Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein alpha-deficient mice

Abstract

Transcription factors are master regulatory switches of differentiation, including the development of specific hematopoietic lineages from stem cells. Here we show that mice with targeted disruption of the CCAAT enhancer binding protein alpha gene (C/EBP alpha) demonstrate a selective block in differentiation of neutrophils. Mature neutrophils and eosinophils are not observed in the blood or fetal liver of mutant animals, while other hematopoietic lineages, including monocytes, are not affected. Instead, most of the white cells in the peripheral blood of mutant mice had the appearance of myeloid blasts. We also observed a selective loss of expression of a critical gene target of CCAAT enhancer binding protein alpha, the granulocyte colony-stimulating factor receptor. As a result, multipotential myeloid progenitors from the mutant fetal liver are unable to respond to granulocyte colony-stimulating factor signaling, although they are capable of forming granulocyte-macrophage and macrophage colonies in methylcellulose in response to other growth factors. Finally, we demonstrate that the lack of granulocyte development results from a defect intrinsic to the hematopoietic system; transplanted fetal liver from mutant mice can reconstitute lymphoid but not neutrophilic cells in irradiated recipients. These studies suggest a model by which transcription factors can direct the differentiation of multipotential precursors through activation of expression of a specific growth factor receptor, allowing proliferation and differentiation in response to a specific extracellular signal. In addition, the c/ebp alpha -/- mice may be useful in understanding the mechanisms involved in acute myelogenous leukemia, in which a block in differentiation of myeloid precursors is a key feature of the disease.

Figure 1

Lack of mature neutrophils in c/ebpα^−/− mice. Blood was collected as described in Table 1. After lysing red blood cells, white blood cells from either c/ebpα^+/− (A) or c/ebpα^−/− (B) newborn mice were cytocentrifuged onto slides and were stained with Wright–Giemsa for analysis of cell morphology. Newborn liver touch preparations from c/ebpα^+/− (C) or c/ebpα^−/− (D) and G-CSF-administered c/ebpα^+/− (E) or c/ebpα^−/− (F) mice were stained with Wright–Giemsa. G-CSF (250 μg per kg of body weight) was injected subcutaneously every 12 hr to pregnant mice from day 17 of pregnancy until birth. Mature neutrophils are indicated with an n. Monocytes are indicated with an m.

Figure 2

Flow cytometry analysis with cell surface markers. Cells obtained from livers of c/ebpα^+/− (A) or c/ebpα^−/− (B) newborns and from wild-type (I) or c/ebpα^−/− (J) day 14 embryos were stained with antibodies to the mature monocyte/granulocyte surface marker Mac-1 (37) and to the mature granulocyte surface marker Gr-1 (38). Cells obtained from spleens of c/ebpα^+/− (C) or c/ebpα^−/− (D) newborns were stained with antibodies to the B lymphocyte marker B220. Cells obtained from thymus of c/ebpα^+/− (E) or c/ebpα^−/− (F) newborns were stained with T lymphocyte surface markers CD4 and CD8. Cells obtained from livers of c/ebpα^+/+ (G) or c/ebpα^−/− (H) day 16 embryos were stained with antibodies to the erythroid surface marker TER-119.

Figure 3

Northern blot analysis of mRNA of growth factor receptors. Total liver RNA (10 μg) from wild-type (lane 1), c/ebpα^+/− (lane 2), or c/ebpα^−/− (lane 3) newborn mice, 10 μg of total brain RNA from wild-type mice (lane 4), or 2 μg of total peritoneal cell RNA from thioglycollate-stimulated wild-type mice (lane 5) were electrophoresed in 1% agarose/formaldehyde gels, transferred to nylon membranes, and probed with murine cDNAs of G-CSF receptor (G-CSFr; ref. 43), erythropoietin receptor (EPOr; ref. 44), GM-CSF receptor α (GM-CSFr; ref. 45), or M-CSF receptor (M-CSFr; ref. 46). The position of each signal is indicated on the left side. GM-CSF receptor α mRNA is expressed at relatively low levels in newborn livers, but densitometry demonstrated that the level of GM-CSF receptor α mRNA is not significantly reduced in −/− liver compared with the +/+ or +/− animals.

Figure 4

c/ebpα^−/− mutant mice do not respond to G-CSF stimulation. Pregnant mice had been injected subcutaneously with either phosphate-buffered saline or G-CSF (250 μg per kg of body weight) every 12 hr since day 17 of pregnancy. Cells obtained from livers of newborn mice were stained with fluorescein isothiocyanate- and phycoerythrin-conjugated IgG (isotype control) or antibodies to the mature monocyte/granulocyte surface marker Mac-1 and antibodies to the mature granulocyte surface marker Gr-1, as described in Fig. 2 legend.

Figure 5

Hematopoietic early progenitor cells from c/ebpα^−/− mice do not undergo granulocytic lineage development in sublethally irradiated recipient mice. Flow cytometry analyses were performed by staining the blood cells collected from mice with antibodies against the mature granulocyte marker Gr-1, the T lymphocyte marker Thy1.2, and the congenic strain marker CD45.2 after 4 weeks of bone marrow transplantation. Donor cells are CD45.2⁺.