Host proinflammatory responses to minute amounts of endotoxins derived from many Gram-negative bacteria require the interaction of lipopolysaccharide-binding protein (LBP), CD14, Toll-like receptor 4 (TLR4) and MD-2. Optimal sensitivity to endotoxin requires an ordered series of endotoxin–protein and protein–protein interactions. At substoichiometric concentrations, LBP facilitates delivery of endotoxin aggregates to soluble CD14 (sCD14) to form monomeric endotoxin–sCD14 complexes. Subsequent interactions of endotoxin–sCD14 with TLR4 and/or MD-2 have not been specifically defined. This study reports the purification of a stable, monomeric, bioactive endotoxin–MD-2 complex generated by treatment of endotoxin–sCD14 with recombinant MD-2. Efficient generation of this complex occurred at picomolar concentrations of endotoxin and nanogram per milliliter doses of MD-2 and required presentation of endotoxin to MD-2 as a monomeric endotoxin–CD14 complex. TLR4-dependent delivery of endotoxin to human embryonic kidney (HEK) cells and cell activation at picomolar concentrations of endotoxin occurred with the purified endotoxin–MD-2 complex, but not with purified endotoxin aggregates with or without LBP and/or sCD14. The presence of excess MD-2 inhibited delivery of endotoxin–MD-2 to HEK/TLR4 cells and cell activation. These findings demonstrate that TLR4-dependent activation of host cells by picomolar concentrations of endotoxin occurs by sequential interaction and transfer of endotoxin to LBP, CD14, and MD-2 and simultaneous engagement of endotoxin and TLR4 by MD-2.

Potent Toll-like receptor 4 (TLR4) activation by endotoxin has been intensely studied, but the molecular requirements for endotoxin interaction with TLR4 are still incompletely defined. Ligandreceptor interactions involving endotoxin and TLR4 were characterized using monomeric endotoxin⅐protein complexes of high specific radioactivity. The binding of endotoxin⅐MD-2 to the TLR4 ectodomain (TLR4 ECD ) and transfer of endotoxin from CD14 to MD-2/TLR4 ECD were demonstrated using HEK293T-conditioned medium containing TLR4 ECD ؎ MD-2. These interactions are specific, of high affinity (K D < 300 pM), and consistent with the molecular requirements for potent cell activation by endotoxin. Both reactions result in the formation of a M r ϳ 190,000 complex composed of endotoxin, MD-2, and TLR4 ECD . CD14 facilitates transfer of endotoxin to MD-2 (TLR4) but is not a stable component of the endotoxin⅐MD-2/TLR4 complex. The ability to assay specific high affinity interactions of monomeric endotoxin⅐protein complexes with TLR4 ECD should allow better definition of the structural requirements for endotoxin-induced TLR4 activation.Essential arms of the innate immune system are the Tolllike receptors (TLRs).2 These receptors link recognition of unique microbial molecules to activation of host defense effector systems by rapidly triggering pro-inflammatory responses (1). Potent host responses toward many Gramnegative bacteria (GNB) are mediated by recognition and response to unique glycolipids (lipopoly-or lipooligosaccharides LOS, endotoxin) of the GNB outer membrane by TLR4. TLR4 does not function alone but requires the accessory protein MD-2, which binds non-covalently to the N-terminal ectodomain of TLR4 (2-6). Maximally potent endotoxin-induced cell activation also requires the extracellular lipopolysaccharide-binding protein (LBP) and membrane (m) or soluble (s) extracellular CD14 (4, 7-9). The sequential action of LBP, CD14, secreted or TLR4-associated MD-2, and TLR4 confers the extraordinary sensitivity of mammalian cells to many GNB endotoxins. This ordered action implies differences in endotoxin binding specificity, with LBP having the highest affinity for endotoxin organized at lipid/water interfaces (e.g. purified endotoxin aggregates and endotoxin in the GNB outer membrane), CD14 for LBP-modified endotoxin-rich interfaces, MD-2 for monomeric endotoxin⅐CD14 and TLR4, apparently, for endotoxin presented as a monomeric complex with MD-2 (8). Together, these proteins can convert one GNB (containing ϳ10 6 endotoxin molecules) to 10 6 TLR4-activating monomeric endotoxin⅐protein complexes (i.e. endotoxin⅐CD14 or endotoxin⅐MD-2), greatly amplifying host responsiveness to endotoxin. At pM concentrations, monomeric complexes of endotoxin⅐CD14 or endotoxin⅐MD-2 activate, respectively, mammalian cells expressing MD-2/TLR4 or TLR4 alone, triggering robust cell activation through engagement of Ͻ10 3 TLR4 molecules.Despite the ability of endotoxin⅐CD14 and endotoxin⅐ MD-2 to activate cells at pM concentrations (half-maximal cell acti...

Potent TLR4-dependent cell activation by Gram-negative bacterial endotoxins depends on sequential endotoxin-protein and protein-protein interactions with LPS-binding protein, CD14, myeloid differentiation protein 2 (MD-2), and TLR4. Previous studies have suggested that reduced agonist potency of underacylated endotoxins (i.e., tetra- or penta- vs hexa-acylated) is determined by post-CD14 interactions. To better define the molecular basis of the differences in agonist potency of endotoxins differing in fatty acid acylation, we compared endotoxins (lipooligosaccharides (LOS)) from hexa-acylated wild-type (wt), penta-acylated mutant msbB meningococcal strains as well as tetra-acylated LOS generated by treatment of wt LOS with the deacylating enzyme, acyloxyacylhydrolase. To facilitate assay of endotoxin:protein and endotoxin:cell interactions, the endotoxins were purified after metabolic labeling with [3H]- or [14C]acetate. All LOS species tested formed monomeric complexes with MD-2 in an LPS-binding protein- and CD14-dependent manner with similar efficiency. However, msbB LOS:MD-2 and acyloxyacylhydrolase-treated LOS:MD-2 were at least 10-fold less potent in inducing TLR4-dependent cell activation than wt LOS:MD-2 and partially antagonized the action of wt LOS:MD-2. These findings suggest that underacylated endotoxins produce decreased TLR4-dependent cell activation by altering the interaction of the endotoxin:MD-2 complex with TLR4 in a way that reduces receptor activation. Differences in potency among these endotoxin species is determined not by different aggregate properties, but by different properties of monomeric endotoxin:MD-2 complexes.

To facilitate studies of the molecular determinants of host-meningococcal lipooligosaccharide (endotoxin) interactions at patho-physiologically relevant endotoxin concentrations (i.e. <10 ng/ml), we have generated acetate auxotrophs NMBACE1 from encapsulated Neisseria meningitidis (serogroup B, strain NMB) and NMBACE2 from an isogenic bacterial mutant lacking the polysialic acid capsule. Growth of the auxotrophs in medium containing [14 C]acetate yielded 14 C-lipooligosaccharides containing ϳ600 cpm/ng. Gel sieving resolved 14 C-lipooligosaccharide-containing aggregates with an estimated molecular mass of >20 ؋ 10 6 Da (peak A) and ϳ1 ؋ 10 6 Da (peak B) from both strains. Lipooligosaccharides in peaks A and B had the same fatty acid composition and SDS-polyacrylamide gel electrophoresis profile. 14 C-Labeled capsule copurified with 14 C-lipooligosaccharides in peak B from NMBACE1, whereas the other aggregates contained only 14 C-lipooligosaccharide. For all aggregates, lipopolysaccharide-binding protein and soluble CD14-induced delivery of lipooligosaccharides to endothelial cells and cell activation correlated with disaggregation of lipooligosaccharides. These processes were inhibited by the presence of capsule but unaffected by the size of the aggregates. In contrast, endotoxin activation of cells containing membrane CD14 was unaffected by capsule but diminished when endotoxin was presented in larger aggregates. These findings demonstrate that the physical presentation of lipooligosaccharide, including possible interactions with capsule, affect the ability of meningococcal endotoxin to interact with and activate specific host targets.

Studies with purified aggregates of endotoxin have revealed the importance of lipopolysaccharide-binding protein (LBP)-dependent extraction and transfer of individual endotoxin molecules to CD14 in Toll-like receptor 4 (TLR4)-dependent cell activation. Endotoxin is normally embedded in the outer membrane of intact Gram-negative bacteria and shed membrane vesicles ("blebs"). However, the ability of LBP and CD14 to efficiently promote TLR4-dependent cell activation by membrane-associated endotoxin has not been studied extensively. In this study, we used an acetate auxotroph of Neisseria meningitidis serogroup B to facilitate metabolic labeling of bacterial endotoxin and compared interactions of purified endotoxin aggregates and of membrane-associated endotoxin with LBP, CD14, and endotoxin-responsive cells. The endotoxin, phospholipid, and protein composition of the recovered blebs indicate that the blebs derive from the bacterial outer membrane. Proteomic analysis revealed an unusual enrichment in highly cationic (pI > 9) proteins. Both purified endotoxin aggregates and blebs activate monocytes and endothelial cells in a LBP-, CD14-, and TLR4/MD-2-dependent fashion, but the blebs were 3-10-fold less potent when normalized for the amount of endotoxin added. Differences in potency correlated with differences in efficiency of LBPdependent delivery to and extraction of endotoxin by CD14. Both membrane phospholipids and endotoxin are extracted by LBP/soluble CD14 (sCD14) treatment, but only endotoxin⅐sCD14 reacts with MD-2 and activates cells. These findings indicate that the proinflammatory potency of endotoxin may be regulated not only by the intrinsic structural properties of endotoxin but also by its association with neighboring molecules in the outer membrane.