Figure 1

HMGB1 in complex with LPS or Pam₃CSK₄ induces cytokines via TLR4 and TLR2, respectively, but not via RAGE signaling. Peritoneal macrophages from wild-type, RAGE^−/−, TLR2^−/− or TLR4^−/− mice were stimulated with preformed HMGB1-LPS or HMGB1-Pam₃CSK₄ complexes, or with individual components separately. Stimulations with 10 µg/mL LPS or Pam₃CSK₄ were used as positive controls. HMGB1-LPS (A) and HMGB1-Pam₃CSK₄ (B) complexes both gave enhanced IL-6 production compared with stimulation with the same dose of LPS or Pam₃CSK₄ alone. (A, B): Results of one representative experiment. Four to five individual experiments were performed with each stimulation. The stimulatory capacity of the complexes (fold change) were calculated by normalizing the raw data in each experiment against 10 ng/mL LPS (n = 5) (C) or Pam₃CSK₄ (n = 4) (D). All values are expressed as mean ± SD. *P < 0.05, **P < 0.01. ND, not detected; ns, not significant; (A, C) , unstimulated; □, LPS (10 ng/mL); , HMGB1 (1 µg/mL); ■, HMGB1 (1 µg/mL) + LPS (10 ng/mL); , LPS (1 µg/mL); (B, D) , unstimulated; □, Pam₃CSK₄ (10 ng/mL); , HMGB1 (20 µg/mL); ■, HMGB1 (20 µg/mL) + Pam₃CSK₄ (10 ng/mL); , Pam₃CSK₄(10 µg/mL).