title: An interplay between scavenger receptor A and CD14 during activation of J774 cells by high concentrations of LPS.
abstract: Lipopolysaccharide (LPS) activates macrophages by binding to the TLR4/MD-2 complex and triggers two pro-inflammatory signaling pathways: one relies on MyD88 at the plasma membrane, and the other one depends on TRIF in endosomes. When present in high doses, LPS is internalized and undergoes detoxification. We found that the uptake of a high concentration of LPS (1000ng/ml) in macrophage-like J774 cells was upregulated upon inhibition of clathrin- and dynamin-mediated endocytosis which, on the other hand, strongly reduced the production of pro-inflammatory mediators TNF-α and RANTES. The binding and internalization of high amounts of LPS was mediated by scavenger receptor A (SR-A) with participation of CD14 without an engagement of TLR4. Occupation of SR-A by dextran sulfate or anti-SR-A antibodies enhanced LPS-induced production of TNF-α and RANTES by about 70%, with CD14 as a limiting factor. Dextran sulfate also elevated the cell surface levels of TLR4 and CD14, which could have contributed to the upregulation of the pro-inflammatory responses. Silencing of SR-A expression inhibited the LPS-triggered TNF-α production whereas RANTES release was unchanged. These data indicate that SR-A is required for maximal production of TNF-α in cells stimulated with LPS, possibly by modulating the cell surface levels of TLR4 and CD14.
authors.affiliation: Department of Cell Biology, Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland.
authors.forename: Maciej
authors.initials: M
authors.lastname: Czerkies
authors.forename: Kinga
authors.initials: K
authors.lastname: Borzęcka
authors.forename: Mykola I
authors.initials: MI
authors.lastname: Zdioruk
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Płóciennikowska
authors.forename: Andrzej
authors.initials: A
authors.lastname: Sobota
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Kwiatkowska
mesh_terms: D000818:Animals; D019138:Antibodies, Blocking / Q000378:metabolism; D002460:Cell Line; D018946:Chemokine CCL5 / Q000378:metabolism; D016264:Dextran Sulfate / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000276:immunology; D008262:Macrophage Activation / Q000235:genetics; D008264:Macrophages / Q000276:immunology*; D051379:Mice; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics; D051117:Scavenger Receptors, Class A / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D019138:Antibodies, Blocking; D018946:Chemokine CCL5; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D034741:RNA, Small Interfering; D051117:Scavenger Receptors, Class A; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D016264:Dextran Sulfate
keywords: CD14; CTX-FITC; Endocytosis; HEPES-buffered saline; LBP; LPS conjugated with Alexa Fluor 488; LPS-AF488; LPS-binding protein; Lipopolysaccharide; PD buffer; SR-A; Scavenger receptor; TLR; poly(I:C); polyinosinic–polycytidylic acid; scavenger receptor A; subunit B of cholera toxin conjugated with FITC; transferrin conjugated with Alexa Fluor 647; transferrin-AF647