GM-CSF increases LPS-induced production of proinflammatory mediators via upregulation of TLR4 and CD14 in murine microglia.
GM-CSF increases LPS-induced production of proinflammatory mediators via upregulation of TLR4 and CD14 in murine microglia.
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DOI：
10.1186/1742-2094-9-268
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发表时间：
2012-12-13
影响因子：
9.3
通讯作者：
Suzumura A
中科院分区：
文献类型：
--
作者：
Parajuli B;Sonobe Y;Kawanokuchi J;Doi Y;Noda M;Takeuchi H;Mizuno T;Suzumura A
小胶质细胞是中枢神经系统（CNS）中常驻的巨噬细胞样细胞，并通过LPS受体、Toll样受体（TLR）4和CD 14引起多种神经炎症性疾病（包括细菌感染、阿尔茨海默病和肌萎缩性侧索硬化症）的先天免疫反应。粒细胞巨噬细胞集落刺激因子（GM-CSF）通过与由两种不同亚基GM-CSF受体α（GM-CSFRα）和共同β链（βc）组成的GM-CSF受体复合物结合，激活小胶质细胞，诱导炎症反应。已证明GM-CSF与多发性硬化症和阿尔茨海默病的神经炎症反应有关。然而，GM-CSF促进神经炎症的机制仍不清楚。用20 ng/ml GM-CSF刺激小胶质细胞，通过RT-PCR和流式细胞术检测TLR 4和CD 14的表达水平。通过流式细胞术分析LPS结合。用免疫细胞化学法分析GM-CSF受体复合物。ELISA法检测GM-CSF刺激后小胶质细胞培养上清中IL-1β、IL-6和TNF-α的水平及NF-κB核转位。用Griess法测定一氧化氮（NO）的产生。Western blotting检测p-ERK 1/2、ERK 1/2、p-p38和p38蛋白的表达。实验组之间的统计学显著性差异通过单因素方差分析确定，然后通过Tukey检验进行多重比较。GM-CSF受体复合物在小胶质细胞中表达。GM-CSF增强了小胶质细胞中TLR 4和CD 14的表达，并随后增强了LPS与细胞表面的结合。此外，GM-CSF预激可增加LPS诱导的NF-κB核转位，并增加小胶质细胞IL-1β、IL-6、TNF-α和NO的产生。GM-CSF上调p-ERK 1/2和p-p38的水平，表明GM-CSF诱导TLR 4和CD 14表达分别通过ERK 1/2和p38介导。这些结果表明GM-CSF分别通过ERK 1/2和p38上调小胶质细胞中TLR 4和CD 14的表达，从而促进CNS中LPS受体介导的炎症。
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 immunocytechemistry. 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.
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