title: TLR4-dependent lipopolysaccharide signalling in epithelial cells is independent of extracellular protease activity.
abstract: Epithelial cells are the first cells that encounter infecting bacteria and, as such, they have developed several mechanisms for microbial protection. We have shown previously that bladder epithelial cells express the lipopolysaccharide (LPS) receptor Toll-like receptor (TLR) 4 that enables a rapid cellular interleukin (IL)-8 response when exposed to Escherichia coli and LPS. TLR4 belongs to a family of receptors that was initially identified in Drosophila, in which Toll is required for the immune response against fungi. Fungal exposure activates a series of serine proteases that process the protein Spaetzle to a cytokine-like form that acts as a ligand for Toll. Here, we investigated whether a similar proteolytic cascade is required for human TLR activation. When screening a set of 18 protease inhibitors, three serine protease inhibitors (TPCK, TLCK and Pefabloc) were shown to inhibit LPS- and peptidoglycan-induced IL-8 production in TLR2- and TLR4-positive bladder epithelial cells. However, they were equally effective inhibitors of IL-1beta-induced signalling, indicating that their target(s) is/are located downstream of the TLRs. Further characterization showed that these inhibitors blocked I kappa B degradation but not phosphorylation in LPS-stimulated cells, which suggests that the serine protease inhibitors target the 26S proteasome. Identical results were obtained on LPS-stimulated monocytes. Based on these data, we find no evidence for the involvement of proteases upstream of TLRs in either epithelial cells or cells of the monocytic lineage.
authors.affiliation: Microbiology and Tumor Biology Center, Box 280, Karolinska Institutet, S-171 77 Stockholm, Sweden.
authors.forename: Fredrik
authors.initials: F
authors.lastname: Bäckhed
authors.forename: Staffan
authors.initials: S
authors.lastname: Normark
authors.forename: Agneta
authors.initials: A
authors.lastname: Richter-Dahlfors
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D029721:Drosophila Proteins; D004347:Drug Interactions; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology*; D004926:Escherichia coli / Q000502:physiology*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007375:Interleukin-1 / Q000494:pharmacology; D016209:Interleukin-8 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D010457:Peptidoglycan / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D001743:Urinary Bladder / Q000166:cytology
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029721:Drosophila Proteins; D020672:I-kappa B Proteins; D007375:Interleukin-1; D016209:Interleukin-8; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D010457:Peptidoglycan; D011956:Receptors, Cell Surface; C419843:TLR protein, bacteria; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014108:Tosylphenylalanyl Chloromethyl Ketone; D012697:Serine Endopeptidases