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Blood, Vol. 113, Issue 15, 3512-3519, April 9, 2009
Inhibition of monocyte-derived inflammatory cytokines by IL-25 occurs via p38 Map kinase–dependent induction of Socs-3
Blood Caruso et al.
113: 3512
Supplemental materials for: Caruso et al
Files in this Data Supplement:
- Figure S1. LPS enhances TNF-α RNA expression in blood CD14+ cells purified by either negative or positive selection (JPG, 30.5 KB)
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CD14+ cells were purified as indicated in methods and cultured with or without (medium= M) 100 ng/ml LPS for 6 hours. RNA was analyzed by real-time PCR. Levels are normalized to β-actin and indicate the mean ± SD of all experiments. M vs LPS-stimulated cells * p=<0.001.
- Figure S2. Representative dot-plots showing TLR-2 and TLR-4 in blood CD14+ cells either left unstimulated (Uns) or stimulated with IL-25 in the presence or absence of PGN (A) or LPS (B) for 6 hours (JPG, 264 KB)
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TLR2 and TLR-4 were evaluated using an anti-human TLR4 PE or TLR2 PE antibody. Numbers in quadrants indicate the percentage of cells in the designated gates. One of four separate experiments is shown. Right insets show the mean fluorescence intensity of TLR-2 and TLR-4 respectively in cells stimulated as indicated above. (C–D) Percentages of TLR-2 (C) and TLR-4 (D) expressing CD14+ cells cultured as indicated in (A–B). Data indicate mean ± SD of all experiments.
- Figure S3. IL-25 inhibits the expression of inflammatory cytokines induced by TNF-α and IFN-γ in human blood CD14+ cells (JPG, 56 KB)
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CD14+ cells were preincubated with or without IL-25 (50 ng/ml) for 30 minutes then either left unstimulated (M= medium) or stimulated with TNF-α (10 ng/ml) (a), or IFN-γ (50 ng/ml) (b) for further 6 hours. Cytokine RNA was analyzed by real-time PCR. Levels are normalized to β-actin and indicate the mean ± SD of all experiments. IFN-γ– or TNF-α–stimulated vs. IFN-γ– or TNF-α– + IL-25–stimulated cells * p=0.003; ** p=0.04.
- Figure S4. SOCS-1 RNA levels in response to IL-25 stimulation in blood CD14+ cells activated with LPS or PGN (JPG, 43.1 KB)
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CD14+ cells were pre-incubated with or without IL-25 for 30 minutes then either left unstimulated or stimulated with LPS or PGN for the indicated time points. SOCS-1 RNA was analyzed by real-time PCR. Levels are normalized to β-actin and indicate the mean ± SD of 5 experiments. Unstimulated vs. LPS or PGN-stimulated cells § p=0.03; LPS-treated vs. LPS+IL-25–treated cells * p=0.01.
- Figure S5 (JPG, 128 KB)
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(A) Induction of Socs-3 by IL-25 does not rely on JNK/ERK activity. CD14+ cells were pre-incubated with 420116 (a JNK inhibitor), PD98059 (an ERK inhibitor), or vehicle (DMSO) then treated with IL-25 and/or LPS for 30 minutes as indicated in methods. Socs-3 RNA was evaluated by real-time PCR. Levels are normalized to β-actin and indicate the mean ± SD of all experiments. (B) Representative Western blots showing both p-ERK, total ERK, p-JNK, and total JNK in total extracts of cells pre-incubation with 420116 or PD98059 and then stimulated with LPS+ IL-25. One of 3 representative experiments in which similar results were obtained is shown. (C) Pre-incubation of cells with SB202190 prevents the phosphorylation of p38, but not that of JNK and ERK1/2, in response to IL-25 and LPS stimulation. Cell culture and Western blots were performed as indicated in materials and methods. One of 4 representative experiments in which similar experiments were obtained is shown.
- Figure S6. Induction of Socs-3 by IL-25 relies on p38 MAPK (JPG, 67.7 KB)
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CD14+ cells were pre-incubated with SB202190 (a p38 Map kinase inhibitor) (A), 420116 (a JNK inhibitor) (B), PD98059 (an ERK inhibitor) (B), or vehicle (DMSO) then treated with IL-25 and/or PGN as indicated in methods. Socs-3 RNA was evaluated by real-time PCR. Levels are normalized to β-actin and indicate the mean ± SD of all experiments. *p=0.001.
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