TLR4-mediated macrophage release of IL-10 and RANTES (but not TNFα) and IRF3 phosphorylation (but not ERK phosphorylation) depend on TLR4 endocytosis. MyD88-independent TLR4-mediated IL-10 and RANTES release requires endocytosis. (A-C) U937 and U1 macrophages were pretreated with a highly specific inhibitor of the endocytosis regulator dynamin GTPase (dynasore, 50μM) for 1 hour and then incubated in the presence or absence of lipid A (10 μg/mL) for 24 hours, and cell-free supernatants were analyzed for IL-10 (A), RANTES (B), or TNFα (C) by ELISA. Data reflect representative experiments (performed in triplicate) of 3 independent experiments with similar results. *P < .01 compared with lipid A alone. (A-C) Error bars indicate SEM. (D) MyD88-independent TLR4-mediated IRF3 phosphorylation requires endocytosis. U937 and U1 macrophages were pretreated with a highly specific dynamin GTPase inhibitor (dynasore, 50μM) for 1 hour and then incubated in the presence or absence of lipid A (10 μg/mL) for 15 minutes, and cell lysates were probed with specific antibodies to phosphorylated ERK1/2 and phosphorylated IRF3 antibody. Protein loading controls used antibody to total ERK1/2 and total IRF3 protein. Representative Western blot from 1 of 3 independent experiments are shown with similar results.