The online version of this article (doi:10.1186/1476-9255-9-11) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
ML: conceived and performed experiments, analyzed data, and helped write the manuscript. YZ: conceived and performed experiments, analyzed data, and helped write the manuscript. TC: conceived and performed experiments, analyzed data, and helped write the manuscript. TZ: analyzed data and helped write the manuscript. JW: conceived experiments, analyzed data, and helped write the manuscript. KD: conceived and performed experiments, analyzed data, and helped write the manuscript. JPH: conceived and performed experiments, analyzed data, and helped write the manuscript. All authors read and approved the final manuscript.
Toll-like receptor 4 (TLR4) is activated by bacterial endotoxin, a pathogen-associated molecular pattern (PAMP). It has been suggested that TLR4 can also be activated by damage-associated molecular pattern (DAMP) proteins such as HSP70. It remains a challenge to provide unequivocal evidence that DAMP proteins themselves play a role in TLR4 activation, as the DAMP proteins used are often contaminated with endotoxin and other TLR ligands introduced during protein expression and/or purification.
Here we report that the activation of TLR4 on primary human macrophage cultures by recombinant HSP70 is not solely due to contaminating endotoxin. Polymyxin B pretreatment of HSP70 preparations to neutralize contaminating endotoxin caused significant reductions in the amount of TNF-α induced by the recombinant protein. However, digestion of HSP70 with Proteinase K-agarose beads also dramatically reduced the TNF-α response of macrophages to HSP70, while leaving levels of contaminating endotoxin largely unchanged relative to controls.
These results indicate that the stimulatory effect of recombinant HSP70 requires both the presence of endotoxin and structural integrity of the heat shock protein itself.
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- Stimulation of TLR4 by recombinant HSP70 requires structural integrity of the HSP70 protein itself
Jill F Wright
J Perry Hall
- BioMed Central
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