Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, ARS, U.S. Department of Agriculture, Beltsville, MD 20705, USA.
Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, ARS, U.S. Department of Agriculture, Beltsville, MD 20705, USA.
Poult Sci. 2020 Feb;99(2):772-782. doi: 10.1016/j.psj.2019.10.023. Epub 2019 Dec 20.
Compared with mammals, the functionality of chicken cytokines is not well understood because of the unavailability of immune reagents. Mammalian interleukin (IL)-13 is an important Th2 type cytokine with well-known biological functions through its 2 receptors, IL-13 receptor (IL-13R)-α1 and IL-13Rα2. In the present study, we developed mouse monoclonal antibodies (mAb) against chIL-13 and further investigated their specificity in detecting endogenously produced chIL-13. Upon characterization of mAb using indirect ELISA and Western blot, the capture ELISA was developed for detecting chIL-13. Neutralizing effects were tested by measuring nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in primary chicken monocytes stimulated with chIL-13, lipopolysaccharide (LPS), chIL-13+LPS, or chIL-13+LPS+mAb. In addition, gene expression of chIL-13Rα1, chIL-13Rα2, and TGF-β1 was tested in chicken monocytes treated with chIL-13 or chIL-13+mAb. Based on indirect ELISA, 5 mAb that detected recombinant chIL-13 were identified, and all of them specifically detected recombinant chIL-13 protein by Western blotting. An optimal signal was obtained with 2 mAb (#9B11 and #10A2) in a pairing assay, and these 2 mAb were used in a capture assay. A neutralization assay further revealed that chIL-13 reduced LPS-stimulated NO production and iNOS expression in monocytes and macrophage cells, and the 2 mAb (#9B11 and #10A2) abrogated these effects. In addition, chIL-13-induced expressions of chIL-13Rα2 and TGF-β1 were neutralized by the 2 mAb. In summary, the present study showed that chIL-13 may be involved in the alternative activation of primary monocytes in chickens and that chIL-13 signaling may be regulated through chIL-13Rα2 binding and TGF-β1 secretion. Importantly, the newly developed anti-chIL-13 mAb will serve as valuable immune reagents for future studies on the biological activity of chIL-13 and its receptors.
与哺乳动物相比，由于缺乏免疫试剂，鸡细胞因子的功能还没有得到很好的理解。哺乳动物白细胞介素（IL）-13 是一种重要的 Th2 型细胞因子，通过其 2 个受体，IL-13 受体（IL-13R）-α1 和 IL-13Rα2，具有众所周知的生物学功能。在本研究中，我们开发了针对鸡白细胞介素（chIL-13）的小鼠单克隆抗体（mAb），并进一步研究了它们在检测内源性产生的 chIL-13 中的特异性。通过间接 ELISA 和 Western blot 对 mAb 进行表征后，开发了用于检测 chIL-13 的捕获 ELISA。通过测量鸡原代单核细胞在 chIL-13、脂多糖（LPS）、chIL-13+LPS 或 chIL-13+LPS+mAb 刺激下产生的一氧化氮（NO）和诱导型一氧化氮合酶（iNOS）的表达，测试了 mAb 的中和作用。此外，在鸡单核细胞中用 chIL-13 或 chIL-13+mAb 处理后，检测了 chIL-13Rα1、chIL-13Rα2 和 TGF-β1 的基因表达。基于间接 ELISA，鉴定出了 5 种可检测重组 chIL-13 的 mAb，所有 mAb 均可通过 Western blot 特异性检测重组 chIL-13 蛋白。在配对试验中，2 种 mAb（#9B11 和 #10A2）获得最佳信号，这 2 种 mAb 用于捕获试验。中和试验进一步表明，chIL-13 降低了单核细胞和巨噬细胞中 LPS 刺激的 NO 产生和 iNOS 表达，而 2 种 mAb（#9B11 和 #10A2）则消除了这些作用。此外，chIL-13 诱导的 chIL-13Rα2 和 TGF-β1 的表达被这 2 种 mAb 中和。总之，本研究表明 chIL-13 可能参与了鸡原代单核细胞的替代激活，并且 chIL-13 信号可能通过 chIL-13Rα2 结合和 TGF-β1 分泌来调节。重要的是，新开发的抗 chIL-13 mAb 将成为未来研究 chIL-13 及其受体生物学活性的有价值的免疫试剂。
