Bile duct epithelia as target cells in primary biliary cirrhosis and primary sclerosing cholangitis

 Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are chronic autoimmune-mediated diseases of the biliary tree, resulting in a loss of bile ducts. There are morphological features that clearly distinguish them from each other: in PBC, there is overt destruction of the bile ducts with disruption of the basement membrane; in PSC there is abundant periductular fibrosis with shrinkage and subsequent loss of the bile ducts. In order to see if the disparate histopathology is paralleled by different immunohistology we looked at a panel of epitopes on bile duct epithelia especially to see if biliary epithelial cells may present as targets for cell mediated immune respone. In PBC bile duct epithelial cells mostly expressed CD58 (lymphocyte function-associated antigen 3), CD80 (B7 BB1), and CD95 (Fas). In PSC, however, these epitopes were only expressed in a few examples to a lower degree. The respective effector T lymphocytes were positive for CD2 and CD28. Subtyping of the lymphocytes in the liver tissue further showed a predominance of CD4 positive T cells over CD8 cells up to 2-to-1 in both diseases. Determination of lymphocytes by cytokines to Th1 or Th2 subtype showed a majority of Th1 lymphocytes in PBC and PSC. We conclude that in PBC bile duct epithelial cells may display features of target cells of a T cell-mediated immune reaction with the Th1 cells predominating. In PSC other mechanisms of bile duct loss may play a role, since in this disease the majority of cells lack essential epitopes that constitute targets of cell mediated immunity. Received: 19 November 1996 / Accepted: 26 February 1997     

Characterization of biliary intra-epithelial lymphocytes at different anatomical levels of intrahepatic bile ducts under normal and pathological conditions: numbers of CD4+CD28- intra-epithelial lymphocytes are increased in primary biliary cirrhosis

Distribution of intra-epithelial lymphocytes along intrahepatic biliary tree (bIEL), and their density and phenotype were examined in normal and diseased livers, particularly in primary biliary cirrhosis (PBC). Immunohistochemically, bIEL were examined in 28 normal livers, 13 cases of chronic viral hepatitis (CVH), 13 cases of PBC, five cases of primary sclerosing cholangitis (PSC), seven cases of extrahepatic biliary obstruction (EBO), and 16 hepatolithiatic livers. In normal livers, bIEL were relatively dense at large and septal bile ducts compared to interlobular ducts. Most of them were positive for CD3 and CD8, while a few were positive for CD4, CD20 and CD57. In CVH, PSC and EBO, neither distribution, phenotype nor density of bIEL differed from normal liver. In hepatolithiasis, numbers of CD8(+)bIEL were increased in stone-containing ducts. In PBC, numbers of CD4(+)CD28(-)bIEL, which are reportedly responsible for target tissue destruction in autoimmune diseases, were markedly increased in damaged interlobular ducts. In conclusion, CD3(+)CD8(+)bIEL may be involved in immune homeostasis of intrahepatic bile ducts in normal livers and in CVH, PSC and EBO. Altered distribution and phenotypes of bIEL in PBC and hepatolithiasis may reflect their participation in biliary lesions. Increased CD4(+)CD28(-)bIEL in damaged bile ducts of PBC may be related to immune-mediated biliary damage.     

Molecular mechanisms of cholangiopathy in primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is histologically characterized by chronic nonsuppurative destructive cholangitis (CNSDC) and the progressive loss of intrahepatic small bile ducts. Cellular immune mechanisms involving T-cell reaction are thought to be significantly involved in the formation of CNSDC and bile duct loss. In inflamed portal tracts of PBC, CD4⁺ T cells of Th1 type expressing IFN-γ or CXCR3 are aggregated and more commonly detected around injured bile ducts than Th2-type CD4⁺ T cells expressing IL-4 or CCR4, indicating that Th1-dominant cellular immunity plays a more-prominent role in recruitment of memory T-cell subsets in PBC and may be responsible for the progressive bile duct damage. Biliary epithelial apoptosis is demonstrated to be a major pathogenic process of bile duct loss in PBC. In CNSDC, several biliary apoptotic cells, an aberrant expression of Fas antigen (proapoptotic molecule) and decreased expression of bcl-2 and mcl-1 (antiapoptotic molecules) are found, although interlobular bile ducts express bcl-2 and mcl-2 but lack Fas. In addition, the upregulation of WAF1 and p53 related to biliary apoptosis is found in biliary epithelial cells of PBC, which may be due to cell senescence in response to genotoxic damage such as oxidative stress. Several steps and mechanisms during induction and progression of cholangitis and biliary apoptosis followed by bile duct loss are now being proposed in PBC, but future analysis of an etiopathogenesis to explain the characteristic histopathogenesis of PBC is required.     

The Immunophysiology and Apoptosis of Biliary Epithelial Cells: Primary Biliary Cirrhosis and Primary Sclerosing Cholangitis

Biliary epithelial cells (BECs) provide the first line of defense against lumenal microbes in the biliary system. BECs express a variety of pathogen recognition receptors and can activate several intracellular signaling cascades to initiate antimicrobial defenses, including production of several anti-microbial peptides, cytokines, chemokines, and adhesion molecules. BECs also secrete immunoglobulin A and interact with other cells through expression and release of adhesion molecules and immune mediators. Recently, several reports suggest a correlation between apoptosis and autoimmunity through ineffective clearance of self-antigens. Primary biliary cirrhosis (PBC) is a slowly progressive, autoimmune cholestatic liver disease characterized by highly specific antimitochondrial antibodies (AMAs) and the specific immune-mediated destruction of BECs. We have demonstrated that the AMA self-antigen, namely the E2 subunit of the pyruvate dehydrogenase complex, is detectable in its antigenically reactive form within apoptotic blebs from human intrahepatic biliary epithelial cells and activates innate immune responses. Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by inflammation and the presence of concentric fibrosis of intrahepatic and/or extrahepatic bile ducts, eventually leading to cirrhosis. However, apoptosis does not appear to play a central role in PSC. Despite both diseases involving immune-mediated injury to bile ducts, apoptosis occurs more commonly overall in PBC where it likely plays a unique role.     

Aberrant expression of stem cell factor on biliary epithelial cells and peribiliary infiltration of c-kit-expressing mast cells in hepatolithiasis and primary sclerosing cholangitis: a possible contribution to bile duct fibrosis

Hepatolithiasis and primary sclerosing cholangitis (PSC) are intractable chronic biliary diseases. In hepatolithiasis, bilirubin-calcium stones are packed in multiple irregularly dilated intrahepatic bile ducts. In PSC, small bilirubin-calcium stones develop terminally. The progressive periductal fibrosis with dilated and stenotic bile ducts in these two diseases may play a role in their incurability. This immunohistochemical study has investigated the expression of some factors that might be involved in fibrogenesis in hepatolithiasis and PSC. Many mast cells positive for c-kit were found in the periductal and ductal fibrosis around the intrahepatic large bile ducts and also around the proliferative peribiliary glands. These mast cells also expressed basic fibroblast growth factor and/or tumour necrosis factor-alpha, which are known as fibrogenetic factors. It was of interest that the aberrant expression of stem cell factor (SCF), a ligand of c-kit, was demonstrated on biliary epithelia of the dilated and stenotic bile ducts showing periductal fibrosis and inflammation and also of the proliferated peribiliary glands in hepatolithiasis and PSC, while no such expression was seen in non-affected bile ducts in hepatolithiasis or in the bile ducts in normal livers. Some of the infiltrating mononuclear cells around the SCF-expressing bile ducts were also positive for SCF. It seems likely that aberrantly expressed SCF on biliary epithelial cells accumulates and stimulates mast cells via the c-kit receptor and that these up-regulated mast cells induce progressive periductal and portal fibrosis by displaying fibrogenetic factors in hepatolithiasis and PSC.     

Periductal interleukin-17 production in association with biliary innate immunity contributes to the pathogenesis of cholangiopathy in primary biliary cirrhosis

An innate immune response to bacterial components is speculated to be involved in the pathogenesis of primary biliary cirrhosis (PBC). Recently, CD4-positive T helper type 17 (Th17) cells, characterized by the secretion of interleukin (IL)-17, have been implicated in the pathogenesis of autoimmune diseases. Human Th17 cells are generated from Th0 cells by IL-6 and IL-1β and maintained by IL-23. In this study, the role of IL-17 in PBC and its association with biliary innate immunity were examined. Using cultured human biliary epithelial cells (BECs), the expression of Th17-related cytokines and chemokines and changes therein on treatment with pathogen-associated molecular patterns (PAMPs) and IL-17 were examined. Immunohistochemistry for IL-17 and Th17-related cytokines was performed using tissue samples of human liver. Consequently, the expression of IL-6, IL-1β, IL-23p19 and IL-23/IL-12p40 mRNAs, and their up-regulation by PAMPs, were found in BECs. Moreover, BECs possessed IL-17-receptors and stimulation with IL-17 induced production of IL-6, IL-1β, IL-23p19 and chemokines. Several IL-17-positive cells had infiltrated damaged bile ducts and the expression of IL-6 and IL-1β was enhanced in the bile ducts of PBC patients. In conclusion, IL-17-positive cells are associated with the chronic inflammation of bile ducts in PBC which is associated causally with the biliary innate immune responses to PAMPs.     

Immunohistochemical analysis of adhesion molecules in the micro-environment of portal tracts in relation to aberrant expression of PDC-E2 and HLA-DR on the bile ducts in primary biliary cirrhosis

We have examined immunohistochemically the expression of adhesion molecules in the micro-environment of portal tracts and their relationship to the expression of the pyruvate dehydrogenase E2 complex (PDC-E2) and HLA-DR in liver biopsy specimens. Ten cases of primary biliary cirrhosis (PBC) and 19 controls were examined, including four cases of extrahepatic biliary obstruction, six of chronic viral hepatitis, and nine normal livers. In PBC, the damaged small bile ducts demonstrated an increased expression of PDC-E2 and an aberrant expression of HLA-DR; about half of these damaged bile ducts also expressed intercellular adhesion molecules (ICAM)-1 and a few expressed vascular adhesion molecule (VCAM)-1. In addition, lymphocyte function-associated antigen (LFA)-1 and very late antigen (VLA)-4 were expressed on infiltrating lymphocytes around these bile ducts. In contrst, in control livers, these alterations in antigen expression on the bile ducts were either not observed or were only focal and weak, when present. These findings suggest that ICAM-1/LFA-1 and also VCAM-1/VLA-4 linkages between the damaged bile ducts and lymphocytes may facilitate antigen-specific reactions such as the presentation of antigens, possibly PDC-E2, to the periductal lymphocytes in PBC. ICAM-1, VCAM-1, and E-selectin were strongly expressed on the endothelial cells of some vessels in the portal tracts in PBC, suggesting the facilitation of the recruitment of lymphocytes around the of some vessels in the portal tracts in PBC, suggesting the facilitation of the recruitment of lymphocytes around the bile ducts of PBC. VCAM-1, a member of the immunoglobulin superfamily, has not hitherto been reported on bile ducts.     

Association analysis of toll-like receptor 4 polymorphisms in Japanese primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is characterized by portal inflammation and immune-mediated destruction of intrahepatic bile ducts that often result in liver failure. Toll-like receptor (TLR) 4 recognizes lipopolysaccharides of Gram-negative bacteria. Infectious agents have been suspected to play a crucial role in PBC pathogenesis since TLR4 expression was found in bile duct epithelial cells and periportal hepatocytes in liver tissues of PBC. To assess the potential contribution of TLR4 SNPs to the development of this disease, we genotyped five SNPs in TLR4 in 261 PBC patients and 359 controls using a TaqMan assay. No significant positive associations with either PBC susceptibility or progression were uncovered. These results indicate that TLR4 polymorphisms do not play a prominent role in the development of PBC in Japanese patients.     

Immunolocalization of putative human liver progenitor cells in livers from patients with end-stage primary biliary cirrhosis and sclerosing cholangitis using the monoclonal antibody OV-6.

The term oval cell describes small cells with oval nuclei that arise in the periphery of the portal tracts in rat models of hepatocarcinogenesis and injury and can differentiate into either hepatocytes or bile duct cells, ie, are bipotential. The presence of such cells in human liver is controversial. Here, immunolocalization of OV-6 and two biliary markers, cytokeratin 19 (CK-19) and human epithelial antigen 125 (HEA-125) is compared in normal adult human livers and in primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) liver sections. CK-19 and HEA-125 stained bile ducts and ductules in normal liver as well as proliferating ductular structures in diseased livers. OV-6 did not label ducts or ductules in normal liver, but in PBC and PSC stained numerous proliferating ductular and periductular cells and lobular hepatocytes. In PBC, discrete OV-6-positive cells with a mature biliary-cell-like morphology were seen integrated into some intact bile ducts as well as occasional small immature oval-like cells. In addition, in PSC, hepatocytes in regenerating lobules were also strongly stained with OV-6, and on close inspection, in both PBC and PSC, oval cells and small hepatocytes at the margins of the lobules were strongly labeled. In contrast to the rat liver, OV-6 and CK-19 staining did not always co-localize. It is proposed that the small OV-6-positive oval cells are analogous to those seen in rat models and may represent human liver progenitor cells that may differentiate into OV-6-positive ductal cells or lobular hepatocytes.     

Monocyte chemotactic protein-1, -2, and -3 are distinctively expressed in portal tracts and granulomata in primary biliary cirrhosis: implications for pathogenesis

The monocyte chemotactic proteins (MCPs) form a distinct structurally related subclass of C-C chemokines. MCPs select specific target cells due to binding to a distinct set of chemokine receptors and because of their effects on monocytes, and may participate in the process of granuloma formation during bacterial and/or mycobacterial infections. The aetiology of primary biliary cirrhosis (PBC) is still unclear, although bacterial infection and autoimmune processes have been implicated. In this study, the expression of three of the most potent monocyte chemoattractants, MCP-1, -2, and -3, was examined in patients with PBC and the data were compared with results for other liver diseases including primary sclerosing cholangitis (PSC), chronic viral hepatitis C, hepatic sarcoidosis, and normal liver. MCP-1 was expressed mainly in biliary epithelial cells of all liver specimens, irrespective of the cause of disease. Some mononuclear leukocytes in the portal tract expressed MCP-1 in all the disease groups examined and there were no significant differences in frequency between these groups. In contrast, more than 80% of PBC livers showed MCP-2- and MCP-3-positive mononuclear leukocyte infiltration in portal tracts, particularly around the bile ducts, whereas such cells were far less frequent in the other disease groups or in normal livers. Epithelioid granulomata of PBC patients contained MCP-2- and MCP-3-positive cells at their edge. In double staining experiments, more than 60% of the MCP-positive mononuclear cells co-expressed CD68, suggesting that a proportion of MCP-2- and MCP-3-positive cells are derived from monocytes. These monocytes expressing MCP-2 and MCP-3 may be responsible for the chemotactic activity of more monocytes. Such an expression pattern of MCP-1, -2 and -3 in portal tracts seems to be distinctive for PBC. This pattern underlines the importance of MCP-1, -2, and -3 in the recruitment of monocytes and possibly T lymphocytes into portal tracts, around the injured bile ducts, and into epithelioid granulomata in PBC. The data further implicate bacterial materials derived from bile in the overall pathogenesis of PBC.     

Diffuse biliary tract involvement mimicking primary sclerosing cholangitis in an experimental model of chronic graft-versus-host disease in mice

Experimental graft-versushost disease (GVHD) across minor histocompatibility antigens was developed by injecting spleen and bone marrow cells (9:1) of congenic B10.D2 mice into sublethally Irradiated BALB/c mice, and the histologic features of the Iiver were studied for up to 14 months after transplantation. Both intrahepatic and extrahepatic bile ducts were severely involved in the GVHD process and showed features of non-suppurative cholangitis. Inflammatory cells, mainly lymphocytes, abutted the bile ducts and Infiltrated Into the duct eplthellal layer together with a variety of degenerative changes in the epithelial cells. The peak Inflammatory activity occurred about 2–3 weeks after transplantation. Thereafter, the inflammatory cell Infiltration around the bile ducts and In the bile duct epithelial layer gradually became reduced in severity, although the infiltration persisted during the entire 14 month observation period. Periductal and duct wall fibroplasia began about 1 week after transplantation and gradually progressed. After 2–3 months post-transplantation, distinct ductal and periductal fibrosis of both intrahepatic and extrahepatic blle ducts was observed. This histologic feature resembled that of primary sclerosing cholangitis (PSC). These results suggest that PSC lesions might develop as a result of chronic cellular Immunologic mechanisms In GVHD across minor histocompatibility barriers.     

Distribution of apoptotic cells and expression of apoptosis-related proteins along the intrahepatic biliary tree in normal and non-biliary diseased liver

AIMS: The cell kinetics and homeostasis of biliary epithelial cells may be maintained differently along the biliary tree. In this study, the role of apoptosis in the maintenance and homeostasis of the intrahepatic biliary tree was evaluated. METHODS AND RESULTS: By counting apoptotic biliary cells and by immunostaining apoptosis-related proteins in normal liver, fatty liver, and those with acute viral hepatitis, chronic viral hepatitis, and hepatitis virus-related cirrhosis, it was found that the larger the intrahepatic bile ducts became, the more biliary epithelial cells underwent apoptosis. bcl-2, an inhibitor of apoptosis, was diffusely expressed in the interlobular bile ducts, but rarely detectable in the large and septal bile ducts. bcl-XL and mcl-1, inhibitors of apoptosis, and bax, a promoter of apoptosis, were diffusely expressed along the intrahepatic biliary tree. CD95, a direct inducer of apoptosis, was present in the large and septal bile ducts, but rarely in the interlobular bile ducts. CONCLUSION: The ratio of bax to bcl-2, as well as the expression of CD95 which differed at the interlobular versus large and septal bile ducts, may be responsible for the unique distribution of apoptotic biliary cells and involved in the homeostasis of the intrahepatic biliary tree.     

Frequent cellular senescence in small bile ducts in primary biliary cirrhosis: a possible role in bile duct loss

The pathogenesis of progressive bile duct loss in primary biliary cirrhosis remains unclear. In this study, the involvement of cellular senescence of biliary epithelial cells was examined in liver tissue samples from patients with primary biliary cirrhosis (n = 33), and compared with control diseased and normal livers (n = 83). In addition, cellular senescence was induced by oxidative stress in cultured mouse biliary epithelial cells. Biliary epithelial cells in small bile ducts in primary biliary cirrhosis, especially those in patients presenting with chronic non-suppurative cholangitis, frequently expressed senescence-associated beta-galactosidase, and senescence-associated p16(INK4) and p21(WAF1/CIP). In contrast, senescence-associated markers were rarely expressed in small bile ducts in control livers. The infiltration of myeloperoxidase-positive inflammatory cells into biliary epithelial cell layers was closely associated with the cellular senescence of biliary epithelial cells in early-stage PBC. Cellular senescence of cultured mouse biliary epithelial cells was induced by treatment with H2O2 via the p38MAPK-dependent pathway and nitric oxide-augmented H2O2-induced cellular senescence. Oxidative stress- and nitric oxide-mediated cellular senescence may be involved in bile duct lesions, which are followed by progressive bile duct loss in primary biliary cirrhosis.     

Expression of HLA-DR antigens on interlobular bile ducts in primary biliary cirrhosis and other hepatobiliary diseases: an immunohistochemical study

Using a monoclonal antibody to alpha chains of HLA-DR antigens, we found that damaged biliary epithelial cells in primary biliary cirrhosis (PBC) and, to a lesser degree and frequency, in other hepatobiliary diseases expressed HLA-DR antigens, while normal bile ducts did not. There was no correlation between biliary epithelial expression of HLA-DR antigens and intraepithelial migration of HLA-DR-positive cells. In PBC, HLA-DR antigens were strongly expressed on the damaged bile ducts surrounded by no or mild inflammatory cell infiltration (nonflorid duct lesion) and on those surrounded by intense lymphoid cells (florid duct lesion). Immunoelectron microscopy confirmed the presence of HLA-DR antigens on the cellular membranes of damaged biliary epithelial cells. Although expression of HLA-DR antigens on bile ducts may itself be a nonspecific epiphenomenon of damaged bile ducts, it seems possible in PBC that biliary epithelial cells are at first damaged in some way and express HLA-DR antigens (and probably self-antigens), and then fall victim to an autoimmune reaction characterized by marked lymphoid cell infiltration (florid duct lesions). The agent causing nonflorid duct lesions is unknown. The reasons why such expression does not lead to immunologic reactions in other hepatobiliary diseases are only speculative.     

Increased expression of nuclear envelope gp210 antigen in small bile ducts in primary biliary cirrhosis

The sustained antibody response to nuclear envelope gp210 antigen indicates a group of primary biliary cirrhosis (PBC) patients at high risk for the progression to end-stage hepatic failure. To address this issue, we immunohistochemically studied the expression of gp210 antigen in needle liver biopsy specimens from PBC patients using a monoclonal antibody specific for gp210 antigen. The specimens from autoimmune hepatitis (AIH), chronic viral hepatitis B (CHB) and C (CHC) patients served as disease controls. The expression of gp210 antigen was apparently increased on the nuclear envelope of biliary epithelial cells (BECs) of small bile ducts in almost all specimens from PBC. In contrast, the expression of gp210 antigen was negative in BECs of small bile ducts in normal liver, while relatively weak anti-gp210 immunostaining was observed in AIH, CHC and CHB. In addition, the degree of gp210 expression in BECs of small bile ducts was positively correlated to that of portal inflammation, interface hepatitis and lobular inflammation in PBC. These results indicate that the increased expression of gp210 in small bile ducts, which is probably associated with damage to BECs by inflammation, is possibly involved in autoimmune response to gp210 leading to the progression to end-stage hepatic failure in PBC.     

The enigma of primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is a chronic cholestatic disease characterized by a striking predominance in female patients (with most cases diagnosed between ages 40 and 60 yr) as well as serum auto-antibodies to mitochondrial antigens, elevated serum immunoglobulin M, progressive destruction of intrahepatic bile ducts, and, ultimately, liver cirrhosis and failure (1). The precise mechanisms leading to selective destruction of biliary epithelial cells lining intrahepatic bile ducts are still unknown, although numerous immunomediated pathways have been proposed. Genetic background appears to be important in determining susceptibility to the disease (2), but no clear association with alleles in the major histocompatibility complex has been identified. Molecular mimicry either by infections (3) or xenobiotics (4) has been proposed to be capable of breaking tolerance in genetically predisposed individuals, thus leading to onset of PBC. This article describes and discusses the available data regarding the immunomediated pathogenesis of PBC (with particular attention to auto-antibodies and autoreactive T-cells) and presents the recent evidence indicating a role for either xenobiotic chemicals or novel infectious agents in the induction of the disease.     

Ultrastructural changes of bile duct epithelium in primary biliary cirrhosis in relation to progression of bile duct loss

Summary Using wedge liver biopsies from patients with primary biliary cirrhosis (PBC), ultrastructural features of the intrahepatic bile ducts in livers with slight or no bile duct loss were compared with those in livers with advanced bile duct loss and in extrahepatic cholestasis (EHC).Most changes in the biliary epithelium in PBC were similar to those in EHC. Microvillous loss and bleb formation, mitochondrial damage and increase in endoplasmic reticulum and ribosomes were found in PBC irrespective of the degree of bile duct loss, and also in EHC. These changes were present almost equally at any level of the biliary tree, and are presumed to represent a variety of non-specific lesions of biliary epithelial cells. As the loss of bile ducts in PBC progressed, cytoskeletal filaments and cytophagosomes increased in number and basement membranes were more thickened and reduplicated. These changes were more or less conspicuous in smaller branches of the biliary tree, and were also prominent in EHC. They might be causally related to the bile flow disturbance in the liver. Lateral intercellular spaces were irregularly dilated and contained osmiophilic membranous and/or granular material, similar to that found in duct lumena, within and without the basement membrane, and in the cytoplasm of periductal macrophages. Furthermore, pinocytotic vesicles were increased in the biliary cytoplasm facing periphery. These findings suggest possible alteration of the permeability of biliary epithelial cells, probably in the direction from the lumena to the periductal tissue. Such changes were found in PBC livers with virtual absence of bile duct loss, and the significance of this phenomenon is discussed.