TLR4-blocking antibodies inhibit macrophage production of TNF-α in response to A2t and LPS, but not Pam₃CSK₄, a TLR2 ligand. Intracellular TNF-α staining of 7-day-old MDM after treatment with indicated stimuli for 6 hours in the presence of brefeldin A (A), or cytokine levels measured by luminex from 24 hours of culture with the same treatments in the absence of brefeldin A (B). Where indicated, cells were pretreated (15 minutes) with 10 μg/mL designated blocking antibody before stimulus addition. (C) Intracellular TNF-α staining of naive (left) or differentiated (right; 7 days with 15nM PMA) THP-1 cells after 6-hour incubation with indicated stimuli. (D) Seven-day PMA-differentiated THP-1 cells were treated with the indicated stimuli and blocking antibodies and stained for intracellular TNF-α, as in panel A. (E) WT or TLR-deficient (TLR4^−/−) 7-day-old murine BMDM were treated with 5nM A2t, 10 ng/mL LPS, or 100 ng/mL Pam₃CSK₄ for 24 hours, and levels of the indicated cytokines (in pg/mL) were measured in the supernatants by luminex. Macrophages were pooled from 5 females per genotype; results are the mean ± SEM of 2 experiments performed in duplicate.