Hypoxia and reoxygenation do not upregulate adhesion molecules and natural killer cell adhesion on human endothelial cells in vitro

Objectives: Ischemia/reperfusion injury is characterized by endothelial cell activation leading to increased expression of adhesion molecules such as inter-cellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, endothelial- and platelet-selectin (E- and P-selectin), and to the subsequent recruitment of leukocytes. The aim of the present study was to investigate the respective effects of a proinflammatory cytokine (tumor necrosis factor alpha , TNF-α), hypoxia and/or reoxygenation on adhesion molecule expression and natural killer (NK) cell adhesion in an in vitro model of I/R. Methods: Human aortic endothelial cells (HAEC) were stimulated in vitro for 8h with TNF-α (1000 U/ml) and exposed to hypoxia (1% O2), reoxygenation (21% O2) or different combinations thereof. Cell surface expression of ICAM-1, VCAM-1 and E-/P-selectin on HAEC was analyzed by flow cytometry, and culture supernatants were tested for soluble adhesion molecules by ELISA. Rolling adhesion of NK cells on HAEC was determined using a rotating assay. Results: Untreated HAEC constitutively expressed ICAM-1 on their surface but neither expressed E-/P-selectin, VCAM-1, nor shedded soluble adhesion molecules. Exposure of HAEC to hypoxia or hypoxia and reoxygenation did not upregulate cell surface expression or shedding of adhesion molecules. In contrast, TNF-α significantly upregulated cell surface expression of ICAM-1, VCAM-1, and E-/P-selectin and led to the shedding of ICAM-1 and E-selectin. Combined treatment of HAEC with TNF-α, hypoxia and reoxygenation reduced E-/P-selectin surface expression and enhanced E-selectin shedding, but did not further influence ICAM-1 and VCAM-1. Soluble VCAM-1 was not detected. NK cell adhesion on HAEC increased 4-fold after TNF-α stimulation, but was not affected by hypoxia or hypoxia and reoxygenation. Conclusions: Both the expression of endothelial adhesion molecules and rolling NK cell adhesion was upregulated by TNF-α but not by hypoxia alone or hypoxia followed by reoxygenation supporting the view that anti-inflammatory treatment may reduce ischemia/reperfusion injur