Kumiko Isse

Lipopolysaccharide activates nuclear factor-kappaB through toll-like receptors and related molecules in cultured biliary epithelial cells

To clarify the innate immunity of the intrahepatic biliary tree, we examined expression of Toll-like receptors and intracellular signalings in biliary epithelial cells in response to bacterial components by using cultured biliary epithelial cells (murine biliary cells and human cholangiocarcinoma cell lines). The expression of Toll-like receptors in cultured cells was examined by reverse transcription and PCR and immunohistochemistry. Intracellular signalings after Toll-like receptors activation by lipopolysaccharide was examined by analysis of nuclear factor (NF)-kappaB activation and inhibition studies using inhibitors for NF-kappaB and mitogen-activated protein kinase and blocking antibody. The mRNAs of Toll-like receptors 2, 3, 4, and 5, and related molecules (MD-2, MyD88, and CD14) were detected, and their proteins were expressed in cultured cells. Lipopolysaccharide was shown to bind to the cell surface of cultured cells. Lipopolysaccharide treatment induced the production of TNF-alpha, and nuclear translocation of NF-kappaB and increased NF-kappaB-DNA binding in cultured cells. This induction of TNF-alpha was partially inhibited by anti–Toll-like receptor 4 antibody. The nuclear translocation and increased binding of NF-kappaB by lipopolysaccharide were blocked by addition of MG132, an inhibitor of NF-kappaB. In conclusion, lipopolysaccharide appears to form a receptor complex of CD14, Toll-like receptor 4, MD-2, and MyD88 in cultured biliary epithelial cells and seems to regulate activation of NF-kappaB and synthesis of TNF-alpha. The recognition of pathogen-associated molecular patterns using Toll-like receptors and related molecules in biliary epithelial cells, which is demonstrated in this in vitro study, may participate in an immunopathology of the intrahepatic biliary tree in vivo.

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.

Fractalkine and CX3CR1 are involved in the recruitment of intraepithelial lymphocytes of intrahepatic bile ducts

Fractalkine is a chemokine with both chemoattractant and cell‐adhesive functions, and in the intestine it is involved with its receptor CX3CR1 in the chemoattraction and recruitment of intraepithelial lymphocytes. We examined the pathophysiological roles of fractalkine and CX3CR1 in normal and diseased bile ducts. Expression of fractalkine and CX3CR1 were examined in liver tissues from patients with primary biliary cirrhosis (17 cases) and controls (9 cases of primary sclerosing cholangitis, 10 cases of extrahepatic biliary obstruction, 20 cases of chronic viral hepatitis C, and 18 cases of histologically normal livers). Expression of fractalkine in biliary epithelial cells (BECs) in response to cytokine treatments was examined using a human cholangiocarcinoma cell line (HuCC‐T1) and human intrahepatic BEC line. The chemotaxis of CX3CR1‐expressing monocytes (THP‐1) toward fractalkine was assayed using chemotaxis chambers. Fractalkine messenger RNA/protein were expressed on BECs of normal and diseased bile ducts, and their expression was upregulated in injured bile ducts of primary biliary cirrhosis. CX3CR1 was expressed on infiltrating mononuclear cells in portal tracts and on CD3+, CD4+, and CD8+ intraepithelial lymphocytes of injured bile ducts in primary biliary cirrhosis. Fractalkine messenger RNA expression was upregulated in two cultured BECs on treatment with lipopolysaccharide and Th1‐cytokines (interleukin 1β, interferon gamma, and tumor necrosis factor α). THP‐1 cells showed chemotaxis toward fractalkine secreted by cultured cells. In conclusion, Th1‐cytokine predominance and lipopolysaccharide in the microenvironment of injured bile ducts resulting from primary biliary cirrhosis induce the upregulation of fractalkine expression in BECs, followed by the chemoattraction of CX3CR1‐expressing mononuclear cells, including CD4+ and CD8+ T cells, and their adhesion to BECs and the accumulation of biliary intraepithelial lymphocytes. (HEPATOLOGY 2005;41:506–516.)

Cyclooxygenase-2–Derived Prostaglandin E2 Activates β-Catenin in Human Cholangiocarcinoma Cells: Evidence for Inhibition of These Signaling Pathways by ω3 Polyunsaturated Fatty Acids

Cholangiocarcinoma is a highly malignant neoplasm of the biliary tree. It has a high rate of mortality, and currently, there is no effective chemoprevention and treatment. This study was designed to investigate the potential effect of omega 3 polyunsaturated fatty acids (omega 3-PUFA) on human cholangiocarcinoma cell growth and to determine their mechanisms of actions. Treatment of three human cholangiocarcinoma cells (CCLP1, HuCCT1, SG231) with two omega 3-PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), for 12 to 72 h resulted in a dose- and time-dependent inhibition of cell growth; in contrast, arachidonic acid, a omega 6-PUFA, had no significant effect. The omega 3-PUFA effect is due to the induction of apoptosis, given that DHA induced the cleaved form of PARP, caspase-3, and caspase-9. DHA and EPA treatment caused dephosphorylation (and hence, the activation) of glycogen synthase kinase-3beta (GSK-3beta) with a decline of beta-catenin protein. Accordingly, DHA treatment also decreased the beta-catenin-mediated T cell factor/lymphoid enhancer factor (TCF/LEF) reporter activity, and inhibited the expression of c-Met, a beta-catenin-controlled downstream gene implicated in cholangiocarcinogenesis. The GSK-3beta inhibitor, SB216763, partially prevented DHA-induced reduction of beta-catenin protein and TCF/LEF reporter activity, and restored cell growth, suggesting the involvement of GSK-3beta dephosphorylation in omega 3-PUFA-induced beta-catenin degradation. In parallel, DHA treatment also induced the formation of the beta-catenin/Axin/GSK-3beta binding complex, further leading to beta-catenin degradation. Moreover, DHA inhibited the expression of cyclooxygenase-2 (COX-2) and enhanced the expression of 15-hydroxyprostaglandin dehydrogenase, a physiologic COX-2 antagonist, in human cholangiocarcinoma cells. These findings suggest that omega 3-PUFAs block cholangiocarcinoma cell growth at least in part through inhibition of Wnt/beta-catenin and COX-2 signaling pathways. Thus, utilization of omega 3-PUFAs may represent an effective and safe therapeutic approach for the chemoprevention and treatment of human cholangiocarcinoma.

Endotoxin tolerance in human intrahepatic biliary epithelial cells is induced by upregulation of IRAK-M

Abstract: Background/Aim: Biliary epithelial cells possess an innate immune system consisting of Toll‐like receptors (TLRs). Although the human bile contains lipopolysaccharide (LPS) in normal as well as diseased livers, LPS physiologically does not elicit an inflammatory response in the biliary tree. This absence of a response to LPS could be due to the ‘endotoxin tolerance’ speculated to maintain innate immune homeostasis in organs. Our aim here is to clarify the presence and molecular mechanisms of endotoxin tolerance of biliary epithelium.

Peptide antibiotic human beta‐defensin‐1 and ‐2 contribute to antimicrobial defense of the intrahepatic biliary tree

Human beta‐defensins (hBDs) are important antimicrobial peptides that contribute to innate immunity at mucosal surfaces. This study was undertaken to investigate the expression of hBD‐1 and hBD‐2 in intrahepatic biliary epithelial cells in specimens of human liver, and 4 cultured cell lines (2 consisting of biliary epithelial cells and 2 cholangiocarcinoma cells). In addition, hBD‐1 and hBD‐2 were assayed in specimens of bile. hBD‐1 was nonspecifically expressed immunohistochemically in intrahepatic biliary epithelium and hepatocytes in all patients studied, but expression of hBD‐2 was restricted to large intrahepatic bile ducts in 8 of 10 patients with extrahepatic biliary obstruction (EBO), 7 of 11 with hepatolithiasis, 1 of 6 with primary biliary cirrhosis (PBC), 1 of 5 with primary sclerosing cholangitis (PSC), 0 of 6 with chronic hepatitis C (CH‐C), and 0 of 11 with normal hepatic histology. hBD‐2 expression was evident in bile ducts exhibiting active inflammation. Serum C reactive protein levels correlated with biliary epithelial expression of hBD‐2. Real‐time PCR revealed that in all of 28 specimens of fresh liver, including specimens from patients with hepatolithiasis, PBC, PSC, CH‐C and normal hepatic histology, hBD‐1 messenger RNA was consistently expressed, whereas hBD‐2 messenger RNA was selectively expressed in biliary epithelium of patients with hepatolithiasis. Immunobloting analysis revealed hBD‐2 protein in bile in 1 of 3 patients with PSC, 1 of 3 with PBC, and each of 6 with hepatolithiasis; in contrast, hBD‐1 was detectable in all bile samples examined. Four cultured biliary epithelial cell lines consistently expressed hBD‐1; in contrast these cell lines did not express hBD‐2 spontaneously but were induced to express hBD‐2 by treatment with Eschericia coli, lipopolysaccharide, interleukin‐1β or tumor necrosis factor‐α. In conclusion, these findings suggest that in the intrahepatic biliary tree, hBD‐2 is expressed in response to local infection and/or active inflammation, whereas hBD‐1 may constitute a preexisting component of the biliary antimicrobial defense system. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;40:925–932.)

Innate immune response to double-stranded RNA in biliary epithelial cells is associated with the pathogenesis of biliary atresia†

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)‐κB and interferon regulatory factor 3 (IRF3)] and the production of interferon‐β1 (IFN‐β1) 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‐κB, 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. (HEPATOLOGY 2007.)

Histologic graft assessment after clinical islet transplantation.

Background. An accurate monitoring would help understanding the fate of islet grafts after transplantation. Methods. This work assessed the feasibility of needle biopsy monitoring after intraportal islet transplantation (n=16), and islet graft morphology was studied with the addition of autopsy samples (n=2). Pancreas autopsy samples from two nondiabetic individuals were used as control. Results. Islet tissue was found in five needle samples (31%). Sampling success was related to size (100% sampling for the four biopsies of 1.8 cm in length or higher, P≤0.01). Mild liver abnormalities included localized steatosis (n=8), mild nodular regenerative hyperplasia and mild portal venopathy (n=3), and hepatocyte swelling (n=2). Endocrine cell composition and distribution were similar between islet grafts and normal islets within the native pancreas. There was no or minimal immune cell infiltrate in patients on and off exogenous insulin, including two patients with ongoing negative metabolic events (increasing HbA1c or insulin requirement). The infiltrate was mainly composed of CD4- and CD8-positive cells. Conclusion. This study demonstrates that needle biopsy is feasible after clinical islet transplantation but with a limited practical value because of its low islet sampling rate using current sampling and analysis methods. Both biopsy and autopsy samples demonstrated the well-preserved islet endocrine composition after transplantation and the presence of focal areas of steatosis. Islet grafts showed no or minimal immune cell infiltration, even in the case of ongoing islet loss. On the basis of the findings, possible reasons for allograft islet loss are discussed.

Th1 cytokine–induced downregulation of PPARγ in human biliary cells relates to cholangitis in primary biliary cirrhosis

Peroxisome proliferator‐activated receptor‐γ (PPARγ) is known to inhibit the production of proinflammatory cytokines. In Th1‐predominant diseases, PPARγ ligands can ameliorate clinical severity by downregulating the expression of proinflammatory cytokines. Primary biliary cirrhosis (PBC) is characterized by chronic destructive cholangitis with a Th1‐predominant cytokine milieu. Unusual immune responses to infectious agents are suspected to underlie its etiopathogenesis. We examined the significance of PPARγ in biliary inflammation in connection to PBC. To this end, we performed immunohistochemistry, quantitative polymerase chain reaction, and nuclear factor‐kappaB (NF‐κB) DNA‐binding assays to clarify the intrahepatic distribution of PPARγ and the regulation of PPARγ by inflammatory cytokines and PPARγ ligand in five cultured biliary cell lines including one derived from PBC liver. In liver specimens from patients with PBC, PPARγ protein was ubiquitously expressed in intrahepatic biliary epithelium, whereas the expression of PPARγ protein and mRNA was reduced in damaged bile ducts. PPARγ expression in cultured cells was upregulated by interleukin‐4 (IL‐4; Th2‐type), but downregulated by IFN‐γ (Th1‐type). PPARγ ligand negatively modulated lipopolysaccharide‐induced NF‐κB activation. Moreover, this inhibitory effect of PPARγ ligand was attenuated by pretreatment with IFN‐γ. In conclusion, PPARγ may be important to maintain homeostasis in the intrahepatic biliary epithelium, and its reduction in the bile ducts of PBC liver may be associated with the Th1‐predominant milieu and with the development of chronic cholangitis in PBC. Immunosuppression using PPARγ ligands may be of therapeutic benefit to attenuate biliary inflammation in PBC. (HEPATOLOGY 2005;41.)

Autoreactive T-Cell Responses in Primary Biliary Cirrhosis Are Proinflammatory Whereas Those of Controls Are Regulatory

BACKGROUND Autoreactive T cells that proliferate in response to autoantigens are found in both autoimmune diseases and controls but have important qualitative differences in relative activation states, costimulation signal requirements and pathogenetic significance. METHODS To understand the differences between autoreactive T cells in PBC versus controls, we have developed autoreactive T-cell clones (TCC) from patients with PBC and healthy controls, and have used a peptide corresponding to the CD4 major autoantigen (Ag) to define the relative proliferative response. Peripheral blood mononuclear cells (PBMC) from PBC respond to the Ag in a costimulation-independent manner, but PBMC from controls respond to the Ag in a costimulation-dependent manner. Next, we established nine autoreactive TCC from patients with PBC and eight from healthy controls. RESULTS Among 17 TCC, eight were the costimulation-dependent type and nine were independent. In addition, costimulation-dependent autoreactive TCC became anergic after stimulation in the presence of APC that did not provide costimulatory signals. Finally, we observed that anergic TCC exhibit regulatory functions. CONCLUSIONS In the case of regulatory dendritic cells, we could not induce TCC anergy. On the other hand, when using peptide analog in a costimulation-deficient manner, we could induce TCC anergy, even though these TCC were costimulation independent.