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DO Zamora, Y Pan, SR Planck, JT Rosenbaum; Expression of Ephrin-B2 Ligand and its Receptor Eph-B4 in Human Iris Tissue, Cultured Iris Endothelial Cells, and Peripheral Blood Mononuclear Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1568.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: At the onset of anterior uveitis, iris vascular endothelial cells (EC) become activated and display increased interaction with circulating leukocytes resulting in the transmigration of leukocytes from vasculature into the stroma. Ephrin-B2, a transmembrane ligand specifically expressed on arterial EC, interacts with receptors of the EphB class. Eph-B4, a specific receptor for Ephrin-B2, is expressed on venous EC and interaction of Ephrin-B2 and Eph-B4 regulates artery/vein boundaries. We report here the localization and expression patterns of Ephrin-B2 and Eph-B4 in human iris tissue, cultured iris EC, and peripheral blood lymphocytes and the effects of TNFα treatment on their expression. Methods: Human eyes were obtained from our local eye bank. Iris explants and purified iris EC cultures were established and stimulated with TNFα (10ng/ml) for various time periods. Total RNA was extracted and assayed for Ephrin-B2 and Eph-B4 expression by RT-PCR. Localization of Ephrin-B2 and Eph-B4 was analyzed in human iris tissue by immunohistology. Iris tissue was dissected from donor eyes, paraffin-embedded, serially sectioned (5µm), and immunostained with Ephrin-B2 and Eph-B4 specific antibodies. Peripheral blood mononuclear cells were isolated from several normal donors by Ficoll-Paque separation and depleted of monocytes by removal of adherent cells. The remaining lymphocytes were cultured in the presence or absence of TNFα for different time periods and assayed for Ephrin-B2 and Eph-B4 mRNA expression. Results: Iris explants and iris endothelial cells demonstrated constitutive expression of Ephrin-B2 and Eph-B4 that was unchanged by TNFα treatment. Immunohistology of human iris revealed vessels distinctly labeled with either Ephrin-B2 or Eph-B4. In contrast, Ephrin-B2 mRNA was undetectable in control peripheral blood lymphocytes, but highly upregulated in response to TNFα. Baseline levels of Eph-B4 mRNA were detected in control lymphocytes and did not change in response to TNFα. Conclusion: Cell-cell interactions via Ephrin-B2/Eph-B4 molecules has been demonstrated to initiate bi-directional signaling in both ligand and receptor bearing cells. Ephrin-B2’s upregulation in circulating lymphocytes in response to TNFα suggests that it could play a role in modulating the transmigration of EC adhering lymphocytes in venules during inflammation. This is the first report of Ephrin-B2 expression in leukocytes and the first to suggest the potential role of this ligand-receptor complex in leukocyte-endothelial interactions.
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