Abstract
A possible function of eukaryotic heat shock protein 60 (Hsp60) as endogenous danger signal has been controversially discussed in the past. Hsp60 was shown to induce the secretion of proinflammatory cytokines in professional antigen-presenting cells and to enhance the activation of T cells in primary stimulation. However, in vitro activation of macrophages by Hsp60 was attributed to contaminating endotoxin in the recombinant Hsp60 protein preparations. Here, we employ low endotoxin recombinant human Hsp60 and murine Hsp60 expressed by eukaryotic cell lines to dissect the Hsp60 protein-mediated effects from biologic effects that are mediated by prokaryotic contaminants in the Hsp60 protein preparation. The induction of tumor necrosis factor-alpha secretion in mouse macrophages is lost after endotoxin removal and is not mediated by Hsp60 expressed in eukaryotic systems. In contrast, the Hsp60-mediated enhancement of antigen-specific T cell activation does not correlate with endotoxin contamination. Moreover, Hsp60 that is expressed on the surface of different eukaryotic cell lines increases the activation of T cells in primary stimulation. Taken together, we provide evidence that endogenous Hsp60, which is thought to be released from dying infected cells in vivo, has a biological function that is not due to contaminating pathogen-associated molecules.
Highlights
Pattern recognition receptors represent an ancient family of receptors on professional antigen-presenting cells (APC),1 sensing pathogenic infection by the presence of conserved prokaryotic molecules, the so-called pathogen-associated molecular pattern (PAMP) [1]
We have shown that the addition of heat shock protein 60 (Hsp60) to cultures containing peritoneal exudate macrophages (PEC) and ex vivo purified T cells expressing transgenic MHC class I [25] or MHC class II [26] restricted TCRs enhanced and accelerated the release of antigen-specific IFN-␥
No Induction of TNF-␣ Release from Murine Macrophages by Low Endotoxin Hsp60 —The human recombinant Hsp60 protein used in the experiments contained an endotoxic activity of 22 EU/mg, as determined by the Limulus amoebocyte lysate (LAL) assay
Summary
Pattern recognition receptors represent an ancient family of receptors on professional antigen-presenting cells (APC),1 sensing pathogenic infection by the presence of conserved prokaryotic molecules, the so-called pathogen-associated molecular pattern (PAMP) [1]. The comparison of the commercially available recombinant murine Hsp60 from StressGen Biotechnologies revealed that only the endotoxin-contaminated (48 EU/mg) Hsp60 preparation induced TNF-␣ release in RAW 264.7 cells, whereas the low endotoxin (Ͻ3 EU/mg) preparation was biologically inactive (Fig. 1C).
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