Molecular Basis of the Functional Differences between Soluble Human Versus Murine MD-2: Role of Val135 in Transfer of Lipopolysaccharide from CD14 to MD-2.

Myeloid differentiation factor 2 (MD-2) is an extracellular protein, associated with the ectodomain of TLR4, that plays a critical role in the recognition of bacterial LPS. Despite high overall structural and functional similarity, human (h) and murine (m) MD-2 exhibit several species-related differences. hMD-2 is capable of binding LPS in the absence of TLR4, whereas mMD-2 supports LPS responsiveness only when mMD-2 and mTLR4 are coexpressed in the same cell. Previously, charged residues at the edge of the LPS binding pocket have been attributed to this difference. In this study, site-directed mutagenesis was used to explore the hydrophobic residues within the MD-2 binding pocket as the source of functional differences between hMD-2 and mMD-2. Whereas decreased hydrophobicity of residues 61 and 63 in the hMD-2 binding pocket retained the characteristics of wild-type hMD-2, a relatively minor change of valine to alanine at position 135 completely abolished the binding of LPS to the hMD-2 mutant. The mutant, however, retained the LPS binding in complex with TLR4 and also cell activation, resulting in a murine-like phenotype. These results were supported by the molecular dynamics simulation. We propose that the residue at position 135 of MD-2 governs the dynamics of the binding pocket and its ability to accommodate lipid A, which is allosterically affected by bound TLR4.

Purified monomeric ligand.MD-2 complexes reveal molecular and structural requirements for activation and antagonism of TLR4 by Gram-negative bacterial endotoxins.

A major focus of work in our laboratory concerns the molecular mechanisms and structural bases of Gram-negative bacterial endotoxin recognition by host (e.g., human) endotoxin-recognition proteins that mediate and/or regulate activation of Toll-like receptor (TLR) 4. Here, we review studies of wild-type and variant monomeric endotoxin.MD-2 complexes first produced and characterized in our laboratories. These purified complexes have provided unique experimental reagents, revealing both quantitative and qualitative determinants of TLR4 activation and antagonism. This review is dedicated to the memory of Dr. Theresa L. Gioannini (1949-2014) who played a central role in many of the studies and discoveries that are reviewed.

Tetraacylated lipid A and paclitaxel-selective activation of TLR4/MD-2 conferred through hydrophobic interactions.

LPS exerts potent immunostimulatory effects through activation of the TLR4/MD-2 receptor complex. The hexaacylated lipid A is an agonist of mouse (mTLR4) and human TLR4/MD-2, whereas the tetraacylated lipid IVa and paclitaxel activate only mTLR4/MD-2 and antagonize activation of the human receptor complex. Hydrophobic mutants of TLR4 or MD-2 were used to investigate activation of human embryonic kidney 293 cells by different TLR4 agonists. We show that each of the hydrophobic residues F438 and F461, which are located on the convex face of leucine-rich repeats 16 and 17 of the mTLR4 ectodomain, are essential for activation of with lipid IVa and paclitaxel, which, although not a structural analog of LPS, activates cells expressing mTLR4/MD-2. Both TLR4 mutants were inactive when stimulated with lipid IVa or paclitaxel, but retained significant activation when stimulated with LPS or hexaacylated lipid A. We show that the phenylalanine residue at position 126 of mouse MD-2 is indispensable only for activation with paclitaxel. Its replacement with leucine or valine completely abolished activation with paclitaxel while preserving the responsiveness to lipid IVa and lipid A. This suggests specific interaction of paclitaxel with F126 because its replacement with leucine even augmented activation by lipid A. These results provide an insight into the molecular mechanism of TLR4 activation by two structurally very different agonists.

The TLR4 antagonist Eritoran protects mice from lethal influenza infection.

There is a pressing need to develop alternatives to annual influenza vaccines and antiviral agents licensed for mitigating influenza infection. Previous studies reported that acute lung injury caused by chemical or microbial insults is secondary to the generation of host-derived, oxidized phospholipid that potently stimulates Toll-like receptor 4 (TLR4)-dependent inflammation. Subsequently, we reported that Tlr4(-/-) mice are highly refractory to influenza-induced lethality, and proposed that therapeutic antagonism of TLR4 signalling would protect against influenza-induced acute lung injury. Here we report that therapeutic administration of Eritoran (also known as E5564)-a potent, well-tolerated, synthetic TLR4 antagonist-blocks influenza-induced lethality in mice, as well as lung pathology, clinical symptoms, cytokine and oxidized phospholipid expression, and decreases viral titres. CD14 and TLR2 are also required for Eritoran-mediated protection, and CD14 directly binds Eritoran and inhibits ligand binding to MD2. Thus, Eritoran blockade of TLR signalling represents a novel therapeutic approach for inflammation associated with influenza, and possibly other infections.

Radioiodination of an endotoxin·MD-2 complex generates a novel sensitive, high-affinity ligand for TLR4.

A purified complex of metabolically labeled [(3)H]lipooligosaccharide (LOS) and recombinant human myeloid differentiation factor 2 (MD-2), [(3)H]LOS·MD-2, has been used to demonstrate pM affinity binding interactions with soluble TLR4 ectodomain (TLR4ecd). For measurement of the binding parameters of membrane-bound TLR4, we took advantage of the stability of endotoxin·MD-2 and tyrosine(s) present on the surface of MD-2 to radioiodinate LOS·MD-2. Radioiodinated LOS·MD-2 generated a reagent with an estimated 1:1 molar ratio of [(125)I] to sMD-2 with 20-fold higher specific radioactivity and TLR4-activating properties comparable to metabolically-labeled LOS·MD-2. LOS·MD-2[(125)I] and [(3)H]LOS·MD-2 have similar affinities for soluble (FLAG) TLR4ecd and for membrane-bound TLR4 in HEK293T/TLR4 cells. In a similar dose-dependent manner, sMD-2 and LOS·MD-2 inhibit LOS·MD-2[(125)I] binding to TLR4 indicating the pM affinity binding of LOS·MD-2[(125)I] is agonist-independent. LOS·MD-2[(125)I] allowed measurement of low levels of cell-surface human or murine TLR4 expressed by stable cell lines (2000-3000 sites/cell) and quantitatively measures low levels of 'MD-2-free' TLR4 (est. 250 molecules/cell) in cells co-expressing TLR4 and MD-2. Occupation of 50-100 TLR4/cell by LOS·MD-2 is sufficient to trigger measurable TLR4-dependent cell activation. LOS·MD-2[(125)I] provides a powerful reagent to measure quantitatively functional human and murine cell-surface TLR4, including in cells where surface TLR4 is potentially functionally significant but not detectable by other methods.

Respiratory syncytial virus fusion protein-induced toll-like receptor 4 (TLR4) signaling is inhibited by the TLR4 antagonists Rhodobacter sphaeroides lipopolysaccharide and eritoran (E5564) and requires direct interaction with MD-2.

UNLABELLED: Respiratory syncytial virus (RSV) is a leading cause of infant mortality worldwide. Toll-like receptor 4 (TLR4), a signaling receptor for structurally diverse microbe-associated molecular patterns, is activated by the RSV fusion (F) protein and by bacterial lipopolysaccharide (LPS) in a CD14-dependent manner. TLR4 signaling by LPS also requires the presence of an additional protein, MD-2. Thus, it is possible that F protein-mediated TLR4 activation relies on MD-2 as well, although this hypothesis has not been formally tested. LPS-free RSV F protein was found to activate NF-κB in HEK293T transfectants that express wild-type (WT) TLR4 and CD14, but only when MD-2 was coexpressed. These findings were confirmed by measuring F-protein-induced interleukin 1ß (IL-1ß) mRNA in WT versus MD-2(-/-) macrophages, where MD-2(-/-) macrophages failed to show IL-1ß expression upon F-protein treatment, in contrast to the WT. Both Rhodobacter sphaeroides LPS and synthetic E5564 (eritoran), LPS antagonists that inhibit TLR4 signaling by binding a hydrophobic pocket in MD-2, significantly reduced RSV F-protein-mediated TLR4 activity in HEK293T-TLR4-CD14-MD-2 transfectants in a dose-dependent manner, while TLR4-independent NF-κB activation by tumor necrosis factor alpha (TNF-α) was unaffected. In vitro coimmunoprecipitation studies confirmed a physical interaction between native RSV F protein and MD-2. Further, we demonstrated that the N-terminal domain of the F1 segment of RSV F protein interacts with MD-2. These data provide new insights into the importance of MD-2 in RSV F-protein-mediated TLR4 activation. Thus, targeting the interaction between MD-2 and RSV F protein may potentially lead to novel therapeutic approaches to help control RSV-induced inflammation and pathology. IMPORTANCE: This study shows for the first time that the fusion (F) protein of respiratory syncytial virus (RSV), a major cause of bronchiolitis and death, particularly in infants and young children, physically interacts with the Toll-like receptor 4 (TLR4) coreceptor, MD-2, through its N-terminal domain. We show that F protein-induced TLR4 activation can be blocked by lipid A analog antagonists. This observation provides a strong experimental rationale for testing such antagonists in animal models of RSV infection for potential use in people.

The role of microRNAs miR-200b and miR-200c in TLR4 signaling and NF-κB activation.

Recognition of microbial products by members of the Toll-like receptor (TLR) family initiates intracellular signaling cascades that result in NF-κB activation and subsequent production of inflammatory cytokines. We explored the potential roles of microRNAs (miRNAs) in regulating TLR pathways. A target analysis approach to the TLR4 pathway adaptor molecules identified several putative targets of miR-200a, miR-200b and miR-200c. miRNA mimics were co-transfected with a NF-κB activity reporter plasmid into HEK293 cells stably expressing TLR4 (HEK293-TLR4). Mimics of both miR-200b and miR-200c, but not miR-200a, decreased NF-κB reporter activity in either untreated cells or in cells treated with endotoxin:MD2 as a TLR4 agonist. Transfection of HEK293-TLR4 cells with miR-200b or miR-200c significantly decreased expression of MyD88, whereas TLR4, IRAK-1 and TRAF-6 mRNAs were unaffected. When miR-200b or miR-200c mimics were transfected into the differentiated monocytic THP-1 cell line, the abundance of MyD88 transcripts, as well as LPS-induced expression of the pro-inflammatory molecules IL-6, CXCL9 and TNF-α were diminished. These data define miRNAs miR-200b and miR-200c as factors that modify the efficiency of TLR4 signaling through the MyD88-dependent pathway and can thus affect host innate defenses against microbial pathogens.

NMR studies of hexaacylated endotoxin bound to wild-type and F126A mutant MD-2 and MD-2·TLR4 ectodomain complexes.

Host response to invasion by many gram-negative bacteria depends upon activation of Toll-like receptor 4 (TLR4) by endotoxin presented as a monomer bound to myeloid differentiation factor 2 (MD-2). Metabolic labeling of hexaacylated endotoxin (LOS) from Neisseria meningitidis with [(13)C]acetate allowed the use of NMR to examine structural properties of the fatty acyl chains of LOS present in TLR4-agonistic and -antagonistic binary and ternary complexes with, respectively, wild-type or mutant (F126A) MD-2 ± TLR4 ectodomain. Chemical shift perturbation indicates that Phe(126) affects the environment and/or position of each of the bound fatty acyl chains both in the binary LOS·MD-2 complex and in the ternary LOS·MD-2·TLR4 ectodomain complex. In both wild-type and mutant LOS·MD-2 complexes, one of the six fatty acyl chains of LOS is more susceptible to paramagnetic attenuation, suggesting protrusion of that fatty acyl chain from the hydrophobic pocket of MD-2, independent of association with TLR4. These findings indicate that re-orientation of the aromatic side chain of Phe(126) is induced by binding of hexaacylated E, preceding interaction with TLR4. This re-arrangement of Phe(126) may act as a "hydrophobic switch," driving agonist-dependent contacts needed for TLR4 dimerization and activation.

Endotoxin{middle dot}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 Ca(2+) and Mg(2+), yields E·albumin complexes (M ( r ) ∼70,000). E·albumin transfers E monomers to sMD-2 or sMD-2·TLR4 ectodomain (TLR4(ecd)) with a 'K (d)' of ∼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.

Hemin and a metabolic derivative coprohemin modulate the TLR4 pathway differently through different molecular targets.

Heme is a prosthetic group in a large number of essential proteins that have a pivotal role in oxygen transport, storage and electron shuttling. High amounts of free heme are associated with pathological states. Recently, it has been suggested that activation of Toll-like receptor 4 (TLR4) is one of the ways in which the 'danger signal' of free heme is detected. Here, we examine the biochemical basis of the modulation of the TLR4 pathway by hemin (iron(III)-protoporphyrin IX) and its metabolic, oxidated derivative coprohemin (iron(III)-coproporphyrin I). High concentrations of hemin (50 µM) triggered TLR4-mediated IL-8 production in the human HEK293/TLR4 cell line in the absence of the co-receptors CD14 and MD-2; the latter an essential co-receptor for TLR4 activation by endotoxin. Hemin and endotoxin have additive effects when co-administrated to HEK/TLR4 cells, suggesting that hemin and endotoxin activate TLR4 by different mechanisms. Coprohemin, in contrast to hemin, is unable to trigger TLR4-dependent activation of HEK/TLR4 cells, but instead causes dose-dependent inhibition of endotoxin-stimulated IL-8 production. The inhibitory effect of coprohemin is paralleled by reduced delivery of endotoxin to MD-2 (-TLR4) that is necessary for activation of TLR4 by endotoxin. Thus, despite their similar chemical structure, hemin and coprohemin have very different effects on the TLR4 pathway, the former acting as a mild agonist of TLR4, the latter as an antagonist selectively targeting the endotoxin-MD-2 interaction.

Expression of functional D299G.T399I polymorphic variant of TLR4 depends more on coexpression of MD-2 than does wild-type 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 endotoxinCD14 and endotoxinMD-2 complexes of high specific radioactivity to measure: 1) interaction of recombinant MD-2TLR4 with endotoxinCD14 and TLR4 with endotoxinMD-2; 2) expression of functional MD-2TLR4 and TLR4; and 3) MD-2TLR4 and TLR4-dependent cellular endotoxin responsiveness. Both wild-type and D299G.T399I TLR4(ecd) demonstrated high affinity (K(d) approximately 200 pM) interaction of endotoxinCD14 with MD-2TLR4(ecd) and endotoxinMD-2 with TLR4(ecd). However, levels of functional TLR4 were reduced up to 2-fold when D299G.T399I TLR4 was coexpressed with MD-2 and >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.

Novel roles of lysines 122, 125, and 58 in functional differences between human and murine MD-2.

The MD-2/TLR4 complex provides a highly robust mechanism for recognition and response of mammalian innate immunity to Gram-negative bacterial endotoxins. Despite overall close structural and functional similarity, human (h) and murine (m) MD-2 show several species-related differences, including the ability of hMD-2, but not mMD-2, to bind endotoxin (E) in the absence of TLR4. Wild-type mMD-2 can support TLR4-dependent cell activation by E only when mMD-2 and mTLR4 are coexpressed in the same cell. However, replacement of Glu122, Leu125, and/or Asn58 of mMD-2 with the corresponding residues (lysines) of hMD-2 was sufficient to yield soluble extracellular MD-2 that reacted with monomeric E . sCD14 complex to form extracellular monomeric E . MD-2 that activated cells expressing TLR4 without MD-2. Moreover, in contrast to wild-type mMD-2, double and triple mMD-2 mutants also supported E-triggered signaling in combination with human TLR4. Conversely, a K125L mutant of hMD-2 reacted with E . CD14 and activated TLR4 only when coexpressed with TLR4, and not when secreted without TLR4. These findings reveal novel roles of lysines 122, 125, and 58 in human MD-2 that contribute to the functional differences between human and murine MD-2 and, potentially, to differences in the sensitivity of humans and mice to endotoxin.

Essential roles of hydrophobic residues in both MD-2 and toll-like receptor 4 in activation by endotoxin.

Gram-negative bacterial endotoxin (i.e. lipopolysaccharide (LPS)) is one of the most potent stimulants of the innate immune system, recognized by the TLR4.MD-2 complex. Direct binding to MD-2 of LPS and LPS analogues that act as TLR4 agonists or antagonists is well established, but the role of MD-2 and TLR4 in receptor activation is much less clear. We have identified residues within the hairpin of MD-2 between strands five and six that, although not contacting acyl chains of tetraacylated lipid IVa (a TLR4 antagonist), influence activation of TLR4 by hexaacylated lipid A. We show that hydrophobic residues at positions 82, 85, and 87 of MD-2 are essential both for transfer of endotoxin from CD14 to monomeric MD-2 and for TLR4 activation. We also identified a pair of conserved hydrophobic residues (Phe-440 and Phe-463) in leucine-rich repeats 16 and 17 of the TLR4 ectodomain, which are essential for activation of TLR4 by LPS. F440A or F463A mutants of TLR4 were inactive, whereas the F440W mutant retained full activity. Charge reversal of neighboring cationic groups in the TLR4 ectodomain (Lys-388 and Lys-435), in contrast, did not affect cell activation. Our mutagenesis studies are consistent with a molecular model in which Val-82, Met-85, and Leu-87 in MD-2 and distal portions of a secondary acyl chain of hexaacylated lipid A that do not fit into the hydrophobic binding pocket of MD-2 form a hydrophobic surface that interacts with Phe-440 and Phe-463 on a neighboring TLR4.MD-2.LPS complex, driving TLR4 activation.

Allergenicity resulting from functional mimicry of a Toll-like receptor complex protein.

Aeroallergy results from maladaptive immune responses to ubiquitous, otherwise innocuous environmental proteins. Although the proteins targeted by aeroallergic responses represent a tiny fraction of the airborne proteins humans are exposed to, allergenicity is a quite public phenomenon-the same proteins typically behave as aeroallergens across the human population. Why particular proteins tend to act as allergens in susceptible hosts is a fundamental mechanistic question that remains largely unanswered. The main house-dust-mite allergen, Der p 2, has structural homology with MD-2 (also known as LY96), the lipopolysaccharide (LPS)-binding component of the Toll-like receptor (TLR) 4 signalling complex. Here we show that Der p 2 also has functional homology, facilitating signalling through direct interactions with the TLR4 complex, and reconstituting LPS-driven TLR4 signalling in the absence of MD-2. Mirroring this, airway sensitization and challenge with Der p 2 led to experimental allergic asthma in wild type and MD-2-deficient, but not TLR4-deficient, mice. Our results indicate that Der p 2 tends to be targeted by adaptive immune responses because of its auto-adjuvant properties. The fact that other members of the MD-2-like lipid-binding family are allergens, and that most defined major allergens are thought to be lipid-binding proteins, suggests that intrinsic adjuvant activity by such proteins and their accompanying lipid cargo may have some generality as a mechanism underlying the phenomenon of allergenicity.

Isolation of monomeric and dimeric secreted MD-2. Endotoxin.sCD14 and Toll-like receptor 4 ectodomain selectively react with the monomeric form of secreted MD-2.

Potent cell activation by endotoxin requires sequential protein-endotoxin and protein-protein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). MD-2 plays an essential role by bridging endotoxin (E) recognition initiated by lipopolysaccharide-binding protein and CD14 to TLR4 activation by presenting endotoxin as a monomeric E.MD-2 complex that directly and potently activates TLR4. Secreted MD-2 (sMD-2) exists as a mixture of monomers and multimers. Published data suggest that only MD-2 monomer can interact with endotoxin and TLR4 and support cell activation, but the apparent instability of MD-2 has thwarted efforts to more fully separate and characterize the individual species of sMD-2. We have taken advantage of the much greater stability of sMD-2 in insect culture medium to fully separate sMD-2 monomer from dimer by gel sieving chromatography. At low nanomolar concentrations, the sMD-2 monomer, but not dimer, reacted with a monomeric complex of E.sCD14 to form monomeric E.MD-2 and activate HEK293/TLR4 cells. The monomer, but not dimer, also reacted with the ectodomain of TLR4 with an affinity comparable with the picomolar affinity of E.MD-2. These findings demonstrate directly that the monomeric form of sMD-2 is the active species both for reaction with E.CD14 and TLR4, as needed for potent endotoxin-induced TLR4 activation.

Functional activity of MD-2 polymorphic variant is significantly different in soluble and TLR4-bound forms: decreased endotoxin binding by G56R MD-2 and its rescue by TLR4 ectodomain.

MD-2 is an essential component of endotoxin (LPS) sensing, binding LPS independently and when bound to the ectodomain of the membrane receptor TLR4. Natural variation of proteins involved in the LPS-recognition cascade such as the LPS-binding protein, CD14, and TLR4, as well as proteins involved in intracellular signaling downstream of LPS binding, affect the cellular response to endotoxin and host defense against bacterial infections. We now describe the functional properties of two nonsynonymous coding polymorphisms of MD-2, G56R and P157S, documented in HapMap. As predicted from the MD-2 structure, the P157S mutation had little or no effect on MD-2 function. In contrast, the G56R mutation, located close to the LPS-binding pocket, significantly decreased cellular responsiveness to LPS. Soluble G56R MD-2 showed markedly reduced LPS binding that was to a large degree rescued by TLR4 coexpression or presence of TLR4 ectodomain. Thus, cells that express TLR4 without MD-2 and whose response to LPS depends on ectopically produced MD-2 were most affected by expression of the G56R variant of MD-2. Coexpression of wild-type and G56R MD-2 yielded an intermediate phenotype with responses to LPS diminished to a greater extent than that resulting from expression of the D299G TLR4 polymorphic variant.

Novel roles in human MD-2 of phenylalanines 121 and 126 and tyrosine 131 in activation of Toll-like receptor 4 by endotoxin.

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 Phe(121) and Tyr(131) 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-2(F126A) binds TLR4 with high affinity (K(d) approximately 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 Phe(121) and Tyr(131) in TLR4-independent interactions of human MD-2 with E.CD14 and, together with Phe(126), 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.

Transfer of monomeric endotoxin from MD-2 to CD14: characterization and functional consequences.

Potent Toll-like receptor 4 (TLR4)-dependent cell activation by endotoxin depends on sequential transfer of monomers of endotoxin from an aggregated form to CD14 via the lipopolysaccharide-binding protein and then to MD-2. We now show that monomeric endotoxin can be transferred in reverse from MD-2 to CD14 but not to lipopolysaccharide-binding protein. Reverse transfer requires an approximately 1000-fold molar excess of CD14 to endotoxin-MD-2. Transfer of endotoxin from MD-2 to extracellular soluble CD14 reduces activation of cells expressing TLR4 without MD-2. However, transfer of endotoxin from MD-2 to membrane CD14 (mCD14) makes cells expressing MD-2.TLR4 sensitive to activation by the endotoxin-MD-2 complex. An endotoxin-mutant (F126A) MD-2 complex that does not activate cells expressing TLR4 alone potently activates cells expressing mCD14, MD-2, and TLR4 by transferring endotoxin to mCD14, which then transfers endotoxin to endogenous wild-type MD-2.TLR4. These findings describe a novel pathway of endotoxin transfer that provides an additional layer of regulation of cell activation by endotoxin.

Regulation of interactions of Gram-negative bacterial endotoxins with mammalian cells.

Host defense against many invading Gram-negative bacteria (GNB) depends on innate immune recognition of endotoxin (lipopolysaccharides, LPS), unique surface glycolipids of GNB. Host responses to endotoxin must be highly sensitive but self-limited. In mammals, optimal sensitivity is achieved by ordered interactions of endotoxin with several different extracellular and cell surface proteins-the LPS-binding protein (LBP), CD14, MD-2, and Toll-like receptor (TLR) 4-reflecting the requirement for specific protein-endotoxin and protein-protein interactions. This complex reaction pathway also provides many ways to attenuate endotoxin-driven inflammation and can explain how differences in endotoxin structure, either intrinsic among GNB or induced by metabolic remodeling, can alter host responsiveness and thus the outcome of host-GNB interactions. Major goals of our research are to better understand: (1) the structural bases of specific host-endotoxin interactions; (2) functional diversity among host endotoxin-binding proteins; and (3) how the actions of various endotoxin-binding proteins are regulated to permit optimal host responses to GNB infection. In addition, the identification of a water-soluble endotoxin:MD-2 complex that, depending on the structure of endotoxin or MD-2, has potent TLR4 agonist or antagonist properties suggests novel pharmacologic approaches to immuno-modulation.

A novel role for the bactericidal/permeability increasing protein in interactions of gram-negative bacterial outer membrane blebs with dendritic cells.

The bactericidal/permeability-increasing protein (BPI) is thought to play an important role in killing and clearance of Gram-negative bacteria and the neutralization of endotoxin. A possible role for BPI in clearance of cell-free endotoxin has also been suggested based on studies with purified endotoxin aggregates and blood monocytes. Because the interaction of BPI with cell-free endotoxin, during infection, occurs mainly in tissue and most likely in the form of shed bacterial outer membrane vesicles ("blebs"), we examined the effect of BPI on interactions of metabolically labeled ([(14)C]-acetate) blebs purified from Neisseria meningitidis serogroup B with either human monocyte-derived macrophages or monocyte-derived dendritic cells (MDDC). BPI produced a dose-dependent increase (up to 3-fold) in delivery of (14)C-labeled blebs to MDDC, but not to monocyte-derived macrophages in the presence or absence of serum. Both, fluorescently labeled blebs and BPI were internalized by MDDC under these conditions. The closely related LPS-binding protein, in contrast to BPI, did not increase association of the blebs with MDDC. BPI-enhanced delivery of the blebs to MDDC did not increase cell activation but permitted CD14-dependent signaling by the blebs as measured by changes in MDDC morphology, surface expression of CD80, CD83, CD86, and MHC class II and secretion of IL-8, RANTES, and IP-10. These findings suggest a novel role of BPI in the interaction of bacterial outer membrane vesicles with dendritic cells that may help link innate immune recognition of endotoxin to Ag delivery and presentation.