The statistical tests were performed using GraphPad Prism version 5.0 b (GraphPad Software, La Jolla, CA) and IBM SPSS Statistics version 21 (IBM Corporation, Armonk, NY). Results Murine cholangiocyte cell lines express CD1d and present lipid antigens to NKT cell hybridomas and primary NKT cells We first explored the CD1d expression of murine small and large cholangiocyte cell lines (19). cell hybridomas and primary NKT cells in a CD1d-dependent manner. A human cholangiocyte cell line, cholangiocarcinoma cell lines and human primary cholangiocytes also presented exogenous CD1d-restricted antigens to iNKT cell clones. CD1d expression was down-regulated in the biliary LB42708 epithelium of patients with late primary sclerosing LB42708 cholangitis (PSC), primary biliary cirrhosis (PBC) and alcoholic cirrhosis compared to healthy controls. Conclusions Cholangiocytes express CD1d, present antigens to NKT cells and CD1d expression is down-regulated in diseased biliary epithelium. These findings show that the biliary epithelium can activate an important lymphocyte subset of the liver. Our study describes the presence of a potentially important immune pathway in the biliary system, which may be capable of regulating inflammation in the context of biliary disease. infection, bacteria which produce -GalCer-like ligands, and LB42708 this was not observed in mice lacking iNKT cells (18). CD1d expression in the biliary epithelium has not been reported in other liver diseases or cholangiopathies such as PSC. In the present study we sought to evaluate the expression of CD1d in murine and human biliary epithelium and to determine whether cholangiocytes are able to present lipid antigens to and activate NKT cells. Our data demonstrate that cholangiocytes can indeed function as antigen presenting cells, and that CD1d is down-regulated in the biliary epithelium of diseased livers. This suggests LB42708 that cholangiocytes exhibit a potential regulatory function through the activation of NKT cells. Materials and methods Cells and cell lines Two murine cholangiocyte cell lines were originally isolated Rabbit Polyclonal to LSHR from the small and large intrahepatic bile ducts of BALB/c mice, respectively small and large cholangiocytes. These murine cell lines and a human cholangiocyte cell line, H69, had previously been immortalized by introduction of LB42708 the SV40 large T antigen (19,20). Cholangiocarcinoma cell lines EGI-1, TFK-1 (21) (DSMZ, Braunschweig, Germany) and HuCCT1 (JCRB Cell Bank, Osaka, Japan) were acquired commercially. Cholangiocarcinoma cell lines KMBC and KMCH-1 (22,23) were kind gifts from Prof Gregory Gores (Mayo Clinic Medical Center, Rochester, MN), while cholangiocarcinoma and gall bladder carcinoma cell lines Sk-ChA-1, Mz-ChA-1 and Mz-ChA-2 (24) were kindly given to us by Prof Alexander Knuth (University Hospital Zrich, Zrich, Switzerland). Murine iNKT cell (DN32.D3, 24.7, 24.8) and niNKT cell hybridomas (14S.6, 14S.7, 14S.10 and 14S.15) and two human iNKT cell clones (JC2.7 and J3N.5) have been previously described (25C27). CD1d transfected murine RMA-S cells and an Epstein-Barr virus (EBV) cell line ectopically expressing human CD1d were used as positive controls (25). Primary murine NKT cells were extracted from the livers of C57BL/6 mice. Primary dendritic cells were isolated from the spleens of C57BL/6 mice using CD11c MicroBeads, (Miltenyi Biotec, Bergisch Gladbach, Germany). Primary cholangiocytes were extracted from explanted livers from liver recipients. Cells were cultured in conditions summarised in the Supplementary material and in Table S1. Mice C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and Taconic Farms (Germantown, NY). The mice were housed in a Minimal Disease Unit at the animal facility at Oslo University Hospital, Rikshospitalet, Oslo. All animal experiments were approved by the Norwegian Animal Research Committee and all animals received human care in line with “Guide for the Care and Use of Laboratory Animals” (National Institutes of Health Publication, 8th Edition, 2011). Flow cytometry All cells were incubated with an antibody against Fc-receptors to avoid nonspecific binding. Cholangiocytes were stained with anti-CD1d antibody or isotype control for 30 minutes. To detect iNKT cells, lymphocytes were stained with anti-TCR and loaded or unloaded PBS-57 CD1d tetramers (kindly provided by the NIH Tetramer Core, Emory, GA) for an hour. Flow cytometric analysis was performed using BD FACS Calibur.