Receptors involved in the oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine-mediated synthesis of interleukin-8. A role for Toll-like receptor 4 and a glycosylphosphatidylinositol-anchored protein.
We demonstrated previously that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC) and, specifically, the component lipid 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine increase interleukin-8 (IL-8) synthesis in aortic endothelial cells. The goal of the current studies was to characterize the receptor complex mediating the increased transcription of IL-8. We demonstrate that scavenger receptor class A, types I and II, lectin-like ox-LDL receptor-1, macrophage receptor with collagenous structure, and CD36 are not responsible for the increase in IL-8. Using dominant-negative constructs and antisense oligonucleotides, we demonstrate a role for Toll-like receptor 4 (TLR4) as the ox-PAPC receptor mediating IL-8 transcription. We demonstrate that a glycosylphosphatidylinositol (GPI)-anchored protein is also necessary because phosphatidylinositol-specific phospholipase C pretreatment inhibited the effect of ox-PAPC. CD14, a GPI-anchored protein that associates with TLR4 in mediating lipopolysaccharide action, did not appear to mediate ox-PAPC action because ox-PAPC-induced IL-8 transcription was not blocked by anti-CD14 neutralizing antibodies nor was it augmented by the addition of soluble CD14 or overexpression of membrane CD14. Instead, anti-TLR4 antibodies immunoprecipitated a 37-kDa protein that also bound ox-PAPC. A protein of this same size was found in aerolysin overlays used to detect GPI-anchored proteins. Therefore, these studies suggest that ox-PAPC may initially bind to a 37-kDa GPI-anchored protein, which interacts with TLR4 to induce IL-8 transcription.
Walton
Kimberly A
KA
Department of Medicine, UCLA, Los Angeles, California 90095, USA.
Hsieh
Xavier
X
Gharavi
Nima
N
Wang
Shirley
S
Wang
Grace
G
Yeh
Michael
M
Cole
Amy L
AL
Berliner
Judith A
JA
D000818:Animals; D001011:Aorta / Q000166:cytology; D015153:Blotting, Western; D018955:CD36 Antigens / Q000096:biosynthesis; D016466:CHO Cells; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D004730:Endothelium, Vascular / Q000166:cytology; D004797:Enzyme-Linked Immunosorbent Assay; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis* / Q000378:metabolism*; D018950:Lipopolysaccharide Receptors / Q000096:biosynthesis; D008077:Lipoproteins, LDL / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D010100:Oxygen / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism*; D011233:Precipitin Tests; D011956:Receptors, Cell Surface / Q000378:metabolism / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014158:Transcription, Genetic; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
C468114:1-palmitoyl-2-arachidonyl-3-phosphorylcholine; D018955:CD36 Antigens; D017261:Glycosylphosphatidylinositols; D016209:Interleukin-8; D018950:Lipopolysaccharide Receptors; D008077:Lipoproteins, LDL; D008562:Membrane Glycoproteins; D010713:Phosphatidylcholines; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D010738:Type C Phospholipases; D010100:Oxygen