Impaired response to GM-CSF and G-CSF, and enhanced apoptosis in C/EBPbeta-deficient hematopoietic cells

Abstract

Transcription factors known as CCAAT enhancer binding proteins (C/EBPs) are involved in hematopoietic differentiation, including myelopoiesis and granulopoiesis. C/EBPbeta-deficient mice develop normally; however, they exhibit defective macrophage function, resulting in increased susceptibility to infection. Little is known about the role of C/EBPbeta in granulopoiesis; therefore, we examined granulopoiesis in C/EBPbeta-deficient mice. Morphology, the number of peripheral blood and bone marrow cells, and the expression of genes specific for the myeloid lineage were normal in C/EBPbeta-deficient mice. Interestingly, the hematopoietic progenitor cells of C/EBPbeta-deficient mice did not respond normally to granulocyte/macrophage-colony stimulating factor and granulocyte colony stimulating factor. In addition, C/EBPbeta-deficient neutrophils displayed enhanced apoptosis compared with wild-type neutrophils. Our present results indicate that C/EBPbeta helps regulate survival of neutrophils, downstream of the granulocyte colony stimulating factor receptor.

Figure 1

Myeloid lineage analysis of bone marrow cells. Bone marrow cells were isolated from wild-type (WT; n = 4) and C/EBPβ-deficient (KO; n = 4) adult mice, stained with fluorescent-labeled antibodies against myeloid lineage-specific antigens (Mac1/CD11b, CD14, and Gr-1), and analyzed by flow cytometry. Results represent the mean plus or minus SD of 4 mice in each cohort.

Figure 2

Analysis of bone marrow cells. Bone marrow cells were isolated from wild-type (n = 4) and C/EBPβ-deficient (n = 4) adult mice. (A) Bone marrow cells (2 × 10⁴) were cultured in 1% methylcellulose supplemented with IL-3 (10 ng/mL), IL-6 (10 ng/mL), SCF (20 ng/mL), and EPO (3 U/mL);or GM-CSF (20 ng/mL); or G-CSF (60 ng/mL) and SCF (20 ng/mL); or G-CSF (60 ng/mL) alone. Colonies were scored if they contained more than 50 cells at day 7. Results in panels A and B represent the mean plus or minus SD of 4 mice in each cohort (*P < .001; **P < .005). (B) Bone marrow cells (2 × 10⁵) were cultured in either 1% methylcellulose or MegaCult-C media (CFU-Meg), supplemented with either EPO (3 U/mL); or EPO (3 U/mL) and SCF (20 ng/mL); or TPO (50 ng/mL), IL-3 (10 ng/mL), IL-6 (20 ng/mL), and IL-11 (10 ng/mL). BFU-E (EPO and SCF) and CFU-Meg (TPO, IL-3, IL-6, and IL-11) were counted on day 7 of culture; CFU-E (EPO alone) was enumerated on day 2. Black and white bars show wild-type and C/EBPβ-deficient mice, respectively. (C) Detection of phospho-STAT3 in bone marrow cells. Bone marrow cells were isolated from C/EBPβ heterozygous mutant and C/EBPβ-deficient adult mice, and the cells were stimulated either with or without G-CSF (100 ng/mL) for 30 minutes. Signals of phospho-STAT3 (pSTAT3) and STAT3 were detected by Western blot analysis.

Figure 3

Differential cytokine gene expression in primary macrophages and neutrophils. Macrophages (A) and neutrophils (B) from wild-type and C/EBPβ-deficient mice were harvested from peritoneal cavity and cultured either with (+) or without (−) LPS (100 ng/mL, 4 hours). Total RNAs were isolated, and expression of cytokine genes was quantified by real-time RT-PCR as a ratio of these transcripts/18S transcripts, and wild-type LPS-untreated samples are calculated as 1. ■ and □ show wild-type and C/EBPβ-deficient mice, respectively. Results represent the mean plus or minus SD.

Figure 4

Apoptosis assay of neutrophils. Neutrophils of wild-type and C/EBPβ-deficient mice were harvested from peritoneal cavity and cultured for 0, 2, 6, and 18 hours with RPMI 1640 and 10% FBS alone (A) or with LPS (100 ng/mL) (B) or GM-CSF (10 ng/mL) (C). Apoptosis was measured by TUNEL assay. Results represent the mean plus or minus SD of 4 mice in each cohort.

Figure 5

Neutrophil counts in mice after 5-FU treatment. 5-FU (150 mg/kg) was given intraperitoneally to the wild-type and C/EBPβ-deficient mice. Blood samples were taken from the ocular sinus at 0, 1, 4, 7, 10, and 14 days, and neutrophils were counted. Black and broken lines show their mean plus or minus SD results from wild-type and C/EBPβ-deficient mice, respectively.