Toll-like receptor 4 functions intracellularly in human coronary artery endothelial cells: roles of LBP and sCD14 in mediating LPS responses.
Endothelial cells are activated by microbial agonists through Toll-like receptors (TLRs) to express inflammatory mediators; this is of significance in acute as well as chronic inflammatory states such as septic shock and atherosclerosis, respectively. We investigated mechanisms of lipopolysaccharide (LPS)-induced cell activation in human coronary artery endothelial cells (HCAEC) using a combination of FACS, confocal microscopy, RT-PCR, and functional assays. We found that TLR4, in contrast to TLR2, is not only located intracellularly but also functions intracellularly. That being the case, internalization of LPS is required for activation. We further characterized the HCAEC LPS uptake system and found that HCAEC exhibit an effective LPS uptake only in the presence of LPS binding protein (LBP). In addition to its function as a catalyst for LPS-CD14 complex formation, LBP enables HCAEC activation at low LPS concentrations by facilitating the uptake, and therefore delivery, of LPS-CD14 complexes to intracellular TLR4-MD-2. LBP-dependent uptake involves a scavenger receptor pathway. Our findings may be of pathophysiological relevance in the initial response of the organism to infection. Results further suggest that LBP levels, which vary as LBP is an acute phase reactant, could be relevant to initiating inflammatory responses in the vasculature in response to chronic or recurring low LPS.
Dunzendorfer
Stefan
S
Scripps Research Institute, Department of Immunology, La Jolla, California 92037, USA.
Lee
Hyun-Ku
HK
Soldau
Katrin
K
Tobias
Peter S
PS
D000209:Acute-Phase Proteins / Q000502:physiology; D000210:Acute-Phase Reaction; D000328:Adult; D000818:Animals; D000951:Antigens, Neoplasm / Q000096:biosynthesis / Q000235:genetics; D000954:Antigens, Surface / Q000096:biosynthesis / Q000235:genetics; D002352:Carrier Proteins / Q000502:physiology; D002451:Cell Compartmentation; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D003331:Coronary Vessels / Q000166:cytology*; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D006017:Glycolipids / Q000494:pharmacology; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism*; D008050:Lipid A / Q000031:analogs & derivatives* / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051218:Lymphocyte Antigen 96; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009000:Monocytes / Q000378:metabolism / Q000648:ultrastructure; D012333:RNA, Messenger / Q000096:biosynthesis; D011956:Receptors, Cell Surface / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D011971:Receptors, Immunologic / Q000502:physiology; D051116:Receptors, Scavenger; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014471:Umbilical Veins / Q000166:cytology; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D002352:Carrier Proteins; D006017:Glycolipids; D008050:Lipid A; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D051116:Receptors, Scavenger; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D000099231:Lipopolysaccharide-Binding Protein; C495377:LY96 protein, human; C495342:TLR2 protein, human; C495345:TLR4 protein, human; C493487:Tlr4 protein, mouse; C040142:lipid A precursors, bacterial; C052646:sarcoma glycoprotein gp96 rejection antigens