Human basophils and eosinophils are the direct target leukocytes of the novel IL-1 family member IL-33

Abstract

In mice, interleukin-18 (IL-18) regulates Th1- or Th2-type immune responses depending on the cytokine environment and effector cells involved, and the ST2-ligand, IL-33, primarily promotes an allergic phenotype. Human basophils, major players in allergic inflammation, constitutively express IL-18 receptors, while ST2 surface expression is inducible by IL-3. Unexpectedly, freshly isolated basophils are strongly activated by IL-33, but, in contrast to mouse basophils, do not respond to IL-18. IL-33 promotes IL-4, IL-13 and IL-8 secretion in synergy with IL-3 and/or FcepsilonRI-activation, and enhances FcepsilonRI-induced mediator release. These effects are similar to that of IL-3, but the signaling pathways engaged are distinct because IL-33 strongly activates NF-kappaB and shows a preference for p38 MAP-kinase, while IL-3 acts through Jak/Stat and preferentially activates ERK. Eosinophils are the only other leukocyte-type directly activated by IL-33, as evidenced by screening of p38-activation in peripheral blood cells. Only upon CD3/CD28-ligation, IL-33 weakly enhances Th2 cytokine expression by in vivo polarized Th2 cells. This study on primary human cells demonstrates that basophils and eosinophils are the only direct target leukocytes for IL-33, suggesting that IL-33 promotes allergic inflammation and Th2 polarization mainly by the selective activation of these specialized cells of the innate immune system.

Figure 1

IL-3 up-regulates ST2 in basophils, while IL-18 receptors are constitutively expressed. (A) Constitutive and inducible expression of IL-18R and ST2. Basophils were cultured overnight in the absence (nil) or presence of IL-3 (10 ng/mL) and analyzed by flow cytometry for cell surface IL-18R and ST2 expression. The histograms show an overlay of unstimulated (nil) and IL-3–stimulated basophils including isotype control. (B) Time course of IL-3–induced ST2 expression. Freshly isolated (f.i.) basophils were analyzed immediately or cultured for the time indicated without (nil) or with IL-3. Cell surface ST2 expression was analyzed by flow cytometry (top panel) and total protein expression of IL-3–treated cells by Western blotting (bottom panel). (C) Kinetics of induction of mRNA of ST2-isoforms. Basophils were cultured with IL-3 for the time indicated and mRNA was quantified by real-time RT-PCR using specific primers for ST2-isoforms: transmembrane/long, ST2L; soluble, sST2; all isoforms ST2, ST2 (all). The expression of ST2 in comparison to the reference gene β2M is given as RQ (relative quantity). Mean values of duplicates are shown. Representative experiments are shown in all panels.

Figure 2

IL-3–induced sST2 is sorted to basophils granules and released by triggers of exocytosis. (A) Immunofluorescence analysis of ST2 expression and release in human basophils. Cytospin preparations of basophils cultured for 20 hours in the presence of IL-3, with or without triggering degranulation by 10 nM C5a for 30 minutes, were stained with anti-ST2 mAb (green, top left panel) and anti-GzB mAb (green, bottom left panel). DNA was stained with Hoechst (blue, middle panels). Images were acquired using constant settings on a Nikon Eclipse E600 fluorescence microscope (Nikon, Tokyo, Japan) equipped with a 60×/1.4 NA oil-immersion objective lens using FITC and DAPI filter sets. A Nikon DXM 1200 digital camera was used to capture images. Merged images (right panels) were created in Adobe Photoshop 8.0.1 (Adobe Systems, San Jose, CA) without any alteration of the original digital images. (B,C) Degranulation of basophils triggers the release of sST2. (B) Protein extracts derived from basophils cultured overnight with or without IL-3 followed by stimulation with C5a or anti-FcϵRIα mAb (100 ng/mL; anti-IgER) for 30 minutes as indicated were analyzed for the presence of ST2 and Granzyme B (GzB) by Western blotting. (C) Purified basophils were cultured in medium, IL-3 or IL-33 (50 ng/mL) for 20 hours and subsequently stimulated with C5a or anti-FcϵRIα mAb as indicated. sST2 was measured in cell supernatants by specific ELISA. Mean values of triplicates are shown. The representative data shown in panels A, B, and C were from separate experiments with cells from different donors.

Figure 3

IL-33, but not IL-18, activates MAP-kinase and NF-kB signaling pathways in blood basophils. (A) IL-33 rapidly activates signaling pathways of the TLR/IL-1R family in freshly isolated blood basophils. Cells were stimulated with IL-33 or IL-18 (50 ng/mL each) for the indicated time periods. Western blot analysis of cell extracts shows the rapid activation of the MAP-kinases, Erk, JNK, and p38, and the phosphorylation and degradation of IκB-α in response to IL-33, but not to IL-18. The lack of responsiveness of human basophils to IL-18 was confirmed with different preparations of IL-18 from 2 different suppliers. (B) Anti-ST2 antibodies block IL-33–induced basophil activation. Basophils were left untreated, or pre-incubated with anti-ST2 antibodies (a mixture of 3 mAbs, clones 2A5, FB9, and HB12; 3 μg/mL each) or anti-CD28 (control mAb; at 10 μg/mL) for 15 minutes at 37°C. Cells were stimulated with 2 ng/mL IL-33 for 15 minutes as indicated. Erk and p38 activation and the degradation of IκB-α were assessed by Western blotting.

Figure 4

IL-33 promotes cytokine production and enhances IgE receptor–induced mediator release as efficiently as IL-3. (A) Direct and synergistic induction of secretion of Th2 cytokines and IL-8 by IL-3 and IL-33. Freshly isolated basophils were cultured in medium alone or with IL-18 (50 ng/mL), IL-33 (50 ng/mL), IL-3 (10 ng/mL) or in combination of IL-3 + IL-33 for 8 hours. Cytokine secretion was measured in the cell supernatants. Means plus SEM of 8 independent experiments are shown. (B,C) IL-33 augments IL-4, IL-13, IL-8, and LTC₄ production in basophils activated by IgE receptor crosslinking. (B) Cells were activated by a maximally effective concentration of anti-FcϵRIα mAb (anti-IgER; 100 ng/mL) alone or with IL-18 (50 ng/mL), IL-33 (50 ng/mL), IL-3 (10 ng/mL), or IL-33 + IL-3, and cytokines were measured in cell supernatants after culture for 8 hours. Mean plus SEM of 5 experiments with cells from different donors are shown. (C) Basophils were stimulated with increasing concentration of anti-IgER in the absence or presence of IL-3, IL-33, or IL-3 + IL-33 for 8 hours. Mean values plus or minus SEM of 2 independent experiments performed in duplicates are shown. Statistical analysis: The statistical significance of differences between the different experimental conditions (marked by lines for each pair of conditions) was analyzed using the Student t test (ns, not significant, *P < .05, **P < .01, ***P < .001).

Figure 5

IL-3 and IL-33 activate distinct signal transduction pathways leading to differential regulation of LTC₄ synthesis. (A) IL-3 and IL-33 activate distinct signaling pathways in basophils. Cells were stimulated with IL-3 or IL-33 at concentrations and for the time periods indicated. Activation of MAP-kinases, Erk, and p38, and transcriptional regulators, Stat3/Stat5 and IκB-α, and phosphorylation of cPLA₂ (P-cPLA₂) were analyzed by Western blotting. A representative experiment is shown. Analysis by densitometry of Western blots of 5 independent experiments showed that ERK activation by IL-3 was at least 9 times higher as compared with that induced by IL-33, while p38 activation in response to IL-33 was at least 4 times higher than that promoted by IL-3. The ratio of cPLA2 phosphorylation of cells treated with IL-3 versus cells treated with IL-33 ranged between 1.5 and 2.1. (B) IL-33 does not efficiently prime basophils for C5a-induced LTC₄ formation. Cells were primed with 10 ng/mL IL-3 or 50 ng/mL IL-33 for the time periods indicted (priming time) followed by stimulation with 10 nM C5a for 30 minutes. NA / none: nonprimed cells stimulated with C5a alone. (C) Culture with IL-33 does not potentiate IgE-dependent or -independent mediator release. Cells were cultured for 24 hours with IL-3 or IL-33 as indicated, followed by stimulation for 30 minutes with C5a or by IgER-crosslinking using anti-IgER mAb. LTC₄ levels (mean values + SEM of 3 experiments) in the cell supernatants are shown in panels B and C.

Figure 6

Biologic activities of IL-33 on leukocyte subpopulations in PBMCs. (A) Basophils are the only direct target cells for IL-33 within the mononuclear leukocyte fraction. Freshly isolated PBMCs were stimulated for 10 minutes with IL-33 (50 ng/mL) or TNF-α (10 ng/mL) and analyzed for p38 phosphorylation by flow cytometry. The top panels show the gating of leukocyte subpopulations in total PBMCs stained with anti-CD3 and anti-CRTH2 Abs in the lymphocyte (left) and monocyte (right) light scatter gates: Ba, CRTH2^high/CD3⁻ basophils; Th2, CD3⁺/CRTH2⁺ Th2 cells; T, CD3⁺ total T cells; CD3⁻Ly, CRTH2⁻/CD3⁻ cells in the lymphocyte scatter gate mainly composed of B cells and NK cells; Mo, CD3⁻ cells in the monocyte scatter gate. Bottom panels: Histograms show overlays of phospho-p38 (P-p38) staining in the gated subpopulations in unstimulated (nil, shaded area) and stimulated (solid line) cells. Only basophils are activated by IL-33 while all other subpopulations, except basophils, respond to TNF-α stimulation to variable degrees. A representative experiment is shown. Identical results were obtained with cells from 10 different donors. (B,C) IL-33 moderately enhances anti-CD3/CD28–induced cytokine release in primary in vivo polarized Th2 cells. Sorted CD4⁺/CRTH2⁻/CCR5⁺ (Th1) and CD4⁺/CRTH2⁺/CCR5⁻ (Th2) memory T cells were stimulated for 24 hours with plate-bound anti-CD3/CD28 mAbs (1 μg/mL each) in the presence of IL-33, IL-1β or IL-18 (all at 50 ng/mL) as indicated, and IL-4, IL-5, IL-13, and IFN-γ was measured in the cell supernatants. Panel B shows an example of CCR5/CRTH2 plots of the sorted Th1 and Th2 (left panels) cells and the cytokine profile in response to CD3/CD28-ligation (right panels; mean of 5 experiments). Panel C shows the effects of IL-33 on anti-CD3/CD28–induced secretion of IL-4, IL-5, and IL-13 by Th2 cells and of IL-33, IL-1β or IL-18 on IFN-γ release by Th1 cells. Shown are the mean of 5 independent experiments with cells isolated from different donors. Data from individual experiments and statistical analysis are shown in Figure S7.

Figure 7

IL-33 activates blood eosinophils. (A) Differential activation of eosinophils and neutrophils by IL-1 family members. Neutrophil and eosinophil granulocytes isolated from the same donors were stimulated with IL-1β, IL-18, IL-33 (all at 50 ng/mL), or TNF-α (100 ng/mL) for 10 minutes. Activation of ERK and p38 and the phosphorylation and degradation of IκB-α was analyzed by Western blotting. (B) IL-33 promotes IL-8 secretion in human eosinophils. Cells were incubated with the cytokines (all at 50 ng/mL) alone or in combination as indicated. After 24 hours, IL-8 was measured in the cell supernatants. Western blots of a representative experiment (of 4) are shown in A, and the mean plus or minus SEM of 4 independent experiments with cells from different donors is shown in panel B. The direct induction of IL-8 by IL-33 alone (control vs IL-33) as well as the synergistic induction by IL-33 in combination with cytokines of the GM-CSF family (IL-33 alone or 1 of the GM-CSF family members alone vs IL-33 combined with IL-3 or IL-5 or GM-CSF) were all statistically significant (P < .01 or P < .001) as assessed by the Student t test.