Allergen activates peripheral blood eosinophil nuclear factor-κB to generate granulocyte macrophage-colony stimulating factor, tumour necrosis factor-α and interleukin-8
Allergen activates peripheral blood eosinophil nuclear factor-κB to generate granulocyte macrophage-colony stimulating factor, tumour necrosis factor-α and interleukin-8
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DOI：
10.1111/j.1365-2222.2004.02003.x
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发表时间：
2004-07-01
影响因子：
6.1
通讯作者：
Church, MK
中科院分区：
文献类型：
--
作者：
Coward, WR;Sagara, H;Church, MK
背景变应性炎症以嗜酸性粒细胞的流入和活化为特征。由驻留细胞和浸润细胞产生的细胞因子负责该发病机制的启动和维持。本研究旨在观察过敏原诱导的嗜酸性粒细胞NF-κ B活化以及粒细胞巨噬细胞集落刺激因子（GM-CSF）、TNF-α和IL-8的产生。方法采用Percoll梯度沉降法和负磁性亲和层析法富集外周血嗜酸性粒细胞至>99.9%。使用抗NF-κ B活性形式（NF-κ B（a））的单克隆抗体，通过免疫细胞化学方法证明了10 μ g/mL屋尘螨（HDM）提取物对NF-κ B的激活。通过Western blot证实NF-κ B的真实性。细胞因子的产生进行了评估，嗜酸性粒细胞的免疫染色和细胞因子的测定在细胞supernaturation.Results活化外周血嗜酸性粒细胞从特应性，但不是非特应性，捐助者诱导激活NF-κ B，在4小时达到峰值，并伴随着IkappaB-α下降。在凝胶迁移试验中证实了真实NF-κ B的活化。超移位分析显示p65是嗜酸性粒细胞NF-κ B的主要亚基。免疫荧光共聚焦显微镜显示NF-κ B（a）定位于细胞核。活化后，在一部分嗜酸性粒细胞中观察到细胞因子免疫反应性，细胞因子释放到上清液中。NF-κ B抑制剂，钙蛋白酶抑制剂1（10 μ m），pentylfylline（0.5 mM），吡咯烷二硫代氨基甲酸酯（PDTC，10 μ m）或gliotoxin（1 μ g/mL）减少的生成GM-CSF，TNF-α和IL-8的平行与他们的抑制NF-κ B.Conclusions HDM过敏原激活人类嗜酸性粒细胞NF-κ B导致的细胞因子GM-CSF，TNF-α和IL-8的生产。我们推测，嗜酸性粒细胞NF-κ B依赖性细胞因子的作用是作为一个自分泌环增加嗜酸性粒细胞在体内的生存。
Background Allergic inflammation is characterized by the influx and activation of eosinophils. Cytokines generated by both resident and infiltrating cells are responsible for the initiation and maintenance of this pathogenesis. This study focuses on allergen-induced activation of eosinophil NF-kappaB and generation of granulocyte macrophage-colony stimulating factor (GM-CSF), TNF-alpha, and IL-8.Methods Peripheral blood eosinophils were enriched to >99.9% by Percoll gradient sedimentation and negative magnetic affinity chromatography. NF-kappaB activation by 10 mug/mL house dust mite (HDM) extract was demonstrated immunocytochemically using a monoclonal antibody against the active form of NF-kappaB (NF-kappaB(a)). The authenticity of NF-kappaB was confirmed by Western blot. Cytokine production was assessed both by immuno-staining of eosinophils and by assay of cytokines in the cell supernatant.Results Activation of peripheral blood eosinophils from atopic, but not non-atopic, donors induced activation of NF-kappaB, which peaked at 4 h and was accompanied by a decline in IkappaB-alpha. The activation of authentic NF-kappaB was confirmed in gel shift assays. Supershift assays showed p65 to be the major subunit of eosinophil NF-kappaB. Immunofluorescent confocal microscopy demonstrated localization of NF-kappaB(a) to the nucleus. Following activation, cytokine immunoreactivity was seen in a fraction of the eosinophils and cytokines were released into the supernatant. The NF-kappaB inhibitors, calpain inhibitor 1 (10 pm), pentoxifylline (0.5 mM), pyrrolidine dithiocarbamate (PDTC, 10 pm) or gliotoxin (1 pg/mL) reduced the generation of GM-CSF, TNF-alpha and IL-8 in parallel with their inhibition of NF-kappaB.Conclusions HDM allergen activates human eosinophil NF-kappaB leading to the production of the cytokines GM-CSF, TNF-alpha and IL-8. We speculate that a role for eosinophil NF-kappaB-dependent cytokines is to act as an autocrine loop augmenting the survival of eosinophils in vivo.