Reperfusion injury involves the adhesion and activation of neutrophils
(PMN) both in affected tissues and distant organs. Cell adhesion mole
cules (CAM) such as endothelial-leukocyte adhesion molecule-1 (ELAM-1)
and intercellular adhesion molecule-1 (ICAM-1) are known to mediate,
at least in part, the adherence of activated PMN to the endothelium. T
o characterize the cellular mechanisms of this phenomenon, we exposed
cultured human umbilical vein endothelial cells (HUVEC) to hypoxia and
reoxygenation (H/R) using an incubator chamber purged of oxygen with
100% nitrogen. Confluent monolayers of HUVEC were subjected to 60 min
of hypoxia followed by variable periods of reoxygenation (120 and 240
min). Flow cytometry was utilized to assess the expression of ELAM-1 a
nd ICAM-1, expressed as percent shift from baseline expression. To det
ermine what role endothelium-derived cytokines such as IL-1 play in th
e expression of CAM after H/R, we performed additional experiments in
the presence of recombinant IL-1 receptor antagonist (IL-1RA). ICAM-1
was present on unstimulated HUVEC while ELAM-1 was not constituitively
expressed. Following exposure of cells to hypoxia and reoxygenation,
significant increases in ELAM expression were seen (8.4 +/- 2.4% at 12
0 min; 19.1 +/- 7.4%, P < 0.05). While there was similar trend in ICAM
expression, this did not achieve statistical significance (0.10 < P <
0.05). The addition of IL-1RA (10 ng/ml) to hypoxic HUVEC consistentl
y attenuated ELAM-1 upregulation during reoxygenation (0.8 +/- 0.7% at
120 min and 5.9 +/- 4.1% at 240 min) such that expression was not sig
nificantly greater than baseline. This in vitro model of ischemia/repe
rfusion demonstrated reproducible increases in the expression of ELAM-
1, an adhesion molecule important in the initial PMN rolling and adhes
ion to endothelial cells in the microcirculation. The data also sugges
t that the cytokine IL-1 plays a central role in the endothelial cell
response to H/R. (C) 1995 Academic Press, Inc.