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D.O. Zamora, B. Babra, Y. Pan, S.R. Planck, J.T. Rosenbaum; Iris Endothelial Cells Potentially Regulate Leukocyte Trafficking During Inflammation via EphB4-EphrinB2 Interactions . Invest. Ophthalmol. Vis. Sci. 2003;44(13):730.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: At the onset of anterior uveitis, endothelial cells (EC) of the iris become activated and increase their interaction with flowing leukocytes, resulting in the transmigration of leukocytes into the iris stroma. EphB4, a tyrosine kinase receptor, specifically interacts with EphrinB2 in a variety of cell types and modulates cell-cell interactions (e.g., cell adhesion, repulsion, and attraction). We hypothesized that EphB4/EphrinB2 interactions may also have a role in mediating leukocyte trafficking during ocular inflammation. Methods: Pure human iris EC cultures were established as previously described (IOVS, 2001; 42:12; 2861-2866). Confluent monolayers of iris EC were stimulated with endotoxin (LPS, 10 µg/ml), TNFα (10 ng/ml), or EphrinB2 (2 µg/ml) for various time periods. Total RNA was then analyzed by RT-PCR for EphB4 mRNA or with Atlas Human Cell Interaction cDNA Arrays. Cell lysates were produced using similar cell culture stimulation parameters and probed for EphB4 by Western blot. Some monolayers of EC were fixed and immunostained for EphB4. Peripheral blood mononuclear cells were isolated from normal donors and depleted of monocytes by removal of adherent cells. In addition, CD4+ T cells were isolated by negative selection. Results: Our studies show four novel findings: 1) EphB4 mRNA and protein is expressed in cultured iris vascular endothelial cells and the expression of these molecules is not regulated by LPS or TNFα after 5hrs stimulation. 2) In contrast, EphrinB2 mRNA and protein are undetectable in isolated peripheral blood lymphocytes, but are markedly upregulated by TNFα. Interestingly, EphB4 mRNA is slightly detectable in unstimulated lymphocytes and is not affected by TNFα. 3) Soluble EphrinB2 upregulated the expression of various genes in iris EC as determined by cDNA array analysis. Upregulated genes include molecules involved in signaling, cytoskeletal integrity and cell adhesion, all of which could potentially modulate leukocyte-EC interactions. 4) CD4+ T cells express EphrinB2 mRNA. Conclusions: These observations indicate that a ligand expressed by TNFα-stimulated lymphocytes activates iris EC. The observations suggest a novel mechanism by which iris EC can regulate lymphocyte trafficking during uveitis.
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