Purchase this article with an account.
Arpita S. Bharadwaj, Hoda A. Ilias, Yuzhen Pan, Justine R. Smith; Migration of Helper T Cell Subsets Across a Simulated Blood-Retinal Barrier. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2931.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Autoimmune posterior uveitis is an inflammatory eye disease that causes vision loss in a majority of affected individuals. Th1 or Th17 cells are initiators of the uveitis in different settings. These cells move from the circulation into the posterior eye across the retinal endothelium, but migratory behavior of the two subsets has not been compared and is the subject of our investigation.
Mononuclear cells were obtained from human peripheral blood by density gradient centrifugation, and CD4+CD45RO+ T cells were isolated by negative selection. T cells were exposed for 5 days to: CD14+ monocytes, isolated from the same peripheral blood mononuclear cells; anti-CD3 antibody; and interleukin (IL)-12 plus anti-IL-4 antibody or IL-23 plus anti-IL-4 and anti-interferon (IFN)-γ antibodies to generate Th1 and Th17 cells, respectively. This protocol was shared by Dr. Alexandre Prat, University of Montreal. Retinal endothelial transmigration assays were performed using a transwell system. 0.3 cm2 diameter transwell membranes with 3 µm pores were coated with collagen I, seeded with human retinal endothelial cells, and incubated for 4 days at 37oC to generate relatively impermeable endothelial monolayers. Polarized T cells were migrated across monolayers from upper to lower chambers for 18 hours. Rate of diffusion of high molecular weight dextran between chambers was measured to confirm intactness of monolayers. T cells were phenotyped before and after migration by intracellular cytokine staining and flow cytometry.
Following selection, surface CD3, CD4 and CD45RO expression indicated purity of memory T cells was ≥96%. Percentages of lymphocytes & memory T cells were equal for both conditions of polarization. Polarization generated means of 27.3% IFN-γ+/IL-17- cells and 11.8% IFN-γ- /IL-17+ cells from total lymphocytes (n=5-6 donors); however, some cells produced both IFN-γ and IL-17 (1.7% or 4.2% for Th1 or Th17 polarizing conditions). There was no significant difference (p>0.05) in the percentage of migrating Th1 (mean=21.1%) or Th17 (mean=15.7%) polarized cells. However, the endothelial barrier significantly limited migration of Th1 cells (IFN-γ+/IL-17-), p=0.016 and p=0.025, but not Th17 cells (IFN-γ±/IL-17+), p > 0.05, generated under conditions of Th1 and Th17 polarization, respectively.
Helper T cells polarized towards Th1 or Th17 phenotypes migrate equally readily across a simulated blood-retinal barrier, but Th1 cells (IFN-γ+/IL-17-), generated by either Th1 or Th17 polarization, exhibit relatively reduced migration.
This PDF is available to Subscribers Only