic liver diseases such as congenital hepatic fibrosis. Major new insights have been gained only in cystic fibrosis. Cystic fibrosis. This lethal autosomal recessive disorder of exocrine glands is caused by the abnormal CFTR, a chloride channel found on chloride-secreting epithelia.60 In adult patients with cystic fibrosis, mucus obstruction of intrahepatic bile ducts is often found, with or without biliary fibrosis. Mutations in CFTR, which is in the apical domain of cholangiocytes, lead to defective ductal Cl0 and water secretion, resulting in biliary disease. No specific therapy is available for cystic fibrosis liver disease; there is some hope for preventive treatment in the near future based on recombinant adenoviruses expressing human CFTR; infusion of these viruses into rat livers temporarily generated CFTR.61 Drug-Induced Cholangiopathies Little progress has been made in the understanding of these conditions although the number of drugs that may cause cholangitis and lead to ductopenia is steadily growing; close to 20 are now known (Table 2). Vascular Cholangiopathies Surgical trauma to hepatic arteries, anastomotic arterial strictures, hepatic artery thrombosis, and foam WBS-Gastro
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Table 2. Drugs That May Cause Nonsuppurative Cholangitis and Ductopenia Generic or chemical name Acetaminophen Amitryptilin Amoxicillin Ampicillin Carbamazepine Chlorpromazine Cromolyn sodium Cyproheptadine Diazepam Erythromycin Haloperidol Imipramine Methyltestosterone Phenylbutazone Prochlorperazine Thiabendazole Tolbutamide Trifluoperazine hydrochloride Troleandomycin
Product classification Analgesic and antipyretic Antidepressant Antibiotic Antibiotic Anticonvulsant Tranquilizer:phenothhiazine Mast cell stabilizer Antihistamine and antiserotonergic Sedative and hypnotic:benzodiazepine Antibiotic Antipsychotic Antidepressant Androgen Anti-inflammatory Tranquilizer:phenothiazine Anthelmintic Oral hypoglycemic Tranquilizer:phenothiazine Antibiotic
cell arteriopathy in allograft rejection are the main causes of vascular cholangiopathies. The low perfusion of the biliary plexus may lead to ductal inflammation with fibrosis and diffuse or segmental degeneration of cholangiocytes (Figure 4C).6 If inflammatory changes are prominent, the condition can be classified as ischemic cholangitis. Neoplasia: Cholangiocarcinoma Cholangiocarcinoma, a malignant tumor of biliary epithelia (Figure 4D), is the most important neoplasm of intrahepatic bile ducts because it is frequent and so far almost universally fatal. Early diagnosis is difficult, and effective therapy is lacking.20 Risk factors for cholangiocarcinoma include PSC, thorium dioxide (Thorotrast) and anabolic steroid administration, biliary cysts such as choledochal cysts, Caroli’s disease and other fibropolycystic liver diseases, and in the Far East, chronic infestation of the bile ducts with the parasitic flukes Opisthorchis viverrini and Clonorchis sinensis. The molecular mechanisms responsible for the malignant transformation of cholangiocytes are unknown, in part because little information is available on the factors regulating cholangiocyte proliferation. Still, several cholangiocarcinoma cell lines prominently express transcript for one subtype of somatostatin receptor SSTR2.20 Based on this observation, investigators have recently shown that a radiolabeled derivative of somatostatin can accurately detect the presence of cholangiocarcinoma in humans and that long-acting somatostatin analogues can inhibit the growth of cholangiocarcinoma cell lines in vitro and in vivo after injection in nude mice.20 Pilot / 5E16$$0062
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studies are now under way in humans to assess somatostatin analogues in the diagnosis and treatment of cholangiocarcinoma.
Summary Our understanding of the pathobiology of biliary epithelia is rapidly growing because of a surge of investigative activity. This became possible after suitable experimental models and techniques were developed with which to study cholangiocyte biology. Although the molecular mechanisms of bile formation by cholangiocytes and the role of these cells as a major cellular target in a variety of severe hepatobiliary diseases are currently being investigated, many questions remain unanswered, particularly regarding cholangiocellular functions, both in normal and abnormal conditions. As current experimental models become more refined, scientists with interests as diverse as cell biology and physiology, morphology, pharmacology, immunology, genetics, and oncology can be expected to further clarify the pathobiology of biliary epithelia.
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Received May 10, 1996. Accepted August 20, 1996. Address requests for reprints to: Nicholas F. LaRusso, M.D., Center for Basic Research in Digestive Diseases, Mayo Medical School, Clinic and Foundation, 200 First Street Southwest, Rochester, Minnesota 55905. Fax: (507) 284-0762. Dr. Roberts’ current address is: Department of Gastroenterology, Alfred Hospital, Prahran, Victoria, Australia.
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