Figure 4. IFN-β neutralization reduces STAT1 phosphorylation and induces hyporesponsiveness in P21^–/– macrophages.

(A) P21 mRNA and protein levels in LPS-rechallenged WT macrophages. (B) STAT1 phosphorylation in WT and P21^–/– macrophages. (C) Increased iNOS and CXCL11 expression in tolerized P21^–/– macrophages. (D and E) P21^–/– peritoneal macrophages were incubated with an IFN-β– or TNF-α–neutralizing antibody or an isotype control during LPS tolerization (20 hours). Cells were washed, cultured in medium (2 hours) and restimulated with LPS (4 hours). (D) Reduction in STAT1 phosphorylation and iNOS expression after antibody treatment at indicated times. (E) RT-PCR analysis of P21^–/– macrophages incubated with appropriate antibodies during LPS tolerization (20 hours) and restimulated with LPS (4 hours). (F) After LPS tolerization, P21^–/– macrophages were restimulated with LPS (4 hours) in the presence of anti–IFN-β or isotype control antibody. Immunoblot shows a reduction in STAT1 phosphorylation and iNOS expression. (G) WT macrophages were treated with IFN-β during LPS tolerization, restimulated with LPS (4 hours), and analyzed by RT-PCR. (H) P21^–/– mice were challenged with 2 LPS doses as in Figure 1. At 2 hours before LPS rechallenge, mice were treated i.p. with anti-IFNAR1 antibody or control IgG. Inhibition of IFNAR1 improved tolerance to LPS, as shown by a Kaplan-Meier survival curve (n = 9), ***P < 0.001, log‑rank (Mantel-Cox) test. For all RT-PCR analyses, results were normalized to β‑actin and show fold induction over unstimulated WT cells. Data shown as the mean ± SEM (n = 3 independent experiments), *P < 0.05, **P < 0.01, ***P < 0.001. For A and E, 1-way ANOVA. For C and G, 2-tailed Student’s t test. NS, not significant. For immunoblots, β-actin was used as a loading control. Western blots in A and B were derived from the same experiment. Representative gels of 3 experiments are shown.