Extensive work has suggested that a number of endogenous molecules such as heat shock proteins (hsp) may be potent activators of the innate immune system capable of inducing proinflammatory cytokine production by the monocyte-macrophage system and the activation and maturation of dendritic cells. The cytokine-like effects of these endogenous molecules are mediated via the Toll-like receptor (TLR) signal-transduction pathways in a manner similar to lipopolysaccharide (LPS; via TLR4) and bacterial lipoproteins (via TLR2). However, recent evidence suggests that the reported cytokine effects of hsp may be a result of the contaminating LPS and LPS-associated molecules. The reasons for previous failure to recognize the contaminant(s) being responsible for the putative TLR ligands of hsp include failure to use highly purified hsp free of LPS contamination; failure to recognize the heat sensitivity of LPS; and failure to consider contaminant(s) other than LPS. Whether other reported putative endogenous ligands of TLR2 and TLR4 are a result of contamination of pathogen-associated molecular patterns is not clear. It is essential that efforts should be directed to conclusively determine whether the reported putative endogenous ligands of TLRs are a result of the endogenous molecules or of contaminant(s), before exploring further the implication and therapeutic potential of these putative TLR ligands.

Heat shock proteins (HSPs) such as HSP 60 (Hsp60), Hsp70, Hsp90, and gp96, have been reported to play important roles in antigen presentation and cross-presentation, activation of macrophages and lymphocytes, and activation and maturation of dendritic cells. HSPs contain peptide-binding domains that bind exposed hydrophobic residues of substrate proteins. As part of their molecular chaperone functions, HSPs bind and deliver chaperoned, antigenic peptides to MHC class I molecules at the cell surface for presentation to lymphocytes. HSPs also bind nonprotein molecules with exposed hydrophobic residues including lipid-based TLR ligands. Recombinant HSP products may be contaminated with pathogen-associated molecules that contain exposed hydrophobic residues such as LPS (a TLR4 ligand), lipoprotein (a TLR2 ligand), and flagellin (a TLR5 ligand). These contaminants appear to be responsible for most, if not all, reported in vitro cytokine effects of HSPs, as highly purified HSPs do not show any cytokine effects. We propose that HSPs are molecular chaperones that bind protein and nonprotein molecules with exposed hydrophobic residues. The reported antigen presentation and cross-presentation and in vitro HSP cytokine functions are a result of molecules bound to or chaperoned by HSPs but not a result of HSPs themselves.

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

Extensive work in the last 10 years has suggested that heat shock proteins (HSPs) may be potent activators of the innate immune system. It has been reported that Hsp60, Hsp70, Hsp90, and gp96 are capable of inducing the production of proinflammatory cytokines by the monocyte-macrophage system and the activation and maturation of dendritic cells (antigen-presenting cells) in a manner similar to the effects of lipopolysaccharide (LPS) and bacterial lipoprotein, e.g., via CD14/Toll-like receptor2 (TLR2) and CD14/TLR4 receptor complex-mediated signal transduction pathways. However, recent evidence suggests that the reported cytokine effects of HSPs may be due to the contaminating LPS and LPS-associated molecules. The reasons for previous failure to recognize the contaminant(s) as being responsible for the reported HSP cytokine effects include failure to use highly purified, low-LPS preparations of HSPs; failure to recognize the heat sensitivity of LPS; and failure to consider contaminant(s) other than LPS. Thus it is essential that efforts should be directed to conclusively determine whether the reported HSP cytokine effects are due to HSPs or to contaminant(s) present in the HSP preparations before further exploring the implication and therapeutic potential of the putative cytokine function of HSPs.