June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Steroid refractory Th17 cells have unperturbed glucocorticoid receptor expression and trafficking
Author Affiliations & Notes
  • Philippa Lait
    Inflammation and Immunotherapy Theme, National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, University Hospitals Bristol NHS Foundation Trust, London, United Kingdom
    Academic Unit of Ophthal, University of Bristol, Bristol, United Kingdom
  • Lauren Schewitz-Bowers
    Inflammation and Immunotherapy Theme, National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, University Hospitals Bristol NHS Foundation Trust, London, United Kingdom
    Academic Unit of Ophthal, University of Bristol, Bristol, United Kingdom
  • Ashwin Dhanda
    Academic Unit of Ophthal, University of Bristol, Bristol, United Kingdom
  • Becky Conway-Campbell
    Academic Unit of Ophthal, University of Bristol, Bristol, United Kingdom
  • Andrew Dick
    Inflammation and Immunotherapy Theme, National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, University Hospitals Bristol NHS Foundation Trust, London, United Kingdom
    Academic Unit of Ophthal, University of Bristol, Bristol, United Kingdom
  • Richard Lee
    Inflammation and Immunotherapy Theme, National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, University Hospitals Bristol NHS Foundation Trust, London, United Kingdom
    Academic Unit of Ophthal, University of Bristol, Bristol, United Kingdom
  • Footnotes
    Commercial Relationships Philippa Lait, None; Lauren Schewitz-Bowers, None; Ashwin Dhanda, None; Becky Conway-Campbell, None; Andrew Dick, Novartis (C), Novartis (F), GSK (F), Abbott (F); Richard Lee, Genentech (C)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 2520. doi:
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      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.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose
 

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.

 
Methods
 

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).

 
Results
 

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

 
Conclusions
 

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.

     
Keywords: 432 autoimmune disease • 487 corticosteroids • 674 receptors  
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