IL-5–dependent expansion of EoPs is blocked in the marrow of MBP-1^−/−/EPX^−/− mice (ie, in vivo) as well as in cell cultures of marrow (ie, ex vivo) derived from these double granule protein gene knockout animals. (A) Constitutive high-level ectopic expression of IL-5 from mature T cells is unable to rescue the significant loss of marrow eosinophil lineage-committed cells observed in MBP-1^−/−/EPX^−/− mice. Immunohistochemical staining (dark-staining cells) of femur sections using a rat anti-mouse Ear mAb (MT3 25.1.1) confirmed our flow cytometric data showing a unique loss of eosinophil lineage-committed cells in MBP-1^−/−/EPX^−/− mice. This immunohistochemical staining also revealed that, unlike the marrow of IL-5 transgenic mice devoid of eosinophils and their progenitors (ie, NJ.1638/PHIL mice), the marrow of NJ.1638/MBP-1^−/−/EPX^−/− mice maintained a finite steady-state population of these eosinophil lineage-committed cells. Isotype control: rat normal serum IgG. Scale bar = 100 µm. (B) Eosinophil differentiation was significantly limited in ex vivo bone marrow cultures using hematopoietic stem cells/progenitors from MBP-1^−/−/EPX^−/− mice. Biphasic bone marrow cultures initially expanding undifferentiated stem cells/progenitors (undifferentiated) prior to IL-5–mediated expansion of EoPs (eosinophil lineage expansion) showed that similar to observations in vivo, EoP cells in PHIL and MBP-1^−/−/EPX^−/− mice were unable to undergo IL-5–dependent expansion. Thus, while bone marrow cultures of wild-type and single granule protein knockout mice (ie, MBP-1^−/− and EPX^−/−) each displayed a significant expansion in total cell number with a resulting compositional shift to >98% eosinophils, the marrow of PHIL and MBP-1^−/−/EPX^−/− mice failed to undergo this expansion.