help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on April 28, 2005, doi:10.1165/rcmb.2005-0034OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2005-0034OCv1
33/2/186    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wong, C. K.
Right arrow Articles by Lam, C. W. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wong, C. K.
Right arrow Articles by Lam, C. W. K.
American Journal of Respiratory Cell and Molecular Biology. Vol. 33, pp. 186-194, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2005-0034OC

Interleukin-25–Induced Chemokines and Interleukin-6 Release from Eosinophils Is Mediated by p38 Mitogen-Activated Protein Kinase, c-Jun N-Terminal Kinase, and Nuclear Factor-{kappa}B

Chun K. Wong, Phyllis F. Y. Cheung, Wai K. Ip and Christopher W. K. Lam

Department of Chemical Pathology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong

Correspondence and requests for reprints should be addressed to Professor C. W. K. Lam, Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong. E-mail: waikeilam{at}cuhk.edu.hk


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Interleukin (IL)-25, a novel Th2 cytokine, is capable of amplifying allergic inflammation. We investigated the modulation of nuclear factor (NF)-{kappa}B and mitogen-activated protein kinases (MAPK) pathways in IL-25–activated eosinophils, the principal effector cells of allergic inflammation, for the in vitro release of chemokines including monocyte chemoattractant protein-1 (MCP-1), IL-8, and macrophage inflammatory protein (MIP)-1{alpha}, and inflammatory cytokine IL-6. Gene expression of chemokines and IL-6 was evaluated by RT-PCR, and concentrations of chemokines and cytokine were measured by cytokine protein array, cytometric bead array, and enzyme-linked immunosorbent assay. NF-{kappa}B, c-Jun amino-terminal kinase (JNK), and p38 MAPK activities in eosinophils were assessed by electrophoretic mobility shift assay and Western blot. IL-25 was found to upregulate the gene expression of chemokines MCP-1, MIP-1{alpha}, and IL-8, and cytokine IL-6, in eosinophils, and to significantly increase the release of the above chemokines and IL-6 from eosinophils. IL-25 could also activate the JNK, p38 MAPK, and NF-{kappa}B activities of eosinophils, while inhibitor of I{kappa}B-{alpha} phosphorylation (BAY11–7082), JNK (SP600125), and p38 MAPK (SB203580) could suppress the release of IL-8, MIP-1{alpha}, MCP-1, and IL-6. Together, the above results showed that the induction of MCP-1, MIP-1{alpha}, IL-8, and IL-6 in IL-25–activated eosinophils are regulated by JNK, p38 MAPK, and NF-{kappa}B pathways.

Key Words: chemokines • eosinophils • interleukin-25 • mitogen-activated protein kinase • nuclear factor-{kappa}B


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Eosinophils are the most important inflammatory effector cells accumulating at the site of allergic inflammation, for example, the airway submucosa (1). The activated eosinophils release cytotoxic molecules such as major basic protein, eosinophil peroxidase, eosinophilic cationic protein (ECP), lipid mediators, and cytokines that cause tissue damage and consequently the manifestation of allergic diseases, for example, allergic asthma (1). Eosinophils are also capable of producing and releasing a variety of proinflammatory cytokines such as interleukin (IL)-3, IL-4, IL-5, IL-6, IL-16, tumor necrosis factor (TNF)-{alpha}, and granulocyte-macrophage colony-stimulating factor (GM-CSF) together with chemokines including eotaxin, IL-8, macrophage inflammatory protein (MIP)-1, monocyte chemoattractant protein (MCP), and regulated upon activation normal T cell expressed and secreted (RANTES) (2). T-helper lymphocyte type 2 (Th2) cytokines, including IL-4 for IgE synthesis and IL-5 for eosinophil proliferation, are crucially involved in the local infiltration and activation of eosinophils (3), whereas other Th2 cytokines IL-10 and IL-13 are important in inducing airway hyperreactivity and allergic inflammation (4).

Th2 cytokines and certain chemokines play essential roles in the pathogenesis of allergic asthma (5). IL-25 (IL-17E) is a novel Th2 proinflammatory cytokine belonging to a newly discovered member of the IL-17 cytokine family with IL-17 receptor homology 1 (Rh1) (6, 7). IL-25 is secreted by CD4+ activated memory (CD45+RO+) T cells (8). Intranasal administration of IL-25 has been shown to induce the production of Th2 cytokines IL-4, IL-5, and IL-13 and the expression of chemokine eotaxin mRNA in the murine lung (9, 10). The induction of IL-4, IL-5, and IL-13 resulted in Th2-like response marked by increased serum IgE, IgG1, and IgA concentrations; eosinophilia in blood, bronchoalveolar lavage, and lung tissue; and the development of pathologic changes including eosinophilic infiltrates, epithelial cell hyperplasia/hypertrophy, increased mucus secretion, and airway hyperreactivity in mice (9, 10). Ikeda and colleagues (11) reported that bone marrow–derived mast cells could generate large amounts of IL-25 when the cells were challenged by IgE cross-linking, thereby indicating that mast cells may affect Th2 immunoresponse through the production of IL-25 (11). Therefore, IL-25 plays an important role in provoking allergic inflammation, especially in IgE-dependent atopic diseases and eosinophil-mediated late phase allergic reactions (8).

Transcription factor nuclear factor (NF)-{kappa}B was found to involve in the expression of many inflammatory cytokines and adhesion molecules of eosinophils during allergic inflammation (12, 13). IL-25 has also been shown to induce the activation of NF-{kappa}B for the production chemokine IL-8 (6). Our previous studies have indicated that the activation of NF-{kappa}B and p38 mitogen-activated protein kinase (MAPK) plays a role in TNF-{alpha}–activated signaling pathways regulating eotaxin release and the expression of adhesion molecules including intercellular adhesion molecule (ICAM)-1 in eosinophils (14–16). However, the IL-25–mediated intracellular signal transduction for the activation of eosinophils has not been fully studied. In an attempt to elucidate the intracellular signaling mechanisms of IL-25–activated eosinophils in allergic inflammation, we investigated the induction of chemokines and cytokines and the modulation of intracellular NF-{kappa}B and MAPK activities on the regulation of the release of chemokines and cytokines.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Reagents
Recombinant human IL-25 was purchased from R&D Systems, Inc. (Minneapolis, MN). Cycloheximide and actinomycin D were purchased from Sigma-Aldrich Co. (St. Louis, MO). I{kappa}B-{alpha} phosphorylation inhibitor BAY11-7082, JNK inhibitor SP600125, and p38 MAPK inhibitor SB203580 were purchased from Calbiochem Corp. (San Diego, CA). SB203580 was dissolved in water, whereas SP600125 and BAY11-7082 were dissolved in dimethyl sulfoxide (DMSO) (17). In all studies, the concentration of DMSO was 0.1% (vol/vol).

Isolation of Human Blood Eosinophils from Buffy Coat and Eosinophil Culture
Fresh human buffy coat obtained from the Hong Kong Red Cross Blood Transfusion Service was diluted 1:2 with phosphate-buffered saline (PBS) at 4°C and centrifuged using an isotonic Percoll solution (density 1.082 g/ml; Amersham and Pharmacia Biotech, Uppsala, Sweden) for 30 min at 1,000 x g. The eosinophil-rich granulocyte fraction was collected and washed twice with cold PBS containing 2% fetal calf serum. The cells were then incubated with anti-CD16 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4°C for 45 min, and CD16-positive neutrophils were depleted by passing through an LS+ column (Miltenyi Biotec) within a magnetic field. With this preparation, the drop-through fraction contained eosinophils with a purity of at least 98% as assessed by 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) and 20 mM Hepes (Gibco).

Endotoxin-Free Solutions
Cell culture medium was purchased from Gibco, free of detectable lipopolysaccharide (LPS) (< 0.1 EU/ml). All other solutions were prepared using pyrogen-free water and sterile polypropylene plasticware. No solution contained detectable LPS, as determined by the Limulus amoebocyte lyase assay (sensitivity limit 12 pg/ml; Biowhittaker Inc., Walkersville, MD).

Assay of Viability
The % viability and necrosis of eosinophils were assessed by propidium iodide assay using a FACSCalibur flow cytometer and MTT tetrazolium colorimetric assay (Becton Dickinson Biosciences Corp., San Jose, CA).

Reverse Transcription–Polymerase Chain Reaction
Total RNA was extracted using Tri-Reagent (Molecular Research Center Inc., Cincinnati, OH).

Extracted RNA was reverse transcribed into first-strand complementary DNA using First-Strand cDNA Synthesis Kit (Amersham Biosciences Corp., Piscataway, NJ). Polymerase chain reaction (PCR) was performed in a reaction mixture containing 3 mM MgCl2, 200 µM dNTPs, 1 unit of AmpliTaq Gold DNA polymerase (Applied Biosystems Ltd., Foster City, CA), 50 pmol of 5' and 3' primers (Invitrogen, Carlsbad, CA) in PCR reaction buffer (1 min each at 94°, 56°, and 72°C) for 30 cycles for ß-actin, IL-6, IL-8, MCP-1, and MIP-1{alpha} after an initial 12 min of denaturation at 94°C. All reverser transcription (RT)-PCR were performed in the linear range of the PCR reaction according to the preliminary experiments. PCR Primers were as the following: IL-8 sense, 5'-CTGTGTGAAGGTGCAGTTTTGCC-3' and antisense, 5'-CTCAGCCCTCTTCAAAAACTTCTCC-3', yielding a 237-bp product (18); MCP-1 sense, 5'-AATGCCCCAGTCACCTGCTGTTAT-3' and antisense, 5'-GCAATTTCCCCAAGTCTCTGTATC-3', yielding a 427-bp product (19); MIP-1{alpha} sense, 5'-GCTGACTACTTTGAGACGAGC-3' and antisense, 5'-CCAGTCCATAGAAGAGGTAGC-3', yielding a 252-bp product; IL-6 sense, 5'-ATGAACTCCTTCTCCACAAGC-3' and antisense, 5'-TGGACTGCAGGAACTCCTT-3', yielding a 610-bp product (18); ß-actin sense, 5'-AGCGGGAAATCGTGCGTG-3' and antisense, 5'-CAGGGTACATGGTGGTGCC-3', yielding a 300-bp product (16). After the amplification reaction using PTC-200 DNA Engine (MJ Research, Inc., Boston, MA), PCR products were electrophoresed on 2% agarose gel in TAE buffer (pH 8.0) and stained with ethidium bromide. The electrophorectic bands were documented with Gene Genius Gel Documentation System (Syngene Inc, Cambridge, UK).

Protein Array Analysis of Chemokines and Cytokines in Culture of Eosinophils
The expression profile of 79 different cytokines in culture supernatant of eosinophils was assessed semiquantitatively using antibody-based RayBio human cytokine array V (RayBiotech Inc., Norcross, GA) (20).

Quantitative Analysis of IL-8, MCP-1, MIP-1{alpha}, and IL-6
IL-8 and MCP-1 concentrations in culture supernatant were measured by cytometric bead array (CBA) using a FACSCalibur flow cytometer (BD) (21, 22), while IL-6 and MIP-1{alpha} were quantitated by ELISA kit from Diaclone Research Inc. (Besançon, France) and R&D Systems, respectively.

Flow Cytometry of Cell Surface Expression of ICAM-1
Eosinophils after preceding treatment were harvested and resuspended with cold PBS supplemented with 0.5% bovine serum albumin. After blocking with 2% human pooled serum for 20 min at 4°C and washed with PBS supplemented with 0.5% bovine serum albumin, cells were incubated either with FITC-conjugated mouse anti-human ICAM-1 monoclonal antibody or fluorescein-conjugated mouse IgG1 isotype for 30 min at 4°C in darkness. After washing, cells were finally resuspended in 1% paraformaldehyde in 1x PBS as fixative. Cell surface expression of ICAM-1 was then analyzed by flow cytometry in terms of mean fluorescence intensity.

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 EDTA, 1 mM EGTA, 0.05% 2-mercaptoethanol, 1x protease inhibitors). Cell debris was removed by centrifugation at 14,000 x 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 sulfate-10% polyacrylamide gel electrophoresis before blotting onto a PVDF 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 h at room temperature, and probed with primary rabbit anti-human ß-actin, anti-human phospho-p38 MAPK, anti-human phospho-JNK, anti-human phospho–I{kappa}B-{alpha}, anti-human total I-{kappa}B antibody (Cell Signaling Technology Inc., Beverly, MA), or anti-human IL-25 receptor (IL-17-Rh1; R&D Systems) at 4°C overnight. After washing, membrane was incubated with secondary donkey anti-rabbit antibody coupled to horseradish peroxidase (Amersham and Pharmacia Biotech) for 1 h at room temperature. Antibody–antigen complexes were then detected using ECL chemiluminescent detection system according to the manufacturer's instructions (Amersham and Pharmacia Biotech) (14).

Electrophoretic Mobility Shift Assay
Eosinophils were harvested and washed, and nuclear proteins were extracted with NE-PER nuclear and cytoplasmic extraction reagents (Pierce Chemical Co, Rockford, IL) according to the manufacturer's instructions. Equal amounts of nuclear protein extracts were subjected to a test for NF-{kappa}B protein/DNA binding using LightShift chemiluminescent EMSA kit (Pierce) with a biotin end-labeled NF-{kappa}B oligonucleotide (5'-AGT TGA GGG GAC TTT CCC AGG C-3') (Research Genetics Invitrogen Co., Huntsville, AL). Briefly, nuclear extracts were incubated with biotin end-labeled NF-{kappa}B oligonucleotide for 20 min at room temperature to allow DNA/protein binding. The DNA/protein complexes were then resolved by a 6% native polyacrylamide gel electrophoresis and transferred to a Hybond-N+ membrane (Amersham and Pharmacia Biotech). The biotin end-labeled DNA was detected using a streptavidin–horseradish peroxidase conjugate and a chemiluminescent substrate (16).

Statistical Analysis
All data were expressed as mean ± SD. Differences between groups were assessed by one-way ANOVA analysis. 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
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Protein Expression of IL-25 Receptor of Human Eosinophils
As shown in Figure 1, IL-25 could upregulate protein expression of IL-25 receptor (IL-17-Rh1) of eosinophils. Similar to the results of Western blot, eosinophils with or without the treatment of IL-25 also expressed mRNA for IL-25 receptor using RT-PCR (data not shown).



View larger version (37K):
[in this window]
[in a new window]
  		Figure 1. Representative Western blot of IL-25 receptor protein expression in eosinophils. Total protein was extracted from eosinophils (1 x 107/well) after treatment with or without IL-25 (50 ng/ml) for 24 h. Samples with equal protein amounts were analyzed using Western blot. Triplicate experiments were performed with essentially identical results.

 
Effect of IL-25 on Release of Chemokines and IL-6 from Eosinophils In Vitro
Figure 2 illustrates the cytokine expression profile using antibody based human cytokine protein membrane array. IL-25 could activate eosinophils to markedly induce the release of chemokines IL-8 (2j), MIP-1ß (3k), MCP-1 (3e), and inflammatory cytokine IL-6 (2h) among the 79 different cytokines being screened after 36 h incubation compared with that of control. Regarding the regulation of gene expression for chemokines and IL-6, IL-25 could also upregulate the mRNA gene expression of IL-8, MIP-1{alpha}, and MCP-1, and IL-6 that of eosinophils, after 4 h incubation (Figure 3). ß-Actin was used as positive control and its amount remained constant with or without IL-25 treatment. Therefore, the gene expression results confirmed the production of the corresponding chemokines and IL-6 in cytokine array. After culture for 36 h, cell viability was ~ 65–70% for IL-25 (10–100 ng/ml)-treated eosinophils comparing to only 35% viability without IL-25 treatment at 36 h using propidium iodide assay with flow cytometry and trypan blue exclusion method. Therefore, IL-25 could actually enhance the viability of eosinophils at 36 h. Moreover, IL-25 could activate eosinophils to release IL-8, MIP-1{alpha}, MCP-1, and IL-6 dose-dependently (0–100 ng/ml) at 8, 16, and 36 h (Figure 4). Moreover, the release at early time was significantly lower than that at the later time points (all P < 0.05) (Figure 4). As shown in Figure 5, release of IL-8, MIP-1{alpha}, MCP-1, and IL-6 induced by IL-25 could be suppressed by protein synthesis inhibitor cycloheximide and transcriptional inhibitor actinomycin D dose-dependently at 36 h incubation.



View larger version (53K):
[in this window]
[in a new window]
  		Figure 2. Representative profile of the release of cytokines from IL-25–activated eosinophils. Eosinophils (1 x 106/well) were treated with or without IL-25 (50 ng/ml) for 36 h. Cell-free culture supernatant was then harvested and 79 different cytokines in culture supernatant were semiquantitated using antibody-based RayBio human cytokine array V. Positive and negative controls were designated at (1a, 1b, 1c, 1 d, 8j, 8k) and (1e, 1f, 1 g, 8i), respectively. Triplicate experiments were performed with essentially identical results. Table listed the format of antibodies on the cytokine membrane array.

 


View larger version (33K):
[in this window]
[in a new window]
  		Figure 3. Representative RT-PCR analysis of ß-actin, IL-6, IL-8, MIP-1{alpha}, and MCP-1 mRNA expression in eosinophils. Total RNA was extracted from eosinophils (1 x 107/well) after treatment with or without IL-25 (50 ng/ml) for 4 h, and then reverse transcribed and analyzed by PCR. The ß-actin housekeeping gene was used as control. M, 100-bp molecular size marker; CTL, control.

 


View larger version (21K):
[in this window]
[in a new window]
  		Figure 4. IL-25–induced release of (A) IL-6, (B) MIP-1{alpha}, (C) IL-8, and (D) MCP-1 from eosinophils. Eosinophils (5 x 105/well) were cultured with or without IL-25 (10–100 ng/ml) for 8, 16, and 36 h in a 24-well plate. Chemokines and cytokines released into the culture supernatant were determined by human chemokine CBA kit using flow cytometry and ELISA as described in MATERIALS AND METHODS. Results are expressed as the arithmetic mean ± SD from three independent experiments. *P < 0.01, **P < 0.001 when compared with the control.

 


View larger version (25K):
[in this window]
[in a new window]
  		Figure 5. Effect of cycloheximide and actinomycin D on IL-25–induced release of (A, B) IL-6, (C, D) MIP-1{alpha}, (E, F) IL-8, and (G, H) MCP-1 from eosinophils. Eosinophils (5 x 105/well) were cultured with or without IL-25 (50 ng/ml) in the presence of actinomycin D or cycloheximide for 36 h in a 24-well plate. Chemokines and cytokines released into the culture supernatant were determined by human chemokine CBA kit using flow cytometry and ELISA as described in MATERIALS AND METHODS. Results are expressed as the arithmetic mean plus SD from three independent experiments. *P < 0.01, **P < 0.001 when compared with the control. Act, actinomycin D; Cyclo, cycloheximide.

 
Effect of IL-25 on the Cell Surface Expression of ICAM-1 on Eosinophils
As shown in Table 1, IL-25 could upregulate the cell surface expression of ICAM-1 on eosinophils dose-dependently from 10–100 ng/ml.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Effect of IL-25 on cell surface expression of ICAM-1 on eosinophils

 
Effect of IL-25 on the Activation of MAPK
Figures 6A and 6B show that IL-25 could activate eosinophils by rapidly inducing the phosphorylation of p38 MAPK and JNK within 1 h, thereby indicating the activation of p38 MAPK and JNK in eosinophils by IL-25. The specific p38 MAPK inhibitor SB203580 could inhibit the p38 MAPK activity by diminishing the enzymatic product phosphorylated ATF-2 (Figure 6C). ß-Actin was used as control to ensure an equal protein amount was loaded at different time points (Figure 6D).



View larger version (38K):
[in this window]
[in a new window]
  		Figure 6. Activation of p38 MAPK and JNK activities in eosinophils. Eosinophils (1 x 107/well) were cultured with or without IL-25 (50 ng/ml) for different indicated incubation times in the presence or absence of SB203580 (10 µM). Total cellular proteins were extracted from eosinophils for the measurement of (A) phosphorylated p38 MAPK and (B) phosphorylated JNK proteins by Western blot analysis, (C) p38 MAPK activity at incubation time 30 min by the detection of phosphorylated ATF-2 using p38 MAP Kinase assay kit (Cell Signaling), and (D) control protein ß-actin by Western blot analysis. Experiments were performed in three independent replicates with essentially identical results, and representative results are shown. SB, SB 203580.

 
Effect of IL-25 on NF-{kappa}B Activity of Eosinophils
As shown in Figure 7A, eosinophils without any treatment exhibited relatively low amount of phospho–I{kappa}B-{alpha}. IL-25–treated eosniophils could activate the NF-{kappa}B activity by inducing the phosphorylation of I{kappa}B-{alpha} at 1 h, while the total I{kappa}B-{alpha} amount remained constant (Figure 7B). Figure 7C illustrates that untreated eosinophils showed a low shifted band, thereby indicating a basal activity of NF-{kappa}B. Eosinophils treated with IL-25 (50 ng/ml) for 4 h were shown to activate NF-{kappa}B for subsequent gene transcription, because there was a significant increase in band shift observed compared with the relative intensity of the shifted bands. Pretreatment with BAY (2.5 µM) for 1 h was found to inhibit the band shift in eosinophils with or without IL-25 treatment. Competitive control at lane 6 using excessive unlabeled p50 NF-{kappa}B binding DNA to totally suppress the IL-25–induced band shift confirmed the specificity of NF-{kappa}B–DNA interaction.



View larger version (41K):
[in this window]
[in a new window]
  		Figure 7. Effect of IL-25 on NF-{kappa}B activity in eosinophils. (A, B) Eosinophils (1 x 107/well) were cultured with or without IL-25 (50 ng/ml). Total protein was then extracted from eosinophils at indicated times. (A) Phosphorylated and (B) total I{kappa}B-{alpha} were then analyzed by Western blot. (C) Eosinophils (1 x 107/well) were cultured with or without IL-25 (50 ng/ml) for 4 h with or without 1 h pretreatment and subsequent 4 h incubation of BAY11-7082 (2.5 µM). Nuclear proteins were extracted from eosinophils; 5 µg protein was then subjected to EMSA and relative intensity of shifted band was detected by densitometry. Experiments were performed in three independent replicates with essentially identical results, and representative results are shown. +, presence; –, absence. Lane 1: negative control (labeled DNA probe only); lane 2: eosinophils only; lane 3: eosinophils + IL-25; lane 4: BAY11-7082 only; lane 5: eoinophils + IL-25 + BAY11-7082; lane 6: eosinophils + IL-25 (competitive control with unlabeled p50 NF-{kappa}B binding DNA).

 
Effect of Different Inhibitors on the Viability of Eosinophils
Figure 8 shows that BAY11–7082 but not SP600125 or SB203580 could suppress the eosinophil viability in a dose-dependent manner at 36 h incubation.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 8. The effect of different inhibitors on the relative viability of eosinophils. Eosinophils (5 x 105/well) were treated with SP600125, BAY11—7082, or SB203580 at various concentrations for 36 h. The relative viability of eosinophils was determined by MTT colorimetric assay. Results are expressed as the arithmetic mean ± SD from three independent experiments.

 
Effect of BAY11-7082, SP600125, and SB203580 on IL-25–Induced Release of IL-8, MIP-1{alpha}, MCP-1, and IL-6 from Eosinophils
As shown in Figure 9, pretreatment of eosinophils with NF-{kappa}B inhibitor BAY11-7082 (2.5 µM), JNK inhibitor SP600125 (3 µM), and p38 MAPK inhibitor SB203580 (10 µM) for 1 h could significantly suppress the IL-25–induced release of IL-6, MIP-1{alpha}, IL-8, and MCP-1 (all P < 0.05).



View larger version (31K):
[in this window]
[in a new window]
  		Figure 9. Effect of BAY11-7082, SP600125, and SB203580 on the IL-25–induced release of (A) IL-6, (B) MIP-1{alpha}, (C) IL-8, and (D) MCP-1 from eosinophils. Eosinophils (5 x 105/well) were pretreated with BAY11–7082 (2.5 µM), SP600125 (3 µM), or SB203580 (10 µM) for 1 h followed by incubation with or without IL-25 (50 ng/ml) in the presence of inhibitors for further 36 h. Release of chemokines in culture supernatant was determined by human chemokine CBA kit or ELISA as described in MATERIALS AND METHODS. Results are expressed as the arithmetic mean ± SD from three independent experiments. DMSO (0.1%) was used as the DMSO control. *P < 0.05, **P < 0.001 when compared with the IL-25 control.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Although IL-25/IL-17E is structurally related to IL-17, the in vivo and in vitro biological activities of IL-25 are different from IL-17 and other IL-17–related family members such as IL-17B and IL-17C (9). Our previous clinical study has revealed the elevation of plasma IL-17 concentration in patients with allergic asthma (23), and the median plasma concentration of IL-25 in patients with allergic asthma was 86 pg/ml, which was higher than that of control subjects (< 50 pg/ml). In animal studies, administration of IL-25 results in the induction of Th2 cytokines IL-4, IL-5, and IL-13, eosinophil chemokine eotaxin, eosinophilia, elevated serum IgE concentration, and the development of striking histologic changes in the lungs, mucus production, and airway hyperreactivity (9, 10). Chemokines and cytokines have been shown to play important pathologic roles in allergic inflammation (21, 24). In the present study, we first used cytokine protein array to screen for the induction of 79 diffferent cytokines and chemokines from eosinophils by IL-25. The results of the array analysis (Figure 2) in conjunction with those of CBA and ELISA (Figure 4) confirmed that IL-25 could selectively induce the significant in vitro release from eosinophils of inflammatory cytokine IL-6 for Th2 cells; CXC chemokine IL-8 for neutrophils; CC chemokine MCP-1 for monocytes, macrophages, eosinophils, and Th2 cells; and chemokine MIP-1{alpha} for monocytes and natural killer (NK) cells. In Figure 5, both the protein synthesis inhibitor cycloheximide and transcriptional inhibitor actinomycin D could dose-dependently suppress the release of chemokines/cytokines upon IL-25 stimulation from eosinophils at 36 h. In conjunction with the upregulation of mRNA expression of chemokines/cyokines by IL-25 (Figure 3), all the above results demonstrated that IL-25 actually induced newly synthesized chemokines and IL-6 rather than preformed chemokines and cytokines in eosinophils.

A recent study has shown that chemokines MIP-1{alpha} and IL-8 could be upregulated in body fluids or tissues 4–12 h after allergen challenge, and IL-6 was consistently increased after 18–24 h (25). MCP-1 can trigger chemotaxis of eosinophils via the CCR2 receptor (26). It has also been shown to provoke aggregation and release of histamines and prostaglandin D2 from mast cells for IgE-mediated hypersensitivity (27), and its production is increased in allergic asthma (28). Our present study of the induction of Th2 chemokine MCP-1 and Th2 cytokine IL-6 from eosinophils confirmed that Th2 cytokine IL-25 could provoke the Th2 response. The induction of IL-8 and MIP-1{alpha} can therefore mediate the infiltration of neutrophils, monocytes, and NK cells into the inflammatory sites at bronchial airway, thereby amplifying inflammatory responses during allergic asthma.

A recent study has shown that eosinophils may upregulate their chemokine receptors upon cytokine activation and use them during the progression of allergic inflammation for eosinophilic migration and activation (29). Because eosinophils express CCR1 and CCR2 for MIP-1{alpha} and MCP-1, respectively, the modulation of chemokine receptors on eosinophils by IL-25 should also be investigated. Besides inducing eosinophilia, IL-25 can also cause neutrophil infiltration (10). Our results showed that IL-25 could induce the secretion of neutrophil chemokine IL-8 from eosinophils. Therefore, this study also provides further explanation of the mechanisms for IL-25–mediated neutrophil migration.

According to our present results, IL-25 could also upregulate the ICAM-1 cell surface expression dose-dependently (Table 1), delay apoptosis of eosinophils, and may enhance the adhesion of eosinophils onto bronchial epithelial cells. Therefore, apart from the induction of chemokines, IL-25 could induce the eosinophilia in bronchoalveolar lavage and lung tissue by the increase of its adhesion onto epithelial cells and the subsequent transmigration into the inflammatory sites. Moreover, IL-25 could induce eotaxin release in lung tissue that can cause eosinophilia (10). Together with the induction of Th2 and eosinophil chemokine MCP-1, Th2 cytokine IL-6, neutrophil chemokine IL-8, and NK and monocyte chemokine MIP-1, IL-25 should play an important role for the eosinophilia, Th2 immune response, and infiltration of inflammatory cells into inflammatory sites that are immunopathologic characteristics during allergic asthma.

Our former study has shown that p38 MAPK and NF-{kappa}B play crucial roles in the TNF-{alpha}–mediated release of eotaxin from eosinophils (14). The present intracellular mechanistic study indicates that IL-25 can activate JNK, p38, and NF-{kappa}B activities (Figures 6 and 7). We used inhibitors BAY11-7082, SP600125, and SB203580 to elucidate the intracellular signaling mechanisms regulating the induction of chemokines. Following previous publications (14, 16, 30, 17, 31) and the toxicity results in Figure 8, we used the optimal concentration of BAY11-7082 (2.5 µM), SP600125 (3 µM), or SB203580 (10 µM) with the highest inhibitory effect without any cell toxicity. The inhibition experiments demonstrated that the production of IL-8, MIP-1{alpha}, MCP-1, and IL-6 was mediated by the intracellular JNK, p38 MAPK, and NF-{kappa}B activities. It is because p38 MAPK, JNK, and NF-{kappa}B are commonly signal transduction molecules by which the expression of cytokines and chemokines can be regulated in eosinophils (32), it is reasonable that the above signaling molecules are commonly involved in IL-25–mediated release of IL-8, MIP-1{alpha}, MCP-1, and IL-6 from eosinophils.

In conclusion, this is a first report on the activation of eosinophils for the release of chemokines and cytokines by IL-25. Our result suggest that IL-25–induced release of MCP-1, MIP-1{alpha}, IL-8, and IL-6 from eosinophils is mediated by the combined activation of MAPK and NF-{kappa}B pathways, thereby providing new clues for the elucidation of immunopathologic mechanisms of Th2- and eosinophil-mediated allergic inflammation in pulmonary disorders. Further investigations are required for other potential intracellular signaling pathways (e.g., Janus-activated kinase [JAK]–signal transducer and activator of transcription [STAT]) for the regulation of the release of chemokines and cytokines. In view of recent advances in the application of p38 MAPK and NF-{kappa}B inhibitors as potential anti-inflammatory agents in asthma (33, 34), our study of IL-25 on eosinophil activation should provide new insights on the development of novel treatment for allergic disorders.


   Footnotes
 
This study was supported by a Chinese University of Hong Kong Direct Grant for Research.

Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form January 19, 2005

Received in final form April 26, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Humbles AA, Lloyd CM, McMillan SJ, Friend DS, Xanthou G, McKenna EE, Ghiran S, Gerard NP, Yu C, Orkin SH, et al. A critical role for eosinophils in allergic airways remodeling. Science 2004;305:1776–1779.[Abstract/Free Full Text]
  2. Giembycz MA, Lindsay MA. Pharmacology of the eosinophil. Pharmacol Rev 1999;51:213–340.[Abstract/Free Full Text]
  3. Broide DH. Molecular and cellular mechanisms of allergic disease. J Allergy Clin Immunol 2001;108:S65–S71.[CrossRef][Medline]
  4. KleinJan A, Dijkstra MD, Boks SS, Severijnen LA, Mulder PG, Fokkens WJ. Increase in IL-8, IL-10, IL-13, and RANTES mRNA levels (in situ hybridization) in the nasal mucosa after nasal allergen provocation. J Allergy Clin Immunol 1999;103:441–450.[CrossRef][Medline]
  5. Romagnani S. Cytokines and chemoattractants in allergic inflammation. Mol Immunol 2002;38:881–885.[CrossRef][Medline]
  6. Lee J, Ho WH, Maruoka M, Corpuz RT, Baldwin DT, Foster JS, Goddard AD, Yansura DG, Vandlen RL, Wood WI, et al. IL-17E, a novel proinflammatory ligand for the IL-17 receptor homolog IL-17Rh1. J Biol Chem 2001;276:1660–1664.[Abstract/Free Full Text]
  7. Moseley TA, Haudenschild DR, Rose L, Reddi AH. Interleukin-17 family and IL-17 receptors. Cytokine Growth Factor Rev 2003;14:155–174.[CrossRef][Medline]
  8. Kempuraj D, Frydas S, Conti P, Kandere-Grzybowska K, Boucher W, Letourneau R, Madhappan B, Huang SH, Sugimoto K, Papadopoulou NG, et al. Interleukin-25 (or IL-17E): a new IL-17 family member with growth factor/inflammatory actions. Int J Immunopathol Pharmacol 2003;16:185–188.[Medline]
  9. Fort MM, Cheung J, Yen D, Li J, Zurawski SM, Lo S, Menon S, Clifford T, Hunte B, Lesley R, et al. IL-25 induces IL-4, IL-5, and IL-13 and Th2-associated pathologies in vivo. Immunity 2001;15:985–995.[CrossRef][Medline]
  10. Hurst SD, Muchamuel T, Gorman DM, Gilbert JM, Clifford T, Kwan S, Menon S, Seymour B, Jackson C, Kung TT, et al. New IL-17 family members promote Th1 or Th2 responses in the lung: in vivo function of the novel cytokine IL-25. J Immunol 2002;169:443–453.[Abstract/Free Full Text]
  11. Ikeda K, Nakajima H, Suzuki K, Kagami S, Hirose K, Suto A, Saito Y, Iwamoto I. Mast cells produce interleukin-25 upon Fc epsilon RI-mediated activation. Blood 2003;101:3594–3596.[Abstract/Free Full Text]
  12. Yang L, Cohn L, Zhang DH, Homer R, Ray A, Ray P. Essential role of nuclear factor kappaB in the induction of eosinophilia in allergic airway inflammation. J Exp Med 1998;188:1739–1750.[Abstract/Free Full Text]
  13. Blackwell TS, Christman JW. The role of nuclear factor-{kappa}B in cytokine gene regulation. Am J Respir Cell Mol Biol 1997;17:3–9.[Abstract/Free Full Text]
  14. Wong CK, Zhang JP, Ip WK, Lam CWK. Activation of p38 mitogen-activated protein kinase and NF-{kappa}B in tumor necrosis factor-{alpha}-induced eotaxin release of human eosinophils. Clin Exp Immunol 2002;128:483–489.[CrossRef][Medline]
  15. Ip WK, Wong CK, Lam CWK. Tumor necrosis factor-{alpha}-induced expression of intercellular adhesion molecule-1 on human eosinophilic leukemia EoL-1 cells is mediated by the activation of nuclear factor-{kappa}B pathway. Clin Exp Allergy 2003;33:241–248.[CrossRef][Medline]
  16. Wong CK, Ip WK, Lam CWK. IL-3, -5, and GM-CSF–induced adhesion molecule expression on eosinophils by p38 MAPK and NF-{kappa}B. Am J Respir Cell Mol Biol 2003;29:133–147.[Abstract/Free Full Text]
  17. Mori N, Yamada Y, Ikeda S, Yamasaki Y, Tsukasaki K, Tanaka Y, Tomonaga M, Yamamoto N, Fujii M. Bay 11–7082 inhibits transcription factor NF-kappaB and induces apoptosis of HTLV-I-infected T-cell lines and primary adult T-cell leukemia cells. Blood 2002;100:1828–1834.[Abstract/Free Full Text]
  18. Wan S, Yim AP, Wong CK. Expression of FHL2 and cytokine messenger RNAs in human myocardium after cardiopulmonary bypass. Int J Cardiol 2002;86:265–272.[CrossRef][Medline]
  19. Jordan NJ, Kolios G, Abbot SE, Sinai MA, Thompson DA, Petraki K, Westwick J. Expression of functional CXCR4 chemokine receptors on human colonic epithelial cells. J Clin Invest 1999;104:1061–1069.[Medline]
  20. Huang RP. Detection of multiple proteins in an antibody-based proteinmicroarray system. J Immunol Methods 2001;255:1–13.[CrossRef][Medline]
  21. Wong CK, Lam CWK. Clinical applications of cytokine assays. Adv Clin Chem 2003;37:1–46.[Medline]
  22. Tarnok A, Hambsch J, Chen R, Varro R. Cytometric bead array to measure six cytokines in twenty-five microliters of serum. Clin Chem 2003;49:1000–1002.[Free Full Text]
  23. Wong CK, Ho CY, Ko FW, Chan CH, Ho AS, Hui DS, Lam CW. Proinflammatory cytokines (IL-17, IL-6, IL-18 and IL-12) and Th cytokines (IFN-gamma, IL-4, IL-10 and IL-13) in patients with allergic asthma. Clin Exp Immunol 2001;125:177–183.[CrossRef][Medline]
  24. Lukacs NW, Tekkanat KK. Role of chemokines in asthmatic airway inflammation. Immunol Rev 2000;177:21–30.[CrossRef][Medline]
  25. Ferreira MA. Cytokine expression in allergic inflammation: systematic review of in vivo challenge studies. Mediators Inflamm 2003;12:259–267.[CrossRef][Medline]
  26. Dunzendorfer S, Kaneider NC, Kaser A, Woell E, Frade JM, Mellado M, Martinez-Alonso C, Wiedermann CJ. Functional expression of chemokine receptor 2 by normal human eosinophils. J Allergy Clin Immunol 2001;108:581–587.[CrossRef][Medline]
  27. Conti P, Boucher W, Letourneau R, Feliciani C, Reale M, Barbacane RC, Vlagopoulos P, Bruneau G, Thibault J, Theoharides TC. Monocyte chemotactic protein-1 provokes mast cell aggregation and [3H]5HT release. Immunology 1995;86:434–440.[Medline]
  28. Rose CE Jr, Sung SS, Fu SM. Significant involvement of CCL2 (MCP-1) in inflammatory disorders of the lung. Microcirculation 2003;10:273–288.[CrossRef][Medline]
  29. Oliveira SH, Lira S, Martinez-A C, Wiekowski M, Sullivan L, Lukacs NW. Increased responsiveness of murine eosinophils to MIP-1beta (CCL4) and TCA-3 (CCL1) is mediated by their specific receptors, CCR5 and CCR8. J Leukoc Biol 2002;71:1019–1025.[Abstract/Free Full Text]
  30. Wong CK, Wang CB, Ip WK, Tian YP, Lam CW. Role of p38 MAPK and NF-kB for chemokine release in coculture of human eosinophils and bronchial epithelial cells. Clin Exp Immunol 2005;139:90–100.[CrossRef][Medline]
  31. Bennett BL, Sasaki DT, Murray BW, O'Leary EC, Sakata ST, Xu W, Leisten JC, Motiwala A, Pierce S, Satoh Y, et al. SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci USA 2001;98:13681–13686.[Abstract/Free Full Text]
  32. Wong CK, Ip WK, Lam CWK. Biochemical assessment of intracellular signal transduction pathways in eosinophils: implications for pharmacotherapy. Crit Rev Clin Lab Sci 2004;41:79–113.[CrossRef][Medline]
  33. Newton R, Holden N. Inhibitors of p38 mitogen-activated protein kinase: potential as anti-inflammatory agents in asthma? BioDrugs 2003;17:113–129.[CrossRef][Medline]
  34. Makarov SS. NF-kappaB as a therapeutic target in chronic inflammation: recent advances. Mol Med Today 2000;6:441–448.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 ChestHome page
J.-S. Jung, B. L. Park, H. S. Cheong, J. S. Bae, J.-H. Kim, H. S. Chang, T. Rhim, J.-S. Park, A.-S. Jang, Y.-M. Lee, et al.
Association of IL-17RB Gene Polymorphism With Asthma
Chest, May 1, 2009; 135(5): 1173 - 1180.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
R. Caruso, C. Stolfi, M. Sarra, A. Rizzo, M. C. Fantini, F. Pallone, T. T. MacDonald, and G. Monteleone
Inhibition of monocyte-derived inflammatory cytokines by IL-25 occurs via p38 Map kinase-dependent induction of Socs-3
Blood, April 9, 2009; 113(15): 3512 - 3519.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Swaidani, K. Bulek, Z. Kang, C. Liu, Y. Lu, W. Yin, M. Aronica, and X. Li
The Critical Role of Epithelial-Derived Act1 in IL-17- and IL-25-Mediated Pulmonary Inflammation
J. Immunol., February 1, 2009; 182(3): 1631 - 1640.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S.-H. Kuo, P.-Y. Yeh, L.-T. Chen, M.-S. Wu, C.-W. Lin, K.-H. Yeh, Y.-S. Tzeng, J.-Y. Chen, P.-N. Hsu, J.-T. Lin, et al.
Overexpression of B cell-activating factor of TNF family (BAFF) is associated with Helicobacter pylori-independent growth of gastric diffuse large B-cell lymphoma with histologic evidence of MALT lymphoma
Blood, October 1, 2008; 112(7): 2927 - 2934.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
E. A. Rickel, L. A. Siegel, B.-R. P. Yoon, J. B. Rottman, D. G. Kugler, D. A. Swart, P. M. Anders, J. E. Tocker, M. R. Comeau, and A. L. Budelsky
Identification of Functional Roles for Both IL-17RB and IL-17RA in Mediating IL-25-Induced Activities
J. Immunol., September 15, 2008; 181(6): 4299 - 4310.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
C. A. Hudson, G. P. Christophi, R. C. Gruber, J. R. Wilmore, D. A. Lawrence, and P. T. Massa
Induction of IL-33 expression and activity in central nervous system glia
J. Leukoc. Biol., September 1, 2008; 84(3): 631 - 643.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. F. Y. Cheung, C. K. Wong, and C. W. K. Lam
Molecular Mechanisms of Cytokine and Chemokine Release from Eosinophils Activated by IL-17A, IL-17F, and IL-23: Implication for Th17 Lymphocytes-Mediated Allergic Inflammation
J. Immunol., April 15, 2008; 180(8): 5625 - 5635.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
C. K. Wong, P. F. Y. Cheung, W. K. Ip, and C. W. K. Lam
Intracellular Signaling Mechanisms Regulating Toll-Like Receptor-Mediated Activation of Eosinophils
Am. J. Respir. Cell Mol. Biol., July 1, 2007; 37(1): 85 - 96.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
W. Matsuyama, H. Mitsuyama, M. Ono, Y. Shirahama, I. Higashimoto, M. Osame, and K. Arimura
Discoidin domain receptor 1 contributes to eosinophil survival in an NF-{kappa}B-dependent manner in Churg-Strauss syndrome
Blood, January 1, 2007; 109(1): 22 - 30.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
S. Lajoie-Kadoch, P. Joubert, S. Letuve, A. J. Halayko, J. G. Martin, A. Soussi-Gounni, and Q. Hamid
TNF-{alpha} and IFN-{gamma} inversely modulate expression of the IL-17E receptor in airway smooth muscle cells
Am J Physiol Lung Cell Mol Physiol, June 1, 2006; 290(6): L1238 - L1246.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2005-0034OCv1
33/2/186    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wong, C. K.
Right arrow Articles by Lam, C. W. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wong, C. K.
Right arrow Articles by Lam, C. W. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2005 American Thoracic Society.