FIG 2.

Tat activates the TLR4 pathway with rapid kinetics. Human monocytes were incubated with 10 nM full-length HIV-1 Tat protein (Tat 1-86) in the presence or absence of various concentrations of anti-TLR4 antibodies as described below. After 24 h of treatment, levels of TNF-α (gray bars) and IL-10 (black bars) in the supernatants of the cells were quantified by ELISA. (A) (Dose-response) Cells were first preincubated with escalating amounts of anti-TLR4 (0.01 to 1 μg/ml) for 60 min before treatment with Tat. (Competition) Anti-TLR4 antibodies (0.01 to 1 μg/ml) and Tat protein (10 nM) were added to the cells at the same time. (Kinetic) Cells were first stimulated with Tat (10 nM) for 5 to 60 min before anti-TLR4 MAbs were added at 0.1 μg/ml or 1 μg/ml. (B) Cells (10⁶ human monocytes) were first preincubated with 0.1 or 1 μg/ml of anti-TLR4 blocking antibodies for 60 min, washed twice or left unwashed, and then stimulated with Tat (10 nM). Cytokine production levels in cell supernatants were quantified after 24 h of treatment. The data represent means and SD for three independent experiments.