Stimulation of TLR4 by recombinant HSP70 requires structural integrity of the HSP70 protein itself

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.TLRs are activated by PAMPs contained in lipids, lipopeptides, proteins, and nucleic acids of microbial origin [1]. There is increasing evidence that TLRs can also be activated by host-derived molecules released from sites of tissue damage, collectively known as damage-associated molecular patterns (DAMPs) [2]. These putative DAMPs can be proteins that are released from necrotic cells or proteins that are expressed in response to inflammatory stimuli. It has been suggested that heat shock protein 70 (HSP70) may function as a DAMP for TLR4 and other receptors (e.g., see [3,4]). However, obtaining proof that HSP70 acts as a DAMP for TLR4 is challenging given that preparations of purified recombinant HSP70 are often contaminated with PAMPs. Indeed, it has been reported that the activation of cultured macrophages by recombinant human HSP70 is the result of contaminating endotoxin [5].We were interested in examining the activation of TLR4 on cultured human macrophages by preparations of commercially-available, low-endotoxin recombinant human HSP70. To evaluate the role of contaminating endotoxin in these preparations, we initially employed a traditional approach using Polymyxin B treatment [6]. We demonstrate here that Polymyxin B treatment to neutralize contaminating endotoxi