Tat protein fails to stimulate TNF-α and IL-10 in macrophages from TLR4−/−MD2−/−, and CD14−/−mice. A-B) Tat induces mTNFα and mIL-10 production in wt C57Bl/6 murine macrophages. Peritoneal macrophages (5.105/well) from wild type C57BL/6 mice were stimulated for 24 h with increasing concentration of GST-Tat 1–101, GST-Tat 1–45, GST-Tat 30–72 or GST as control. LPS was used as a positive control. C-F) TLR4, CD14 and MD2 cell surface membrane proteins are essential for TNF-α and IL-10 production by Tat. Macrophages were isolated from wt mice or mice KO for TLR4, TLR2 (C-D) or CD14 or MD2 (E-F). The cells were stimulated with increasing concentrations of GST-Tat 1–101, GST-Tat 1–45 or GST as control. Positive control experiments were performed by using the following TLR ligands: LPS (TLR4-CD14-MD2) and Pam3CsK4 (TLR2-CD14). G-H) Human monocytes were pretreated with blocking antibodies, anti-CD14 or anti-MD2 (0.1 - 1 μg/ml) for 60 min. Cells were then stimulated with GST or GST-Tat 1–101 (100 nM). I-J) TNF-α and IL-10 production in peritoneal washes from wt (I) or TLR4 KO mice (J) injected with Tat protein. The data represent means +/− SD of three independent experiments.