© 1999 American Heart Association, Inc.
Original Contributions |
Molecular Mechanisms of Neutrophil-Endothelial Cell Adhesion Induced by Redox Imbalance
From the Department of Molecular and Cellular Physiology (S.K., D.N.G., T.Y.A.), Louisiana State University Medical Center, Shreveport, La; Center of Excellence in Arthritis and Rheumatism (R.E.W.), Louisiana State University Medical Center, Shreveport, La; and First Department of Internal Medicine (T.Y.), Kyoto Prefectural University of Medicine, Kyoto, Japan.
Correspondence to Tak Yee Aw, PhD, Department of Molecular and Cellular Physiology, LSU Medical Center, 1501 Kings Highway, Shreveport, LA 71130-3932. E mail TAW@lsumc.edu
Abstract—Previous
studies have implicated a role for intracellular thiols in the
activation of nuclear factor-
B and transcriptional regulation of
endothelial cell adhesion molecules. This study was
designed to determine whether changes in endothelial
cell glutathione (GSH) or oxidized glutathione (GSSG) can alter
neutrophil adhesivity and to define the molecular mechanism that
underlies this GSSG/GSH-induced adhesion response. Treatment of human
umbilical vein endothelial cell (HUVEC) monolayers for
6 hours with 0.2 mmol/L diamide and 1 mmol/L buthionine
sulfoximine (BSO) decreased GSH levels and increased the ratio of GSSG
to GSH without cell toxicity. These redox changes are similar to those
observed with anoxia/reoxygenation. Diamide plus
BSO–induced thiol/disulfide imbalance was associated with a biphasic
increase in neutrophil adhesion to HUVECs with peak responses observed
at 15 minutes (phase 1) and 240 minutes (phase 2).
N-Acetylcysteine treatment attenuated neutrophil
adhesion in both phases, which indicated a role for GSH in the adhesion
responses. Interestingly, phase 1 adhesion was inversely correlated
with GSH levels but not with the GSSG/GSH ratio, whereas phase 2
neutrophil adhesion was positively correlated with GSSG/GSH ratio but
not with GSH levels. Intercellular adhesion molecule-1 and
P-selectin–specific monoclonal antibodies attenuated the increased
neutrophil adhesion during both phases, whereas an anti–E-selectin
monoclonal antibody also attenuated the phase 2 response. Pretreatment
with actinomycin D and cycloheximide or with competing
ds-oligonucleotides that contained
nuclear factor-B or activator protein-1 cognate DNA
sequences significantly attenuated the phase 2 response, which
implicated a role for de novo protein synthesis. Surface expression of
intercellular adhesion molecule-1, P-selectin, and E-selectin on HUVECs
correlated with the phase 1 and 2 neutrophil adhesion responses. This
study demonstrates that changes in endothelial cell
GSSG/GSH cause transcription-independent and transcription-dependent
surface expression of different endothelial cell
adhesion molecules, which leads to a 2-phase
neutrophil–endothelial adhesion response.
Key Words: neutrophil • glutathione • endothelium • oxidation-reduction • anoxia • adhesion • diamide
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