Endotoxin.albumin complexes transfer endotoxin monomers to MD-2 resulting in activation of TLR4

Response to Gram-negative bacteria (GNB) is partially mediated by the recognition of GNB-derived endotoxin by host cells. Potent host response to endotoxin depends on the sequential interaction of endotoxin with lipopolysaccharide binding protein (LBP), CD14, MD-2 and TLR4. While CD14 facilitates the efficient transfer of endotoxin monomers to MD-2 and MD-2.TLR4, activation of MD-2.TLR4 can occur in the absence of CD14 through an unknown mechanism. Here, we show that incubation of purified endotoxin (E) aggregates (E-agg, M-r >= 20 million) in PBS with >= 0.1% albumin in the absence of divalent cations Ca2+ and Mg2+, yields E.albumin complexes (M-r similar to 70,000). E.albumin transfers E monomers to sMD-2 or sMD-2.TLR4 ectodomain (TLR4(ecd)) with a 'K-d' of similar to 4 nM and induces MD-2.TLR4-dependent, CD14-independent cell activation with a potency only 10-fold less than that of monomeric E.CD14 complexes. Our findings demonstrate, for the first time, a mechanistic basis for delivery of endotoxin monomers to MD-2 and for activation of TLR4 that is independent of CD14.