Tobias P S, Soldau K, Kline L, Lee J D, Kato K, Martin T P, Ulevitch R J
Scripps Research Institute, La Jolla, CA 92037.
J Immunol. 1993 Apr 1;150(7):3011-21.
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.
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是携带LPS颗粒的调理素，且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的单克隆抗体可抑制LPS依赖的LPS与MO的结合以及LPS-LBP依赖的MO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，但并未证明LPS与CD14有直接关联。使用骨化三醇在人单核细胞样细胞系THP-1中诱导高水平的CD14表达，导致这些细胞对LPS-LBP复合物的反应增强，表现为LPS结合增强以及诱导IL-8释放所需的LPS量减少。一种含有放射性碘化、光反应性苯叠氮化物的Re595 LPS衍生物（125I-ASD-LPS）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。在有或无LBP存在的情况下，将125I-ASD-LPS添加到骨化三醇诱导的THP-1细胞中，混合物进行光解，然后通过SDS-PAGE和放射自显影分析所得的放射性碘化蛋白。当存在LBP时，我们观察到125I-ASD-LPS与一种55 kDa的膜蛋白有强烈交联，但未观察到任何其他表观分子量不同于CD14的蛋白被放射性标记。通过用抗人CD14单克隆抗体进行免疫沉淀，将交联产物鉴定为CD14。对表达人CD14的鼠B细胞系70Z/3转染子的研究也显示125I-ASD-LPS与CD14有LBP依赖的交联。这些数据证明LPS与作为LPS受体的特定膜蛋白结合。
