Intracellular signaling mechanisms regulating the activation of human eosinophils by the novel Th2 cytokine IL-33: implications for allergic inflammation

Abstract

The novel interleukin (IL)-1 family cytokine IL-33 has been shown to activate T helper 2 (Th2) lymphocytes, mast cells and basophils to produce an array of proinflammatory cytokines, as well as to mediate blood eosinophilia, IgE secretion and hypertrophy of airway epithelium in mice. In the present study, we characterized the activation of human eosinophils by IL-33, and investigated the underlying intracellular signaling mechanisms. IL-33 markedly enhanced eosinophil survival and upregulated cell surface expression of the adhesion molecule intercellular adhesion molecule (ICAM)-1 on eosinophils, but it suppressed that of ICAM-3 and L-selectin. In addition, IL-33 mediates significant release of the proinflammatory cytokine IL-6 and the chemokines CXCL8 and CCL2. We found that IL-33-mediated enhancement of survival, induction of adhesion molecules, and release of cytokines and chemokines were differentially regulated by activation of the nuclear factor (NF)-kappaB, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathways. Furthermore, we compared the above IL-33 activities with two structurally and functionally related cytokines, IL-1beta and IL-18. IL-1beta, but not IL-18, markedly upregulated cell surface expression of ICAM-1. IL-1beta and IL-18 also significantly enhanced eosinophil survival, and induced the release of IL-6 and chemokines CXCL8 and CCL2 via the activation of the NF-kappaB, p38 MAPK and ERK pathways. Synergistic effects on the release of IL-6 were also observed in combined treatment with IL-1beta, IL-18 and IL-33. Taken together, our findings provide insight into IL-33-mediated activation of eosinophils via differential intracellular signaling cascades in the immunopathogenesis of allergic inflammation.

Figure 1

Representative western blot of the protein expression of the IL-33 receptor ST2 in human eosinophils, neutrophils, HaCaT cells, HMC-1 cells and T lymphocytes. Total proteins were extracted from eosinophils, neutrophils, HaCaT cells, HMC-1 cells and T lymphocytes (1×10⁷ cells). Equal amounts of protein (15 μg) were analyzed by western blot. Experiments were performed in triplicate with essentially identical results, and a representative blot is shown. HMC, human mast cell line; IL, interleukin.

Figure 2

Effects of IL-1β, IL-18 and IL-33 on the viability of human eosinophils. Eosinophils (5×10⁵ per well) were cultured with or without (a) IL-1β, (b) IL-18 or (c) IL-33 (10–100 ng/ml) for 48 h in a 24-well plate. Percentage of viability of eosinophils was assessed by the TACS Annexin V-FITC assay using flow cytometry. *P<0.05, **P<0.01 when compared with medium control. FITC, fluorescein isothiocyanate; IL, interleukin.

Figure 3

Effects of IL-1β, IL-18 and IL-33 on surface expression of ICAM-1, ICAM-3 and L-selectin on human eosinophils. Eosinophils (5×10⁵ cells) were cultured with or without IL-1β, IL-18 or IL-33 (10–100 ng/ml) for 16 h. The surface expression of (a) ICAM-1, (b) ICAM-3 and (c) L-selectin on 10 000 cells was analyzed by flow cytometry as MFI, which was normalized by subtracting the appropriate isotypic control and shown as the arithmetic mean±SEM of three independent experiments. *P<0.05, **P<0.01, ***P<0.001 when compared with medium control. ICAM, intercellular adhesion molecule; IL, interleukin; MFI, mean fluorescence intensity.

Figure 4

Effects of IL-1β, IL-18 and IL-33 on CCL2, CXCL8 and IL-6 release from human eosinophils. Eosinophils (1×10⁶ cells) were cultured with or without IL-1β, IL-18 or IL-33 (50 ng/ml) for 12 and 24 h. Cell-free culture supernatant was collected and (a) CCL2, (b) CXCL8 and (c) IL-6 released into the supernatant were quantified using ELISA. Results are expressed as the arithmetic mean±SEM of three independent experiments. *P<0.05, **P<0.01 when compared with medium control. ELISA, enzyme-linked immunosorbent assay; IL, interleukin.

Figure 5

Synergistic effects of IL-1β, IL-18 and IL-33 on IL-6 release from human eosinophils. Eosinophils (1×10⁶ cells) were cultured with different concentrations of IL-1β, IL-18 and IL-33 in combination for 24 h. Cell-free culture supernatant was collected and IL-6 released into the supernatant was quantified using ELISA. Results are expressed as the arithmetic mean±SEM of three independent experiments. **P<0.01 when compared with medium control. ^#P<0.05, ^{# #}P<0.01 when compared with the sum of IL-6 production in treatment with individual cytokines (IL-33, IL-1β or IL-18) added alone. Ctrl, medium control; IL, interleukin; 1β(10), IL-1β (10 ng/ml); 1β(20), IL-1β (20 ng/ml); 18(10), IL-18 (10 ng/ml); 18(20), IL-18 (20 ng/ml); 33(10), IL-33 (10 ng/ml); 33(20), IL-33 (20 ng/ml).

Figure 6

Activation of ERK, p38 MAPK, NF-κB in human eosinophils upon IL-1β, IL-18 and IL-33 stimulation. Eosinophils (5×10⁵ cells) were cultured with or without IL-1β, IL-18 or IL-33 (50 ng/ml) for 15 min. After fixation and permeabilization, the intracellular contents of phosphorylated (a) ERK, (b) p38 MAPK and (c) IκB-α in 10 000 permeabilized eosinophils were measured by intracellular staining and flow cytometry. Results were normalized by subtracting the appropriate isotypic control and shown as the arithmetic mean±SEM of three independent experiments. *P<0.05, **P<0.01 when compared with medium control. Ctrl, medium control; ERK, extracellular signal-regulated kinase; IL, interleukin; MAPK, mitogen-activated protein kinase; NF, nuclear factor.

Figure 7

Effects of different inhibitors on IL-1β-, IL-18- and IL-33-induced survival enhancement of human eosinophils. Eosinophils (5×10⁵ cells) were pre-treated with inhibitors for 30 min followed by incubation with or without (a) IL-1β, (b) IL-18 and (c) IL-33 (50 ng/ml) in the presence of inhibitors for a further 48 h. Percent viability of eosinophils was assessed by the TACS Annexin V-FITC assay using flow cytometry and are expressed as the arithmetic mean±SEM of three independent experiments. *P<0.05, **P<0.01 when compared between groups denoted by the horizontal lines. AG, AG490; BAY, BAY11-7082; Ctrl, medium control; FITC, fluorescein isothiocyanate; IL, interleukin; LY, LY294002; U, U0126; SB, SB203580; SP, SP600125.

Figure 8

Effects of different inhibitors on IL-1β- and IL-33-induced adhesion molecule expression on human eosinophils. Eosinophils (5×10⁵ cells) were pre-treated with inhibitors for 30 min followed by incubation with or without IL-1β or IL-33 (50 ng/ml) in the presence of inhibitors for a further 16 h. (a, b) ICAM-1, (c) ICAM-3, (d) L-selectin expression on 10 000 cells was analyzed by flow cytometry as MFI, which was normalized by subtracting the appropriate isotypic control and shown as the arithmetic mean±SEM of three independent experiments. *P<0.05, **P<0.01, ***P<0.001 when compared between groups denoted by the horizontal lines. AG, AG490; BAY, BAY11-7082; Ctrl, medium control; LY, LY294002; U, U0126; SB, SB203580; SP, SP600125.

Figure 9

Effects of different inhibitors on IL-1β-, IL-18- and IL-33-induced CCL2, CXCL8 and IL-6 release from human eosinophils. Eosinophils (5×10⁵ cells) were pre-treated with inhibitors for 45 min followed by incubation with or without (a) IL-1β, (b) IL-18 and (c) IL-33 (50 ng/ml) in the presence of inhibitors for a further 24 h. Cell-free culture supernatant was collected and CCL2, CXCL8 and IL-6 released into the supernatant were quantified using ELISA. Results are expressed as the arithmetic mean±SEM of three independent experiments. *P<0.05, **P<0.01, ***P<0.001 when compared between groups denoted by the horizontal lines. AG, AG490; BAY, BAY11-7082; Ctrl, medium control; ELISA, enzyme-linked immunosorbent assay;LY, LY294002; U, U0126; SB, SB203580; SP, SP600125.