Specific High Affinity Interactions of Monomeric Endotoxin·Protein Complexes with Toll-like Receptor 4 Ectodomain
2006; Elsevier BV; Volume: 282; Issue: 2 Linguagem: Inglês
10.1074/jbc.m609400200
ISSN1083-351X
AutoresPolonca Prohinar, Fabio Re, Richard Widstrom, Desheng Zhang, Athmane Teghanemt, Jerrold Weiss, Theresa L. Gioannini,
Tópico(s)Antimicrobial Peptides and Activities
ResumoPotent 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. Ligand-receptor interactions involving endotoxin and TLR4 were characterized using monomeric endotoxin·protein complexes of high specific radioactivity. The binding of endotoxin·MD-2 to the TLR4 ectodomain (TLR4ECD) and transfer of endotoxin from CD14 to MD-2/TLR4ECD were demonstrated using HEK293T-conditioned medium containing TLR4ECD ± MD-2. These interactions are specific, of high affinity (KD < 300 pm), and consistent with the molecular requirements for potent cell activation by endotoxin. Both reactions result in the formation of a Mr ∼ 190,000 complex composed of endotoxin, MD-2, and TLR4ECD. CD14 facilitates transfer of endotoxin to MD-2 (TLR4) but is not a stable component of the endotoxin·MD-2/TLR4 complex. The ability to assay specific high affinity interactions of monomeric endotoxin·protein complexes with TLR4ECD should allow better definition of the structural requirements for endotoxin-induced TLR4 activation. 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. Ligand-receptor interactions involving endotoxin and TLR4 were characterized using monomeric endotoxin·protein complexes of high specific radioactivity. The binding of endotoxin·MD-2 to the TLR4 ectodomain (TLR4ECD) and transfer of endotoxin from CD14 to MD-2/TLR4ECD were demonstrated using HEK293T-conditioned medium containing TLR4ECD ± MD-2. These interactions are specific, of high affinity (KD < 300 pm), and consistent with the molecular requirements for potent cell activation by endotoxin. Both reactions result in the formation of a Mr ∼ 190,000 complex composed of endotoxin, MD-2, and TLR4ECD. CD14 facilitates transfer of endotoxin to MD-2 (TLR4) but is not a stable component of the endotoxin·MD-2/TLR4 complex. The ability to assay specific high affinity interactions of monomeric endotoxin·protein complexes with TLR4ECD should allow better definition of the structural requirements for endotoxin-induced TLR4 activation. Essential arms of the innate immune system are the Toll-like receptors (TLRs). 2The abbreviations used are: TLR, Toll-like receptor; TLR4ECD, Toll-like receptor 4 ectodomain; AOAH, acyloxyacyl hydrolase; GNB, Gram-negative bacteria; HEK, human embryonic kidney; HSA, human serum albumin; LBP, lipopolysaccharide-binding protein; LOS, lipooligosaccharide; LOSagg, LOS aggregate; PBS, phosphate-buffered saline; sCD14, soluble CD14; cpm, counts/min. These receptors link recognition of unique microbial molecules to activation of host defense effector systems by rapidly triggering pro-inflammatory responses (1Beutler B. Jiang Z. Georgel P. Crozat K. Croker B. Rutschmann S. Du X. Hoebe K. Annu. Rev. Immunol. 2006; 24: 353-389Crossref PubMed Scopus (668) Google Scholar). Potent host responses toward many Gram-negative 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 (2Akashi S. Nagai Y. Ogata H. Oikawa M. Fukase K. Kusumoto S. Kawasaki K. Nishijima M. Hayashi S. Kimoto M. Miyake K. Int. Immunol. 2001; 13: 1595-1599Crossref PubMed Scopus (209) Google Scholar, 3Akashi S. Saitoh S. Wakabayashi Y. Kikuchi T. Takamura N. Nagai Y. Kusumoto Y. Fukase K. Kusumoto S. Adachi Y. Kosugi A. Miyake K. J. Exp. Med. 2003; 198: 1035-1042Crossref PubMed Scopus (330) Google Scholar, 4Miyake K. Int. Immunopharmacol. 2003; 3: 119-128Crossref PubMed Scopus (102) Google Scholar, 5Nagai Y. Akashi S. Nagafuku M. Ogata M. Iwakura Y. Akira S. Kitamura T. Kosugi A. Kimoto M. Miyake K. Nat. Immunol. 2002; 3: 667-672Crossref PubMed Scopus (844) Google Scholar, 6Shimazu R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M. J. Exp. Med. 1999; 189: 1777-1782Crossref PubMed Scopus (1749) Google Scholar). Maximally potent endotoxin-induced cell activation also requires the extracellular lipopolysaccharide-binding protein (LBP) and membrane (m) or soluble (s) extracellular CD14 (4Miyake K. Int. Immunopharmacol. 2003; 3: 119-128Crossref PubMed Scopus (102) Google Scholar, 7Beutler B. Annu. Rev. Pharmacol. Toxicol. 2003; 43: 609-628Crossref PubMed Scopus (71) Google Scholar, 8Gioannini T.L. Teghanemt A. Zhang D. Levis E.N. Weiss J.P. J. Endotoxin Res. 2005; 11: 117-123Crossref PubMed Google Scholar, 9Ulevitch R.J. Tobias P.S. Curr. Opin. Immunol. 1999; 11: 19-22Crossref PubMed Scopus (487) Google Scholar). 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 endotoxin·CD14 and TLR4, apparently, for endotoxin presented as a monomeric complex with MD-2 (8Gioannini T.L. Teghanemt A. Zhang D. Levis E.N. Weiss J.P. J. Endotoxin Res. 2005; 11: 117-123Crossref PubMed Google Scholar). Together, these proteins can convert one GNB (containing ∼106 endotoxin molecules) to 106 TLR4-activating monomeric endotoxin·protein complexes (i.e. endotoxin·CD14 or endotoxin·MD-2), greatly amplifying host responsiveness to endotoxin. At pm concentrations, monomeric complexes of endotoxin·CD14 or endotoxin·MD-2 activate, respectively, mammalian cells expressing MD-2/TLR4 or TLR4 alone, triggering robust cell activation through engagement of <103 TLR4 molecules. Despite the ability of endotoxin·CD14 and endotoxin· MD-2 to activate cells at pm concentrations (half-maximal cell activation is 20 × 106) was obtained after hot phenol extraction of [3H]LOS followed by ethanol precipitation of [3H]LOSagg and ultracentrifugation. Monomeric [3H]LOS·CD14 complexes (Mr ∼60,000) were prepared by treatment of [3H]LOSagg for 30 min at 37 °C with substoichiometric LBP (molar ratio 200:1 of LOS:LBP) and equimolar sCD14 followed by gel exclusion chromatography (Sephacryl S200, 1.6 × 70-cm column) in PBS, pH 7.4, 0.03% HSA to isolate monomeric [3H]LOS·sCD14 complex. [3H]LOS·MD-2 (Mr ∼25,000) was generated by treatment of [3H]LOS·sCD14 (30 min at 37 °C) with High Five insect cell medium containing MD-2-His6 followed by isolation of [3H]LOS·MD-2 by S200 chromatography. Tetraacylated LOS (specific activity ∼16,000 cpm/pmol) was prepared by partial deacylation of [3H]LOS with AOAH according to the method of Munford and Erwin (20Munford R.S. Erwin A.L. Methods Enzymol. 1992; 209: 485-492Crossref PubMed Scopus (25) Google Scholar), and [3H]LOSAOAH·MD-2 was prepared and isolated as described previously (21Teghanemt A. Zhang D. Levis E.N. Weiss J.P. Gioannini T.L. J. Immunol. 2005; 175: 4669-4676Crossref PubMed Scopus (128) Google Scholar). The extent of deacylation of [3H]LOS by AOAH was monitored by separation of released [3H]-free fatty acids from partially deacylated and remaining intact [3H]LOS by ethanol precipitation. Ethanol-soluble radioactivity representing released [3H]fatty acids was analyzed by liquid scintillation spectroscopy (20Munford R.S. Erwin A.L. Methods Enzymol. 1992; 209: 485-492Crossref PubMed Scopus (25) Google Scholar, 21Teghanemt A. Zhang D. Levis E.N. Weiss J.P. Gioannini T.L. J. Immunol. 2005; 175: 4669-4676Crossref PubMed Scopus (128) Google Scholar); ≥ 90% [3H]LOS was deacylated (21Teghanemt A. Zhang D. Levis E.N. Weiss J.P. Gioannini T.L. J. Immunol. 2005; 175: 4669-4676Crossref PubMed Scopus (128) Google Scholar). Radiochemical purity of [3H]LOSagg, [3H]LOS·sCD14, and [3H]LOS·MD-2 was confirmed by Sephacryl S500 (LOSagg) or S200 ([3H]LOS·sCD14, [3H]LOS·MD-2) chromatography (17Gioannini T.L. Teghanemt A. Zhang D. Coussens N.P. Dockstader W. Ramaswamy S. Weiss J.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4186-4191Crossref PubMed Scopus (297) Google Scholar, 18Giardina P.C. Gioannini T. Buscher B.A. Zaleski A. Zheng D.S. Stoll L. Teghanemt A. Apicella M.A. Weiss J. J. Biol. Chem. 2001; 276: 5883-5891Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Reaction of Secreted TLR4ECD and MD-2/TLR4ECD with [3H]LOS·Protein Complexes—[3H]LOSagg, [3H]LOS·sCD14, or [3H]LOS·MD-2 (1 nm or as indicated) was incubated with concentrated (8–10×) conditioned medium ± proteins diluted to a final volume of 0.5 or 1 ml in PBS, pH 7.4, for 30 min at 37°C. In most experiments, conditioned medium harvested after a 48-h culture of transfected HEK293T cells in serum-free medium was used for incubations with [3H]LOSagg, [3H]LOS·sCD14, or [3H]LOS·MD-2. However, in selected experiments, the serum-free medium was spiked with [3H]LOS·sCD14 (1 nm) at the time of the addition of medium to the transfected cells to permit reaction of MD-2 with [3H]LOS·sCD14 upon secretion. Medium harvested without [3H]LOS·sCD14 is expressed (see Figs. 1 and 2, 3, 4, 5) as recombinant protein secreted in cm, whereas medium spiked with [3H]LOS·sCD14 during cell culture are represented as HEK/recombinant protein(s) secreted + [3H]LOS·sCD14 (see Fig. 4). Reaction products were analyzed by Sephacryl HR S200 or S300 (1.6 × 70 cm) chromatography in PBS, pH 7.4, ±0.5 mm Mg2+, 1 mm Ca2+, 0.03% HSA. Divalent cations and HSA were used in columns with LOSagg to improve recoveries of LOSagg. Fractions (0.5 ml) were collected at a flow rate of 0.3 ml/min at room temperature using AKTA Purifier or Explorer 100 fast protein liquid chromatography (GE Healthcare). Radioactivity in collected fractions was analyzed by liquid scintillation spectroscopy (Beckman LS liquid scintillation counter). Recoveries of [3H]LOS were ≥70% in all cases. All solutions used were pyrogen-free and sterile-filtered. After chromatography, selected fractions were sterile-filtered (0.22 μm) and kept at 4 °C for 3–6 months with no detectable changes in chromatographic or functional properties. The same conditioned medium was used for all concentrations of [3H]LOS·sCD14 or [3H]LOS·MD-2 used for Scatchard analysis. Multiple preparations of conditioned medium contained similar amounts of TLR4ECD or MD-2/TLR4ECD, i.e. 1–2 and 3–4 pmol/ml, respectively.FIGURE 2Specific high affinity interactions of [3H]LOS·sCD14 with MD-2/TLR4ECD. Concentrated control-conditioned medium from HEK293T cells, (HEK293)cm, or medium containing MD-2-FLAG-His6/FLAG-TLR4ECD (MD-2/TLR4ECD)cm) was incubated 30 min at 37 °C with 1 nm [3H]LOS as indicated. Reactants and products were resolved by Sephacryl S200 (A and B) or S300 (C) as described under “Experimental Procedures.” D, fractions containing encircled peak (A) were analyzed on Sephacryl S300, and the apparent Mr determined by comparison to protein standards (see inset and “Experimental Procedures”). The profile of the product from Fig. 1D is shown for comparison. Profiles shown are representative of ≥3 experiments. Recovery of [3H]LOS was ≥80% endotoxin. Co-capture of [3H]LOS from Mr 190,000 complex by adsorption to Ni2+ chelating resin (E) or streptavidin-agarose (F) after incubation of the Mr 190,000 complex with biotinylated or non-biotinylated anti-TLR4 monoclonal antibody HTA125 as described under “Experimental Procedures.” Results shown are representative of two or more similar experiments each in duplicate. Conditioned medium containing MD-2/TLR4ECD (∼75 pm) was incubated for 30 min at 37 °C with varying concentrations of [3H]LOS·sCD14. Formation of the Mr ∼ 190,000 complex was monitored by gel-sieving chromatography (selected doses of [3H]LOS·sCD14 (G)) and converted to molar amounts based on specific radioactivity of [3H]LOS (H).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Monomeric complexes of [3H]LOS with full-length or truncated (amino acids 1–156) sCD14 form the 190,000 complex after incubation with MD-2/TLR4ECD. Concentrated conditioned medium containing MD-2-FLAG-His6/FLAG-TLR4ECD was incubated 30 min at 37 °C with 1 nm [3H]LOS·sCD14 or [3H]LOS·sCD14-(1–156) as indicated. Products were resolved by Sephacryl S200 chromatography. Profiles shown are representative of ≥3 experiments. Recovery of [3H]LOS was ≥80%.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Increased stability of endotoxin binding activity of secreted MD-2 by co-expression with TLR4ECD in HEK293 cells. HEK293T cells were transfected with expression vector (pEF-BOS/pFLAG-CMV) encoding MD-2-FLAG-His/FLAG-TLR4ECD (A), MD-2-FLAG-His6 (B), or vectors alone (C); after 12 h, medium was replaced with 1 nm [3H]LOS·sCD14 in serum-free medium, 0.1% HSA. Conditioned medium was collected after an additional 24 h, and 0.5 ml applied directly to a Sephacryl S200 column. D, conditioned medium from HEK293T cells transfected with pEF-BOS encoding MD-2-FLAG-His was harvested at 48 h, concentrated 10×, and an aliquot incubated 30 min at 37 °C with 1 nm [3H]LOS·sCD14 as indicated. Reactants and products were resolved by chromatography on Sephacryl S200. Profiles shown are representative of ≥3 experiments; overall recoveries were >80%.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Comparison of the reactivity of hexaacylated [3H]LOS·MD-2 and tetraacylated [3H]LOSAOAH·MD-2 complexes with TLR4ECD. Concentrated conditioned medium from HEK293T cells containing TLR4ECD was incubated 30 min at 37 °C with ∼1 nm 3H wild-type LOS·MD-2 or tetraacylated LOSAOAH·MD-2. Products were resolved by Sephacryl S200 chromatography. Profiles shown are representative of two or more profiles for each experimental condition.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Determination of Apparent Size—Sephacryl S300 used for determination of apparent Mr was calibrated with the following proteins: blue dextran (2 × 106, V0), thyroglobulin (650,000), ferritin (440,000), catalase (232,000), IgG (158,000), HSA (66,000), ovalbumin (44,500), myoglobin (17,500), vitamin B12 (1200, Vi). For size determination, the complex containing [3H]LOS was resolved in the presence of at least three protein standards. Protein standards were detected by A280 and the [3H]LOS-containing complex by liquid scintillation spectroscopy. Mr was calculated using GraphPad Prism version 4. Co-capture Analyses—The protein composition of [3H]LOS-containing complexes generated from incubation of [3H]LOS·MD-2-His6 with medium containing FLAG-TLR4ECD or of [3H]LOS·sCD14 with medium containing MD-2-FLAG-His6 and FLAG-TLR4ECD was determined by monitoring [3H]LOS adsorption to resins that specifically interacted with either TLR4 or MD-2. Co-capture of [3H]LOS associated with MD-2-FLAG-His6 (HEK293T cell-derived) or MD-2-His6 (insect cell-derived) was performed using Ni2+ FF-Sepharose resin pre-equilibrated in either PBS (product containing MD-2-FLAG-His6) or 20 mm phosphate and 0.5 m NaCl, pH 7.4, (product containing MD-2-His6). Resin (∼50 μl) was incubated with 0.2 pmol [3H]LOS-containing complexes for 1 h at room temperature. The resin was spun down, supernatant was removed, and the resin was washed with PBS, pH 7.4, or 20 mm phosphate, 0.5 m NaCl, pH 7.4, before elution with 2% SDS or 0.5 m imidazole. [3H]LOS absorbed to the resin was evaluated by liquid scintillation spectroscopy. Detection of FLAG-TLR4ECD in [3H]LOS-containing complexes was determined by adsorption to anti-FLAG-agarose (for the product of the reaction of [3H]LOS·MD-2-His6 + (FLAG-TLR4ECD)cm) or by reaction with biotinylated HTA125 anti-TLR4 antibody followed by adsorption to streptavidin-agarose (for the product of the reaction of [3H]LOS·sCD14 with (MD-2-FLAG-His6/FLAG-TLR4)cm). [3H]LOS-containing complex (0.2 pmol) was incubated with 75 μl of anti-FLAG-agarose in 0.5 ml of buffer (PBS, pH 7.4, or 20 mm phosphate and 0.5 m NaCl, pH 7.4) overnight at 4 °C on a rotating wheel. The supernatant was collected after spinning (2000 revolutions/min, 5 min, 4 °C), and the sedimented beads were washed three times with 20 mm phosphate and 0.5 m NaCl before [3H]LOS adsorbed to beads was determined by liquid scintillation spectroscopy. Immunocapture by anti-TLR4 antibody of [3H]LOS-containing complex (0.2 pmol) was performed by incubation of the complex with 2 μg of biotinylated HTA125 anti-TLR4 antibody (or non-biotinylated HTA125 as a negative control) in PBS, pH 7.4. After overnight incubation at 4 °C with the sample, 50 μl of PBS-equilibrated streptavidin-coupled agarose beads were added for an additional 1 h at room temperature. Adsorbed [3H]LOS-containing complex was determined by measuring [3H]LOS associated with the washed agarose. Soluble TLR4ECD Binds [3H]LOS·MD-2 with Picomolar Affinity—We have previously described the generation and isolation of a monomeric [3H]LOS·MD-2 complex that potently activates cells expressing TLR4 without MD-2 (17Gioannini T.L. Teghanemt A. Zhang D. Coussens N.P. Dockstader W. Ramaswamy S. Weiss J.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4186-4191Crossref PubMed Scopus (297) Google Scholar, 21Teghanemt A. Zhang D. Levis E.N. Weiss J.P. Gioannini T.L. J. Immunol. 2005; 175: 4669-4676Crossref PubMed Scopus (128) Google Scholar, 22Jia H.P. Kline J.N. Penisten A. Apicella M.A. Gioannini T.L. Weiss J. McCray Jr., P.B. Am. J. Physiol. 2004; 287: L428-L437Crossref PubMed Scopus (140) Google Scholar). [3H]LOS·MD-2, but not the same [3H]LOS (25,000 cpm/pmol) presented as LOS aggregates (LOSagg) or monomeric LOS·sCD14, associates with these cells in a TLR4-dependent fashion (17Gioannini T.L. Teghanemt A. Zhang D. Coussens N.P. Dockstader W. Ramaswamy S. Weiss J.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4186-4191Crossref PubMed Scopus (297) Google Scholar). However, detection of cell-associated [3H]LOS·MD-2 required several hours of incubation (17Gioannini T.L. Teghanemt A. Zhang D. Coussens N.P. Dockstader W. Ramaswamy S. Weiss J.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4186-4191Crossref PubMed Scopus (297) Google Scholar), suggesting that levels of surface-expressed TLR4 in these cells were too low to measure direct binding. To circumvent these limitations and permit more direct assay of the molecular requirements for endotoxin-TLR4 interactions, we transiently expressed, in HEK293T cells, an N-terminal fragment of recombinant human TLR4 (residues 24–634) corresponding to the predicted ectodomain of TLR4 (TLR4ECD) and containing an N-terminal FLAG tag. Harvested control and TLR4ECD-containing culture media were incubated with 1 nm of purified [3H]LOS aggregates (LOSagg), monomeric [3H]LOS·sCD14, or [3H]LOS·MD-2-His6. Interaction of the various forms of [3H]LOS with TLR4ECD was monitored by gel filtration analysis to assay for TLR4ECD-dependent changes in the physical state of [3H]LOS. Under these experimental conditions, only [3H]LOS·MD-2 reacted with TLR4ECD (Fig. 1). Incubation of [3H]LOS·MD-2 with conditioned medium containing TLR4ECD (but not control medium) yielded a novel [3H]LOS-containing product (Fig. 1A, encircled peak) whose formation depended upon TLR4ECD and the presentation of [3H]LOS as [3H]LOS·MD-2 (Fig. 1, compare A to B and C). Neither [3H]LOSagg nor [3H]LOS·sCD14 showed interaction with TLR4ECD (Fig. 1, B and C), even when added at 200× greater LOS concentrations (data not shown). These findings strongly suggest a specific and direct interaction of [3H]LOS·MD-2 with TLR4ECD. Rechromatography on Sephacryl S300 of the newly formed [3H]LOS-containing product recovered after incubation of [3H]LOS·MD-2-His6 with medium containing FLAG-TLR4ECD (Fig. 1A, encircled fractions) yielded a single symmetrical peak that, by comparison to elution of protein standards, gave a predicted Mr ∼ 190,000 (Fig. 1D). [3H]LOS in the product could be captured by anti-FLAG (Fig. 1F) antibodies as well as by Ni+2-chelating resin (Fig. 1E), indicating the presence of MD-2-His6 as well as [3H]LOS and FLAG-TLR4ECD in the Mr ∼ 190,000 complex. The high specific radioactivity of [3H]LOS·MD-2 permitted quantitative assay of the formation of the Mr ∼ 190,000 complex at pm concentrations of [3H]LOS·MD-2 and under conditions where the concentration of TLR4ECD was limiting. Formation of the Mr ∼ 190,000 complex was dependent on [3H]LOS·MD-2 concentration and saturated at ∼1 nm [3H]LOS·MD-2 with half-maximal formation at ∼200–300 pm [3H]LOS·MD-2 (Fig. 1H). Equilibrium conditions were met after 30 min at 37 °C as equal amounts of product were formed at 30 and 120 min. Scatchard analysis (Fig. 1H) indicated an apparent KD of ∼300 pm for the reaction of [3H]LOS·MD-2 with TLR4ECD. The interaction of [3H]LOS·MD-2 (25,000 cpm/pmol) with TLR4ECD could be competed with weakly labeled [14C]LOS·MD-2 (625 cpm/pmol) but not [14C]LOSagg or LOS·sCD14 (data not shown), further demonstrating the specificity of the interaction of LOS·MD-2 with TLR4ECD. Transfer of [3H]LOS from [3H]LOS·sCD14 to MD-2/TLR4ECD—The inability of [3H]LOS·sCD14 to react with TLR4ECD (Fig. 1B) is consistent with the requirements of co-expression of MD-2 and TLR4 for sensitive cellular responses to monomeric endotoxin·sCD14 complex (22Jia H.P. Kline J.N. Penisten A. Apicella M.A. Gioannini T.L. Weiss J. McCray Jr., P.B. Am. J. Physiol. 2004; 287: L428-L437Crossref PubMed Scopus (140) Google Scholar). To demonstrate more directly the requirement of MD-2 for interaction of [3H]LOS·sCD14 with complexes containing TLR4ECD, we co-transfected HEK293T cells with expression plasmids for FLAG-TLR4ECD and MD-2-FLAG-His6. In contrast to incubations with control medium or medium containing TLR4ECD alone, incubation of [3H]LOS·sCD14 with harvested culture medium containing both secreted MD-2 and TLR4ECD yielded an [3H]LOS-containing product that eluted at Mr > [3H]LOS·sCD14 (Fig. 2A). In contrast to the efficient reaction of [3H]LOS·sCD14 with MD-2 and TLR4ECD, there
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