Clofazimine Induced Suicidal Death of Human Erythrocytes

Abstract

Background/Aims: The antimycobacterial riminophenazine clofazimine has previously been shown to up-regulate cellular phospholipase A₂ and to induce apoptosis. In erythrocytes phospholipase A₂ stimulates eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Phospholipase A₂ is in part effective by fostering formation of prostaglandin E₂, which triggers Ca²⁺ entry. Stimulators of Ca²⁺ entry and eryptosis further include oxidative stress and energy depletion. The present study tested, whether and how clofazimine induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, cytosolic Ca²⁺ activity ([Ca²⁺]_i) from Fluo3-fluorescence, reactive oxygen species (ROS) from 2′, 7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, and cytosolic ATP level utilizing a luciferin-luciferase assay kit. Results: A 24-48 hours exposure of human erythrocytes to clofazimine (≥1.5 µg/ml) significantly increased the percentage of annexin-V-binding cells without appreciably modifying forward scatter. Clofazimine significantly increased [Ca²⁺]_i, significantly decreased cytosolic ATP, but did not significantly modify ROS. The effect of clofazimine on annexin-V-binding was significantly blunted, but not fully abolished by removal of extracellular Ca²⁺, and by phospholipase A₂ inhibitor quinacrine (25 µM). Clofazimine further augmented the effect of Ca²⁺ ionophore ionomycin (0.1 µM) on eryptosis. The clofazimine induced annexin-V-binding was, however, completely abrogated by combined Ca²⁺ removal and addition of quinacrine. Conclusion: Clofazimine stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect in part dependent on entry of extracellular Ca²⁺, paralleled by cellular energy depletion and sensitive to phospholipase A₂ inhibitor quinacrine.