Myelopoiesis is highly regulated by transcription factors including C/EBPα, C/EBPβ and C/EBPε. These transcription factors play important roles in myelopoiesis as highlighted by the studies of single knockout murine models 
, providing several unexpected findings. For example, C/EBPα-deficient mice, which normally do not have neutrophils, can produce them in the presence of a high concentration of cytokines with rapid upregulation of C/EBPβ in myeloid progenitors. Also, C/EBPβ-deficient mice are unable to produce “emergency” neutrophils when stimulated by either cytokines or infection, suggesting that C/EBPβ is important for the production of these neutrophils 
C/EBP family proteins share a high degree of amino acid sequence similarity in their dimerization and DNA-binding domains, and potentially bind the same cis-elements in promoters 
. This may allow for one family member to compensate for the loss of another. For example, we previously showed that C/EBPα-deficient mice expressing C/EBPβ from the Cebpa
gene locus had normal hematopoiesis, indicating that C/EBPβ can substitute for functions of C/EBPα in hematopoietic cells 
. C/EBPα-deficient mice have a severe loss of liver function; in contrast, the C/EBPα-deficient mice expressing C/EBPβ from the Cebpa
locus displayed normal liver function 
. Double knockout mice of C/EBPα and C/EBPβ die between E10 and E11 and have a defective placenta; contrary, single knockout mice of C/EBPα and C/EBPβ develop normally at that embryonic stage 
. In addition, the activities of C/EBPα, C/EBPβ and C/EBPδ are redundant in LPS-induced expression of IL-6 and monocyte chemoattractant protein-1 
C/EBPε is involved in the functional maturation of neutrophils and macrophages both of which represent major elements of the innate immune system. A recent study showed that C/EBPβ and C/EBPε regulate the expression of cytokines including IL-8 in human neutrophils 
, demonstrating that C/EBP proteins play important roles for induction of cytokine expression in neutrophils. Importantly, a highly immature state of neutrophils was detected in the bbee
mice with a potential differentiation arrest at the myelocytes/metamyelocytes stage. Taken together, C/EBPβ and C/EBPε are required not only for cytokine expression, but also for the terminal differentiation of the neutrophil.
Our colony formation assay revealed that bbee
mononuclear bone marrow cells formed decreased numbers of myeloid colonies compared with the single knockout and wild-type mice in the presence of various cytokines including G-CSF, although expression of G-CSF receptor mRNA in the bbee
cells was intact. Regulation of G-CSF receptor expression is controlled by C/EBPα 
, and bbee
bone marrow cells expressed C/EBPα (data not shown). In addition, STAT3 activation, which is one of the downstream signals of G-CSF, also appeared to be intact, since the phosphorylation status of STAT3 stimulated with G-CSF was comparable among all 4 types of mice. The molecular mechanism of the impaired growth response to cytokines including G-CSF remains to be explored.
A slightly impaired response to cytokines was also found in bbEE bone marrow cells (current study and references 19 and 20), but bbee bone marrow cells showed a severe lack of response. One possible explanation for this finding is obtained from our hematopoietic cell fraction (KSL) analysis. KSL cells dramatically accumulated in the bone marrow of bbee mice. We assume that the accumulation is in part the result of impaired responses to differentiation-related cytokines. Therefore, these data strongly suggested that C/EBPβ and C/EBPε are required for commitment of hematopoietic progenitor cells under cytokine stimulation to myeloid cells.
C/EBPβ is dramatically induced during macrophage differentiation 
. C/EBPβ-deficient macrophages have a defect in their ability to kill bacteria 
, and the C/EBPβ isoform 34 kDa protein is responsible for induction of inflammatory cytokines in macrophages 
. Expression of C/EBPε is upregulated during granulocyte differentiation, but not macrophage differentiation 
; however, macrophages from C/EBPε-deficient mice have functional abnormalities 
. Importantly, our current study revealed that the expression of several important proinflammatory cytokines and immune mediators is significantly repressed in activated bone marrow-derived macrophages from bbee
mice as compared to single deficient mice. Some promoter regions of proinflammatory cytokine genes such as Marco
contain C/EBP binding sites, and both C/EBPβ and C/EBPε can bind and regulate their expression. In single knockout mice, either C/EBPβ or C/EBPε might compensate for the loss of the other transcription factor allowing expression of the target gene. In contrast, bbee
mice have neither transcriptional activators, C/EBPβ and C/EBPε, resulting in highly dysregulated gene expression in their macrophages.
In summary, the findings from the present study demonstrated that in contrast to the single knockout mice, synergistic effects of the absence of both genes were found in the double knockout mice. Severe aberrations involving the hematopoietic system and the innate immune response were present in the double knockout mice that were found either as a comparatively mild abnormality or even normal in the single knockout mice, indicating that C/EBPβ and C/EBPε can in part substitute for each other in the BBee or bbEE mice, respectively.