title: Astrocyte-Derived CCL2 is Associated with M1 Activation and Recruitment of Cultured Microglial Cells.
abstract: OBJECTIVE
  Microglia are an essential player in central nervous system inflammation. Recent studies have demonstrated that the astrocytic chemokine, CCL2, is associated with microglial activation in vivo. However, CCL2-induced microglial activation has not yet been studied in vitro. The purpose of the current study was to understand the role of astrocyte-derived CCL2 in microglial activation and to elucidate the underlying mechanism(s).
  
  
  METHODS
  Primary astrocytes were pre-treated with CCL2 siRNA and stimulated with TNF-α. The culture medium (CM) was collected and added to cultures of microglia, which were incubated with and without CCR2 inhibitor. Microglial cells were analyzed by quantitative RT-PCR to determine whether they polarized to the M1 or M2 state. Microglial migratory ability was assessed by transwell migration assay.
  
  
  RESULTS
  TNF-α stimulated the release of CCL2 from astrocytes, even if the culture media containing TNF-α was replaced with fresh media after 3 h. CM from TNF-α-stimulated astrocytes successfully induced microglial activation, which was ascertained by increased activation of M1 and enhanced migration ability. In contrast, CM from astrocytes pretreated with CCL2 siRNA showed no effect on microglial activation, compared to controls. Additionally, microglia pre-treated with RS102895, a CCR2 inhibitor, were resistant to activation by CM from TNF-α-stimulated astrocytes.
  
  
  CONCLUSIONS
  This study demonstrates that the CCL2/CCR2 pathway of astrocyte-induced microglial activation is associated with M1 polarization and enhanced migration ability, indicating that this pathway could be a useful target to ameliorate inflammation in the central nervous system.
authors.affiliation: Department of Anesthesiology, the First Affiliated Hospital of Nanjing Medical University, Nanjing, P. R. China.
authors.forename: Mingfeng
authors.initials: M
authors.lastname: He
authors.forename: Hongquan
authors.initials: H
authors.lastname: Dong
authors.forename: Yahui
authors.initials: Y
authors.lastname: Huang
authors.forename: Shunmei
authors.initials: S
authors.lastname: Lu
authors.forename: Shu
authors.initials: S
authors.lastname: Zhang
authors.forename: Yanning
authors.initials: Y
authors.lastname: Qian
authors.forename: Wenjie
authors.initials: W
authors.lastname: Jin
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects; D051379:Mice; D017628:Microglia / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: C491768:Ccl2 protein, mouse; D018932:Chemokine CCL2; D003470:Culture Media; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha