title: The chemokine, macrophage inflammatory protein-2γ, reduces the expression of glutamate transporter-1 on astrocytes and increases neuronal sensitivity to glutamate excitotoxicity.
abstract: 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 μg/ml lipopolysaccharide (LPS) or 50 ng/ml tumor necrosis factor α (TNF-α) for 24 hours. Soluble macrophage inflammatory protein-2γ (MIP-2γ) in culture supernatants was determined using a sandwich ELISA. The MIP-2γ 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-2γ by mouse cortical astrocytes increased significantly after stimulation with LPS or TNF-α in vitro. Astrocytes over-expressing MIP-2γ 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-2γ activity. We also found complementary results by knocking down MIP-2γ activity in astrocytes with MIP-2γ small interfering RNA (siRNA). MIP-2γ overexpression in astrocytes enhanced the neuronal toxicity of glutamate by decreasing GLT-1 activity, but MIP-2γ itself was not toxic to neurons.
  
  
  CONCLUSIONS
  These results suggest that MIP-2γ mediates the pathogenesis of central nervous system disorders associated with neutrophil infiltration in the brain and decreased GLT-1 activity.
authors.affiliation: First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, 350005, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Fang
authors.forename: Deping
authors.initials: D
authors.lastname: Han
authors.forename: Jinsheng
authors.initials: J
authors.lastname: Hong
authors.forename: Qi
authors.initials: Q
authors.lastname: Tan
authors.forename: Yeping
authors.initials: Y
authors.lastname: Tian
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000166:cytology; D051242:Caveolin 1 / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D054426:Chemokine CXCL2 / Q000235:genetics / Q000378:metabolism*; D027341:Excitatory Amino Acid Transporter 1 / Q000378:metabolism; D027342:Excitatory Amino Acid Transporter 2 / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism / Q000633:toxicity*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D021962:Membrane Microdomains / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D051242:Caveolin 1; D054426:Chemokine CXCL2; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D005904:Glial Fibrillary Acidic Protein; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D034741:RNA, Small Interfering; C493393:Slc1a3 protein, mouse; D013778:Tetrazolium Salts; D013844:Thiazoles; D014409:Tumor Necrosis Factor-alpha; C106354:flotillins; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid; D007770:L-Lactate Dehydrogenase; C022616:thiazolyl blue