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Myeloid differentiation factor (MD-2) is an extracellular protein, associated with the ectodomain of Toll-like receptor 4 (TLR4), that plays a critical role in the recognition of bacterial lipopolysaccharide (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 LPS binding pocket have been attributed to this difference. In this study, site-directed mutagenesis was used to explore the hydrophobic residues within MD-2 binding pocket, as the source of functional differences between hMD-2 and mMD-2. While decreased hydrophobicity of residues 61 and 63 in 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.

It is unknown if endotoxin reactivity in the LAL assay varies by its presentation or if this parallels differences in potency in vitro and in vivo.We compared endotoxin responses in the LAL assay, in cell cultures and in vivo when presented in different forms.

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.

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.

Rationale: Maximum host cell responsiveness to endotoxin requires at least four extracellular and cell surface host proteins (LBP, CD-14, MD-2 and TLR4). These proteins engage endotoxin in a series of protein-protein and protein-endotoxin interactions initiating a signal transduction cascade leading to the release of proinflammatory mediators. We sought to compare the ability of two different monomeric endotoxin:MD-2 complexes in inducing lung inflammatory responses. Methods: BALB/c mice were nasally instilled with monomeric complexes of penta- (LOSmsbB) and hexa-acylated (LOSwt) N. meningitidis endotoxin:MD-2 in doses of 0, 3, 30 and 300 EU/mouse. After 4 hours, mice were euthanized and processed for bronchoalveolar lavage (BAL). Concentration of MIP-1α , TNF-α , IL-6 and G-CSF were determined in BAL and lungs were evaluated for the accumulation of neutrophils. Results: At lower doses (3 and 30 EU/mouse) LOSmsbB:MD-2-induced inflammatory responses were markedly reduced compared with LOSwt:MD-2-treated mice in the number of BAL cells (2 to 10-fold) and concentration of IL-6, G-CSF (4 to 6-fold), TNF-α and MIP-1α (9 to 18-fold). At the higher dose (300 EU/mouse) the LOSwt:MD-2-treated group had 2-fold higher number of total cells, neutrophils and concentrations of TNF-α and G-CSF than LOSmsbB:MD-2, while IL-6 and MIP-1α reached similar levels in BAL. Conclusions: Differences in the intensity of lung inflammatory responses between two types of monomeric complexes of lipooligosaccharides with MD-2 reflected differences in the structure and bioactivity of hexa- and penta-acylated lipooligosaccharides. Complexes of endotoxin with MD-2 serve as ligands and activators of TLR-4 signaling and their activity in vivo can be modulated by using endotoxin species differing in fatty acid content. Funded By: NIH P30 ES05605

Rationale: Myeloid-secreted protein (MD-2) is required for Toll-like receptor (TLR) 4-dependent pro-inflammatory signaling by Gram-negative bacterial endotoxin. Our goal in this study was to determine whether MD-2 is necessary for endotoxin responses in lungs in vivo. For this purpose, we used MD-2 knockout and TLR-4 mutant mice. Methods: MD-2 null (C57/BL6 MD-2-/-), TLR-4 mutant (C3H/HeJ) and endotoxin-responsive (BALB/c) mice were exposed by nasal aspiration to purified aggregates of Neisseria meningitidis endotoxin (lipooligosaccharide ; LOSagg) or to a monomeric complex of LOS with MD-2 (LOS:MD-2), each at 300 EU/mouse. Four hours after the administration, total cell count, differentials and concentration of TNF-α and MIP-1α were evaluated in bronchoalveolar lavage. Results: The number of total cells and neutrophils was significantly higher in LOS treated BALB/c mice (1240x103 and 1144 x103) than in TLR-4 mutants (176x103 and 38.5x103) and MD-2 null mice (150x103 and 24.1x103). Moreover, the concentration of TNF-α and MIP-1α was markedly reduced in TLR-4 mutants and MD-2 null mice (115 pg/mL and 200 pg/mL vs. 85 pg/mL and 42 pg/mL) in comparison with BALB/c mice (6107 pg/mL and 5925 pg/mL). When treated with LOS:MD-2 complex MD-2 null mice showed stronger inflammatory responses than TLR-4 mutant in the number of neutrophils (495x103 vs. 75x103), concentration of TNF-α (1012 vs. 106 pg/mL) and MIP-1α (892 vs. 176 pg/mL). Conclusions: Mice with the TLR-4 mutation or without expression of MD-2 were hyporesponsive to nasally instilled endotoxin compared with wild type mice. The defect in endotoxin responsiveness of MD-2 null but not TLR4 mutant mice could be partially overcome by presentation of endotoxin as a preformed complex with MD-2. These data demonstrate that lung inflammatory responses depend on the presence of MD-2 as well as TLR4. Funded By: NIH P30 ES05605

Rationale: Endotoxins can induce airway inflammation and exacerbate asthma. A major determinant of endotoxin potency is the fatty acid composition of the bioactive lipid A moiety. We sought to determine the potency in inducing airway inflammation of two forms of Neisseria meningitidis (N.m.) strain B (NMB) endotoxin (LOS) that differ in fatty acid structure: hexa-acylated LOSwt from a wild type strain of NMB and penta-acylated endotoxin (LOSmsbB) from a mutant strain derived from wt NMB. Methods: LOSwt and LOSmsbB were metabolically labeled, extracted, and purified from wild type NMB (NMBACE1) and mutant NMB (NMBA11K3cap-), respectively. Groups of BALB/c mice were intranasally instilled with varying doses of LOSwt and LOSmsbB (0, 30, 300 EU). At 4 and 24 hr post-exposure, concentrations of total cells, neutrophils and cytokines/chemokines (TNF-α , MIP-1α , IL-6, IL-1β , G-CSF and IL-17) were measured in bronchoalveolar lavage (BAL). Results: Both the hexa- and penta-acylated endotoxins generated dose-related lung inflammation where neutrophils peaked at 24 h and concentration of TNF-α and MIP-1α at 4 h post-exposure. The inflammatory potency of LOSwt in comparison with LOSmsbB was significantly higher in the concentration of total cells, neutrophils, TNF-α and MIP-1 α in BAL fluid. At 4 h neutrophils were 5 to 6-fold higher and TNF-α was 19 to 21-fold higher in mice exposed to LOSwt compared to LOSmsbB. The concentration of MIP-1α was over 20-fold higher in BAL fluid of LOSwt-exposed mice. At 24 h the number of neutrophils was still 3 to 6-fold higher. Conclusion: Markedly reduced inflammatory lung responses were observed in mice exposed to penta-acylated LOSmsbB as compared to LOSwt. Funded By: NIEHS P30 ES05605

Rationale: MD-2 is a secreted protein that associates with TLR4 and represents the link between endotoxin binding and TLR4 signaling. In the absence of MD-2, preformed monomeric endotoxin:MD-2 complex is the only form of endotoxin that can activate TLR4. MD-2 expression is very low in resting human airway epithelia. We tested the hypothesis that airway levels of MD-2 are the limiting factor in the response to inhaled endotoxin by comparing the ability of purified aggregates vs. endotoxin:MD-2 complex to induce lung inflammation. Methods: MD-2 cDNA was inserted into baculovirus with polyhistidine-tagged recombinant DNA from MD-2. Monomeric complexes of Neisseria meningitidis LOS (LOS:MD-2) and LOS aggregates were generated by gel sieving chromatography. BALB/c mice were exposed to LOS:MD-2 or LOS by nasal aspiration (30-300 EU). Inflammation was evaluated in bronchoalveolar lavage (BAL) fluid 4 hr later by the concentration of total cells, neutrophils, and 6 cytokines. Results: At both doses mice treated with the LOS:MD-2 complex had more total cells, neutrophils, TNF-α and MIP-1α than those treated with LOS alone. The 30 EU LOS:MD2-treated mice had 3.3-fold higher total cells, 4.2-fold higher neutrophils and 2.6-fold higher TNF-α than mice treated with 30 EU LOS. At the 300 EU dose responses to the complex were higher than LOS but the difference was less dramatic. Conclusion: LOS:MD-2 complex enhanced the activation of inflammatory responses and generated more intensive neutrophilic inflammation than LOS alone. This suggests that normal airway levels of MD-2 limit epithelial responses to endotoxin and demonstrate the importance of MD-2 in the airway responsiveness to inhaled endotoxin. Funded By: NIH P30 ES05605

Rationale: Endotoxin is a potent inflammatory agent capable of triggering asthma episodes as well as inducing organic dust toxic syndrome. Our goal was to test the inflammatory potency of one form of endotoxin (lipooligosaccharide or LOS) to establish the dose-response curve, the time course and histopathological changes in the lungs for future studies of MD-2 and TLR4 signalling. Methods: Endotoxin-responsive 6-wk old BALB/c mice were exposed to selected doses of LOS from N. meningitidis (0, 5, 10, 30, 300 and 3000 EU/mouse) by nasal aspiration. Concentrations of total and differential cells and cytokines (TNF-α , MIP-1α , IL-1β , IL-6, G-CSF, IL-17) were measured at 1, 4, 8, 16, 24 and 48 hr in bronchoalveolar lavage fluid (BAL). Results: BAL neutrophils, proinflammatory cytokines, especially TNF-α , MIP-1α , IL-1β , IL-6 and histopathological changes in the lungs clearly showed a dose-response relationship with increasing doses producing increased inflammation and cytokine concentrations. Time-course studies demonstrated maximal neutrophilia in BAL at 24 hours followed by histopathological changes in the lungs. Assay of BAL supernatant for TNF-α and MIP-1α concentration showed significant increases compared to controls 4 hr after LOS exposure. After 8 hr, their concentration began to decline toward baseline. Conclusions: We characterized the early inflammatory events associated with nasal aspiration of LOS. A similar pattern of cytokine release was shown after aspiration of 30 and 300 EU/mouse. The dose response and time course of LOS response now make possible more detailed studies of the mechanism of proinflammatory cytokine signaling and neutrophil recruitment in the lungs. Funded By: NIH P30 ES05605-15

Rationale: NF-kappaB is necessary for coordinating the activation of many genes involved in inflammatory responses in the lungs. We explored the use of NF-kB-luciferase BALB/c transgenic mice to evaluate the relationship between inhaled endotoxin and the activation of NF-kB signals in lungs in vivo. Methods: Xenogen NF-kB BALB/c mice with 3 luciferase reporter elements were exposed to N. meningitidis endotoxin (0-300, 000 EU/mouse) and saline (controls) by nasal aspiration. Before exposure and after certain time points (4, 8hr) luciferin was injected intraperitoneally and mice were imaged with an ultrasensitive CCD camera (IVIS-100, Xenogen). After imaging the thoracic region, mice were euthanized and lungs were rapidly excised and imaged. The oxyluciferin signals from the thoracic region and lungs were quantified using Living Image Software. Results: The imaging of exposed transgenic NF-kB-luc BALB/c mice demonstrated a quantifiable endotoxin induced signal in the thoracic region in vivo over controls at doses of endotoxin above 3, 000 EU/mouse. The signal in the lungs of exposed mice imaged ex vivo showed high values (5 to 12-fold increases) over control and provided documentation of NF-kB activation in the lung. Conclusions: Markers of lung inflammation from bronchoalveolar lavage such as neutrophil recruitment and cytokine concentrations were more sensitive biomarkers than imaging oxyluciferin photonic yield in vivo. A lower dose of inhaled endotoxin was capable of inducing measurable lung luminescence compared with controls when lungs were imaged ex vivo. This study provides new data regarding the sensitivity of NF-kB-luc transgenic mice and their utility for in vivo monitoring of lung inflammation. Funded By: NIH P30 ES05605-15