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Background: MUC2 and MUC5AC overproduction is considered to be associated with hepatolithiasis and related to inflammation. However, mechanisms underlying MUC upregulation under inflammatory stimulation in human intrahepatic biliary epithelial cells (HIBECs) are not completely understood., Material and Methods: Expression of MUC2 and MUC5AC mRNA in HIBECs was detected by real-time PCR. Expression of COX-2, EP4, and phosphorylated ERK, JNK and p38MAPK protein was detected by Western blot. Concentrations of PGE2, IL-1β and TNF-α in cell culture supernatants were measured using the Quantikine Elisa kit., Results: COX-2 expression as well as PGE2 production in HIBECs was upregulated significantly by LPS, which was completely blocked by either TLR4 antagonist or NFκB inhibitor. Selective COX-2 inhibitor suppressed LPS-induced MUC2 and MUC5AC mRNA expression remarkably. Exogenous PGE2 increased MUC2 and MUC5AC mRNA expression in a dosage-dependent manner independent of IL-1β and TNF-α. PGE2 receptor EP4 agonist elevated MUC2 and MUC5AC expression, whereas EP4 antagonist had the opposite effect. Expression of phosphorylated p38MAPK was upregulated by exogenous PGE2, and p38MAPK inhibitor reduced MUC2 and MUC5AC expression in HIBECs. In addition, it was found that levels of PGE2, MUC2 and MUC5AC in bile samples from the hepatic ducts affected by intrahepatic stones were significantly higher than those from the unaffected hepatic ducts of patients with hepatolithiasis., Conclusions: Our findings indicate that PGE2 induces MUC2 and MUC5AC expression in HIBECs via EP4-p38MAPK signaling.

Primary biliary cirrhosis (PBC) is an autoimmune disease characterized by clinical homogeneity among patients, an overwhelming female predominance, production of a multilineage immune response to mitochondrial autoantigens, inflammation of small bile ducts, and in some patients the development of fibrosis and cirrhosis. The targets in this disease are small bile ducts, and the prototypic serologic response includes antimitochondrial antibodies (AMAs). Several key observations have greatly advanced our understanding of PBC. First, the multilineage immune response, including AMAs, is directed at the E2 component of the 2-oxo-dehydrogenase pathway, particularly PDC-E2. Second, such autoantibodies may be identified years before the clinical diagnosis of disease. Third, the autoreactive T cell precursor frequency for both CD4 and CD8 cells is significantly higher in liver and regional lymph node than in blood, so the multilineage antimitochondrial response may be required for the development of this disease. Fourth, the apotope of biliary cells contains intact PDC-E2; this apotope, in a setting that includes granulocyte macrophage colony-stimulating factor-stimulated macrophages and AMAs, produces an intense proinflammatory response. Fifth, several mouse models of PBC highlight the importance of loss of tolerance to PDC-E2 as well as a critical role for the interleukin (IL)-12 signaling pathway. Finally, genome-wide association studies suggest an important role for the IL-12 pathway in disease susceptibility. Taken together, these findings have resulted in a better understanding of the mechanism for selective biliary cell destruction and have also suggested unique pathways for therapeutic intervention.

CD14 is a glycoprotein that recognizes gram-negative bacterial lipopolysaccharide (LPS) and exists in both membrane-bound and soluble forms. Infectious and/or inflammatory diseases induce CD14 expression, which may be involved in the pathology of endotoxin shock. We previously found that the expression of CD14 protein differs among the endotoxin shock models used, although the reasons for these differences are unclear. We hypothesized that the differences in CD14 expression might be due to liver injury, because the hepatic tissue produces CD14 protein. We investigated CD14 expression in the plasma and liver in the carrageenan (CAR)-primed and D-galN-primed mouse models of endotoxin shock. Our results showed that severe liver injury was not induced in CAR-primed endotoxin shock model mice. In this CAR-primed model, the higher mRNA and protein expression of CD14 was observed in the liver, especially in the interlobular bile duct in contrast to D-galN-primed-endotoxin shock model mice. Our findings indicated that the molecular mechanism(s) underlying septic shock in CAR-primed and D-galN-primed endotoxin shock models are quite different. Because CD14 expression is correlated with clinical observations, the CAR-primed endotoxin shock model might be useful for studying the functions of CD14 during septic shock in vivo.

Biliary innate immunity is involved in the pathogenesis of cholangiopathies in patients with various biliary diseases. Biliary epithelial cells possess an innate immune system consisting of the Toll-like receptor (TLR) family and recognize pathogen-associated molecular patterns (PAMPs). Recently, regulatory mechanisms by intracellular negative regulators including peroxisome proliferator-activated receptor-γ and micro-RNA have been clarified. In primary biliary cirrhosis (PBC) and primary sclerosing cholangitis, dysregulated biliary innate immunity, namely hyper-responsiveness to PAMPs, is associated with the histopathogenesis of cholangiopathy. Moreover, biliary epithelial cells produce monocyte chemotactic protein-1 (MCP-1/CCL2) as a result of the innate immune response and bile ductules play a role in hepatic fibrosis caused by hepatic stellate cells (HSCs). Also, biliary innate immune responses induce the production of two chemokines, fractalkine and macrophage inflammatory protein-3α (MIP-3α), causing the migration of inflammatory cells and a population of antigen-presenting cell found in epithelium, Langerhans cell, and involve chronic cholangitis associated with biliary epithelium-specific innate and acquired immunity in PBC.

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.

Cholangiocytes, or bile duct epithelia, were once thought to be the simple lining of the conduit system comprising the intra- and extrahepatic bile ducts. Growing experimental evidence demonstrated that cholangiocytes are in fact the first line of defense of the biliary system against foreign substances. Experimental advances in recent years have unveiled previously unknown roles of cholangiocytes in both innate and adaptive immune responses. Cholangiocytes can release inflammatory modulators in a regulated fashion. Moreover, they express specialized pattern-recognizing molecules that identify microbial components and activate intracellular signaling cascades leading to a variety of downstream responses. The cytokines secreted by cholangiocytes, in conjunction with the adhesion molecules expressed on their surface, play a role in recruitment, localization, and modulation of immune responses in the liver and biliary tract. Cholangiocyte survival and function is further modulated by cytokines and inflammatory mediators secreted by immune cells and cholangiocytes themselves. Because cholangiocytes act as professional APCs via expression of major histocompatibility complex antigens and secrete antimicrobial peptides in bile, their role in response to biliary infection is critical. Finally, because cholangiocytes release mediators critical to myofibroblastic differentiation of portal fibroblasts and hepatic stellate cells, cholangiocytes may be essential in the pathogenesis of biliary cirrhosis.

Notwithstanding numerous evidences implicating toll-like receptor-4 (TLR4) in the pathogenesis of chronic hepatitis C virus (HCV) infection, the localization and level of TLR4 expression in the liver of patients with hepatitis C have never been investigated. We aimed to evaluate, by means of immunohistochemistry and real-time PCR (rt-PCR), hepatic TLR4 expression in patients with chronic HCV infection. Fifty patients who had undergone liver biopsy and 11 patients transplanted because of chronic HCV infection, and 12 controls free of liver disease, were included in the study. Each case was analyzed by immunohistochemistry for TLR4, α-smooth muscle actin and cytokeratin-7 (CK-7), and a subgroup of patients and all controls by rt-PCR for TLR4. Immunohistochemistry for α-smooth muscle actin was used to derive a score of activation of hepatic stellate cells and portal/septal myofibroblasts, while immunohistochemistry for CK-7 was used to evaluate and count hepatic progenitor cells, interlobular bile ducts and intermediate hepatocytes. In patients, the parenchymal elements responsible for the highest TLR4 level of expression were hepatic progenitor cells and biliary epithelial cells of interlobular bile ducts. Double-labeling experiments between anti-TLR4 and anti-CK7, anti-CD133, anti-CD44, anti-neural cell adhesion molecule, anti-epithelial cell adhesion molecule and anti-sex determining region Y-box 9, confirmed these findings. TLR4-positive hepatic progenitor cells and interlobular bile ducts were significantly correlated with the stage of liver disease (P<0.001), the grade of inflammation (P<0.001), and the activity of portal/septal myofibroblasts (P<0.001). rt-PCR study confirmed an increased TLR4 expression in the 26 patients analyzed with respect to controls (P<0.001). TLR4 expression positively correlated with fibrosis (P<0.05) and inflammation (P<0.05). The present results suggest that TLR4 expression by hepatic progenitor cells and biliary epithelial cells contributes to the progression of liver damage in the course of chronic HCV-related infection.

Galectins are increasingly recognised as important mediators of immune homeostasis and disease regulation, but comparatively little is known about their role in parasite infection. This study investigates the interaction between two ovine galectins, galectin-11 and galectin-14, and the parasitic liver fluke, F. hepatica. Galectin-14 was found in eosinophils infiltrating the tissue surrounding infected bile ducts and secreted in the connective tissue, while galectin-11 was specifically induced in epithelial cells of bile ducts from infected sheep. Strong nuclear staining was observed for galectin-11. Both galectins were found to be secreted into the bile fluid of parasite infected sheep, and were also detected in the excretory/secretory products of adult flukes, following their removal from the ovine host. Recombinant galectin-14, but not recombinant galectin-11, was found to bind specifically to the surface tegument of adult flukes in a carbohydrate dependent manner. This study shows for the first time that both galectin-14 and galectin-11 are produced in liver tissue after chronic liver fluke infection and that they can directly interact with the parasite in the bile ducts. Galectin-11 may also be involved in epithelial cell turnover and cancerogenesis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background & Aims: Loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) in the biliary epithelium reduces bile flow and alkalinization in patients with cystic fibrosis (CF). Liver damage is believed to result from ductal cholestasis, but only 30% of patients with CF develop liver defects, indicating that another factor is involved. We studied the effects of CFTR deficiency on Toll-like receptor 4 (TLR4)-mediated responses of the biliary epithelium to endotoxins., Methods: Dextran sodium sulfate (DSS) was used to induce colitis in C57BL/6J-Cftrtm1Unc (Cftr-KO) mice and their wild-type littermates. Ductular reaction and portal inflammation were quantified by keratin-19 and CD45 immunolabeling. Cholangiocytes isolated from wild-type and Cftr-KO mice were challenged with lipopolysaccharide (LPS); cytokine secretion was quantified. Activation of nuclear factor κB (NF-κB), phosphorylation of TLR4, and activity of Src were determined. HEK-293 that expressed the secreted alkaline phosphatase reporter and human TLR4 were transfected with CFTR complementary DNAs., Results: DSS-induced colitis caused biliary damage and portal inflammation only in Cftr-KO mice. Biliary damage and inflammation were not attenuated by restoring biliary secretion with 24-nor-ursodeoxycholic acid but were significantly reduced by oral neomycin and polymyxin B, indicating a pathogenetic role of gut-derived bacterial products. Cftr-KO cholangiocytes incubated with LPS secreted significantly higher levels of cytokines regulated by TLR4 and NF-κB. LPS-mediated activation of NF-κB was blocked by the TLR4 inhibitor TAK-242. TLR4 phosphorylation by Src was significantly increased in Cftr-KO cholangiocytes. Expression of wild-type CFTR in the HEK293 cells stimulated with LPS reduced activation of NF-κB., Conclusions: CFTR deficiency alters the innate immunity of the biliary epithelium and reduces its tolerance to endotoxin, resulting in an Src-dependent inflammatory response mediated by TLR4 and NF-κB. These findings might be used to develop therapies for CF-associated cholangiopathy., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Objectives: Primary biliary cirrhosis (PBC) is an autoimmune disease, characterized by antimitochondrial antibodies and autoreactive T cells causing destruction of the primary bile ducts. The molecular mechanisms regulating the autoreactive T cells remain elusive. β-Arrestins (βarr) are multifunctional signaling molecules that are crucial to T cell survival. We hypothesized that βarr plays a critical regulatory function in the autoreactive T cells of PBC patients., Methods: Patients with hepatic biliary cirrhosis (n=60) were evaluated. Cytokine expression, T cell proliferation, and transcription factors were evaluated to assess regulatory functions in autoreactive T cells from the patient., Results: Our studies showed that expression of βarr1 was elevated significantly in T lymphocytes from patients with PBC. Moreover, the level of βarr1 mRNA positively correlated with Mayo risk score in PBC patients. Based on modulation of βarr in autoreactive T cell lines, overexpression of βarr1 increased T cell proliferation, augmented interferon production, downregulated activities of nuclear factor κB and AP-1, promoted acetylation of histone H4 in the promoter regions of CD40L, LIGHT, IL-17 and interferon-γ, while downregulating acetylation of histone H4 in the promoter regions of TRAIL, Apo2, and HDAC7A, thereby regulating expression of these genes., Conclusions: Our findings suggest that βarr1 contributes to the pathogenesis of PBC, having significant implications for novel therapy strategy, further providing information for investigating the mechanisms of autoimmune disease.

Both liver epithelial and oval cells are believed to be liver stem cells. We investigated the identification by producing monoclonal antibodies against liver epithelial cells. Monoclonal antibodies against hepatic stem-like cells (HSL cells) have been selected to follow the hepatic stem cells during hepatic regeneration and developmental changes in the liver. Monoclonal antibodies were induced by immunization of BALB/c mice with HSL cells established from the epithelial cells of the adult rat liver. The hybridomas were screened by indirect immunofluorescence staining of HSL cells. We produced a unique monoclonal antibody against HSL cells, MabH, which specifically recognizes liver epithelial cells. MabH did not react with liver parenchymal cells but did react with bile ductule cells under normal conditions in the adult liver. This antibody also reacted with oval cell lines and with the oval cells that appeared during liver regeneration. In addition, fetal liver cells showed immunoreactivity with MabH. Although the level of staining decreased after birth, some cells in the portal area remained highly reactive. These results suggested that liver epithelial cells, oval cells, and fetal liver cells possess a common cell marker of liver stem cells.

Unlabelled: Primary biliary cirrhosis (PBC) is characterized by chronic nonsuppurative destructive cholangitis (CNSDC) associated with destruction of small bile ducts. Although there have been significant advances in the dissection of the adaptive immune response against the mitochondrial autoantigens, there are increasing data that suggest a contribution of innate immune mechanisms in inducing chronic biliary pathology. We have taken advantage of our ability to isolate subpopulations of liver mononuclear cells (LMC) and examined herein the role of Toll-like receptors (TLRs), their ligands, and natural killer (NK) cells in modulating cytotoxic activity against biliary epithelial cells (BECs). In particular, we demonstrate that Toll-like receptor 4 ligand (TLR4-L)-stimulated NK cells destroy autologous BECs in the presence of interferon alpha (IFN-α) synthesized by TLR 3 ligand (TLR3-L)-stimulated monocytes (Mo). Indeed, IFN-α production by hepatic Mo is significantly increased in patients with PBC compared to disease controls. There were also marked increases in the cytotoxic activity of hepatic NK cells from PBC patients compared to NK cells from controls but only when the NK cells were prepared following ligation of both TLR3-L- and TLR4-L-stimulated LMC. These functional data are supported by the immunohistochemical observation of an increased presence of CD56-positive NK cells scattered around destroyed small bile ducts more frequently in liver tissues from PBC patients than controls., Conclusion: These data highlight critical differences in the varied roles of Mo and NK cells following TLR3-L and TLR4-L stimulation., (2011 American Association for the Study of Liver Diseases.)

Background: IgG4-related sclerosing cholangitis (IgG4-SC) needs to be differentiated from primary sclerosing cholangitis (PSC). In this study, we performed a retrospective study to reveal cases in which liver needle biopsy was useful for differential diagnosis., Methods: Nineteen patients with IgG4-SC and 22 patients with PSC were studied. All patients underwent endoscopic retrograde cholangiography and liver needle biopsy. We defined small bile duct involvement of IgG4-SC histologically as damage to the small bile duct associated with infiltration of ≥10 IgG4+ plasma cells per high power field (HPF). Clinicopathological characteristics were compared between IgG4-SC patients with and without small bile duct involvement., Results: Small bile duct involvement was observed in 5 (26%) of the patients with IgG4-SC. Patients with small bile duct involvement showed a higher incidence of intrahepatic biliary strictures on cholangiography (80 vs. 21%, p = 0.038). Conversely, 4 of 7 (57%) patients with intrahepatic biliary strictures on cholangiography had histologically evident small duct involvement. The number of IgG4+ plasma cells was significantly correlated with the site of the most proximal stricture on cholangiograms (p = 0.021). The number of IgG4+ plasma cells per HPF was significantly higher in IgG4-SC patients with intrahepatic biliary strictures than in those with PSC (13.4 vs. 0.4 cells/HPF, p < 0.001)., Conclusions: Involvement of small bile ducts is more frequent in patients with intrahepatic biliary strictures on cholangiography, and liver needle biopsy is especially useful for these patients.

Background/aims: To clarify the primary biliary cirrhosis (PBC)-specific antigen-presenting mechanism, we examined the distribution and phenotypic characteristics of infiltrating dendritic cells (DCs) with respect to bile ducts and the mechanism of migration in terms of the periductal cytokine milieu and biliary innate immunity., Methods and Results: Immunohistochemistry using liver sections from patients with PBC and controls revealed that blood dendritic cell antigen (BDCA)-2(+) plasmacytoid DCs were found mainly in the portal tracts in PBC and the controls, but their distribution was not related to bile ducts. BDCA-1(+) and CD19(-) myeloid DCs were also found in portal tracts in PBC and the controls and, in particular, Langerin+Langerhans cells (LCs) were dominantly scattered around or within biliary epithelial layers of the damaged bile ducts in PBC. Moreover, experiments with cultured human biliary epithelial cells (BECs) showed that an LC-attracting chemokine, macrophage inflammatory protein-3α, was produced by BECs in the response to cytokines [interleukin (IL)-1β, tumour necrosis factor-α and IL-17] and pathogen-associated molecular patterns., Conclusions: LCs existing around or within biliary epithelial layers are important as periductal antigen-presenting cells in PBC and the migration of LCs into bile ducts is closely associated with the periductal cytokine milieu and biliary innate immunity in PBC., (© 2010 John Wiley & Sons A/S.)

We previously described the NOD.c3c4 mouse, which is protected from type 1 diabetes (T1D) because of protective alleles at multiple insulin-dependent diabetes (Idd) genes, but develops autoimmune biliary disease (ABD) resembling primary biliary cirrhosis (PBC). In this paper, we characterize the NOD.ABD strain, which is genetically related to the NOD.c3c4 strain but develops both ABD and T1D. Histologically, NOD.ABD biliary disease is indistinguishable from that in NOD.c3c4 mice. The frequency of effector memory (CD44(+)CD62L(-)) and central memory (CD44(+)CD62L(+)) CD8 T cells is significantly increased in the intrahepatic lymphocyte fraction of NOD.ABD mice, and NOD.ABD CD8 T cells produce more IFN-γ and TNF-α, compared with controls. NOD.ABD splenocytes can transfer ABD and T1D to NOD.c3c4 scid mice, but only T1D to NOD scid mice, suggesting that the genetic origin of the target organ and/or its innate immune cells is critical to disease pathogenesis. The disease transfer model, importantly, shows that biliary duct damage (characteristic of PBC) and inflammation precede biliary epithelial cell proliferation. Unlike T1D where both CD4 and CD8 T cells are required for disease transfer, purified NOD.ABD CD8 T cells can transfer liver inflammation into NOD.c3c4 scid recipients, and disease transfer is ameliorated by cotransferring T regulatory cells. Unlike NOD.c3c4 mice, NOD.ABD mice do not develop anti-nuclear or anti-Smith autoantibodies; however, NOD.ABD mice do develop the antipyruvate dehydrogenase Abs typical of human PBC. The NOD.ABD strain is a model of immune dysregulation affecting two organ systems, most likely by mechanisms that do not completely coincide.

Biliary atresia is the most common cholangiopathy of childhood. With complete obstruction of segments or the entire length of extrahepatic bile ducts, the timely pursuit of hepatoportoenterostomy is the best strategy to restore bile drainage. However, even with prompt surgical intervention, ongoing injury of intrahepatic bile ducts and progressive cholangiopathy lead to end-stage cirrhosis. The pace of disease progression is not uniform; it may relate to clinical forms of disease and/or staging of liver pathology at diagnosis. Although the etiology of disease is not yet defined, several biological processes have been linked to pathogenic mechanisms of bile duct injury. Among them, there is increasing evidence that the immune system targets the duct epithelium and disrupts bile flow. We discuss how careful clinical phenotyping, staging of disease, and basic mechanistic research are providing insights into clinical trial designs and directions for development of new therapies to block progression of disease.

Non-suppurative destructive cholangitis (NSDC) is characterized by T-cell infiltration of the biliary epithelia of small-to medium-caliber bile ducts, causing apoptosis of biliary epithelial cells (BEC) and, ultimately, ductopenia. NSDC is the primary histopathologic process in the autoimmune disease known as primary biliary cirrhosis (PBC) and in alloimmune graft-versus-host disease (GVHD) and hepatic allograft rejection. The onset of NSDC in the B10.D2→BALB/c murine model of hepatic GVHD is preceded by hepatic production of pro-inflammatory cytokines, accumulation of lipopolysaccharide (LPS), and expression of chemokine genes. To explain the curious restriction of NSDC to small- and medium-caliber intrahepatic bile ducts, we hypothesized that BEC lining these bile ducts secrete chemokines and cytokines that chemoattract, activate, and polarize the effector T cells mediating NSDC. To test this hypothesis we stimulated BALB/c immortalized BEC (IBEC) in vitro with pro-inflammatory mouse recombinant cytokines with and without LPS and determined the expression of chemokines and cytokines by IBEC using a polymerase chain reaction (PCR), quantitative protein enzyme-linked immunosorbent assays (ELISAs), and microarrays. The capacity of stimulated IBEC to chemoattract activated T cells was assessed in the presence and absence of inhibitors of specific chemokine receptors. We found that pro-inflammatory cytokines, especially the combination of IFNγ and TNFα, induced IBEC gene expression and the secretion of chemokine ligands for the chemokine receptors CCR1, CCR3, CCR5, and CXCR3. Chemokines secreted by IBEC stimulated with IFNγ plus TNFα chemoattracted activated T cells. Inhibition of CCR1, CCR3, CCR5, or CXCR3 significantly reduced the chemoattraction of activated T cells. We conclude that BEC probably play an active role in the immunopathogenesis of NSDC by mediating the chemoattraction and terminal activation of effector T cells responsible for apoptosis of BECs and ductopenia. Selective chemokine expression by BEC lining small- and medium-caliber bile ducts could explain the restriction of NSDC to ducts of this caliber. Inhibition of CCR1, CCR3, CCR5, and CXCR3 to block the chemoattraction and terminal activation of alloreactive T cells represents a potential therapeutic strategy for preventing NSDC after hematopoietic stem-cell transplantation or orthotopic liver transplantation.

Biliary tract infection with the Group I carcinogenic liver fluke Opisthorchis viverrini is associated with severe inflammation leading to cholangiocarcinoma--a major biliary cancer in Southeast Asia. However, mechanism(s) by which the liver fluke induces host mucosal immune/inflammatory responses is unclear. In the present study we address whether a normal immortalized human cholangiocyte cell line (H69 cells) recognizes and responds to O. viverrini excretory/secretory products (OVES). Expression of multiple TLRs, activation of NF-κB, and expression of pro-inflammatory cytokines were monitored in the presence and absence of OVES. Our results showed that OVES induced increased cholangiocyte TLR4 mRNA expression, induced IκB-α degradation in a MyD88-dependent manner, and activated NF-κB nuclear translocation. Moreover, OVES induced expression and secretion of the strong chemoattractant chemokine interleukin 8 (IL-8) and pro-inflammatory cytokine IL-6. These results demonstrate that secreted/excreted products of O. viverrini are recognized by human cholangiocytes and initiate innate mucosal immunity/inflammatory cascades, a primary event in the pathogenesis of opisthorchiasis and cholangiocarcinoma., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Background/aim: Plasma cells infiltrate in the liver is a prototype lesion of autoimmune liver diseases. The possible role of plasma cells isotyping (IgM and IgG) in the liver in the diagnostic definition of autoimmune liver disease, and particularly in variant syndromes such as autoimmune cholangitis and the primary biliary cirrhosis/autoimmune hepatitis overlap syndrome, is less defined., Methods: We analysed the clinical, serological and histological features of 83 patients with autoimmune liver disease (40 primary biliary cirrhosis, 20 autoimmune hepatitis, 13 primary sclerosing cholangitis, 4 autoimmune cholangitis and 6 overlap syndrome) compared to 34 patients with chronic hepatitis C and evaluated the expression of IgM and IgG plasma cells in their liver by immunostaining., Results: By Spearman's correlation, the mean-counts of IgM plasma cells in portal tracts were significantly correlated with female gender, serum alkaline phosphatase, gamma-glutamyl transferase and IgM values, positivity for anti-mitochondrial antibody-M2 and, on liver biopsy, with bile duct changes, orcein-positive granules and granulomas. Whereas IgG plasma cells resulted more correlated with alanine aminotransferase levels. IgG/IgM ratio lower than 1 was found no only in primary biliary cirrhosis but also in all patients with autoimmune cholangitis. Conversely, all patients with overlap syndrome showed IgG/IgM ratio higher than 1., Conclusion: Immunostaining for IgM and IgG plasma cells on liver tissue can be a valuable parameter for better diagnosis of autoimmune liver disease and also for variant or mixed syndromes., (Copyright 2009 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Objectives: Patients with autoimmune pancreatitis (AIP) characteristically show elevated serum levels of immunoglobulin G4 (IgG4) and abundant infiltration of IgG4-positive plasmacytes in the involved organs. The most common involved organ showing extrapancreatic lesions is the bile duct, which exhibits sclerosing cholangitis (SC). However, the role of IgG4 in the development of IgG4-related SC (IgG4-SC) remains unclear. To clarify the role of IgG4 in IgG4-SC, we have performed an immunohistochemical analysis of the bile duct., Methods: Laboratory and immunohistochemical findings of liver biopsy specimens obtained from patients with IgG4-SC, primary sclerosing cholangitis (PSC), autoimmune hepatitis (AIH), and primary biliary cirrhosis (PBC) were compared. The biopsy specimens were first stained with anti-IgG1, anti-IgG4, and anti-Foxp3 (forkhead box P3) antibodies, and the ratio of IgG4-, IgG1-, and Foxp3-positive cells, respectively, to infiltrated mononuclear cells (IgG4/Mono, IgG1/Mono, Foxp3/Mono) was assessed., Results: The ratio of IgG4/IgG1-positive plasma cells was significantly higher in specimens obtained from patients with IgG4-SC than in those from patients with PSC, AIH, and PBC. The Foxp3/Mono ratio in patients with PBC was significantly higher than that in patients with IgG4-SC and PSC. In patients with IgG4-SC, the number of Foxp3-positive cells was significantly correlated with the number of IgG4-positive cells; in the other patient groups, there was no correlation., Conclusions: The IgG4/IgG1 ratio in the liver may be a useful marker for differential diagnosis of IgG4-SC and PSC. In IgG4-SC, abundant infiltration of regulatory T cells (Tregs) may affect the switching of B cells to IgG4-producing plasmacytes, and there is a possibility that the function of Tregs is different in IgG4-SC and other liver diseases (PSC, AIH, and PBC).

An innate immune response to bacterial and viral components is thought to be involved in the pathogenesis of cholangiopathies in case of primary biliary cirrhosis (PBC) and biliary atresia. Biliary epithelial cells possess the Toll-like receptor (TLR) family which recognizes pathogen-associated molecular patterns (PAMPs) and plays a pivotal role in the innate immune response. In PBC, disordered regulations of TLRs and a negative regulator of intracellular signaling, peroxisome proliferator-activated receptor-gamma (PPARgamma), with Th1-predominant cytokine milieu are involved in the pathogenesis of cholangitis such as chronic non-suppurative destructive cholangitis (CNSDC). Moreover, CD4-positive Th17 cells characterized by the secretion of IL-17, are implicated in the chronic inflammation of bile ducts in PBC and the induction of Th17 cells around bile ducts is causally associated with the biliary innate immune responses to PAMPs. In biliary atresia characterized by a progressive, inflammatory, and sclerosing cholangiopathy, dsRNA viruses could directly induce the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and apoptosis in biliary epithelial cells as a result of the biliary innate immune response via dsRNA-recognizing receptors such as TLR3 and retinoic acid inducible gene I (RIG-I). Moreover, as a mechanism behind the sclerosing cholangiopathy in biliary atresia, epithelial-mesenchymal transition (EMT) has been proposed and the biliary innate immune response to dsRNA viruses is demonstrated to induce biliary epithelial cells to undergo EMT. Biliary innate immunity is associated with the pathogenesis of various cholangiopathies in biliary diseases as well as biliary defense systems.

Biliary atresia is a rare pediatric disorder that results in obstructive jaundice in early infancy. Liver biopsies are characterized histologically by cholestasis, portal inflammatory infiltrate, and marked bile ductular proliferation. We studied the distribution of neutrophils in biliary atresia in patients who have undergone the Kasai procedure. Thirteen different liver biopsies were accompanied by hilar section in eight cases. Control liver specimens were from two patients with choledochal cysts, three patients with neonatal hepatitis, and three patients with alpha-1 antitrypsin deficiency. We also studied the expression of CAP37, an antimicrobial protein present in neutrophils, using immunohistochemistry in biopsy and hilar sections. Neutrophils were present in the portal tracts in all cases and widely distributed in the parenchyma in nine cases. In six cases, infiltration of neutrophils was associated with discrete foci of liver cell necrosis. There was a significantly higher number of portal tract neutrophils in biliary atresia and choledochal cyst cases than in neonatal hepatitis and alpha-1 antitrypsin deficiency (p<0.05). All neutrophils stained diffusely and strongly with CAP37. Medium staining was also seen in the proliferating portal bile ductules in six cases. This study confirms the wide distribution of neutrophils in biliary atresia, occurring as a result of bile duct obstruction. Infiltrating neutrophils are better highlighted with CAP37 immunostain. Proliferating bile ductules may play a role in the inflammatory response in biliary atresia through expression of CAP37., ((c) 2010 Elsevier GmbH. All rights reserved.)

Loss of bile duct epithelium is characteristic of early chronic rejection following liver transplantation. Recent studies have suggested that intrahepatic biliary epithelial cells can transform into myofibroblasts. This study examines the induction and molecular regulation of this transition during allograft rejection. Immortalized human cholangiocytes were stimulated with either transforming growth factor beta1 (TGFbeta1) or a T cell line, and they were examined for morphological, proteomic, and functional features. Posttransplant liver biopsy sections were also examined. Treatment of cholangiocytes with TGFbeta1 or TGFbeta-presenting T cells induced a bipolar morphology, reduced expression of E-cadherin and zona occludens 1 (ZO-1), and increased vimentin, fibronectin, matrix metalloproteinase 2 (MMP-2), MMP-9, and S100 calcium binding protein A4 (S100A4); treated cells invaded a model basement membrane. Chemokines induced T cell penetration of 3-dimensional, cultured bile duct-like structures and bile ducts in liver biopsy sections. A spatial association was observed between duct-infiltrating T cells and cholangiocyte expression of mesenchymal markers, including S100A4. Inhibition of S100A4 expression in vitro blocked TGFbeta1-mediated loss of E-cadherin and ZO-1 but did not reduce induction of fibronectin, MMP-2, or MMP-9. This study demonstrates the potential for T cells to induce an intrahepatic biliary epithelial-to-mesenchymal cell transition during chronic rejection. Furthermore, S100A4 expression by cholangiocytes was identified as a crucial regulator of this transition., (2010 AASLD.)

Unlabelled: The mechanisms mediating hepatic accumulation of inflammatory cells in cholestatic liver disease remain enigmatic. Our thesis is that Hedgehog (Hh) pathway activation promotes hepatic accumulation of immune cells that interact with cholangiocytes. We believe that myofibroblastic hepatic stellate cells (MF-HSCs) release soluble Hh ligands that stimulate cholangiocytes to express chemokines that recruit mononuclear cell types with cognate receptors for these chemokines, thereby orchestrating a repair-related mechanism for liver inflammation. To address this thesis, we used three experimental systems that allow the definition of Hh-dependent mechanisms that induce phenotypic changes in cholangiocytes. First, cholangiocytes were cultured alone or in the presence of Hh-producing MF-HSCs in a transwell coculture system and/or treated with MF-HSC-conditioned medium with or without Hh-neutralizing antibodies. Changes in the cholangiocyte phenotype were then evaluated by microarray analysis, quantitative reverse-transcriptase polymerase chain reaction (QRT-PCR), and/or enzyme-linked immunosorbent assay for chemokine (C-X-C) motif ligand 16 (Cxcl16). Bile duct ligation was chosen to model biliary fibrosis in mice with an overly active Hh pathway, control littermates, and healthy rats, and the gene profile was evaluated by QRT-PCR in whole liver tissue. Second, a transwell chemotaxis assay was used to examine natural killer T (NKT) cell migration in response to cholangiocytes and particularly cholangiocyte-derived Cxcl16. Finally, we studied liver samples from primary biliary cirrhosis patients and controls by QRT-PCR to compare differences in the Hh pathway and Cxcl16. Co-immunostaining of cytokeratin-7 and Cxcl16 was then performed to localize the phenotypic source of Cxcl16. We found that MF-HSCs release soluble Hh ligands that stimulate cholangiocytes to produce Cxcl16 and recruit NKT cells. Hh pathway activation during cholestatic liver injury also induces cholangiocyte expression of Cxcl16., Conclusion: During biliary injury, Hh pathway activation induces cholangiocyte production of chemokines that recruit NKT cells to portal tracts.

Lymphocyte accumulation is characteristic of chronic hepatitis, but the mechanisms regulating lymphocyte numbers and their roles in liver disease progression are poorly understood. The Hedgehog (Hh) pathway regulates thymic development and lymphopoeisis during embryogenesis, and is activated in fibrosing liver disease in adults. Our objective was to determine if Hh ligands regulate the viability and phenotype of NKT cells, which comprise a substantial sub-population of resident lymphocytes in healthy adult livers and often accumulate during liver fibrosis. The results demonstrate that a mouse invariant NKT cell line (DN32 iNKT cells), mouse primary liver iNKT cells, and human peripheral blood iNKT cells are all responsive to sonic hedgehog (Shh). In cultured iNKT cells, Shh enhances proliferation, inhibits apoptosis, induces activation, and stimulates expression of the pro-fibrogenic cytokine, IL-13. Livers of transgenic mice with an overly active Hh pathway harbor increased numbers of iNKT cells. iNKT cells also express Shh. These results demonstrate that iNKT cells produce and respond to Hh ligands, and that Hh pathway activation regulates the size and cytokine production of liver iNKT cell populations. Therefore, Hh pathway activation may contribute to the local expansion of pro-fibrogenic iNKT cell populations during certain types of fibrosing liver damage.

An indirect enzyme-linked immunosorbent assay (iELISA) was evaluated for its diagnostic capability in detecting antibodies against Fasciola gigantica infection in cattle, sheep and donkeys sera using crude worm, excretory-secretory and glutathione S-transferase antigens prepared from adult liver fluke. Presence of F. gigantica worms at post-mortem examination of cattle, sheep and donkey's livers was taken as a gold standard for the evaluation of the assay. The diagnostic sensitivity, specificity and accuracy percentages of iELISA were determined for each antigen. Excretory-secretory antigen gave the best results for the serodiagnosis of F. gigantica infection in cattle, sheep and donkeys using iELISA with diagnostic sensitivity percentages of 93.3%, 94.9% and 93.3%, respectively, while the specificity percentages were 96.7%, 97.2% and 96.3%, respectively, whereas the accuracy percentages were 95%, 96% and 95.7%, respectively. The diagnostic sensitivity percentages of iELISA using crude worm antigen were 96.7%, 100% and 93.3%, respectively, while the specificity percentages were 80%, 83.3% and 85.2%, respectively, whereas the accuracy percentages were 88.3%, 86.7% and 87%, respectively. The diagnostic sensitivity percentages of iELISA using glutathione S-transferase antigen were 66.7%, 71.8% and 60%, respectively, while the specificity percentages were 70%, 77.8% and 77.8%, respectively, whereas the accuracy percentages were 68.3%, 74.7% and 73.9%, respectively. Conclusively, excretory-secretory antigen dependent iELISA can be used as a reliable serodiagnostic test for F. gigantica infection in cattle, sheep and donkeys.

Biliary cryptosporidiosis is associated with acquired immunodeficiency syndrome (AIDS) cholangiopathy and occurs almost exclusively in adult patients with AIDS. Infection of biliary epithelial cells (cholangiocytes) with Cryptosporidium parvum induces Toll-like receptor (TLR) 4 expression and stimulates a TLR-dependent response against infection. Here, we tested whether human immunodeficiency virus type 1 (HIV-1) Tat affects TLR expression and, hence, anti-C. parvum defense responses. Using an in vitro model of human biliary cryptosporidiosis, we found that recombinant Tat protein increased TLR4 mRNA expression in both uninfected and C. parvum-infected cholangiocytes. Conversely, Tat decreased TLR4 protein levels and suppressed C. parvum-induced TLR4 protein expression. Using actinomycin to inhibit transcription, we found that Tat increased the half-life of TLR4 mRNA from approximately 25 to 60 min, and RNA gel-shift assays demonstrated direct binding of Tat to TLR4 mRNA. In vitro transcription/translation studies suggested that Tat does not affect transcription but does decrease TLR4 translation. Importantly, more parasites were found in Tat-treated cells than in control cells 48 h after infection. These findings suggest that Tat inhibits cholangiocyte TLR4 protein expression through translational inhibition. These events appear to diminish the ability of cholangiocytes to initiate an innate immune response to C. parvum. We suggest that these findings may contribute to the unusual susceptibility of HIV-infected individuals to biliary cryptosporidiosis.

A complex interplay of liver, bile ducts, and the gut critically determines functioning of these organs in health and disease. Biochemical and physiological aspects of their interaction and the role of inflammatory cells, cytokines, bile acids, visceral hormones, neuropeptides and other messengers in the pathogenesis of model immune-mediated hepatobiliary disorders are discussed in the actual issue of Clinical Reviews in Allergy and Immunology. Potential molecular targets for future therapeutic approaches are also addressed.

Biliary epithelial cells (cholangiocytes) respond to proinflammatory cytokines such as IFN-gamma and actively participate in the regulation of biliary inflammatory response in the liver. B7-H1 (also known as CD274 or PD-L1) is a member of the B7 costimulatory molecules and plays a critical immunoregulatory role in cell-mediated immune responses. In this study, we show that resting human cholangiocytes in culture express B7-H1 mRNA, but not B7-H1 protein. IFN-gamma induces B7-H1 protein expression and alters the microRNA (miRNA) expression profile in cholangiocytes. Of those IFN-gamma-down-regulated miRNAs, we identified microRNA-513 (miR-513) with complementarity to the 3'-untranslated region of B7-H1 mRNA. Targeting of the B7-H1 3'-untranslated region by miR-513 results in translational repression. Transfection of cholangiocytes with an antisense oligonucleotide to miR-513 induces B7-H1 protein expression. Additionally, transfection of miR-513 precursor decreases IFN-gamma-induced B7-H1 protein expression and consequently influences B7-H1-associated apoptotic cell death in cocultured Jurkat cells. Thus, miR-513 regulates B7-H1 translation and is involved in IFN-gamma-induced B7-H1 expression in human cholangiocytes, suggesting a role for miRNA-mediated gene silencing in the regulation of cholangiocyte response to IFN-gamma.

Cholangiocytes, the epithelial cells lining bile ducts, provide the first line of defense against lumenal microbes in the biliary system. Recent advances in biliary immunity indicate that cholangiocytes express a variety of pathogen-recognition receptors and can activate a set of intracellular signaling cascades to initiate a profound antimicrobial defense, including release of proinflammatory cytokines and chemokines, production of antimicrobial peptides and maintenance of biliary epithelial integrity. Cholangiocytes also interact with other cell types in the liver (for example, lymphocytes and Kupffer cells) through expression and release of adhesion molecules and immune mediators. Subsequently, through an intricate feedback mechanism involving both epithelial and other liver cells, a set of intracellular signaling pathways are activated to regulate the functional state of cholangiocyte responses during microbial infection. Thus, cholangiocytes are actively involved in mucosal immunity of the biliary system and represent a fine-tuned, integral component of liver immunity.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease of unknown etiology but lymphocytic portal tract infiltration is suggestive of an immune-mediated basis for this disease. Associations with inflammatory bowel disease (IBD) especially ulcerative colitis (UC), and with particular autoimmune diseases, as well as the genetic associations further suggest PSC may be an immune-mediated disease. The immunogenetics of PSC have been the subject of active research and several HLA and non-HLA associated genes have been implicated in the development of the disease. Lymphocytes derived from the inflamed gut may enter the liver via the enterohepatic circulation to cause hepatic disease. PSC may be triggered in genetically susceptible individuals by infections or toxins entering the portal circulation through a permeable colon and hence evoking an abnormal immune response.

Background & Aims: Immunoglobulin (Ig)G4-associated cholangitis (IAC) is the biliary manifestation of a steroid-responsive multisystem fibroinflammatory disorder in which affected organs have a characteristic lymphoplasmacytic infiltrate rich in IgG4-positive cells. We describe clinical features, treatment response, and predictors of relapse in IAC and compare relapse rates in IAC with intrapancreatic vs proximal bile duct strictures., Methods: We reviewed clinical, serologic, and imaging characteristics and treatment response in 53 IAC patients., Results: IAC patients generally were older (mean age, 62 y) men (85%), presenting with obstructive jaundice (77%) associated with autoimmune pancreatitis (92%), increased serum IgG4 levels (74%), and abundant IgG4-positive cells in bile duct biopsy specimens (88%). At presentation, biliary strictures were confined to the intrapancreatic bile duct in 51%; the proximal extrahepatic/intrahepatic ducts were involved in 49%. Initial presentation was treated with steroids (n = 30; median follow-up period, 29.5 months), surgical resection (n = 18; median follow-up period, 58 months), or was conservative (n = 5; median follow-up period, 35 months). Relapses occurred in 53% after steroid withdrawal; 44% relapsed after surgery and were treated with steroids. The presence of proximal extrahepatic/intrahepatic strictures was predictive of relapse. Steroid therapy normalized liver enzyme levels in 61%; biliary stents could be removed in 17 of 18 patients. Fifteen patients treated with steroids for relapse after steroid withdrawal responded; 7 patients on additional immunomodulatory drugs remain in steroid-free remission (median follow-up period, 6 months)., Conclusions: IAC should be suspected in unexplained biliary strictures associated with increased serum IgG4 and unexplained pancreatic disease. Relapses are common after steroid withdrawal, especially with proximal strictures. The role of immunomodulatory drugs for relapses needs further study.

The polycystic kidney (PCK) rat is an animal model of Caroli's disease with congenital hepatic fibrosis, in which the mechanism of progressive hepatic fibrosis remains unknown. This study aimed to clarify the mechanism of hepatic fibrosis of the PCK rat from the viewpoint of the contribution of pathological cholangiocytes. In liver sections of the PCK rats, intrahepatic bile ducts were constituted by two different phenotypes: bile ducts lined by cuboidal-shaped and flat-shaped cholangiocytes. The flat-shaped cholangiocytes showed reduced immunohistochemical expression of the biliary epithelial marker cytokeratin 19 and positive immunoreactivity for vimentin and fibronectin. When cultured cholangiocytes of the PCK rat were treated with transforming growth factor (TGF)-beta1, a potent inducer of epithelial-mesenchymal transition, induction of vimentin, fibronectin, and collagen expression occurred in the PCK cholangiocytes. Although the TGF-beta1 treatment reduced cytokeratin 19 expression, the epithelial cell features characterized by the expression of E-cadherin and zonula occludens-1 was maintained, and alpha-smooth muscle actin expression was not induced in the cholangiocytes. Cholangiocytes of the PCK rat may acquire mesenchymal features in response to TGF-beta1 and participate in progressive hepatic fibrosis by producing extracellular matrix molecules, which seems to be a different event from epithelial-mesenchymal transition.

Unlabelled: Infections of Reoviridae consisting of a double-stranded RNA (dsRNA) genome are a possible cause of biliary atresia (BA). The aim of the present study is to clarify the pathophysiological function of dsRNA viruses in the pathogenesis of BA. The expression of dsRNA pattern-recognizing receptors, Toll-like receptor 3 (TLR3), retinoic acid inducible gene I (RIG-I), melanoma differentiation-associated gene-5 (MDA-5), and dsRNA-activated protein kinase R (PKR) was constitutively detected in cultured human biliary epithelial cells (BECs). Stimulation with polyinosinic-polycytidylic acid [poly(I:C), a synthetic analog of viral dsRNA] induced the activation of transcription factors [nuclear factor (NF)-kappaB and interferon regulatory factor 3 (IRF3)] and the production of interferon-beta1 (IFN-beta1) and MxA as potent antiviral responses. Moreover, poly(I:C) up-regulated the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and both poly(I:C) and TRAIL reduced the viability of cultured human BECs by enhancing apoptosis. Experiments in vivo using tissue sections of extrahepatic bile ducts from patients with BA and controls (choledochal cysts and nonbiliary diseases) showed that the activation of NF-kappaB, interferon regulatory factor-3 (IRF-3), and PKR, and the enhancement of TRAIL and single-stranded DNA (ssDNA)-positive apoptosis were significant in BA, although extrahepatic bile ducts diffusely and constantly expressed TLR3 in all diseases., Conclusion: dsRNA viruses could directly induce the expression of TRAIL and apoptosis in human biliary epithelial cells as a result of the biliary innate immune response, supporting the notion that Reoviridae infections are directly associated with the pathogenesis of cholangiopathies in cases of BA.

Toll-like receptors (TLRs) are important pathogen recognition molecules and are key to epithelial immune responses to microbial infection. However, the molecular mechanisms that regulate TLR expression in epithelia are obscure. Micro-RNAs play important roles in a wide range of biological events through post-transcriptional suppression of target mRNAs. Here we report that human biliary epithelial cells (cholangiocytes) express let-7 family members, micro-RNAs with complementarity to TLR4 mRNA. We found that let-7 regulates TLR4 expression via post-transcriptional suppression in cultured human cholangiocytes. Infection of cultured human cholangiocytes with Cryptosporidium parvum, a parasite that causes intestinal and biliary disease, results in decreased expression of primary let-7i and mature let-7 in a MyD88/NF-kappaB-dependent manner. The decreased let-7 expression is associated with C. parvum-induced up-regulation of TLR4 in infected cells. Moreover, experimentally induced suppression or forced expression of let-7i causes reciprocal alterations in C. parvum-induced TLR4 protein expression, and consequently, infection dynamics of C. parvum in vitro. These results indicate that let-7i regulates TLR4 expression in cholangiocytes and contributes to epithelial immune responses against C. parvum infection. Furthermore, the data raise the possibility that micro-RNA-mediated post-transcriptional pathways may be critical to host-cell regulatory responses to microbial infection in general.

Background: The aim of this study was to determine the existence and extent of maternal microchimerism in the livers of biliary atresia (BA) patients., Methods: Two series of investigations were performed based on the sex of our subjects. Subjects for series I were men, of which 6 had BA. Livers were analyzed using X and Y chromosome probes and fluorescent in situ hybridization. Subjects for series II were woman. Nine BA cases and their mothers were HLA typed (class I). Daughter livers were also tested for antibodies to maternal and other HLA. Two cases of neonatal hepatitis, 2 cases of Alagille syndrome, and 1 case of Byler syndrome acted as controls., Results: All male BA livers were found to contain a mixture of cells with 1 and 2 X chromosomes (ie, XY or XX). All livers from male controls had only 1 X chromosome (ie, XY). All female BA subjects had varying intensities of antimaternal HLA class I (HLA-A) antibodies in their bile duct epithelium and hepatocytes (strong, 5; mild, 3; weak, 1). The liver from the female control did not display any antimaternal HLA class I antibodies (HLA-Ab)., Conclusion: Our preliminary data appear to show that maternal microchimerism is present within the livers of patients with progressive postnatal type BA. We suggest that BA could in fact be a graft-vs-host disease masquerading as an autoimmune reaction triggered by maternal microchimerism, and we intend to pursue this hypothesis further to clarify the etiology of BA.

Biliary atresia is an inflammatory fibrosclerosing lesion of the bile ducts that leads to biliary cirrhosis and is the most frequent indication for liver transplantation in children. The pathogenesis of biliary atresia is not known; one theory is that of a virus-induced, subsequent autoimmune-mediated injury of bile ducts. The aim of this study was to determine whether autoreactive T cells and autoantibodies specific to bile duct epithelia are present in the rotavirus (RRV)- induced murine model of biliary atresia and whether the T cells are sufficient to result in bile duct inflammation. In vitro analyses showed significant increases in IFN-gamma-producing T cells from RRV-diseased mice in response to bile duct epithelial autoantigen. Adoptive transfer of the T cells from RRV-diseased mice into naïve syngeneic SCID recipients resulted in bile duct-specific inflammation. This induction of bile duct pathology occurred in the absence of detectable virus, indicating a definite response to bile duct autoantigens. Furthermore, periductal immunoglobulin deposits and serum antibodies reactive to bile duct epithelial protein were detected in RRV-diseased mice. In conclusion, both cellular and humoral components of autoimmunity exist in murine biliary atresia, and the progressive bile duct injury is due in part to a bile duct epithelia-specific T cell-mediated immune response. The role of cellular and humoral autoimmunity in human biliary atresia and possible interventional strategies therefore should be the focus of future research.

Primary biliary cirrhosis (PBC) is an autoimmune disease of the liver, characterized by lymphocytic infiltrates in portal tracts, selective destruction of biliary epithelial cells, and anti-mitochondrial Abs (AMAs). The elucidation of early events in the induction of tissue inflammation and autoimmunity in PBC has been hampered by the cryptic onset of the disease, the practical limitations in accessing the target tissue, and the lack of a suitable animal model. We demonstrate in this study that a mouse transgenic for directed expression of a dominant-negative form of TGF-beta receptor type II (dnTGFbetaRII), under the direction of the CD4 promoter, mimics several key phenotypic features of human PBC, including spontaneous production of AMAs directed to the same mitochondrial autoantigens, namely PDC-E2, BCOADC-E2, and OGDC-E2. The murine AMAs also inhibit PDC-E2 activity. Moreover, there is lymphocytic liver infiltration with periportal inflammation analogous to the histological profile in human PBC. Additionally, the serum cytokine profile of affected mice mimics data in human PBC. The concomitant presence of these immunopathological features in the transgenic mice suggests that the TGF-betaRII pathway is implicated in the pathogenesis of PBC. Finally, these data point away from initiation of autoimmunity by mechanisms such as molecular mimicry and more toward activation of an intrinsically self-reactive T cell repertoire in which necessary regulatory T cell influences are lacking.

Mucosal tissues require constant immune surveillance to clear harmful pathogens while maintaining tolerance to self Ags. Regulatory T cells (Tregs) play a central role in this process and expression of alpha(E)beta(7) has been reported to define a subset of Tregs with tropism for inflamed tissues. However, the signals responsible for recruiting Tregs to epithelial surfaces are poorly understood. We have isolated a subset of CCR10-expressing CD25+CD4+Foxp3+ Tregs with potent anti-inflammatory properties from chronically inflamed human liver. The CCR10+ Tregs were detected around bile ducts that expressed increased levels of the CCR10 ligand CCL28. CCL28 was secreted by primary human cholangiocytes in vitro in response to LPS, IL-1beta, or bile acids. Exposure of CCR10+ Tregs to CCL28 in vitro stimulated migration and adhesion to mucosal addressin cell adhesion molecule-1 and VCAM-1. Liver-derived CCR10+ Tregs expressed low levels of CCR7 but high levels of CXCR3, a chemokine receptor associated with infiltration into inflamed tissue and contained a subset of alpha(E)beta7(+) cells. We propose that CXCR3 promotes the recruitment of Tregs to inflamed tissues and CCR10 allows them to respond to CCL28 secreted by epithelial cells resulting in the accumulation of CCR10+ Tregs at mucosal surfaces.

Background: Lymphocyte-mediated inflammatory damage of the bile ducts has been proposed as a potential mechanism in the pathogenesis of biliary atresia (BA). Chemokines regulate leukocyte migration and act as critical organizers of cell distribution in inflammatory responses. The aim of this study was to analyze the infiltration of T lymphocytes and the expression of a chemokine receptor, CXCR3, predominantly expressed on type 1 polarized T cells (T(H)1, T(C)1) in the liver and excised biliary remnants in infants with BA., Methods: Immunohistochemistry for CD3, CD8, and CXCR3 was performed using liver biopsy specimens collected from the following 3 age-matched groups of patients: group 1, BA (nonsyndromic) at the time of Kasai portoenterostomy (n = 10); group 2, congenital choledochal dilatation (n = 2); and group 3, other cholestatic diseases including paucity of intrahepatic bile ducts and cholestasis (n = 3) related to total parenteral nutrition. Cellular staining on each section was graded from 0 to 4 and compared using nonparametric statistics., Results: Infiltrating CD3+ and CD8+ lymphocytes in the portal tracts were significantly increased in group 1 (3.1 +/- 0.4, 2.8 +/- 0.4), compared with groups 2 (1.0 +/- 0.0, 1.0 +/- 0.0) and 3 (1.7 +/- 0.3, 1.5 +/- 0.5) (P < .01, P < .05). CXCR3+ mononuclear cells were significantly increased in group 1 (2.6 +/- 0.3) compared with groups 2 (0.5 +/- 0.5) and 3 (0.7 +/- 0.3) (P < .05). They were mainly found in the portal tracts with a similar distribution to CD3+ cells. CXCR3+ cells and CD3+ cells also showed a similar distribution in specimens of biliary remnants from just below the portal plate., Conclusions: Increased expression of CXCR3 associated with a significantly increased CD3 and CD8 T-cell infiltration suggests that CXCR3+ lymphocytes in a type 1 (T(H)1, T(C)1) cytokine milieu may play a role in the pathogenesis of BA.

Infection of epithelial cells by Cryptosporidium parvum triggers a variety of host-cell innate and adaptive immune responses including release of cytokines/chemokines and up-regulation of antimicrobial peptides. The mechanisms that trigger these host-cell responses are unclear. Thus, we evaluated the role of TLRs in host-cell responses during C. parvum infection of cultured human biliary epithelia (i.e., cholangiocytes). We found that normal human cholangiocytes express all known TLRs. C. parvum infection of cultured cholangiocytes induces the selective recruitment of TLR2 and TLR4 to the infection sites. Activation of several downstream effectors of TLRs including IL-1R-associated kinase, p-38, and NF-kappaB was detected in infected cells. Transfection of cholangiocytes with dominant-negative mutants of TLR2 and TLR4, as well as the adaptor molecule myeloid differentiation protein 88 (MyD88), inhibited C. parvum-induced activation of IL-1R-associated kinase, p-38, and NF-kappaB. Short-interfering RNA to TLR2, TLR4, and MyD88 also blocked C. parvum-induced NF-kappaB activation. Moreover, C. parvum selectively up-regulated human beta-defensin-2 in directly infected cells, and inhibition of TLR2 and TLR4 signals or NF-kappaB activation were each associated with a reduction of C. parvum-induced human beta-defensin-2 expression. A significantly higher number of parasites were detected in cells transfected with a MyD88 dominant-negative mutant than in the control cells at 48-96 h after initial exposure to parasites, suggesting MyD88-deficient cells were more susceptible to infection. These findings demonstrate that cholangiocytes express a variety of TLRs, and suggest that TLR2 and TLR4 mediate cholangiocyte defense responses to C. parvum via activation of NF-kappaB.

Studies to determine the role of preformed antibodies to biliary epithelial cells (BECs) in liver transplant rejections have been initiated. However, the clinical importance of these antibodies in the posttransplantation period still remains to be elucidated. Reactivity to BECs isolated from a normal healthy liver was investigated in sera of 56 patients before and after liver transplantation (LTX) using flow cytometry. Functional capacity of BEC antibodies was determined by the ability to induce expression of Toll-like receptors (TLRs) on BECs. Cytokine and chemokine production induced by BEC antibodies was determined by enzyme-linked immunosorbent assay. In all, 7 patients (13%) had BEC antibodies only pre-LTX, 14 (25%) only after LTX, 18 (32%) both before and after LTX, and 17 (30%) had no detectable antibodies. Presence of preformed BEC antibodies correlated with acute rejections (P < 0.03). Deposition of immunoglobulins in bile ducts was detected in biopsies of patients during rejections. Significantly higher numbers of patients with post-LTX antibodies (9 of 32) developed cholangitis, compared with 0 of 17 without antibodies (P < 0.02). Specificity studies indicated that these antibodies were both non-HLA- and HLA-specific. Normal BECs expressed mRNA but not the proteins for the TLRs. However, treatment with F(ab')2 fragments of BEC antibodies induced protein expression of TLRs 2 and 3 and significantly high production of interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha, epithelial neutrophil activating peptide (ENA)-78, and IL-8. In conclusion, BEC antibodies via induction of TLR2 and TLR3 expression, as well as inflammatory cytokine and chemokine production may induce epithelial cell inflammatory responses to bacterial components and contribute to posttransplantation cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease of unknown etiology;however, lymphocytic portal tract infiltration is suggestive of an immune-mediated basis for PSC. Associations with inflammatory bowel disease--especially ulcerative colitis--and with other auto-immune diseases, together with genetic associations, further suggest that PSC may be an immune-mediated disease. The immunogenetics of PSC have been the subject of active research, and several human leukocyte antigen (HLA)- and non-HLA-associated genes have been implicated in the development of the disease. Lymphocytes derived from the inflamed gut may enter the liver via the enterohepatic circulation to cause hepatic disease.PSC may be triggered in genetically susceptible individuals by infections or toxins entering the portal circulation through a permeable colon and, therefore, evoking an abnormal immune response.

Unlabelled: Many diseases are associated with cytokine release after inflammatory infiltration, which perturbs organ function. Radioligands capable of noninvasive tracking to assess the integrity of specific biochemical pathways offer potent ways to establish such perturbing mechanisms., Methods: To demonstrate regulation of hepatobiliary transport in disease, we used (99m)Tc-mebrofenin in a carbon tetrachloride-induced liver injury model in Fischer 344 rats. Healthy rats served as control animals. Image analysis was used to determine (99m)Tc-mebrofenin handling. Liver tests and histologic analysis were used for grading liver injury and hepatic fibrosis. To address the role of inflammatory cytokines, we used in vitro assays with (99m)Tc-mebrofenin-loaded primary rat hepatocytes., Results: In healthy rats, (99m)Tc-mebrofenin was promptly excreted, and after 1 h only 20% +/- 5% (mean +/- SD) of peak (99m)Tc-mebrofenin activity remained in the liver. In contrast, rats treated with carbon tetrachloride for 1 or 3 mo showed 84% +/- 5% and 80% +/- 7% (mean +/- SD), respectively, of peak (99m)Tc-mebrofenin activity in the liver after 1 h (P < 0.001). Abnormal (99m)Tc-mebrofenin transport was associated with necroinflammatory activity and not hepatic fibrosis. This was examined directly in animals, where withdrawal of carbon tetrachloride for 2 wk after significant liver injury produced loss of inflammatory activity without affecting hepatic fibrosis. In this situation, (99m)Tc-mebrofenin transport returned to normal, indicating a central role of inflammatory activity in this process. In vitro assays showed impairment in (99m)Tc-mebrofenin excretion after incubation of cultured hepatocytes with interleukin-6 and further impairment with interleukin-6 plus tumor necrosis factor-alpha., Conclusion: The findings indicate that inflammatory cytokines regulate (99m)Tc-mebrofenin transport. This cytokine-mediated process establishes a paradigm for identifying and monitoring organ inflammation, including in viral or alcoholic hepatitis, fatty liver disease, allograft rejection, and responses to gene therapy vectors.

Background & Aims: Previous work has suggested that CD4+ CD28- or costimulation-independent T cells are increased in autoimmune diseases. In this study, we compared frequency and qualitative characteristics of autoreactive costimulation-independent or CD4+ CD28- T cells in primary biliary cirrhosis (PBC) by taking advantage of the well-defined immunodominant autoepitope of the E2 component of pyruvate dehydrogenase (PDC-E2)., Methods: We determined the frequency of costimulation-independent autoreactive T cells that respond to PDC-E2 163-176 and the frequency of CD4+ CD28- T cells. Finally, we determined the role of biliary epithelial cells (BEC) as both an antigen-presenting cell or, alternatively, as a target cell for T-cell-mediated cytotoxicity., Results: The precursor frequency of costimulation-independent CD4+ T cells that respond to PDC-E2 163-176 and the frequency of CD4+ CD28- T cells were dramatically elevated in PBC. Furthermore, 2 types of T-cell clones that respond to PDC-E2 163-176 emerged from this study. One type was costimulation dependent and the other costimulation independent. Both types of clones lyse BEC in a similar effector target (E/T) ratio distribution. However, BEC did not help the proliferation of any T-cell clones. Furthermore, costimulation-independent T-cell clones do not become anergic by BEC., Conclusions: In PBC, costimulation-independent autoreactive T cells, which do not become anergic, increase and maintain the autoimmune response. In controls, although autoantigens are expressed on BEC and autoantigen-reactive T cells exist around BEC, autoantigen-reactive T cells are costimulation dependent and will become anergic and maintain peripheral tolerance.

We investigated the immune responses of patients with cholestatic and hepatitis C virus-positive (HCV-positive) liver cirrhosis by analysing T-cell subsets and cytokine levels in the portal and peripheral veins, using flow cytometry and enzyme-linked immunosorbent assay. In cholestatic liver cirrhosis, the proportion of natural-killer (NK) T cells and interleukin (IL) 6 and IL-18 levels in the portal venous blood were significantly higher than those in the peripheral venous blood. In HCV-positive liver cirrhosis, the proportions of NK T cells and Fas+ T cells and IL-6 and soluble Fas levels in the portal venous blood were significantly higher than those in the peripheral venous blood. These results suggest that in these diseases, activated T cells and soluble molecules in portal venous blood may promote Fas/FasL-mediated apoptosis of the bile-duct cells and hepatocytes, and contribute to the deterioration in liver function as an inevitable result of positive feedback.

Background: Autoimmune pancreatitis is a unique clinical entity proposed recently, and is sometimes associated with inflammation of other organs., Aims: To examine the pathophysiology of the pancreas and other organs in patients with autoimmune pancreatitis., Patients and Methods: We evaluated clinicopathological findings in six resected and one autopsied patient with autoimmune pancreatitis. The pancreas, peripancreatic tissue, bile duct, and gall bladder were examined histologically and immunohistochemically. Biopsied salivary gland and cervical lymph node of one patient were also examined. We also performed similar immunohistochemical examinations in pancreatectomy specimens from 10 patients with alcoholic chronic pancreatitis and biopsied salivary glands from five patients with Sjögren's syndrome., Results: Stenosis of the extrahepatic bile duct was detected in all patients. Histological findings were characterised by diffuse lymphoplasmacytic infiltration with marked interstitial fibrosis and acinar atrophy, obliterated phlebitis of the pancreatic veins, and involvement of the portal vein. Immunohistochemically, diffusely infiltrating cells consisted predominantly of CD4 or CD8 positive T lymphocytes and IgG4 positive plasma cells. Similar inflammatory processes also involved the peripancreatic tissue, extrahepatic bile duct, gall bladder, and salivary gland. Lymph nodes were swollen with infiltration of IgG4 positive plasma cells. None of these findings was seen in alcoholic chronic pancreatitis or Sjögren's syndrome., Conclusions: The development of the specific inflammations in extensive organs as well as the pancreas in patients with autoimmune pancreatitis strongly suggests a close relationship between autoimmune pancreatitis and multifocal fibrosclerosis.