Majed Abed

Dynamic adhesion of eryptotic erythrocytes to endothelial cells via CXCL16/SR-PSOX

Suicidal death of erythrocytes, or eryptosis, is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the cell surface. Eryptosis is triggered by increase of cytosolic Ca2+ activity, which may result from treatment with the Ca2+ ionophore ionomycin or from energy depletion by removal of glucose. The present study tested the hypothesis that phosphatidylserine exposure at the erythrocyte surface fosters adherence to endothelial cells of the vascular wall under flow conditions at arterial shear rates and that binding of eryptotic cells to endothelial cells is mediated by the transmembrane CXC chemokine ligand 16 (CXCL16). To this end, human erythrocytes were exposed to energy depletion (for 48 h) or treated with the Ca2+ ionophore ionomycin (1 μM for 30 min). Phosphatidylserine exposure was quantified utilizing annexin-V binding, cell volume was estimated from forward scatter in FACS analysis, and erythrocyte adhesion to human vascular endothelial cells (HUVEC) was determined in a flow chamber model. As a result, both, ionomycin and glucose depletion, triggered eryptosis and enhanced the percentage of erythrocytes adhering to HUVEC under flow conditions at arterial shear rates. The adhesion was significantly blunted in the presence of erythrocyte phosphatidylserine-coating annexin-V (5 μl/ml), of a neutralizing antibody against endothelial CXCL16 (4 μg/ml), and following silencing of endothelial CXCL16 with small interfering RNA. The present observations demonstrate that eryptotic erythrocytes adhere to endothelial cells of the vascular wall in part by interaction of phosphatidylserine exposed at the erythrocyte surface with endothelial CXCL16.

Sphingomyelinase-induced adhesion of eryptotic erythrocytes to endothelial cells

Eryptosis, the suicidal erythrocyte death, leads to cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptotic erythrocytes adhere to the vascular wall by binding of phosphatidylserine to the CXC chemokine ligand 16 (CXCL16). Stimulators of eryptosis include increased cytosolic Ca(2+) activity, energy depletion, and activation of ceramide-producing sphingomyelinase. The present study explored whether sphingomyelinase triggers erythrocyte adhesion to endothelial cells. To this end, human erythrocytes were exposed for 6 h to bacterial sphingomyelinase (1-10 mU/ml) and phosphatidylserine exposure was estimated from fluorescent annexin-V-binding, cell volume from forward scatter in FACS-analysis, erythrocyte adhesion to human umbilical vein endothelial cells (HUVEC) from trapping of labeled erythrocytes in a flow chamber under flow conditions at arterial shear rates, and CXCL16 protein abundance utilizing Western blotting and FACS analysis of fluorescent antibody binding. As a result, sphingomyelinase (≥1 mU/ml) triggered cell shrinkage, phosphatidylserine exposure and erythrocyte adhesion to HUVEC, effects blunted by Ca(2+) removal. Adhesion was significantly blunted by phosphatidylserine-coating annexin-V (5 μl/ml), following addition of neutralizing antibodies against endothelial CXCL16 (4 μg/ml) and following silencing of the CXCL16 gene with small interfering RNA. Pretreatment of HUVEC with sphingomyelinase upregulated CXCL16 protein abundance. Six hours pretreatment of HUVEC with sphingomyelinase (10 mU/ml) or C6-ceramide (50 μM) augmented erythrocyte adhesion following a 30-min treatment with Ca(2+) ionophore ionomycin (1 μM) or following energy depletion by 48-h glucose removal. Thus exposure to sphingomyelinase or C6-ceramide triggers eryptosis followed by phosphatidylserine- and CXCL16-sensitive adhesion of eryptotic erythrocytes to HUVEC.

Stimulation of Erythrocyte Cell Membrane Scrambling by Mitotane

Background: Mitotane (1,1-dichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethane), a cytostatic drug used for the treatment of adrenocortical carcinomas, is effective by triggering tumor cell apoptosis. In analogy to apoptosis of nucleated cells, eryptosis is the suicidal death of erythrocytes, which is typically paralleled by cell shrinkage and breakdown of cell membrane phosphatidylserine asymmetry with subsequent phosphatidylserine exposure at the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca2+ concentration ([Ca2+]i). The present study tested, whether treatment of human erythrocytes with mitotane is followed by eryptosis. Methods: [Ca2+]i was estimated from Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, and hemolysis from hemoglobin release. Results: Exposure to mitotane (≥ 5 µg/ml ≈ 16 µM) significantly increased [Ca2+]i, increased annexin V binding and triggered hemolysis, but did not significantly modify forward scatter. The effect on annexin V binding was significantly blunted in the absence of extracellular Ca2+. Within 30 min Ca2+ ionophore ionomycin (1 µM) decreased forward scatter, an effect virtually abolished in the presence of mitotane (15 µg/ml). Conclusions: Mitotane increases [Ca2+]i with subsequent phosphatidylserine translocation. By the same token mitotane inhibits Ca2+ induced cell shrinkage.

The Uremic Toxin Acrolein Promotes Suicidal Erythrocyte Death

Background: Anemia is a major complication of end stage renal disease. The anemia is mainly the result of impaired formation of erythrocytes due to lack of erythropoietin and iron deficiency. Compelling evidence, however, points to the contribution of accelerated erythrocyte death, which decreases the life span of circulating erythrocytes. Erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine-exposure at the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i). Erythrocytes could be sensitized to cytosolic Ca2+ by ceramide. In end stage renal disease, eryptosis may possibly be stimulated by uremic toxins. The present study explored, whether the uremic toxin acrolein could trigger eryptosis. Methods: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fluorescence, and ceramide from fluorescent antibodies. Results: A 48 h exposure to acrolein (30 － 50 µM) did not significantly modify [Ca2+]i but significantly decreased forward scatter and increased annexin-V-binding. Acrolein further triggered slight, but significant hemolysis and increased ceramide formation in erythrocytes. Acrolein (50 µM) induced annexin-V-binding was significantly blunted in the nominal absence of extracellular Ca2+. Acrolein augmented the annexin-V-binding following treatment with Ca2+ ionophore ionomycin (1 µM). Conclusion: Acrolein stimulates suicidal erythrocyte death or eryptosis, an effect at least in part due to stimulation of ceramide formation with subsequent sensitisation of the erythrocytes to cytosolic Ca2+.

Sensitization of erythrocytes to suicidal erythrocyte death following water deprivation.

Background/Aims: Klotho deficiency results in excessive formation of 1,25(OH)2D3, accelerated ageing and early death. Moreover, klotho deficiency enhances eryptosis, the suicidal erythrocyte death characterized by phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i), glucose depletion, hyperosmotic shock and oxidative stress. Klotho expression is decreased and 1,25(OH)2D3-formation enhanced by dehydration. The present study thus explored whether dehydration influences eryptosis. Methods: Blood was drawn from hydrated or 36h dehydrated mice. Plasma osmolarity was determined by vapour pressure method, plasma 1,25(OH)2D3 and aldosterone concentrations using ELISA, and plasma Ca2+-concentration utilizing photometry. Erythrocytes were exposed to Ca2+-ionophore ionomycin (1 µM, 30 min), energy depletion (12 h glucose removal), hyperosmotic shock (500 mM sucrose added, 2 h) and oxidative stress (100 µM tert-butyl-hydroperoxide, 30 min) and phosphatidylserine exposure at the erythrocyte surface estimated from annexin V binding. Results: Dehydration increased plasma osmolarity and plasma 1,25(OH)2D3 and aldosterone concentrations. Dehydration did not significantly modify phosphatidylserine-exposure of freshly drawn erythrocytes but significantly enhanced the increase of phosphatidylserine-exposure under control conditions and following treatment with ionomycin, glucose-deprivation, hyperosmolarity or tert-butyl-hydroperoxide. Conclusions: Dehydration sensitizes the erythrocytes to spontaneous eryptosis and to the triggering of eryptosis by excessive Ca2+-entry, energy depletion, hyperosmotic shock and oxidative stress.

Stimulation of Suicidal Erythrocyte Death by Increased Extracellular Phosphate Concentrations

Background/Aim: Anemia in renal insufficiency results in part from impaired erythrocyte formation due to erythropoietin and iron deficiency. Beyond that, renal insufficiency enhances eryptosis, the suicidal erythrocyte death characterized by phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be stimulated by increase of cytosolic Ca2+-activity ([Ca2+]i). Several uremic toxins have previously been shown to stimulate eryptosis. Renal insufficiency is further paralleled by increase of plasma phosphate concentration. The present study thus explored the effect of phosphate on erythrocyte death. Methods: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, and [Ca2+]i from Fluo3-fluorescence. Results: Following a 48 hours incubation, the percentage of phosphatidylserine exposing erythrocytes markedly increased as a function of extracellular phosphate concentration (from 0-5 mM). The exposure to 2 mM or 5 mM phosphate was followed by slight but significant hemolysis. [Ca2+]i did not change significantly up to 2 mM phosphate but significantly decreased at 5 mM phosphate. The effect of 2 mM phosphate on phosphatidylserine exposure was significantly augmented by increase of extracellular Ca2+ to 1.7 mM, and significantly blunted by nominal absence of extracellular Ca2+, by additional presence of pyrophosphate as well as by presence of p38 inhibitor SB203580. Conclusion: Increasing phosphate concentration stimulates erythrocyte membrane scrambling, an effect depending on extracellular but not intracellular Ca2+ concentration. It is hypothesized that suicidal erythrocyte death is triggered by complexed CaHPO4.

Tannic Acid Induced Suicidal Erythrocyte Death

Background: The polyphenol tannic acid with antioxidant and antimicrobial potency may trigger suicidal death of nucleated cells or apoptosis and thus may counteract tumor growth. In analogy to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, a suicidal death characterized by cell shrinkage and cell membrane scrambling with appearance of phosphatidylserine at the erythrocyte surface. A major trigger of eryptosis is increase of cytosolic Ca2+-activity ([Ca2+]i). Erythrocytes could be sensitized to the eryptotic effect of cytosolic Ca2+ by ceramide. Methods: Cell volume has been estimated from forward scatter, phosphatidylserine abundance at the erythrocyte surface from annexin V binding, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fuorescence and ceramide utilizing fluorescent antibodies. Results: A 48 h treatment with tannic acid was followed by significant decrease of forward scatter (≥ 1 µg/ml) and significant increase of annexin-V-binding (≥ 10 µg/ml). Tannic acid did not significantly modify [Ca2+]i (up to 50 µM) but significantly increased ceramide formation (50 µM). The annexin-V-binding following tannic acid treatment (50 µM) was significantly blunted in the nominal absence of extracellular Ca2+. Conclusions: Tannic acid stimulates eryptosis, an effect at least partially due to ceramide formation with subsequent sensitization of erythrocytes to cytosolic Ca2+.

Stimulation of Suicidal Erythrocyte Death by Artesunate

Background: The artemisinin derivative artesunate is effective in the treatment of severe malaria and is considered for the treatment of malignancy. Artesunate triggers tumor cell apoptosis, an effect at least in part mediated by mitochondria. Even though lacking mitochondria, erythrocytes may similarly enter suicidal death or eryptosis, which is characterized by cell shrinkage and breakdown of the phospholipid asymmetry of the cell membrane with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i), ceramide formation, and oxidative stress. The present study explored whether artesunate stimulates eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ceramide abundance from binding of specific antibodies, and oxidative stress from 2′,7′-dichlorodihydrofluorescein-diacetate fluorescence. Results: A 48 h exposure of human erythrocytes to artesunate significantly increased the percentage of annexin-V-binding cells (≥ 9 µg/ml) without significantly influencing forward scatter. Artesunate significantly increased [Ca2+]i. The stimulation of annexin-V-binding by artesunate (15 µg/ml) was significantly blunted but not abolished by removal of extracellular Ca2+. Artesunate increased the ceramide abundance at the cell surface and the 2′,7′-dichlorodihydrofluorescein-diacetate fluorescence. Conclusions: Artesunate stimulates phosphatidylserine translocation at the erythrocyte cell membrane, an effect at least partially due to increase of [Ca2+]i, stimulation of ceramide formation and generation of oxidative stress.

Effect of nitazoxanide on erythrocytes.

Nitazoxanide, a drug effective against a variety of pathogens, triggers apoptosis and is thus considered to be employed against malignancy. Similar to nucleated cells, erythrocytes may undergo an apoptosis‐like suicidal cell death or eryptosis. Hallmarks of eryptosis include cell shrinkage and phospholipid scrambling of the cell membrane with translocation of phosphatidylserine to the erythrocyte surface. Stimulators of eryptosis include increase in cytosolic Ca2+‐activity ([Ca2+]i). The Ca2+‐sensitivity of eryptosis is increased by ceramide. This study explored whether nitazoxanide triggers eryptosis. [Ca2+]i was estimated from Fluo3‐fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin‐V‐binding, ceramide abundance utilizing fluorescent antibodies and haemolysis from haemoglobin release. A 48‐hr exposure to nitazoxanide (1–50 μg/ml) did not significantly modify [Ca2+]i but significantly increased ceramide formation, decreased forward scatter (≥10 μg/ml), increased the percentage of annexin‐V‐binding erythrocytes (≥10 μg/ml) and, at higher concentrations (≥20 μg/ml), stimulated haemolysis. The stimulation of annexin‐V‐binding was significantly blunted in the absence of calcium. Nitazoxanide thus stimulates eryptosis, an effect in part due to ceramide formation.

Stimulation of Suicidal Erythrocyte Death by Ribavirin

Ribavirin is widely used in the treatment for viral disease such as chronic viral hepatitis. Side effects limiting the use of the drug include haemolytic anaemia. If challenged by stimulators of haemolysis, erythrocytes may enter suicidal death or eryptosis, thus preventing the release of haemoglobin into circulating blood. Eryptosis is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase in cytosolic Ca2+ activity ([Ca2+]i). This study explored whether ribavirin modifies [Ca2+]i and elicits eryptosis. Cell volume has been estimated from forward scatter, phosphatidylserine abundance at the erythrocyte surface from annexin V binding, haemolysis from haemoglobin release and [Ca2+]i from Fluo‐3 fluorescence. A 48‐hr exposure to ribavirin (≥8 μg/ml) was followed by a significant increase in [Ca2+]i, a significant decrease in forward scatter and a significant increase in annexin V binding. The annexin V binding after ribavirin treatment was significantly blunted but not abolished in the nominal absence of extracellular Ca2+. In conclusion, ribaverin stimulates eryptosis, an effect at least in part due to entry of extracellular Ca2+.