C/EBP transcription factors are crucial for the regulation of granulopoiesis in vitro and in vivo. C/EBPa is considered to be the master regulator of “steady state” granulopoiesis via upregulation of several myeloid genes (e.g. ELA2, CSFR3, etc.). The absence of C/ EBPa results in a complete loss of neutrophils. We were able to show that in patients with severe congenital neutropenia (CN) harbouring HAX-1 or ELA2 mutations C/EBPa is severely abrogated secondary to defective expression of LEF-1 (

Skokowa J, et al. Nat Med. 12, 1191–7 (2006)
). Therefore, we were interested, whether other transcription factors are capable of substituting C/EBPa, since these patients respond to G-CSF with slight increase in neutrophils from less than 200/ul to above 1500/ul depending on the dose of G-CSF. C/EBPβ has recently been shown to be required for cytokine induced “emergency” granulopoiesis (
Hirai H, et al. Nat Immunol. 7, 732-9 (2006)
). Therefore, we investigated the expression pattern of C/EBPβ during G-CSF treatment of CN patients. Indeed, C/EBPβ mRNA was upregulated 2.8-fold in CD33+ myeloid cells from CN patients by G-CSF treatment, as compared to healthy individuals. It was associated with upregulation of G-CSFR mRNA and –protein expression as well as ligand binding to G-CSFR in myeloid cells, and elevated levels of biologically active G-CSF in serum from CN patients. To confirm C/EBPβ-dependent activation of G-CSFR and G-CSF gene expression, we performed reporter gene assays in CD34+ bone marrow hematopoietic progenitor cells from two CN patients co-transfected with C/EBPβ and reporter constructs containing upstream regulatory regions of G-CSF or G-CSFR genes, −1470bp and −670bp, respectively. Indeed, C/EBPβ activated G-CSFR promoter 3.2-fold and G-CSF promoter 5.4-fold. These data demonstrate that in CN patients, G-CSF induces C/EBPa independent granulopoiesis and that C/EBPβ is required for the response to G-CSF treatment in these patients. C/EBPβ leads in response to G-CSF to induction of differentiation of neutrophil precursors to mature neutrophils in vivo. Our hypothesis is that in CN the steady state granulopoiesis is abrogated whereas the emergency granulopoiesis still leads to sufficient numbers of neutrophils.

Topics:
congenital neutropenia, granulocyte colony-stimulating factor, granulocyte production, recombinant granulocyte colony stimulating factor, receptors, colony-stimulating factor, rna, messenger, transcription factor, cd33 antigen, cd34 antigens, cytokine

Disclosures: No relevant conflicts of interest to declare.

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2008, The American Society of Hematology
2008
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