Figure 3.

TLR2 mediates endothelial cell permeability to HMGB1. HDMVECs were transfected with siRNA to a nonspecific target, TLR2 or TLR4. Twenty-four hours after transfection, cells were seeded into trans-wells at 10⁵ cells per well to form a monolayer. Cells were stimulated with LPS, HMGB1 or Pam3CSK4 (1 μg/mL final concentration). Six hours after stimulation, FITC dextran was added to the trans-well to assess its transport through the cell monolayer. Medium was collected from the bottom well, and the level of fluorescence was measured. (A) LPS, Pam3CSK4 and HMGB1 all resulted in increased permeability through the transwells (*p < 0.003; **p < 0.03). Increased permeability to LPS (B) (*p < 0.005 compared with control) and Pam3CSK4 (C) (*p < 0.001 compared with control) was attenuated with treatment of cells with siRNA to TLR4 (B) and TLR2 (C), respectively. Increased permeability to HMGB1 was attenuated by treatment of cells with siRNA to TLR2 (D), but not with siRNA to TLR4 (**p < 0.05 HMGB1 versus control buffer) or nonspecific target (*p < 0.01 HMGB1 versus control buffer). N = 3 for each condition.