Figure 1
Figure 1. Specific effect of IL-36 in inducing IL-6 production in BMDCs. (A) Determination of IL-36 mRNA levels in different cell types. Total mRNA was isolated from primary keratinocytes, BMDCs, bone marrow–derived macrophages, bone marrow–derived neutrophils, CD4+ T cells, CD8+ T cells, and B cells for analyses by quantitative RT-PCR. Results represent IL-36R mRNA expression levels relative to GAPDH. Data are shown from one of 3 independent experiments with similar results. Error bars represent the SD of the mean of triplicates in the same experiment. (B) Dose-dependent effect of IL-36 in BMDCs from WT C57BL/6 mice. BMDCs (2.5 × 105 cells/well) were plated in 48-well plates and cultured in the absence (Unstimulated) or the presence of the indicated concentrations of IL-36Ra, IL-36α, IL-36β, IL-36γ, IL-1β, or LPS for 72 hours. IL-6 levels were measured in culture supernatants by ELISA. Data are shown from one of 3 independent experiments with similar results. Error bars represent the SD of the means of triplicates in the same experiment. *P < .05 (Student t test), IL-36, IL-1β, and LPS significantly differ from unstimulated cells. (C) Stimulatory effects of IL-36α, IL-36β, and IL-36γ are dependent on IL-36R. BMDCs from WT (black bars) or IL-36R−/− (gray bars) C57BL/6 mice were either left unstimulated or stimulated with 1 μg/mL of IL-36α, IL-36β, IL-36γ, or 100 ng/mL of IL-1β or LPS for 72 hours. IL-6 levels were measured in culture supernatants by ELISA. Data are shown from one of 3 independent experiments with similar results. Error bars represent the SD of the means of triplicates in the same experiment. IL-36 and LPS stimulation significantly differs from unstimulated cells (Student t test, P < .05). (D) IL-36β specifically activates the pathway leading to p38 MAPK phosphorylation in BMDCs. Histograms show overlays of phospho-p38 (P-p38) staining in the WT (left panels) or IL-36R−/− BMDCs (right panels) unstimulated (thin gray line) and stimulated (thick black line) with 100 ng/mL of IL-36β or LPS for 15 minutes. Data are shown from 1 of 3 independent experiments with similar results.

Specific effect of IL-36 in inducing IL-6 production in BMDCs. (A) Determination of IL-36 mRNA levels in different cell types. Total mRNA was isolated from primary keratinocytes, BMDCs, bone marrow–derived macrophages, bone marrow–derived neutrophils, CD4+ T cells, CD8+ T cells, and B cells for analyses by quantitative RT-PCR. Results represent IL-36R mRNA expression levels relative to GAPDH. Data are shown from one of 3 independent experiments with similar results. Error bars represent the SD of the mean of triplicates in the same experiment. (B) Dose-dependent effect of IL-36 in BMDCs from WT C57BL/6 mice. BMDCs (2.5 × 105 cells/well) were plated in 48-well plates and cultured in the absence (Unstimulated) or the presence of the indicated concentrations of IL-36Ra, IL-36α, IL-36β, IL-36γ, IL-1β, or LPS for 72 hours. IL-6 levels were measured in culture supernatants by ELISA. Data are shown from one of 3 independent experiments with similar results. Error bars represent the SD of the means of triplicates in the same experiment. *P < .05 (Student t test), IL-36, IL-1β, and LPS significantly differ from unstimulated cells. (C) Stimulatory effects of IL-36α, IL-36β, and IL-36γ are dependent on IL-36R. BMDCs from WT (black bars) or IL-36R−/− (gray bars) C57BL/6 mice were either left unstimulated or stimulated with 1 μg/mL of IL-36α, IL-36β, IL-36γ, or 100 ng/mL of IL-1β or LPS for 72 hours. IL-6 levels were measured in culture supernatants by ELISA. Data are shown from one of 3 independent experiments with similar results. Error bars represent the SD of the means of triplicates in the same experiment. IL-36 and LPS stimulation significantly differs from unstimulated cells (Student t test, P < .05). (D) IL-36β specifically activates the pathway leading to p38 MAPK phosphorylation in BMDCs. Histograms show overlays of phospho-p38 (P-p38) staining in the WT (left panels) or IL-36R−/− BMDCs (right panels) unstimulated (thin gray line) and stimulated (thick black line) with 100 ng/mL of IL-36β or LPS for 15 minutes. Data are shown from 1 of 3 independent experiments with similar results.

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