Lipopolysaccharide Activates Nuclear Factor-KappaB through Toll-Like Receptors and Related Molecules in Cultured Biliary Epithelial Cells

Abstract

SUMMARY: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. (Lab Invest 2003, 83:1657-1667.

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

“…14,17,18,29,30 In the TLR family, TLR3 recognizes dsRNA and is located in the endosomal membranes with the ligand-binding domain facing the lumen of the endosomes and the signaling domain positioned on the cytoplasmic side. Therefore, TLR3 allows cells to detect dsRNA that is phagocytosed from the extracellular space, where it is released by infected cells that are undergoing lysis or necrosis.…”

“…For the evaluation of messenger ribonucleic acid (mRNAs) of TLR3, RIG-I, MDA-5, PKR, NF-B, interferon regulatory factor-3 (IRF-3), IFN-␤, MxA, TRAIL, TRAIL receptors (TRAIL-DR4, TRAIL-DR5, TRAIL-DcR1, and TRAIL-DcR2), CD95, and CD95 ligand in cultured BECs, total RNA was isolated from BECs, and 1 g total RNA was reverse-transcribed with an oligo-(dT) primer and reverse transcriptase to synthesize complementary deoxyribonucleic acid. Human cultured cell lines (HuCCT1, HuH7, HepG2, and WI38) and normal tissues (pancreas and liver) [18][19][20][21][22][23][24][25][26][27] were used as controls, and the complementary deoxyribonucleic acid was amplified by polymerase chain reaction (PCR) using the specific primers ( Table 1). The PCR products were subjected to electrophoresis on 1.5% agarose gels containing ethidium bromide.…”

Reovirus type-2–triggered autoimmune cholangitis in extrahepatic bile ducts of weanling DBA/1J mice

“…In addition, it has been reported that bacterial components (e.g., intestinal lipopolysaccharides) may stimulate macrophages to produce enough TGF-β from those cells, resulting in induction of severe fibrosis (29). It seems likely, because biliary epithelial cells constantly express at least Toll-like receptors 2-5 and can respond to the Toll-like receptor ligands (lipopolysaccharides) derived from the duodenum (30). In addition, it has been pointed out that IL-17A promotes the development of several autoimmune diseases as one of the factors (31).…”

Pattern recognition receptors: A contemporary view on liver diseases

“…64,65 TLR2, 3, 4 and 5, but not TLR7, 8,9, were detected at the protein level and appeared functionally active based on ligand-induced IL-6, IL-8 and MCP-1 production in biliary epithelial cell lines. 66,67 In vitro TLR mRNA expression and, to a lesser extent, protein levels were increased by Th1 cytokines (IFN␥ and TNF␣) in biliary cells. 66,67 In vivo, however, LPS failed to elicit an inflammatory response in intrahepatic cholangiocytes.…”