The chemokine, macrophage inflammatory protein-2γ, reduces the expression of glutamate transporter-1 on astrocytes and increases neuronal sensitivity to glutamate excitotoxicity

Background: Changes in glutamatergic neurotransmission via decreased glutamate transporter (GLT) activity or expression contributes to multiple neurological disorders. Chemokines and their receptors are involved in neurological diseases but the role of chemokines in the expression of glutamate transporters is unclear. Methods: Primary astrocytes were prepared from neonatal (< 24 hours old) SJL/J mouse brains and incubated with 5 mu g/ml lipopolysaccharide (LPS) or 50 ng/ml tumor necrosis factor alpha (TNF-alpha) for 24 hours. Soluble macrophage inflammatory protein-2 gamma (MIP-2 gamma) in culture supernatants was determined using a sandwich ELISA. The MIP-2 gamma effect on the expression of GLT-1 was measured by quantitative RT-PCR, flow cytometric analysis or western blot assay. Detergent-resistant membranes from astrocytes were isolated on the basis of their ability to float in density gradients. Raft-containing fractions were tracked by the enrichment of caveolin-1 and the dendritic lipid raft marker, flotillin-1. Cell viability was determined by measuring either the leakage of lactate dehydrogenase or the reduction of 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide by viable cells and confirmed by visual inspection. Results: The production of the chemokine MIP-2 gamma by mouse cortical astrocytes increased significantly after stimulation with LPS or TNF-alpha in vitro. Astrocytes over-expressing MIP-2 gamma down-regulated the expression of GLT-1 at the mRNA and protein level and caused redistribution of GLT-1 out of the lipid rafts that mediate glutamate uptake. We used pharmacological inhibitors to identify the downstream signaling pathways underlying MIP-2 gamma activity. We also found complementary results by knocking down MIP-2 gamma activity in astrocytes with MIP-2 gamma small interfering RNA (siRNA). MIP-2 gamma overexpression in astrocytes enhanced the neuronal toxicity of glutamate by decreasing GLT-1 activity, but MIP-2 gamma itself was not toxic to neurons. Conclusions: These results suggest that MIP-2 gamma mediates the pathogenesis of central nervous system disorders associated with neutrophil infiltration in the brain and decreased GLT-1 activity.