Inhibition by Teriflunomide of Erythrocyte Cell Membrane Scrambling Following Energy Depletion, Oxidative Stress and Ionomycin

Abstract

Background/Aims: Teriflunomide, an inhibitor of pyrimidine synthesis and thus proliferation of activated T and B lymphocytes, is successfully used for treatment of inflammatory disease. Teriflunomide has further been shown to trigger apoptosis of tumor cells and has thus been considered for the treatment of malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and phospholipid scrambling of the cell membrane with translocation of phosphatidylserine to the erythrocyte surface. Triggers of cell membrane scrambling include energy depletion, oxidative stress and increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored whether teriflunomide modifies eryptosis. Methods: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, and [Ca2+]i from Fluo3 fluorescence. Results: Oxidative stress (60 min exposure to 0.3 mM tert-butylhydroperoxide), energy depletion (removal of glucose for 48 hours), and exposure to the Ca2+ ionophore ionomycin (1 µM, 60 min) all increased annexin-V-binding, decreased forward scatter and enhanced Fluo3 fluorescence. Teriflunomide (5 µg/ml) did not significantly influence Fluo3 fluorescence, forward scatter and annexin-V-binding under control conditions but significantly blunted the increase of annexin-V-binding following oxidative stress, energy depletion and ionomycin exposure. Teriflunomide further blunted the increase of Fluo3 fluorescence following energy depletion, but did not significantly interfere with increase of Fluo3 fluorescence following oxidative stress and ionomycin exposure. Conclusion: Teriflunomide is a novel inhibitor of suicidal erythrocyte death.