Anti-A IgG antibodies have previously been shown to stimulate Ca2+ entry into red blood cells. Increased cytosolic free Ca2+ concentration is known to trigger eryptosis, i.e. suicidal erythrocyte death, characterized by exposure of phosphatidylserine at the erythrocyte surface. As macrophages are equipped with phosphatidylserine receptors, they bind, engulf and degrade phosphatidylserine exposing cells. The present experiments have been performed to explore whether anti-A IgGs trigger phosphatidylserine exposure of erythrocytes. Phosphatidylserine exposure was estimated from annexin-V binding as determined in FACS analysis. Exposure to anti-A IgGs (0.5 µg/ml) indeed significantly increased annexin-V binding in erythrocytes with blood group A, but not in erythrocytes with blood group 0. According to Fluo3 fluorescence, anti-A IgGs increased cytosolic Ca2+ concentration. Whole cell patch clamp recordings revealed the activation of a Ca2+-permeable cation channel following treatment with anti-A-IgGs. Annexin-V binding following anti-A IgG exposure was blunted by Ca2+ removal while anti-A IgG-stimulated cation channel activity was not dependent on extracellular Ca2+. Osmotic shock (exposure of erythrocytes to 850 mOsm) increased annexin binding, an effect further enhanced by exposure to anti-A IgGs. In conclusion, anti-A IgGs activate erythrocyte cation channels leading to Ca2+ entry and subsequent erythrocyte cell membrane scrambling. The effect most likely contributes to the elimination of erythrocytes following an immune reaction against the A antigen.
The prostaglandin PGE2, a metabolite of the cyclooxygenase pathway, activates Ca2+-permeable cation channels in erythrocyte cell membranes leading to entry of Ca2+ with subsequent eryptosis, i.e. cell shrinkage, breakdown of phosphatidylserine (PS) asymmetry and membrane blebbing, all features typical for apoptosis in nucleated cells. PS exposing cells are recognized by macrophages, engulfed, degraded and thus cleared from circulating blood. The present study explored whether the specific lipoxygenase inhibitor Bay-Y5884 influences eryptosis. As determined by competitive ELISA, Bay-Y5884 (20 µM) enhanced the release of PGE2 from human erythrocytes. According to whole-cell patch-clamp, Bay-Y5884 (20 µM) activated nonselective cation channels. The effect of Bay-Y5884 on cation channels was abolished by the cyclooxygenase inhibitor diclophenac (10 µM). Bay-Y5884 (30-40 µM) significantly increased erythrocyte free Ca2+ concentration and PS exposure as analyzed in flow cytometry by Fluo3 fluorescence and annexin-V binding, respectively. PS exposure triggered by 20 µM (but not by 40 µM) Bay-Y5884 was blunted by cyclooxygenase inhibitors acetylsalicylic acid (50 µM) and diclophenac (10 µM). In conclusion, the lipoxygenase inhibitor Bay-Y5884 enhances erythrocyte PGE2 formation with subsequent activation of non-selective cation channels, Ca2+ entry and phospholipid scrambling.
Genetic defects of anion exchanger 1 (AE1) may lead to spherocytic erythrocyte morphology, severe hemolytic anemia, and/or cation leak. In normal erythrocytes, osmotic shock, Cl(-) removal, and energy depletion activate Ca(2+)-permeable cation channels with Ca(2+)-induced suicidal erythrocyte death, i.e., surface exposure of phosphatidylserine, cell shrinkage, and membrane blebbing, all features typical for apoptosis of nucleated cells. The present experiments explored whether AE1 deficiency favors suicidal erythrocyte death. Peripheral blood erythrocyte numbers were significantly smaller in gene-targeted mice lacking AE1 (AE1(-/-) mice) than in their wild-type littermates (AE1(+/+) mice) despite increased percentages of reticulocytes (AE1(-/-): 49%, AE1(+/+): 2%), an indicator of enhanced erythropoiesis. Annexin binding, reflecting phosphatidylserine exposure, was significantly larger in AE1(-/-)erythrocytes/reticulocytes ( approximately 10%) than in AE1(+/+) erythrocytes ( approximately 1%). Osmotic shock (addition of 400 mM sucrose), Cl(-) removal (replacement with gluconate), or energy depletion (removal of glucose) led to significantly stronger annexin binding in AE1(-/-) erythrocytes/reticulocytes than in AE1(+/+) erythrocytes. The increase of annexin binding following exposure to the Ca(2+) ionophore ionomycin (1 muM) was, however, similar in AE1(-/-) and in AE1(+/+) erythrocytes. Fluo3 fluorescence revealed markedly increased cytosolic Ca(2+) permeability in AE1(-/-) erythrocytes/reticulocytes. Clearance of carboxyfluorescein diacetate succinimidyl ester-labeled erythrocytes/reticulocytes from circulating blood was more rapid in AE1(-/-) mice than in AE1(+/+) mice and was accelerated by ionomycin treatment in both genotypes. In conclusion, lack of AE1 is associated with enhanced Ca(2+) entry and subsequent scrambling of cell membrane phospholipids.
Zn(2+) triggers suicidal erythrocyte death, an effect partially due to ceramide formation and an increase of cytosolic Ca(2+) activity.
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