mTLR4–MD-2 specificity of 1A6 and effective TLR4 and TLR2 blockade on murine macrophages infected with Gram-negative bacteria. (A) Macrophages incubated with anti-TLR4 mAb (1A6) shown by Nomarski differential interference contrast microscopy (DIC), fluorescence recording (middle), and superimposition of both recordings (right). Bar, 10 μm. (B) Lysates of HEK293 cells that overexpressed murine TLR4 and MD-2 were subjected to immunoprecipitation (IP; α, anti) and analyzed using flag-specific antiserum (lys., total lysates). (C and D) HEK293 cells overexpressing mTLR4–MD-2 transiently or RAW264.7 macrophages, respectively, were challenged as indicated upon preincubation with anti-TLR2 mAb (T2.5), 1A6, or isotype control (i.c.) for 30 min. HEK293 cells were lysed 16 h after challenge and assayed for NF-κB–driven relative (Rel.) luciferase activity (C), whereas RAW264.7 macrophage supernatants were sampled 16 h after challenge (Pam₃CSK₄, tripalmitoylated hexapeptide) and analyzed by ELISA (D). *, P < 0.003. (E) RAW264.7 macrophages were infected 30 min after incubation with 1A6 (•), T2.5 (□), 1A6 and T2.5 (▴), or isotype control (⋄) mAb. Antibiotic therapy started 1 h after infection, and supernatants were analyzed by ELISA 6 h after infection. Results illustrated represent similar results (A, B, and E) or summarize the results (C, D) of at least three independent experiments.