Increased expression of Toll-like receptor 3 in intrahepatic biliary epithelial cells at sites of ductular reaction in diseased livers

Background Toll-like receptors (TLRs) may play active roles in both innate and adaptive immune responses in human intrahepatic biliary epithelial cells (HIBECs). The role of TLR3 expressed by HIBECs, however, remains unclear. Methods We determined the in vivo expression of TLRs in biopsy specimens derived from diseased livers immunohistochemically using a panel of monoclonal antibodies against human TLRs. We then examined the response of cultured HIBECs to a TLR3 ligand, polyinosinic–polycytidylic acid (polyI:C). Using siRNAs specific for Toll-IL-1R homology domain-containing adaptor molecule 1 (TICAM-1) and mitochondrial antiviral signaling protein (MAVS), we studied signaling pathways inducing IFN-β expression. Results The expression of TLR3 was markedly increased in biliary epithelial cells at sites of ductular reaction in diseased livers, including primary biliary cirrhosis (PBC), autoimmune hepatitis (AIH), and chronic viral hepatitis (CH) as compared to nondiseased livers. Although cultured HIBECs constitutively expressed TLR3 at both the protein and mRNA levels in vitro, the addition of polyI:C to culture media induced only minimal increases in IFN-β mRNA. In contrast, transfection of HIBECs with polyI:C induced a marked increase in mRNAs encoding a variety of chemokines/cytokines, including IFN-β, IL-6, and TNF-α. The induction of IFN-β mRNA was efficiently inhibited by an siRNA against MAVS but not against TICAM-1, indicating that the main signaling pathway for IFN-β induction following polyI:C transfection is via retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5) in HIBECs. Conclusions TLR3 expression by biliary epithelial cells increased at sites of ductular reaction in diseased livers; further study will be necessary to characterize it’s in vivo physiological role.     

The role of TRAIL in mediating autophagy in myositis skeletal muscle: A potential nonimmune mechanism of muscle damage

Objective

Multinucleated cells are relatively resistant to classic apoptosis, and the factors initiating cell death and damage in myositis are not well defined. We hypothesized that nonimmune autophagic cell death may play a role in muscle fiber damage. Recent reports indicate that TRAIL may induce both NF‐κB activation and autophagic cell death in other systems. We undertook this study to investigate the role of TRAIL in cell death and pathogenesis in vitro and in vivo, using myositis muscle tissues from humans and mice.

Methods

Gene expression profiling was performed in myositis patient and control muscle specimens. Immunohistochemistry analysis was performed to confirm the gene array findings. We also analyzed TRAIL‐induced cell death (apoptosis and autophagy) and NF‐κB activation in vitro in cultured cells.

Results

TRAIL was expressed predominantly in myositis muscle fibers, but not in biopsy specimens from normal or other dystrophic‐diseased muscle. Autophagy markers were up‐regulated in humans with myositis and in mouse models of myositis. TRAIL expression was restricted to regenerating/atrophic areas of muscle fascicles, blood vessels, and infiltrating lymphocytes. TRAIL induced NF‐κB activation and IκB degradation in cultured cells that are resistant to TRAIL‐induced apoptosis but that undergo autophagic cell death.

Conclusion

Our data demonstrate that TRAIL is expressed in myositis muscle and may mediate both activation of NF‐κB and autophagic cell death in myositis. Thus, this nonimmune pathway may be an attractive target for therapeutic intervention in myositis.

     

Significance of periductal Langerhans cells and biliary epithelial cell‐derived macrophage inflammatory protein‐3α in the pathogenesis of primary biliary cirrhosis

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.     

Therapy-induced expression of NF-κB portends poor prognosis in patients with localized esophageal cancer undergoing preoperative chemoradiation

Activated nuclear factor‐kappa B (NF‐κB) in the pretreatment cancer tissue of patients with localized esophageal adenocarcinoma (LEA) undergoing preoperative chemoradiation is associated with poor prognosis. It is known that constitutively activated NF‐κB prior to any therapy portends poor prognosis, and it is also known that activated NF‐κB in the treated specimen is associated with poor prognosis. However, the prognosis of patients who have treatment‐induced activation of NF‐κB (meaning their cancers activate NF‐κB during or after therapy) is not been reported. We hypothesized that the treatment‐induced activation of NF‐κB would impart poor prognosis similar to that imparted by constitutively activated NF‐κB cancer. Patients with LEA who had undergone preoperative chemoradiation plus surgery and had pre‐ and post‐therapy cancer tissue available were selected. Pre‐ and post‐therapy cancer tissues were stained by immunohistochemistry for nuclear staining of NF‐κB. The overall survival (OS) and disease‐free survival were assessed and compared for patients who had intrinsic constitutively activated NF‐κB cancer with those who had induced activation of NF‐κB only post‐therapy. A total of 41 patients with LEA were investigated. Twenty‐five patients had NF‐κB positive cancer at baseline, and 16 had NF‐κB negative cancer at baseline but became positive post‐therapy. There was no difference in the location, histology grade, clinical stage, or the curative resection (RO) resection rate in the two populations. OS (P = 0.71), disease‐free survival (P = 0.86), and median survivals (Converters: 24 months [95% confidence intervals: 7.78 to 40.22]vs. Nonconverters: 34.13 months [95% confidence intervals: 3.54 to 64.27]) were not different between the two groups. Our data suggest that activation of NF‐κB in response to stress/injury of therapy leads to poor OS. These results need to be confirmed in a larger number of patients. It may be that only pre‐therapy evaluation of NF‐κB is insufficient to assess prognosis of patients with LEA. Additional implications include that when effective anti‐NF‐κB therapies become available, they may have to be considered in patients whose cancers do not have constitutively activated NF‐κB or cancer may have to be monitored during therapy with biomarker assessments.     

Isolation,culture and characterization of biliary epithelial cells from different anatomical levels of the intrahepatic and extrahepatic biliary tree from a mouse

ABSTRACT— We developed methods to isolate biliary epithelial cells (BECs) from the gallbladder (GB), common bile duct (CBD), intrahepatic large bile duct (ILBD) and small bile duct (ISBD) of a mouse, simultaneously. ILBD and ISBD were cut from the biliary tree after collagenase perfusion of the liver. BECs from all of these biliary segments were cultured as explants on collagen gel. BECs spread from the explants and formed cellular sheets. Areas of these sheets composed entirely of BECs were cut and placed on other gels as subculture, and this continued for 10 passages. Primary and passage cultured BECs on gel were composed of a monolayer of epithelial cells. Passaged cultured BECs in gel formed a spherical cyst lined by a single epithelial layer. Ultrastructurally, microvilli were dense on the luminal surface, and junctional complex and interdigitation was identifiable on the lateral surfaces. These features were similar in both primary and passaged cultured BECs, irrespective of their anatomical origin. Major histocompatibility complex antigens and intercellular adhesion molecule-1 were induced on the basolateral cell membranes of primary and passaged cultured BECs, by interferon-γ. Although several phenotypic, structural and probable biological features of BECs inherent to each anatomical level may be lost after culture on gel, a combination of this method, several immunological modifications in experimental animals, and addition of immunologically active substances to the culture medium will make the immunopathologic analysis of biliary diseases possible.     

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

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. METHODS AND RESULTS: In nuclear factor-kappaB (NF-kappaB)-DNA binding assays using three-cultured human intrahepatic biliary epithelial cell (HIBEC) lines, all the cells responded to LPS (TLR4 ligand) by activating NF-kappaB, but pretreatment with LPS for 24 h effectively induced tolerance against any subsequent stimulation with LPS (endotoxin tolerance). This tolerance was also induced by pretreatment with Pam(3)Cys-Ser-(Lys)(4) trihydrochloride (Pam(3)CKS(4), TLR1/2 ligand). Then, real-time polymerase chain treaction and Western blotting revealed that LPS treatment upregulated the expression of IRAK-M (a negative regulator of TLR signaling), but did not affect interleukin-1 receptor-associated kinase-1 (IRAK-1, an essential molecule of TLR signaling), in HIBECs. Moreover, immunohistochemistry revealed that IRAK-M was diffusely expressed in intrahepatic bile ducts. CONCLUSIONS: This study showed that the mechanism of endotoxin tolerance exists in the intrahepatic biliary tree and is possibly induced by the expression of IRAK-M in the intrahepatic biliary epithelium, suggesting that the endotoxin tolerance is important in maintaining innate immune biliary homeostasis.     

Inhibition of nuclear factor κB and phosphatidylinositol 3‐kinase/Akt is essential for massive hepatocyte apoptosis induced by tumor necrosis factor α in mice

Background/aims: Tumor necrosis factor (TNF)‐α itself does not induce liver injury in normal mice or hepatocytes. Rather, this event, especially in vitro, is explained by the fact that the TNF‐α/TNF receptor system not only triggers downstream signals leading to apoptosis but also induces an antiapoptotic pathway through the activation of nuclear factor (NF)‐κB. The aim of this study was to determine whether inhibition of antiapoptotic pathways influences the susceptibility of mice to TNF‐α. Here, we focused on the roles of NF‐κB and phosphatidylinositol 3‐kinase (PI3K)‐regulated serine/threonine kinase Akt. Methods: TNF‐α was administered to BALB/c mice after treatment with an adenovirus expressing a mutant form IκBα (Ad5IκB), the PI3K inhibitor wortmannin, or both. Liver injury was assessed biochemically and histologically. The expression of Bcl‐2 family members and caspase activity were examined. Results: In the mice livers, treatment with Ad5IκB or the wortmannin suppressed the activation of NF‐κB or Akt, respectively. Suppression of either NF‐κB or Akt showed a slight increase in transaminase levels and focal liver cell death after TNF‐α administration. However, in mice treated with both Ad5IκB and wortmannin, TNF‐α administration resulted in massive hepatocyte apoptosis and hemorrhagic liver destruction in mice. The combination of Ad5IκB, wortmannin, and TNF‐α markedly increased the activation of caspase‐3 and ‐9, and activated caspase‐8 to a lesser degree, suggesting that TNF‐α‐induced hepatocyte apoptosis is dependent on type II cell death signaling pathway, probably through the mitochondria. Inhibition of the NF‐κB and PI3K/Akt pathways had no effect on expression of Bcl‐2 families. Conclusion: The inducible activation of NF‐κB and constitutive activation of Akt regulate hepatocyte survival against TNF‐α, which occurs independent of Bcl‐2 families.     

Immunohistochemical study of nuclear factor‐κB expression in esophageal squamous cell carcinoma: prognostic significance and sensitivity to treatment with 5‐FU

Nuclear factor‐κB (NF‐κB) is expressed in many types of cancers. It has been suggested that the expression of NF‐κB is associated with poor prognosis and resistance to chemoradiation therapies. This study evaluated the relationship between the expression of NF‐κB and the prognosis and sensitivity of esophageal squamous cell carcinoma (ESCC) to chemotherapy. One hundred and nine ESCC specimens, from patients who had undergone radical esophagectomy, were divided into two groups depending on the expression of NF‐κB. Surgical data and prognosis were compared between the two groups. NF‐κB‐positive tumors were detected in 61.5% of the cases. In 69 patients with stage II and III disease, 41 patients who were NF‐κB‐positive showed poor survival. The sensitivity of esophageal squamous cell carcinoma cell lines to 5‐fluorouracil (5‐FU) was analyzed by their NF‐κB expression, and the effect of 5‐FU was evaluated on the proliferation and activity of two cell lines of cultured ESCCs expressing NF‐κB. ESCCs with activated NF‐κB had poor sensitivity to 5‐FU. These results suggest that the increased expression of NF‐κB is associated with poor prognosis in patients with ESCC. NF‐κB may be a target for ESCC therapy because of its selective expression in this type of cancer.     

Prophylaxis with mesna prevents oxidative stress induced by ischemia reperfusion in the intestine via inhibition of nuclear factor-kappaB activation

Background and Aim: Mesna (2‐mercaptoethane‐sulfonate) has been shown to attenuate oxidative injury induced by ischemia reperfusion (I/R) in the kidneys, the liver, and the intestine; however, its mechanism of action has not been fully elucidated. We sought to determine a prophylactic admininstration schedule of mesna that would confer optimal antioxidant protection on the intestinal mucosa following I/R and to investigate whether mesna's action is mediated via inhibition of nuclear factor‐κB (NF‐κB) activity. Methods: Wistar rats were subjected to one of the following: (a) induction of 30 min ischemia followed by 60 min reperfusion (I30/R60) of the intestine, (b) pretreatment with intraperitoneal or oral mesna at various time‐ and dose‐ administration schedules plus I30/R60, (c) sham operation, (d) no operation (controls), or (e) oral mesna alone. At the end of the reperfusion period or at various time points after mesna alone administration, the oxidative state of the intestinal mucosa was assessed in terms of glutathione to glutathione disulfide ratio, malondialdehyde concentration, and superoxide dismutase activity. In addition, NF‐κB activity in the intestinal mucosa was assessed immunohistochemically in the oral mesna plus I/R and in the oral mesna alone groups. Results: Sham operation caused mild stress, while I/R caused substantial oxidative stress in the intestinal mucosa. Mesna pretreatment had an antioxidant effect which varied from attenuation to prevention of oxidative stress. Over the two routes of administration, the oral proved to be more effective and had a time‐ and dose‐ dependent effect. The antioxidant action of mesna was not related to enhancement of the intestinal mucosa oxidative state. Furthermore, I/R induced NF‐κB activation in the intestinal mucosa which was inhibited by mesna pretreatment. In the absence of oxidative damage, mesna led to downregulation of activated NF‐κB. Conclusions: Prophylaxis with mesna prevents oxidative stress induced by I/R in the intestine via inhibition of NF‐κB activation.     

Early development of primary biliary cirrhosis in female C57BL/6 mice because of poly I:C administration.

BACKGROUND: Primary biliary cirrhosis (PBC) is one of the organ-specific autoimmune diseases characterized by destruction of the biliary epithelial cells, cholestasis, liver cirrhosis, and liver failure. With the postulation that induction of the autoimmune process might induce PBC-like cholangitis, here we used polyinosinic polycytidylic acid (poly I:C), an inducer of type-1 interferon (IFN), to generate an autoimmune cholangitis animal model. METHODS: Female C57BL/6 mice were injected with 5 mg/kg of poly I:C twice a week for 28 consecutive weeks. Liver specimens were collected to evaluate the degree of cell infiltration. Autoantibodies, including antimitochondrial antibody (AMA), were assayed by immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and immunoblotting. IFN-alpha was estimated in the sera by an ELISA method. Poly I:C injection induced IFN-alpha. RESULTS: Mononuclear cells were detected at the portal areas 8 weeks after the start of poly I:C injection, which progressed up to 16 weeks. Autoantibodies, including AMA, were detected in the sera from all poly I:C-injected mice. CONCLUSIONS: In conclusion, we show an early development of a PBC-like cholangitis in a genetically susceptible mouse strain because of poly I:C administration. This model would be helpful to study PBC immunopathogenesis and to evaluate the effectiveness of newly developed therapeutic regimens for PBC.     

Epidemiological features of biliary atresia in Taiwan, a national study 1996-2003

Background and Aim: The incidence of biliary atresia (BA) varies among different countries. It is supposed to be higher in Asian countries than in Western countries; however, the incidence of BA in Taiwan has not been well investigated. The aim of this study was to analyze the epidemiological characteristics and the incidence of BA in Taiwan. Methods: National Health Insurance (NHI) was implemented in Taiwan in 1995, and covers most of the population (>99%). We use the NHI database to investigate the epidemiological features of BA and compare Taiwan's annual BA incidence with that of other countries. Results: We identified 327 new BA cases during the period from 1996 to 2003. The overall incidence of BA was 1.46 cases per 10 000 live births (0.89–1.90 per 10 000). The estimation was 1.32–1.65 per 10 000 after adjustment for the misdiagnosis rate. The peak incidence occurred in 2002 (1.90 per 10 000), accompanying Taiwan's dengue fever epidemic in 2002. The 5‐year overall survival rate during 1999–2003 was higher than that during 1996–1998 (74.8% vs 61.1%, P = 0.014). Conclusion: Taiwan has the second‐highest incidence of BA reported in world literature. Viral infection outbreaks remain a potential candidate as a cause of BA. The management of BA has been improving, with a better 5‐year overall survival rate.