title: Extracellular heat shock protein-70 induces endotoxin tolerance in THP-1 cells.
abstract: Recent data suggest that heat shock protein-70 (HSP-70), an intracellular protein, can exist in the extracellular compartment and signal through the CD14/TLR4 pathway. In this study, we tested the hypothesis that extracellular HSP-70 induces endotoxin (LPS) tolerance. Using human monocyte cell line (THP-1), initial dose-response experiments were conducted to determine a subthreshold concentration of HSP-70 that does not induce NF-kappaB activity. Differentiated THP-1 cells were preconditioned with subthreshold concentration (0.03 microg/ml HSP-70) for 18 h, followed by LPS stimulation (1 microg/ml) for 4 h. Preconditioning with HSP-70 decreased subsequent LPS-mediated NF-kappaB-dependent promoter activity and was accompanied by significant decreases of supernatant TNF levels. Furthermore, human monocytes isolated from human volunteers, subsequently preconditioned with HSP-70, demonstrated LPS tolerance as evidenced by abrogated supernatant TNF levels. Additional experiments were conducted to exclude the possibility of endotoxin contamination of HSP-70 by boiling HSP-70 at 100 degrees C for 1 h or preconditioning with equivalent concentrations of endotoxin as present in the HSP-70 preparation. These experiments indicated that induction of tolerance was not secondary to endotoxin contamination. Neutralization experiments with an anti-HSP-70 Ab confirmed the specificity of HSP-70 in tolerance induction. Preconditioning with HSP-70 attenuated cytosolic degradation of inhibitor kappaB-alpha and inhibited activation of inhibitor kappaB kinase following LPS stimulation. HSP-70 preconditioning decreased phosphorylation of the p65 subunit of NF-kappaB following LPS stimulation. These data suggest a novel role for extracellular HSP-70 in modifying mononuclear cell responses to subsequent LPS challenge.
authors.affiliation: Department of Critical Care Medicine, University of Pittsburgh School of Medicine and Children's Hospital of Pittsburgh, Pittsburgh, PA 15213, USA. anejar@upmc.edu
authors.forename: Rajesh
authors.initials: R
authors.lastname: Aneja
authors.forename: Kelli
authors.initials: K
authors.lastname: Odoms
authors.forename: Katherine
authors.initials: K
authors.lastname: Dunsmore
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Shanley
authors.forename: Hector R
authors.initials: HR
authors.lastname: Wong
mesh_terms: D019138:Antibodies, Blocking / Q000494:pharmacology; D002454:Cell Differentiation / Q000276:immunology; D045744:Cell Line, Tumor; D002474:Cell-Free System / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D004306:Dose-Response Relationship, Immunologic; D045604:Extracellular Fluid / Q000276:immunology* / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000276:immunology / Q000502:physiology*; D006358:Hot Temperature; D006801:Humans; D007108:Immune Tolerance; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000633:toxicity*; D009000:Monocytes / Q000276:immunology / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism
chemical_list: D019138:Antibodies, Blocking; D018840:HSP70 Heat-Shock Proteins; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha