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 ␣ (TNF-␣) 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-␣ release from murine macrophages at concentrations of up to 10 g/ml. In contrast, a less purified rhHSP60 preparation (designated rhHSP60-2) was able to induce a marked TNF-␣ release at concentrations as low as 1 g/ml. Failure of rhHSP60-1 to induce TNF-␣ 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-␣-inducing activity of rhHSP60-2. However, both the endotoxin activity and the TNF-␣-inducing activity of rhHSP60-2 were essentially eliminated after passing through a polymyxin Bagarose column that removes LPS and LPS-associated molecules from the rhHSP60 preparation. The TNF-␣-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-␣ release from macrophages. Approximately 50% of the observed TNF-␣-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).The 60-kDa heat shock proteins (HSPs) 1 (HSP60 or chaperonins) are highly conserved intracellular proteins expressed in all organisms, both constitutively and under stress conditions.

Lipopolysaccharide is a frequent and significant contaminant in microglia‐activating factors

“…In light of the findings presented here for microglia, and consistent with what has been previously suggested in the macrophage/monocyte literature (Gao and Tsan, 2003b), we propose that the following considerations be taken into account for future studies involving microglia. First, whenever possible, pharmaceutical-grade, endotoxin-free reagents be used.…”

“…Interestingly, the findings not only confirmed the functionally significant levels of LPS contamination in the plasmaderived thrombin preparations, but also demonstrated the scope and severity of the LPS contamination problem for a host of other factors and compounds relevant to CNS pathophysiology. The results are also consistent with several isolated findings from the immunology literature describing the effects of LPS contamination on macrophage/ monocyte activation by a few selected commercial-grade protein preparations including heat-shock protein-60 (Gao and Tsan, 2003b), −70 (Gao and Tsan, 2003) and the lectin pokeweed mitogen (Yang et al, 2006).…”

Heat-shock proteins induce T-cell regulation of chronic inflammation

“…18 19 However, such reports have been questioned by studies indicating that proinflammatory activity of HSP via such receptors was solely due to bacterial contaminants. [20][21][22] Even though this cannot be entirely excluded, because regular tests to exclude that contamination by lipopolysaccharide (LPS) is responsible for the observed effects (such as polymyxin B preincubation and/or protein denaturing by boiling HSP), might not affect all other bacterial compounds, several reports show that highly purified HSP can still activate dendritic cells and macrophages. For example, HSP derived from murine liver and kidney has been shown to be able to activate dendritic cells and macrophages.…”

Pathogen-Induced Apoptotic Neutrophils Express Heat Shock Proteins and Elicit Activation of Human Macrophages

“…Numerous investigations have shown that both HSP60 and HSP70 potently provoke proinflammatory responses (29,(33)(34)(35)(36), but two recent studies of murine M have suggested that the TNF-␣-inducing activities of recombinant HSP preparations are due entirely to contaminating LPS (37,38). Accordingly, to clarify the role of HSP in activation of human M , we used highly purified recombinant HSP60 and HSP70 preparations (ESP-540 and ESP-555; StressGen Biotechnologies, Victoria, Canada), which contain low levels of endotoxin and do not induce release of TNF-␣ from murine M (37,38).…”

“…Accordingly, to clarify the role of HSP in activation of human M , we used highly purified recombinant HSP60 and HSP70 preparations (ESP-540 and ESP-555; StressGen Biotechnologies, Victoria, Canada), which contain low levels of endotoxin and do not induce release of TNF-␣ from murine M (37,38). In our experiments, both recombinant HSP60 and HSP70, at concentrations up to 5 g/ml, caused only a slight increase in TNF-␣ production by human M (Fig.…”