Pathology and pathogenesis of intrahepatic bile duct loss

Transcription

1 J Hepatobiliary Pancreat Surg (2001) 8: Pathology and pathogenesis of intrahepatic bile duct loss Yasuni Nakanuma, Koichi Tsuneyama, and Kenichi Harada Second Department of Pathology, Kanazawa University School of Medicine, Kanazawa , Japan Abstract In recent years, the pathology and pathogenesis of bile duct loss have been extensively studied, and a number of hepatobiliary diseases have been added to the list of ductopenic diseases. In addition, the biology of biliary epithelial cells is now being studied with respect to bile duct loss, as well as biliary epithelial neoplasia. In this review, recent advances in pathogenetic and pathological studies of intrahepatic bile duct loss are described, with an emphasis on immune-mediated cholangiopathies. The bile duct loss, an acquired and pathologic process that occurs in the biliary tree, is recognizable as an absence of bile duct in an individual portad tract, and also as such absence in the vicinity of parallel running hepatic arterial branches that constitute the portal triad. Immunostaining with biliary cytokeratin and other carbohydrate materials is useful for the identification of biliary elements in the inflamed portal tracts or fibrous septa. The underlying processes responsible for bile duct loss include immunological, ischemic, infectious, metabolic, and toxic processes. Bile duct loss in primary biliary cirrhosis and primary sclerosing cholangitis is immune-mediated, that in interventional radiology using hepatic arterial branches is related to biliary ischemia, while that in hepatic allograft rejection is related to both immunological and ischemic insults. Bacterial and viral cholangitis with bile duct loss is an example of infectious cholangitis. The biliary tree maintains its homeostasis by renewal and dropout, and bile duct loss occurs mainly via biliary apoptosis. In some patients with bile duct loss, such as occurs in drug-induced injuries, the bile ducts regenerate and finally redistribute in the liver, while in other types of bile duct loss, the loss is progressive and is followed by vanishing bile duct syndrome, leading to biliary cirrhosis or liver transplantation. More analysis of the biology of biliary epithelial cells is mandatory for the evaluation of the pathobiology of bile duct loss, as well as for the effective restoration of biliary epithelial cells, in ductopenic liver diseases. Offprint requests to: Y. Nakanuma Received: October 12, 2000 / Accepted: January 10, 2001 Key words Bile duct loss Vanishing bile duct syndrome Immune-mediated cholangiopathy Biliary apoptosis Introduction Bile duct loss (obliteration or disappearance of bile duct) is one of basic pathologic changes of the biliary tree, and occurs at different anatomical levels of the intrahepatic biliary tree, and also with different degrees of involvement in various clinical settings. 1 Bile duct loss is defined as an acquired process in the biliary tree, thus being different from agenesis or hypoplasia of the bile ducts. This bile duct loss, is recognizable as an absence of bile duct in an individual portal tract (Fig. 1) and is also recognizable as absence of bile duct in the vicinity of parallel running hepatic arterial branches that constitute the portal triad. A number of pathogenetic processes, of immunologic, ischemic, and infectious etiologies, have been proposed to be involved in the pathogenesis of bile duct loss, depending on the type of insult or agent. 1,2 The consequence of bile duct loss is also variable in individual diseases and patients cases; some of such patients undergo progressive and irreversible duct loss followed by the development of extensive ductopenia and biliary fibrosis or cirrhosis, while others show biliary epithelial regeneration, with clinical recovery finally occurring in several months or years later. In the past two decades, a number of new hepatobiliary diseases with bile duct loss, such as hepatic allograft rejection (HAR) and idiopathic adulthood ductopenia, 3,4 have been added to the list of classical ductopenic diseases, such as primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). In this review, we will describe the recent advances in determining the pathology and pathogenesis of bile duct loss in various cholangiopathies, especially in immune-

2 304 Y. Nakanuma et al.: Pathobiology of bile duct loss Fig. 1. In this portal tract, a hepatic arterial branch and portal vein branches are found, although the bile duct is missing, suggesting interlobular bile duct loss. Arrow, Hepatic arterial branch. P, Portal vein branch. Primary biliary cirrhosis. H&E, 250 mediated cholangiopathies. First, the anatomy of the intrahepatic biliary tree and evaluation of bile duct loss in the liver are briefly described. Fig. 2. Schema of the intrahepatic biliary tree and expression of Bcl-2 family proteins at the anatomical levels of the intrahepatic biliary tree in normal liver. The intrahepatic biliary tree is classified into large bile duct, septal bile duct, interlobular bile duct, and bile ductule (see text). The immunoreactivity of the Bcl-2 family proteins was semiquantitatively graded as,,, and (from reference 17, with permission) Anatomy of the intrahepatic biliary tree and evaluation of bile duct loss Anatomy of the intrahepatic biliary tree The intrahepatic biliary tree is defined as the biliary tree distal to the right and left hepatic ducts and is composed of segmental ducts, area ducts, and their finer branches. 2 The segmental and area ducts and their first and second branches which are grossly visible, are collectively termed intrahepatic large bile ducts. The wall of the intrahepatic large bile duct consists of a hypocellular, collagenous band lined by a single layer of high columnar biliary epithelium. The periductal tissue is a loose connective tissue around the bile duct wall. Peribiliary glands showing lobular patterns are consistently present along the large intrahepatic bile ducts. 5 Bile ductules are tubular structures, with a diameter of less than 20µm, in the peripheral zone of the portal tract; interlobular bile ducts have a diameter of 20 to 80µm. Septal bile ducts are larger than the interlobular bile ducts. Septal bile ducts have their own bile duct wall and are lined by a single layer of high columnar epithelium, while the interlobular bile ducts and bile ductules are lined by a single layer of low columnar or cuboidal epithelium and have no bile duct walls. A schema is shown in Fig. 2. The biliary tree, in particular, the perihilar extrahepatic and intrahepatic biliary tree, is exclusively supplied by the hepatic arterial branches, which are known as the peribiliary vascular plexus (PVP). 6 The small vessels of the PVP are easily recognizable by immunostaining and lectin-histochemistry of endothelial markers, such as CD34, factor VIII-related substance, or Ulex europaeus agglutinin-1. The small vessels of the PVP are well organized as three layers (inner, middle and outer) around the perihilar extrahepatic bile duct and intrahepatic large and septal bile ducts. The small portal vein branches and small arteries around the bile ducts (peribiliary arteries) may be included in the outer layer. The PVP becomes less organized at the level of interlobular bile duct and bile ductules. 7 The PVP is actively involved in the pathophysiology of the intrahepatic biliary tree, such as ischemia and cholangitis, providing recruiting vessels. Identification of biliary elements Bile ducts or biliary elements are identifiable as ductal, tubular, or cord-like epithelial structures in portal tracts and fibrous septa. In the portal tracts of any size in normal livers, hepatic arterial branches, portal veins, and bile ducts compose a triad, and hepatic arterial branches and interlobular bile ducts of similar sizes run parallel in individual portal tracts. The parallelism of the hepatic arterial branches and bile ducts is useful for the understanding of bile duct pathology: the size of affected bile ducts and the degree of bile duct loss are evaluable with respect to the hepatic arterial branches. 8,9 Immunostaining with biliary cytokeratin; in particular, cytokeratin 7 and 19; epithelial membrane

3 Y. Nakanuma et al.: Pathobiology of bile duct loss 305 antigen; and blood group-related antigen 10 is useful for the identification of bile ducts and biliary elements, particularly in inflamed portal tracts where the identification of bile ducts is more difficult with routine stainings. along the whole biliary tree. 16 The distribution of Bcl-2 family proteins on the intrahepatic biliary tree is shown in Fig. 2. The cellular threshold for apoptosis in biliary epithelium is highly regulated by the Bcl-2 family of proteins. 13,17 Evaluation of bile duct loss Semiquantitative assessment of bile duct loss is done by calculating the proportion of portal tracts without bile ducts. This is a practical approach for the evaluation of bile duct loss and its degree. More than ten portal tracts in the liver specimen are necessary for this method, and ductopenia is defined by the absence of interlobular bile ducts in at least 50% of small portal tracts. 11 It is also possible to determine the level and degree of missing bile ducts quantitatively by counting the ratio of the number of the hepatic arterial branches running parallel to intrahepatic bile ducts to the number of hepatic arterial branches alone in portal tracts. 9 Basic pathogenetic processes of bile duct loss Several pathogenetic processes of biliary epithelial destruction followed by bile duct loss have been proposed, 12 with biliary epithelial apoptosis and necrosis being representative, although necrosis of biliary epithelial cells is controversial in terms of differentiation from apoptosis. Homeostasis of biliary epithelia and biliary apoptosis Homeostasis of the biliary epithelial layer is maintained through a balance between cell death and cell renewal or proliferation of the biliary epithelial cells themselves. 13 As for the process of cell death, apoptosis has been most studied. In addition, a liver stem cell or bipotential cell, residing in the canal of Hering, may continuously feed the biliary tree with new cells. These offspring may migrate toward the larger branches of the bile duct and then toward the hepatic hilum. In addition, the intrahepatic peribiliary glands, in particular, the conduits of these glands before the opening into the bile ductal lumen, could be a place where new or regenerating cells or stem cells develop. 14 The significance of stem cells in biliary epithelial homeostasis and biliary diseases remains unexplored. Reliable and easier methods of identifying such stem cells may lead to rapid advances in this field. The maintenance of biliary homeostasis is mainly regulated by Bcl-2 family proteins. 15 Bcl-2, which counteracts apoptosis, is known to be diffusely expressed on the bile ductules and interlobular bile ducts in normal livers, while Bax, a promoter of apoptosis, is expressed Apoptosis and cholangiopathies The role of apoptosis in the pathophysiology of the biliary tree may have been underestimated, because apoptotic biliary epithelial cells (BECs) are probably rapidly shed into bile and eliminated from the biliary tree. 11 While apoptosis is very difficult to accurately identify and quantitate in H&E-stained sections, shrunken slender cells with pyknotic nuclei and fragmented and condensed nuclei in the biliary epithelial layer and bile duct lumen are regarded as apoptotic (Fig. 3). 18 Ultrastructurally, the shrinkage of cell volume and density, and the compacting of cytoplasmic organelles suggestive of apoptosis, are occasionally encountered in acute and chronic biliary diseases, and also in the normal biliary tree. 15 By in situ nick-end labeling detecting DNA fragmentation, apoptosis is easily detectable in BECs lining the intrahepatic biliary tree. 18 Biliary apoptosis has been described as important to several cholangiopathies, in which bile duct loss develops as a result of apoptosis rather than renewal or proliferation. That is, excessive apoptosis that exceeds the proliferative response or renewal of biliary epithelial cells may result in the progressive bile duct loss and extensive ductopenia in the processes of autoimmune, infectious, developmental, and genetic cholangiopathies. 13,19,20 The process of apoptosis may differ among various biliary diseases, and may also at different stages of individual diseases. In contrast, inhibition of the biliary apoptotic process may cause hyperplasia and neo- Fig. 3. Shrunken biliary cells with pyknotic nuclei (arrows) in the interlobular bile duct suggest apoptosis. Primary biliary cirrhosis. H&E, 350

4 306 Y. Nakanuma et al.: Pathobiology of bile duct loss plastic transformation of bile ducts and biliary epithelial cells. 13 Initiation or induction of biliary apoptosis Activation of cell death receptors (e.g., Fas, tumor necrosis factor [TNF]-α receptor), nitric oxide (NO), pathogens, toxins, and immunological assaults, may have the effect of initiating biliary epithelial apoptosis in diverse human biliary diseases. The following apoptotic processes of initiation or induction have been proposed to occur in biliary apoptosis. Fas/FasL system. At present, this system remains the best characterized model of apoptosis. In PBC, hepatic allograft rejection, and PSC, Fas is strongly expressed on the damaged BEGs (Fig. 4a) and on FasL-expressing cytotoxic T lymphocytes (CTL) of Th1 subset around them, as well as within the biliary epithelial layer. 18 In addition, CD-68-positive cells strongly expressing FasL have also been noted around the damaged bile ducts of PBC. 21 The precise stimuli for Fas expression on the biliary epithelium may vary in individual immune-mediated diseases or at different stages of these diseases. Perforin and granzyme B system. This system, which is committed by CD8 CTL and natural killer (NK) cells, is important in the bile duct apoptosis in HAR, and, to a lesser degree, in PBC, and messages for CTL granzyme are detectable in many rejected allograft livers. 22 This system is also operative in the physiologic functions of the intrahepatic biliary tree. 15 TNF-α/TNF-α receptor system. This system may use a signal pathway with features common to Fas/FasL. This may be commonly involved in immune-mediated ductopenia. 20 TNF-α is a cytokine that is produced by many cells, including lymphocytes and macrophages around the bile ducts. Expression of mrna and protein for the TNF receptor and for TNF on BECs occurs in PBC and PSC, suggesting the occurrence of apoptotic cell death of bile ducts with this system in these diseases. TNF receptor is actually expressed by BECs in PBC 23 and can induce apoptosis when triggered. 13 a Oxidative stress. This has been implicated in biliary apoptosis in immune-mediated cholangitis. Following activation, lymphocytes produce increased levels of reactive oxygen species. Oxidative stress may lead to DNA damage, and has been implicated in the induction of apoptosis in many ductopenic disease models, such as HAR. 24 In BECs of PBC, the expression of wild-type p53-activated fragment 1 (WAF 1) is increased, and this is related to the p53 expression caused by DNA damage (Harada et al., manuscript in submission). Disturbance of Bcl-2 expression on BECs. Downregulation of Bcl-2 in the small bile ducts with variable damage is shown in hepatic allograft rejection (HAR) and PBC (Fig. 4b). This reduction or loss of Bcl-2 may play a role in the increased apoptosis of BECs and subsequent bile duct loss. 13,20 In these ducts with reduced expression of Bcl-2, the expression of annexin V is increased. b Fig. 4. a Fas expression is increased on biliary epithelial cells in damaged interlobular bile duct (arrows) showing chronic nonsuppurative destructive cholangitis. Immunostaining for Fas and counterstaining with hematoxylin. b Bcl-2 expression is reduced or lost on biliary epithelial cells of the interlobular bile duct (arrow) in primary biliary cirrhosis. Immunostaining for Bcl-2 and counterstaining with hematoxylin. a 350; b 350 Detachment-induced apoptosis (anoikis). This is a kind of trigger of apoptosis of epithelium. Recently, it has been reported that detachment of adherent epithelial cells from the extracellular matrix (ECM) promotes their apoptosis, in a process termed anoikis. 12 In PBC, decreased expression of cell-cell and cell-matrix connecting molecules, such as integrin-α6, was recently shown in biliary epithelium. 25 These alterations in cell-

5 Y. Nakanuma et al.: Pathobiology of bile duct loss 307 cell and cell-matrix adhesion may play a role in anoikis. Ultrastructurally, the detachment of biliary epithelial cells from the basement membrane, and widened intercellular spaces, are reported in damaged bile ducts in PBC. Regenerative recovery of bile duct loss Bile duct loss may not be an irreversible process. That is, reportedly, regrowth of the bile ducts can occur, and a ductular reaction proceeds the reappearance of the bile ducts, this is occasionally observed in patients with drug-induced ductopenia after the cessation of drug use. Hepatic stem cells or progenitor cells which migrate from the periportal areas into the biliary tree, 14,16 may be involved in this reappearance and restoration of the bile duct. However, in other diseases with extensive bile duct loss, re-establishment of the biliary tree by such stem cells does not work successfully. It remains unclear why there are such differences in biliary restoration among many ductopenic diseases; this issue is very important and should be clarified in the near feature. Pathogenesis of bile duct loss There may be several pathogenetic mechanisms working in bile duct destruction, with more than one such mechanism being involved in any individual disease or patient. The anatomical level of the biliary tree affected also differs according to the individual disease. 1,2 Intrahepatic bile ducts are affected focally or heterogeneously in PBC and PSC, while they are affected rather diffusely and homogeneously in HAR and graft-versushost disease (GVHD). The following mechanisms are representative and well studied. Immune-mediated cholangitis and bile duct loss Cholangitides in PBC, PSC, GVHD, and HAR are autoimmune or alloimmune-mediated cholangiopathies 1,2,26 in which BECs and, more precisely, target antigens on BECs, are subjected to immunological injury such as humoral, and acute and/or chronic cellular injury. A mixture of immunological competent cells, particularly CD3, and CD4, or CD8 T cells, bearing the T-cell receptor α/β, surround the damaged bile ducts 27 suggesting a role for CD3 T cells in exerting cytotoxic activity or cytokine release in the pathogenesis of these biliary diseases. The proportions of CD4 and CD8 T cells differ according to the individual disease or the disease stage. For example, CD4 cells are rather predominant around the damaged bile ducts in portal tracts in early stages of PBC, while CD8 cells are predominant in acute HAR, 28 suggesting different immunopathogenetic mechanisms among these cholangitides, reflecting different etiologies or pathogeneses. Humoral immune mechanisms may also be operative to some degree. Suspected target antigens and their presentation and recognition Immune-mediated reaction is against peptide antigens presented by allogeneic major histocompatibility complex (MHC) or allogeneic MHC themselves expressed on BECs in these diseases. Target antigens. The pathogenesis of HAR and GVHD is mainly related to the alloimmune response. That is, the recipient s immune response is operative against allogeneic antigens of the donor liver in HAR, 26 and MHC-related antigens induced on the BECs, facilitated by locally released cytokines or viral infections, may be a target antigen recognized by graft lymphoid cells in GVHD. 26 By contrast, in the autoimmune response, antimitochondrial antibody (AMA)-related antigens, in particular, pyruvate dehydrogenase complex E2- subunit (PDC-E2) and E3-binding protein (protein X), 29 as well as unidentified biliary epithelial antigens, may be involved in PBC. In fact, T-cell clones reacting with PDC-E2 and/or branched-chain ketoacid dehydrogenase E2 subunit are obtainable from the liver biopsy specimens of patients with PBC. 30 T-Cell clones established from PBC liver tissue are usually oligoclonal, suggesting that the epitopes recognized by these T cells may not be single in individual lesions. A 40-kD colonocyte protein (an intestinal isoform of tropomyosin) or other cross-reactive peptides, shared by BECs and colonic mucosa, may be a target antigen in ulcerative colitis and PSC. 31 TcRV β3 usage is predominant in liver tissue of PSC, suggesting the presence of a specific antigen, with the capacity to select driving T cells, utilizing the V β3 segment product. Further, other antigens may be involved secondarily in these immune-mediated cholangitides, and they may aggravate immune processes, such as heat shock protein (HSP), blood-group-related substances, carbonic anhydrase II, mucus core protein-1, and bacterial antigens Actually, increased expression of HSP-60 is demonstrated in the biliary epithelium of patients with PBC and, to a lesser extent, in those with PSC. 33 Antigen presentation. Two kinds of antigen-presenting cells (APCs), professional APCs and BECs themselves, are thought to be involved in immune-mediated cholangitis. Professional APC, especially dendritic cells (DCs) and, to a lesser degree, macrophages, are the most important regulators of immune responses in the initial events that occur during the T-cell-mediated response. DCs, which are scattered around the damaged bile duct,

6 308 Y. Nakanuma et al.: Pathobiology of bile duct loss with a few located in the biliary epithelial layer, in immune-mediated cholangiopathies, are positive for B- 7, MHC-class II, or S ,35 At least two molecules, B7-1 and B7-2, are said to work as costimulatory ligands for CD28 on T-cell surfaces. T cells expressing CD28 and CAT are found around the damaged bile ducts. Recently, in DCs derived from peripheral blood, dysfunction due to increased NO production in the DCs themselves, was noted in PBC. 36 Furthermore, BECs in the damaged bile ducts also become strongly positive for MHC-class I and aberrantly express MHC-class II antigens in PBC and HAR, 37 while the expression of these molecules is relatively weak in PSC. The expression of these immune molecules occurs mainly on the cell membranes, 38 and could make these BECs eligible as APCs. Immunoregulation around the bile ducts The up- and downregulation of cytokines and their receptors in the portal tracts are central to the progression of immune-mediated bile duct lesions. In PBC and PSC, a triggering event that damages the BECs or changes the inflammatory microenvironment of the portal tracts may be required in genetically susceptible persons. As potential triggers, treatment with interferon (IFN)-α, toxic effects, drug-induced injury, bacterial or viral infection, and pregnancy may be important in the development or aggravation of immune-mediated cholangiopathies such as PBC and PSC Th1 and Th2 balance and cytokine networks. The relative predominance of Th1 and Th2 responses and the resultant cytokine milieu, along with the breakdown of self-tolerance, are important determining factors in the immunopathologic processes of these cholangitides. 38 In inflamed portal tracts of PBC, PSC, and HAR livers, Th1 subsets produce interleukin (IL)-2 and INF-γ, and help in the proliferation of CTLs and in the local secretion of IFN-γ and TNF-α. The local secretion of IFN-γ and TNF-α is known to up-regulate or induce the increased expression of MHC class I, the aberrant expression of MHC class II, and the expression of adhesion molecules on BECs. 23,38 This Th1 predominance is highly correlated with the immunopathologic changes in portal tracts: accumulation of macrophages and granuloma formation, and the cell-mediated cytotoxicity against bile ducts. 42 However, several cytokine effects are not fully explained by the Th1 predominance in the liver in these immune-mediated cholangiopathies. That is, mrnas for IL-5, IL-6, and transforming growth factor-β are also detectable in the majority of cases of immune-mediated cholangitis. 38 Both Th-1 (IFN-γ) and Th-2 type (IL10) cytokine secretion is described in these patients, particularly in PBC. Altered portal microenvironments and aberrant expression of immune molecules on the damaged bile ducts. The damaged bile ducts themselves express several cytokines, such as IL-6 and TNF-α, and regulated upon activation, normal T-cells expressed and secreted (RANTES), and also their receptors. 23,38 Such expression may be induced by locally released cytokines, such as IL- 6, in the microenvironment of the portal traces. The aberrant expression of TNF receptor and, to a lesser de-gree, IL-6 receptor α-chain, on these damaged bile ducts suggests an autocrine effect. 23 TNF-α increases the proliferation and migration of intraepithelial lymphocytes, which may expand their number in the biliary epithelial layer. Both TNF-α and IL-6 are further involved in the production of bile duct lesions, particularly biliary epithelial proliferation and apoptotic destruction in PBC. 18,43 The immune-mediated bile duct injury is also mediated or aggravated by IL-8 and monocyte chemotactic protein (MCP). 44 In the portal tracts of PBC, MCP-2- and MCP-3-positive mononuclear cell infiltration is frequent, whereas such cells are less frequent in other chronic liver diseases. Co-expression of CD68 was frequent in MCP-positive mononuclear leukocytes. In PBC livers, MCP-2- and MCP-3-positive mononuclear cells infiltrate close to damaged bile ducts and also constitute part of the epithelioid granuloma, suggesting that these cells are activated monocytes involved in the actual granuloma formation. These results underscore the importance of MCP-2 and MCP-3 in the recruitment of monocytes and in granuloma formation. In the production of bile duct lesions in PSC, other cytokines, such as IL-8, may be involved. In immune-mediated bile duct damage, the inflammatory microenvironment of portal tracts, associated with T-cell activation and proliferation, and the secretion of cytokines, further induces and upregulates an expression of MHC class II, intercellular adhesion molecule-1 (ICAM-1), lymphocyte-associated antigen (LFA)-3, and vascular adhesion molecule (VCAM-1), and also of constitutive surface immune molecules, such as MHC class I and very late antigen (VLA)-2, 3 and 6, on BECs, 33 also inducing and upregulating immune molecules on the nearby microvasculature. In turn, this leads to the recruitment of more inflammatory cells from the portal microvasculature. These immune molecules may serve as ligands or receptors for inflammatory cells and matrix proteins. 25,38 These changes may further lead to the facilitation of immune reactions in bile ducts, such as cell positioning or adhesion, antigen presentation, costimulatory signaling, and a direct role in the activation of antigen-specific CD4 T cells. Epitheliotropism in bile duct lesions The migration and penetration of immunocompetent cells in the portal tracts and into the intraepithelial layer

7 Y. Nakanuma et al.: Pathobiology of bile duct loss 309 Fig. 5. Chronic nonsuppurative destructive cholangitis (arrow) in primary biliary cirrhosis. The biliary epithelial cells show swelling and disordered polarity, and the intraepithelial migration of lymphoid cells is also recognizable. There are many epithelioid cells in the vicinity of the damaged bile duct, H&E, 300 of bile ducts (epitheliotropism) are a key process in immune-mediated cholangitis (Fig. 5) and eventual bile duct loss. Epitheliotropism is related to a specific and efficient function of lymphocytes in the target tissue. 25 In PBC and HAR, the presence of a basement membrane of bile ducts that contains binding sites for receptors present on inflammatory cells, and the upregulation of integrin and other adhesion molecules, facilitate the interaction of inflammatory cells. Recruitment of inflammatory cells and PVP. The leukocytes accumulated in the portal tracts and around the bile ducts may be recruited from small vessels of PVP which are connected to the hepatic arterial branches, as well as portal venous branches. 6 These vessels are increased around bile ducts showing cholangitis. Chemokines released in portal tracts induce the expression of adhesion molecules involved in the trafficking, extravasation, and localization of inflammatory cells. ICAM-1, VCAM-1, and E-selectin are strongly expressed on the endothelial cells of PVP in immunemediated cholangitis, suggesting the facilitation of lymphocyte recruitment around the bile ducts. 25 For the migration of lymphocytes from the blood vessels, the expression of integrin-α4 on lymphocytes is believed to be essential, 25 compatible with the finding that almost all infiltrating cells within portal tracts express integrin-α4 (very late antigen [VLA]-4) on their surface, after perivascular migration in PBC and chronic viral hepatitis (CVH). E-selectin is detected on vascular endothelium in association with lymphocytic infiltrates in immune-mediated cholangitis, but not on normal endothelium. 45 E-selectin, which acts as an addressin for T-cell cutaneous lymphocyte antigen, can be detected on endothelium in inflammatory liver diseases. Interaction of lymphoid cells and BECs. ICAM-1/LFA- 1 and also VCAM-1/VLA-4 linkages between the BECs of damaged bile ducts and infiltrating lymphocytes facilitate antigen-specific reactions, such as antigen presentation to periductal lymphocytes and effector mechanisms. Cell-to-cell and cell-to-ecm contacts may also play an important role in the development of chronic nonsuppurative destructive cholangitis (CNSDC). In PBC, most of the infiltrating lymphoid cells expressed integrin-α4. These results suggest that increased fibronectin expression on the biliary basement membrane and integrin-α4/fibronectin interaction facilitate the adhesion and penetration of infiltrating integrin-α4-expressing lymphocytes into the biliary epithelial layer in PBC. 25 Effector mechanism Several effector mechanisms are responsible for immune-mediated cholangitis that is followed by bile duct destruction and ductopenia. Autoreactive or alloreactive T cells, which infiltrate the liver, may play a major role. 38 In particular, CTLs play a dominant role in the biliary epithelial cytolysis (necrosis or apoptosis). Apoptosis and T-cell-mediated cytotoxicity. Disturbed balance of cellular renewal and dropout is important in immune-mediated cholangitis, and apoptosis of BECs is considered a major cellular degrading pathway. 13 In bile duct injuries in PBC and acute HAR, the proliferative responses that counteract apoptotic cell loss are, eventually, insufficient, resulting in vanishing bile duct syndrome (VBDS). 38 This progression is rapid in HAR, while it is slow or variable in PBC. The immunemediated apoptosis may be initiated through the following mechanisms: (i) direct T-cell cytotoxicity; (ii) activation of the Fas/Fas ligand (FasL) system; (iii) induction of cytokines, such as TNF-α and IFN-γ; and (iv) other mechanisms such as oxidative stress generated by chronic inflammation. As to T-cell cytotoxicity, CD4 and CD8 CTLs can be injurious to BECs. The former are mainly dependent on the Fas-FasL interaction, while the latter are mainly dependent on the perforin-granzyme exocytosis pathway. 18 While most CTLs in HAR are MHC class I- restricted CD8 T cells and play a dominant role in the disease, the effector cells in PBC and PSC are CD4 cells belonging mainly to the Th1 subset. 19 Cytokines may directly trigger the apoptosis of BECs, followed by the loss of bile duct. TNF-α and INF-γ have been shown to induce an increase in Fas expression and to enhance Fas-related apoptosis. Autoantibody-mediated effector process. Several autoantibodies are detectable in PBC and PSC patients, some being specific to certain diseases. For example, AMA and antinuclear antibodies, particularly of the

8 310 Y. Nakanuma et al.: Pathobiology of bile duct loss centromere type, are frequently detected in PBC, whereas ANA and P-antineutrophil cytoplasmic antibodies are found in PSC. IgA and IgM of AMA, which are secreted through the cytoplasm of BECs via secretory component (SC)- mediated transport, may be injurious to mitochondria or to their transported enzymes. 46 A majority of the plasma cells that infiltrate the portal tracts in PBC secrete antipyruvate dehydrogenase complex (PDC) of the IgG, IgA, and IgM classes, and these antibodies may be involved in antibody-dependent cell-mediated cytotoxicity. CD20 B cells and immunoglobulin-positive cells, which may result from the proliferation of autoreactive B cells stimulated by CD4 of the Th2 subset, are detectable in portal tracts in these diseases. 47 Immunoglobulins (IgG and IgM, in addition to IgA) and complements are, reportedly, deposited on the surface of BECs in PBC. 47 These immunoglobulins may bind to antigens on the biliary epithelium, and provoke antibody-mediated attack by IgA and IgG antibodies present in bile. 46 Other mechanisms. Eosinophils and their primary granule proteins, such as major basic proteins, are also detectable in portal tracts with peripheral eosinophilia in acute HAR and, to a lesser degree, in PBC and PSC at early stages. 48 In particular, patients with severe rejection in the first month after liver transplantation often have blood eosinophilia and marked eosinophilic infiltration of portal tracts with eosinophil degranulation. It is known that the granule proteins of eosinophils are cytotoxic for bile ducts. These findings underline the importance of the eosinophils in acute HAR and, probably to a lesser degree, in bile duct damage in PBC and PSC. 44 These eosinophils are correlated with the upregulation of eosinophilic chemotactic factors, such as IL-5, and several other chemokines, such as eotaxin and MCP3, in infiltrating cells in portal tracts. Drug-induced, toxic, and metabolic biliary injury and bile duct loss In addition to the loss caused by various types of cholangitis and biliary epithelial damage, bile duct loss is also reportedly caused by drug-induced, toxic, and metabolic damage Although the precise mechanisms of such biliary damage, followed by the loss of bile ducts, remain unclarified, some consist of cytopathic effects to the BECs themselves. Others may be related to the allergic process. Predictable toxic biliary injury Endogenous and exogenous toxins are known to damage the bile ducts, directly or indirectly. Toxic materials, or materials injurious to cells, in bile or delivered from the PVP could expose the BECs of the intrahepatic biliary tree to various concentrations of such materials, followed by damage and destruction. For example, paraquat produces severe cytopathic changes in BECs lining the biliary tree, from the bile ductules to the intrahepatic large bile ducts. 52 4,4 - Diaminodiphenylmethane causes necrotic cholangitis in small bile ducts with infiltration of eosinophils. 49 As for the pathology, vacuolation and swelling of BECs and even sloughing into the bile ductal lumen are known. Hepatic arterial infusion including anticancer drugs is also known to frequently produce hyperplastic and degenerative, and even bizarre, epithelial changes in intrahepatic bile ducts and peribiliary glands, in some cases resembling biliary malignancy. 1,51 Unpredictable toxic biliary injury Idiosyncratic metabolic and immune-mediated mechanisms have been suggested in biliary injury, particularly in drug-induced VBDS. 50 A drug (or its metabolites) may act as a hapten to bind proteins, and then trigger an immune-mediated or allergic response against the biliary epithelium, resulting in bile duct damage, bile duct loss, and cholestasis. Epithelioid granuloma is also occasionally seen in portal tracts and/or hepatic parenchyma. The precise pathogenetic mechanisms leading to bile duct loss remain unclear. Infectious cholangitis and bile duct loss In infectious cholangitis caused by bacterial or viral infection, progressive bile duct loss occurs infrequently. Bacteria and their products may be involved in the biliary epithelial damage and the bile duct loss. Infectious agents also seem to be involved in other types of bile duct loss, such as immune-mediated cholangitis, as a trigger or aggravating factor. Bacterial infection and bile duct loss In acute ascending cholangitis or pyogenic cholangitis, cholangitic abscess with biliary epithelial damage or obliteration is occasionally seen, suggesting the destruction of bile ducts caused by bacterial infection or toxin. It is unclear whether or not the obliterated bile ducts regenerate. In long-standing bacterial cholangitis, such as secondary sclerosing cholangitis and long-standing hepatolithiasis, the interlobular and septal bile ducts, and even the intrahepatic large bile ducts, are obliterated and replaced by a fibrous core with rich elastic fibers. Such bile duct loss is also seen in the liver peripheral to intrahepatic cholangiocarcinoma or biliary papillomatosis. This type of bile duct loss is likely to occur in severely affected areas of the liver. Usually, histologic findings of cholestasis are not likely to occur. Although this features of bile duct obliteration resembles PSC,

9 Y. Nakanuma et al.: Pathobiology of bile duct loss 311 the bile duct loss is not usually associated with inflammatory cell infiltration. Ischemic and toxic factors, in addition to the infectious agents, may be involved secondarily in this type of bile duct loss. Bacterial infection is also a cause of paucity of interlobular bile ducts or obliterative cholangiopathies in neonates or infants. 53,54 Viral infection and duct injuries Injury to the bile-duct epithelium in cytomegalovirus (CMV) hepatitis is not unusual in neonates and adults. In the former, viral inclusions in the biliary epithelium are seen as a feature of neonatal hepatitis. 4 In the latter, CMV is associated with an infectious mononucleosislike lesion and occasional epithelioid granuloma. In immunocompromised hosts with CMV infection, viral inclusions are encountered in the biliary epithelium. It is plausible that the paucity of interlobular bile ducts in neonates or infants may have been a sequela of CMV infection of the liver followed by progressive destruction of bile ducts. 54 Reovirus infection has long been considered a possible etiologic agent inciting the inflammatory process that leads to the infantile obstructive cholangiopathies with bile duct loss. That is, reovirus RNA was demonstrated in the liver tissue of about half the patients with extrahepatic biliary atresia and in the liver tissue of more than half the patients with choledochal cysts, by polymerase chain reaction (PCR). 55 The precise process of bile duct loss with respect to viral infection in this bile duct damage remains unexplored. Infections as a trigger of or aggravating factor in immune-mediated cholangitis Bacterial or viral infections and their products may play a role, either primarily or secondarily, in the progression of immune-mediated biliary diseases. 32 Modern molecular and genetic studies, using the bacterial 16S ribosomal RNA gene and other specific genes, have disclosed that hepatic Helicobacter DNA is frequently detectable in bile and gallbladder tissue with chronic cholangitis. 56 Certain other bacteria can live and proliferate in bile and may form a specific bacterial microecology. Microbiology in bile and the biliary tree should be reconsidered in terms of cultural and genetic studies. Although the biliary tree is said to be sterile, it could have its own normal and pathologic bacterial flora (biliary microbiology). In PBC and hepatic sarcoidosis, bacterial infection may be determined to be the etiology when examined by such modern approaches, but the clinicopathological features may be overshadowed by other phenomena, such as the immunologic features. PBC. Retroviral infection is also speculated to be part of the etiology of hepatobiliary diseases, including PBC. 40 As a primary factor, mycobacteria have been implicated in the pathogenesis of granulomatous diseases, including PBC, although the data supporting this idea are controversial. 12,41 In addition, bacterial participation in the pathogenesis of bile duct lesions in PBC is now being tested, and gram-positive and gram-negative bacterial DNA has been recovered from gallbladder bile and liver tissue of PBC patients by PCR. 41 Tiny fragments of the gram-positive bacterial ribosomal RNA gene have also been recovered from periductal granuloma of PBC patients by PCR (Harada et al., manuscript in preparation). PSC. Bacteria are frequently isolated in the bile duct tissues from explanted livers, and may be related to the progression and clinical manifestations of this disease: the most frequently encountered bacteria were alphahaemolytic streptotocci, enterococci, and staphylococci. Bacteria, particularly enterobacteria, may be involved in the progression of PSC. GVHD and HAR. As a secondary factor, bacterial or viral infections may aggravate or initiate the alloimmune-mediated bile duct lesions in GVHD and HAR. 60 CMV infection has been shown to increase the risk of ductopenic rejection, although doubt remains as to a pathogenetic role for CMV. Hepatitis C infection and treatment with INF-α may cause a predisposition to the development of ductopenic rejection after liver transplantation. The degree of immunologic mismatch is a risk factor for HAR and GVHD. Ischemic biliary injury and bile duct loss Histologic and radiologic studies have disclosed that bile ducts are preferentially damaged by the ischemic processes such as interventional radiology (IVR) therapy and HAR. 1 The main reason for such damage is the fact that biliary tree is exclusively supplied by the hepatic arterial branches, which are known as PVP. 6 Small PVP vessels are well organized, although at the level of interlobular bile ducts and bile ductules, PVP is less organized. Underlying pathologic changes and biliary manifestations The hepatic arterial branches or PVP are primarily and secondarily damaged by the processes of thrombotic occlusion or numerical reduction; vasculitis or thrombosis of small PVP vessels; and vasculitis, thrombosis, and the occlusion of peribiliary arteries or the hepatic artery, itself, including occlusive foam cell arteriopathy. 1 As manifestations of ischemic biliary injury, biliary epithelial damage, cholangitis without necrosis, bile duct fibrosis, erosion, focal necrosis or infarction of bile duct with extravasation of bile (biloma) or infec-

10 312 Y. Nakanuma et al.: Pathobiology of bile duct loss tion, ductopenia, and biliary stricture or cholangiectasis are known to develop. In the event of biliary ischemia, portal tracts are also edematous or fibrotic, and fibrous thickening of the intima and adventitia of arteries, thrombosis, and stenosis of portal vein branches occur. 51 Interventional radiology (IVR) Ischemic biliary injury related to IVR is seen in the livers of patients receiving transcatheter arterial embolization therapy (TAE) and hepatic arterial infusional chemotherapy (HAI). Usually, the emboli of TAE contain gelatin sponge with lipiodol and anticancer drugs. One example is fluoxuridine-related sclerosing cholangitis, which may develop after HAI of this antineoplastic agent, which causes drug-induced occlusion or intravascular thrombosis, in PVP. Semiquantitative assessment disclosed that the PVP vessels were considerably reduced in number or absent in these cases. These injuries are more significant in noncirrhotic livers than in cirrhotic livers, probably because the latter show numerous anastomoses or collaterals involving PVP, and are, therefore, resistant to ischemia. Recent technological advances in IVR have greatly reduced the incidence of these types of ischemic biliary injuries. Late complication of abdominal radiation Irradiation-induced bile duct injury includes vascular changes, such as arteriolar intimal thickening responsible for luminal narrowing, dilated capillary vessels and arterioles with fibrous thickening, and, also, dense inflammatory fibrosis replacing the muscular layer, including variable degrees of stenosis. 1 The biliary epithelium is usually normal. The common bile duct could be palpated in the hepatoduodenal ligament: it was thick and firm and felt like a pencil. Bile duct changes observed after irradiation resemble those that may occur after orthotopic liver transplantation, as the result of reduced arterial supply to the graft and/or chronic rejection. 58 This late complication is now quite rare. Pathologic processes in PVP in other biliary diseases Necrotizing arteritis, such as periarteritis nodosa involving arterial branches and PVP, also causes variable ischemic bile duct damage. 59 Other examples are sclerosing cholangitis, including PSC, and also sclerotic bile ducts in hepatolithiasis, which show marked reductions in the PVP in the sclerotic areas. 51 It remains unclear whether these changes in the PVP are secondary to pathologic processes in bile ducts or whether they are responsible for bile duct sclerosis or loss. Hepatic allografts and bile duct loss Bile duct damage and bile duct loss caused by hepatic allograft rejection are described in detail in this issue of the Journal. Humoral rejection primarily affects the microvasculature, and is often mediated by complementfixing, preformed anti-donor MHC class I antibodies, and antibodies directed against major ABO blood group antigens, all of which are normally expressed on the hepatic artery, portal vein, and PVP endothelium. 1 In acute HAR, recipient T cells enter the allograft and spontaneously cluster with donor DCs that are present in the portal tract. 1 This results in T-cell activation, proliferation, and secretion of cytokines that upregulate the nearby microvasculature. In turn, this leads to the recruitment of more inflammatory cells, and tissue destruction begins. In chronic HAR, two mechanisms are operative in the bile duct loss: direct lymphocytotoxicity and ischemic damage. 60 As to the latter, the alloimmune response preferentially injures the endothelial cells of the intrahepatic arteries and their branches, including the PVP, as well as veins. Ischemic biliary injury, followed by duct loss, may also be an important cause of cholestatic graft failure. Cholangitis and bile duct infarction or necrosis of the intrahepatic large or septal bile ducts, 61 and extensive loss of smaller bile ducts (ductopenic rejection) are examples of such injury. These ischemic biliary injuries may be further affected by bacterial or viral infection during antiimmune therapy. These changes, along with alterations in cytokine networks and in the expression of immune molecules, may lead to further attack of the biliary tree by alloimmune or autoimmune processes. In addition, the allograft biliary tree may be more susceptible to ischemia because of a lack of vascular collaterals in the early posttransplant period. 1 Other diseases with bile duct loss There are many other diseases with bile duct loss, such as sarcoidosis, Hodgkin s disease, and idiopathic adulthood ductopenia. 1 The pathogenesis of bile duct loss remains unexplored in these diseases. In addition to these acquired diseases, there are a number of infantile and neonatal disorders with extensive bile duct loss. In neonates, the pathogenesis is related to failure of the highly cooperative and complicated process of apoptosis and cell proliferation in BECs. Syndromatic and nonsyndromatic paucity of interlobular bile ducts Syndromatic paucity of interlobular bile ducts, also called Alagille syndrome, shows an autosomal dominant mode of transmission with specific congenital features which preferentially affect the biliary tree. 61 The bile duct paucity is a result of the destruction of previously existing bile ducts. This syndrome varies in severity. The responsible gene has been identified; it is the human Jagged 1 gene, which is located within the short

11 Y. Nakanuma et al.: Pathobiology of bile duct loss 313 arm of chromosome 20 and encodes a ligand in the Notch intercellular signaling pathway. The interactions of Notch receptors and their ligands are crucial to the control of cell fate in a variety of developmental processes, although it remains uncertain how this defective gene leads to the bile duct paucity, and, in some instances, to progressive cholestatic liver disease. Nonsyndromatic paucity of interlobular bile ducts is heterogeneous in etiology. 1 Progressive familial intrahepatic cholestasis Normally, the biliary epithelial cells are protected from 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 effects of bile acids increase with increasing relative hydrophobicity of bile acids. 62 The cell membrane of BECs is protected from the detergent effects of hydrophobic bile acids by phospholipids, which are secreted by the multiple drug resistance (MDR) gene. 62 In patients with progressive familial intrahepatic cholestasis, a defect of the MDR3 gene is suspected. As a consequence, the biliary epithelium lacks the protection conferred by phospholipids against the detergent bile acid effects. A form of nonsuppurative destructive cholangitis develops with progressive ductopenia and leads to VBDS. Vanishing bile duct syndrome (VBDS) VBDS is a common and terminal clinicopathological symptom complex that follows extensive and irreversible ductopenia. 1 3 VBDS consists of heterogeneous diseases, such as the immune-mediated cholangitides, toxin- and drug-induced bile duct damage, infectious cholangitis, sarcoidosis, Hodgkin s disease, and Alagille syndrome. While restoration of the biliary tree in livers with bile duct loss can occur by renewal or regeneration of the biliary epithelial cells in some ductopenic diseases, in VBDS patients, the bile duct loss is usually followed by progressive cholestasis, biliary fibrosis with atypical ductular proliferation, and, finally, liver cirrhosis or liver failure. To prevent liver failure, liver transplantation is the only treatment in such patients. The deposition of copper granules, Mallory body formation, and feathery degeneration or cholate stasis in hepatocytes are seen in patients with prolonged VBDS. Overview Bile duct loss is a feature of hepatobiliary diseases and characterizes a number of ductopenic liver diseases. Although the bile duct loss itself is not a major hepatobiliary disease, the progressive bile duct destruction is always followed by VBDS and then by liver failure, or to prevent this, liver transplantation. The stimulation or promotion of biliary epithelial regeneration, and the activation of presumed stem cells may lead to the restoration of the biliary tree in these ductopenic diseases. Further studies of the biliary biology and also of biliary stem cells are mandatory if we are to restore the loss in ductopenic diseases; in particular, the stimulation of hepatic stem cells, followed by their maturation to biliary epithelial cells, seems to be critical in these diseases. References 1. Portmann BC, Nakanuma Y (2001) Biliary disorders. In: Portmann BC, Anthony PP, Scheuer PJ (eds) Pathology of liver, 4th edn. Churchill Livingstone, Edinburgh, London, New York 2. Nakanuma Y, Hoso M, Sanzen T, Sasaki M (1997) Microstructure and development of the normal and pathologic biliary tract in humans, including blood supply. Microscopy Res Tech 38: Ludwig J, Wiesner RH, Batts KP, Perkins JD, Krom RA (1987) Vanishing bile duct syndrome (acute irreversible) after orthotopic liver transplantation. 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