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. 2010 Feb;38(2):90-103.
doi: 10.1016/j.exphem.2009.11.004. Epub 2009 Dec 5.

C/EBPepsilon directs granulocytic-vs-monocytic lineage determination and confers chemotactic function via Hlx

Stephanie Halene  1 , Peter Gaines, Hong Sun, Theresa Zibello, Sharon Lin, Arati Khanna-Gupta, Simon C Williams, Archibald Perkins, Diane Krause, Nancy Berliner
Affiliations

C/EBPepsilon directs granulocytic-vs-monocytic lineage determination and confers chemotactic function via Hlx

Stephanie Halene et al. Exp Hematol. 2010 Feb.

Abstract

Objective: Mutations in the CCAAT enhancer binding protein epsilon (C/EBPepsilon) 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/EBPepsilon(-/-) (knockout [KO]) mice, the mechanisms by which C/EBPepsilon mutations act to decrease neutrophil function are not entirely clear.

Materials and methods: In order to determine the role of C/EBPepsilon in neutrophil differentiation and migration, we generated immortalized progenitor cell lines from C/EBPepsilon 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/EBPepsilon 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/EBPepsilon KO cells, rescued chemotaxis, but not the other defects of C/EBPepsilon KO neutrophils.

Conclusions: We show two new regulatory functions of C/EBPepsilon in myelopoiesis: in the absence of C/EBPepsilon, 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/EBPepsilon KO cells.

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Conflict of interest statement

Conflict of Interest Disclosure

No financial interest/relationships with financial interest relating to the topic of this article have been declared.

Figures

FIGURE 1
FIGURE 1. C/EBPε−/− cells fail to become fully mature neutrophils
EML cells derived from C/EBPε WT (left panels) and KO (right panels) bone marrow are sequentially induced to the promyelocyte (EPRO) and neutrophil stage (EPRO + ATRA) and stained with Wright-Giemsa stain. C/EBPε cells lack secondary granules and nuclei do not fully mature.
FIGURE 2
FIGURE 2. C/EBPε−/− cells fail to express lactoferrin
RNA was harvested from C/EBPε WT and KO EML clones after differentiation into neutrophils. The Northern blots were probed with cDNA probes as labeled. Equal RNA loading was verified by 28S band on the ethidium bromide-stained gel (bottom).
FIGURE 3
FIGURE 3. Respiratory burst is abnormal in C/EBPε −/− cells
Respiratory burst was assayed by the luminol enhanced chemiluminescence method on uninduced EPRO and on EPRO cells before (“0 min”) and after (“3min”) stimulation with PMA. A representative experiment of at least 3 is shown.
FIGURE 4
FIGURE 4. Functional defects of C/EBPε −/− cells can be rescued by restoration of C/EBPε expression
KO EPRO cells were transduced with C/EBPε containing and control vectors and assayed for C/EBPε and lactoferrin expression by RT-PCR (A). Respiratory burst was assayed by the luminol enhanced chemiluminescence method before (“0 min”) and after (“4 min”) stimulation with PMA (B).
FIGURE 5
FIGURE 5. Cell Surface marker expression is abnormal in C/EBPε KO neutrophils
(A) Gr1 expression on BM (top left) and PB (top right) from C/EBPε WT (green) and KO (black) mice, and (B) on WT (green) and KO (black) uninduced EML cells (top left), ATRA/IL-3 induced EML cells (top right), EPRO cells (bottom left), and ATRA induced EPRO cells (bottom right). Percentages of Gr1+ cells are shown. Gating was based on the appropriate isotype (blue). A representative experiment of at least 3 independent experiments is shown.
FIGURE 6
FIGURE 6. ATRA induced C/EBPε KO EPRO cells abnormally retain monocytic cell surface marker expression
(A) C/EBPε WT and KO EPRO cells were differentiated with ATRA and stained for Gr-1 and F4/80. Analysis of F4/80 expression on Gr-1 positive cells is shown for C/EBPε WT (filled histogram) and C/EBPε KO (thick black line) compared to isotype (thin blue line). Analysis of F4/80 and 7/4 co-expression in BM (B) and PB (C) from WT (left) and KO (right) mice. Analysis of 7/4 and Gr1 expression in BM from WT (left) and KO (right) mice (D). F4/80 expression in Gr1/7/4 subsets as shown in D are analyzed for F4/80 expression (E): 7/4hiGr1hi (left), 7/4hiGr1lo (center), and 7/4loGr1lo (right) myeloid cells in BM from WT (blue) and KO (black) mice. Gating is based on the appropriate isotype controls (pink).
FIGURE 6
FIGURE 6. ATRA induced C/EBPε KO EPRO cells abnormally retain monocytic cell surface marker expression
(A) C/EBPε WT and KO EPRO cells were differentiated with ATRA and stained for Gr-1 and F4/80. Analysis of F4/80 expression on Gr-1 positive cells is shown for C/EBPε WT (filled histogram) and C/EBPε KO (thick black line) compared to isotype (thin blue line). Analysis of F4/80 and 7/4 co-expression in BM (B) and PB (C) from WT (left) and KO (right) mice. Analysis of 7/4 and Gr1 expression in BM from WT (left) and KO (right) mice (D). F4/80 expression in Gr1/7/4 subsets as shown in D are analyzed for F4/80 expression (E): 7/4hiGr1hi (left), 7/4hiGr1lo (center), and 7/4loGr1lo (right) myeloid cells in BM from WT (blue) and KO (black) mice. Gating is based on the appropriate isotype controls (pink).
FIGURE 6
FIGURE 6. ATRA induced C/EBPε KO EPRO cells abnormally retain monocytic cell surface marker expression
(A) C/EBPε WT and KO EPRO cells were differentiated with ATRA and stained for Gr-1 and F4/80. Analysis of F4/80 expression on Gr-1 positive cells is shown for C/EBPε WT (filled histogram) and C/EBPε KO (thick black line) compared to isotype (thin blue line). Analysis of F4/80 and 7/4 co-expression in BM (B) and PB (C) from WT (left) and KO (right) mice. Analysis of 7/4 and Gr1 expression in BM from WT (left) and KO (right) mice (D). F4/80 expression in Gr1/7/4 subsets as shown in D are analyzed for F4/80 expression (E): 7/4hiGr1hi (left), 7/4hiGr1lo (center), and 7/4loGr1lo (right) myeloid cells in BM from WT (blue) and KO (black) mice. Gating is based on the appropriate isotype controls (pink).
FIGURE 7
FIGURE 7. Chemotaxis C/EBPε WT and KO neutrophils
ATRA induced KO and WT EPRO cells were assayed for chemotaxis in response to human IL-8 using the transwell assay. The cell number migrated to the bottom well was counted and normalized as described.
FIGURE 8
FIGURE 8. Granulocytes from C/EBPε KO mice fail to fully differentiate and do not upregulate expression of Hlx1
Mature (B220−SSChi7/4hiGr1hi) and immature (B220−SSClo7/4hiGr1hi) cells from C/EBPε WT and KO BM was sorted (A) and cell purity and cellular morphology verified by Wright Giemsa staining (B). QRT-PCR of Hlx1 in whole bone marrow (WBM) and sorted mature (B220-SSChi7/4hiGr1hi) and immature (B220-SSClo7/4hiGr1hi) granulocytes from C/EBPε WT and KO bone marrow.
FIGURE 8
FIGURE 8. Granulocytes from C/EBPε KO mice fail to fully differentiate and do not upregulate expression of Hlx1
Mature (B220−SSChi7/4hiGr1hi) and immature (B220−SSClo7/4hiGr1hi) cells from C/EBPε WT and KO BM was sorted (A) and cell purity and cellular morphology verified by Wright Giemsa staining (B). QRT-PCR of Hlx1 in whole bone marrow (WBM) and sorted mature (B220-SSChi7/4hiGr1hi) and immature (B220-SSClo7/4hiGr1hi) granulocytes from C/EBPε WT and KO bone marrow.
FIGURE 8
FIGURE 8. Granulocytes from C/EBPε KO mice fail to fully differentiate and do not upregulate expression of Hlx1
Mature (B220−SSChi7/4hiGr1hi) and immature (B220−SSClo7/4hiGr1hi) cells from C/EBPε WT and KO BM was sorted (A) and cell purity and cellular morphology verified by Wright Giemsa staining (B). QRT-PCR of Hlx1 in whole bone marrow (WBM) and sorted mature (B220-SSChi7/4hiGr1hi) and immature (B220-SSClo7/4hiGr1hi) granulocytes from C/EBPε WT and KO bone marrow.
FIGURE 9
FIGURE 9. HLX1 over-expression rescues chemotaxis in C/EBPε KO ATRA induced EPRO cells
KO and WT EPRO cells were transduced with Hlx1 expressing or control retroviral vectors and assayed for chemotactic response to human IL-8 (left) and murine KC (right) in the transwell assay.
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