title: Recombinant human heat shock protein 60 does not induce the release of tumor necrosis factor alpha from murine macrophages.
abstract: Recent studies have shown that commercially available recombinant human heat shock protein 60 (rhHSP60) could induce tumor necrosis factor alpha (TNF-alpha) release from macrophages and monocytes in a manner similar to that of lipopolysaccharide (LPS), e.g. via CD14 and Toll-like receptor 4 complex-mediated signal transduction pathway. In this study, we demonstrated that a highly purified rhHSP60 preparation with low endotoxin activity (designated rhHSP60-1) was unable to induce TNF-alpha release from murine macrophages at concentrations of up to 10 microg/ml. In contrast, a less purified rhHSP60 preparation (designated rhHSP60-2) was able to induce a marked TNF-alpha release at concentrations as low as 1 microg/ml. Failure of rhHSP60-1 to induce TNF-alpha release was not due to defective physical properties because rhHSP60-1 and rhHSP60-2 contained a similar amount of HSP60 as determined by SDS gels stained with Coomassie Blue and Western blots probed with an anti-rhHSP60 antibody. Both rhHSP60 preparations also had similar enzymatic activities as judged by their ability to hydrolyze ATP. Polymyxin B added in the incubation media abolished the endotoxin activity but inhibited only about 50% of the TNF-alpha-inducing activity of rhHSP60-2. However, both the endotoxin activity and the TNF-alpha-inducing activity of rhHSP60-2 were essentially eliminated after passing through a polymyxin B-agarose column that removes LPS and LPS-associated molecules from the rhHSP60 preparation. The TNF-alpha-inducing activities of both rhHSP60-2 and LPS with equivalent endotoxin activity present in rhHSP60-2 were equally sensitive to heat inactivation. These results suggest that rhHSP60 does not induce TNF-alpha release from macrophages. Approximately 50% of the observed TNF-alpha-inducing activity in the rhHSP60-2 preparation is due to LPS contamination, whereas the rest of the activity was due to the contamination of LPS-associated molecule(s).
authors.affiliation: Institute for Clinical Research, Washington, D. C. 20422, USA. baochong.gao@med.va.gov
authors.forename: Baochong
authors.initials: B
authors.lastname: Gao
authors.forename: Min-Fu
authors.initials: MF
authors.lastname: Tsan
mesh_terms: D000818:Animals; D002460:Cell Line; D018834:Chaperonin 60 / Q000235:genetics* / Q000494:pharmacology / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D011994:Recombinant Proteins / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D018834:Chaperonin 60; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha