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.

A New Isoform of Interleukin-3 Receptor α with Novel Differentiation Activity and High Affinity Binding Mode

“…5A) in agreement with previous findings (19). Evaluation of surface markers by FACS showed significant increases in expression of Mac-1 and Gr-1 but little increase in Mar-1 (Fc⑀RI␣) when compared with cells treated with low levels of mIL-3 (Fig.…”

“…It remains controversial whether cytokine receptors in general can play an instructive role in lineage specification in hematopoiesis, and steady-state blood cell formation appears to largely involve stochastic processes (18). However, there is evidence that the GM-CSF receptor (GM-CSFR) can play an instructive role in the differentiation of multipotential cells (19).…”

Multilineage gene expression precedes commitment in the hemopoietic system.

“…Expression of cytokine receptors is also low; this is evident from Northern blot analyses and also from experiments attempting to detect receptor proteins using labeled ligands or antireceptor antibodies (Nicola and Metcalf 1988, McKinstry et al 1997). Using radiolabeled growth factors in binding assays on FDCP-mix cells it is possible to detect lov\^-level expression of receptors for GM-CSF and G-CSF, but not for EPO (Heyworth et al 1991(Heyworth et al , 1995A.M. OTarell and CM.…”

LRIG1 Extracellular Domain: Structure and Function Analysis

“…In our initial experiments, we could not express the native LRIG1-LRR domain (41-494) [23] in our baculovirus system; however, by mutating four residues at the N-terminus of the human LRR domain to the equivalent hydrophilic residues present in the zebrafish LRIG1 homologue (UniProt E7EZ01), to produce the analogue Pro42Ser43Arg44Ser69L-RIG1-LRR, we obtained excellent expression in Hi5 insect cells (i.e., N 3 mg/L). Although none of these residues are conserved in either LRIG2 † or LRIG3 ‡ , the crystal structure of the Pro42Ser43Arg44Ser69-L-RIG1-LRR domain (coding sequences 41-494) could be solved by molecular replacement using the atomic coordinates of decorin [24] as a search model.…”