Hypoxia/reoxygenation of human endothelium activates PMNs to detach endothelial cells via a PAF mechanism.
Our previous in vivo studies have implicated phospholipase A2 activation and platelet-activating factor (PAF) production as an important mediator of neutrophil (PMN) priming after mesenteric ischemia/reperfusion. Furthermore, our in vitro studies demonstrate that PAF priming of PMN enhances PMN respiratory burst and increases PMN adherence to human umbilical vein endothelial cell cultures (HUVEC). Others have shown that cytokine stimulated HUVEC can activate quiescent PMNs to provoke endothelial cell (EC) monolayer disruption via EC detachment by a noncytolytic PMN protease mechanism. Hypoxia and reoxygenation (H/R) of HUVEC can also directly stimulate PAF production. Consequently, we hypothesized that HUVEC H/R can activate quiescent PMNs to disrupt the EC monolayer (detachment) through a PAF mediated mechanism. HUVEC were labeled with 51 chromium (51Cr) and subjected to 45 min hypoxia (95% N2/5% CO2). PMNs freshly isolated by Percoll gradient centrifugation were added to HUVEC and reoxygenated for 120 min. Additionally, H/R HUVEC with PMN pretreated with WEB2170 (a PAF receptor antagonist) was compared to control (non-H/R HUVEC incubated with PMNs). Wells were washed at end incubation, and adherent ECs counted. Detachment = [total counts - sample counts]/total counts X 100. H/R HUVEC plus PMN provoked a 29.3 +/- 1.6% detachment of EC compared to 9.3 +/- 2.9% detachment in control (non-H/R HUVEC with PMNs). In contrast, H/R HUVEC with PMNs preincubated with WEB2170 had 9.9 +/- 3.8% detachment of EC. In summary, HUVEC H/R activated quiescent PMNs to disrupt an EC monolayer (detachment) via a PAF mechanism.
Franciose
R J
RJ
Department of Surgery, Denver General Hospital, Colorado 80204-4507, USA.
Moore
E E
EE
Moore
F A
FA
Read
R A
RA
Carl
V S
VS
Banerjee
A
A
D002448:Cell Adhesion; D015687:Cell Hypoxia; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D018375:Neutrophil Activation; D010972:Platelet Activating Factor / Q000502:physiology*
D010972:Platelet Activating Factor