Histone H4 interacts with TLR4/MD-2 on monocytes.

(A) Human blood was incubated with histone H4 (red curves) or PBS (blue curves) for 30 min at 37°C. Cells were fixed and stained with a primary antibody against histone H4 followed by a FITC-conjugated secondary antibody. Cells were subjected to flow cytometry analysis and gated for platelets (upper left), monocytes (upper middle), lymphocytes (upper right) and neutrophils (lower left). Histone stimulated unstained cells are shown in black curves. (B) THP-1 cells were incubated with histone H4 for 30 min at 37°C. Cells were then fixed and prepared for transmission electron microscopy. Samples were first labeled with rabbit anti-histone H4 and rat anti-TLR4 followed by gold-conjugated goat anti-rabbit (5 nm) and goat anti-rat (15 nm). Arrows indicate co-localization of histone H4 (5 nm gold particles) and TLR4 (15 nm gold particles). Scale bar: 500 nm. (C) Histone H4 was immobilized on a CM5 sensor chip and TLR4/MD-2 complex was applied in a flow. (D) The TLR4/MD-2 complex was injected and after a 580 s dissociation time, an antibody against TLR4 (black) or an isotype control antibody (grey) was applied.