Purpose: To develop a method to isolate human iris microvascular endothelial cells (HIECs) for exploring their constitutive and inflammatory agent-modulated expression of intercellular adhesion molecules (ICAM)-1 and -2, vascular cell adhesion molecule (VCAM)-1, and E-selectin.
Methods: Endothelial cells from collagenase-digested irises were isolated on the basis of their expression of platelet endothelial cell adhesion molecule (PECAM)-1, using antibody-coupled magnetic beads. Cells were characterized as endothelial based on morphologic criteria, their expression of PECAM-1 and von Willebrand factor, their uptake of acetylated low-density lipoprotein, and their ability to form capillary-like networks on a synthetic basement membrane. Constitutive and inflammatory agent-modulated expression of ICAM-1 and -2, VCAM-1, and E-selectin was evaluated by the reverse transcription-polymerase chain reaction, enzyme-linked immunocellular assays (ELICAs), 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 stimulated with endotoxin- or tumor necrosis factor (TNF)-alpha, ICAM-1, VCAM-1, and E-selectin were potently and time- and dose-dependently upregulated at both the message and protein levels. By contrast, ICAM-2 message and protein were slowly downregulated by inflammatory agents over time, but nonetheless remained present and functional. Overall, cytokine- or endotoxin-activation 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 selective isolation of HIECs, with a demonstration of differential expression and regulation of these adhesion molecules in them. In addition, this is the first demonstration of the regulated expression of ICAM-2 in any ocular microvascular cells.