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

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 ␣ (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. In the current study, we demonstrated that a highly purified rhHsp70 preparation (designated as rhHsp70 -1) with a LPS content of 1.4 pg/ g was unable to induce TNF␣ release by RAW264.7 murine macrophages at concentrations up to 5 g/ml. In contrast, a less purified rhHsp70 preparation (designated as rhHsp70 -2) 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. 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␣-inducing activity of rhHsp70 -2. The addition of LPS at the concentration found in rhHsp70 -2 to rhHsp70 -1 resulted in the same TNF␣-inducing activity as observed with rhHsp70 -2. The TNF␣-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␣ release from murine macrophages and that the observed TNF␣-inducing activity in the rhHsp70 -2 preparation is entirely due to the contaminating LPS.The 70-kDa heat shock proteins (Hsp70s) 1 are highly conserved proteins expressed both constitutively (Hsc70) and under stressful conditions (Hsp70) in all prokaryotes and eukaryotes (1, 2). Members of the Hsp70 protein family play essential roles as molecular chaperones in the cytosol, mitochondria, and endoplasmic reticulum. 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 (7-9). These molecular chaperone functions of Hsp70s require the enzymatic hydrolysis of ATP and cycles of bound nucleotide exchange.Recently, Hsp70s were found to be present in circulation, and their levels were increased in a number of pathological conditions (10 -12). Studies also show that Hsp70 is a potent

The heat shock protein Hsp70 enhances antigen-specific proliferation of human CD4+ memory T cells

“…As mentioned above, HSP themselves can stimulate the maturation of APC and the production of proinflammatory cytokines [10,30,31], and the HSP effect has to be carefully distinguished from effects of contaminations like LPS [32,33]. In our experiments, complexing Hsp70 with the antigenic peptide was crucial for an enhanced proliferation of CD4 + T cells.…”

“…Different mechanisms explaining the effect of HSP have been discussed, including an increased uptake and enhanced intracellular processing of chaperoned peptide by APC [18,28,29], a direct effect of HSP on APC [6, 10,[29][30][31] or an effect only due to microbial contaminations of the HSP preparation [32,33]. HSP:peptide complexes can be released by necrotic cells [2] and taken up by APC via specific receptors (reviewed in [34]).…”

CD4+CD25+ Treg induction by an HSP60‐derived peptide SJMHE1 from Schistosoma japonicum is TLR2 dependent

“…LPS has been reported to activate various types of lymphocytes via TLR2 signaling [57]. To exclude possible LPS contamination, we pre-treated the SjHSP60, SJMHE1, and OVA 323-339 with polymyxin B-agarose as described previously [58].…”

Integrating the cell stress response: a new view of molecular chaperones as immunological and physiological homeostatic regulators

“…and Figueiredo and coworkers have recently reported that 'Hsp70' expressed in eukaryotic cells is still active as a T lymphocyte stimulator [106], refuting the work of Gao and Ye [103,104]. In addition, the work of Lehner and co-workers has comprehensively ruled out LPS as a contributor to the cellular effects produced by M. tuberculosis Hsp70 [84].…”

“…Post-translational modifications (which will not be present in recombinant proteins prepared in E. coli) could influence activity. A more serious issue hindering the study of the Hsp70 system is a small number of papers which claim that the activity of 'Hsp70' protein is due to either contaminating E. coli components such as LPS [103] or flagellin [104], or to nucleotides left over as part of the isolation procedure [105]. The flagellin paper [104] focused on Hsp70 stimulation of T cells.…”