HSP70 protects against experimental stroke through the inhibition of postischemic inflammatory reaction

We and others previously showed that the inducible 70 kDa heat shock protein (HSP70) confers neuroprotection in the brain from ischemia. While the mechanism of such protection is believed to be through reduced protein malfolding and aggregation, recent evidence suggests that HSP70 may also modulate the inflammatory response in certain of pathological settings. Whether this is relevant following ischemic insults is unclear, however. Here we evaluated the effects of HSP70 overexpression on glial cell activation and nuclear factor kappa-B (NFkB) activity using in vivo and in vitro ischemia models. Primary cultures of astrocytes and microglia were prepared from neonatal pups and subjected to simulated ischemia (oxygen-glucose deprivation), then assessed for cell death. Sister cultures were activated by exposure to lipopolysaccharide (LPS) and assessed for NFkB translocation and nitric oxide (NO) production. Transgenic mice overexpressing rat HSP70 or wildtype mice underwent 2 hours of middle cerebral artery occlusion (MCAO) plus 24 hours of reperfusion (n=6/group), or were given 5 mg/kg lipopolysaccharide (LPS) intraperitoneally (n=6/group). Infarct size was assessed by using cresyl violet stains; the neurological deficit was evaluated using a semiquantitative scale (Bederson et al, 1986); microglial activation was assessed using isolectin B4 (IB4) and an antibody against MHC-classII. The DNA-binding capacity of NFkB was assayed using the Trans-AM NFKb p65 transcription factor assay kit (Active Motif, Carlshad, CA). Phosphoralation of IkB was detected using western blots; nuclear NF-kB translocation was assessed by fluorescent immunohistochemistry. In transgenic mice, compared to wildtype, infarct size was significantly reduced by 48% (P<0.01) after experimental stroke and this effect was related to 51% reduction of activated microglia (P<0.01). In LPS-treated transgenic mice, microglia activation was also decreased by 32% (p<0.05), compared with wildtype. HSP 70 transgenic mice had improved neurological deficits compared with wildtype littermates 24 h after ischemia onset (p<0.05). Chemiluminescent detection revealed that the DNA-binding capacity of NFkB was significantly decreased in ischemic brain regions among transgenic mice compared with wildtype mice (p<0.05). Western blots showed that HSP70 overexpression did not change IkB expression, but IkB phosphorylation was remarkably inhibited among HSP70 transgenic mice. Co-immunoprecipitation of HSP70 and NFkB p65 subunit indicated that the two proteins were bound to one another, especially within the cytosol. In cultures, HSP70 overexpression led to improved survival of both transgenic astrocytes and microglia (90% and 48% reduced cell death, respectively, P<0.01). Transgenic astrocyte cultures also expressed 82% less NO following LPS stimulation (P<0.05) and suppressed nuclear NF-kB translocation. These data suggest that HSP70 protects against ischemia and simulated ischemia followed by reperfusion, and this protection may be associated with the inhibition of the postischemic inflammatory response.