Increased adherence of oxidant‐treated human and bovine erythrocytes to cultured endothelial cells

Abstract

Bovine erythrocytes, which normally lack phosphatidyl choline in their membranes, when treated with either H₂O₂ or diamide (1–3 mM), showed a partial appearance of phosphatidyl ethanolamine (PE 40%) and phosphatidyl serine (PS, 30–33%) in the external leaflet of the bilayer and a concomitant increased (four‐ to five‐fold) propensity to adhere to cultured bovine aortic endothelial cells. Similar treatment of normal human erythrocytes caused an alteration in the organization of the phospholipid bilayer and also resulted in their increased adherence to endothelial cells derived either from human umbilical vein or bovine aorta. Treatment of RBCs with H₂O₂ at low concentration (0.5 mM) resulted in cross‐linking of spectrin without significant changes in the orientation of aminophospholipids but the RBCs exhibited 15–20% increase in adherence to endothelial cells. Pretreatment of either human or bovine erythrocytes with antioxidants such as vitamin E (2 mM) prevented both oxidant‐induced reorganization of phospholipids in the bilayer and enhancement of adherence to endothelial cells. Introduction of either phosphatidyl serine or phosphatidyl ethanolamine but not phosphatidyl choline into erythrocyte membranes increased their adherence to endothelial cells threefold. Oxidant‐treated RBCs exhibited enhanced binding and fluorescence of Merocyanine 540 dye (MC‐540), which is sensitive to the packing of lipids in the lipid bilayer. On flow cytometric analysis, 78% of H₂O₂ (0.5 mM)‐treated erythrocytes compared to 30% of untreated RBCs exhibited MC‐540 binding and fluorescence, indicating differences in the lipid packing in the outer leaflet of the bilayer. Oxidant‐treated erythrocytes adhere preferentially to endothelial cells rather than to bovine aortic smooth muscle cells and skin fibroblasts. It is suggested that the alterations in the erythrocyte membrane surface due to spectrin cross‐linking and the organization of the phospholipids concomitant with less ordered packing in the external leaflet of the bilayer, either induced by oxidative manipulation in normal RBC or in pathological erythrocytes, play a role in erythrocyte‐endothelial cell interaction.