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Involvement of Protein Tyrosine Kinases in Activation of Human Eosinophils by Platelet-Activating Factor

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Involvement of Protein Tyrosine Kinases in Activation of Human Eosinophils by Platelet-Activating Factor Immunology 2000 100 231±237 Involvement of protein tyrosine kinases in activation of human eosinophils by platelet-activating factor G. DENT,*{ N. M. MUNÄ OZ,{ X. ZHU,{ E. RUÈ HLMANN,*1 H. MAGNUSSEN,* A. R. LEFF{ & K. F. RABE*1 *Krankenhaus Groûhansdorf, Zentrum fuÈr Pneumologie und Thoraxchirurgie, LVA Hamburg, D-22927 Groûhansdorf, Germany, and {Departments of Medicine and Pharmacologic and Physiologic Sciences, Division of Biological Sciences, University of Chicago, Chicago, IL, USA SUMMARY Activation of human eosinophils by platelet-activating factor (PAF) involves multiple signal transduction pathways. Among these, protein kinase C has been demonstrated both to mediate respiratory burst and to suppress an alternative pathway of activation of respiratory burst and arachidonic acid metabolism in eosinophils. We utilized inhibitors of protein tyrosine kinases (PTK) to elucidate the role of PTK in PAF-induced activation of eosinophils. Eosinophils were isolated from peripheral blood of atopic donors and stimulated with PAF in the absence or presence of broad-spectrum PTK inhibitors ± genistein or lavendustin A; an inhibitor of mitogen-activated protein (MAP) kinase activation ± tyrphostin AG126; or an inhibitor of Janus kinase 2 (Jak2) ± Å. tyrphostin B42 (AG490). PAF induced superoxide anion (O2 ) generation, leukotriene C4 (LTC4) release, intracellular calcium ion mobilization and tyrosine phosphorylation of multiple eosinophil proteins in a concentration-dependent manner. All of these responses were concentration- dependently inhibited by genistein; lavendustin A also exhibited potent inhibition of PAF-induced Å. LTC4 release. AG126 had no effect on either O2 generation or LTC4 release, while AG490 inhibited both responses, albeit less effectively than genistein. We conclude that PAF activates PTK in human eosinophils and that this signalling pathway is involved in eliciting respiratory burst and leukotriene production. The speci®c PTK(s) involved are unknown but may include Jak2. INTRODUCTION derived basic proteins, lipid mediators, reactive oxygen species (ROS) and cytokines have been implicated in allergic and Eosinophils are immune effector cells that are particularly asthmatic reactions.2 associated with immunoglobulin E (IgE) -dependent reactions, The products of eosinophils stimulated with platelet- such as parasite killing and allergy.1 They are the distinctive activating factor (PAF) have been demonstrated to induce in®ltrating cells in human airways in asthma; eosinophil- contraction of airway smooth muscle3,4 and to damage respiratory epithelium in vitro.5,6 The former effect is mediated Received 18 October 1999; revised 24 January 2000; accepted largely through the release of the cysteinyl leukotriene, 3,7 31 January 2000. LTC4. Activated eosinophils have also been shown to increase the responsiveness of bronchial smooth muscle to Abbreviations: CI, con®dence interval; IC , median inhibitory 50 other contractile agents,8 an action that can be mimicked in concentration; Jak2, Janus kinase 2; LTC4, leukotriene C4; MAPK, Å. vivo by inhaled LTC .9 The toxic actions of activated mitogen-activated protein kinase; O2 , superoxide anion radical; PAF, 4 platelet-activating factor; PKC, protein kinase C; PTK, protein eosinophils on respiratory epithelium appear to be mediated tyrosine kinase; ROS, reactive oxygen species; RT90, 90% recovery largely through a combination of ROS and granule proteins, time; SDS, sodium dodecyl sulphate; SOD, superoxide dismutase. particularly eosinophil peroxidase.5,10 Thus, both eicosanoid Present addresses: {Division of Respiratory Cell and Molecular lipid mediator production and other aspects of eosinophil Biology, University of Southampton School of Medicine, activation, including ROS generation, are important in the Southampton, SO16 6YD, UK, 1Department of Pulmonology, eosinophilic in¯ammation occurring in asthmatic airways. Leiden University Medical Centre, NL-2300 Leiden RC, the The cell signalling pathways through which in¯ammatory Netherlands. mediators activate eosinophils have only recently begun to be 11 Correspondence: Dr G. Dent, Medical Specialities, Mail Point elucidated. We have recently identi®ed the role of protein 0810, Southampton General Hospital, Tremona Road, Southampton, kinase C (PKC) in the activation of eosinophil respiratory SO16 6YD, UK. E-mail: [email protected] burst by PAF, measured as production of the ROS, superoxide # 2000 Blackwell Science Ltd 231 232 G. Dent et al. Å. (a) actions of PTK inhibitors were assessed on O2 generation and Baseline LTC4 release, as well as on PAF-induced intracellular calcium 2+ PAF 1 µM concentration ([Ca ]i) elevations and protein tyrosine phos- phorylations, to determine the involvement of PTK both in early cell signalling events and in the release of active molecules from the cells. MATERIALS AND METHODS Cells Eosinophils were isolated from acid citrate phosphate-antic- oagulated blood of atopic, non-asthmatic donors by negative immunomagnetic selection using anti-CD16-coated microbe- ads, as described previously.12 Cell preparations contained >90% eosinophils [95.5t0.50% (mean tSEM), n=37 from Genistein (µM) 20 donors; contaminants mostly mononuclear cells] which were (b) >97% viable at the time of experimentation. Eosinophils were cells) 6 suspended in sterile-®ltered HEPES-bovine serum albumin (BSA) buffer, as described previously.12 Cell suspensions were stored on ice for up to 20 min before experimentation. All experiments were performed in HEPES-BSA buffer. Respiratory burst measurements Å. Superoxide anion (O2 ) generation was measured as the superoxide dismutase (SOD)-inhibitable reduction of ferricy- tochrome c, as described previously.12 Results are expressed as nanomoles of cytochrome c reduced per 106 cells in 15 min. Leukotriene production generation (nmol cytochrome c reduced/10 Cysteinyl leukotriene release was measured as described − 2 12 O previously. As cell suspensions were almost exclusively AG126 (µM) eosinophils, all cysteinyl leukotriene measured was assumed to be LTC .7 Results are expressed as femtomoles of LTC (c) 4 4 released per 106 cells in 5 min. Measurement of intracellular Ca2+ 2+ 2+ Intracellular Ca concentration ([Ca ]i) was measured in Fura-2-loaded eosinophils, as described previously.12 Measurement of protein phosphorylation Phosphorylation of tyrosine residues on eosinophil proteins was assessed by Western blotting. Eosinophils (106) were incubated for 10 min at 37u in the absence or presence of genistein prior to the addition of 1 mM PAF or an equal volume of HEPES-BSA buffer. Mixtures were incubated for 1±20 min, after which cells were precipitated by centrifugation. Super- natants were discarded; cell pellets were disrupted in 100 ml AG490 (µM) denaturing lysis buffer (composition: Tris±HCl, 20 mM; NaCl, 40 mM; Nonidet P-40, 1% v/v; deoxycholic acid, 0.5% w/v; Figure 1. Effects of (a) genistein (n=6), (b) tyrphostin AG126 (n=3) ethylenediaminetetraacetic acid, 5 mM; leupeptin, 10 mg/ml; and (c) AG490 (n=3) on basal and PAF-induced OÅ. generation in 2 aprotinin, 5 mg/ml; phenylmethylsulphonyl ¯uoride, 2 m ; human eosinophils. Data are mean tSEM from the indicated numbers M Å. NaF, 50 mM; sodium pyrophosphate, 30 mM; sodium ortho- of experiments conducted in duplicate. PAF-stimulated O2 generation . was signi®cantly higher than baseline in all cases (P<0.05). *P<0.05, vanadate, 2 mM;pH74) on ice for 10 min. Lysates were **P<0.01, compared to control cells preincubated without inhibitors. centrifuged at 1000 g for 5 min to precipitate unbroken nuclei and cell debris; supernatants were mixed 1 : 1 with 4% sodium Å. anion radical (O2 ), but also found PKC activation to be dodecyl sulphate (SDS) sample buffer (composition: Tris±HCl, responsible for damping of alternative pathways of activation 250 mM; SDS, 9.2% w/v; glycerol, 40% v/v; 2-mercaptoethanol, for both respiratory burst and eicosanoid production.12 The 20% v/v; bromophenyl blue, 0.004% w/v; pH 6.8) and boiled aim of the present study was to determine the role of an for 5 min. Proteins in cell lysates (approx. 25 mg per sample) alternative set of signal transduction enzymes ± the protein were separated by 7.5% polyacrylamide gel electrophoresis and tyrosine kinases (PTK) ± in these responses in eosinophils. The blotted onto polyvinylidene di¯uoride membranes (400 mA for # 2000 Blackwell Science Ltd, Immunology, 100, 231±237 Eosinophil activation by platelet-activating factor 233 (a) (b) Control Control Genistein 10 µM Lavendustin A 1 µM Lavendustin A 10 µM cells) 6 (c) (d) release (fmol/10 4 Control Control LTC AG126 10 µM AG490 10 µM PAF (M) Figure 2. Effects of (a) genistein (n=6), (b) lavendustin A (n=3), (c) tyrphostin AG126 (n=3) and (d) AG490 (n=3) on PAF-induced . LTC4 release from human eosinophils. Data are mean tSEM. *P<0 05, **P<0 01, ***P<0 001, compared to responses to the same concentration of PAF in the absence of inhibitors. 1 hr). Tyrosine-phosphorylated protein bands were stained Novabiochem GmbH [Bad Soden (Taunus), Germany]. using anti-phosphotyrosine antibody 4G10 (1 mg/ml for 1 hr) Genistein and lavendustin A methyl ester were supplied by and detected by enhanced chemiluminescence (ECL+, Amer- Alexis GmbH (GruÈnberg, Germany). Anti-FccRIII (CD16)- sham Corp., Arlington Heights, IL). coated microbeads and magnetic separation apparatus (MACS system) were purchased from Miltenyi Biotec GmbH (Bergisch Statistical analysis Gladbach, Germany). Cysteinyl leukotriene enzyme immuno- Data are expressed as arithmetic
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