The prophagocytic effect of MD-2 is mediated by TLR4. (A) 293 cells expressing TLR4 and a NF-κB-luciferase reporter plasmid were stimulated with live Nm (MC58) or with Nm lpxA⁻ that had been treated as indicated (y-axis). Luciferase readings were normalized by the activity of unstimulated cells (RLU = 1). Shown are the averages of duplicate points plus the range. LPS served as control. A similar experiment was performed with 293 cells (bottom panel). (B) pMφ from TLR4^−/− (□) or wild-type (■) mice were infected with GFP-Nm or YFP-Yp that were processed for the phagocytosis assay as in Figure 4A. Note that MD-2 coating exerts no effect on TLR4^−/− cells. (C) pMφ from the BALB/C (Lps^wt) and C3H/HeJ (Lps^d) mice were infected with nonopsonized (▩) or MD-2^6xHis–coated YFP-Yp and subjected to fluorescent opsonophagocytosis assay as in panel B. These experiments were repeated at least 3 times with similar results. Data are averages of triplicates plus SD.