Fig. 5.

CD14-independent, MD-2 dependent activation of HEK/TLR4 by LOS·albumin. HEK293 cells stably expressing TLR4 (HEK TLR4) or the parental HEK293 cell line (HEK) were incubated with LOS·albumin (2 nM) and increasing amounts of either sMD-2-containing insect cell conditioned medium (sMD-2 CM) or insect cell conditioned medium lacking sMD-2 (Ctrl CM) (A) for 18 h at 37°C. Alternatively, HEK TLR4 cells were transiently transfected with either an expression vector encoding human MD-2 (HEK TLR4 + pMD-2) or an empty control vector (HEK TLR4 + Empty Vector). Following transfection, cells were incubated with increasing amounts of LOS·albumin for 18 h at 37°C (B). Accumulation of IL-8 in culture supernatants was quantified by ELISA as a marker of activation. *** = p < 0.0001, ** = p < 0.005, as calculated by t-test. Data shown are representative of > 3 independent experiments.