Age sensitivity of NFkappaB abundance and programmed cell death in erythrocytes induced by NFkappaB inhibitors

Abstract

BACKGROUND/AIMS: Erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte outer membrane. Susceptibility to eryptosis is enhanced in aged erythrocytes and stimulated by NFkappaB-inhibitors Bay 11-7082 and parthenolide. Here we explored whether expression of NFkappaB and susceptibility to inhibitor-induced eryptosis is sensitive to erythrocyte age. METHODS: Human erythrocytes were separated into five fractions, based on age-associated characteristics cell density and volume. NFkappaB compared to ss-actin protein abundance was estimated by Western blotting and cell volume from forward scatter. Phosphatidylserine exposure was identified using annexin-V binding. RESULTS: NFkappaB was most abundant in young erythrocytes but virtually absent in aged erythrocytes. A 24h or 48h exposure to Ringer resulted in spontaneous decrease of forward scatter and increase of annexin V binding, effects more pronounced in aged than in young erythrocytes. Both, Bay 11-7082 (20 microM) and parthenolide (100 microM) triggered eryptosis, effects again most pronounced in aged erythrocytes. CONCLUSION: NFkappaB protein abundance is lowest and spontaneous eryptosis as well as susceptibility to Bay 11-7082 and parthenolide highest in aged erythrocytes. Thus, inhibition of NFkappaB signalling alone is not responsible for the stimulation of eryptosis by parthenolide or Bay 11-7082.