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Comparative Study
. 2005 Dec 13;102(50):18105-10.
doi: 10.1073/pnas.0509148102. Epub 2005 Dec 5.

Developmental checkpoints of the basophil/mast cell lineages in adult murine hematopoiesis

Yojiro Arinobu  1 , Hiromi Iwasaki, Michael F Gurish, Shin-ichi Mizuno, Hirokazu Shigematsu, Hidetoshi Ozawa, Daniel G Tenen, K Frank Austen, Koichi Akashi
Affiliations
Comparative Study

Developmental checkpoints of the basophil/mast cell lineages in adult murine hematopoiesis

Yojiro Arinobu et al. Proc Natl Acad Sci U S A. .

Abstract

Basophils and mast cells, which are selectively endowed with the high-affinity IgE receptor and mediate a range of adaptive and innate immune responses, have an unknown developmental relationship. Here, by evaluating the expression of the beta7 integrin, a molecule that is required for selective homing of mast cell progenitors (MCPs) to the periphery, we identified bipotent progenitors that are capable of differentiating into either cell type in the mouse spleen. These basophil/mast cell progenitors (BMCPs) gave rise to basophils and mast cells at the single-cell level and reconstituted both mucosal and connective tissue mast cells. We also identified the basophil progenitor (BaP) and the MCP in the bone marrow and the gastrointestinal mucosa, respectively. We further show that the granulocyte-related transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) plays a primary role in the fate decision of BMCPs, being expressed in BaPs but not in MCPs. Thus, circulating basophils and tissue mast cells share a common developmental stage at which their fate decision might be controlled principally by C/EBPalpha.

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Figures

Fig. 1.
Fig. 1.
Identification of BMCPs in C57BL/6 murine hematopoiesis. (A) The expression of β7 and T1/ST2 in the bone marrow myeloerythroid progenitors and peritoneal mast cells (MC). A fraction of myeloerythroid progenitors expressed a low level of β7. (B) The surface phenotype of β7hi BMCPs in the spleen. (C)(Left) Morphology of purified Lin-c-Kit+FcγRII/IIIhiβ7hi BMCPs. These cells gave rise exclusively to mast cells and basophils (Right) in the presence of a panel of myeloerythroid cytokines (see Materials and Methods). May-Giemsa staining was used. (Magnification: ×1,000.) (D) A basophil/mast cell colony developed from a single BMCP in vitro. May-Giemsa staining was used. (Magnification: ×1,000.) (E) Clonogenic analysis of β7lo bone marrow myeloerythroid progenitors and β7hi BMCP. Note that a fraction of the spleen BMCP and the bone marrow β7lo GMP gave rise to both basophils and mast cells at the single-cell level. (F) FACS analysis of day-7 progeny of the bone marrow β7- and β7lo GMP. Both populations gave rise to FcεRIα+CD11c+ basophils and FcεRIα+CD11c- mast cells expressing c-Kit.
Fig. 2.
Fig. 2.
Identification of BaPs in the bone marrow. (A) FACS analysis of Lin-CD34+FcεRIαhic-Kit- BaPs in the bone marrow for β7, T1/ST2, CD13, and Thy-1. (B) The BaPs possessed a blastic morphology and differentiated only into basophils. May-Giemsa staining was used. (Magnification: ×1,000.)
Fig. 3.
Fig. 3.
Identification of MCPs in the intestine. (A) FACS analysis of intestinal MCPs in normal (Top), W/Wv (Middle), and OVA-sensitized (Bottom) mice. (B) (Left) Morphology of purified intestinal MCPs. Intestinal MCPs formed colonies, which contained only mast cells (Right). May-Giemsa staining was used. (Magnification: ×1,000.) (C) Expression of mast cell-related antigens in purified intestinal MCPs. (D) Changes in the absolute numbers of BMCPs and BaPs in mice infected with T. spiralis and in uninfected mast cell-deficient W/Wv mice. The error bars represent the standard deviation.
Fig. 4.
Fig. 4.
Lineal relationship of BMCPs, BaPs, and MCPs. (A) A single GMP-derived colony contained neutrophils, basophils, and mast cells. May-Giemsa staining was used. (Magnification: ×600.) (B) FACS analysis of the day-3 progeny of GMPs cultured in the presence of stem cell factor (SCF), IL-3, IL-5, IL-6, and IL-9. In addition to Lin-CD34+IL-5Rα+c-Kitlo EoPs (14), BMCPs, BaPs, and MCPs were isolatable within the GMP progeny. (C) FACS analysis of day-2 progeny of spleen BMCPs. BMCPs exclusively developed BaPs and MCPs. Morphology of BMCP-derived BaP and MCP populations and of their progeny after 7-day cultures are also shown. May-Giemsa staining was used. (Magnification: ×1,000.) (D) Proliferation curve of single BMCPs, BaPs, and MCPs in the presence of SCF, IL-3, and IL-6. (E) FACS analysis of the reconstitution of peritoneal mast cells 8 weeks after i.p. transplantation of 400 BMCPs or 400 intestinal MCPs purified from C57BL/6 (Ly5.1) mice into W/Wv mice (Ly5.2). (F) Successful reconstitution of mast cells in the spleen and the gastrointestinal submucosa of W/Wv mice 12 weeks after i.v. injection of 3,000 BMCPs.
Fig. 5.
Fig. 5.
C/EBPα plays a key role in the bifurcation of mast cell vs. basophil lineage fates. (A) RT-PCR analyses of lineage-related gene expression in purified progenitor populations. GMPs and BaPs were purified from the bone marrow, and BMCPs were from the spleen of C57BL/6 mice. MCP, GMP-derived MCPs after in vitro culture (see text and Fig. 3B); Int MCP, intestinal MCPs; PMC, peritoneal mast cells. (B) A quantitative real-time PCR assay for C/EBPα mRNA in purified populations. (C) FACS analyses of progeny of spleen BMCPs with or without C/EBPα. BMCPs isolated from the spleen of C/EBPαF/F mice were infected with retroviruses carrying control GFP (Left), Cre recombinase (Center), or C/EBPα (Right). GFP+ cells were phenotyped after 7 days in culture. (D) FACS analyses of progeny of bone marrow BaPs disrupted with C/EBPα. BaPs differentiated only into c-Kit-CD11b+ basophils in vitro, irrespective of C/EBPα disruption. (E) Analysis of intestinal MCPs transduced with C/EBPα. MCPs infected with control retroviruses differentiated exclusively into CD11b-c-Kit+ mature mast cells, whereas those transduced with C/EBPα converted into CD11b+c-Kit- basophils. The morphology of progeny of GMP-derived MCPs transduced with control retroviruses (Lower Left) and with C/EBPα (Lower Right) is also shown. May-Giemsa staining was used. (Magnification: ×1,000.) (F) RT-PCR analyses of basophil-related genes in progeny of MCPs with or without ectopic expression of C/EBPα. P, positive control.
Fig. 6.
Fig. 6.
The proposed developmental scheme of basophil and mast cell development.
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