Sustained Interleukin-1β Exposure Modulates Multiple Steps in Glucocorticoid Receptor Signaling, Promoting Split-Resistance to the Transactivation of Prominent Anti-Inflammatory Genes by Glucocorticoids

Figure 1

Sustained IL-1β conditions reduce DEX-driven nuclear GR translocation in a dose-dependent manner, mediated by IL-1R1. Cells were pretreated with either vehicle (veh) or 5 ng/mL IL-1β for 16 h and then treated for 1 h with either veh, 10⁻⁸ M DEX or 5 ng/mL IL-1β + 10⁻⁸ M DEX, in the absence or presence of 1 μg/mL IL-1ra, as detailed in the table below the histogram. After culture, the cells were fixed, permeabilized, and stained with β-actin- and GR-specific antibodies. They were then labeled with secondary fluorescent antibodies and DAPI, mounted, and subjected to HCA three-color fluorescence microscopy. β-actin and DAPI staining were used to demarcate the cytoplasmic and nuclear regions of interest (ROI), respectively. (a) Histogram bars represent the HCA results for each culture condition, numbered 1–6. Changes in the levels of nuclear (gray column) and cytoplasmic (white column) GR are expressed as % mean fluorescence intensity (MFI)/nuclear or cytoplasmic ROI ± SD (), relative to GR content in vehicle-treated cells, which are set as 100%. ^##, differences in either nuclear or cytoplasmic GR between veh/DEX-treated (a2) and veh/veh-treated cells (a1). , difference in nuclear GR between IL-1β/IL-1β + DEX-treated (a5) and DEX-treated cells (a2). and , differences between IL-1β + IL-1ra/IL-1β + IL-1ra + DEX-treated (a6) and IL-1β/IL-1β + DEX-treated cells (a5) in their nuclear or cytoplasmic GR level, respectively. (b) Dose-dependent inhibition of nuclear GR translocation was assayed in cells pretreated with titrated concentrations of IL-1β (50 pg/mL to 25 ng/mL) for 16 h and then treated for 1 h with the corresponding IL-1β concentration +10⁻⁸ M DEX. The results show the mean percentage of inhibition of GR nuclear translocation observed under sustained IL-1β conditions, compared to veh/10⁻⁸ M DEX-alone conditions and their 95% confidence intervals.

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