Surface TLR4 expression in monocytes is specifically up-regulated by superantigens via MHC class II ligation. (A) Membrane TLR4 expression following incubation of purified monocytes with medium alone (resting monocytes; dotted line) or with different microbial products (stimulated monocytes; solid line). Left panel, superantigens; right panel, other microbial products. Representative flow cytometry histograms are shown at the superantigen concentrations found to induce maximal TLR4 up-regulation. Cells stained with isotype-matched control (ITMC) antibody are shown in shaded region. Other microbial products, in particular LTA and E coli LPS 0111:B4, did not support TLR4 up-regulation at any concentration tested and in some cases down-regulated TLR4 expression. (B) Dose-dependence of SEB (⬡) and LPS (E coli 0111:B4; ○) effects on membrane TLR4 expression relative to unstimulated cells (ΔMFI): mean ± SD of 4 separate donor monocyte preparations. ^*Denotes difference from TCM-only controls. (C) Membrane expression of TLR4 in monocytes from expanded donor pool stimulated with 1 ng/mL SEB (n = 35), SEA (n = 16), SMEZ-1 (n = 9), LPS (n = 12), and lipid A (n = 9). Additional results are shown for cells stimulated with 10 ng/mL LPS, 10 ng/mL lipid A, 1 μg/mL LTA, and 10⁵ cfu/mL HKSA (n = 9) Median and 10th, 25th, 75th, and 90th centiles are shown: concentrations shown are those used in subsequent cytokine induction experiments. (D) In contrast to WT SEA, MHC nonbinding mutant constructs fail to support TLR4 up-regulation. Mean and SD of 3 experiments. ^*Denotes significant difference from TCM-only controls. (E) Genestein (37 μM) inhibits SEB-induced TLR4 up-regulation. Mean and SD of 3 experiments. ^**Denotes significant difference from SEB-only TLR4 up-regulation.