Objective: The objectives of this study were to 1) determine the time-course of T-lymphocyte adhesion to monolayers of human umbilical vein endothelial cell (HUVEC) that were exposed to 60 min of anoxia followed by 24 h of reoxygenation, and 2) define the mechanisms responsible for the hyperadhesivity of postanoxic HUVEC to human T-lymphocytes.
Methods: Human peripheral blood mononuclear leukocytes were isolated from heparinized peripheral blood. T-lymphocytes were obtained by negative selection using a MACS column. HUVEC monolayers were exposed to anoxia/reoxygenation (A/R), and then reacted with 51Cr -labeled T-lymphocytes in adhesion assays.
Results: A/R leads to an increased adhesion of T-lymphocytes to HUVEC monolayers, with peak responses occurring at 8 h after reoxygenation. This adhesion response was largely attributed to the CD4+ T-cell subset. The hyperadhesivity of A/R-exposed HUVEC was inhibited by monoclonal antibodies directed against either LFA-1, VLA-4, ICAM-1, or VCAM-1, indicating a contribution of these adhesion molecules and their ligands. Moreover, T-cell hyperadhesivity was attenuated by anti- IL-8. consistent with a role for this chemokine in the adhesion response. Protein synthesis inhibitors (actinomycin D and cycloheximide) as well as chemical inhibitors of (and binding ds-oligonucleotides to) NFkappaB and AP-1 significantly attenuated the A/R-induced T-lymphocyte adhesion responses. The kinetics of VCAM-1 on post-anoxic HUVEC correlated with the T-lymphocyte adhesion response.
Conclusions: A/R elicits a T-lymphocyte-endothelial cell adhesion response that involves transcription-dependent surface expression of VCAM-1.