SiglecF+Gr1hi eosinophils are a distinct subpopulation within the lungs of allergen-challenged mice

Abstract

Although eosinophils as a group are readily identified by their unique morphology and staining properties, flow cytometry provides an important means for identification of subgroups based on differential expression of distinct surface Ags. Here, we characterize an eosinophil subpopulation defined by high levels of expression of the neutrophil Ag Gr1 (CD45⁺CD11c^-SiglecF⁺Gr1^hi). SiglecF⁺Gr1^hi eosinophils, distinct from the canonical SiglecF⁺Gr1^- eosinophil population, were detected in allergen-challenged wild-type and granule protein-deficient (EPX^-/- and MBP-1^-/-) mice, but not in the eosinophil-deficient ΔdblGATA strain. In contrast to Gr1⁺ neutrophils, which express both cross-reacting Ags Ly6C and Ly6G, SiglecF⁺Gr1^hi eosinophils from allergen-challenged lung tissue are uniquely Ly6G⁺ Although indistinguishable from the more-numerous SiglecF⁺Gr1^- eosinophils under light microscopy, FACS-isolated populations revealed prominent differences in cytokine contents. The lymphocyte-targeting cytokines CXCL13 and IL-27 were identified only in the SiglecF⁺Gr1^hi eosinophil population (at 3.9 and 4.8 pg/10⁶ cells, respectively), as was the prominent proinflammatory mediator IL-13 (72 pg/10⁶ cells). Interestingly, bone marrow-derived (SiglecF⁺), cultured eosinophils include a more substantial Gr1⁺ subpopulation (∼50%); Gr1⁺ bmEos includes primarily a single Ly6C⁺ and a smaller, double-positive (Ly6C⁺Ly6G⁺) population. Taken together, our findings characterize a distinct SiglecF⁺Gr1^hi eosinophil subset in lungs of allergen-challenged, wild-type and granule protein-deficient mice. SiglecF⁺Gr1^hi eosinophils from wild-type mice maintain a distinct subset of cytokines, including those active on B and T lymphocytes. These cytokines may facilitate eosinophil-mediated immunomodulatory responses in the allergen-challenged lung as well as in other distinct microenvironments.

Keywords: allergy; cytokines; flow cytometry; inflammation.

Figure 1.. SiglecF⁺Gr1^hi eosinophils are an independent subpopulation detected in lungs of wild-type and eosinophil–peroxidase gene-deleted (EPX^−/−) mice.

(A) Protocol I: Af sensitization on d 0 and d 14, followed by Af or PBS challenge (d 25, 26, 27). Mice were evaluated on d 30 as indicated. SiglecF⁺Gr1⁻ eosinophils (CD45⁺CD11c⁻SiglecF⁺Gr1⁻) (B) and SiglecF⁺Gr1^hi cells (CD45⁺CD11c⁻SiglecF⁺Gr1^hi) (C) in single-cell lung suspensions from Af-sensitized and Af- or PBS-challenged mice as indicated in (A). ***P < 0.001, **P < 0.01 Af-sensitization and Af challenge vs. Af-sensitization and PBS challenge, 2-way ANOVA, n = 5–13 mice/group. (D) SiglecF⁺Gr1^hi cells, as distinct from SiglecF⁺Gr1⁻ eosinophils, represent ∼2% of the total CD45⁺CD11c⁻ leukocyte population isolated from the lungs of allergen-sensitized and -challenged mice. (E and F) SiglecF⁺Gr1^hi cells represent ∼10% of the CD45⁺CD11c⁻Gr1^hi cells isolated from lungs of Af-sensitized and -challenged, wild-type mice and EPX^−/− mice, respectively. CD45⁺CD11c⁻SiglecF⁺Gr1^hi cells (G), CD45⁺CD11c⁻SiglecF⁻Gr1^hi (polymorphonuclear neutrophils; neutrophils) (H), and CD45⁺CD11c⁻SiglecF⁺Gr1⁻ (Eos; eosinophils) (I) isolated from lung single-cell suspensions from Af-sensitized and -challenged mice by flow cytometry and cell sorting (FACS); original magnification ×100.

Figure 2.. SiglecF⁺Gr1^hi eosinophils are detected in lungs of eosinophil MBP-1^−/− mice.

(A) SiglecF⁺Gr1^hi eosinophils in single-cell lung suspensions from Af-sensitized and -challenged MBP-1^−/− mice both with and without subsequent virus infection; n = 5–6 mice/group. (B) SiglecF⁺Gr1^hi eosinophils represent >10% of the CD45⁺CD11c⁻Gr1^hi cells isolated from lungs of Af-sensitized and -challenged, wild-type mice and MBP-1^−/− mice.

Figure 3.. SiglecF⁺Gr1^hi eosinophils are detected in response to Af-sensitization and challenge in wild-type (WT) BALB/c, but not eosinophil-deficient, ΔdblGATA mice.

SiglecF⁺Gr1⁻ eosinophils (A) and SiglecF⁺Gr1^hi eosinophils (B) in single-cell lung suspensions from Af-sensitized and -challenged mice, as described in Fig. 1; **P < 0.01 Af-sensitization and Af-challenge vs. Af-sensitization and PBS challenge, ^††P < 0.01 PVM infected vs. diluent control, 2-way ANOVA, n = 3–5 mice/group. C. SiglecF⁺Gr1^hi eosinophils are an independent population and represent ∼5% of the CD45⁺CD11c⁻Gr1⁺ cells isolated from lungs of Af-sensitized and -challenged BALB/c mice. (D) SiglecF⁺Gr1^hi cells cannot be detected in Af-sensitized and -challenged, eosinophil-deficient ΔdblGATA mice.

Figure 4.. SiglecF⁺Gr1^hi eosinophils are detected in response to intranasal challenge with Alternaria alternata in wild-type BALB/c mice.

(A) Protocol II: Aa intranasal challenge on d 0, 3, and 6; mice were evaluated on d 10 as indicated. SiglecF⁺Gr1⁻ eosinophils (B) and SiglecF⁺Gr1^hi eosinophils (C) in single-cell lung suspensions from mice challenged with Aa, **P < 0.01 Mann-Whitney U test, n = 4–6 mice/group. (D) SiglecF⁺Gr1^hi eosinophils represent 50% of the CD45⁺CD11c⁻Gr1⁺ cells isolated from lungs of the Aa-challenged BALB/c mice. (E) CD45⁺CD11c⁻SiglecF⁺Gr1^hi eosinophils isolated from lung single-cell suspensions from Aa-challenged mice by flow cytometry; original magnification ×100.

Figure 5.. Ly6C/Ly6G Ag profiles of bmEos and Gr1⁺ eosinophils from allergen-challenged lung.

Eosinophils generated in culture from unselected bmEos from C57BL/6 mice are SiglecF⁺ (99%) (A), Gr1⁺ (54%) (B), and Ly6C⁺ or Ly6C⁺Ly6G⁺ (C) SiglecF⁺Gr1^hi (D) eosinophils identified in lung suspensions from mice challenged with Aa (see Fig. 4A) are Ly6G⁺ (E) and Ly6C⁻ (F).