Burnett R J, Lyden C A, Tindal C J, Cave C M, Marra M N, Solomkin J S
Department of Surgery, University of Cincinnati, Ohio, College of Medicine, USA.
Arch Surg. 1996 Feb;131(2):200-5; discussion 206. doi: 10.1001/archsurg.1996.01430140090023.
Bactericidal/permeability-increasing protein (BPI) binds lipopolysaccharide and neutralizes its toxic effects in vitro and in endotoxemic animals. Our recent work identified physiologically significant interactions between BPI, lipopolysaccharide, and mononuclear cells.
To determine whether the interaction between BPI and mononuclear cells is receptor mediated.
Labeled BPI was incubated with THP-1 cells in the presence of up to 100-fold excess of unlabeled BPI. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting were performed to evaluate competitive binding and total uptake of BPI. Crosslinking was performed to determine whether BPI binds to a single protein entity. Acid washing experiments and flow cytometric analysis were performed to determine whether BPI remains on the cellular surface. Finally, flow cytometry analysis was used to determine whether BPI incubation with THP-1 cells affects the surface expression of the lipopolysaccharide-binding protein-lipopolysaccharide receptor CD14.
Labeled BPI uptake was not inhibited by the presence of 100-fold excess of unlabeled BPI at 37 degrees C or 4 degrees C in the presence of azide. Uptake was not saturable under either condition with incubation concentrations up to 10 microgram/mL. Cross-linking did not show BPI bound to a single entity. Acid washing and flow cytometry experiments disclosed rapid internalization of BPI. Finally, BPI uptake by THP-1 cells had no effect on the surface expression of CD14.
Bactericidal/permeability-increasing protein is rapidly internalized by mononuclear cells in a nonspecific fashion not saturable at very high doses, which is consistent with pinocytosis. This process may represent a disposal mechanism for lipopolysaccharide in closed-space infections and may be partially responsible for the rapid clearance of BPI from the peripheral circulation.
杀菌/通透性增加蛋白（BPI）可结合脂多糖并在体外和内毒素血症动物体内中和其毒性作用。我们最近的研究确定了BPI、脂多糖和单核细胞之间具有生理意义的相互作用。
确定BPI与单核细胞之间的相互作用是否由受体介导。
将标记的BPI与THP-1细胞在存在高达100倍过量未标记BPI的情况下孵育。进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和蛋白质印迹以评估BPI的竞争性结合和总摄取。进行交联以确定BPI是否与单一蛋白质实体结合。进行酸洗实验和流式细胞术分析以确定BPI是否保留在细胞表面。最后，使用流式细胞术分析来确定BPI与THP-1细胞孵育是否会影响脂多糖结合蛋白-脂多糖受体CD14的表面表达。
在37℃或4℃存在叠氮化物的情况下，100倍过量未标记BPI的存在不会抑制标记BPI的摄取。在孵育浓度高达10μg/mL的任何一种条件下，摄取都不饱和。交联未显示BPI与单一实体结合。酸洗和流式细胞术实验揭示了BPI的快速内化。最后，THP-1细胞摄取BPI对CD14的表面表达没有影响。
杀菌/通透性增加蛋白被单核细胞以非特异性方式快速内化，在非常高的剂量下不饱和，这与胞饮作用一致。这个过程可能代表了封闭空间感染中脂多糖的一种清除机制，并且可能部分负责BPI从外周循环中的快速清除。
