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

Baochong Gao,Min-Fu Tsan

doi:10.1074/jbc.m208742200

Abstract

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 (TNFalpha) 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/microg was unable to induce TNFalpha release by RAW264.7 murine macrophages at concentrations up to 5 microg/ml. In contrast, a less purified rhHsp70 preparation (designated as rhHsp70-2) at 1 microg/ml with a LPS content of 0.2 ng/microg was able to induce TNFalpha release to the same extent as that induced by 0.2 ng/ml LPS. Failure of rhHsp70-1 to induce TNFalpha 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 TNFalpha-inducing activity of rhHsp70-2. The addition of LPS at the concentration found in rhHsp70-2 to rhHsp70-1 resulted in the same TNFalpha-inducing activity as observed with rhHsp70-2. The TNFalpha-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 TNFalpha release from murine macrophages and that the observed TNFalpha-inducing activity in the rhHsp70-2 preparation is entirely due to the contaminating LPS.

Highlights

Using commercially available recombinant human heat shock protein 70, recent studies have shown that rhHsp70 could induce the production of tumor necrosis factor ␣ (TNF␣) by macrophages and monocytes in a manner similar to lipopolysaccharide (LPS) e.g. via CD14 and Toll-like receptor 4-mediated signal transduction pathway
A less purified rhHsp70 preparation at 1 ␮g/ml with a LPS content of 0.2 ng/␮g was able to induce TNF␣ release to the same extent as that induced by 0.2 ng/ml LPS
Failure of rhHsp70 –1 to induce TNF␣ 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

Summary

The abbreviations used are

Heat shock protein 70; rhHsp, recombinant human Hsp; TNF, tumor necrosis factor; LPS, lipopolysaccharide; EU, endotoxin units; LAL, Limulus amebocyte lysate. Hsp70s are required for nascent or misfolded protein folding [3, 4], protein translocation into endoplasmic reticulum and mitochondria [5, 6], and uncoating of clathrin-coated vesicles [7,8,9] These molecular chaperone functions of Hsp70s require the enzymatic hydrolysis of ATP and cycles of bound nucleotide exchange. Using recombinant human Hsp (rhHsp70), it has been demonstrated that Hsp induces the production of pro-inflammatory cytokines such as tumor necrosis factor-␣ (TNF␣) and interleukin-6 via the CD14 and Toll-like receptor (TLR)-mediated signal transduction pathway [12, 15,16,17]. We demonstrated that the ability of commercially available rhHsp preparations to induce TNF␣ production by murine macrophages is solely due to the contaminating LPS

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION