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.
Harada
Kenichi
K
Department of Human Pathology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan.
Ohira
Shusaku
S
Isse
Kumiko
K
Ozaki
Satoru
S
Zen
Yoh
Y
Sato
Yasunori
Y
Nakanuma
Yasuni
Y
D000328:Adult; D000368:Aged; D000818:Animals; D001646:Bile / Q000737:chemistry; D001650:Bile Duct Neoplasms / Q000378:metabolism / Q000473:pathology; D001653:Bile Ducts, Intrahepatic / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D018281:Cholangiocarcinoma / Q000378:metabolism / Q000473:pathology; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004731:Endotoxins / Q000032:analysis / Q000494:pharmacology*; D004848:Epithelium / Q000187:drug effects* / Q000378:metabolism; D004926:Escherichia coli; D005260:Female; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008875:Middle Aged; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D017931:DNA Primers; D004731:Endotoxins; D007976:Leupeptins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; C044101:endotoxin, Escherichia coli; D004247:DNA; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde