The cAMP Pathway Amplifies Early MyD88-Dependent and Type I Interferon-Independent LPS-Induced Interleukin-10 Expression in Mouse Macrophages

Figure 1

The cAMP pathway synergistically stimulates early, but not late, LPS-induced IL-10 transcription. (a–d) Early synergism at the IL-10 promoter reporter and IL-10 secretion levels. RAW264.7 macrophages, transfected with a full mouse IL-10 promoter reporter construct, were incubated with LPS (10 ng/ml) and/or isoproterenol (Iso, 1 μM) for 3 h (a, b) or for up to 24 h (c, d). (b, d) The medium was collected, and the secretion of endogenous IL-10 was measured by ELISA. Data represent the (). (a, c) Luciferase reporter data expressed as the () of values normalized against Renilla luciferase activity. (a, b) and relative to resting cells. (c) relative to resting cells for all treatments except for LPS at 3 h and Iso at 24 h. (d) Values of isoproterenol treatment were indistinguishable from control values. relative to resting cells for LPS and for LPS+Iso at all time points. (c, d) for LPS+Iso relative to LPS only at 3 h and 8 h. The experiments shown in (a–d) were carried out 45, 48, 14, and 12 times, respectively, with similar results. (e) Decay rate of the reporter. RAW264.7 macrophages, transfected with a full mouse IL-10 promoter reporter construct, were incubated with LPS (10 ng/ml) and isoproterenol (Iso, 1 μM) for 3 h, the stimuli-containing medium was removed, and decay of the reporter was measured at the indicated time points along the dashed line. Luciferase reporter data expressed as the () of values normalized against Renilla luciferase activity, relative to unstimulated control cells. relative to resting cells at all time points. The experiment was carried out twice. (f) Early synergism at the IL-10 mRNA level. RAW264.7 macrophages were stimulated with LPS (10 ng/ml) and/or isoproterenol (Iso, 1 μM) or the PDE4 inhibitor rolipram (20 μM) for the indicated time. Total RNA was isolated from the cells, and IL-10 mRNA levels were assessed by real-time PCR. The intensity of IL-10 mRNA in unstimulated cells, normalized by HPRT mRNA, was set to 1. Data represent the (). relative to resting cells for Iso at 1 h only and for all other treatments at all time points. for LPS relative to LPS+Iso or LPS+rolipram only at 1-4 h. The experiment was carried out twice.

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