Dual TLR blockade inhibits TNF-α release, whereas TLR4-specific blockade impedes IFN-γ release from hPBMCs upon Gram-negative bacterial infection and IFN-γ enhances TLR2-specific, but not TLR4-specific responsiveness of hPBMCs. (A) hPBMCs were preincubated with mAbs for 30 min (•, 15C1; □, T2.5; ▴, 15C1 and T2.5; ⋄, isotype control), infected with the indicated doses of S. enterica or E. coli, and treated by application of antibiotics after 1 h. Supernatant was analyzed 6 h after infection by ELISA. Illustration represents one out of three equivalent results of three independent experiments. (B) 30 min before infection with 10⁵ CFU/ml or 10⁶ CFU/ml E. coli and subsequent antibiotic therapy, hPBMCs were pretreated with mAbs (i.c., isotype control). Nonpretreated cells were challenged with 100 ng/ml or 1 μg/ml LPS or tripalmitoylated hexapeptide (Pam₃CSK₄). Triangles indicate smaller and larger doses. IFN-γ in the supernatants was analyzed 16 h after challenge by ELISA (ND, not detected; illustration represents summarized results of three independent experiments; *, P = 0.027). (C) hPBMCs were primed with IFN-γ for 3 h (shaded bars) or left untreated (open bars). Subsequently, cells were washed twice by centrifugation and challenged with TLR agonists (Pam₃CSK₄, lipopeptide) or infected with 10⁶ CFU/ml E. coli and subjected to antibiotic therapy after 1 h. Supernatants sampled 5 h after challenge were analyzed by ELISA (summarized result of five independent experiments; *, P < 0.02; **, P = 0.26).