Extracellular heat shock protein-70 induces endotoxin tolerance in THP-1 cells

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-kappa B activity. Differentiated THP-1 cells were preconditioned with subthreshold concentration (0.03 mu g/ml HSP-70) for 18 h, followed by LPS stimulation (1 mu g/ml) for 4 h. Preconditioning with HSP-70 decreased subsequent LPS-mediated NF-kappa B-dependent promoter activity and was accompanied by significant decreases of supernatant TNF levels. Furthermore, human monocytes isolated from human volunteers, subsequently preconditioned with HSP70, 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 kappa B-alpha and inhibited activation of inhibitor kappa B kinase following LPS stimulation. HSP-70 preconditioning decreased phosphorylation of the p65 subunit of NF-kappa B following LPS stimulation. These data suggest a novel role for extracellular HSP-70 in modifying mononuclear cell responses to subsequent LPS challenge.