Effect of LPS prestimulation on cytokine production after a second challenge with LPS, antigen, or a combination of both. Reduction of TNFα (A) and IL-6 (B) production after stimulation with 1 μg/ml LPS in BMMC prestimulated with 10 ng/ml LPS (p = 0.18) and 100 ng/ml LPS (p = 0.01). No significant reduction of TNFα (C) or IL-6 (D) production was observed after stimulation with 20 ng/ml Ag in cells prestimulated with 10 ng/ml LPS (p = 0.057) and 100 ng/ml LPS (p = 0.17). Attenuation of potentiation of TNFα (E) and IL-6 (F) production by simultaneous stimulation with 20 ng/ml Ag and 1 μg/ml LPS when BMMC were prestimulated with 10 ng/ml LPS (p = 0.16) and 100 ng/ml LPS (p = 0.01). The TLR4 KO BMMC graphs (A-F) demonstrate that potentiation and attenuation with the FCεR1 agonist and LPS is mediated by TLR4 because there is no significant difference between the non-prestimulated and prestimulated groups. Of note, the synergy with the combination of LPS+Ag for TNFα (E) and for IL-6 (F) is no longer observed in TLR4 KO BMMC. Values are mean and SEM from 3 independent experiments.