Cross-linking of lipopolysaccharide (LPS) to CD14 on THP-1 cells mediated by LPS-binding protein.
Cross-linking of lipopolysaccharide (LPS) to CD14 on THP-1 cells mediated by LPS-binding protein.
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DOI：
10.4049/jimmunol.150.7.3011
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发表时间：
1993-04
影响因子：
4.4
通讯作者：
Peter S. Tobias;K. Soldau;L. Kline;Jae Lee;K. Kato;T. Martin;R. Ulevitch
中科院分区：
文献类型：
--
作者：
Peter S. Tobias;K. Soldau;L. Kline;Jae Lee;K. Kato;T. Martin;R. Ulevitch
最近的研究表明，细菌内毒素（LPS）与血浆蛋白LPS结合蛋白（LBP）结合形成高亲和力复合物（LPS-LBP）， LBP是一种含LPS颗粒的调理素，LPS-LBP复合物是单核细胞（MO）的强效激动剂。针对MO质膜蛋白CD14的单抗可抑制lbp依赖性的LPS与MO结合，以及LPS- lbp依赖性的细胞因子释放刺激。这些数据表明，CD14作为LPS的膜受体发挥作用，但未证明LPS与CD14之间存在直接关联。骨化三醇在人单核细胞样细胞系THP-1中诱导CD14高水平表达，导致这些细胞对LPS- lbp复合物的反应增强，表现为LPS结合增强，诱导IL-8释放所需的LPS量减少。一种含有放射性碘化、光反应性苯叠氮的Re595 LPS衍生物（125I-ASD-LPS）被用于交联实验，以鉴定骨化三醇处理的THP-1细胞中与LPS相互作用的膜蛋白。将125I-ASD-LPS加入到骨化三醇诱导的THP-1细胞中，无论是否存在LBP，混合物进行光解，并通过SDS-PAGE和放射自显影分析得到的放射性碘化蛋白。当LBP存在时，我们观察到125I-ASD-LPS与55-kDa膜蛋白的强交联，但没有观察到任何其他明显与CD14不同的分子质量的蛋白质的放射性标记。用抗人CD14单抗免疫沉淀鉴定交联产物为CD14。对表达人CD14的小鼠B细胞系70Z/3的转染物的研究也发现了lbp依赖性的125I-ASD-LPS与CD14的交联。这些数据证明了LPS与作为LPS受体的特定膜蛋白的结合。
Recent work has established that bacterial endotoxin (LPS) binds to the plasma protein LPS-binding protein (LBP) forming high affinity complexes (LPS-LBP), that LBP is an opsonin for LPS-bearing particles, and that LPS-LBP complexes are potent agonists for monocytic cells (MO). mAb to the MO plasma membrane protein, CD14, inhibit LBP-dependent binding of LPS to MO, and LPS-LBP-dependent stimulation of cytokine release from MO. These data suggest that CD14 functions as a membrane receptor for LPS but do not demonstrate a direct association of LPS with CD14. Calcitriol was used to induce a high level of CD14 expression in the human monocyte-like cell line THP-1, resulting in enhanced responses of these cells to LPS-LBP complexes manifested by enhanced binding of LPS and a decrease in the amount of LPS needed to induce IL-8 release. An Re595 LPS derivative containing a radioiodinated, photoreactive, phenyl azide (125I-ASD-LPS) was used in cross-linking experiments to identify membrane proteins in calcitriol-treated THP-1 cells that interact with LPS. 125I-ASD-LPS was added to calcitriol-induced THP-1 cells in the presence or absence of LBP, the mixture photolyzed, and the resultant radioiodinated proteins analyzed by SDS-PAGE and autoradiography. We observed strong cross-linking of 125I-ASD-LPS to a 55-kDa membrane protein when LBP was present, but failed to observe radiolabeling of any other proteins with apparent molecular masses distinct from CD14. The cross-linked product was identified as CD14 by immunoprecipitation with anti-human CD14 mAb. Studies with human CD14 expressing transfectants of the murine B cell line 70Z/3 also revealed LBP-dependent cross-linking of 125I-ASD-LPS to CD14. These data document binding of LPS to a specific membrane protein that serves as an LPS receptor.