Antagonistic antibody prevents toll-like receptor 2–driven lethal shock-like syndromes
J. Clin. Invest. Guangxun Meng, et al. 113:1473 doi:10.1172/JCI20762 [Go to this article.]

Figure 3
Inhibitory effect of mAb T2.5 on cell activation in vitro. (A–D) NF-κB–dependent luciferase activities in HEK293 cells overexpressing either murine (A) or human TLR2 (B), as well as TNF-α concentrations in supernatants of RAW264.7 (C) or primary murine macrophages (D) challenged with inflammatory agonists. Rel. lucif. activity, relative luciferase activity; ND, not detectable. Cells were incubated with T2.5 or conT2 only (white bars), or additionally challenged with IL-1β (A and B, light gray bars), ultrapure LPS (C and D, medium gray bars), P₃CSK₄ (black bars), or h.i. B. subtilis (A–D, dark gray bars). (E) NF-κB/p65 nuclear translocation dependent on mAb, P₃CSK₄ challenge, or LPS challenge in human macrophages was analyzed by cytochemical staining. Unstim., unstimulated. Scale bar: 20 ∝m; magnification was equal for all recordings. (F and G) NF-κB–dependent EMSA was analyzed by application of nuclear extracts from RAW264.7 macrophages, and phosphorylation of MAPKs Erk1/2 (pErk1/2), p38 (pP38), and Akt (pAkt) was analyzed by application of total extracts from RAW264.7 macrophages. Cells were preincubated with the indicated amounts of mAb T2.5 or conT2 (∝g/ml) and challenged with P₃CSK₄ or LPS subsequently for 90 minutes (F; arrows indicate specific NF-κB–DNA complexes) or 30 minutes (G; phosphorylation-independent p38-specific immunoblot analysis as positive control). Untreated cells were analyzed as controls (Control).