Signalling mechanisms regulating the activation of human eosinophils by mast-cell-derived chymase: implications for mast cell-eosinophil interaction in allergic inflammation

Abstract

Allergic diseases such as asthma and allergic dermatitis are associated with the degranulation of mast cells. Chymase, a mast-cell-specific protease, is the major component in mast cell granules that can induce eosinophil infiltration into inflammatory sites. We examined the immunopathological mechanisms for the activation of eosinophils by chymase in allergic inflammation. Cytokines were measured by cytometric bead array Flex Sets multiplex assay using flow cytometry and enzyme-linked immunosorbent assay. Adhesion molecules, migration and intracellular signalling pathways were assessed by flow cytometry, Boyden chamber assay and Western blot, respectively. Chymase suppressed the apoptosis of eosinophils and induce the release of the cytokine interleukin-6 (IL-6) and chemokines CXCL8, CCL2 and CXCL1 by eosinophils dose-dependently. It also up-regulated the surface expression of adhesion molecule CD18 and stimulated the chemokinetic migration of eosinophils. The expressions of adhesion molecules, cytokines and chemokines, and chemokinetic migration were differentially regulated by the activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, Akt, Janus-activated kinase and nuclear factor-kappaB pathways. Chymase therefore plays a pivotal immunological role in the interaction between mast cells and eosinophils in allergic diseases such as allergic dermatitis by inducing adhesion molecule-mediated chemokinetic migration and inflammatory cytokines and chemokines of eosinophils, through multiple intracellular signalling molecules and transcription factor. Our results therefore provide a further biochemical basis for the pathogenesis of allergic inflammation consequent on the interaction between mast cells and eosinophils, and give insight for the development of new therapies.

Figure 1

Effects of chymase on the viability of eosinophils. Eosinophils (5 × 10⁵ cells) were treated with or without chymase (0·2–1 μg/ml) for 48 hr. The percentage of viable cells was assessed by the TACS™ Annexin V-FITC assay using flow cytometry. Representative dot plots of (a) medium control and (b) chymase-treated (1 μg/ml) eosinophils were shown from triplicate experiments. The lower left region denotes the viable eosinophils. (c) Bar chart for the dosage-dependent effect of chymase on the percentage viability of eosinophils was calculated from triplicate experiments. *P < 0·01. AV: annexin V.

Figure 2

Effects of chymase on the surface expression of CD18, intercellular adhesion molecule 1 (ICAM-1), ICAM-3 and CD62L. Eosinophils (5 × 10⁵ cells) were treated with or without chymase (1 μg/ml) for 24 hr. Surface expression of (a) CD18, (b) ICAM-1, (c) ICAM-3 and (d) CD62L on eosinophils was determined by flow cytometry. Isotypic control is denoted by a down arrow while medium-treated and chymase-treated cells are shown as black and grey lines, respectively. Results are expressed as histograms of relative cell counts with mean fluorescence intensity (MFI). These figures are representatives from three independent experiments with similar results. (e) Eosinophils (5 × 10⁵ cells) were cultured with or without chymase (0·2–1 μg/ml) for 8, 16 and 24 hr. Surface expression of CD18 of 10 000 cells was analysed by flow cytometry. Results have been normalized by subtracting the appropriate isotypic control and are expressed as the arithmetic mean ± SD of MFI from three independent experiments. *P < 0·05 when compared with medium control.

Figure 3

Chymase-induced releases of (a) CCL2, (b) CXCL8, (c) CXCL1 and (d) interleukin-6 (IL-6) from eosinophils. Eosinophils (5 × 10⁵ cells) were cultured with or without chymase (0·2–1 μg/ml) for 16, 24 and 48 hr. Release of chemokines and IL-6 from eosinophils into the culture supernatant was determined by CBA FlexSet kit using flow cytometry and enzyme-linked immunosorbent assay. Results are expressed as the mean ± SD. *P < 0·05, **P< 0·01 when compared with the medium control.

Figure 4

Effects of chymase on activation of (a) p38 mitogen-activated protein kinase (MAPK), (b) extracellular signal-regulated kinase (ERK), (c) Janus-activated kinase 2 (JAK2), (d) Akt and (e) inhibitor of nuclear factor-κB (IκB). Eosinophils (1 × 10⁷ cells) were treated with or without chymase (1 μg/ml) for the indicated incubation time. Total cellular proteins were extracted for the detection of total and phosphorylated signalling proteins by Western blot analysis. Experiments were performed in three independent experiments with essentially identical results, and representative blots are shown. Total protein was used as protein loading control.

Figure 5

Effects of AG490, SB203580, PD98059, LY294002, SP600125 and BAY117082 on the chymase-induced (a) cell surface expression of CD18, (b) chemokinetic migration, (c) CCL2, (d) CXCL8, (e) CXCL1 and (f) interleukin-6 (IL-6) of eosinophils. Eosinophils (5 × 10⁵ cells) were pretreated with inhibitors for 35 min, followed by incubation with or without chymase (1 μg/ml) in the presence of inhibitors for a further 24 hr. Induction of CD18 on the cell surface, of chemokinetic migration, and of chemokines and IL-6 in the culture supernatant were determined by flow cytometry, Boyden chamber assay, and CBA FlexSet by flow cytometer and enzyme-linked immunosorbent assay, respectively. Results are expressed as the mean ± SD from three independent experiments. Dimethyl sulphoxide (DMSO; 0·1%) was used as the DMSO control. *P< 0·05, **P < 0·01, ***P < 0·001 when compared with the chymase control. AG: AG490 (3 μm). SB: SB203580 (7·5 μm). PD: PD98059 (10 μm). LY: LY294002 (5 μm). SP: SP600125 (3 μm). BAY: BAY117082 (1 μm).