Purchase this article with an account.
Philippa Lait, Lauren Schewitz-Bowers, Ashwin Dhanda, Becky Conway-Campbell, Andrew Dick, Richard Lee; Steroid refractory Th17 cells have unperturbed glucocorticoid receptor expression and trafficking. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2520.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We have previously reported that both human and murine Th17 cells maintain phenotype and proliferation in the face of steroid treatment corroborated by the observations that CD4+ T cells from patients with steroid refractory (SR) uveitis are biased to express IL-17 following TCR engagement. Such IL-17 bias in the presence of steroids may be due to failure of glucocorticoid receptor (GR) nuclear translocation and over-expression of the receptor isoform GRβ. The purpose of this study was to extend our previous observations to determine whether these mechanisms confer the steroid refractory Th17 phenotype we observe in uveitis.
We first optimised the quantification of GR trafficking in T cells by testing a panel of GR antibodies and image analysis techniques in freshly isolated CD4+ cells from normal volunteers. Subsequently, CD4+ CCR6+ (Th17) and CD4+CCR6- (Th0) cells were sorted (BD Influx) from the PBMCs of normal volunteers (n=4) and cultured for 2 weeks in Th17 and Th0 (unpolarised) conditions respectively, with or without exposure to the synthetic glucocorticoid dexamethasone (Dex) or RU486 (which induces GR translocation). Intracellular IL-17 and IFN-γ expression was determined by flow cytometry (BD LSR II). Nuclear translocation of GR was quantified using laser scanning confocal microscopy of paraformaldehyde fixed cells after 30 minutes exposure to Dex (anti-GR mAb, Santa Cruz 3D5). Full depth z-stack images were acquired and fluorescence analysed using Volocity 6.2 (Perkin Elmer) within delineation via nuclear staining (DAPI) to facilitate quantification of red (GR) staining and expressed as total red divided by nucleus volume (nuclear density). Matched cell samples from the same volunteers were collected into RNAlater after Dex treatment and the relative expression of GR isoforms was quantified when normalised to GAPDH (Applied Biosystems).
Fig 1 Nuclear density of GR increased significantly in both Th0 and Th17 cells after treatment with dex or positive control RU486 (multivariate ANOVA). Fig 2 Expression of total GR and the GRβ isoform is equal in human Th0 and Th17 cells, and this is not affected by exposure to Dex
There was no significant difference in GR translocation or the expression of GR isoforms in human Th17 and Th0 cells and neither mechanism explains the glucocorticoid refractory Th17 phenotype observed clinically.
This PDF is available to Subscribers Only