| Summary: | <p>Abstract</p> <p>Background</p> <p>Toll-like receptor 4 (TLR4) is activated by bacterial endotoxin, a prototypical 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.</p> <p>Results</p> <p>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 as determined by ELISA. 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.</p> <p>Conclusions</p> <p>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.</p>
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