Article Figures & Data
Figures
- FIGURE 1.
p43 induces IL-12 production in primary mouse macrophages. A, Primary macrophages were incubated for 48 h with various concentrations of p43 (10, 50, 100, 250, 500, and 1000 nM) or LPS. Culture supernatants were harvested, and IL-12 levels were evaluated by ELISA. The results are expressed as means ± SEM (n = 3). ∗, p < 0.01, relative to an untreated group. B, Effect of p43 on the expression of IL-12p40 and IL-12p35 genes at the mRNA level. The macrophages were incubated for 6 h with p43 (500 nM) or LPS (0.5 μg/ml), and total RNA was prepared from the cells. RT-PCR products for IL-12p40, IL-12p35, and β-actin were analyzed on 1.5% agarose gels. C, Effect of a neutralizing anti-p43 Ab on IL-12 production in p43-treated macrophages. Primary mouse macrophages were cultured with p43 (500 nM) in the presence of anti-p43 Ab. Culture supernatants were harvested 48 h later and assayed for IL-12 levels by ELISA. The results are presented as means ± SEM (n = 3). ∗∗, p < 0.01, relative to p43-treated group in the absence of anti-p43.
- FIGURE 2.
p43-induced IL-12 production is not due to endotoxin contamination. RAW264.7 cells were stimulated for 48 h with heat-treated or proteinase K-digested p43 (500 nM) or LPS (0.5 μg/ml), and IL-12 levels were determined by ELISA. The results are presented as means ± SEM (n = 3). ∗, p < 0.001, relative to the untreated group.
- FIGURE 3.
Analysis of p43-mediated transcriptional induction of IL-12 p40 promoter constructs. A, Mouse IL-12 p40 promoter constructs and a linker-scanning mutant of the NF-κB site are depicted schematically, along with the Ets and NF-κB binding sites. The nucleotide sequence numbers are represented for each of the constructs. B and C, Transient transfection of RAW264.7 cells with each of the p40 promoter constructs, followed by treatment with p43 (0.5 and 1.0 μM) or LPS (0.5 μg/ml). The results are expressed as induction over the values obtained with the unstimulated RAW264.7 cells transfected with each of the promoter constructs, which was assigned an arbitrary value of 1. The data are representative of three similar experiments.
- FIGURE 4.
p43 increase NF-κB DNA-binding activity. RAW264.7 cells were incubated for 2 h with various concentrations (50–1000 nM) of p43, or with 500 nM p43 at various time periods (5, 20, 60, 120 min), and the nuclear extracts from the treated cells were evaluated with regard to NF-κB-binding activity in EMSA using labeled oligonucleotides containing a consensus NF-κB site (A) or an IL-12p40 NF-κB site (B), as indicated. S and NS designate the presence of an unlabeled, identical oligonucleotide and nonspecific oligonucleotide, respectively. The specific NF-κB complexes are as indicated. C, In vivo NF-κB binding to the IL-12p40 promoter. RAW264.7 cells were incubated for 2 h with 500 nM p43, and cross-linked chromatin was subjected to immunoprecipitation with anti-p65, anti-p50, anti-Rel B, or without Ab (No Ab). Immunoprecipitated DNA was amplified by PCR using primers specific for IL-12p40 or α-actin promoter as a negative control. The data are representative of two independent experiments.
- FIGURE 5.
NF-κB inhibitors suppress p43-induced NF-κB-binding activity and IL-12 production in macrophages. A, RAW264.7 cells were cultured for 2 h in the absence or presence of various concentrations of CAPE (1–100 μM) or BAY11-7082 (5–30 μM), and then treated with 500 nM p43. After 1 h of culture, nuclear extracts were prepared from the RAW264.7 cells, and examined for NF-κB-binding activity in EMSAs, using a labeled oligonucleotide containing a consensus NF-κB site, as indicated. S and NS indicate the presence of an unlabeled, identical oligonucleotide and nonspecific oligonucleotide, respectively. The specific NF-κB complexes are as indicated. B, RAW264.7 cells were cultured for 2 h in the absence or presence of various concentrations of CAPE (0.01–1 μM) or BAY11-7082 (0.1–2.5 μM), and then treated with 500 nM p43. After 48 h of culture, IL-12 levels in the cell supernatants were evaluated via sandwich ELISA. The results are presented as means ± SEM (n = 3). ∗, p < 0.0005, relative to the p43-treated group in the absence of NF-κB inhibitors.
- FIGURE 6.
Effects of p38-, PKC-, PLC-, and MEK-specific inhibitors on p43-stimulated IL-12 production. A, RAW264.7 cells were cultured in the absence or presence of the p38 inhibitors (SB203580, SB202190) and their inactive analog (SB202474), the PKC inhibitor (chelerythrin), the PLC inhibitor (U73122), and the MEK inhibitor (PD98059) for 2 h, followed by treatment with 500 nM p43. After 48 h of culture, the IL-12 protein levels in the cell supernatants were determined via sandwich ELISA. The results are presented as means ± SEM (n = 3). ∗, p < 0.05, relative to a group incubated with p43 alone. B, The p38 MAPK levels were determined via Western blot analyses at 45 min after the treatment with p43. The cell lysates were then immunoprecipitated using anti-phosphorylated tyrosine mAb and blotted with anti-p38 MAPK mAb (p-p38), or directly probed with anti-p38 MAPK mAb (p38).
- FIGURE 7.
p43-mediated IFN-γ production in CD4+ T cells is mediated by IL-12 in Ag-primed lymph node cells. A, Effects of p43 on the production of IFN-γ, IL-4, and IL-12 in KLH-primed lymph node cells. The footpads of mice were injected with KLH (100 μg) in alum. Seven days later, the lymph node cells were collected and stimulated in vitro for 4 days with KLH (100 μg/ml), in the presence of p43 (500 nM). The cell culture supernatants were then harvested and assayed for IFN-γ, IL-4, and IL-12 by ELISA. The KLH-primed lymph node cells were treated for 6 h with 500 nM p43, after which RT-PCR was conducted. The results are presented as means ± SEM (n = 3). ∗, p < 0.01; ∗∗, p < 0.0001, relative to an untreated group. B, The addition of neutralizing IL-12p40 mAb reduces p43-induced IFN-γ production in KLH-primed lymph cell cultures. The KLH-primed cells were cultured with 500 nM p43 in the presence of anti-IL-12p40 (C17.8; 0.01–5 μg/ml) or isotype control mAb (JES3-19F1; 0.01–5 μg/ml). The culture supernatants were harvested 4 days later, and assayed for IFN-γ by ELISA. The results are presented as means ± SEM (n = 3). ∗, p < 0.005; ∗∗, p < 0.001, relative to the p43-treated group in the absence of anti-IL-12 mAb. C, Macrophages pretreated with p43 enhance IFN-γ production by KLH-primed CD4+ T cells. Macrophages (2 × 105 cells/well) were pretreated with 500 nM p43. After 6 h, the cells were washed and incubated for 4 days with KLH-primed CD4+ T cells (1 × 106 cells/well). The culture supernatants were harvested, and the IFN-γ levels were determined by ELISA. The results are presented as the mean ± SEM (n = 3). ∗, p < 0.005, relative to an untreated group.
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