Search

Epithelial response to injury is critical to the pathogenesis of biliary cirrhosis, and IL-6 has been suggested as a mediator of this phenomenon. Several liver cell types can secrete IL-6 following activation by various signaling molecules including circulating adenosine. The aims of this study were to assess whether adenosine can induce IL-6 secretion by cholangiocytes via the A2b adenosine receptor (A2bAR) and to determine the effect of A2bAR-sensitive IL-6 release on injury response in biliary cirrhosis. Human normal cholangiocyte H69 cells were used for in vitro studies to determine the mechanism by which adenosine and the A2bAR induce release of IL-6. In vivo, control and A2bAR-deficient mice were used to determine the roles of A2bAR-sensitive IL-6 release in biliary cirrhosis induced by common bile duct ligation (BDL). Additionally, the response to exogenous IL-6 was assessed in C57BL/6 and A2bAR-deficient mice. Adenosine induced IL-6 mRNA expression and protein secretion via A2bAR activation. Although activation of A2bAR induced cAMP and intracellular Ca2+ signals, only the Ca2+ signals were linked to IL-6 upregulation. After BDL, A2bAR-deficient mice have impaired survival, which is further impaired by exogenous IL-6; however, decreased survival is not due to changes in fibrosis and no changes in inflammatory cells. Exogenous IL-6 is associated with the increased presence of bile infarcts. Extracellular adenosine induces cholangiocyte IL-6 release via the A2bAR. This signaling pathway is important in the pathogenesis of injury response in biliary cirrhosis but does not alter fibrosis. Adenosine upregulates IL-6 release by cholangiocytes via the A2bAR in a calcium-sensitive fashion. Mice deficient in A2bAR experience impaired survival after biliary cirrhosis induced by common bile duct ligation independent of changes in fibrosis.

Sortilin, a member of the vacuolar protein sorting 10 domain receptor family, traffics newly synthesized proteins from the trans-Golgi network to secretory pathways, endosomes, and cell surface. Sortilin-trafficked molecules, including IL-6 and acid sphingomyelinase (aSMase), mediate cholangiocyte proliferation and liver inflammation, hepatic stellate cell activation, hepatocyte apoptosis, and fibrosis. Based on these sortilin-regulated functions, we investigated its role in biliary damage leading to hepatocellular injury and fibrosis. Sortilin -/- mice displayed impaired inflammation and ductular reaction 3 days after bile duct ligation (BDL), as demonstrated by reduced cholangiocyte proliferation and activation and reduced serum IL-6. Interestingly, liver fibrosis was reduced in Sortilin -/- mice after both BDL and carbon tetrachloride treatment, in line with attenuated in vitro activation of Sortilin -/- hepatic stellate cells. Sortilin -/- hepatic aSMase activity was reduced in the BDL and carbon tetrachloride models and accompanied by reduced in vivo hepatocyte apoptosis. In addition, wild type (WT), but not Sortilin -/- hepatocytes, had increased aSMase-dependent susceptibility to bile acid-induced apoptosis in vitro. Mechanistically, short-term IL-6 neutralization in bile duct-ligated WT mice decreased hepatic inflammation and reactive cholangiocyte-derived cytokines and chemokines, without affecting fibrosis, whereas pharmacological inhibition of aSMase activity was not sufficient to attenuate hepatic fibrosis. Only combined IL-6 and aSMase inhibition significantly reduced fibrosis in bile duct-ligated WT mice. We conclude that sortilin regulates cholestatic liver damage and fibrosis via effects on both aSMase activity and serum IL-6., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Bile duct epithelia are the target of a number of "cholangiopathies" characterized by disordered bile ductular proliferation. Although mechanisms for bile ductular proliferation are unknown, recent evidence suggests that extracellular nucleotides regulate cell proliferation via activation of P2Y receptors. Portal fibroblasts may regulate bile duct epithelial P2Y receptors via expression of the ecto-nucleotidase NTPDase2. Thus, we tested the hypothesis that portal fibroblasts regulate bile duct epithelial proliferation via expression of NTPDase2. We generated a novel co-culture model of Mz-ChA-1 human cholangiocarcinoma cells and primary portal fibroblasts. Cell proliferation was measured by bromodeoxyuridine uptake. NTPDase2 expression was assessed by immunofluorescence and quantitative real-time reverse transcription PCR. NTPDase2 expression in portal fibroblasts was blocked using short interfering RNA. NTPDase2 overexpression in portal myofibroblasts isolated from bile duct-ligated rats was achieved by cDNA transfection. Co-culture of Mz-ChA-1 cells with portal fibroblasts decreased their proliferation to 26% of control. Similar decreases in Mz-ChA-1 proliferation were induced by the soluble ecto-nucleotidase apyrase and the P2 receptor inhibitor suramin. The proliferation of Mz-ChA-1 cells returned to baseline when NTPDase2 expression in portal fibroblasts was inhibited using NTPDase2-specific short interfering RNA. Untransfected portal myofibroblasts lacking NTPDase2 had no effect on Mz-ChA-1 proliferation, yet portal myofibroblasts transfected with NTPDase2 cDNA inhibited Mz-ChA-1 proliferation. We conclude that portal fibroblasts inhibit bile ductular proliferation via expression of NTPDase2 and blockade of P2Y activation. Loss of NTPDase2 may mediate the bile ductular proliferation typical of obstructive cholestasis. This novel cross-talk signaling pathway may mediate pathologic alterations in bile ductular proliferation in other cholangiopathic conditions.