Fig. 3.
Fig. 3. Measurement of apoptosis by annexin-V detection in erythroblasts after their overnight incubation with autologous myeloma cells. / (A) The experiment included 8 cocultures from group A patients and 7 from group B patients. A great increase in the annexin-V+/GpA+ cell population was recorded within the cocultures from the group A patients. This was particularly evident in cocultures incubating erythroblasts with untreated myeloma cells, whereas the selective disablement of either Fas-L or TRAIL in parallel cultures whose myeloma cells were inhibited by the soluble Fas-Fc or the anti-TRAIL antibody, respectively, revealed the effect separately induced by each apoptogenic receptor of myeloma cells. These effects were almost equivalent to those obtained in control erythroblasts directly stimulated by either the CH11 anti-Fas mAb or the recombinant TRAIL. On the contrary, the effect induced in the cocultures from group B patients by the autologous plasma cells was modest and lower than the respective values observed in group A patients (P < .02 in all instances). Values are mean ± SD of double-fluorescent (annexin-V+/GpA+) cells. (B) Flow cytometry analysis of cocultured cells from a patient with severe anemia. Incubation of erythroblasts (red) with myeloma cells (green) induced a dramatic increase of annexin-V+cells within the GpA+ population. This effect, presumably induced by the concurrent expression of both Fas-L and TRAIL by myeloma cells (top), was then separately measured by inhibiting either TRAIL (middle) or Fas-L (bottom) and was still higher than that recorded in control erythroblasts treated with the anti-Fas mAb or TRAIL (right). (C) Erythroblasts from a patient with preserved erythropoiesis were only moderately driven to apoptosis by their autologous plasma cells and the alternate inhibitions abolished the feeble cytotoxic effect in both cases. (D) Morphologic pattern in the coculture from patient A-1. The malignant myeloma cells among the erythroblasts whose nuclei are stained in blue by DAPI show a high content of TRAIL (red) detected by the PE-conjugated antibody in both cytoplasm and membrane and are adjacent to apoptotic erythroblasts characterized by nuclear fragmentation. Original magnification, × 50 (top), × 100 (bottom).

Measurement of apoptosis by annexin-V detection in erythroblasts after their overnight incubation with autologous myeloma cells.

(A) The experiment included 8 cocultures from group A patients and 7 from group B patients. A great increase in the annexin-V+/GpA+ cell population was recorded within the cocultures from the group A patients. This was particularly evident in cocultures incubating erythroblasts with untreated myeloma cells, whereas the selective disablement of either Fas-L or TRAIL in parallel cultures whose myeloma cells were inhibited by the soluble Fas-Fc or the anti-TRAIL antibody, respectively, revealed the effect separately induced by each apoptogenic receptor of myeloma cells. These effects were almost equivalent to those obtained in control erythroblasts directly stimulated by either the CH11 anti-Fas mAb or the recombinant TRAIL. On the contrary, the effect induced in the cocultures from group B patients by the autologous plasma cells was modest and lower than the respective values observed in group A patients (P < .02 in all instances). Values are mean ± SD of double-fluorescent (annexin-V+/GpA+) cells. (B) Flow cytometry analysis of cocultured cells from a patient with severe anemia. Incubation of erythroblasts (red) with myeloma cells (green) induced a dramatic increase of annexin-V+cells within the GpA+ population. This effect, presumably induced by the concurrent expression of both Fas-L and TRAIL by myeloma cells (top), was then separately measured by inhibiting either TRAIL (middle) or Fas-L (bottom) and was still higher than that recorded in control erythroblasts treated with the anti-Fas mAb or TRAIL (right). (C) Erythroblasts from a patient with preserved erythropoiesis were only moderately driven to apoptosis by their autologous plasma cells and the alternate inhibitions abolished the feeble cytotoxic effect in both cases. (D) Morphologic pattern in the coculture from patient A-1. The malignant myeloma cells among the erythroblasts whose nuclei are stained in blue by DAPI show a high content of TRAIL (red) detected by the PE-conjugated antibody in both cytoplasm and membrane and are adjacent to apoptotic erythroblasts characterized by nuclear fragmentation. Original magnification, × 50 (top), × 100 (bottom).

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