Adhesion of sickle red blood cells and damage to interleukin-1β stimulated endothelial cells under flow in vitro

Abstract

Two factors that are hypothesized to contribute to vasoocclusive crises in sickle cell anemia are increased sickle red blood cell-endothelial cell interactions and damage to endothelium. Despite considerable study, the mechanisms by which erythrocyte-endothelial interactions occur and the role of endothelial damage have not yet been fully elucidated. In this report, we demonstrate that adhesion and damage may be related in a model of vasoocclusion in sickle cell anemia. Phase contrast microscopy coupled to digital image processing was used to determine the adhesion of sickle red blood cells to 1-, 4-, and 24-hour interleukin-1β (IL-1β) stimulated endothelial cells in a parallel plate flow chamber. Morphological alterations to activated endothelial cells after the perfusion of sickle erythrocytes were also identified. Pretreatment of monolayers with 50 pg/mL of IL-1β for 1, 4, and 24 hours caused approximately 16-fold increases in adhesion of sickle cells to activated endothelium at all time points. Results with an Arginine-glycine aspartic acid (RGD) peptide and monoclonal antibodies indicated a role for three different endothelial cell receptors: α(v)β3 after 1 hour of IL-1β stimulation; E-selectin after 4 hours of IL-1β stimulation; and vascular cell adhesion molecule-1 after prolonged exposure to cytokines. Perfusion of sickle, but not normal, erythrocytes resulted in alteration of endothelial morphology. Approximately 6% to 8% damage was observed on 4- and 24-hour IL-1β stimulated endothelial cells after the perfusion of sickle cells. Damage to 24-hour activated endothelial cells showed a positive correlation (r = .899) with the number of adherent sickle erythrocytes.