Identification of eosinophil lineage-committed progenitors in the murine bone marrow

Abstract

Eosinophil lineage-committed progenitors (EoPs) are phenotypically isolatable in the steady-state murine bone marrow. Purified granulocyte/monocyte progenitors (GMPs) gave rise to eosinophils as well as neutrophils and monocytes at the single cell level. Within the short-term culture of GMPs, the eosinophil potential was found exclusively in cells activating the transgenic reporter for GATA-1, a transcription factor capable of instructing eosinophil lineage commitment. These GATA-1-activating cells possessed an IL-5Ralpha(+)CD34(+)c-Kit(lo) phenotype. Normal bone marrow cells also contained IL-5Ralpha(+)CD34(+)c-Kit(lo) EoPs that gave rise exclusively to eosinophils. EoPs significantly increased in number in response to helminth infection, suggesting that the EoP stage is physiologically involved in eosinophil production in vivo. EoPs expressed eosinophil-related genes, such as the eosinophil peroxidase and the major basic protein, but did not express basophil/mast cell-related mast cell proteases. The enforced retroviral expression of IL-5Ralpha in GMPs did not enhance the frequency of eosinophil lineage read-outs, whereas IL-5Ralpha(+) GMPs displayed normal neutrophil/monocyte differentiation in the presence of IL-5 alone. Thus, IL-5Ralpha might be expressed specifically at the EoP stage as a result of commitment into the eosinophil lineage. The newly identified EoPs could be the cellular target in the treatment of a variety of disorders mediated by eosinophils.

Figure 1.

Eosinophils develop from GMPs together with neutrophils and monocytes. (A) Frequency of eosinophil read-outs from purified HSCs stem cells or from myeloid- or lymphoid-committed progenitors determined by limiting dilution assays. (B) Single GMP-derived colony contained eosinophils (Eo) and neutrophils (N). (C) Isolation of EoPs within GMP cultures was achieved by using a transgenic GFP reporter for GATA-1 transcription. GFP is expressed in MEPs, but not in GMPs. The eosinophil potential was found exclusively in the GATA-1–GFP⁺ fraction of day 3 GMP progeny that expressed IL-5Rα and CD34. (D) Purified day 3 GATA-1–GFP⁺IL-5Rα⁺ cells (left) exclusively generated mature eosinophils (right).

Figure 2.

EoPs are prospectively isolatable in the murine bone marrow. (A) FACS analysis of the normal bone marrow demonstrated the existence of IL-5Rα⁺Lin⁻Sca-1⁻CD34⁺c-Kit^lo EoPs. (B) Purified EoPs were blastic cells (left, top), and formed homogenous compact colonies (left, middle) that contained only eosinophils (bottom left). The results of the methylcellulose colony assay also are shown (top right). Cytokine cocktails used in this study contained Slf, IL-3, IL-5, IL-9, GM-CSF, Epo, and Tpo. RT-PCR analyses of purified Gr-1⁺CD11b⁺ neutrophils and progeny of EoPs are shown (bottom right). HPRT, hypoxanthine phosphoribosyltransferase; MPO, myeloperoxidase. (C) FACS analysis of the bone marrow from T. spiralis–infected mice. (D) Percentages of EoPs and CMPs plus GMPs in the bone marrow of mice with or without T. spiralis infection.

Figure 3.

RT-PCR analyses of lineage-affiliated genes in purified EoPs and other myeloid progenitors. (A) Conventional RT-PCR analyses of lineage-affiliated genes. Mast, peritoneal mast cells. HPRT, hypoxanthine phosphoribosyltransferase. (B) A quantitative real-time PCR assay for GATA-1 mRNA. Eo, purified IL-5Rα⁺Gr-1⁺ eosinophils; Neutro, Gr-1^hiCD11b^hi neutrophils.

Figure 4.

The enforcement of IL-5 signaling did not affect eosinophil lineage read-outs at the GMP stage. (A) The construct of MSCV-mIL-5Rα-ires-GFP retrovirus. LTR, long terminal repeat. (B) GMP infected with GFP-tagged mIL-5Rα retroviruses expressed IL-5Rα protein detected by anti–mIL-5Rα monoclonal antibodies (H7). (C) The effect of IL-5 or other cytokines on myeloid differentiation of control-GFP⁺ and IL-5Rα-GFP⁺ GMPs. Mac, macrophage. (D) Cells derived from IL-5Rα-GFP⁺ GMPs in the presence of IL-5 alone on day 6. IL-5Rα-GFP⁺ GMPs generate mainly neutrophils and macrophages. (E) Frequency of eosinophil read-outs from control-GFP⁺ and IL-5Rα-GFP⁺ GMPs determined by limiting dilution assays.