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-κB 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.

Introduction

Upon allergen provocation, cross-linkage of immunoglobulin E (IgE) bound on mast cells via the high-affinity receptors triggers the release of an array of inflammatory mediators including histamine, proteases and heparin sulphate, prostaglandins and cysteinyl leukotrienes, as well as various cytokines and chemokines that are involved in the recruitment and activation of mast cells and other leucocytes such as eosinophils.¹ In allergic diseases, mast cells can interact with infiltrated eosinophils at the inflammatory tissues to orchestrate late-phase chronic inflammation.

There is accumulating evidence that human mast cells contribute to the pathophysiology of asthma.² Mast cells, but not T lymphocytes or eosinophils, are abundant within the bronchial smooth muscle bundles in asthmatic patients but not in normal subjects or those with eosinophilic bronchitis. The mechanism of mast cell recruitment by asthmatic airway smooth muscle involves the CXCL10/CXCR3 axis, and several mast cell mediators have profound effects on airway smooth muscle function.³ The mast-cell-derived proteases tryptase and chymase have been shown to play an important immunological role in inflammation, matrix destruction, tissue remodelling, and bronchial hyperresponsiveness by destroying procoagulant, matrix, growth and differentiation factors and by activating proteinase-activated receptors (PAR), urokinase, metalloproteinases and angiotensin.^2,4 Tryptase is the most abundant secretory granule-derived serine proteinase contained in mast cells which have been shown to enhance macrophage and neutrophil chemokines CCL2 and CXCL8 production from human endothelial cells, transforming growth factor-β and stem cell factor from smooth muscle cells, and interleukin-6 (IL-6) and CXCL8 release from eosinophils.^5–7 Mast cell chymase is a chymotrypsin-like serine protease exclusively stored in mast cells and released by degranulation.⁸ It has been found to induce eosinophil migration and expression of CXCL8 in human eosinophilic leukaemia EoL-1 cells and mouse peritonitis eosinophils via the activation of extracellular signal-regulated kinase (ERK) pathway.^9,10 In animal models, chymase can increase microvascular permeability and marked accumulation of inflammatory cells including eosinophils, monocytes, neutrophils and macrophages.^11,12 Tryptase seems to participate in proinflammatory mast cell function, whereas chymase seems to be more involved in inflammatory reactions.⁸ Both of them can modulate the immune responses by hydrolysing chemokines and cytokines.⁴

The mast cell subtype MC_TC that expresses both chymase and tryptase is predominant in skin and peritoneum but not in lungs.¹ Moreover, the gene encoding chymase could be a susceptible gene that is related to the development of allergic dermatitis.¹³ Accumulation of chymase-containing mast cells has been found in skin biopsies of patients with allergic dermatitis.¹⁴ The chymase inhibitors SUN13350 and SUN C8257 were effective in treating the symptoms of allergic dermatitis.^15,16 Therefore, chymase activity can play a pathological role in the development of the allergic dermatitis, probably by facilitating eosinophil infiltration.¹⁶ The PAR are widely distributed on the cells of the airways to trigger inflammation stimulated by mast cell tryptase or chymase in allergic diseases because chymase and tryptase are potent activators of PAR-1 and PAR-2, respectively. Stimulation of epithelial cells by PAR opens tight junctions, causes desquamation, produces cytokines, chemokines and growth factors, and causes degranulation of eosinophils and mast cells.¹⁷

Previous studies of the effect of chymase on eosinophils were solely based on the eosinophil leukaemic cell line and mouse peritonitis eosinophils, the effects of chymase on primary human eosinophils have not been investigated. In an attempt to elucidate the detailed intracellular mechanisms regulating the activation of human eosinophils, we investigated the intracellular signalling mechanisms regulating the expression profile of adhesion molecules including: (1) integrin family: leucocyte function-associated antigen (LFA-1) (CD11a/ CD18); (2) selectin family: l-selectin (CD62L); and (3) immunoglobulin superfamily: intercellular adhesion molecule 1 (ICAM-1; CD54) and ICAM-3 (CD50);¹⁸ release of cytokines and chemokines, and cell migration of eosinophils activated by chymase.

Materials and methods

Reagents

Human recombinant chymase from human skin mast cells, cycloheximide and actinomycin D were purchased from Sigma-Aldrich Co (St Louis, MO); the inhibitor of nuclear factor-κB (NF-κB; i.e. IκBα) phosphorylation inhibitor BAY117082, ERK inhibitor PD98059, Jun N-terminal kinase (JNK) inhibitor SP600125, p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, phosphatidyl-inositol 3′-kinase (PI3K) inhibitor LY294002, and Janus-activated kinases (JAK) inhibitor AG490 were purchased from Calbiochem Corp., San Diego, CA. SB203580 and LY294002 were dissolved in water, while PD98059, SP600125, AG490 and BAY117082 were dissolved in dimethyl sulphoxide (DMSO). In all studies, the concentration of DMSO was 0·1% (v/v).

Isolation of human blood eosinophils from buffy coat and eosinophil culture

Fresh human buffy coat obtained from healthy volunteers from the Hong Kong Red Cross Blood Transfusion Service was diluted 1 : 2 with phosphate-buffered saline (PBS) at 4° and centrifuged using an isotonic Percoll solution (density 1·082 g/ml; Amersham and Pharmacia Biotech, Uppsala, Sweden) for 30 min at 1000 g. The eosinophil-rich granulocyte fraction was collected and washed twice with cold PBS containing 2% fetal bovine serum. The cells were then incubated with anti-CD16 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4° for 45 min and CD16-positive cells including neutrophils were depleted by passing through a LS⁺ column (Miltenyi Biotec) within a magnetic field. With this preparation, the drop-through fraction contained eosinophils with a purity of at least 99% as assessed by a Hemacolor rapid blood smear stain (E Merck Diagnostica, Darmstadt, Germany). The isolated eosinophils were cultured in RPMI-1640 medium (Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco Laboratories) and 20 mm HEPES (Gibco Laboratories).

Endotoxin-free solutions

Cell culture medium was purchased from Gibco Laboratories, free of detectable lipopolysaccharide (< 0·1 EU/ml). All other solutions were prepared using pyrogen-free water and sterile polypropylene plastic ware. No solution including chymase solution (2 μg/ml) contained detectable lipopolysaccharide, as determined by the Limulus amoebocyte lysate assay (sensitivity limit 12 pg/ml; Biowhittaker Inc., Walkersville, MD).

Apoptosis assay

Apoptosis of eosinophils was assessed by the TACS™ Annexin V-FITC assay (Trevigen Inc., Gaithersburg, MD) using flow cytometry (FACSCalibur, BD Biosciences Corp, San Jose, CA) on eosinophils gated on the basis of their forward and side light scatter with any cell debris excluded from analysis. The population of viable cells was characterized by low mean fluorescence intensity of both Annexin V-FITC and propidium iodide.

Protein array analysis of chemokines and cytokines in culture supernatant of eosinophils

The expression profile of 79 different cytokines in the culture supernatant of eosinophils was assessed semi-quantitatively using an antibody-based RayBio™ human cytokine array V (RayBiotech Inc., Norcross, GA).^19,20

Quantitative analysis of IL-6, CXCL8, CCL2 and CXCL1

Concentrations of cytokine IL-6, and chemokines CXCL8 and CCL2 in culture supernatant were measured simultaneously by bead-based multiplex assay with a BD cytometric bead array (CBA) Flex Set (BD Pharmingen Corp., San Diego, CA) using a four-colour FACSCalibur flow cytometer (BD Biosciences Corp).²¹ Concentration of CXCL1 was measured using an enzyme-linked immunsorbent assay kit from R&D Systems, Minneapolis, MN.

Flow cytometry of cell surface expression of adhesion molecules

Eosinophils (5 × 10⁵ cells/0·5 ml), after preceding treatments, were washed and resuspended in cold PBS supplemented with 0·5% bovine serum albumin. After blocking with 2% human pooled serum for 20 min at 4° and washing with PBS supplemented with 0·5% bovine serum albumin, cells were incubated either with fluorescein isothiocyanate-conjugated mouse anti-human adhesion molecule monoclonal antibody or fluorescein-conjugated mouse IgG1 and IgG2b isotype for 30 min at 4° in the dark. After washing, the cells were resuspended in 1% paraformaldehyde in 1 × PBS as fixative. Expression of surface adhesion molecule on 10 000 viable cells was then analysed by flow cytometry (BD Biosciences Corp) and presented as mean fluorescence intensity, which represents the changes of adhesion molecule expression on individual cells. We have excluded the dead cells by gating the viable cells with large size (forward scatter/FSC) and granularity (side scatter/SSC) in SSC–FSC dot plots for the analysis of the cell surface expression of adhesion molecules. Therefore, the results of the cell surface expression of adhesion molecules on eosinophils actually represented the adhesion molecule expression only on viable eosinophils.

Assay of cell migration

The chemokinetic assay was performed in a 48-well microchamber (Neuro Probe, Bethesda, MD). Briefly, eosinophils (1 × 10⁶ cells) were treated with chymase (0·01–1 μg/ml) for 16 hr. Cells were then added to the upper wells of the Boyden chamber, which was separated from the lower wells which contain chymase (0·01–1 μg/ml) in 25 μl RPMI-1640 by a 5-μm pore-size nitrocellulose membrane (Neuro Probe). The chamber was incubated for 6 hr at 37° in an atmosphere containing 5% CO₂. The cells that had migrated to the lower wells were counted at × 400 magnification. For each of the duplicate wells, the numbers of cells in four random fields were determined, and the arithmetic mean and SD were calculated.^11,20

Western blot analysis

Eosinophils were washed with ice-cold PBS, and lysed in 0·2 ml lysis buffer (20 mm Tris–HCl, pH 8·0, 120 mm NaCl, 1% Triton X-100, 10 mm ethylenediaminetetraacetic acid, 1 mm ethyleneglycoltetraacetic acid, 0·05% 2-mercaptoethanol, 1 × protease inhibitors). Cell debris was removed by centrifugation at 14 000 g for 15 min, and the supernatant was boiled in Laemmli sample buffer (Bio-Rad Laboratory, Hercules, CA) for 5 min. An equal amount of proteins was subjected to sodium dodecyl sulphate–10% polyacrylamide gel electrophoresis before blotting onto a polyvinylidene difluoride membrane (Amersham and Pharmacia Biotech). The membrane was blocked with 5% skimmed milk in Tris-buffered saline with 0·05% Tween-20, pH 7·6 for 1 hr at room temperature, and probed with anti-human total or phosphorylated-ERK, anti-human total or phosphorylated-p38 MAPK, anti-human total or phosphorylated-JAK2, anti-human total or phosphorylated-IκB-α, and anti-human total or phosphorylated-Akt antibody (Cell Signaling Technology Inc., Beverly, MA) at 4° overnight. After washing, the membrane was incubated with secondary donkey anti-rabbit antibody coupled to horseradish peroxidase (Amersham and Pharmacia Biotech) for 1 hr at room temperature. Antibody–antigen complexes were then detected using an enhanced chemiluminescent (ECL) detection system according to the manufacturer's instructions (Amersham and Pharmacia Biotech). The blots were visualized by exposing the membrane with the Hyperfilm™ ECL™ which is a high-performance chemiluminescence film used for Western blot analysis. The band signals on the hyperfilm were developed using the Kodax automatic film processor.¹⁹

Statistical analysis

All data were expressed as mean ± SD. Differences between groups were assessed by one-way analysis of variance and a P value < 0·05 was considered significantly different. All analyses were performed using the Statistical Package for the Social Sciences (spss) statistical software for Windows, version 10.1.4 (SPSS Inc., Chicago, IL).

Results

Chymase-enhanced eosinophil survival

As shown in Fig. 1, the in vitro viability of eosinophils after 48 hr of incubation was significantly enhanced from 25% to 55% by chymase at 1 μg/ml (P < 0·01).

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.

Chymase up-regulated cell surface expression of adhesion molecule CD18 on eosinophils

Figure 2 shows that chymase could up-regulate the surface expression of CD18 both dose (0·2–1 μg/ml) and time (8–24 hr) dependently but not CD62L/L-selectin, ICAM-1 and ICAM-3.

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.

Chymase enhanced the chemokinetic migration of eosinophils

To investigate the effect of chymase on the migration of eosinophils, checkerboard analysis was used to assess whether chymase treatment (0·01–1 μg/ml) could increase the migration of eosinophils to the lower wells of the Boyden chamber. As shown in Table 1, eosinophils treated with chymase (0·1 and 1 μg/ml) could facilitate the migration of eosinophils. The increase of migrated cell number was correlated to the chymase concentration on the upper compartment but not the lower compartment, indicating that chymase could exert a chemokinetic rather than a chemotactic effect on eosinophils.

Table 1.

Chemokinetic effect of chymase on eosinophils

	Upper compartment (μg/ml)
	0	0·01	0·1	1
Lower compartment (μg/ml)
0	16·6 ± 7·4	20·4 ± 8·5	22·3 ± 15·7	31·1 ± 7·7**
0·01	21·2 ± 6·2	20·7 ± 6·8	30·3 ± 9·8*	26·6 ± 12·6
0·1	21·2 ± 7·1	23·4 ± 12·9	29·7 ± 11·1*	33·4 ± 9·2*
1	18·1 ± 8·4	22·1 ± 5·3	28·1 ± 9·4*	34·4 ± 5·8***

Eosinophils (1 × 10⁶ cells) were treated with chymase (0·01–1 μg/ml) for 16 hr. They were then added to the upper wells of a Boyden chamber while the RPMI-1640 medium with chymase (0·01–1 μg/ml) was added to the lower wells. Migration was carried out for 6 hr and the numbers of cells migrating to the lower wells were counted. The experiments were repeated five times and the mean ± SD are shown.

^*P < 0·05,

^**P < 0·01,

^***P < 0·001 when compared with the medium control.

Chymase induced the release of CCL2, CXCL1, CXCL8 and IL-6 from eosinophils

Cytokine expression profiles induced by chymase were first screened using an antibody-based human cytokine protein membrane array. Results indicated that chymase could activate eosinophils to markedly induce the release of chemokines CCL2 (monocyte chemotactic protein-1), CXCL1 (growth-regulated oncogene-α), CXCL8 (IL-8) and inflammatory cytokine IL-6 among the 79 different cytokines being screened after a 48-hr incubation when compared with that of control (data not shown). Figure 3 shows that the release of the IL-6, CCL2 and CXCL8 from eosinophils upon chymase treatment was both concentration-dependent and time-dependent while the induction of CXCL1 was only concentration-dependent.

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.

Chymase activated the activities of ERK, p38 MAPK, JAK, Akt and NF-κB in eosinophils

As shown in Fig. 4, chymase (1 μg/ml) could rapidly induce the phosphorylation of p38 MAPK and Akt at 5 min. Phosphorylation of ERK, JAK2 and IκB was detected after 15 min of chymase treatment and sustained to 30 min.

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.

Chymase-induced adhesion molecules, chemokinetic migration, cytokines and chemokines were differentially regulated by intracellular MAPK, JAK, Akt and NF-κB of eosinophils

As shown in Fig. 5(a,b), pretreatment of eosinophils (5 × 10⁵ cells) with ERK inhibitor PD98059 and PI3K inhibitor LY294002 for 35 min could significantly suppress the chymase-induced up-regulation of CD18 and chemokinetic migration. JAK inhibitor AG490, JNK inhibitor SP600125 and NF-κB inhibitor BAY117082 did not exhibit any effect on the chymase-induced CD18 up-regulation. Figure 5(c, d) shows that except for the JNK inhibitor SP600125, pretreatment of the other five inhibitors could significantly reduce the chymase-induced release of CXCL8 and CCL2. While for CXCL1, only the p38 MAPK inhibitor SB203580 and NF-κB inhibitor BAY117082 could significantly reduce the chymase-induced secretion (Fig. 5e). As shown in Fig. 5(f), p38 MAPK inhibitor SB203580, NF-κB inhibitor BAY117082 and ERK inhibitor PD98059 all suppressed the chymase-induced IL-6 secretion.

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).

Discussion

The co-localization of MC_TC and eosinophils in inflamed sites such as skin lesions in allergic dermatitis suggests that the interaction and cross-talk of two leucocytes could play important roles in allergic inflammation. Actually, mast cells can prolong eosinophil survival and activate eosinophils to release cytokines and undergo degranulation for the release of granular eosinophil peroxidase and β-hexosaminidase, probably through tryptase.^6,22–24 However, the effect of chymase on primary human eosinophils has not been studied.

Since the serum concentration of chymase is less than 10 ng/ml in normal subjects and up to 100 ng/ml in patients suffering from anaphylaxis, the concentration of chymase in the microenvironment of the inflammatory sites during allergic inflammation should be around 10 times higher than the circulating level.²⁵ Therefore, we adopted the physiological concentration range of chymase for the activation of eosinophils, which should be ranged from 0·01 to 1 μg/ml in the microenvironment at the inflammatory sites. In the present study, we found that chymase could significantly delay the apoptosis of eosinophils and up-regulate the surface expression of adhesion molecule CD18 on eosinophils. CD18–ICAM-1-dependent adhesion of eosinophils to bronchial epithelial cells has been demonstrated.²⁶ Moreover, checkerboard analysis showed that chymase could promote chemokinetic migration of eosinophils in a dose-dependent manner that is similar to the chemoattractive effect of chymase on neutrophils and monocytes.¹¹ Results therefore indicated that chymase plays a crucial role for the induction of eosinophil transmigration and accumulation at local inflammatory sites, probably through the up-regulation of CD18.

The release of chemokines and inflammatory cytokines has been shown to play important pathological roles in allergic inflammation such as atopic dermatitis.²⁷ Release of chemokines CXCL1, CXCL8, CCL2 and the proinflammatory cytokine IL-6 from eosinophils was elevated upon chymase activation. The above IL-6 and chemokines were also released by eosinophils in allergic conditions upon stimulation by the allergen house dust mite Der p I protein and the T helper type 2 (Th2) cytokine IL-25.^19,28,29 Moreover, both the protein synthesis inhibitor cycloheximide and transcription inhibitor actinomycin D could significantly suppress the release of chemokines/cytokines upon chymase stimulation from eosinophils at 24 hr (data not shown). The above results therefore demonstrated that chymase actually induced newly synthesized IL-6 and chemokines rather than the release of preformed chemokines and cytokines in eosinophils.

Interleukin-6 is a crucial cytokine for mast cell maturation because it causes increases in cell size, frequency of chymase-positive cells and intracellular histamine levels.³⁰ The CXC chemokines CXCL1 and CXCL8 could provoke the accumulation of neutrophils and T lymphocytes in inflamed skin.³¹ The CC chemokine CCL2 could recruit monocytes and eosinophils.³² Besides, chymase could increase the microvascular permeability.¹¹ Together with other Th2 cytokines such as IL-5 and IL-4, and the eosinophil chemokines eotaxin and CCL5, increased secretion of mast cell chymase could further enhance eosinophilia and accumulation of other leucocytes, such as T cells, neutrophils and monocytes, to orchestrate allergic inflammation upon interaction between mast cells and eosinophils.

Progression of allergic diseases such as allergic dermatitis is a biphasic reaction, the Th2-mediated humoral response is dominant during the early acute phase, while the Th1-mediated inflammatory response occurs during the late chronic phase of development.³³ The proinflammatory cytokine IL-6 was shown to increase in chymase-stimulated eosinophils (up to 100-fold) in the present study. The concentration of IL-6 was also correlated with allergic dermatitis disease severity.³⁴ In dermal inflammation, eosinophils might collaborate with T lymphocytes, endothelial cells and keratinocytes to up-regulate the concentration of both local and circulating IL-6 during the early phase of inflammation.³⁵ Chymase might therefore resemble the stimulating effect of cytokines IL-17 and IL-25 by increasing CXCL1 and CXCL8 production to exacerbate the inflammation.³⁶

Another mast cell protease tryptase has also been well characterized for its pathological roles in allergic asthma.^4,17 Compared to chymase, tryptase could also induce the production of CCL2, CXCL8 and IL-6 in human endothelial cells and eosinophils, enhance the chemotaxis of mast cells, and induce neutrophil infiltration.^4–6,37

Previous studies have shown that p38 MAPK and ERK, but not JNK, are involved in chymase-induced chemotaxis and degranulation in eosinophils.^9,10 Tryptase induces IL-6 and CXCL8 release through the MAPK–AP-1 pathway.⁷ Our former study showed that p38 MAPK and NF-κB play crucial roles in the Th2 cytokine IL-25, leptin and allergen Der p I-mediated release of cytokines, chemokines and expression of adhesion molecules from eosinophils.^19,20,28,29 In this study, we have found that chymase could activate Akt, ERK, p38 MAPK, JAK2 and NF-κB but not JNK within 15 min. We used signalling molecule inhibitors to elucidate the intracellular signalling mechanisms regulating the induction of adhesion molecules and cytokines/chemokines. Following previous publications,^19,20,28,29 we used the optimal concentrations of AG490 (3 μm), PD98059 (10 μm), SB203580 (7·5 μm), LY294002 (5 μm), SP600125 (3 μm) and BAY117082 (1 μm) and an incubation time of 35 min which had the highest inhibitory effect without any cell toxicity. The inhibition experiments demonstrated that CD18 expression, chemokinetic migration, chemokines and IL-6 secretion were differentially regulated by JAK-signal transducer and activator of transcription factor, ERK, p38 MAPK, Akt-PI3K and NF-κB pathways. Other studies have also found that JNK was not involved in chymase-mediated activation of eosinophils.

In conclusion, the present report is the first demonstration of the activation of human blood eosinophils by chymase for the expression of chemokines, cytokines and adhesion molecules, and chemokinesis. Our results suggest that chymase-regulated apoptosis, expression of adhesion molecule LFA-1, chemokinesis and the release of IL-6 and chemokines from eosinophils are mediated by the combined activation of intracellular Akt, MAPK, JAK and NF-κB pathways. Together, these results provide new clues to the immunopathological mechanisms of mast-cell-mediated activation of eosinophils for allergic inflammation. In view of the recent development of chymase inhibitor for treating allergic dermatitis through the dual inhibition of the chymase-dependent IgE production pathway and itching sensation^38,39 and the application of MAPK and NF-κB inhibitors as potential anti-inflammatory agents and treatment of allergic diseases,⁴⁰ our study of intracellular mechanisms on the activation of eosinophils upon interacting with mast cells should provide new insights into the development of therapeutic interventions for allergic inflammatory diseases.