Recombinant human heat shock protein 60 does not induce the release of tumor necrosis factor α from murine macrophages

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 mug/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 mug/ 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).