Functional expression of TLR4 on human MCs. (A) TNF-α release from MCs after LPS stimulation or FcϵRI aggregation. Human MCs were precultured with (•) or without (▴) IFN-γ and then exposed to LPS. Control cells were incubated with (○) or without (▵) IFN-γ for 24 hours, but LPS was omitted. Positive control cells were precultured with IgE and then activated with anti-IgE (▪). Control cells were incubated with IgE, but anti-IgE was omitted (□). Cell supernatants were harvested at various times up to 12 hours for ELISA of TNF-α. (B) The source of TNF-α produced by LPS was MCs. MCs were precultured with IFN-γ as described, and monensin was added to the MCs suspension simultaneously with LPS. Cells were incubated with LPS for 6 hours. Double-intracellular staining was performed with antitryptase and anti-TNF-α in human MCs (lower panels) following stimulation with IFN-γ alone (lower left) or IFN-γ LPS (lower right). The dot plots are representative of 7 separate analyses. As a positive control, human monocytes were precultured with IFN-γ and then activated with LPS for 6 hours. Double-intracellular staining with anti-CD14 and antitryptase in monocytes (upper panels) following IFN-γ (upper left) or LPS plus IFN-γ (upper right) was performed.