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M. A. Cunningham, Z. Li, B. Liu, S. P. Mahesh, R. N. Fariss, R. B. Nussenblatt; OX40 Ligand (OX40L) and Its Potential Role in Ocular Inflammation: An in vitro Approach. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2523. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the role of OX40L in ocular inflammation, using an in vitro expression approach.
OX40L cDNA was PCR amplified from an HTLV-1 infected T cell lymphoma (MT-2) cell line and cloned into an eYFP fusion vector. Cultured retinal pigment epithelial cells (ARPE-19) were transfected with this recombinant OX40L-expressing vector, and OX40L surface expression was assessed using flow cytometry and fluorescence microscopy. To investigate regulation of OX40L in RPE cells by inflammatory stimuli, total RNAs from unstimulated or stimulated ARPE cells (with TNF-alpha, IL-1, and IFN-gamma) were isolated and analyzed for OX40L expression by RT-PCR. To investigate the potential effect of OX40L on ARPE cells on leukocyte proliferation, peripheral blood mononuclear cells (PBMC) were isolated from healthy human donor using the Ficoll gradient centrifugation. Human ARPE cells (± OX40L expression) and PBMCs were co-cultured for in vitro proliferation studies.
Polymerase Chain Reaction confirmed insertion of the OX40L gene into the fusion vector. Flow cytometry and fluorescence microscopy further confirmed surface expression of OX40L on ARPE cells as early as within 4 hours after transient transfection of OX40L expressing vector.There was no constitutive expression of OX40L by RT-PCR analysis of the unstimulated RPE cells, while OX40L expression was observed in the RPE cells stimulated with pro-inflammatory cytokines. In the co-culture studies, there was a significant reversal (between 20-30%) of the RPE-induced suppression of activated PBMCs when the ARPE cells were transfected with OX40L. The reversal was not as dramatic as seen in GITRL expressing ARPE, but when both OX40L and GITRL were concomitantly transfected into ARPE cells, there was a slight additive reversal of RPE-mediated T cell suppression, when comparing it to the reversal caused by RPE cells expressing either OX40L alone or GITRL alone.
Using an in vitro approach, we found that OX40L causes an abrogation of the RPE-mediated immunosuppression, a critical mechanism in maintaining the ocular immune privilege. OX40L appears to be regulated in the ARPE-19 cell line, and may play an important role in the pathogenesis of various ocular inflammatory conditions.
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