An essential role for albumin in the interaction of endotoxin with lipopolysaccharide-binding protein and sCD14 and resultant cell activation

Experiments utilizing endotoxin aggregates, lipooligosaccharides (LOS) isolated from metabolically labeled Neisseria meningitidis serotype group B, demonstrate that albumin is an essential component of lipopolysaccharide binding protein- (LBP) and sCD14-dependent 1) disaggregation of LOS and 2) LOS activation of human umbilical vein endothelial cells (HUVEC). Aggregates of LOS (LOSagg) with an apparent M(r)greater than or equal to2x10(7) were isolated by gel sieving on Sephacryl HR S500 in buffered balanced salts solution plus albumin. Incubation of LOSagg with LBP and sCD14 promoted LOSagg disaggregation in an albumin-dependent fashion to complexes that contain LOS and sCD14, but no LBP, with an apparent M(r)similar to60,000 (LOS:sCD14) as determined by Sephacryl S200 chromatography. Isolation by gel filtration of LOSagg:protein aggregates formed by the interaction of LOSagg with either LBP or sCD14 alone revealed that the sequence of LOS-protein interactions as well as the step(s) at which albumin is necessary for the production of bioactive LOS: sCD14 were specific. Efficient generation of LOS:sCD14 required 1) interaction of LOSagg with LBP before interaction with CD14 and 2) the presence of albumin during the interaction of LBP with LOSagg. Activation of HUVEC by LOSagg, as measured by IL-8 production, required both LBP and sCD14 and was thirty times more potent in the presence of albumin. In contrast, LOS:sCD14 did not require additional LBP, sCD14, or albumin to activate HLTVEC but depended on the presence of albumin for optimal solubility/stability once formed. The albumin effect is apparently specific, because neither ovalbumin nor gelatin substituted for albumin in facilitating LBP:sCD14-dependent disaggregation of LOSagg or activation of endothelial cells. These results indicate that albumin is an essential facilitator of LBP/sCD14-induced LOS disaggregation that is required for activation of endothelial cells by LOSagg.