FIGURE 8.

MDP tolerance is mediated by Nod2 and Rip2 down-regulation in vivo. Wild-type or Nod2-deficient mice were injected with 2 ml of autoclaved 4% thioglycollate intraperitoneally. Five days after thioglycollate treatment, MDP (35 mg/kg) was administered to the mice intraperitoneally for the indicated times, and then the mice were reinjected with MDP. Three hours after the second MDP injection, peritoneal fluid and macrophages were collected (n = 3 per group). A, IL-6 and TNF-α in peritoneal fluid were measured by ELISA. Mean value is indicated by a bar. B and C, cell extracts were subjected to Western blot analysis for TNF-α, Nod2, Rip2, Hsp90, and p38. RAW264.7 cells treated with IFN-γ (100 units/ml) were used as a positive control. D, model of MDP tolerance mediated by rapid Nod2 degradation. MDP stimulation causes rapid dissociation of the Nod2·Hsp90 complex (1). MDP-induced SOCS-3 binds to Nod2 (2). Nod2 undergoes polyubiquitination and proteasome-mediated degradation, resulting in reduced responsiveness to subsequent MDP. inj., injection.