Cell adhesion molecule expression in cultured human iris endothelial cells

PURPOSE. To develop a method to isolate human iris microvascular endothelia l cells (HIECs) for exploring their constitutive and inflammatory agent-mod ulated expression of intercellular adhesion molecules (ICAM)-1 and -2, vasc ular cell adhesion molecule (VCAM)-1, and E-selectin. METHODS. Endothelial cells from collagenase-digested irises were isolated o n the basis of their expression of platelet endothelial cell adhesion molec ule (PECAM)-1, using antibody-coupled magnetic beads. Cells were characteri zed as endothelial based on morphologic criteria, their expression of PECAM -1 and von Willebrand factor, their uptake of acetylated low-density lipopr otein, and their ability to form capillary-like networks on a synthetic bas ement membrane. Constitutive and inflammatory agent-modulated expression of ICAM-1 and -2, VCAM-1, and E-selectin was evaluated by the reverse transcr iption-polymerase chain reaction, enzyme-linked immunocellular assays (ELIC As), Western blot analysis, and functional studies of leukocyte adhesion to HIEC monolayers. RESULTS. HIECs constitutively expressed mRNA and protein for ICAM-1 and -2, but only low to nondetectable levels of VCAM-1 or E-selectin. When Stimula ted with endotoxin- or tumor necrosis factor (TNF)-alpha, ICAM-1, VCAM-1, a nd E-selectin were potently and time- and dose-dependently upregulated at b oth the message and protein levels. By contrast, ICAM-2 message and protein were slowly downregulated by inflammatory agents over time, but nonetheles s remained present and functional, Overall, cytokine- or endotoxin-activati on of HIECs resulted in enhanced adhesiveness for leukocytes. CONCLUSIONS. ICAM-1, VCAM-1, and E-selectin have been previously implicated in mediating anterior ocular inflammation. This is a report of the selecti ve isolation of HIECs, with a demonstration of differential expression and regulation of these adhesion molecules in them. In addition, this is the fi rst demonstration of the regulated expression of ICAM-2 in any ocular micro vascular cells.