Microglial activation and amyloid-β clearance induced by exogenous heat-shock proteins

SPECIFIC AIMS We addressed the hypothesis that extracellular heat shock proteins (HSPs) induce microglial activation resulting in cytokine production and clearance of amyloid-β peptide (Aβ). The effects of exogenous HSP90, HSP70, and HSP32 on the production of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) and the phagocytosis of Aβ(1-42) were studied in isolated microglial cultures from rat and Toll-like receptor 4 (TLR4) mutant mouse brains; levels of expression and localization of HSP90 were investigated in the brains of patients with Alzheimer's disease (AD). PRINCIPAL FINDINGS 1. Exogenous HSPs induce cytokine production In rat microglial culture, addition of HSP90, HSP70, or HSP32 markedly induced production of IL-6 or TNF-α in a concentration-dependent manner whereas HSP27 did not, even at 10 μg/ml (Fig. 1 ⤻ A, B). To exclude the possibility of endotoxin contamination, samples of HSPs and lipopolysaccharide (LPS) from E. coli were heat treated at 100°C for 20 min to inactivate protein but not LPS activity before incubation with the microglia. Heat treatment completely abolished HSP90-, HSP70-, and HSP32-related induction of IL-6 and TNF-α (Fig. 1C, D ⤻ ) but, as expected, did not affect the induction of cytokine production by LPS. In contrast, 10 μg/ml of polymyxin B, which is an LPS trapper and blocks LPS-induced cellular activation, completely abolished LPS-induced production of IL-6 and TNF-α but had no effect on cytokine production induced by HSP90, HSP70, or HSP32 (Fig. 1C, D ⤻ ). Thus, microglial activation by HSPs is unlikely to be due to endotoxin contamination. [Figure: see text] 2. Exogenous HSPs enhanced phagocytosis and clearance of Aβ(1-42) At 1 day after addition of Aβ(1-42) into the culture, it was phagocytosed into rat microglia in a concentration-dependent manner. At that time, aggregated peptides of Aβ(1-42) were also detected by immunoblotting with anti-Aβ antibody. Laser confocal microscopy showed Aβ immunoreactivity in small vesicles in some microglia. The amount of Aβ(1-42) in the microglia was significantly increased after the administration of 5 μg/ml of HSP90 (56 nM), HSP70 (71 nM), or HSP32 (156 nM) but not after administration of IL-6 or TNF-α (Fig. 2 ⤻ ). The number of microglia that phagocytosed Aβ(1-42) was markedly increased: after 3 days, the amount of Aβ(1-42) detected in the microglia was significantly decreased by treatment with these HSPs vs. vehicle (Fig. 2) ⤻ . Thus, exogenous HSPs significantly facili