Hypersensitivity of IRF-4^-/- mice to LPS in vivo.(A) IRF-4^-/- mice and their littermates were injected i.p. with LPS. (B) IRF-4^-/-, C57BL/6 (IRF-4^+/+), and IRF-4^-/- TNF-α receptor^-/- DKO mice were injected i.p. with LPS. (C) Serum from IRF-4^+/+ and IRF-4^-/- mice was collected 2 h after LPS challenge. TNF-α and IL-12 p70 levels were measured by ELISA. Each circle represents an individual mouse, and the bars represent the average of each group.

Hyperresponse of IRF-4^-/- macrophages to other TLR ligands. Peritoneal macrophages (2 × 10⁵ per well) from IRF-4^+/+ (open bars) and IRF-4^-/- (filled bars) mice were stimulated with zymosan A (1.5 × 10⁶ bioparticles per ml), bacterial peptidoglycan (PGN) (1.5 × 10⁶ bioparticles per ml), or CpG (300 nM) for 24 h. The TNF-α levels in the supernatant were determined by ELISA.

LPS tolerance can be induced in IRF-4^-/- macrophages. (A) Peritoneal macrophages were treated with LPS (1 μg/ml). The expression levels of SOCS-3, IRAK-M, and G3PDH were analyzed by Northern blotting. The relative levels of SOCS-1 mRNA were determined by real-time RT-PCR (open bars, IRF-4^+/+; gray bars, IRF-4^-/-). (B) Peritoneal macrophages were treated with LPS (100 ng/ml) or left untreated. After 24 h, cells were washed and restimulated with LPS (100 ng/ml) for an additional 24 h. N.D., not detected.

Altered LPS signaling in IRF-4^-/- macrophages. (A) Peritoneal macrophages from IRF-4^+/+ and IRF-4^-/- mice were stained with FITC-anti-TLR4/MD2 mAb (solid line) or without Ab (gray). (B) Peritoneal macrophages were stimulated with LPS (1 μg/ml) for 0–60 min, and the nuclear extract was prepared. Mobility-shift assay was performed with NF-κB and Oct-1 probes. (C) Peritoneal macrophages from IRF-4^+/+ (open bars) and IRF-4^-/- (filled bars) mice were treated with the indicated concentration of SP600125 (JNK-specific inhibitor) for 2 h and then stimulated with LPS (1 μg/ml) for an additional 24 h in the presence of the inhibitor. The relative TNF-α levels in the supernatant were expressed as the ratio to the TNF-α levels in the absence of the inhibitor. (D) Peritoneal macrophages were stimulated with LPS (1 μg/ml) for 0–60 min. Cell lysates (30 μg per lane) were separated by 12.5% SDS/PAGE and blotted. The blot was probed with anti-phospho-JNK Ab, stripped, and reprobed with anti-JNK Ab.

The levels of IRF-4 expression in macrophages affect the production of TNF-α. (A) Peritoneal macrophages were transfected with IRF-4-specific siRNA, control siRNA, or Lipofectamine 2000 only. Eight hours later, the expression of IRF-4 mRNA was determined by real-time RT-PCR. (B) Peritoneal macrophages were transfected with control siRNA (open bars) or IRF-4 specific siRNA (filled bars), cultured for 5 h, washed, and cultured in the presence of LPS for an additional 24 h. (C) Peritoneal macrophages transfected with control or IRF-4-specific siRNA were cultured with LPS (1 μg/ml) for the indicated period. The expression levels of TNF-α and IL-12 mRNA were determined by real-time RT-PCR analysis. (D) RAW264.7 cells were transfected with pcDNA3 or pcDNA3-mIRF-4. After5hof culture, cell lysate was separated by 12.5% SDS/PAGE. The blot was probed with anti-IRF-4 Ab, stripped, and reprobed with anti-Flag mAb. The same sample was separated by SDS/PAGE, blotted, and probed with anti-actin Ab. (E) RAW264.7 cells were transfected with pcDNA3 or pcDNA3-mIRF-4, cultured for 5 h, and stimulated with LPS for 8 h. (F) RAW-264.7 cells transfected with empty vector (pcDNA3) or with IRF-4 (pcDNA3-mIRF4) were stimulated with LPS. Cell lysate was separated by 12.5% SDS/PAGE. The blot was probed with anti-phospho-JNK Ab, stripped, and reprobed with anti-JNK Ab. The same blot was stripped and reprobed with anti-IRF-4 Ab.

Increased macrophages and their hyperresponse to LPS in vitro in IRF-4^-/- mice. (A)(Upper) CD11b and F4/80 expression of spleen cells is shown after gating on the Gr-1^- population. The number in the upper right corner of each profile indicates the percentage of Gr-1^-CD11b⁺F4/80⁺ cells in total spleen cells. (Lower) Peritoneal adherent macrophages were stained for CD11b and F4/80. The proportion of the cells within the box is indicated. (B) Graded numbers of peritoneal macrophages (2.5–20 × 10⁴) from IRF-4^+/+ (open bars) and IRF-4^-/- (gray bars) mice were stimulated with LPS (1 μg/ml). The production of cytokines in the supernatant was determined by ELISA. (C) Peritoneal macrophages (1 × 10⁵ per well for TNF-α and IL-6 and 2 × 10⁵ per well for IL-10) from IRF-4^+/+ (open circles) and IRF-4^-/- (filled circles) mice were stimulated with varying amounts of LPS for 24 h. (D) The expression levels of TNF-α, IL-12, and IL-6 were determined by real-time RT-PCR in IRF-4^+/+ or IRF-4^-/- peritoneal macrophages 0–4 h after stimulation with LPS (1 μg/ml).