The nucleotide sequence of human MD-2 and its similarity to human MD-1. (A) The nucleotide sequence is shown with a deduced amino acid sequence. The signal peptide and canonical N-glycosylation sites are underlined. The stop codon is denoted by an asterisk. The nucleotide sequence of MD-2 will appear in the EMBL/GenBank/DDBJ nucleotide sequence databases under accession no. AB018549. (B) Amino acid sequences of human MD-1 and MD-2 were aligned using the BLAST2 program at the National Center for Biological Information (Bethesda, MD).

Expression of the MD-2 transcript. (A) Total RNA (20 μg/lane) from the indicated human cell lines was electrophoresed and hybridized with probes for human MD-2, TLR4, and GAPDH as indicated. Nalm-6, Ramos, Daudi, and RPMI8866 are of B lymphocyte lineage. CEM and Molt4 are of T cell origin. U937 and THP-1 are of monocytic origin. K562 is the erythroleukemia line. (B) Total RNA (20 μg/lane) from the indicated mouse tissues was used for Northern hybridization. The cDNA clone encoding mouse MD-2 was used as a probe. For GAPDH, the rat cDNA clone was used.

Membrane anchoring of MD-2 via TLR4. A stable line expressing MD-2 with the flag epitope was stained with the anti-flag mAb, followed by goat anti–mouse IgG–FITC (a and d). Staining with cell permeabilization is shown in d. Another stable transfectant was used that expressed MD-2 with the protein C epitope and TLR4 in b, c, and e. Cell surface MD-2 and TLR4 are shown with the rat anti–protein C mAb or the mouse anti–human TLR4 mAb HTA125 (b and c, respectively). MD-2 expression in permeabilized cells is shown in e. The second reagents used were goat anti–rat IgG–PE or goat anti–mouse IgG–FITC, respectively. Control histograms stained with isotype-matched mouse or rat mAb are shown (dotted lines).

MD-2 is coprecipitated with TLR4. (A) Stable transfectants expressing MD-2 alone or with TLR4 were stained with or without cell permeabilization. The intracellular MD-2 precursor was stained with the anti-protein C mAb. The specificity of the anti-TLR4 mAb was shown by cell surface staining of stable transfectants. Goat anti– mouse IgG–FITC was used as the second reagent. Dotted lines correspond to histograms stained with the second reagent alone. Panels correspond to the original Ba/F3 line, a transfectant expressing TLR4 alone, and a transfectant expressing TLR4 + MD-2, respectively. (B) Stable transfectants expressing TLR4 and MD-2 were subjected to immunoprecipitation with control mouse IgG (lane 1) or the anti-TLR4 mAb, HTA125 (lanes 2 and 3). After blotting, precipitated molecules were detected with an anti-flag mAb, M2. The precipitates shown in lanes 1 and 2 are from the transfectant expressing TLR4 and MD-2, both of which were tagged with the flag epitope. In lane 3, we used the control line in which the flag epitope was on TLR4 but not on MD-2.

MD-2 enhances ligand-independent signaling via TLR4. A human kidney cell line, 293T, was transfected with expression vectors encoding the molecules indicated. A reporter plasmid for NF-κB activity and an expression vector encoding β-galactosidase was also transfected (see Materials and Methods). Relative NF-κB activity was calculated by normalizing luciferase activity with β-galactosidase activity. Data are shown as mean values from triplicate wells. Error bars, SD.

MD-2 confers LPS signaling on TLR4. (A) Stable transfectants (see Materials and Methods for details) expressing TLR4 alone (▪) or TLR4 and MD-2 (•) were stimulated with LPS from E. coli 055:B5, S. minnesota Re595, or lipid A at the concentrations indicated. After a 4-h culture, cells were harvested, and luciferase activity was determined and expressed as relative light units. (B) The transfectant expressing TLR4 and MD-2 was stimulated with LPS from E. coli 055: B5, LPS from S. minnesota Re595, or lipid A at 100 ng/ml. The mAb HTA125 (black bars) or a control mAb (gray bars) were included in the indicated groups (10 μg/ml). After 4 h, NF-κB activity was determined. Data represent mean values from triplicate wells.