GM-CSF increases LPS-induced production of proinflammatory mediators via upregulation of TLR4 and CD14 in murine microglia

• Published in: Journal of neuroinflammation 2012-12, Vol.9 (1), p.268-268, Article 268
• Main Authors: Parajuli, Bijay, Sonobe, Yoshifumi, Kawanokuchi, Jun, Doi, Yukiko, Noda, Mariko, Takeuchi, Hideyuki, Mizuno, Tetsuya, Suzumura, Akio
• Format: Article
• Language: English
• Subjects: Alzheimer's disease; Amyotrophic lateral sclerosis; Analysis; Analysis of Variance; Animals; Animals, Newborn; Bacterial infections; CD14; Cells, Cultured; Cerebral Cortex - cytology; Culture Media, Conditioned - pharmacology; Cytokines - genetics; Cytokines - metabolism; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Enzyme Inhibitors - pharmacology; Enzyme-linked immunosorbent assay; Flow Cytometry; GM-CSF; Granulocyte-Macrophage Colony-Stimulating Factor - metabolism; Granulocyte-Macrophage Colony-Stimulating Factor - pharmacology; Health aspects; Immune response; Infection; Inflammation; Lipopolysaccharide Receptors - genetics; Lipopolysaccharide Receptors - metabolism; Lipopolysaccharides - pharmacology; Macrophages; Mice; Mice, Inbred C57BL; Microglia; Microglia - chemistry; Microglia - drug effects; Microglia - metabolism; Multiple sclerosis; Neurons - drug effects; Neurons - metabolism; NF-kappaB-Inducing Kinase; NF-κB; Nitric oxide; Nitrites - metabolism; Protein Biosynthesis - drug effects; Protein Serine-Threonine Kinases - metabolism; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor - genetics; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor - metabolism; RNA, Messenger - metabolism; Signal Transduction - drug effects; TLR4; Toll-Like Receptor 4 - genetics; Toll-Like Receptor 4 - metabolism; Up-Regulation - drug effects
• ISSN: 1742-2094; 1742-2094
• DOI: 10.1186/1742-2094-9-268

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. 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. 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. 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.