Endothelial permeability and IL-6 production during hypoxia: role of ROS in signal transduction.
Prolonged hypoxia produces reversible changes in endothelial permeability, but the mechanisms involved are not fully known. Previous studies have implicated reactive oxygen species (ROS) and cytokines in the regulation of permeability. We tested whether prolonged hypoxia alters permeability to increasing ROS generation, which amplifies cytokine production. Human umbilical vein endothelial cell (HUVEC) monolayers were exposed to hypoxia while secretion of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1alpha, IL-6, and IL-8 was measured. IL-6 and IL-8 secretion increased fourfold over 24 h in a pattern corresponding to changes in HUVEC permeability measured by transendothelial electrical resistance (TEER). Addition of exogenous IL-6 to normoxic HUVEC monolayers caused time-dependent changes in TEER that mimicked the hypoxic response. An antibody to IL-6 significantly attenuated the hypoxia-induced changes in TEER (86 +/- 4 vs. 63 +/- 3% with hypoxia alone at 18 h), whereas treatment with anti-IL-8 had no effect. To determine the role of hypoxia-induced ROS on this response, HUVEC monolayers were incubated with the antioxidants ebselen (50 microM) and N-acetyl-L-cysteine (NAC, 1 mM) before hypoxia. Antioxidants attenuated hypoxia-induced IL-6 secretion (13 +/- 2 pg/ml with ebselen and 19 +/- 3 pg/ml with NAC vs. 140 +/- 15 pg/ml with hypoxia). Ebselen and NAC prevented changes in TEER during hypoxia (94 +/- 2% with ebselen and 90 +/- 6% with NAC vs. 63 +/- 3% with hypoxia at 18 h). N-nitro-L-arginine (500 microM) did not decrease hypoxia-induced changes in dichlorofluorescin fluorescence, IL-6 secretion, or TEER. Thus ROS generated during hypoxia act as signaling elements, regulating secretion of the proinflammatory cytokines that lead to alterations of endothelial permeability.
Ali
M H
MH
Pritzker School of Medicine, The University of Chicago, Chicago, Illinois 60637, USA.
Schlidt
S A
SA
Chandel
N S
NS
Hynes
K L
KL
Schumacker
P T
PT
Gewertz
B L
BL
D000906:Antibodies / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D001393:Azoles / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D000860:Hypoxia / Q000276:immunology / Q000378:metabolism*; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000276:immunology; D054833:Isoindoles; D016566:Organoselenium Compounds / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D010100:Oxygen / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000276:immunology*; D014471:Umbilical Veins / Q000166:cytology
D000906:Antibodies; D000975:Antioxidants; D001393:Azoles; D015850:Interleukin-6; D054833:Isoindoles; D016566:Organoselenium Compounds; D016877:Oxidants; D017382:Reactive Oxygen Species; C042986:ebselen; D006861:Hydrogen Peroxide; D010100:Oxygen