Stimulation of suicidal death of erythrocytes by rifampicin

The antibiotic rifampicin is widely used in the treatment of tuberculosis. Side effects of rifampicin include hemolytic anemia. Loss of circulating erythrocytes resembling hemolytic anemia could result from stimulation of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure at the cell surface. Stimulators of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i) and formation of ceramide. The present study explored, whether and, if so, how rifampicin triggers eryptosis. To this end, [Ca2+]i was estimated from Fluo3 fluorescence, cell volume from forward scatter in flow cytometry, PS exposure from annexin binding, ceramide formation from binding of fluorescent antibodies and hemolysis from hemoglobin release. As a result, a 48 h exposure to rifampicin (≥24 μg/ml) significantly increased Fluo3 fluorescence, ceramide abundance and annexin binding, and significantly decreased forward scatter. Rifampicin triggered slight, but significant hemolysis. Removal of extracellular Ca2+ significantly blunted, but did not fully abolish rifampicin induced annexin binding. In conclusion, exposure of human erythrocytes to rifampicin is followed by suicidal erythrocyte death or eryptosis, an effect at least partially due to increase of cytosolic Ca2+ concentration and stimulation of ceramide formation.