MOLECULAR MECHANISMS OF ANOXIA REOXYGENATION-INDUCED NEUTROPHIL ADHERENCE TO CULTURED ENDOTHELIAL-CELLS/

The objectives of this study were to (1) determine the time course of neutrophil adhesion to monolayers of human umbilical vein endothelial cells (HUVECs) that were exposed to 60 minutes of anoxia followed by 3 0 to 600 minutes of reoxygenation and (2) define the mechanisms respon sible for both the early (minutes) and late (hours) hyperadhesivity of postanoxic HUVECs to human neutrophils. The results clearly demonstra te that anoxia/reoxygenation (A/R) leads to a biphasic increase in neu trophil adhesion to HUVECs, with peak responses occurring at 30 minute s (phase 1) and 240 minutes (phase 2) after reoxygenation. Oxypurinol and catalase inhibited phase-1 adhesion, suggesting a role for xanthin e oxidase and H2O2. In comparison, platelet activating factor (PAF) co ntributed to both phases of neutrophil adhesion. Anti-intercellular ad hesion molecule-1 (ICAM-1) and anti-P-selectin antibodies (monoclonal antibodies [mAbs]) attenuated phase-1 neutrophil adhesion, consistent with roles for constitutively expressed ICAM-1 and enhanced surface ex pression of preformed P-selectin. Phase-2 neutrophil adhesion was atte nuated by an anti-E-selectin mAb, indicating a dominant role of this a dhesion molecule in the late phase response. Pretreatment with actinom ycin D and cycloheximide or with competing ds-oligonucleotides contain ing the nuclear factor-kappa B or activator protein-1 cognate DNA sequ ences significantly attenuated phase-2 response, suggesting a role for de novo macromolecule synthesis. Surface expression of ICAM-1, P-sele ctin, and E-selectin on HUVECs correlated with the phase-1 and -2 neut rophil adhesion responses. Collectively, these findings indicate that A/R elicits a two-phase neutrophil-endothelial cell adhesion response that involves transcription-independent and transcription-dependent su rface expression of different endothelial cell adhesion molecules.