Peschle C, Gabbianelli M, Testa U, Pelosi E, Barberi T, Fossati C, Valtieri M, Leone L
Department of Hematology-Oncology, Istituto Superiore di Sanità, Rome, Italy.
Blood. 1993 Jan 15;81(2):328-36.
We have analyzed the reactivation of fetal hemoglobin (HbF) synthesis under rigorous in vitro conditions, ie, in mature erythroblasts generated by erythroid burst-forming units (BFU-E) stringently purified from normal adult peripheral blood and grown in fetal calf serum(FCS)-free semisolid or liquid phase culture. In clonogenetic dishes, graded amounts of c-kit ligand (KL) were added together with saturating levels of erythropoietin (Ep) and variable amounts of interleukin-3 and granulocyte-macrophage colony stimulating factor (IL-3/GM-CSF), ie, high or low level, or no IL-3/GM-CSF addition. In all conditions, KL induced a sharp, dose-dependent increase in the percentage of F cells and HbF content from nearly normal levels (< 10% and < 2.5%, respectively, at 0.1 and 1 ng/mL) up to 40% to 50% and 10% to 15% at 100 to 200 ng/mL. This increase was not associated with significant differences of burst number or stage of maturation at the time of analysis (as evaluated on the basis of percent mature erythroblasts and Hb content per cell). However, the KL-induced reactivation of HbF synthesis was strictly and directly correlated with a sharp increase of colony size, ie, cell number per burst. Addition of large amounts of IL-3 and GM-CSF (10 to 100 U and 1 to 10 ng/mL, respectively) significantly potentiated the KL-induced reactivation of HbF, as compared with low levels (0.1 U and 0.01 to 0.1 ng) or no addition of these growth factors: this increase was highly significant at low KL doses (ie, 1 to 10 ng/mL). Single-burst analysis showed that the KL-induced HbF reactivation occurs homogeneously in the erythroid colonies within each of these culture conditions. We have analyzed the effect of KL in liquid phase BFU-E culture treated with the IL-3/GM-CSF/Ep combination at sequential times until terminal erythroid maturation: KL causes a sharp increase in the percentage of F cells and HbF content in all stages of maturation, whereas the IL-3/GM-CSF/Ep combination alone has a markedly lower effect. These results suggest that KL plays a key role in the reactivation of HbF synthesis in adult life, whereas IL-3/GM-CSF potentiate this effect at low KL levels. The KL-induced HbF reactivation is seemingly related to an enhanced proliferation of erythroid progenitors in the erythropoietic differentiation pathway.
我们在严格的体外条件下分析了胎儿血红蛋白（HbF）合成的重新激活情况，即在由从正常成人外周血中严格纯化的红系爆式集落形成单位（BFU-E）产生的成熟成红细胞中进行分析，这些细胞在无胎牛血清（FCS）的半固体或液相培养基中培养。在克隆培养皿中，加入不同剂量的c-kit配体（KL），同时加入饱和水平的促红细胞生成素（Ep）以及不同量的白细胞介素-3和粒细胞-巨噬细胞集落刺激因子（IL-3/GM-CSF），即高剂量、低剂量或不添加IL-3/GM-CSF。在所有条件下，KL均诱导F细胞百分比和HbF含量呈急剧的剂量依赖性增加，从接近正常水平（分别在0.1和1 ng/mL时<10%和<2.5%）增加到100至200 ng/mL时的40%至50%和10%至15%。这种增加与分析时爆式集落数量或成熟阶段的显著差异无关（根据成熟成红细胞百分比和每个细胞的Hb含量评估）。然而，KL诱导的HbF合成重新激活与集落大小的急剧增加严格且直接相关，即每个爆式集落中的细胞数量。与低水平（0.1 U和0.01至0.1 ng）或不添加这些生长因子相比，添加大量的IL-3和GM-CSF（分别为10至100 U和1至10 ng/mL）显著增强了KL诱导的HbF重新激活：在低KL剂量（即1至10 ng/mL）时这种增加非常显著。单爆式集落分析表明，在每种培养条件下的红系集落中，KL诱导的HbF重新激活均均匀发生。我们分析了KL在依次用IL-3/GM-CSF/Ep组合处理直至终末红系成熟的液相BFU-E培养中的作用：KL在所有成熟阶段均导致F细胞百分比和HbF含量急剧增加，而单独的IL-3/GM-CSF/Ep组合的作用明显较低。这些结果表明，KL在成体中HbF合成的重新激活中起关键作用，而IL-3/GM-CSF在低KL水平时增强这种作用。KL诱导的HbF重新激活似乎与红系祖细胞在红系分化途径中的增殖增强有关。
