Hypoxia and reoxygenation do not upregulate adhesion molecules and natural killer cell adhesion on human endothelial cells in vitro.
OBJECTIVE
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-alpha), 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-alpha (1000 U/ml) and exposed to hypoxia (1% O(2)), reoxygenation (21% O(2)) 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-alpha 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-alpha, 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-alpha 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-alpha but not by hypoxia alone or hypoxia followed by reoxygenation supporting the view that anti-inflammatory treatment may reduce ischemia/reperfusion injury.
Maurus
Christine F
CF
Clinic for Cardiovascular Surgery, University Hospital Zürich, Rämistr. 100, F LAB 39, CH-8091, Zürich, Switzerland. christine.maurus@usz.ch
Schmidt
Dörthe
D
Schneider
Mårten K J
MK
Turina
Marko I
MI
Seebach
Jörg D
JD
Zünd
Gregor
G
D001011:Aorta; D015415:Biomarkers / Q000032:analysis; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000276:immunology* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007694:Killer Cells, Natural / Q000473:pathology*; D036904:Leukocyte Rolling; D015427:Reperfusion Injury / Q000276:immunology* / Q000378:metabolism; D013268:Stimulation, Chemical; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015854:Up-Regulation
D015415:Biomarkers; D015815:Cell Adhesion Molecules; D014409:Tumor Necrosis Factor-alpha