Critical role of P1-Runx1 in mouse basophil development

Abstract

Runx1(P1N/P1N) mice are deficient in the transcription factor distal promoter-derived Runt-related transcription factor 1 (P1-Runx1) and have a > 90% reduction in the numbers of basophils in the BM, spleen, and blood. In contrast, Runx1(P1N/P1N) mice have normal numbers of the other granulocytes (neutrophils and eosinophils). Although basophils and mast cells share some common features, Runx1(P1N/P1N) mice have normal numbers of mast cells in multiple tissues. Runx1(P1N/P1N) mice fail to develop a basophil-dependent reaction, IgE-mediated chronic allergic inflammation of the skin, but respond normally when tested for IgE- and mast cell-dependent passive cutaneous anaphylaxis in vivo or IgE-dependent mast cell degranulation in vitro. These results demonstrate that Runx1(P1N/P1N) mice exhibit markedly impaired function of basophils, but not mast cells. Infection with the parasite Strongyloides venezuelensis and injections of IL-3, each of which induces marked basophilia in wild-type mice, also induce modest expansions of the very small populations of basophils in Runx1(P1N/P1N) mice. Finally, Runx1(P1N/P1N) mice have normal numbers of the granulocyte progenitor cells, SN-Flk2(+/-), which can give rise to all granulocytes, but exhibit a > 95% reduction in basophil progenitors. The results of the present study suggest that P1-Runx1 is critical for a stage of basophil development between SN-Flk2(+/-) cells and basophil progenitors.

Figure 1

Runx1^P1N/P1N mice have markedly reduced numbers of basophils. (A) BM, spleen, and blood were isolated from WT and Runx1^P1N/P1N mice and stained with anti-IgE and anti-DX5 mAbs. Data shown are representative of 5 independent experiments, each of which gave similar results. (B) The numbers of basophils are shown as means + SEM. ***P < .0001; no asterisks, P > .05. (C) Semiquantitative RT-PCR analysis for Mcpt8, which encodes mMCP-8, was performed using RNA prepared from total BM cells from WT or Runx1^P1N/P1N mice. cDNA was diluted 3-fold. Data shown are from 1 of 3 independent experiments, each of which gave similar results.

Figure 2

Phenotypic analysis of other granulocytes and leukocytes in Runx1^P1N/P1N mice. (A) Representative flow cytometric plots of neutrophils (Gr-1^high SiglecF⁻), eosinophils (Gr-1^int SiglecF⁺), monocytes (Gr-1^int SiglecF⁻), NK cells (NK1.1⁺CD3⁻), NKT cells (NK1.1⁺CD3⁺), B cells (B220⁺), conventional dendritic cells (DCs; CD11c⁺B220⁻), plasmacytoid dendritic cells (CD11c⁺B220⁺), and T cells (CD3⁺), and their cell counts (B) from BM and spleens from WT or Runx1^P1N/P1N mice. Data shown are from 1 of 3 independent experiments, each of which gave similar results. Data in panel B show means + SEM.

Figure 3

Runx1^P1N/P1N mice have normal numbers of mast cells in multiple anatomic sites. (A) Cells from peritoneal lavage fluid were stained with anti-mIgE and anti–c-Kit mAbs. Data shown are from 1 of 5 independent experiments, each of which gave similar results. The numbers of peritoneal mast cells are shown as means + SD. ns indicates not significant (P > .05). (B) Toluidine blue staining for mast cells (some indicated by solid arrows) in 4-mm-thick paraffin sections of ear pinnae from WT (top) and Runx1^P1N/P1N mice (bottom). The numbers of mast cells in the ear pinnae, back skin, or stomach are shown as means + SD. ns indicates not significant (P > .05).

Figure 4

Runx1^P1N/P1N mice have normal mast-cell functions. (A) Analysis of passive cutaneous anaphylaxis reactions in WT and Runx1^P1N/P1N mice that received intradermal injections of IgE anti-DNP into the right ear pinnae and of saline into the left ear pinnae (control; none). After sensitization, mice were challenged intravenously with DNP-BSA. Data show means + SD of the extravasation of Evans blue into the ears. (B) Degranulation of WT and Runx1^P1N/P1N BMCMCs, assessed as the release of β-hexosaminidase. BMCMCs were sensitized with anti-DNP IgE and stimulated with the indicated concentrations of DNP-HSA (0, 6.25, 12.5, 25, 50, and 100 ng/mL). Data show the means + SD. (C) ELISA of IL-6 in BMCMCs from WT and Runx1^P1N/P1N mice sensitized with anti-DNP IgE and stimulated with DNP-HSA (10 ng/mL). nd indicates not detected. ***P < .0001; **P < .001; no asterisks, P > .05 relative to the corresponding WT mice. Data are from 1 of 3 independent experiments, each of which gave similar results.

Figure 5

Runx1^P1N/P1N mice have markedly reduced basophil- and IgE-dependent chronic allergic inflammation. (A) WT (■) or Runx1^P1N/P1N mice (○) were sensitized passively by an IV injection of TNP-specific IgE 1 day before being challenged with an intradermal injection of TNP-OVA into the left ear pinna and OVA into the right ear pinna as a control. Ear swelling at each time point is shown (means + SEM, n = 3 each). (B) Immunohistochemical staining with an anti-mMCP8 Ab (DAB substrate) to visualize basophils (some indicated with solid arrowheads) and Giemsa counterstaining (4-μm-thick, paraffin-embedded sections) to demonstrate leukocytes in ear pinnae from WT or Runx1^P1N/P1N mice 4 days after challenge with OVA or TNP-OVA. Scale bars indicate 100 μm (top panel) or 25 μm (bottom panel). Data shown are from 1 of 2 independent experiments, each of which gave similar results. ***P < .0001; **P < .001; no asterisk, P > .05 relative to the corresponding WT mice.

Figure 6

Changes in basophil numbers after S venezuelensis infection or IL-3 injection in Runx1^P1N/P1N versus WT mice. (A) WT or Runx1^P1N/P1N mice were infected with 10 000 S venezuelensis larvae and basophils (mIgE⁺DX5⁺) in the BM and spleen were analyzed 8 days after infection. Data shown are from 1 of 3 independent experiments, each of which gave similar results. (B) Recombinant IL-3 (200 ng/mouse/d) was injected into WT or Runx1^P1N/P1N mice for 7 consecutive days. Basophils in the BM and spleen from each mouse were stained the day after the 7th injection. Data shown in panels B and D are means + SEM.

Figure 7

Impaired BaPs in Runx1^P1N/P1N mice. (A-C) Representative flow cytometry plots and percentage of indicated gates of SN-Flk2⁺ (Sca-1⁻Lin⁻c-Kit⁺CD150⁻Flk2⁺CD27⁺) cells and SN-Flk2⁻ (Sca-1⁻Lin⁻c-Kit⁺CD150⁻Flk2⁻ CD27⁺) cells (A), and BaPs (Lin⁻CD34⁺c-Kit⁻FcϵRIα⁺) in BM from WT or Runx1^P1N/P1N mice (B), and BMCPs (Lin⁻c-Kit⁺β7⁺FcγRII/III⁺) in spleen from WT or Runx1^P1N/P1N mice (C). Data shown are from 1 of 3 independent experiments, each of which gave similar results.