March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
ICAM-1 Regulates Migration of Th1 and Th17 Cells across Simulated Human Retinal Endothelium
Author Affiliations & Notes
  • Arpita S. Bharadwaj
    Casey Eye Institute, Oregon Health & Science University, Portland, Oregon
  • Yuzhen Pan
    Casey Eye Institute, Oregon Health & Science University, Portland, Oregon
  • Lauren P. Schewitz-Bowers
    School of Clinical Sciences, University of Bristol, Bristol, United Kingdom
    NIHR Biomedical Research Centre for Ophthalmology, University College London Institute of Ophthalmology & Moorfields Eye Hospital, London, United Kingdom
  • Richard W. Lee
    School of Clinical Sciences, University of Bristol, Bristol, United Kingdom
    NIHR Biomedical Research Centre for Ophthalmology, University College London Institute of Ophthalmology & Moorfields Eye Hospital, London, United Kingdom
  • Lai Wei
    National Eye Institute, National Institutes of Health, Bethesda, Maryland
  • Justine R. Smith
    Casey Eye Institute, Oregon Health & Science University, Portland, Oregon
  • Footnotes
    Commercial Relationships  Arpita S. Bharadwaj, None; Yuzhen Pan, None; Lauren P. Schewitz-Bowers, None; Richard W. Lee, None; Lai Wei, None; Justine R. Smith, None
  • Footnotes
    Support  NIH/NEI R01 EY019875, Collins Medical Trust & Research to Prevent Blindness
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 4268. doi:
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      Arpita S. Bharadwaj, Yuzhen Pan, Lauren P. Schewitz-Bowers, Richard W. Lee, Lai Wei, Justine R. Smith; ICAM-1 Regulates Migration of Th1 and Th17 Cells across Simulated Human Retinal Endothelium. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4268.

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

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Abstract

Purpose: : Autoimmune posterior uveitis causes vision loss in a majority of affected individuals. Th1 or Th17 cells are initiators of the disease, and they traffic from circulation to posterior eye across the retinal endothelium. We investigated the role of IgG superfamily members, ICAM-1, VCAM-1 and ALCAM, in this process.

Methods: : Mononuclear cells were obtained from human peripheral blood by density gradient centrifugation. Th1 and Th17 cells were generated from negatively selected (RosetteSep) CD4+ T cells, following FACS sorting for CCR6- and CCR6+ cells, respectively. T cells were cultured on anti-human α-CD3 and α-CD28 antibody-coated plates in either Th1 [recombinant human (rh)IL-2, rhIL-12 and α-IL-4 antibody] or Th17 [rhIL-2, rhIL-6, rhIL-1β, rhIL-23, α-IFN-γ and α-IL-4 antibodies] polarizing conditions for 14 days. Retinal endothelial transmigration assays were performed using a transwell system. PET transwell membranes (0.3 cm2, 3 μm pores) were pre-coated with collagen I, seeded with human retinal endothelial cells, and incubated 4 days at 37oC to generate a relatively impermeable monolayer. Polarized T cells (5x105) were migrated across the simulated retinal endothelium from upper to lower wells for 18 hours, in the presence of α-ICAM-1 (10 μg/ml), α-VCAM-1 (30 μg/ml) or α-ALCAM (30 μg/ml) blocking antibodies (R&D Systems), or isotype-matched negative control antibody. Diffusion of high molecular weight dextran between wells was measured to assess intactness of endothelial monolayers. T cells were immunophenotyped before and after migration.

Results: : CCR6- Th1 and CCR6+ Th17 polarized cells migrated equally efficiently across simulated human retinal endothelium (22.64% ± 1.37% versus 24.14% ± 1.63% cells, n = 5 donors, p > 0.05). There was no difference in the phenotype of migrated Th1 polarized cells in comparison to pre-migrated and non-migrated cells. However, there was a significant reduction (p <0.0001) in percentage of IL-17+ IFN-γ- Th17 polarized cells following migration, in comparison to pre-migrated cells and non-migrated cells. Blocking endothelial ICAM-1 significantly (p < 0.05) reduced migration of Th1 and Th17 polarized cells for a majority of human donors (4/5 Th1, 8/10 Th17). Conversely, VCAM-1 or ALCAM blockade reduced Th1 and Th17 polarized cell migration in a minority of donors (VCAM-1: 0/5 Th1, 1/9 Th17; ALCAM: 1/5 Th1, 1/6 Th17).

Conclusions: : Helper T cells polarized towards Th1 or Th17 phenotypes migrate equally readily across a simulated blood-retinal barrier, although IL-17 expression by Th17 polarized cells is reduced over time and as a consequence of migration. In most individuals, migration is mediated in part by ICAM-1, but not VCAM-1 or ALCAM.

Keywords: inflammation • retinal adhesion • flow cytometry 
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