Role of Mitogen-Activated Protein Kinases in CpG DNA-Mediated IL-10 and IL-12 Production: Central Role of Extracellular Signal-Regulated Kinase in the Negative Feedback Loop of the CpG DNA-Mediated Th1 Response

Ae-Kyung Yi, Jae-Geun Yoon, Seon-Ju Yeo, Soon-Cheol Hong, B. Keith English and Arthur M. Krieg
J Immunol May 1, 2002, 168 (9) 4711-4720; DOI: https://doi.org/10.4049/jimmunol.168.9.4711

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    Activation of MAPK pathways by CpG DNA in RAW264.7 cells. RAW264.7 cells (2 × 106 cells/ml) were stimulated with medium, CpG DNA (3 μg/ml), LPS (50 ng/ml), or PMA (50 ng/ml) for 30 min. Equal amounts of whole-cell lysates (15 mg/lane) were subjected to electrophoresis on a 10% polyacrylamide gel containing 0.1% SDS (SDS-PAGE), and then Western blots were performed using a specific Ab against the phosphorylated form of JNK (pJNKs), MKK4 (pMKK4), ERK (pERK1 and pERK2), MEK1/2 (pMEK1/2), Raf (pRaf), p38 (pp38), or MKK3/6 (pMKK3/6). Total p38 or a nonspecific protein (NS) in each sample was used as the equal loading control. The same blot was used for each different Ab after stripping of the previous Ab. The experiment was done three times with similar results.

  • FIGURE 2.
    FIGURE 2.

    Specific inhibitory effects of U0126 and SB202190. A and B, RAW264.7 cells (2 × 106 cells/ml) were pretreated with DMSO, U0126 (2.5 μM), or SB202190 (2.5 μM) for 15 min. Cells were then stimulated with medium, CpG DNA (3 μg/ml), or LPS (50 ng/ml) for 30 min. Equal amounts of whole-cell lysates (15 μg/lane) were subjected to electrophoresis on a 10% polyacrylamide gel containing 0.1% SDS (SDS-PAGE), and then Western blots were performed using a specific Ab against the phosphorylated form of JNK (pJNKp52), ERK (pERK1 and pERK2), p38 (pp38), ATF2 (pATF2), or MAPKAPK-2 (pMAPKAPK2). Total p38 in each sample was used as the equal loading control. The experiment was done three times with similar results. C, RAW264.7 cells (107 cells) were pretreated with DMSO or SB202190 (2.5 μM) for 15 min. Cells were then stimulated with medium or CpG DNA (3 μg/ml) for 30 min. Whole-cell lysates (600 μg/lane) were immunoprecipitated with agarose bead-bound anti-p38 Abs. In vitro kinase assays were done at 30°C for 15 min using polyhistidine-tagged ATF2 (rATF2) as a substrate. The experiment was done twice with similar results.

  • FIGURE 3.
    FIGURE 3.

    Contribution of MEK/ERK and p38 to CpG DNA-mediated cytokine production. RAW264.7 cells (A, 5 × 105 cells/ml; B–D, 2 × 106 cells/ml) were stimulated with medium, CpG DNA (3 μg/ml), LPS (50 ng/ml), or PMA (50 ng/ml) plus ionomycin (1 μM) for 6 (A) or 24 h (B–D) in the presence or absence of DMSO (▦), U0126 (2.5 μM; ▪), or SB202190 (2.5 μM; □). The levels of TNF-α, IL-10, IL-12p40, and IL-12p70 in culture supernatants were determined by ELISA. Data represent the mean ± SD of triplicates. The experiment was done five times with similar results.

  • FIGURE 4.
    FIGURE 4.

    Effects of MEK1/2 inhibitor and p38 inhibitor on the CpG DNA-induced cytokine mRNA expression. RAW264.7 cells (2 × 106 cells/ml) were pretreated with DMSO (open bar), U0126 (2.5 μM; filled bar), or SB202190 (2.5 μM, grey bar) for 15 min. Cells were then stimulated with medium, CpG DNA (6 μg/ml), or LPS (50 ng/ml) for 1 (A and C), 2 (F), or 4 h (B, D, and E). Total RNA was isolated and the presence of mRNA for IL-10, IL-12p40, IL-12p35, TNF-α, and GAPDH in each sample was detected by real-time PCR using SYBR green. GAPDH was used for endogenous control. Data represent the mean (fold induction from unstimulated control) ± SD of triplicates. The experiment was done two to four times with similar results.

  • FIGURE 5.
    FIGURE 5.

    Inhibition of MEK1/2 or p38 lead to the suppression of CpG DNA-induced DNA binding activities of AP-1 but not NF-κB. RAW264.7 cells (2 × 106 cells/ml) were pretreated with DMSO, U0126 (2.5 μM), or SB202190 (2.5 μM) for 15 min. Cells were then stimulated with medium, CpG DNA (3 μg/ml), or LPS (50 ng/ml) for 2 (A) or 19 h (B). DNA-binding activities of transcription factor NF-κB, AP-1, NFAT, or NF-IL-6 in equal amounts of nuclear extracts (3 μg/lane) were analyzed by EMSA using 32P-labeled double-stranded oligodeoxynucleotides containing the AP-1, NFAT, NF-IL-6, or NF-κB consensus DNA-binding sequence as a probe. Specificity of bands was determined by cold competition and supershift EMSA. The experiment was done three times with similar results.

  • FIGURE 6.
    FIGURE 6.

    Effects of MEK inhibitor or p38 inhibitor on the CpG DNA-mediated activation of transcription factors. RAW264.7 cells were transiently transfected with NF-κB-luciferase + pRL-TK-luciferase (A), or AP1-β-galactosidase (B) constructs using LipofectAMINE PLUS. Transfected cells were pooled and washed three times with culture media. Cells (105 cells/200 μl/well) were stimulated with medium, CpG DNA (3 μg/ml), LPS (50 ng/ml), or PMA (50 ng/ml) for 12 h in the presence or absence of DMSO (▦), U0126 (2.5 μM; ▪), or SB202190 (2.5 μM; □). NF-κB-luciferase activities in cell extracts were analyzed by Dual-Luciferase Reporter Assay System and normalized using pRL-TK-luciferase activity in each sample. AP1-β-galactosidase activities in cell extracts were analyzed using Galacto-Light Plus Reporter gene assay for β-galactosidase and normalized by equal concentrations of cell lysates used in each sample. Data represent the mean ± SD of triplicates. The experiment was done more than four times with similar results.

  • FIGURE 7.
    FIGURE 7.

    CpG DNA induces phosphorylation of CREB via a p38-dependent pathway. RAW264.7 cells (2 × 106 cells/ml) were pretreated with DMSO, U0126 (2.5 μM), or SB202190 (2.5 μM) for 15 min. Cells were then stimulated with medium, CpG DNA (3 μg/ml), or LPS (50 ng/ml) for 2 h. Equal amounts of whole-cell lysates (15 μg/lane) were subjected to SDS-PAGE, and then Western blots were performed using a specific Ab against the phosphorylated form of CREB (pCREB). The experiment was done three times with similar results.

  • FIGURE 8.
    FIGURE 8.

    Addition of exogenous rIL-10 suppresses IL-12 production enhanced by MEK/ERK inhibition. RAW264.7 cells (1 × 106 cells/ml) were treated with BSA or rmIL-10 (1 or 10 ng/ml) for 5 min, and then stimulated with medium or CpG DNA (3 μg/ml) in the presence or absence of DMSO or U0126 (2.5 μM) for 24 h. The levels of IL-12p40 and IL-12p70 in culture supernatants were determined by ELISA. Data represent the mean ± SD of triplicates. The experiment was done five times with similar results.

  • FIGURE 9.
    FIGURE 9.

    Effects of U0126 and SB202190 on the CpG DNA-induced expression of SOCS1 and SOCS3. RAW264.7 cells (2 × 106 cells/ml) were pretreated with DMSO (open bar), U0126 (2.5 μM; filled bar), or SB202190 (2.5 μM, grey bar) for 15 min. Cells were then stimulated with medium, CpG DNA (6 μg/ml), or LPS (50 ng/ml) for 2 h. Total RNA was isolated and the presence of mRNA for SOCS1, SOCS3, and GAPDH in each sample was detected by real-time PCR using SYBR green. GAPDH was used for endogenous control. Data represent the mean (fold induction from unstimulated control) ± SD of triplicates. The experiment was done three times with similar results.

  • FIGURE 10.
    FIGURE 10.

    CpG DNA induces phosphorylation of STAT1 which is potentiated by inhibition of ERK activation. A, RAW264.7 cells (2 × 106 cells/ml) were stimulated with medium, CpG DNA (3 μg/ml), or LPS (50 ng/ml) for 1, 3, or 24 h. B and C, RAW264.7 cells (2 × 106 cells/ml) were pretreated with DMSO, U0126 (2.5 μM), or SB202190 (2.5 μM) for 15 min. Cells were then stimulated with medium, CpG DNA (3 μg/ml), or LPS (50 ng/ml) for 2 h (B) or for 8 h (C). D, RAW264.7 cells (2 × 106 cells/ml) were pretreated with DMSO or U0126 (2.5 μM) for 15 min. Cells were then stimulated with medium or CpG DNA (3 μg/ml) for 2 h in the presence or absence of BSA or rmIL-10 (10 ng/ml). Equal amounts of whole-cell lysates (15 μg/lane) were subjected to electrophoresis on a SDS-PAGE, and then Western blots were performed using a specific Ab against the phosphorylated forms of STAT1. The same blot was used for each different Ab after stripping of the previous Ab. The experiment was done three times with similar results.

Tables

  • Table I.

    MEK1/2 inhibitor failed to enhance CpG DNA-mediated IL-12 production in IL-10 gene deficient spleen cellsa

    TreatmentIL-10 (pg/ml)IL-12p40 (pg/ml)IL-12p70 (pg/ml)IL-6 (pg/ml)
    C57BL/6IL-10 KOC57BL/6IL-10 KOC57BL/6IL-10 KOC57BL/6IL-10 KO
    DMSO
    Medium180 ± 49162 ± 63UDUDUD235 ± 98219 ± 65184 ± 85
    CpG DNA913 ± 28201 ± 156,498 ± 17138,205 ± 714546 ± 718,651 ± 1,72211,179 ± 2,05715,824 ± 781
    nCpG DNA170 ± 12286 ± 124834 ± 892,498 ± 148UDUD439 ± 97446 ± 22
    U0126
    Medium155 ± 31185 ± 8UDUDUDUD278 ± 138304 ± 16
    CpG DNA218 ± 51169 ± 2412,814 ± 64929,796 ± 1,1521,166 ± 242865 ± 4561,427 ± 1123,676 ± 234
    nCpG DNA204 ± 35211 ± 2446 ± 42561 ± 42UD892 ± 84162 ± 32223 ± 44

    • a Spleen cells (1.5 × 106 cells/200 μl/well) from C57BL/6 or IL-10 KO mice were stimulated with medium, CpG DNA (6 μg/ml), or control non-CpG DNA (nCpG DNA, 6 μg/ml) for 24 h in the presence or absence of U0126 (2.5 μM). The levels of IL-6, IL-10, IL-12p40, and IL-12p70 in culture supernatants were determined by ELISA. Data present the mean (picograms per milliliter) ± SD of triplicates. Sensitive range of cytokine ELISA was ≤10 pg/ml. UD, undetected. The experiment was done twice with similar results.

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