Figure 5.

TLR4-SNP macrophages exhibit decreased responsiveness to weak TLR4 agonists and antagonists. (A) WT and TLR4-SNP macrophages were stimulated with LPS and sMPL at the indicated concentrations and Il1b mRNA quantified by qRT-PCR. The results represent the mean ± SEM of four separate experiments. Data were analyzed by two-way ANOVA and Tukey’s post hoc test. WT vs. TLR4-SNP: 0.1 ng/ml LPS, **, P = 0.005; 1.0 ng/ml LPS, **, P = 0.0079; 10 ng/ml LPS, P = not significant (N.S.); 1.0 ng/ml sMPL, **, P = 0.0032; 10 ng/ml sMPL, ***, P < 0.0001. (B) WT and TLR4-SNP macrophages were pretreated for 20 min with Eritoran (10-fold dilutions ranging from 100 pg/ml to 1 µg/ml) and then stimulated with LPS (10 ng/ml) for 2 h. The ID₅₀ was calculated as described in Materials and methods. Results represent the mean ± SEM of four separate experiments. LogID₅₀ data were analyzed by a paired Student’s t test; Il1b: *, P = 0.013; Il12b: *, P = 0.022. (C) WT and TLR4-SNP macrophages were pretreated for 30 min with 4BB peptide (dilutions ranging from 0.8 to 26 µM in experiment 1 or from 0.08 to ∼20 µM in experiments 2 and 3) and then stimulated with LPS (10 ng/ml) for 2 h. The ID₅₀ for each genotype was calculated as described in Materials and methods. Results represent the mean ± SEM of three separate experiments. LogID₅₀ data were analyzed by a paired Student’s t test; IL1b: *, P = 0.014; Il12b: **, P = 0.008.