Ionomycin-induced Ca Cytosolic Increase Is Not Inducing Massive Apoptosis of Ba/f3 Cells

IONOMYCIN-INDUCED Ca CYTOSOLIC INCREASE IS NOT INDUCING MASSIVE APOPTOSIS OF Ba/F3 CELLS (Abstract): It was shown that a novel immunotherapy using ex vivo activated splenocytes is capable of promoting survival and hematopoietic recovery after syngeneic and semiallogeneic bone marrow transplantation in BALB/c mice subjected to a lethal dose of total body irradiation. We tested the hypothesis that the low number of B cells obtained in the above conditions might be due to the effects of calcium overload, induced by calcium ionophore ionomycin, through the apoptosis of the early B cells. We used flow cytometry, together with calcein and propidium iodide as indicators of mitochondrial permeability transition pore (MPTP) activation and apoptosis. From the normal Ba/F3 cells 69.83±3.90% were associating high calcein fluorescence of mitochondria in the presence of 80 M cobaltous chloride. In some experiments Ba/F3 cells were treated with 2 M ionomycin and 2.5 mM Ca in culture medium for 24 hours. In this case, 85.68±2.66% of the Ba/F3 cells were associating high calcein fluorescence of mitochondria, thus a large proportion of them remaining living cells, being unaffected by the calcium overloading induced by ionomycin. Moreover, only about 8% of cells are necrotic as shown by propidium iodide. Thus, calcium ionophore ionomycin induced higher mitochondrial calcein fluorescence than in normal cells. For comparison we used as positive control the effects of staurosporine 10 M, a well known inducer of apoptosis. After 24 h of treatment only 5.98±1.32% of cells were having mitochondria loaded with calcein (live cells), more than 80% being necrotic. To demonstrate the involvement of MPTP activation we administered the inhibitor cyclosporine A (CsA), also administered in parallel in some experiments (1 M) for 24 hours. CsA dramatically reduced the staurosporine apoptotic effects on Ba/F3 cells, having no significant influence on ionomycin treatment. Thus, we can consider that the calcium ionophore ionomycin is not inducing MPTP opening and apoptosis of Ba/F3 cells, although is increasing [Ca]c and [Ca]mt. This conclusion is further supported by the propidium iodide staining, since a very low proportion of Ba/F3 cells treated with ionomycin for 24 h were accumulating specific red fluorescence. These results are also in contrast to those obtained by staurosporine treatment, which induced accumulation of specific red fluorescence characteristic to propidium iodide (necrotic and apoptotic) in more than 80% of Ba/F3 cells. We can conclude that the calcium overload induced by ionophore ionomycin is not responsible for the low numbers of B cells obtained through selective selection of splenocytes used for hematopoietic recovery after syngeneic and semiallogeneic bone marrow transplantation in BALB/c mice.