Responsiveness of intestinal epithelial cell lines to lipopolysaccharide is correlated with Toll-like receptor 4 but not Toll-like receptor 2 or CD14 expression.
OBJECTIVE
Luminal bacteria have been implicated in the pathogenesis of inflammatory bowel diseases. Exposure of intestinal epithelial cells (IEC) to bacterial components potentially initiates intestinal inflammation by release of chemokines and recruitment of inflammatory cells. We analyzed receptor expression and signaling pathways involved in activation of human primary IEC and carcinoma-derived cell lines by lipopolysaccharide (LPS).


METHODS
HT-29/p, HT-29/MTX, and Caco-2 cells were stimulated by LPS. IL-8 content in supernatants was analyzed by ELISA, and expression of CD14, Toll-like receptor (TLR) 2 and TLR 4 was determined by RT-PCR. Presence of TLR 4 protein was assessed by western blot analysis. LPS response was modulated by sCD14, LPS-binding protein, neutralization of CD14, and inhibitors of early signal activation.


RESULTS
LPS dose-dependently induced secretion of IL-8 in undifferentiated HT-29/p cells while Caco-2 and permanently differentiated HT-29/MTX cells were unresponsive. Differently to HT-29/MTX, both HT-29/p and Caco-2 cells constitutively expressed transcripts for CD14. However, CD14 was not required for LPS-mediated induction of IL-8 in HT-29/p cells since neutralizing anti-CD14 antibodies left IL-8 levels unchanged. Unresponsiveness of Caco-2 and HT-29/MTX cells to LPS persisted in the presence of sCD14 and/or LPS-binding protein. Neither cell line expressed TLR 2 transcripts while only responsive HT-29/p cells expressed TLR 4 mRNA and TLR 4 protein. Butyrate down-regulated TLR 4 expression and significantly diminished LPS-dependent IL-8 secretion. Inhibition of G protein dependent kinase activation reduced IL-8 levels to 50%; the phosphatidyl-inositol-3'-kinase inhibitor LY294002 abrogated the response.


CONCLUSIONS
Responsiveness of IEC lines to LPS is positively correlated with TLR 4 expression. Strategies targeting TLR 4 expression or TLR 4 mediated signaling may antagonize IEC activation by LPS.
Böcker
Ulrich
U
Department of Medicine II and Medical Research Center, University of Heidelberg, Medical Faculty of Mannheim, Theodor-Kutzer-Ufer, 68167 Mannheim, Germany. ulrich.boecker@med.ma.uni-heidelberg.de
Yezerskyy
Oleksandr
O
Feick
Peter
P
Manigold
Tobias
T
Panja
Asit
A
Kalina
Uwe
U
Herweck
Frank
F
Rossol
Siegbert
S
Singer
Manfred V
MV
D003429:Cross Reactions / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D029721:Drosophila Proteins; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism*; D019073:HT29 Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007375:Interleukin-1 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D007413:Intestinal Mucosa / Q000166:cytology*; D018950:Lipopolysaccharide Receptors / Q000096:biosynthesis* / Q000187:drug effects*; D008070:Lipopolysaccharides / Q000008:administration & dosage*; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000187:drug effects*; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000187:drug effects / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects; D013223:Statistics as Topic; D013997:Time Factors; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha / Q000187:drug effects / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases
D029721:Drosophila Proteins; D007375:Interleukin-1; D016209:Interleukin-8; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D019869:Phosphatidylinositol 3-Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases