C/EBPε directs granulocytic-vs-monocytic lineage determination and confers chemotactic function via Hlx
Received 28 August 2009; received in revised form 11 November 2009; accepted 12 November 2009. published online 18 November 2009.
Objective
Mutations in the CCAAT enhancer binding protein epsilon (C/EBPε) gene have been identified in the cells of patients with neutrophil specific granule deficiency, a rare congenital disorder marked by recurrent bacterial infections. Their neutrophils, in addition to lacking specific granules required for normal respiratory burst activity, also lack normal phagocytosis and chemotaxis. Although the specific granule deficiency phenotype has been replicated in C/EBPε−/− (knockout [KO]) mice, the mechanisms by which C/EBPε mutations act to decrease neutrophil function are not entirely clear.
Materials and Methods
In order to determine the role of C/EBPε in neutrophil differentiation and migration, we generated immortalized progenitor cell lines from C/EBPε KO and wild-type mice and performed expression and flow cytometric analysis and functional studies.
Results
Expression of lineage-specific cell surface antigens on our in vitro differentiated cell lines revealed persistent expression of monocytic markers on KO granulocytes. We verified this in primary murine peripheral blood and bone marrow cells. In addition, KO bone marrow had an increase in immature myeloid precursors at the common myeloid progenitor and granulocyte/monocyte progenitor levels, suggesting a critical role for C/EBPε not only in granulocyte maturation beyond the promyelocyte stage, but also in the monocyte/granulocyte lineage decision. We found that restoration of Hlx (H2.0-like homeo box 1) expression, which was decreased in C/EBPε KO cells, rescued chemotaxis, but not the other defects of C/EBPε KO neutrophils.
Conclusions
We show two new regulatory functions of C/EBPε in myelopoiesis: in the absence of C/EBPε, there is not only incomplete differentiation of granulocytes, but myelopoiesis is disrupted with the appearance of an intermediate cell type with monocyte and granulocyte features, and the neutrophils have abnormal chemotaxis. Restoration of expression of Hlx provides partial recovery of function; it has no effect on neutrophil maturation, but can completely ameliorate the chemotaxis defect in C/EBPε KO cells.
aSection of Hematology, Department of Internal Medicine and Yale Cancer Center, Yale University School of Medicine, New Haven, Conn., USA
bDepartment of Biological Sciences, University of Massachusetts Lowell, Lowell, Mass., USA
cDivision of Hematology, Brigham and Women's Hospital, Boston, Mass., USA
dDepartments of Laboratory Medicine, Pathology, and Cell Biology, Yale University School of Medicine, New Haven, Conn., USA
eDepartment of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center and Southwest Cancer Treatment and Research Center, Lubbock, Tex., USA
fDepartment of Pathology and Lab Medicine, University of Rochester, Rochester, NY., USA
Offprint requests to: Stephanie Halene, M.D., Section of Hematology, Yale Cancer Center and Department of Internal Medicine, Yale University School of Medicine, P.O. Box 208021, New Haven, CT 06520-8073
ABSTRACT