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

Abstract

Recent studies have shown that commercially available recombinant human heat shock protein 60 (rhHSP60) could induce tumor necrosis factor α (TNF-α) 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-α release from murine macrophages at concentrations of up to 10 μg/ml. In contrast, a less purified rhHSP60 preparation (designated rhHSP60-2) was able to induce a marked TNF-α release at concentrations as low as 1 μg/ml. Failure of rhHSP60-1 to induce TNF-α 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-α-inducing activity of rhHSP60-2. However, both the endotoxin activity and the TNF-α-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-α-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-α release from macrophages. Approximately 50% of the observed TNF-α-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).