The expression of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TRAIL receptors was investigated in resting and cytokine-activated purified primary human natural killer (NK) and CD8 ؉ T cells. Resting NK and CD8 ؉ T cells expressed the mRNA for all TRAIL receptors, but TRAIL-R4 was the only receptor clearly detectable on the surface of both cell types. NK cells were activated by interleukin 2 (IL-2) or IL-15, whereas CD8 ؉ T cells were activated by phytohemagglutinin (PHA) ؉ IL-2 followed by IL-2 alone for up to 10 days. On activation, both cell types rapidly expressed TRAIL-R2 and TRAIL-R3, whose expression peaked at day 10 of culture. TRAIL-R1, however, was never expressed at any time point examined, whereas the expression of TRAIL-R4, which showed a progressive increase in CD8

Apoptosis plays a central role in the regulation of the size of the hematopoietic stem cell pool as well as in the processes of cell differentiation along the various hematopoietic lineages. TRAIL is a member of the TNF family of cytokines with a known apoptogenic role against a variety of malignant cells and an emerging role in the modulation of normal hematopoiesis. Here we worked on the hypothesis that PKC⑀ could act as a switch of the cellular response to TRAIL during erythropoiesis.We demonstrate that EPO-induced erythroid CD34 cells are insensitive to the apoptogenic effect of TRAIL at day 0 due to the lack of specific receptor expression. From day 3 onward, erythroid cells express surface death receptors and become sensitive to TRAIL up to day 7/8 when, notwithstanding death-receptor expression, the EPO-driven up-regulation of PKC⑀ intracellular levels renders differentiating erythroid cells resistant to TRAIL likely via Bcl-2 up-regulation. Our conclusion is that in human CD34 cells, EPO promotes a series of events that, being finely regulated in their kinetics, restricts the sensitivity of these cells to TRAIL to a specific period of time, which therefore represents the "TRAIL window" for the negative regulation of erythroid-cell numbers. ( IntroductionIt is now well established that apoptosis plays a central role in the regulation of the size of the hematopoietic stem cell pool 1 as well as in the processes of cell differentiation along the various hematopoietic lineages. Fetal erythropoiesis can be negatively regulated by Fas ligand, 2,3 although bone marrow hematopoiesis does not appear to be affected by Fas deficiency. 3 However, Fas expression on hematopoietic stem cells or hematopoietic progenitors can be induced by certain cytokines such as IFN␥ or tumor necrosis factor ␣ (TNF␣), [3][4][5] reducing hematopoietic repopulating potential. Relatively little is known on the effects of other members of the TNF family on hematopoietic progenitors. We have demonstrated that TNF-related apoptosis-inducing ligand (TRAIL) acts as a negative regulator of adult erythropoiesis, selectively reducing the number of erythroblasts in liquid culture, as well as reducing the number and size of erythroid colonies in semisolid assays. 6 Recently, Secchiero et al demonstrated that TRAIL inhibited the generation of mature erythroblasts in liquid culture through the activation of an ERK 1/2-mediated signaling pathway. 7 TRAIL is a member of the TNF family of cytokines, which are structurally related proteins playing important roles in regulating cell death, immune response, and inflammation. 8,9 The unique feature of TRAIL, compared with other members of the TNF family, is its ability to induce apoptosis in a variety of malignant cells both in vitro and in vivo, displaying minimal toxicity on normal cells and tissues. 10,11 TRAIL interacts with 4 high-affinity transmembrane receptors belonging to the apoptosis-inducing TNF-receptor (R) family. TRAIL-R1 (DR4) and TRAIL-R2 (DR5) transduce apoptotic signals on binding of TR...