Human endothelial as well as epithelial cells were shown to respond to lipopolysaccharides (LPSs). However, the expression and release of CD14 by these so-called CD14-negative cells have not been studied in detail. We investigated three human intestinal epithelial cell lines (ECLs), SW-480, HT-29, and Caco-2, for their expression of CD14 and CD11c/CD18 as well as their responsiveness to endotoxins. Fluorescence-activated cell sorter analysis revealed no expression of CD11c/CD18, but there was low expression of membrane-bound CD14 on HT-29, Caco-2, and SW-480 ECLs. Both Western blotting and reverse transcription-PCR confirmed the CD14 positivity of all three intestinal ECLs. No substantial modulation of CD14 expression was achieved after 6, 8, 18, 24, and 48 h of cultivation with 10-fold serial dilutions of LPS ranging from 0.01 ng/ml to 100 microg/ml. Interestingly, soluble CD14 was found in the tissue culture supernatants of all three ECLs. Finally, only HT-29 and SW-480, and not Caco-2, cells responded to LPS exposure (range, 0.01 ng/ml to 100 microg/ml) by interleukin 8 release. Thus, we show that HT-29, SW-480, and Caco-2 human intestinal ECLs express membrane-bound CD14. As Caco-2 cells did not respond to LPS, these cell lines might be an interesting model for studying the receptor complex for LPS. The fact that human intestinal epithelial cells are capable not only of expression but also of release of soluble CD14 may have important implications in vivo, e.g., in shaping the interaction between the mucosal immune system and bacteria in the gut and/or in the pathogenesis of endotoxin shock.

• MeSH
• Cell Line MeSH
• HT29 Cells MeSH
• Caco-2 Cells MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Epithelial Cells immunology   MeSH
• Humans MeSH
• Lipopolysaccharide Receptors genetics   metabolism   MeSH
• Lipopolysaccharides pharmacology   MeSH
• RNA, Messenger metabolism   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Flow Cytometry MeSH
• Intestines cytology   MeSH
• Blotting, Western MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Lipopolysaccharide Receptors MeSH
• Lipopolysaccharides MeSH
• RNA, Messenger MeSH

According to a recently proposed hypothesis, initiation of signal transduction via immunoreceptors depends on interactions of the engaged immunoreceptor with glycosphingolipid-enriched membrane microdomains (GEMs). In this study, we describe a novel GEM-associated transmembrane adaptor protein, termed phosphoprotein associated with GEMs (PAG). PAG comprises a short extracellular domain of 16 amino acids and a 397-amino acid cytoplasmic tail containing ten tyrosine residues that are likely phosphorylated by Src family kinases. In lymphoid cell lines and in resting peripheral blood alpha/beta T cells, PAG is expressed as a constitutively tyrosine-phosphorylated protein and binds the major negative regulator of Src kinases, the tyrosine kinase Csk. After activation of peripheral blood alpha/beta T cells, PAG becomes rapidly dephosphorylated and dissociates from Csk. Expression of PAG in COS cells results in recruitment of endogenous Csk, altered Src kinase activity, and impaired phosphorylation of Src-specific substrates. Moreover, overexpression of PAG in Jurkat cells downregulates T cell receptor-mediated activation of the transcription factor nuclear factor of activated T cells. These findings collectively suggest that in the absence of external stimuli, the PAG-Csk complex transmits negative regulatory signals and thus may help to keep resting T cells in a quiescent state.

• MeSH
• Adaptor Proteins, Signal Transducing MeSH
• Lymphocyte Activation * MeSH
• CD3 Complex metabolism   MeSH
• CSK Tyrosine-Protein Kinase MeSH
• Phosphoproteins genetics   metabolism   MeSH
• Glycosphingolipids metabolism   MeSH
• Cloning, Molecular MeSH
• DNA, Complementary genetics   MeSH
• Humans MeSH
• Membrane Proteins genetics   metabolism   MeSH
• Molecular Sequence Data MeSH
• Receptors, Antigen, T-Cell metabolism   MeSH
• Amino Acid Sequence MeSH
• Signal Transduction MeSH
• src-Family Kinases MeSH
• T-Lymphocytes immunology   MeSH
• Protein-Tyrosine Kinases metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adaptor Proteins, Signal Transducing MeSH
• CD3 Complex MeSH
• CSK Tyrosine-Protein Kinase MeSH
• CSK protein, human MeSH Browser
• Phosphoproteins MeSH
• Glycosphingolipids MeSH
• DNA, Complementary MeSH
• Membrane Proteins MeSH
• PAG1 protein, human MeSH Browser
• Receptors, Antigen, T-Cell MeSH
• src-Family Kinases MeSH
• Protein-Tyrosine Kinases MeSH