Figure 3
Stimulation of fetal human cortical astrocytes with interleukin-1β (IL-1β) and interferon-γ (IFN-γ). Fetal cultured human cortical astrocytes stimulated with recombinant human IL-1β (10 ng ml−1) and IFN-γ (200 ng ml−1), a prototypical indoleamine 2,3-dioxygenase 1 (IDO1) activator, as positive control. Protein levels of IDO1 and tryptophan 2,3-dioxygenase-2 (TDO2) analyzed at baseline and after exposure to IL-1β or IFN-γ for 48 h. IDO and TDO immunopositive bands are normalized to β-actin. Bar graphs here represent IDO1 and TDO2 protein levels expressed as % of vehicle-treated control cultures. All cells were analyzed for kynurenic acid (KYNA) at time points 1, 3 and 24 h. Bar graphs here represent % of vehicle (t=1 h) at these time points. All data are reported as mean±s.e.m. All experiments were performed in triplicate and repeated twice. Representative western blots of IDO1 and TDO2 are shown below each bar graph (see Supplementary Figures S7–S9 for full size western blots). (a) Low levels of IDO1 protein expression were detected in unstimulated cells, but despite the marked increase of IDO1 mRNA levels following IL-1β stimulation, no changes in protein levels were observed after 48 h of IL-1β exposure (90±5.13% vs 100±7.85%, P=0.41). Stimulation with IFN-γ was associated with a marked increase in protein levels of IDO1 (1084±117% vs 100±7.85%, P=0.001). (b) Stimulation with IL-1β for 48 h increased protein levels of TDO2 compared with vehicle-treated cells (139±11.1% vs 100±2.70%, P=0.006), whereas stimulation with IFN-γ did not affect protein levels of TDO2 (90±11.2% vs 100±8.70% P=0.21). Immunofluorescent staining of IDO1 (c) protein visualized in green and TDO2 (d) protein visualized in red following 48 h of IL-1β stimulation confirmed low levels of IDO protein in most cells and scattered cells expressing high levels of TDO2. Images captured at × 40 magnification. Nuclear staining was performed using 4',6-diamidino-2-phenylindole (DAPI). (e) At 24 h, cells stimulated with IFN-γ had more than 85 times higher KYNA concentrations (10.8±0.34 nM) than vehicle-treated cells (0.14±0.04 nM, P<0.0001). (f) At 24 h, cells stimulated with IL-1β showed 4 times higher KYNA concentrations (mean±s.e.m.: 1.70±0.11 nM) than vehicle-treated cells (0.42±0.10 nM, p<0.0001). Two-sided P-values, statistical significance set to P<0.05, **P<0.01, ***P<0.001.
