1. Expression of the secondary granule proteins major basic protein 1 (MBP-1) and eosinophil peroxidase (EPX) is required for eosinophilopoiesis in mice
by Alfred D. Doyle, Elizabeth A. Jacobsen, Sergei I. Ochkur, Michael P. McGarry, Kevin G. Shim, David T. C. Nguyen, Cheryl Protheroe, Dana Colbert, Jake Kloeber, Joseph Neely, Kelly P. Shim, Kimberly D. Dyer, Helene F. Rosenberg, James J. Lee, and Nancy A. Lee
Blood
Volume 122(5):
August 1, 2013
©2013 by American Society of Hematology

2. The targeted disruptions of the mouse eosinophil MBP-1 and EPX genes.
The targeted disruptions of the mouse eosinophil MBP-1 and EPX genes. Restriction maps of the targeting constructs and the MBP-1 and EPX loci before and after the homologous recombination events occurring in the embryonic stem cells that were used to generate the corresponding knockout strains of MBP-1−/− and EPX−/− mice, respectively. As noted, the genes encoding these abundant secondary granule proteins are located on different mouse chromosomes, facilitating the production of double knockout mice through selective breeding strategies of the single knockout strains of mice; offspring genotypes are subject to Mendelian inheritance patterns based on the genotype of the breeding dam and sire. Various polymerase chain reaction (PCR) primer combinations used to identify and confirm the genotype of individual mice are shown for each loci (P1-P5, MBP-1 and P1-P4, EPX).
Alfred D. Doyle et al. Blood 2013;122:
©2013 by American Society of Hematology

3. The loss of both MBP-1 and EPX gene expression in MBP-1−/−/EPX−/− mice leads to a peripheral blood eosinophil deficiency that is both definitive and specific.
The loss of both MBP-1 and EPX gene expression in MBP-1−/−/EPX−/− mice leads to a peripheral blood eosinophil deficiency that is both definitive and specific. (A) Cell counts and hematologic cell differentials showed that MBP-1−/−/EPX−/− double knockout mice have an eosinophil deficiency (without effects on the composition of the other prominent leukocytes; mean ± standard error of the mean; n = 5 to 8 animals per group) that was equivalent to the deficiency observed in an engineered transgenic mouse model congenitally devoid of eosinophils (ie, PHIL mice). (B) Flow cytometric assessments of circulating WBCs derived from individual mice confirm that eosinophils are virtually absent in the blood of MBP-1−/−/EPX−/− mice, similar to PHIL mice, and are significantly lower than eosinophil numbers observed in either wild-type controls or MBP-1−/− or EPX−/− single knockout animals. (C) The loss of eosinophils in MBP-1−/−/EPX−/− mice had no effect on basophils. Flow cytometric analysis (supplemental Figure 2) of bone marrow–derived leukocytes demonstrated again that the loss of eosinophils in MBP-1−/−/EPX−/− mice, similar to PHIL mice, had no effect on basophil populations. *P < .05.
Alfred D. Doyle et al. Blood 2013;122:
©2013 by American Society of Hematology

4. Peripheral blood leukocytes that have staining characteristics and subcellular morphologies consistent with eosinophils deficient in MBP-1 and EPX are present in MBP-1−/−/EPX−/− mice.
Peripheral blood leukocytes that have staining characteristics and subcellular morphologies consistent with eosinophils deficient in MBP-1 and EPX are present in MBP-1−/−/EPX−/− mice. Flow cytometric assessments of WBCs (supplemental Figure 3) from mice of each genotype (ie, wild-type, MBP-1−/−/EPX−/−, MBP-1−/−, and EPX−/−) were used to sort cells, isolating eosinophils that were subsequently cytospun onto slides and stained with a Romanowsky dye set (top panels). Scale bar = 5 µm. The electron microscopic morphology of the eosinophils that remain in MBP-1−/−/EPX−/− mice (lower panels) showed that these cells retain the membrane-bound vesicles (ie, granules) found in wild-type, MBP-1−/−, or EPX−/− mice. As expected, these granules appear devoid of their electron-dense cores (MBP-1) and much of the electron-translucent matrices (characteristic of EPX) of these granules. Scale bars in panels highlighted by a single eosinophil = 1 µm. Scale bars in the insert panels representative of the cytoplasm = 200 nm.
Alfred D. Doyle et al. Blood 2013;122:
©2013 by American Society of Hematology

5. The loss of both MBP-1 and EPX gene expression in MBP-1−/−/EPX−/− mice significantly reduces the steady-state number of eosinophils and their progenitors in the bone marrow.
The loss of both MBP-1 and EPX gene expression in MBP-1−/−/EPX−/− mice significantly reduces the steady-state number of eosinophils and their progenitors in the bone marrow. (A) Flow cytometric assessments of bone marrow–derived leukocytes from individual mice showed that the number of eosinophils (ie, CCR3+ and IL-5Rα+ cells) is significantly lower in MBP-1−/−/EPX−/− mice relative to either wild-type controls or MBP-1−/− or EPX−/− single knockout animals. *P < .05. (B) Immunohistochemical staining (dark-staining cells) of femur sections using a rat mAb (MT ) recognizing Ear-1, -2, -6/7, and -5/11 confirmed our flow cytometric data showing that the number of Ear+ eosinophils in MBP-1−/−/EPX−/− mice was significantly lower than the number of eosinophils observed in the marrow of either wild-type, MBP-1−/−, or EPX−/− mice. However, unlike the marrow of mice devoid of eosinophils and their progenitors (ie, PHIL mice), the marrow of MBP-1−/−/EPX−/− mice maintained a finite steady-state population of these eosinophil lineage-committed cells. Isotype control: rat normal serum immunoglobulin G (IgG). Scale bar = 100 µm.
Alfred D. Doyle et al. Blood 2013;122:
©2013 by American Society of Hematology

6. The concomitant loss of MBP-1 and EPX led to a targeted reduction in EoP cells without similar effects on other less committed myeloid progenitor populations.
The concomitant loss of MBP-1 and EPX led to a targeted reduction in EoP cells without similar effects on other less committed myeloid progenitor populations. (A) Hematopoietic differentiation pathways leading to eosinophils in mice (adapted from Lee et al3). The unique and specific loss of eosinophils displayed by MBP-1−/−/EPX−/− mice suggested the blockade in eosinophilopoiesis (?) occurred at the EoP stage as opposed to earlier myeloid progenitors (ie, common myeloid progenitors [CMP] or granulocyte monocyte progenitors [GMP]). (B) Flow cytometric assessments of marrow leukocytes confirmed that the defect in eosinophilopoiesis observed in MBP-1−/−/EPX−/− mice is limited to the EoP population with no observed effects on either the CMP or GMP populations. (C) Flow cytometric assessments showed that the EoP populations of both PHIL (n = 3) and MBP-1−/−/EPX−/− (n = 7) mice each displayed significant increases in apoptosis (eightfold and threefold increases, respectively) relative to wild-type (n = 7). *P < .05; **P < .002.
Alfred D. Doyle et al. Blood 2013;122:
©2013 by American Society of Hematology

7. 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.
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 (MT ) 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.
Alfred D. Doyle et al. Blood 2013;122:
©2013 by American Society of Hematology

8. Partial bone marrow engraftment of wild-type recipient mice with marrow from MBP-1−/−/EPX−/− marrow donors showed that the loss of eosinophilopoiesis induced by the concomitant loss of both MBP-1 and EPX was a cell-autonomous effect.
Partial bone marrow engraftment of wild-type recipient mice with marrow from MBP-1−/−/EPX−/− marrow donors showed that the loss of eosinophilopoiesis induced by the concomitant loss of both MBP-1 and EPX was a cell-autonomous effect. (A) Representative flow cytometric assessments of peripheral WBC populations from the resulting mixed-marrow chimeric mice are shown; they demonstrated that while non-eosinophil leukocytes displayed a 70% donor/30% recipient distribution of CD45.2/CD45.1, respectively, contributions to overall steady-state eosinophil populations in these mixed-marrow chimeric animals by donor MBP-1−/−/EPX−/− (CD45.2) eosinophils was limited. (B) Flow cytometric assessments of peripheral blood from mixed-marrow chimeras generated by γ irradiation of CD45.1+ recipient wild-type mice and subsequent adoptive transfer of either CD45.2+ wild-type or MBP-1−/−/EPX−/− donor marrow. These data showed that the non-eosinophil blood leukocytes of donor origin (% of total WBC) remained unchanged when using wild-type vs MBP-1−/−/EPX−/− donor marrow. However, the eosinophil populations of these mixed-marrow chimeras displayed a differential distribution of donor origin (>80% lower) when comparing recipient mice that had received wild-type vs MBP-1−/−/EPX−/− marrow. *P < .05.
Alfred D. Doyle et al. Blood 2013;122:
©2013 by American Society of Hematology