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(Journal of Leukocyte Biology. 2002;72:1046-1053.)
© 2002 by Society for Leukocyte Biology

IL-5-induced integrin adhesion of human eosinophils caused by ERK1/2-mediated activation of cPLA2

Xiangdong Zhu*, Benjamin Jacobs*, Evan Boetticher*, Shigeharu Myou*, Angelo Meliton*, Hiroyuki Sano{dagger}, Anissa T. Lambertino*, Nilda M. Muñoz* and Alan R. Leff*

* Section of Pulmonary and Critical Care Medicine, Department of Medicine and Department of Neurobiology, Pharmacology and Physiology, Pediatrics, Anesthesia and Critical Care, and Committees on Clinical Pharmacology, Cell Physiology and Molecular Medicine, Division of the Biological Sciences, The University of Chicago, Illinois; and
{dagger} Third Department of Internal Medicine, Tottori University, Japan

Correspondence: Dr. Alan R. Leff, Section of Pulmonary and Critical Care Medicine, Department of Medicine, MC6076, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637. E-mail: aleff{at}medicine.bsd.uchicago.edu


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ABSTRACT
 
We examined the mechanism by which interleukin (IL)-5 causes ß2-integrin adhesion of human eosinophils. IL-5 caused time-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38{alpha} in eosinophils as detected by their phosphorylation. Preincubation of eosinophils with U0126, a mitogen-activated protein kinase/ERK kinase inhibitor, suppressed IL-5-induced activation of cytosolic phospholipase A2 (cPLA2) and eosinophil adhesion, and p38 inhibition by SB203580 had neither effect. ERK1/2 phosphorylation and eosinophil adhesion were blocked by inhibition of the src-family tyrosine kinase, Janus tyrosine kinase (JAK)2, or phosphoinositide-3 kinase (PI3K). Coimmunoprecipitation assay demonstrated that Lyn, a src-family tyrosine kinase, was constitutively associated with PI3K. Inhibition of src-tyrosine kinase but not JAK2 suppressed PI3K activation. Our data suggest that IL-5 induces ß2-integrin adhesion of human eosinophils by regulation of cPLA2 activation caused by ERK1/2 phosphorylation. This phosphorylation results from activation of PI3K and protein tyrosine kinases. We also find that src-family tyrosine kinase, possibly Lyn, is the upstream kinase causing PI3K activation.

Key Words: adhesion molecules • signal transduction • arachidonic acid


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INTRODUCTION
 
Eosinophils, which are major effector granulocytes in allergic inflammation such as asthma, remain inactive and nonadherent while circulating in the blood but are activated and recruited to airways in response to the cytokine, interleukin (IL)-5 [1 ]. Integrins of the ß2 subfamily (CD11b/CD18) are required for eosinophil adhesion to endothelial cells and migration into tissues [2 ]; however, the signal transduction pathways linking the IL-5 membrane receptor to ß2-integrin activation have not been defined. We recently reported that cytosolic phospholipase A2 (cPLA2) is the critical enzyme in the regulation of integrin-mediated eosinophil adhesion and that cPLA2-mediated adhesion is regulated by its downstream production of platelet-activating factor (PAF) [3 , 4 ]. The role of cPLA2 in airway infiltration of granulocytes also has been demonstrated in vivo [5 , 6 ]. Pharmacological inhibition of cPLA2 blocked eosinophil infiltration and airway hyperresponsiveness in antigen-sensitized guinea pigs [5 ]. However, the signal transduction leading to cPLA2 phosphorylation in eosinophil adhesion has not been elucidated.

Four distinct mitogen-activated protein kinase (MAPK) cascades have been described in mammalian cells, including the extracellular signal-regulated kinases 1 and 2 (ERK1/2), the c-Jun N-terminal kinase (JNK), the p38 MAPK, and ERK5. Several reports have analyzed cPLA2 phosphorylation in vivo, and MAPK has been implicated as a possible upstream kinase for cPLA2 phosphorylation and activation [7 ]. However, the specific MAPK isoforms that critically mediate cPLA2 phosphorylation remain controversial [8 9 10 11 ]. IL-5 has been shown to induce cPLA2 phosphorylation; however, the mechanism by which this phosphorylation occurs has not been established [3 ]. Activation of all MAPKs occurs through tyrosine and threonine phosphorylation by the dual-specificity kinase termed MAPK/ERK kinases (MEKs). Using a pharmacological inhibitor of MEK and p38 MAPK, we recently demonstrated a role for ERK and p38 in the formyl-Met-Leu-Phe (fMLP)-induced arachidonic acid (AA) metabolism caused by cPLA2 in eosinophils [12 ]. However, cPLA2 has also been shown to regulate IL-5-induced adhesion of eosinophils to endothelial ligands by a mechanism unrelated to the AA metabolism [3 ]. In contrast to AA hydrolysis, the mechanism of regulation of eosinophil adhesion and the specific role of MAPK isoforms remain to be elucidated.

Recent studies have examined the role of phosphoinositide-3 kinase (PI3K) in cytokine signaling [13 ]. PI3K phosphorylates the D3 position of phosphoinositide and phosphoinositide phosphates. At least three families of PI3K have been described in mammalian cells [14 ]. Among them, one of these families is activated by tyrosine kinase and consists of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit. Tyrosine kinase-mediated activation of PI3K has been identified in human eosinophils, and its activation has been implicated in the up-regulation of cytokine-stimulated Fc{alpha}R affinity and in zymosan-stimulated respiratory burst activity [13 , 15 ]. The role of PI3K activation and its role in regulating eosinophil adhesion have not been investigated previously.

The objective of this study was to determine the pathways mediating IL-5-induced adhesion of eosinophils. The protein tyrosine kinase (PTK), PI3K, MAPK, and their interaction were examined. We found that IL-5-induced eosinophil adhesion to intercellular adhesion molecule-1 (ICAM-1) was regulated by activation of cPLA2 by ERK1/2 through the activation of PI3K and PTKs. We also found that src-family tyrosine kinase but not Janus tyrosine kinase (Jak)2 kinase was the upstream kinase for PI3K activation.


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MATERIALS AND METHODS
 
Materials
IL-5 and soluble ICAM-1 were purchased from R&D Systems (Minneapolis, MN). Eosinophil isolation materials were obtained from Miltenyi Biotec (Sunnyvale, CA). Polystyrene 96-well microtiter plates were obtained from Costar (Cambridge, MA). U0126, phosphorylation-specific ERK1/2 polyclonal antibody (Ab), phosphorylation-specific ERK5 Ab, and phosphorylation-specific and nonspecific JNK1/2 Ab were obtained from Promega Co. (Madison, WI). T180/Y182 phosphorylation-specific and nonspecific p38{alpha} MAPK Ab, S505 phosphorylation-specific cPLA2 Ab, S473 phosphorylation-specific and nonspecific protein kinase B (PKB) Ab, ERK1/2 Ab, and phosphorylated JNK1/2 control cell extracts were purchased from New England BioLabs Inc. (Beverly, MA). Wortmannin, LY294002, SB203580, AG490, SP600125, and PP2 were purchased from Calbiochem (San Diego, CA). The polyclonal anti-p85 subunit of PI3K and anti-Jak2 Ab were purchased from Upstate Biotechnology (Lake Placid, NY). Phosphorylation nonspecific polyclonal anti-cPLA2 and anti-Lyn Ab were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). 1-Palmitoyl-2-[14C]arachidonyl phosphatidylcholine (PAPC) was purchased from New England Nuclear (Boston, MA).

Isolation of human eosinophils
Eosinophils were isolated by a modification of the negative immunomagnetic selection technique [16 ]. The method is based on percoll centrifugation (density 1.089 g/ml) to isolate granulocytes, hypotonic lysis of red blood cells, and finally, immunomagnetic depletion of neutrophils by a magnetic cell separation system using anti-CD16-coated magnetic cell sorter particles. Eosinophil purity of >=98% was routinely obtained. Cells were kept on ice until use.

Eosinophil adhesion assay
Eosinophil adhesion was assessed as residual eosinophil peroxidase (EPO) activity of adherent cells [3 , 17 ]. Briefly, 96-well microplates were coated with 10 µg/ml ICAM-1 and blocked with heat-inactivated fetal bovine serum. Eosinophils [1x104/100 µl Hanks’ balanced saline solution (HBSS)/0.1% gelatin] were preincubated with different concentrations of U0126, PD98059, SB203580, SP600125, PP2, wortmannin, and LY 294002 for 30 min or AG490 for 60 min at 37°C. Cells were then added to ICAM-1-coated wells and allowed to settle for 10 min on ice. Plates were rapidly warmed to 37°C and incubated for the indicated times. After 3x washing with HBSS, 100 µl HBSS/0.1% gelatin was added to the reaction wells, and serial dilutions of original cell suspension were added to the empty wells to generate a standard curve. EPO substrate (100 µl; 1 mM H2O2, 1 mM O-phenylenediamine, and 0.1% Triton X-100 in Tris buffer, pH 8.0) was then added to the wells. After 30 min incubation at room temperature, 50 µl 4 M H2SO4 was added to stop the reaction. Absorbance was measured at 490 nM in a microplate reader (Thermomax, Molecular Devices, Palo Alto, CA).

Western blot analysis of MAPK, PKB, and cPLA2 phosphorylation
Eosinophils (2x106/group) were stimulated with IL-5 for various times, and the reaction was stopped by brief centrifugation. The pellets were lysed in 80 µl lysis buffer (20 mM Tris-HCl, 30 mM Na4P2O7, 50 mM NaF, 40 mM NaCl, 5 mM EDTA, pH 7.4) containing 1% Nonidet P-40, 10 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM Na3VO4, and 0.5% deoxycholic acid. After 20 min on ice, samples were centrifuged at 12,000 g for 1 min to remove nuclear and cellular debris. Afterward, 14 µl 6x loading buffer was added to the collected supernatants, boiled for 5 min, and then saved at -80°C. Samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using 10% acrylamide gels under reducing condition (15 mA/gel). Electrotransfer of proteins from the gels to polyvinylidene fluoride (PVDF) membrane was achieved using a semi-dry system (400 mA, 60 min). The membrane was blocked with 1% bovine serum albumin for 60 min and then was incubated with 1:5000 antiphosphorylated ERK1/2 Ab, 1:1000 antiphosphorylated p38 MAPK mAb, 1:1000 antiphosphorylated PKB, 1:1000 antiphosphorylated cPLA2, 1:1000 anti-ERK1/2, 1:1000 anti-p38{alpha}, 1:1000 anti-PKB, or 1 µg/ml anti-cPLA2 Ab diluted in Tris-buffered saline plus Tween-20 (TBS-T) for 60 min. The membranes then were washed three times for 20 min with TBS-T. Goat antirabbit immunoglobulin (Ig) conjugated with horseradish peroxidase was diluted 1:3000 in TBS-T and incubated with PVDF membrane for 60 min. The membranes were washed again three times with TBS-T and assayed by an enhanced chemiluminescence (ECL) system (Amersham, Arlington Hts., IL).

Coimmunoprecipiation assay
To assess the physical association between PTK and PI3K, aliquots of eosinophils (5x106) were stimulated with IL-5 or diluent for 10 min, and reactions were stopped by brief centrifugation. The pellets were lysed in 500 µl lysis buffer and incubated on ice for 20 min. The lysates were centrifuged at 11,000 g for 10 min, and 2 µl anti-Jak2 or 5 µl anti-Lyn Ab was added to the supernatants and shaken at 4°C overnight. The lysates were further incubated with protein A/G agrose beads for 1 h and washed four times with lysis buffer, and the pellets were resuspended in loading buffer and boiled for 5 min. The immunoprecipitated proteins were separated on 7.5% SDS-PAGE and transferred to membranes by semidry transfer. After incubation with 1:5000 polyclonal anti-p85 subunit of PI3K, blots were amplified and visualized using antirabbit IgG and ECL.

Determination of cPLA2 enzyme activity
The cPLA2 activity assay was modified from Kim et al. [18 ]. Briefly, eosinophils (2x106) were pretreated with U0126, SB203580, or SP600125 for 30 min and then stimulated with or without 10 ng/ml IL-5 for 30 min. The reaction was stopped by centrifugation, and the pellets were resuspended in 70 µl sonication buffer (20 mM Tris, pH 8.0, 2.5 mM EDTA, 10 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM PMSF, 2 mM Na3VO4, 50 mM NaF, and 5 µg/ml pepstatin) and sonicated briefly (4x10 s at a power setting of 3). Lysates were pretreated with 5 mM dithiothreitol on ice for 5 min to inactivate sPLA2, and 10 µl 50 mM CaCl2 was then added to each sample. Substrate (10 µl; [14C]-PAPC) was dried under a stream of N2 and resuspended in 200 µl 10% ethanol in H2O with vigorous votex mixing. The reaction was initiated by adding a 10-µl portion of the substrate (final concentration 9 µM) to the cell lysate. The reaction was carried out for 30 min at 37°C and was stopped by adding 560 µl Dole’s reagent (heptane:isopropyl alcohol:1 N H2SO4 400:390:10 by vol), followed by 110 µl H2O, votexed for 20 s, and then centrifuged at 12,000 g. Upper layer (180 µl) was transferred to 800 µl hexane containing 25 mg silica gel. Samples (750 µl) were then mixed with 2 ml scintillation fluids, and the radioactivity was counted in a liquid scintillation counter.

Determination of eosinophil viability after treatment with pharmacological inhibitors
To determine if pharmacological inhibitors used in this study affected eosinophil viability, trypan blue exclusion was assessed in eosinophils incubated with either inhibitor. Aliquots of 104 eosinophils were incubated with the greatest (see below) inhibitory concentrations of U0126, PD98059, SB203580, SP600125, PP2, wortmannin, and LY294002 for 30 min or AG490 for 60 min at 37°C. Eosinophils then were centrifuged at 400 g, and pellets were resuspended in 10 µl HBSS. An equal volume of 0.01% trypan blue was added, and viable eosinophils were counted in a hemacytometer.

Statistical analysis
All data are expressed as mean ± SEM. Differences between groups were assessed by paired t-tests. Where more than two groups were compared, differences among groups were assessed by one-way ANOVA. Where differences were found, comparisons among groups were made by Fisher’s least-protected difference test. Statistical significance was claimed where P < 0.05.


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RESULTS
 
IL-5 stimulation of MAPK activation in human eosinophils
We have shown previously that IL-5-induced adhesion of ß2-integrin (CD11b/CD18) to ICAM-1 occurred after 5 min and plateaued after 20–30 min [3 , 17 ]. To determine the role of MAPK in IL-5-stimulated eosinophil adhesion, the kinetics of MAPK isoform activation was determined in temporal association with eosinophil adhesion. IL-5 induced a rapid increase in ERK1/2 phosphorylation, which began at 5 min, peaked at 15 min, and declined thereafter (Fig. 1A ); this phosphorylation preceded eosinophil adhesion. Using phospho-specific ERK5 Ab, which cross-reacts with ERK1/2, ERK1/2 phosphorylation but not ERK5 phosphorylation was detected in IL-5-stimulated eosinophils (same as Fig. 1A ; data not shown). Neither p38{alpha} MAPK nor JNK activation corresponded temporally to eosinophil adhesion (see below). IL-5-caused p38{alpha} phosphorylation was detectable at 15 min and was maximal at 30–60 min after eosinophil adhesion occurred (Fig. 1B) . A JNK1/2 phosphorylation band was not detected by JNK1/2 phosphorylation-specific Ab in eosinophils, which constitutively express JNK2 and JNK1 (Fig. 1C) . Using this same phosphorylation-specific JNK1/2 Ab, JNK1/2 phosphorylation was detected in UV light-treated control cell extracts (second from right end, upper panel of Fig. 1C ).



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  		Figure 1. MAPK phosphorylation in IL-5-stimulated eosinophils. Time-dependent effects of IL-5 on ERK1/2 (A), p38{alpha} (B), and JNK (C) phosphorylation. Eosinophils were incubated with 10 ng/ml IL-5 for indicated times. Eosinophils were lysed, and the lysates were mixed with sample buffer and loaded on 10% SDS-PAGE, followed by immunoblotting with antiphosphorylation-specific ERK1/2 (A, upper panel), anti-ERK1/2 (A, lower panel), antiphosphorylation-specific p38 Ab (B, upper panel), anti-p38{alpha} (B, lower panel), antiphosphorylation-specific JNK1/2 Ab (C, upper panel), or anti-JNK1/2 (C, lower panel) as described in Materials and Methods. + or -, UV light-treated or nontreated 293 cell extracts. These results are representative of three different experiments.

Effect of MAPK isoform inhibition on IL-5-stimulated eosinophil adhesion
To establish specifically the role of MAPK in eosinophil adhesion, pharmacological inhibitors of the MAPK pathway were used. U0126 and PD98059, two structurally different inhibitors of MEK1/2/5 [19 20 21 ], suppressed IL-5-stimulated eosinophil adhesion in a concentration-dependent manner (Fig. 2 ). IL-5-stimulated adhesion was decreased from 21.0 ± 1.3% to 7.2 ± 2.7% with 10 µM U0126 and to 10.1 ± 0.1% with 50 µM PD98059 (P<0.01 vs. stimulated controls without inhibitor; both comparisons). By contrast, neither SB203580, a specific p38 MAPK inhibitor [22 ], nor SP600125, a JNK-specific inhibitor [23 ], had any inhibitory effect on IL-5-stimulated adhesion. These data, in conjunction with above results, suggest that activation of ERK1/2 but neither ERK5, p38{alpha}, nor JNK is required for IL-5-stimulated eosinophil adhesion.



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  		Figure 2. Effect of MAPK inhibition on IL-5-stimulated adhesion of eosinophils to plated ICAM-1. Eosinophils were preincubated with indicated concentrations of U0126, PD98059, SB203580, or SP600125 for 30 min and were then stimulated by IL-5 for 30 min. Eosinophil adhesion was measured by residual eosinophil peroxidase activity (n=5). Results are presented as the mean ± SEM, and backgroud adhesion was 6.4 ± 1.2%.

Role of ERK1/2 in activation of cPLA2
We have shown previously that cPLA2 activation is required for IL-5-stimulated eosinophil adhesion [3 ]. As ERK1/2 and cPLA2 are required for eosinophil adhesion, we explored the sequential activation of these two enzymes. IL-5 caused phosphorylation of cPLA2 at S505 in eosinophils (Fig. 3A ), which was prevented by U0126, a MEK1/2/5 pathway inhibitor. By contrast, neither SB203580, a p38 MAPK inhibitor, nor SP600125, a JNK inhibitor, suppressed IL-5-induced cPLA2 phosphorylation. Experiments were also performed to examine the effects of MAPK inhibitors on IL-5-stimulated cPLA2 activity. Eosinophils were pretreated for 30 min with 10 µM U0126, 10 µM SP600125, or 30 µM SB203580, followed by stimulation with 10 ng/ml IL-5 for 30 min. cPLA2 activity as measured by AA production increased from 0.44 ± 0.01 pM AA/30 min/106 eosinophils for nonstimulated eosinophils to 1.21 ± 0.06 pM/30 min/106 eosinophils after IL-5 stimulation (P<0.01; Fig. 3B ). This increased activity was blocked substantially by U0126 (P<0.01 vs. IL-5 alone). By contrast, neither SB203580 nor SP600125 prevented IL-5-induced cPLA2 activity (P=NS). These results correspond to their effects on eosinophil adhesion (Fig. 2) .



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  		Figure 3. (A) Effect of MAPK inhibition on cPLA2 phosphorylation. Eosinophils were preincubated with 10 µM U0126, 30 µM SB203580, or 10 µM SP600125 for 30 min and were then stimulated by IL-5 for 30 min. cPLA2 phosphorylation was measured by Western blot using S505 phosphorylation-specific antibody (upper panel). Equal loading of cPLA2 protein in lanes was verified by phosphorylation-nonselective cPLA2 Ab (lower panel). A representative blot is shown (n=3). (B) Effect of MAPK inhibition on cPLA2 activity. Eosinophils were treated as above, and cPLA2 activity in cell lysate was measured using [14C]-PAPC as substrate. Results are presented as the mean ± SEM from three separate experiments. (C) Reversal of U0126-caused inhibition of eosinphil adhesion by lyso-PAF or PAF. Eosinophils were incubated with 10 µM U0126 for 30 min and further incubated with or without 10 µM AA, 10 µM lyso-PAF, or 1 µM PAF for another 5 min, followed by stimulation with 10 ng/ml IL-5. Eosinophil adhesion was measured by residual eosinophil peroxidase activity, and results are presented as the mean ± SEM from three separate experiments.

To confirm further the role of ERK1/2-cPLA2 activation in IL-5-stimulated eosinophil adhesion to ICAM-1, experiments were designed to determine if the addition of cPLA2 catalytic products, exogenous lyso-PAF, PAF, or AA could reverse the effect of the MEK1/2/5 inhibitor on adhesion. Adhesion of nonstimulated eosinophils was 4.4 ± 0.9%. IL-5 caused 18.1 ± 1.7% adhesion of eosinophils. Pretreatment with 10 µM U0126 suppressed IL-5-stimulated eosinophils to 6.6 ± 1.6% (P<0.01). Addition of 10 µM lyso-PAF or 1 µM PAF reversed the inhibition of IL-5-induced adhesion caused by U0126 to 22.3 ± 0.7% and 15.3 ± 0.5%, respectively (P=NS vs. IL-5 alone; Fig. 3C ). By contrast, addition of exogenous AA did not restore IL-5-induced eosinophil adhesion (3.9±1.3% for U0126+AA) versus IL-5 alone (18.1±1.7%; P<0.01). These data are consistent with our previous finding that production of lysophospholipid but not AA metabolites is involved in integrin-mediated adhesion of eosinophils [3 , 5 ].

Role of PI3K in ERK1/2 activation and eosinophil adhesion
Previous studies have suggested that PI3K mediates G-protein-stimulated activation of Raf and ERK1/2 in neutrophils and monocytes [24 , 25 ]. Accordingly, we examined whether PI3K mediates eosinophil adhesion via stimulation of ERK1/2. PI3K activation was measured by its downstream kinase PKB phosphorylation [26 ]. IL-5-stimulated PKB phosphorylation was observed at 5 min and plateaued at 10–15 min (Fig. 4A ). Preincubation with wortmannin, a PI3K inhibitor at concentrations sufficient to inhibit PI3K activity [27 , 28 ], attenuated IL-5-stimulated ERK1/2 phosphorylation (Fig. 4B) . Similarly, LY294002 [29 ], a structurally dissimilar PI3K inhibitor, also blocked ERK1/2 phosphorylation. These data suggest that IL-5 stimulates ERK1/2 phosphorylation through a PI3K-dependent pathway.



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  		Figure 4. The role of PI3K in ERK1/2 activation. Kinetics of PI3K activation (A). Eosinophils were incubated with 10 ng/ml IL-5 for indicated times and were lysed. The lysates were loaded on 10% SDS-PAGE, followed by immunoblotting with antiphosphorylation-specific PKB (A, upper panel) or anti-PKB (A, lower panel) as described in Materials and Methods. These results are representative of three different experiments. Effect of PI3K inhibition on ERK1/2 phosphorylation (B, C). Eosinophils were preincubated with various concentrations of wortmannin (B) or LY294002 (C) for 30 min and were then stimulated by IL-5 for 10 min. ERK1/2 phosphorylation and expression were detected as described in Figure 1A . A representative blot is shown (n=3).

Wortmannin and LY294002 suppressed IL-5-stimulated eosinophil adhesion in a concentration-dependent manner (Fig. 5 ). IL-5-stimulated adhesion decreased from 25.2 ± 2.5% to 6.0 ± 0.2% with 100 nM wortmannin and from 22.2 ± 1.4% to 6.3 ± 1.5% with 100 µM LY294002 (P<0.01 vs. stimulated controls without inhibitor; both comparisons). These results suggest that activation of PI3K and subsequent ERK1/2 activation are required for IL-5-stimulated eosinophil adhesion.



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  		Figure 5. Effect of PI3K inhibition on IL-5-stimulated adhesion of eosinophils to plated ICAM-1. Eosinophils were preincubated with indicated concentrations of wortmannin (nM) or LY294002 (µM) for 30 min and were then stimulated by IL-5 for 30 min. Eosinophil adhesion was measured by residual eosinophil peroxidase activity (n=5). Results are presented as the mean ± SEM, and background adhesion was 4.4 ± 2.3%.

Role of PTK in IL-5-stimulated ERK1/2 activation and eosinophil adhesion
To examine the possible involvement of PTKs in IL-5-induced ERK1/2 activation, eosinophils were preincubated with the src-family tyrosine kinase inhibitor PP2 [30 ] or the Jak2 inhibitor AG490 [31 32 33 ]. IL-5-induced ERK1/2 phosphorylation was attenuated by these two inhibitors in a concentration-dependent manner (Fig. 6 ). ERK1/2 phosphorylation was blocked substantially by 30 µM PP2 or 100 µM AG490. These results suggest that cytoplasmic PTKs also are upstream kinases for ERK1/2 in adhesion mediated by ß2-integrins.



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  		Figure 6. Effects of PTK inhibition on ERK1/2 activation. Eosinophils were preincubated with src-tyrosine kinase inhibitor PP2 for 30 min (A) or with JAK2 inhibitor AG490 for 60 min (B) and were then stimulated by IL-5 for 10 min. ERK phosphorylation and expression were detected as in Figure 1A . A representative blot is shown (n=3).

To establish the role of PTK in ERK1/2-mediated eosinophil adhesion, IL-5-stimulated adhesion of eosinophils was measured in the presence of PP2 or AG490. IL-5-stimulated adhesion decreased from 21.7 ± 1.1% to 5.5 ± 1.2% with 30 µM PP2 and to 6.9 ± 3.6% with 100 µM AG490 (P<0.01 vs. stimulated controls without inhibitor; Fig. 7 ). These data suggest that activation of PTKs and subsequent ERK1/2 activation are required for IL-5-stimulated eosinophil adhesion.



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  		Figure 7. Effect of PTK inhibition on IL-5-stimulated adhesion of eosinophils to plated ICAM-1. Eosinophils were preincubated with indicated concentrations of src-family PTK inhibitor PP2 for 30 min or Jak2 kinase inhibitor AG490 for 60 min and were then stimulated by IL-5 for 30 min. Eosinophil adhesion was measured by residual eosinophil peroxidase activity (n=5). Results are presented as the mean ± SEM, and background adhesion is 3.2 ± 1.8%.

Role of PTK in PI3K activation
As PTK and PI3K were required for IL-5-stimulated ERK activation and eosinophil adhesion, we next examined the causal relationship of these two types of kinases. IL-5-induced PI3K activation, as demonstrated by PKB phosphorylation, was inhibited by the src-family PTK inhibitor PP2 in a concentration-dependent manner (Fig. 8 ). Herbimycin A, another unrelated src-family PTK inhibitor [30 , 34 ], also suppressed PI3K activation (data not shown). By contrast, Jak2 inhibition by AG490 had no inhibitory role on PI3K activation.



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  		Figure 8. Effect of PTK inhibition on PI3K activation. Eosinophils were preincubated with src-tyrosine kinase inhibitor PP2 for 30 min (A) or with JAK2 inhibitor AG490 for 60 min (B) and were then stimulated by IL-5 for 10 min. PKB phosphorylation was detected by phosphorylation-specific PKB Ab (upper panel), and an equal amount of PKB in all lanes was confirmed by phosphorylation-nonselective PKB Ab (lower panel). These results are representative of three different experiments.

We next examined the possible candidates of cytoplasmic PTK in IL-5-stimulated PI3K activation. Lyn (a src-family kinase) and Jak2—two PTKs that are activated by IL-5 in eosinophils [35 ]—have been shown to be physically associated with the p85 subunit of PI3K upon stimulation in other cells [36 , 37 ]. To determine the physical association of PI3K with these two PTKs, eosinophils were stimulated with IL-5 or diluent for 10 min at 37°C, lysed, and immunoprecipitated by anti-Lyn or anti-Jak2. The p85 subunit of PI3K was present in the Lyn immunoprecipitates prior to stimulation by IL-5 (Fig. 9 ). Conversely, no p85 was detected in Jak2 immunoprecipitates in control cells or after treatment with IL-5. These results suggest Lyn but not Jak2 is constitutively associated with PI3K in human eosinophils.



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  		Figure 9. The physical association between Lyn PTK and PI3K. Eosinophils were stimulated with or without IL-5 for 10 min, and cells were lysed and immunoprecipitated by anti-Lyn or anti-Jak2 antibodies. The immunoprecipitated proteins were probed by an anti-p85 subunit of PI3K as described in Materials and Methods (n=5).


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DISCUSSION
 
In this study, we found that ERK1/2 activation caused by IL-5 preceded eosinophil adhesion, and p38{alpha} activation occurred after adhesion; ERK pathway inhibition suppressed eosinophil adhesion and cPLA2 activation; PTK or PI3K inhibition blocked IL-5-stimulated ERK1/2 activation and eosinophil adhesion; src-family PTK inhibition by PP2 but not Jak2 inhibition by AG490 suppressed IL-5-stimulated PI3K activation; and Lyn tyrosine kinase but not Jak2 was physically associated with the p85 subunit of PI3K. Taken together, these results indicate that IL-5-induced eosinophil adhesion to ICAM-1 is regulation by ERK1/2 activation of cPLA2 through the activation of PI3K and cytoplasmic PTKs. We also have demonstrated that src-family tyrosine kinase (possibly Lyn) but not Jak is an upstream kinase for PI3K activation.

We have reported previously that cPLA2 phosphorylation is essential for ß1 and ß2 integrin-dependent adhesion of eosinophils. However, the upstream kinases regulating cPLA2 activation and eosinophil adhesion were not defined in that study. The S505 of cPLA2 is a potential phosphorylation site for MAPK, which has been associated with its activation [7 ]. Prior investigations have shown that three MAPK families (ERK1/2, p38, and JNK) can phosphorylate cPLA2 in vivo and in vitro [7 , 9 , 10 ]. Immunoblotting for phosphorylated MAPK demonstrated that IL-5 caused activation of ERK1/2 and p38{alpha} but neither ERK5 nor JNK1/2; however, only ERK1/2 activation preceded IL-5-stimulated adhesion (Fig. 1) . It is interesting to note that ERK1/2 and p38 are involved in cPLA2 activation in the fMLP- or zymosan-stimulated AA metabolism [8 , 12 ]. These different results may be explained by the nature of the stimuli used in these studies. Alternately, different signal components may be used for adhesion and AA metabolism.

In addition to ERK1/2 activation, PI3K has been shown to be activated in IL-5-stimulated eosinophils and to play an important role in the up-regulation of Fc receptor affinity [13 ]. Recent studies have also suggested that PI3K is important for cell-cell and cell-matrix adhesion in T cells, neutrophils, and mast cells [38 39 40 ]. To study the role of PI3K in eosinophil adhesion, kinetics of PI3K activation and the effects of two commonly used PI3K inhibitors on eosinophil adhesion were examined. We found that PI3K activation precedes eosinophil adhesion (Fig. 4A) and that two unrelated PI3K inhibitors suppressed eosinophil adhesion (Fig. 5) . Thus, our data indicate that PI3K is involved in IL-5-stimulated eosinophil adhesion. In this study, we also showed that PI3K is an important upstream kinase mediating IL-5-induced activation of ERK1/2 in eosinophils, as PI3K inhibition suppressed IL-5-stimulated ERK activation (Fig. 4B and 4C ). The mechanism by which PI3K induces ERK1/2 activation in eosinophils is currently unknown.

Despite the lack of consensus sequences of the IL-5 receptor for tyrosine kinase activity [35 ], studies have shown that IL-5 stimulates cytoplasmic PTK Jak2 and src-family tyrosine kinase Lyn activation [33 , 41 ]. Previous studies have shown that neither Lyn nor Jak2 is involved in IL-5-induced up-regulation of surface CD11b [33 ]. However, recent investigations have shown that CD11b up-regulation is not essential for eosinophil adhesion [3 , 42 ]. In this study, we show that src tyrosine kinase (possibly Lyn) and Jak2 are required for IL-5-induced activation of ERK and subsequent adhesion of eosinophils to ICAM-1. The role of Jak2 in eosinophil adhesion and transmigration into lungs has been confirmed by a recent in vivo study showing that AG490 prevents antigen-induced eosinophil infiltration into the airways of sensitized mice [43 ].

The p85 regulatory subunit of PI3K is tyrosine-phosphorylated after IL-5 treatment, and this phosphorylation is an essential step in activating PI3K [15 ]. However, the identity of the tyrosine kinases involved in this activation remains unknown. Our results indicate that src tyrosine kinase (possibly Lyn) but not Jak2 acts upstream of PI3K. Two lines of evidence suggest this conclusion. First, we observed that Lyn but not Jak2 was constitutively associated with PI3K. Second, inhibition of src-PTK but not Jak2 abolished PI3K activation. IL-5 has been shown previously to activate Lyn within 1 min, which is compatible with a subsequent activation of PI3K and ERK1/2 (10–15 min). Taken together, our results indicate that one pathway of IL-5-stimulated ERK1/2 activation uses Lyn-PI3K activation. The intermediate signals involved in Jak2-mediated ERK1/2 activation were not investigated in this study.

It is important to note some limitations of this study. Our data rely substantially on pharmacological inhibitory experiments as assessed by immunoblotting techniques. In our studies, inhibition/blockade of adhesion was, however, achieved without nonspecific toxicity. At the greatest concentrations used for these studies, cell viability exceeded 95% by trypan blue exclusion (data not shown). At the concentration that blocked IL-5-stimulated eosinophil adhesion, other eosinophil functions remained intact. CD11b surface up-regulation was not prevented by AG490 [33 ] or U0126, and actin polymerization was not affected by PD98059 or wortmannin [44 ]. Other studies also have reported that neither the PI3K inhibitors, wortmannin and LY294002, nor PD98059 have any inhibitory role in granulocyte macrophage-colony stimulating factor-induced eosinophil survival at concentrations used in these studies [32 ]. Although reasonable pharmacological selectivity was established in these studies, dominant negative protein transduction or development of upstream kinase-specific knockout mice will need to be developed to confirm these findings.

In summary, our data suggest a complex coordinated activation of PTKs, PI3K, ERK1/2, and cPLA2, which may mediate upstream components of the firm adhesion process caused by IL-5 in human eosinophils. ERK1/2 phosphorylation, which causes cPLA2 activation, occurs by PI3K-dependent and PI3K-independent mechanisms. We find that src-family tyrosine kinase-mediated ß2-integrin adhesion of human eosinophils is likely dependent on PI3K. By contrast, we find that Jak2-mediated ERK1/2 activation and subsequent eosinophil adhesion do not depend on PI3K activation.


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ACKNOWLEDGEMENTS
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-46368, by NHLBI SCOR Grant HL-56399 (A. R. L.), by American Lung Association Research Grant RG-003-N (X. Z.), and by a grant from GlaxoSmithKline, Inc.

Received March 21, 2002; revised June 27, 2002; accepted July 9, 2002.


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