Expression of TLR4 by human MCs. (A) More than 99% of 12-week-old human MC populations stained for FcϵRI and kit (bottom panel) and the isotype controls (mIgG1 and mIgG2b; top panel). (B) Time-course of TLR4 mRNA expression by IFN-γ-treated human MCs (n = 3 donors). Total RNA (100 ng) was used for cDNA synthesis, and the fold change in the TLR4 mRNA level was evaluated by TaqMan analysis as described in “Materials and methods.” The results were expressed as the fold change in the TLR4 mRNA level of IFN-γ-treated cells (•) and untreated cells (○). ^*P < .05, for the comparison of MCs incubated with IFN-γ versus cells incubated without IFN-γ. Dagger indicates that the experimental number is 2. As a positive control, the fold change in the TLR4 mRNA level of IFN-γ-treated monocytes (▪) and untreated cells (□) is shown. (C) Surface and intracellular expression of TLR4 by human MCs in the presence or absence of IFN-γ. MCs and monocytes were cultured in medium with or without IFN-γ for 24 hours. TLR4 was detected in FcϵRI+ gated cells using biotin-conjugated anti-TLR4 mAb and streptavidin-PE and analyzed by flow cytometry. The isotype control is shown as a light line. As a positive control, monocytes were cultured similarly, and surface and intracellular TLR4 expressions were analyzed by FACS. (D-E) Localization of TLR4 in human cultured MCs (D) and human lung MCs (E). IFN-γ-treated MCs were first incubated with anti-FcϵRI (green) and anti-TLR4 (red), then stained with DAPI (blue). TLR4 is shown alone or merged with DAPI, whereas FcϵRI is shown merged with DAPI. A differential interference image is also shown. Original magnification, × 800.