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...

Acyloxyacyl hydrolase (AOAH) is an eukaryotic lipase that partially deacylates and detoxifies Gram-negative bacterial lipopolysaccharides and lipooligosaccharides (LPSs orLOSsTissue invasion by even minute quantities of many Gramnegative bacteria (GNB) 2 initiates rapid mobilization of the innate immune responses of the host. In these circumstances, both GNB recognition and many responses depend upon activation of the exquisitely sensitive Toll-like receptor 4 (TLR4) by endotoxins, structurally unique and abundant glycolipids that occupy much of the outer leaflet of the GNB outer membrane (3). Maximal TLR4-dependent host responses to endotoxin are orchestrated through a sequential set of interactions of endotoxin with lipopolysaccharide-binding protein (LBP), membrane or soluble CD14, and soluble or TLR4-associated MD-2 (3-5). Although timely mobilization of host responses is essential, equally important is the regulation of the duration and intensity of host responses to endotoxin to prevent over-exuberant and sustained responses that can result in severe pathological consequences (6).One mechanism of dampening host responses to endotoxin is for the host to modify endotoxin itself, converting it from a potent TLR4 agonist to a much weaker agonist with antagonistic properties. To date, the best described host endotoxin-detoxifying enzyme is acyloxyacyl hydrolase (AOAH) (6). AOAH reduces endotoxin activity by catalyzing the release of secondary fatty acyl chains that are attached to primary 3-hydroxy fatty acyl chains within the bioactive lipid A region (7,8). AOAH thus converts hexaacylated endotoxin species that are potent TLR4 agonists to pentaacylated or tetraacylated forms that have reduced or no agonist properties (9 -13) and, at least in vitro, possess the ability to inhibit TLR4 activation by intact, hexaacylated species (10,11,13, 14). Recent studies indicate that hexaacylated and partially deacylated or underacylated endotoxin react similarly with LBP, CD14, and MD-2 to form monomeric complexes with MD-2 (13). However, only hexaacylated endotoxin-human MD-2 is a potent TLR4 agonist (13, 14).AOAH-dependent deacylation of endotoxin has been demonstrated in vivo (1,15,16) and in complex in vitro settings that roughly simulate the extracellular and intracellular conditions of inflammatory fluids (1, 2, 15,

Potent mammalian cell activation by Gram-negative bacterial endotoxin requires sequential protein-endotoxin and protein-protein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). TLR4 activation requires simultaneous binding of MD-2 to endotoxin (E) and the ectodomain of TLR4. We now describe mutants of recombinant human MD-2 that bind TLR4 and react with E·CD14 but do not support cellular responsiveness to endotoxin. The mutants F121A/K122A MD-2 and Y131A/K132A MD-2 react with E·CD14 only when co-expressed with TLR4. Single mutants K122A and K132A each react with E·CD14 ± TLR4 and promote TLR4-dependent cell activation by endotoxin suggesting that Phe121 and Tyr131 are needed for TLR4-independent transfer of endotoxin from CD14 to MD-2 and also needed for TLR4 activation by bound E·MD-2. The mutant F126A MD-2 reacts as well as wild-type MD-2 with E·CD14 ± TLR4. E·MD-2F126A binds TLR4 with high affinity (Kd ∼ 200 pm) but does not activate TLR4 and instead acts as a potent TLR4 antagonist, inhibiting activation of HEK/TLR4 cells by wild-type E·MD-2. These findings reveal roles of Phe121 and Tyr131 in TLR4-independent interactions of human MD-2 with E·CD14 and, together with Phe126, in activation of TLR4 by bound E·MD-2. These findings strongly suggest that the structural properties of E·MD-2, not E alone, determine agonist or antagonist effects on TLR4.

Two missense variants (D299G and T399I) of TLR4 are cosegregated in individuals of European descent and, in a number of test systems, result in reduced responsiveness to endotoxin. How these changes within the ectodomain (ecd) of TLR4 affect TLR4 function is unclear. For both wild-type and D299G.T399I TLR4, we used endotoxin⋅CD14 and endotoxin⋅MD-2 complexes of high specific radioactivity to measure: 1) interaction of recombinant MD-2⋅TLR4 with endotoxin⋅CD14 and TLR4 with endotoxin⋅MD-2; 2) expression of functional MD-2⋅TLR4 and TLR4; and 3) MD-2⋅TLR4 and TLR4-dependent cellular endotoxin responsiveness. Both wild-type and D299G.T399I TLR4ecd demonstrated high affinity (Kd ~ 200 pM) interaction of endotoxin⋅CD14 with MD-2⋅TLR4ecd and endotoxin⋅MD-2 with TLR4ecd. However, levels of functional TLR4 were reduced up to 2-fold when D299G.T399I TLR4 was coexpressed with MD-2 and &gt;10-fold when expressed without MD-2, paralleling differences in cellular endotoxin responsiveness. The dramatic effect of the D299G.T399I haplotype on expression of functional TLR4 without MD-2 suggests that cells expressing TLR4 without MD-2 are most affected by these polymorphisms.

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