Lipopolysaccharide interaction with cell surface Toll-like receptor 4-MD-2: higher affinity than that with MD-2 or CD14

Abstract

Toll-like receptors (TLRs) are innate recognition molecules for microbial products, but their direct interactions with corresponding ligands remain unclarified. LPS, a membrane constituent of gram-negative bacteria, is the best-studied TLR ligand and is recognized by TLR4 and MD-2, a molecule associated with the extracellular domain of TLR4. Although TLR4-MD-2 recognizes LPS, little is known about the physical interaction between LPS and TLR4-MD-2. Here, we demonstrate cell surface LPS-TLR4-MD-2 complexes. CD14 greatly enhances the formation of LPS-TLR4-MD-2 complexes, but is not coprecipitated with LPS-TLR4-MD-2 complexes, suggesting a role for CD14 in LPS loading onto TLR4-MD-2 but not in the interaction itself between LPS and TLR4-MD-2. A tentative dissociation constant (Kd) for LPS-TLR4-MD-2 complexes was approximately 3 nM, which is approximately 10-20 times lower than the reported Kd for LPS-MD-2 or LPS-CD14. The presence of detergent disrupts LPS interaction with CD14 but not with TLR4-MD-2. E5531, a lipid A antagonist developed for therapeutic intervention of endotoxin shock, blocks LPS interaction with TLR4-MD-2 at a concentration 100 times lower than that required for blocking LPS interaction with CD14. These results reveal direct LPS interaction with cell surface TLR4-MD-2 that is distinct from that with MD-2 or CD14.

Figure 1.

No detectable LPS binding to cells expressing TLR4-MD-2 without CD14. Ba/F3 stable transfectants were stained with anti–TLR4-MD-2 mAb (MTS510, left) or anti-CD14 mAb (4C1, middle), followed by goat anti–rat IgG-PE. (right) Ba/F3 transfectants were stimulated with 1 μg/ml LPS at 37°C for 30 min, washed, and stained with anti-LPS mAb, followed by goat anti–mouse IgG-FITC. All the open histograms depict staining with the second reagent alone.

Figure 2.

LPS down-regulates MTS510 binding to cell surface TLR4-MD-2. Ba/F3 cells expressing TLR4-MD-2 (left) or CD14⁺TLR4-MD-2 (middle), or a macrophage line RAW264 (right) were stimulated with medium alone, 1 μg/ml lipid A, 10 μg/ml peptidoglycan (PGN), or 100 μM CpG DNA as indicated. After washing, cells were stained with biotinylated MTS510 followed by streptavidin-PE.

Figure 3.

A novel mAb to TLR4-MD-2 reveals the LPS-triggered change of cell surface TLR4-MD-2. (a) Immunoprecipitation with anti-flag (top) or Sa15-21 (bottom) was conducted with Ba/F3 transfectants expressing the indicated molecules (Materials and Methods). The precipitates were probed with rabbit anti–mouse TLR4 sera followed by goat anti–rabbit alkaline phosphatase. Only immature, smaller TLR4 is detected in cells expressing TLR4 alone (top, TLR4f and CD14/TLR4f), because TLR4 without MD-2 cannot reach the cell surface. (b) Ba/F3 transfectants expressing CD14 and TLR4-MD-2 were stimulated with medium alone, 1 μg/ml lipid A, or 1 μg/ml LPS at 37°C for 30 min. Cells were stained with biotinylated MTS510 mAb or Sa15-21 as indicated, followed by streptavidin-PE. Open histograms depict staining with streptavidin-PE alone. (c) Ba/F3 cells expressing TLR4-MD-2 and CD14 (top) or RAW264 (bottom) were stimulated with medium, 2 μg/ml LPS, or 2 μg/ml lipid A antagonist E5531 as indicated at 37°C for 30 min. After washing, cells were subjected to cell surface biotinylation, detergent lysis, immunoprecipitation with MTS510 mAb (right three lanes) or Sa15-21 mAb (left three lanes), SDS-PAGE (7.5% polyacrylamide under nonreducing conditions), and electroblotting. Precipitated cell surface TLR4 was probed with streptavidin–alkaline phosphatase conjugate.

Figure 4.

Lipid A–TLR4-MD-2 complexes. (a) Ba/F3 cells expressing TLR4-MD-2 and CD14 (10⁸/10 ml sample) were incubated with 5 μg/ml lipid A at 37°C for 30 min. Cells were subjected to washing, detergent lysis, immunoprecipitation (with mAbs to TLR4-MD-2, MTS510, and Sa15-21, or an mAb to CD14, Sa2-8, and SDS-PAGE [17.5% for lipid A, 10% for CD14, and 7.5% for TLR4; under nonreducing conditions]), and electroblotting. Precipitates were probed with anti-TLR4 polyclonal Ab, anti-CD14 mAb (Sa2-8), or anti–lipid A mAb, followed by alkaline phosphatase–conjugated secondary antibodies. Nonspecific signals detecting IgG heavy chains were observed in the precipitates probed with anti-CD14 mAb (left four lanes). (b) A variety of Ba/F3 stable transfectants (2.5 × 10⁷/sample) were stimulated with ³H-lipid A (0.75 μCi/sample) at 37°C for 30 min. Cells were subjected to washing, detergent lysis, and immunoprecipitation with MTS510, Sa15-21, anti-flag, or anti-CD14 mAbs. Precipitated radioactivity was counted by scintillation counter. Similar results were obtained from three independent experiments using ³H-lipid A.

Figure 5.

LPS–CD14 complexes are disrupted by detergents. Supernatant from Ba/F3 cells expressing 1 ml CD14 (cultured up to 4–5 × 10⁶/ml) was incubated with 3 μg/ml LPS at 37°C for 30 min; sCD14 in the supernatant from Ba/F3 cells expressing CD14 was immunoprecipitated with anti-CD14 mAb (4C1). The indicated detergents (1% Triton X-100, 0.5% N-octyl-β-d-glucoside, and 1% Brij98) were included before immunoprecipitation. Precipitated LPS or CD14 was probed with anti-LPS mAb and anti-CD14 mAb (Sa2-8), followed by alkaline phosphatase–conjugated secondary antibodies.

Figure 6.

Binding properties of lipid A interaction with TLR4-MD-2. Ba/F3 stable transfectant expressing CD14 and TLR4-MD-2 (10⁸ cells/10 ml medium) were stimulated with various concentrations of ³H-lipid A (0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μCi) for 30 min at 37°C. After washing, cells were subjected to immunoprecipitation with Sa15-21 or with MTS510. Precipitated radioactivity was counted by a liquid scintillation counter (Aloka). Specific binding was obtained by subtracting bound cpm with MTS510 from that with Sa15-21. Bound lipid A (cpm) was plotted against input lipid A (cpm) in panel a. Scatchard plot is shown in panel b. Two independent experiments were conducted and similar results were obtained.

Figure 7.

E5531 acts on LPS interaction with TLR4-MD-2 at a concentration that does not affect LPS binding to mCD14. (a) Ba/F3 cells expressing TLR4-MD-2 and CD14 were pretreated with or without E5531 (indicated concentration) at 37°C for 30 min. Cells were stimulated with medium alone or 3 μg/ml LPS at 37°C for 30 min. After washing, cells were stained with biotinylated MTS510 mAb followed by streptavidin-PE (left and middle columns), or with anti-LPS followed by goat anti–mouse IgG-FITC (right column). Open histograms depict staining with the secondary reagent alone. (b) 3 μg/ml LPS with indicated concentrations of E5531 was added to the supernatant from Ba/F3 cells expressing CD14. sCD14 in the supernatant was precipitated with anti-CD14 mAb, followed by immunoprobing with anti-LPS (top) or anti-CD14 (bottom). (c) After treatment with E5531 and LPS as in panel a, cells were subjected to cell surface biotinylation, washing, detergent lysis, immunoprecipitation with Sa15-21, SDS-PAGE (polyacrylamide gel:18.0% for LPS and 7.5% for TLR4; under nonreducing conditions), and electroblotting. Precipitated LPS (top) and cell surface TLR4 (bottom) were probed with anti-LPS mAb or alkaline phosphatase–conjugated streptavidin, respectively. (d) After treatment with LPS and E5531 as in panel c, cells were subjected to detergent lysis, SDS-PAGE, electroblotting, and immunoprobing IkBα (top) or actin (bottom).

Figure 7.

E5531 acts on LPS interaction with TLR4-MD-2 at a concentration that does not affect LPS binding to mCD14. (a) Ba/F3 cells expressing TLR4-MD-2 and CD14 were pretreated with or without E5531 (indicated concentration) at 37°C for 30 min. Cells were stimulated with medium alone or 3 μg/ml LPS at 37°C for 30 min. After washing, cells were stained with biotinylated MTS510 mAb followed by streptavidin-PE (left and middle columns), or with anti-LPS followed by goat anti–mouse IgG-FITC (right column). Open histograms depict staining with the secondary reagent alone. (b) 3 μg/ml LPS with indicated concentrations of E5531 was added to the supernatant from Ba/F3 cells expressing CD14. sCD14 in the supernatant was precipitated with anti-CD14 mAb, followed by immunoprobing with anti-LPS (top) or anti-CD14 (bottom). (c) After treatment with E5531 and LPS as in panel a, cells were subjected to cell surface biotinylation, washing, detergent lysis, immunoprecipitation with Sa15-21, SDS-PAGE (polyacrylamide gel:18.0% for LPS and 7.5% for TLR4; under nonreducing conditions), and electroblotting. Precipitated LPS (top) and cell surface TLR4 (bottom) were probed with anti-LPS mAb or alkaline phosphatase–conjugated streptavidin, respectively. (d) After treatment with LPS and E5531 as in panel c, cells were subjected to detergent lysis, SDS-PAGE, electroblotting, and immunoprobing IkBα (top) or actin (bottom).