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

Abstract

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.

FIGURE 1.

Insect-derived conditioned medium containing secreted MD-2-His contains more active MD-2 and more MD-2 in a monomeric state than HEK-derived MD-2-His. A, functional MD-2 was assayed by measuring activation of HEK/TLR4 cells induced by incubating cells with [¹⁴C]LOS·sCD14 (2 nm) and increasing amounts of conditioned medium (insect, ○; HEK-293, •) for 24 h. Cell activation was monitored by extracellular accumulation of IL-8, which was measured by ELISA. The results shown represent the means ± S.E. of three experiments, each in triplicate. B, composition of secreted MD-2 determined by SDS-PAGE ± dithiothreitol (DTT)/immunoblots (25 μl/lane) harvested at 48 h from HEK293T cells transfected with expression vector (pEF-BOS) encoding MD-2-FLAG-His₆ and cultured in serum-free medium with 0.4% HSA (lanes 1 and 2) or from insect cells infected with baculovirus containing MD-2-His (lanes 3 and 4). MD-2 was detected using anti-(His)₄ antibody. Lane 1 represents molecular weight markers (Perfect Protein; Novagen). The blot shown is representative of more than three experiments.

FIGURE 2.

Monomeric MD-2 can be isolated in good yield on Sephacryl S200 using insect medium, but not PBS, as eluant. A and B, concentrated (10×) conditioned insect medium was applied to Sephacryl S200 (1.6 ×∼70 cm) equilibrated and eluted in PBS (A) or Express Five™ medium (B). Aliquots (25 μl) of individual fractions (1 ml) were resolved by SDS-PAGE under nonreducing conditions and after transfer to nitrocellulose probed for MD-2-His by reactivity with an anti-(His)₄ antibody. C, fractions from Sephacryl S200 eluted in Express Five™ medium (B) corresponding to dimer and monomer forms of MD-2 as determined by the immunoblot were pooled. Pooled fractions of dimer (lanes D; fractions 73–77) and monomer (lanes M; fractions 92–96) and concentrated conditioned medium (lanes CM) were treated with sample buffer ± dithiothreitol, electrophoresed, transferred to nitrocellulose, and then probed for MD-2 using an anti-(His)₄ antibody. The blots shown are representative of fractions derived from two different chromatographic separations for each eluant. Note that testing of fractions shown required two separate blots; however, the gels were electrophoresed, transferred, and probed with anti-(His)₄ antibody simultaneously.

FIGURE 3.

Only the monomeric form of MD-2 reacts with LOS·sCD14, but not LOS aggregates, to generate LOS. MD-2. A, pooled monomer (10 μl) or dimer (100 μl) was incubated with [¹⁴C]LOS·sCD14 (2 nm, 10 ng of LOS) for 30 min at 37 °C in PBS with 0.1% HSA. The reaction mixture was separated by chromatography on Sephacryl S200 in PBS. The elution profile of untreated [¹⁴C]LOS·sCD14 is shown for reference. The profiles shown are representative of at least two experiments. B and C, conditioned medium containing MD-2-His (2 ml, 10× concentrated) was incubated for 30 min at 37 °C with either (2 μm, 10 μg of LOS) [¹⁴C]LOS·sCD14 or [¹⁴C]LOS aggregates and the reaction mixture was chromatographed through Sephacryl S200 in Express Five™ medium. The fractions were analyzed for [¹⁴C]LOS content by liquid scintillation spectroscopy and the presence of MD-2 by immunoblot using anti-(His)₄ antibody. Note fractions 42–72 and 76–102 were analyzed by separate blots. However, the gels were electrophoresed, transferred, and probed with anti-(His)₄ antibody simultaneously. The blots shown are representative of fractions derived from two different chromatographic separations from two separate experiments.

FIGURE 4.

Monomeric MD-2 competes with [³H]LOS·MD-2 for reaction with the ectodomain of TLR4. A, concentrated (10×) HEK293 conditioned medium containing the ectodomain of TLR4 (100μl) was incubated with [³H]LOS·MD-2 (1 nm)(•) alone or with pooled MD-2 monomer (15μl, ▵) or MD-2 dimer (100μl, *) for 30 min at 37 °C, and the reaction mixture was applied and eluted on Sephacryl S200 in PBS. The fractions were analyzed for [³H]LOS content and formation of the M_r 190 K product by liquid scintillation spectroscopy. B, concentrated (10×) HEK293 conditioned medium containing the ectodomain of TLR4 (100 μl) was incubated with [³H]LOS·MD-2 (1 nm) ± varying amounts of MD-2 for 30 min at 37 °C, and the amount of radiolabeled 190K product formed was determined by liquid scintillation spectroscopy of the chromatographed reaction mixture on Sephacryl S200. The data are expressed as ratios of radiolabeled 190 K complex formed to LOS·MD-2 added in the presence of increasing amounts of MD-2 monomer added. C, dose-dependent inhibition by sMD-2 monomer but not by sMD-2 dimer of HEK/TLR4 cell activation by 60 pm LOS·MD-2. Cell activation was determined by measuring extracellular accumulation of IL-8 by ELISA.

FIGURE 5.

Activation of HEK/TLR4 cells by incubation of the monomeric form of MD-2 with LOS·sCD14. The HEK/TLR4 cells were incubated overnight with increasing amounts of conditioned medium containing MD-2 (•), isolated dimer (*), or monomer (▵) sMD-2 in the presence of [¹⁴C]LOS·sCD14 (2 nm)(A) or with increasing amounts of monomer sMD-2 in the presence of [¹⁴C]LOS·sCD14 (60 pm) or [¹⁴C]LOS aggregates (20 nm)(B). Cell activation was measured by determining accumulation of extracellular IL-8. Extracellular IL-8 was measured by ELISA. The results shown represent the means ± S.E. of three experiments, each in triplicate.

FIGURE 6.

Time-dependent decay of bioactive sMD-2 monomer at 37 °C (A) and at 4 °C (B and C). A, insect cell conditioned medium containing sMD-2 was diluted 20-fold in PBS with 0.1% albumin or in Express Five™ medium. Bioactive sMD-2 was measured by assay of inhibition of [³H]LOS·MD-2 binding to TLR4_ECD (as in Fig. 4) before and after 24 h of preincubation of sMD-2 at 37 °C. The results represent the means ± S.E. of three separate determinations. B, a sample of pooled sMD-2 monomer (10 μl) stored at 4 °C was taken at days 2, 21, and 33 after isolation in Express Five™ medium and incubated with [¹⁴C]LOS·sCD14 (2 nm) for 30 min at 37 °C in PBS with 0.1% HSA. The fractions were analyzed for [¹⁴C]LOS content by liquid scintillation spectroscopy. The reaction mixture was separated by chromatography on Sephacryl S200 in PBS. The profiles shown are representative of at least two experiments and show reduced activity of isolated sMD-2 monomer (i.e. formation of [¹⁴C]LOS·MD-2) during prolonged storage of 4 °C. C, samples (40 μl) of pooled sMD-2 monomer taken 33 days after isolation and storage at 4 °C and of sMD-2-rich conditioned insect cell medium (CM) were treated with SDS-PAGE sample buffer under nonreducing conditions, electrophoresed, transferred to nitrocellulose, and then probed for MD-2 using an anti-(His)₄ antibody. The markers are Perfect Protein markers (Novagen).