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Interleukin-3, -5, and Granulocyte Macrophage Colony-Stimulating Factor–Induced Adhesion Molecule Expression on Eosinophils by p38 Mitogen-Activated Protein Kinase and Nuclear Factor-B

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

Address correspondence to: Professor C. W. K. Lam, Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N.T., Hong Kong. E-mail: waikeilam{at}cuhk.edu.hk

	Abstract

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We investigated the intracellular signaling mechanisms for cytokine interleukin (IL)-3, IL-5, or granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced expression of adhesion molecules including very late antigen 4 (CD49 d), macrophage antigen-1 (CD11b), leukocyte function–associated antigen-1 (CD11a/CD18), intercellular adhesion molecule (ICAM)-1, and ICAM-3 on eosinophils. The expression of adhesion molecules and nuclear factor (NF)-B pathway was measured by flow cytometry and cDNA expression array, respectively. The phosphorylation of inhibitor B- and p38 mitogen-activated protein kinase (MAPK) was detected by Western blot, whereas NF-B activity was measured by electrophoretic mobility shift assay. IL-3, IL-5, and GM-CSF could enhance p38 MAPK and NF-B activity and induce ICAM-1, CD11b, and CD18 expressions on eosinophils. They could suppress ICAM-3 expression, but had no effect on CD49 d expression. Either SB 203580 or MG-132 was able to offset the cytokine-induced expression of ICAM-1. Only SB 203580 could reverse the effect on CD11b, CD18, and ICAM-3 expressions. Therefore, the expression of ICAM-1 might involve both p38 MAPK and NF-B activities, whereas the regulation of CD11b, CD18, and ICAM-3 expressions might be mediated through p38 MAPK but not NF-B. These cytokines therefore play a crucial role, via the p38 MAPK and NF-B pathways, in the expression of important adhesion molecules on eosinophils in allergic inflammation.

Abbreviations: dimethyl sulfoxide, DMSO • eosinophilic cationic protein, ECP • electrophoretic mobility shift assay, EMSA • fluorescein isothiocyanate, FITC • granulocyte macrophage colony-stimulating factor, GM-CSF • inhibitor B-, IB- • intercellular adhesion molecule, ICAM • immunoglobulin, Ig • interleukin, IL • leukocyte function-associated antigen, LFA • lipopolysaccharide, LPS • macrophage antigen-1, Mac-1 • mitogen-activated protein kinase, MAPK • monocyte chemotactic protein, MCP • macrophage inflammatory protein, MIP • nuclear factor-B, NF-B • phosphate-buffered saline, PBS • tumor necrosis factor-, TNF- • vascular cell adhesion molecule, VCAM • very late antigen-4, VLA-4

	Introduction

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Eosinophils are the most important inflammatory effector cells accumulating at the site of allergic inflammation, e.g., 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 manifestion of allergic diseases, e.g., allergic asthma (1). Eosinophils express several kinds of adhesion molecules on their cell membrane including (i) ß1 integrin family: very late antigen-4 (VLA-4, CD49 d/CD29); (ii) ß2 integrin family: leukocyte function–associated antigen (LFA)-1 (CD11a/CD18) and macrophage antigen (Mac)-1 (CD11b); (iii) selectin family: L-selectin; and (iv) immunoglobulin (Ig) superfamily: intercellular adhesion molecule (ICAM)-1 (CD54) and ICAM-3 (CD50) (2). These adhesion molecules have been proposed to be involved in the release of ECP (3), chemotaxis, and transendothelial migration of eosinophils (4). Such processes can be mediated and enhanced by several cytokines including interferon-, tumor necrosis factor- (TNF-) and interleukin-1 (IL-1) (5), and chemotactic factors including eotaxin, regulated upon activation normal T cell expressed and secreted (RANTES), IL-8, monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1 (MIP-1) (6). These inflammatory cytokines activate intracellular signaling cascades including mitogen-activated protein kinases (MAPK) (7) and nuclear factor-kappa B (NF-B) signal transduction pathways (8).

ICAM-1 belongs to the Ig superfamily cellular adhesion molecules, and is expressed by many airway cells including eosinophils, bronchial epithelium cells, endothelial cells, T cells, mast cells, and alveolar macrophages (9). It can facilitate intercellular interaction and potentiate inflammatory process in childhood asthma (10). ICAM-1 can bind to LFA-1, Mac-1, fibrinogen, hyaluronan, and CD43 on leukocytes (11), whereas ICAM-3 can bind to LFA-1 but not Mac-1 (12). LFA-1 expression on eosinophils has been shown to increase in individuals with atopic asthma, and to modulate the enhanced eosinophil migration (13). The ß1 integrin VLA4 on eosinophils can bind to the vascular cell adhesion molecule (VCAM)-1 on endothelial for chemotaxis. Elevation of VLA4-positive eosinophils is correlated with eosinophils in the induced sputum of patients with atopic asthma, and VLA4 expression is related to the disease severity (14). Mac-1 (_Mß₂) expression on monocytes, granulocytes can be elevated and interacted with ICAM on endothelial cells during inflammatory reaction, thereby activating the transendothelial migration of eosinophils (15, 16).

Recent studies have reported that activation of NF-B can mediate the transcription of ICAM-1, VCAM-1, and cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and eotaxin in leukocytes (8, 17, 18). NF-B comprises inducible transcription factors that serve as important regulators in allergic inflammation (8). In quiescent cells, NF-B is bound to an inhibitory protein known as IB. Upon stimulation, IB kinase is activated to phosphorylate IB which is then degraded by the proteasome (19). This results in the translocation of NF-B from cytoplasm to the nucleus, where it binds to DNA response elements and activates the transcription of specific genes (19). MAPK are serine and threonine kinases that can be activated by phosphorylation in kinase cascades. p38 MAPK is activated by osmotic stress, ultraviolet irradiation, and proinflammatory cytokines including TNF- to phosphorylate both cytoplasmic and nuclear targets (18). We previously reported that the activation of NF-B and p38 MAPK play a role in TNF-–activated signaling pathways regulating eotaxin release in eosinophils (18).

Elevated T cell production of eosinophil-active cytokines (IL-5, IL-3, GM-CSF) is thought to be fundamental in asthma pathogenesis (20). Although some previous studies reported that hematopoietic cytokine IL-3, IL-5, and GM-CSF can induce ICAM-1 expression on eosinophils (21), detailed mechanisms of IL-3, IL-5, and GM-CSF–mediated different adhesion molecule expressions on eosinophils are not well established. In the present study, we investigated the effect of IL-3, IL-5, and GM-CSF on NF-B pathway–related gene expression, and the modulation of NF-B and MAPK activity on the regulation of the expression of adhesion molecules including ICAM-1, ICAM-3, CD18, CD11b, and CD49 d on human blood eosinophils.

	Materials and Methods

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Reagents and Antibodies
Human recombinant IL-3, IL-5, and GM-CSF were obtained from PeproTech Inc. (Rocky Hill, NJ). Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human ICAM-1 monoclonal antibody and its corresponding fluorescein-conjugated IgG₁ isotype were purchased from R&D Systems Inc. (Minneapolis, MN). FITC-conjugated mouse anti-human ICAM-3, CD18, CD11b, and CD49 monoclonal antibody, and fluorescein-conjugated mouse IgG₁ and IgG_2b isotype, were purchased from BD Pharmingen (San Diego, CA). Rabbit anti–phospho-p38 MAPK and anti–phospho-IB- polyclonal antibodies were supplied by Cell Signaling Technology Inc. (Beverly, MA). NF-B proteasome inhibitor N-cbz-Leu-Leu-Leucinal, MG-132, and specific p38 MAPK inhibitor, SB 203580, were purchased from Calbiochem Corp (San Diego, CA).

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) and centrifuged using an isotonic Percoll solution (density 1.082 g/ml; Amersham and Pharmacia Biotech, Uppsala, Sweden) at 4°C for 30 min at 1,000 x g. The eosinophil-rich granulocyte fraction was collected and washed twice with 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 a 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% defined fetal bovine serum (Gibco) and 20 mM Hepes (Gibco). MG-132 (Calbiochem) (25) was dissolved in dimethyl sulfoxide (DMSO) and added to eosinophil culture at a concentration of 20 µM for 1 h. In all studies, the concentration of DMSO was 0.2% (vol/vol).

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; Associates of Cape Cod, Woods Hole, MA).

cDNA Expression Array Analysis
Total RNA was extracted from eosinophils (1 x 10⁷) with different treatments using Tri-reagent (Molecular Research Center Inc., Cincinnati, OH). Nonradioactive NF-B Signaling Pathway GEArray Q-series Kit (SuperArray Inc., Bethesda, MD) was used to analyze the gene expression profile of NF-B pathway. Briefly, 5 µg total RNA was used as template for biotinylated cDNA probe synthesis. RNA was reverse-transcribed by gene-specific primers with biotin-16-dUTP. Biotinylated cDNA probes were denatured and hybridized to NF-B pathway gene-specific cDNA fragments spotted on the membranes. The GEArray membranes were then washed and blocked with GEA blocking solution, and incubated with alkaline phosphatase–conjugated streptavidin. The hybridized biotinylated probes were detected by chemiluminescent method using the alkaline phosphatase substrate, CDP-Star. The results were analyzed using Bio-Rad Quantity One software (Bio-Rad Laboratories, Hercules, CA). Each array membrane comprised 96 marker genes in quatraplicate; four positive controls including ß-actin, glyceraldehyde-3-phosphate dehydrogenase, cyclophilin A, and ribosomal protein L13a; and a negative control, bacterial plasmid pUC18. The relative expression levels of different genes were estimated by comparing its signal intensity with that of internal control ß-actin.

Reverse Transcription-Polymerase Chain Reaction
Total RNA from eosinophils was extracted using Tri-Reagent (Molecular Research Center).

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 (Perkin Elmer, Fremont, CA), 50 pmol of 5' and 3' primers (Invitrogen, Carlsbad, CA) in PCR reaction buffer (1 min each at 94°, 60°C and 72°C) for 28 and 35 cycles for ß-actin and IL-8, and ICAM-1, NF-B, and IB, respectively, after an initial 12 min of denaturation at 94°C. All reverse transcription (RT)-PCR were performed in the linear range of the PCR reaction according to the preliminary experiment. PCR Primers were as the following: NF-B sense, 5'-ATGGGGCATTTTGTTGAGAG-3' and antisense, 5'-ACAAATGGGCTACACCGAAG-3', yielding a 254-bp product; I-B sense, 5'-GCAGAAAAAGGATCGTGAGC-3' and antisense, 5'-ATAGTGAGCCGAAACCCCTT-3', yielding a 272-bp product; ICAM-1 sense, 5'-CGTGCCGCACTGAACTGGAC-3' and antisense, 5'-CCTCACACTTCACTGTCACCT-3', yielding a 447-bp product; IL-8 sense, 5'-CTGTGTGAAGGTGCAGTTTTGCC-3' and antisense, 5'-CTCAGCCCTCTTCAAAAACTTCTCC-3', yielding a 237-bp product; ß-actin sense, 5'-AGCGGGAAATCGTGCGTG-3' and antisense, 5'-CAGGGTACATGGTGGTGCC-3', yielding a 300-bp product. 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).

Western Blot Analysis
Eosinophils (5 x 10⁶) with different treatments were washed with PBS and lysed in 0.1 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 were removed by centrifugation at 10,000 x g for 10 min and the supernatants were boiled in Laemmli sample buffer (Bio-Rad) for 5 min. An equal amount of proteins was subjected to sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis, and the proteins were blotted onto a PVDF membrane (Amersham and Pharmacia Biotech). Membranes were blocked with 5% skimmed milk in Tris-buffered saline with 0.05% Tween 20, pH 7.6 for overnight at 4°C and probed with different primary rabbit anti-human antibodies for 1 h at room temperature. After washing, membranes were 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).

Electrophoretic Mobility Shift Assay
After eosinophils (1 x 10⁷) were harvested and washed, 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 extracts were subjected to a test for NF-B protein/DNA binding using LightShift chemiluminescent EMSA kit (Pierce Chemical Co.) with a biotin end-labeled NF-B oligonucleotide (5'-AGT TGA GGG GAC TTT CCC AGG C-3') (Research Genetics, Huntsville, AL). Briefly, nuclear extracts were incubated with biotin end-labeled NF-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.

Flow Cytometric Analysis
Eosinophils (5 x 10⁵) after different treatments were washed 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 adhesion molecule monoclonal antibody or fluorescein-conjugated mouse IgG₁ and IgG_2b Isotype for 30 min at 4°C in dark. After washing, the cells were finally resuspended in 1% paraformaldehyde in 1x PBS as fixative. Expression of surface adhesion molecule was then analyzed by flow cytometry (FACSCalibur, Becton Dickinson, Palo Alto, CA) as mean fluorescence intensity.

Statistical Analysis
All data were expressed as mean ± SEM. Differences between groups were assessed by the nonparametric Mann-Whitney rank sum test. A probability P < 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

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Effect of IL-3, IL-5, and GM-CSF on the Expression Profile of NF-B Pathway–Related Genes in Eosinophils
Figure 1A shows that IL-3, IL-5, and GM-CSF modulated the expression profile of genes involved in NF-B signal transduction pathway in eosinophils. The locations of the upregulated genes are shown in Table 1. The expression of positive controls ß-actin (111, 112), GAPDH (103, 104), cyclophilin A (105, 106, 107, 108), and ribosomal protein L13a (109, 110) remained constant in both the presence and absence of cytokines. As also shown in Table 1, the mRNAs of NF-B (p105), NF-B (p49/p100) and its inhibitor IB- were upregulated in IL-3, IL-5, and GM-CSF–treated eosinophils. The mRNA level of ICAM-1 and several proinflammatory cytokines and chemokines such as IL-1, IL-1ß, IL-8, and monocyte chemotactic protein (MCP)-1 were also significantly increased with IL-3, IL-5, and GM-CSF stimulation. The results of RT-PCR in Figure 1B confirm that IL-3, IL-5, and GM-CSF could increase the mRNA expression of NF-B, I-B, ICAM-1, and IL-8 in eosinophils, which was similar to the results of cDNA expression array analysis.

View larger version (138K): [in this window] [in a new window]   Figure 1. (A) Representative expression profile of NF-B pathway–related genes in human eosinophils detected by cDNA expression array system. RNA was extracted from human blood eosinophils (1 x 107) after overnight incubation (12 h) with or without IL-3 (20 ng/ml), IL-5 (20 ng/ml), and GM-CSF (50 ng/ml). Total RNA was reverse-transcribed and labeled with biotin, and gene expressions were detected using Nonrad-GEArray Q series kit. Blanks: (100–102); negative control genes: PUC18 DNA (97–99); positive control genes: glyceraldehyde-3-phosphate dehydrogenase (103, 104), cyclophilin A (PPIA) (105–108), ribosomal protein L13a (109, 110), and ß-actin (111, 112). (B) Representative RT-PCR analysis of NF-B, I-B, ICAM-1, and IL-8 mRNA expression in eosinophils. Total RNA was extracted from eosinophils (1 x 107) treated with or without different cytoines for 12 h, and then reverse-transcribed and analyzed by PCR. The ß-actin housekeeping gene was used as the control. M: 100-bp DNA ladder molecular weight maker; lane 1: control (without cytokine); lane 2: 20 ng/ml IL-3; lane 3: 20 ng/ml IL-5; lane 4: 50 ng/ml GM-CSF; lane 5: negative PCR control (without cDNA).

View this table: [in this window] [in a new window]   TABLE 1 Differential expression of genes related to human NF-B pathway in control or IL-3–, IL-5–, and GM-CSF–treated eosinophils

Effect of IL-3, IL-5, and GM-CSF on Phosphorylation of IB- and p38 MAPK in Eosinophils

View larger version (42K): [in this window] [in a new window]   Figure 2. Effects of IL-3, IL-5, and GM-CSF on (A) phosphorylation of IB- and (B) activation of p38 MAPK. Eosinophils (5 x 106) were treated with or without IL-3 (20 ng/ml), IL-5 (20 ng/ml), GM-CSF (50 ng/ml) for the period of time indicated. Total cellular proteins were extracted for the detection of (A) phosphorylated IB- or (B) phosphorylated p38 MAPK by Western blot analysis. Experiments were performed in three independent experiments with essentially identical results, and representative blots are shown.

Effect of MG-132 and SB 203580 on IL-3–, IL-5–, and GM-CSF–Induced NF-B Activity

View larger version (43K): [in this window] [in a new window]   Figure 3. Effects of MG 132 and SB 203580 on IL-3–, IL-5–, and GM-CSF–induced NF-B translocation and DNA binding activities. Eosinophils (1 x 107) were treated with or without MG-132 (20 µM) or SB 203580 (20 µM) for 1 h followed by stimulation with (A) IL-3 (20 ng/ml), (B) IL-5 (20 ng/ml), or (C) GM-CSF (50 ng/ml) for further 18 h. Nuclear protein was extracted and 5 µg was subjected to EMSA. Experiments were performed in three independent experiments with essentially identical results, and representative blots are shown. (A) Lane 1: negative control (labeled DNA probe only); lane 2: control; lane 3: IL-3 (20 ng/ml); lane 4: MG-132 (20 µM)+IL-3; lane 5: SB 203580 (20 µM)+IL-3; lane 6: competitive control. (B) Lane 1: control; lane 2: IL-5 (20 ng/ml); lane 3: MG-132 (20 µM)+IL-5; lane 4: SB 203580 (20 µM)+IL-5; lane 5: MG-132 only; lane 6: SB 203580 only; lane 7: competitive control; lane 8: negative control (labeled DNA probe only). (C) Lane 1: negative control (labeled DNA probe only); lane 2: control; lane 3: GM-CSF (50 ng/ml); lane 4: MG-132 (20 µM)+GM-CSF; lane 5: SB 203580 (20 µM)+GM-CSF; lane 6: competitive control.

View larger version (60K): [in this window] [in a new window]   Figure 4. Effects of MG-132 and SB 203580 on the (A) IL-3–, (B) IL-5–, and (C) GM-CSF–induced ICAM-1 expression on eosinophils. Eosinophils (5 x 105/ml) were treated with or without SB 203580 (20 µM) or MG-132 (20 µM) for 1 h followed by stimulation with or without IL-3 (20 ng/ml), IL-5 (20 ng/ml), and GM-CSF (50 ng/ml) for further 16 h. Results are expressed as the arithmetic mean ± SEM of three independent experiments. *P < 0.02 versus medium control. #P < 0.05 versus cytokine treatment.

View larger version (58K): [in this window] [in a new window]   Figure 5. Effects of MG-132 and SB 203580 on the (A) IL-3–, (B) IL-5–, and (C) GM-CSF–induced ICAM-3 expression on eosinophils. Eosinophils (5 x 105/ml) were treated with or without SB 203580 (20 µM) or MG-132 (20 µM) for 1 h followed by stimulation with or without IL-3 (20 ng/ml), IL-5 (20 ng/ml), and GM-CSF (50 ng/ml) for further 16 h. Results are expressed as the arithmetic mean ± SEM of three independent experiments. *P < 0.04 versus medium control.

View larger version (62K): [in this window] [in a new window]   Figure 6. Effects of MG-132 and SB 203580 on the (A) IL-3–, (B) IL-5–, and (C) GM-CSF–induced CD18 expression on eosinophils. Eosinophils (5 x 105/ml) were treated with or without SB 203580 (20 µM) or MG-132 (20 µM) for 1 h followed by stimulation with or without IL-3 (20 ng/ml), IL-5 (20 ng/ml), and GM-CSF (50 ng/ml) for further 16 h. Results are expressed as the arithmetic mean ± SEM of three independent experiments. *P < 0.03 versus medium control. #P < 0.03 versus cytokine treatment.

View larger version (59K): [in this window] [in a new window]   Figure 7. Effects of MG-132 and SB 203580 on the (A) IL-3–, (B) IL-5–, and (C) GM-CSF–induced CD11b expression on eosinophils. Eosinophils (5 x 105/ml) were treated with or without SB 203580 (20 µM) or MG-132 (20 µM) for 1 h followed by stimulation with or without IL-3 (20 ng/ml), IL-5 (20 ng/ml), and GM-CSF (50 ng/ml) for further 16 h. Results are expressed as the arithmetic mean ± SEM of three independent experiments. *P < 0.03 versus medium control. #P < 0.03 versus cytokine treatment.

	Discussion

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The transcription factor NF-B and the protein kinase p38 MAPK have been shown to serve as important regulators in allergic inflammation (8, 22). NF-B was found to regulate adhesion molecules including ICAM-1 and VCAM-1, and a number of proinflammatory cytokines including IL-1, IL-2, IL-6, IL-8, GM-CSF, and RANTES at transcription level in many inflammatory diseases (8). p38 MAPK could regulate the expression of inflammatory cytokine and adhesion molecules (23), eosinophil degranulation, and chemotaxis (24). Eosinophils have been shown to express IL-3, IL-5, and GM-CSF receptor chain and common ß chain (2). IL-3, IL-5, and GM-CSF can trigger intracellular biochemical cascades leading to overlapping responses such as adhesion, proliferation, survival, and differentiation of eosinophils due to the common ß chain (25). We and other researchers have previously found that IL-3, IL-5, and GM-CSF can activate MAPK cascades in eosinophils (26, 27).

In our present cDNA expression array analysis of NF-B pathway–related genes, the expression of ICAM-1 gene was greatly upregulated by IL-3, IL-5, and GM-CSF at transcription level. As shown in Figure 1 and Table 1, IL-3, IL-5, and GM-CSF were found to enhance the mRNA expression of NF-B and its inhibitor IB- in eosinophils. In addition, the gene expression of a group of proinflammatory cytokines and chemokines including IL-1, IL-8, and MCP-1 was also upregulated by cytokine treatment. In conjunction with the results of EMSA (Figure 3) and phosphorylation of IB- (Figure 2), the above results further indicate the critical role of NF-B in the regulation of inflammatory gene transcriptions in IL-3–, IL-5–, and GM-CSF–activated eosinophils.

A previous report has demonstrated that p38 MAPK is required for NF-B–dependent cytokine gene expression by the modulation of DNA binding of TATA-binding protein to the TATA box (28). Inhibition of p38 MAPK may potentially attenuate NF-B–dependent transcription (29). Our previous results have also shown that the activation of p38 MAPK and NF-B is required for TNF-–induced eotaxin release in eosinophils (18). In the present study, we found that IL-3, IL-5, and GM-CSF could rapidly induce the phosphorylation of p38 MAPK in eosinophils (Figure 2), thereby confirming the activation of both NF-B and p38 MAPK signaling pathways in IL-3–, IL-5–, and GM-CSF–stimulated eosinophils. In an attempt to further investigate the relationship between the activation of p38 MAPK and NF-B in IL-3–, IL-5–, and GM-CSF–treated eosinophils, we used a specific p38 MAPK inhibitor, SB 203580, a pyridinyl imidazole class inhibitors of p38 MAPK that competitively binds to the ATP-binding pocket of p38 MAPK, thus inhibiting its downstream kinase activity (22). The proteasome inhibitor MG-132 is a substrate analog for inhibiting proteasome activity on the degradation of phosphorylated IB protein, thereby inhibiting the dissociation and activation of NF-B (18). We found that SB 203580 did not show any significant effect on IL-3–, IL-5–, or GM-CSF–activated NF-B activity, whereas MG-132 could inhibit cytokine-induced NF-B activity (Figure 3); therefore, the activation of p38 MAPK may not directly relate to NF-B activation in IL-3–, IL-5–, and GM-CSF–treated eosinophils. According to the concentration of MG-132 and SB 203580 reported in previous other studies (including our study [18]), we used the optimal concentration of MG-132 (20 µM) and SB 203,580 (20 µM) with the highest inhibitory effect without any cell toxicity.

As shown in Figure 4, untreated eosinophils expressed low levels of ICAM-1, but IL-3, IL-5, and GM-CSF could significantly enhance cell surface ICAM-1 expression on eosinophils in vitro. Inhibition of NF-B with MG-132 could significantly inhibit the surface expression of cytokine-induced ICAM-1 on eosinophils (Figure 4). This finding concurs with our recent data on an eosinophilic leukemic cell line, EoL-1 cells (30). Moreover, inhibition of p38 MAPK with SB 203580 could inhibit cytokine-induced ICAM-1 expression, suggesting that both p38 MAPK and NF-B signaling pathway regulate the post-transcription level of ICAM-1 in eosinophils.

Those three cytokines could, however, suppress ICAM-3 expression in vitro that was regulated by p38 MAPK but not NF-B activation (Figure 5). Previous studies have indicated that dexamethasone could suppress the expression of ICAM-3 on eosinophils (31), and it could activate p38 MAPK activity in eosinophils (32). Therefore, it is reasonable that the activation of p38 MAPK activity can probably downregulate the ICAM-3 expression in eosinophils. As shows in Figures 6 and 7, IL-3, IL-5, and GM-CSF could upregulate both CD18 and CD11b expression in vitro that was regulated by the activation of p38 MAPK activity but not NF-B activation. In allergic asthma, an early recruitment of blood eosinophils overexpressing LFA-1 (CD18) occurs after allergen challenge (16), whereas in allergic dermatitis, CD11b expression on eosinophils is elevated (33). CD11b has also been shown to be elevated after eosinophil adhesion and transmigration (6). Therefore, IL-3, IL-5, and GM-CSF may play an important role for adhesion and transmigration of eosinophils during allergic inflammation. Moreover, we also found that these three cytokines could enhance the in vitro chemotaxis and transmigration of eosinophils through inserts with polycarbonate membrane filter (3 µm) coated with 20 ng/ml human fibronectin and adhesion onto human bronchial epithelial cell, BEAS-2B (data not shown). However, we found that IL-3, IL-5, and GM-CSF did not exert any significant effect on the expression of CD49 d in vitro on eosinophils (data not shown). Previous results have shown that VLA4 expression on eosinophils decreased in patients with eosinophilia (34), whereas another report indicated that VLA4 on eosinophils did not change in atopic asthmatic children (13). Therefore the actual change of VLA4 expression on eosinophils in allergic inflammation should require further investigation.

In view of the above results regarding the differential expression of different adhesion molecules and activation of p38 MAPK and NF-B pathways, different adhesion molecules may be regulated by various intracellular signal transduction mechanisms. Further studies are required to elucidate other signaling pathways such as Janus kinases-signal transducers and activators of transcription and phosphatidylinositol 3-kinase pathways in the expression of adhesion molecules and chemotaxis of eosinophils. Recent accumulating results for adhesion molecule expression have provided insights into the structural basis and clinical relevance of adhesion molecule inhibition by peptides and small molecules as adhesion-based therapeutic strategies for inflammation (35). In fact, potent p38 MAPK inhibitor has been shown to inhibit inflammatory cytokine production and airway eosinophil infiltration (23). Our present study reveals the important role of several intracellular signal transduction molecules regulating the expression of crucial adhesion molecules on eosinophils, thereby shedding light for the future development of more effective agents for allergic and inflammatory diseases.

	Acknowledgments

 
The study was supported by a Chinese University of Hong Kong Direct Grant for Research and a donation from Zindart (De Zhen) Foundation Ltd, Hong Kong.

Received in original form December 5, 2002

Received in final form February 7, 2003

	References

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