title: Bacterial lipopolysaccharide augments febrile-range hyperthermia-induced heat shock protein 70 expression and extracellular release in human THP1 cells.
abstract: Sepsis, a devastating and often lethal complication of severe infection, is characterized by fever and dysregulated inflammation. While infections activate the inflammatory response in part through Toll-like receptors (TLRs), fever can partially activate the heat shock response with generation of heat shock proteins (HSPs). Since extracellular HSPs, especially HSP70 (eHSP70), are proinflammatory TLR agonists, we investigated how exposure to the TLR4 agonist, bacterial lipopolysaccharide (LPS) and febrile range hyperthermia (FRH; 39.5°C) modify HSP70 expression and extracellular release. Using differentiated THP1 cells, we found that concurrent exposure to FRH and LPS as well as TLR2 and TLR3 agonists synergized to activate expression of inducible HSP72 (HSPA1A) mRNA and protein via a p38 MAP kinase-requiring mechanism. Treatment with LPS for 6 h stimulated eHSP70 release; levels of eHSP70 released at 39.5°C were higher than at 37°C roughly paralleling the increase in intracellular HSP72 in the 39.5°C cells. By contrast, 6 h exposure to FRH in the absence of LPS failed to promote eHSP70 release. Release of eHSP70 by LPS-treated THP1 cells was inhibited by glibenclamide, but not brefeldin, indicating that eHSP70 secretion occurred via a non-classical protein secretory mechanism. Analysis of eHSP70 levels in exosomes and exosome-depleted culture supernatants from LPS-treated THP1 cells using ELISA demonstrated similar eHSP70 levels in unfractionated and exosome-depleted culture supernatants, indicating that LPS-stimulated eHSP70 release did not occur via the exosome pathway. Immunoblot analysis of the exosome fraction of culture supernatants from these cells showed constitutive HSC70 (HSPA8) to be the predominant HSP70 family member present in exosomes. In summary, we have shown that LPS stimulates macrophages to secrete inducible HSP72 via a non-classical non-exosomal pathway while synergizing with FRH exposure to increase both intracellular and secreted levels of inducible HSP72. The impact of increased macrophage intracellular HSP70 levels and augmented secretion of proinflammatory eHSP70 in the febrile, infected patient remains to be elucidated.
authors: Division of Pulmonary and Critical Care, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
authors: Mohan E
authors: ME
authors: Tulapurkar
authors: Division of Pulmonary and Critical Care, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
authors: Aparna
authors: A
authors: Ramarathnam
authors: Division of Pulmonary and Critical Care, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States of America; Cytokine Core Laboratory, University of Maryland School of Medicine, Baltimore, Maryland, United States of America; Research Services of the Baltimore Veteran Affairs Medical Center, Baltimore, Maryland, United States of America.
authors: Jeffrey D
authors: JD
authors: Hasday
authors: Division of Pulmonary and Critical Care, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States of America; Research Services of the Baltimore Veteran Affairs Medical Center, Baltimore, Maryland, United States of America.
authors: Ishwar S
authors: IS
authors: Singh
mesh_terms: D002460:Cell Line; D015870:Gene Expression; D050883:HSC70 Heat-Shock Proteins / Q000096:biosynthesis* / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D015398:Signal Transduction; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors
chemical_list: D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C490786:HSPA1A protein, human; C494371:HSPA8 protein, human; D008070:Lipopolysaccharides; D048051:p38 Mitogen-Activated Protein Kinases