title: GM-CSF increases LPS-induced production of proinflammatory mediators via upregulation of TLR4 and CD14 in murine microglia.
abstract: BACKGROUND
  Microglia are resident macrophage-like cells in the central nervous system (CNS) and cause innate immune responses via the LPS receptors, Toll-like receptor (TLR) 4 and CD14, in a variety of neuroinflammatory disorders including bacterial infection, Alzheimer's disease, and amyotrophic lateral sclerosis. Granulocyte macrophage-colony stimulating factor (GM-CSF) activates microglia and induces inflammatory responses via binding to GM-CSF receptor complex composed of two different subunit GM-CSF receptor α (GM-CSFRα) and common β chain (βc). GM-CSF has been shown to be associated with neuroinflammatory responses in multiple sclerosis and Alzheimer's disease. However, the mechanisms how GM-CSF promotes neuroinflammation still remain unclear.
  
  
  METHODS
  Microglia were stimulated with 20 ng/ml GM-CSF and the levels of TLR4 and CD14 expression were evaluated by RT-PCR and flowcytometry. LPS binding was analyzed by flowcytometry. GM-CSF receptor complex was analyzed by immunocytochemistry. The levels of IL-1β, IL-6 and TNF-α in culture supernatant of GM-CSF-stimulated microglia and NF-κB nuclear translocation were determined by ELISA. Production of nitric oxide (NO) was measured by the Griess method. The levels of p-ERK1/2, ERK1/2, p-p38 and p38 were assessed by Western blotting. Statistically significant differences between experimental groups were determined by one-way ANOVA followed by Tukey test for multiple comparisons.
  
  
  RESULTS
  GM-CSF receptor complex was expressed in microglia. GM-CSF enhanced TLR4 and CD14 expressions in microglia and subsequent LPS-binding to the cell surface. In addition, GM-CSF priming increased LPS-induced NF-κB nuclear translocation and production of IL-1β, IL-6, TNF-α and NO by microglia. GM-CSF upregulated the levels of p-ERK1/2 and p-p38, suggesting that induction of TLR4 and CD14 expression by GM-CSF was mediated through ERK1/2 and p38, respectively.
  
  
  CONCLUSIONS
  These results suggest that GM-CSF upregulates TLR4 and CD14 expression in microglia through ERK1/2 and p38, respectively, and thus promotes the LPS receptor-mediated inflammation in the CNS.
authors.affiliation: Department of Neuroimmunology, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Bijay
authors.initials: B
authors.lastname: Parajuli
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Sonobe
authors.forename: Jun
authors.initials: J
authors.lastname: Kawanokuchi
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Doi
authors.forename: Mariko
authors.initials: M
authors.lastname: Noda
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Takeuchi
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Mizuno
authors.forename: Akio
authors.initials: A
authors.lastname: Suzumura
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism / Q000494:pharmacology*; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects*; D000097782:NF-kappaB-Inducing Kinase
chemical_list: D017077:Culture Media, Conditioned; D016207:Cytokines; D004791:Enzyme Inhibitors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D009573:Nitrites; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D051197:Toll-Like Receptor 4; D000097782:NF-kappaB-Inducing Kinase; C493487:Tlr4 protein, mouse