IL-33 promotes eosinophilia in vivo and antagonizes IL-5-dependent eosinophil hematopoiesis ex vivo

Abstract

IL-33 is an IL-1 family cytokine that elicits IL-5-dependent eosinophilia in vivo. We show here that IL-33 promotes minimal eosinophil hematopoiesis via direct interactions with mouse bone marrow progenitors ex vivo and that it antagonizes eosinophil hematopoiesis promoted by IL-5 on SCF and Flt3L primed bone marrow progenitor cells in culture. SCF and Flt3L primed progenitors respond to IL-33 by acquiring an adherent, macrophage-like phenotype, and by releasing macrophage-associated cytokines into the culture medium. IL-33-mediated antagonism of IL-5 was reproduced in part by the addition of GM-CSF and was inhibited by the actions of neutralizing anti-GM-CSF antibody. These findings suggest that the direct actions of IL-33 on bone marrow progenitors primed with SCF and Flt3L are antagonistic to the actions of IL-5 and are mediated in part by GM-CSF.

Figure 1. Administration of IL-33 alters Th2 serum cytokine levels in mice and promotes bone marrow eosinophilia

Mice received IL-33 (0.1 μg/day for 7 days) via intraperitoneal injection (closed symbols) or were left untreated (open symbols); serum was collected from individual BALB/c mice on day 8; bone marrow was collected on day 9 from wild-type BALB/c, eosinophil-deficient ΔdblGATA, and interleukin-5 gene-deleted mice. (A) Serum levels of IL-4, IL-5, IL-13; n = 5 – 14 mice per group. Groups were compared with the Mann-Whitney test, **p < 0.01. (B) Total number of bone marrow cells recovered from each mouse; (C) Percent eosinophils were determined by counting of Diff-Quik stained cells recovered from the bone marrow; (D) Cell surface expression of Siglec F was evaluated by flow cytometry. Each symbol represents data derived from a single mouse. Groups were compared with ANOVA followed by Bonferroni’s multiple comparison post-test, *** p < 0.001.

Figure 2. IL-33 antagonizes IL-5-mediated eosinophil hematopoiesis ex vivo.

Bone marrow cells were cultured for 4 days in stem cell factor (SCF) and Flt3 ligand (Flt3L), then transferred into media containing IL-5, IL-33 or the combination of IL-5 + IL-33. (A) Total number of cells in suspension at various time points, day 4 and onward, n = 3 – 6 cultures from individual mice per time point. (B) Percentage eosinophils at each time point; groups were compared by ANOVA followed by Bonferroni’s multiple comparison post-test, *** p < 0.001, representative experiment from a total of 4. (C – E) Cells fixed and stained with Diff-Quik; shown are cells in suspension (Su) from cultures transferred to IL-5; those in suspension from cultures transferred to IL-33; and cells adherent to the culture flask from cultures transferred to IL-33, all at day 8. Original magnification was 63x. (F) Expression of Siglec F at day 8 on suspension cells (Su) and adherent cells (Ad) as indicated. Groups compared with ANOVA followed by Bonferroni’s multiple comparison post-test, **** p<0.0001.

Figure 3. Detection of cytokines in bone marrow progenitor cultures

Culture supernatants were collected at day 8 and evaluated for the expression of proinflammatory cytokines. Cultures with IL-5 only open bar; with IL-33 only, grey bars; with IL-5 + IL-33, black bars. Cytokines detected include: (A) IL-1α, (B) IL-1β, (C) IL-6, (D) IL-12p40, (E) IL-12p70, (F) G-CSF, (G) MCP-1, (H) MIP-1α, (I) MIP-1β (J) TNF-α (K) eotaxin-1 and (L) IL-13. Groups were compared by ANOVA followed by Bonferroni’s multiple comparison post-test, *p<0.05, **p<0.01, *** p<0.001; Data pooled from two experiments, n = 7 mice per data point.

Figure 4. GM-CSF but not M-CSF partially mediates the antagonistic effects of IL-33 on IL-5 dependent eosinophil differentiation

Cells were cultured as indicated and cell supernatants were collected and assayed by ELISA for the presence of: (A) M-CSF or (B) GM-CSF. (C) IL-5 (10 ng/ml) was supplemented with IL-33, M-CSF or GM-CSF or the combination of M-CSF and GM-CSF; percent eosinophils was determined on day 12 of culture. (D) Antibodies (5 μg/ml) directed against either M-CSF or GM-CSF or isotype controls (IgG2a for anti-GM-CSF antibody and IgG2b for anti-M-CSF antibody) were added to IL5 + IL-33 supplemented cultures and the percent eosinophils was determined on day 11 of culture. Groups were compared with unpaired t-test, ns = not significant; *** p<0.001, n = 4 – 7 mice per group.