Figure 1.
Time-and dose-dependent induction of interleukin-7 (IL-7) expression by IL-12p70, p402, p40, and IL-23. Mouse microglia cells were stimulated with different concentration (5 and 10 ng/ml) of IL-12p70, p402, p40, IL-23 (p23), and other stimuli under serum-free conditions. After 4 hr of stimulation, the mRNA expression of IL-7 was monitored by semi-quantitative RT-PCR (a) and quantitative real-time PCR (b). Results are means ± SD of three different experiments.*P < 0·001 versus control IL-7 mRNA; **P < 0·01 versus control; ***P < 0·01 versus control IL-7 mRNA; NS, non significant. Cells were stimulated with (10 ng/ml) of IL-12p70 under serum-free conditions. At different time-points of stimulation, total RNA was analysed for the expression of IL-7 by semi-quantitative RT-PCR (c) and quantitative real-time PCR (d). Results are means ± SD of three different experiments.*P < 0·001 versus control IL-7 mRNA. Cells were stimulated with different concentration of IL-12p70 under serum-free conditions. After 4 hr of stimulation, total RNA was analysed for the expression of IL-7 by semi-quantitative RT-PCR (e) and quantitative real-time PCR (f). *P < 0·001 versus control IL-7 mRNA; **P < 0·01 versus control; ***P < 0·01 versus control. After 18 hr of stimulation, the expression of IL-7 protein was monitored by immunofluorescence (g). Human primary microglia cells were stimulated with (10 ng/ml) of p402, IL-12p70, p40 and IL-23 under serum-free conditions. After 4 hr of stimulation, the mRNA expression of IL-7 was monitored by semi-quantitative RT-PCR (h) and quantitative real-time PCR (i). *P < 0·001 versus control IL-7 mRNA. NS, non significant. After 18 hr of stimulation, the expression of IL-7 protein was monitored by immunofluorescence (j). DAPI was used to visualize the nucleus. Cells were plated at 50–60% confluence in 12-well plates and transfected with 0·25 μg pIL-7-Luc and 25 ng pRL-TK (Renilla luciferase control) by Lipofectamine Plus (Invitrogen) as described earlier.30,33 Twenty-four hours after transfection, cells were stimulated with different concentrations of IL-12p70 (k) or different stimuli in mouse BV-2 (l) for 6 hr under serum-free condition. Firefly and Renilla luciferase activities were determined by Dual Luciferase Kit (Promega) following the manufacturer's protocol. Data are mean ± SD of three separate experiments. *P < 0·001 versus control (k); *P < 0·001 versus control and**P < 0·01 versus control (l). Concentrations of different stimuli are as follows: p402, 10 ng/ml; lipopolysaccharide (LPS), 1 μg/ml; IL-12p70, 10 ng/ml; IL-23, 10 ng/ml; tumour necrosis factor-α (TNF-α), 20 ng/ml; gp120, 200 pg/ml; IL-16, 10 ng/ml; IL-1β, 10 ng/ml; p40, 10 ng/ml.