Macrophage receptors of polysaccharide isolated from a marine filamentous fungus Phoma herbarum YS4108
Macrophage receptors of polysaccharide isolated from a marine filamentous fungus Phoma herbarum YS4108
复制标题
从海洋丝状真菌 Phoma herbarum YS4108 中分离的多糖的巨噬细胞受体
DOI：
10.1038/aps.2009.93
复制
发表时间：
2009-07-01
影响因子：
8.2
通讯作者：
Gao, Xiang-dong
中科院分区：
文献类型：
--
作者：
Chen, Song;Yin, Deng-ke;Gao, Xiang-dong
目的：从海洋丝状真菌Phoma herbarum YS 4108的菌丝体中分离得到一种新的（1，4）-α-D-葡聚糖YCP。本工作研究了巨噬细胞表达的YCP结合细胞受体以及YCP诱导巨噬细胞活化的细胞内信号转导途径。方法：用CDAP活化法制备荧光标记的YCP（fl-YCP）。荧光共聚焦显微镜和荧光激活细胞分选术（FACS）用于分析fl-YCP对巨噬细胞的作用。为了表征YCP受体的性质，使用碳水化合物和抗体来抑制fl-YCP与巨噬细胞的结合。此外，我们研究了膜受体Toll样受体2（TLR 2）、Toll样受体4（TLR 4）、Toll样受体6（TLR 6）和补体受体3（CR 3）的作用。结果：YCP在体外对巨噬细胞释放NO有促进作用，且与巨噬细胞表面的受体结合呈时间和剂量依赖性。竞争性研究表明，LPS、海带多糖、抗TLR 4抗体和抗CD 11b（CR 3）抗体可抑制fl-YCP与巨噬细胞的结合。甘露糖、抗TLR 2和抗TLR 6抗体则不能。用YCP处理RAW264.7细胞导致p38以时间依赖性方式显著活化。特异性p38抑制剂SB 203580可阻断YCP诱导的NO生成。用抗TLR 4抗体和抗CR 3抗体处理RAW264.7细胞显著降低YCP诱导的NO产生和p38激活。结论：我们已经证明YCP诱导的NO产生是通过TLR 4和CR 3膜受体以p38激酶依赖的方式在巨噬细胞中发生的。
Aim: YCP, a novel (1,4)-alpha-D-glucan, was isolated from the mycelium of the marine filamentous fungus Phoma herbarum YS4108. In this work, we investigated a YCP-binding cellular receptor expressed by macrophages and the intracellular signal transduction pathways involved in YCP-induced macrophage activation.Methods: Fluorescence-labeled YCP (fl-YCP) was prepared using the CDAP-activation method. Fluorescence confocal laser microscopy and fluorescence-activated cell sorting (FACS) were used to analyze the effect of fl-YCP on macrophages. To characterize the properties of the YCP receptor, carbohydrates and antibodies were used to inhibit the binding of fl-YCP to macrophages. Moreover, we investigated the role of membrane receptors Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), Toll-like receptor 6 (TLR6) and complement receptor 3 (CR3). We also examined the role of the p38 kinase pathway in mediating nitric oxide (NO) production.Results: YCP had an in vitro stimulatory effect on the release of NO in macrophage, and fl-YCP can bind directly to receptors on the surface of macrophages in a time-and dose-dependent manner. Competition studies show that LPS, laminarin, anti-TLR4 antibody and anti-CD11b (CR3) antibody could inhibit fl-YCP binding to macrophages. Conversely, mannose, anti-TLR2 and anti-TLR6 antibody could not. Treatment of RAW264.7 cells with YCP resulted in significant activation of p38 in a time-dependent manner. The specific p38 inhibitor SB203580 abrogated YCP-induced NO generation. Treatment of RAW264.7 cells with anti-TLR4 antibody and anti-CR3 antibody significantly reduced YCP-induced NO production and p38 activation.Conclusion: We have demonstrated that YCP-induced NO production occurs through the TLR4 and CR3 membrane receptors in a p38 kinase-dependent manner in macrophages.