Analysis of the function of IL-10 in chickens using specific neutralising antibodies and a sensitive capture ELISA.
Analysis of the function of IL-10 in chickens using specific neutralising antibodies and a sensitive capture ELISA.
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DOI：
10.1016/j.dci.2016.04.016
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发表时间：
2016-10
影响因子：
2.9
通讯作者：
Hume DA
中科院分区：
文献类型：
--
作者：
Wu Z;Hu T;Rothwell L;Vervelde L;Kaiser P;Boulton K;Nolan MJ;Tomley FM;Blake DP;Hume DA
在哺乳动物中，可诱导的细胞因子白介素10是炎症的负反馈调节因子。为了确定这一功能在鸟类中的保守程度，将重组鸡IL-10表达为分泌型人免疫球蛋白Fc融合蛋白(ChIL-10-Fc)，并用于免疫小鼠。制备了5株特异性识别鸡IL-10的单抗并对其进行了鉴定。建立了两种捕捉法检测脂多糖(LPS)刺激的鸡骨髓巨噬细胞(ChBMMs)分泌的天然ChIL-10。其中3株单抗检测到细胞内IL-10。这只在相同的脂多糖刺激的chBMM中检测到了这一点。ELISA法还检测到感染柔嫩艾美耳球虫的鸡循环中IL-10的大量增加。相同的单抗中和了重组CHIL-10的生物活性。体外检测IL-10在反馈控制中的作用。中和抗体可阻止IL-10抑制有丝分裂原激活的淋巴细胞的干扰素-γ合成，并增加内毒素刺激的chBMM中一氧化氮的产生。结果证实，IL-10是鸡免疫反应的可诱导反馈调节因子，可作为提高疫苗效力或育种策略的靶标。制备了5株抗鸡IL-10的鼠单抗。建立了两种捕捉法测定天然IL-10的方法。三种单抗(ROS-AV162、163和164)中和了CHIL-10的生物活性。抗IL-10抗体仅在脂多糖刺激的巨噬细胞亚群中检测到细胞内IL-10。雏鸡感染艾美耳球虫后，循环中IL-10水平显著升高。
In mammals, the inducible cytokine interleukin 10 is a feedback negative regulator of inflammation. To determine the extent to which this function is conserved in birds, recombinant chicken IL-10 was expressed as a secreted human Ig Fc fusion protein (chIL-10-Fc) and used to immunise mice. Five monoclonal antibodies (mAb) which specifically recognise chicken IL-10 were generated and characterised. Two capture ELISA assays were developed which detected native chIL-10 secreted from chicken bone marrow-derived macrophages (chBMMs) stimulated with lipopolysaccharide (LPS). Three of the mAbs detected intracellular IL-10. This was detected in only a subset of the same LPS-stimulated chBMMs. The ELISA assay also detected massive increases in circulating IL-10 in chickens challenged with the coccidial parasite, Eimeria tenella. The same mAbs neutralised the bioactivity of recombinant chIL-10. The role of IL-10 in feedback control was tested in vitro. The neutralising antibodies prevented IL-10-induced inhibition of IFN-γ synthesis by mitogen-activated lymphocytes and increased nitric oxide production in LPS-stimulated chBMMs. The results confirm that IL-10 is an inducible feedback regulator of immune response in chickens, and could be the target for improved vaccine efficacy or breeding strategies. Five mouse mAbs to chicken IL-10 was produced. Two capture ELISA assays were developed to quantify native IL-10. Three mAbs (ROS-AV162, 163 and 164) neutralised the bioactivities of chIL-10. Anti-IL-10 antibodies detected intracellular IL-10 in only a subset of LPS-stimulated macrophages. Eimeria infection of chickens produced a massive increase in circulating IL-10.