Development of Multipotential Haemopoietic Stem Cells to Neutrophils is Associated with Increased Expression of Receptors for Granulocyte Macrophage Colony-Stimulating Factor: Altered Biological Responses to GM-CSF during Development

Abstract

Interleukin-3 (IL-3) dependent multipotent haemopoietic stem cells FDCP-Mix A4 (A4) were induced to differentiate and develop into mature neutrophils in response to Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) plus granulocyte CSF (G-CSF). This resulted in an increase in cell number over seven days of culture, following which the cells lost the ability to undergo further proliferation. The effect of GM-CSF on these cells has been assessed at various stages of development. Clonogenic cells, able to respond to GM-CSF, were generated only at days 3, 4 post-induction. From day 5 onwards, mature post-mitotic neutrophils are produced and clonogenic cells are lost. Loss of proliferative potential, in response to GM-CSF, was confirmed using [3H]-thymidine incorporation. Receptors for GM-CSF, were also measured during development using [125I]-GM-CSF binding assays. Although the dissociation constant for GM-CSF binding sites did not vary considerably, the number of such sites increased dramatically from about 20 (day 0, when the cells have a primitive morphology) to about 1000 by day 6 (when the cells are predominantly mature neutrophils). GM-CSF-stimulated Na+/H+ antiport activation was also determined. Although few GM-CSF receptors are expressed at day 0, there is a significant response (63% of maximal) to GM-CSF in terms of intracellular alkalinisation: this response increased markedly until, by day 4 (700 GM-CSF binding sites/cell), there is a maximal activation of the antiport by GM-CSF. By day 7 (greater than 900 GM-CSF binding sites/cell), however, there is significant reduction in activation of the Na+/H+ antiport by GM-CSF. Nonetheless, increased viability of these mature cells is still seen in response to GM-CSF. These results suggest that not only does expression of GM-CSF receptors alter during development of multipotential cells to mature neutrophils, but that these receptors are coupled to different intracellular effector mechanisms as the cells progressively mature.