The inhibitory effect of IL-25 on CD14⁺ cell cytokine response requires Socs-3. (A) Socs-3 RNA levels in response to IL-25 stimulation in blood CD14⁺ cells induced by LPS and PGN. CD14⁺ cells were preincubated with or without IL-25 (50 ng/mL) for 30 minutes then stimulated with LPS or PGN for the indicated time points. Data indicate the mean ± SD of 4 separate experiments. Untreated versus LPS-treated cells, §P = .04; untreated versus PGN-treated cells, §P = .03, §§P = .02; LPS-treated versus LPS + IL-25–treated cells, *P = .001; PGN-treated versus PGN + IL-25–treated cells, **P = .03. (B) Representative dot plots showing the percentages of PI-positive and fluorescent (FITC)–labeled siRNA-transfected CD14⁺ cells. Blood CD14⁺ cells were transfected with a fluorescent siRNA as indicated in “Methods,” and the percentages of PI-positive and FITC-labeled cells were then evaluated at the indicated time points by flow cytometry. One of 3 representative experiments is shown. (C,D) Silencing of Socs-3 expression in human blood CD14⁺ cells. Cells were cultured with control or Socs-3 siRNA. After 2 days, cells were washed and cultured with IL-25 for 30 minutes followed by stimulation with LPS for 2 hours. Socs-3 RNA expression was evaluated by real-time PCR (C). Levels are normalized to β-actin and indicate the mean (± SD) of all experiments. *P = .001. (D) Representative Western blots showing Socs-3 and β-actin protein in extracts of cells cultured as indicated in panel C. One of 3 experiments in which similar results were obtained is shown. (E) IL-25 fails to inhibit the cytokine expression induced by LPS and PGN in Socs-3–deficient cells. Cells were transfected with control or Socs-3 siRNA. After 2 days, cells were washed and then stimulated with IL-25 for 30 minutes followed by LPS/PGN for a further 6 hours. Data indicate mean (± SD) of 5 separate experiments in which cells purified from 5 healthy donors were used. LPS/PGN-treated versus LPS/PGN + IL-25–treated cells, *P < .04.