Endotoxin Contamination in Recombinant Human Heat Shock Protein 70 (Hsp70) Preparation Is Responsible for the Induction of Tumor Necrosis Factor [alpha] Release by Murine Macrophages

Using commercially available recombinant human heat shock protein 70 (rhHsp70), recent studies have shown that rhHsp70 could induce the production of tumor necrosis factor [alpha] (TNF[alpha]) by macrophages and monocytes in a manner similar to lipopolysaccharide (LPS) e.g. via CD14 and Toll-like receptor 4-mediated signal transduction pathway. In the current study, we demonstrated that a highly purified rhHsp70 preparation (designated as rhHsp70-1) with a LPS content of 1.4 pg/ mu g was unable to induce TNF[alpha] release by RAW264.7 murine macrophages at concentrations up to 5 mu g/ml. In contrast, a less purified rhHsp70 preparation (designated as rhHsp70-2) at 1 mu g/ml with a LPS content of 0.2 ng/ mu g was able to induce TNF[alpha] release to the same extent as that induced by 0.2 ng/ml LPS. Failure of rhHsp70-1 to induce TNF[alpha] release was not because of defective physical properties since rhHsp70-1 and rhHsp70-2 contained identical hsp70 content as determined by SDS gels stained with Coomassie Blue and Western blots probed with an anti-rhHsp70 antibody. Both rhHsp70 preparations also had similar enzymatic activities as judged by their ability to remove clathrin from clathrin-coated vesicles. Removal of LPS from rhHsp70-2 by polymyxin B-agarose column or direct addition of polymyxin B to the incubation medium essentially eliminated the TNF[alpha]-inducing activity of rhHsp70-2. The addition of LPS at the concentration found in rhHsp70-2 to rhHsp70-1 resulted in the same TNF[alpha]-inducing activity as observed with rhHsp70-2. The TNF[alpha]-inducing activities of rhHsp-2, LPS alone, and LPS plus rhHsp70-1 were all equally sensitive to heat inactivation. These results suggest that rhHsp-70 does not induce TNF[alpha] release from murine macrophages and that the observed TNF[alpha]-inducing activity in the rhHsp70-2 preparation is 3ntirely due to the contaminating LPS.