Effects Of HSP70 On Release And Proinflamatory Function Of Wnt5a

Recent study found that Wnt5a is a highly specific autocrine and paracrine macrophaged-derived effector molecule which triggers inflammation. Wnt5a secreted outside the cells becomes an important cytokine that mediates partial or full inflammation, so it has been a novel molecular target for the prevention and treatment of inflammation-related diseases. Heat shock proteins(HSPs) are a kind of proteins that are induced by heat shock response(HSR), of which heat shock protein 70 is the most important member and the most induced one. In this study the change of Wnt5a in level in burn patients are first investigated, and then the effects of HSP70 on expression and release and proinflammatory function of Wnt5a are also studied for the first time.Firstly, the changes of Wnt5a levels in sera of burn patients with sepsis were investigated. The results were: (1) the levels of serum Wnt5a of burn patients with sepsis are found to be greater than those of controls; dynamic observation showed that the serum Wnt5a concentrations were associated with the severity of burn patients, and Wnt5a levels going up or down with the severity of the burn patients. (2) the similar results were obtained when serum HSP70 levels were investigated. These results suggest that Wnt5a may take part in the process of sepsis in burn patients with sepsis and may be as a diagnostic parameter to severity of sepsis.Inflammatory cytokines are produced by monocytes/macrophages stimulated with mycobacteria or conserved bacterial structures. The use of LPS to induce macrophages to produce inflammatory cytokines is a widely applied model. In this study the investigation of the effects of LPS and TNF-αon the expression and release of Wnt5a was then performed. It was found that LPS could induce the expression and release of wnt5a from macrophage Raw264.7, and at the same time the release of the proinflammatory cytokine TNF-α; the release of TNF-αfrom raw264.7 was also induced by wnt5a, and vise versa. These results are consistent with the reports from other literatures. Therefore we demonstrated that wnt5a is a new inflammatory mediator, functioning in the processes of sepsis of burn patients. Previous study reported that the anti-inflammatory cytokine interleukin (IL)-10 and activated protein C (APC) can inhibit the expression and release of wnt5a, suggesting that wnt5a may be a new target molecule for treatment and prevention of sepsis. HSR and HSP70 were studied in the processes of sepsis. Some researchers think they inhibit the expression and release of inflammatory cytokines while others think they promote their release. As a new inflammatory mediator little is known about wnt5a in the processes of sepsis. The effects of HSR and HSP70 on wnt5a are unknown.HSPs are a superfamily of highly conserved proteins distributing throughout prokaryotic cells and eukaryotic cells, of which HSP70 is the most important member. Their basic functions are to take part in folding, disaggregation and translocalization of neonate proteins, so they are called molecular chaperone. HSPs are thought to be intracellular proteins that function only within cells during the long-term period. In order to investigate the effects of intracellular HSP70 on the expression and release of wnt5a, and based upon the facts that wnt5a can be expressed and released from macrophages by induction of LPS and TNF-α, and that wnt5a can induce the release of other cytokines such as TNF-α, this study for the first time investigated the effects of intracellular HSP70 on the expression, release and pro-inflammation of wnt5a. We first constructed HSP70 expression vector, then the expression vector was transduced into macrophage Raw264.7 for overexpression. Using HS pretreatment and gene transffection it was found: (1) overexpression of hsp70 can inhibit the expression and release of wnt5a induced by LPS; (2) when the experiment was done using TNF-αin stead of LPS the similar results were obtained; (3) the overexpression of hsp70 can inhibit pro-inflammatory function of wnt5a by inhibiting the release of other pro-inflammatory cytokines including TNF-αby induction of wnt5a. All these results demonstrated that intracellular hsp70 can significantly inhibit the expression and release of wnt5a induced by LPS and TNF-α, and also inhibit the pro-inflammatory function of wnt5a.Recent study shows that hsp70 can be released to external milieu that was called extracellular hsp70,and extracellular hsp70 may regulate the function of immune cells as a "dangerous signal" in immune systems, participating in the processes of inflammatory diseases. In recent years it was found that the levels of serum hsp70 in patients with sepsis or septic shock were higher than those in normal controls. In this study recombinant human hsp70 was used to stimulate macrophage raw264.7 so as to investigate the effects of extracellular hsp70 on wnt5a.The results showed that the recombinant purified hsp70 itself did not affect the release of wnt5a, but it can markedly inhibit the release of wnt5a induced by LPS. The anti-inflammatory cytokine IL-10 was detected at the same time, and it was found that the pretreatment of the recombinant hsp70 can promote the release of IL-10 from macrophage raw264.7. The receptors TLR2 and TLR4 were studied with the corresponding antibodies, and it was found that the recombinant hsp70 promoted the release of IL-10 in a TLR4 dependent manner.Taken together, the levels of serum wnt5a and hsp70 in burn patients significantly increased, and the concentrations of serum wnt5a may be related to the severity of sepsis of the burn patients. LPS and TNF-αincreased the production of wnt5a, and wnt5a in return stimulated the release of the proinflammatory cytokine TNF-α. HS and gene transfection can lead to the increasing expression of HSP70 in macrophages, and the intracellular hsp70 can inhibit both the expression and release of wnt5a induced by LPS and TNF-α, and the release of TNF-αinduced by wnt5a. Recombinant hsp70 can also inhibit the release of Wnt5a induced by LPS possibly by promoting the release of anti-inflammatory cytokine IL-10 in a TLR4 dependent manner.