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 microbeads, 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

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 mean tSEM or geometric assay (EIA) kits were obtained from Cayman Chemical Co. mean with 95% con®dence interval (CI) from the indicated (Ann Arbor, MI). The 4G10 anti-phosphotyrosine monoclonal numbers of experiments. antibody was purchased from Upstate Biotechnology Inc. 1 All statistical analyses were performed using INSTAT (Lake Placid, NY). Acid citrate phosphate, cytochrome c (type 2 (GRAPHPAD Software, San Diego, CA). Groups were compared VI, from horse heart) and SOD were supplied by Sigma Chemie by repeated-measures ANOVA. Comparisons between untreated (Deisenhofen, Germany). All other reagents were of analytical (control) cells and cells pretreated with inhibitors were grade. performed using Dunnett's test for multiple comparisons; Genistein, tyrphostins, lavendustin A and Fura-2/AM were comparisons between points on concentration±response curves dissolved in dimethyl sulphoxide. Lyophilized PAF was obtained in the absence and presence of inhibitors were made dissolved at 1 mM in deionized water 15±30 min before use. 2+ using Bonferroni-corrected Student's t-tests. Indices of [Ca ]i All compounds were diluted to the desired concentration in changes were compared using paired Student's t-tests. A HEPES-BSA buffer. probability <0.05 was de®ned as signi®cant.

Materials RESULTS Fura-2 acetoxymethyl ester (Fura-2/AM), lyophilized PAF, Effects of PTK inhibitors on PAF-induced OÅ. generation lavendustin A (RG14355), tyrphostin A10 (AG126) and 2 Å. tyrphostin B42 (AG490) were purchased from Calbiochem- PAF (1 mM) induced a production of O2 by human eosinophils

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PAF 1 µM Table 1. Effects of genistein pretreatment on indices of PAF-induced Ca2 mobilization in human eosinophils

Baseline (nM) Peak (nM) `Plateau' (nM)RT (seconds) Control 90 Genistein 10 µM Control 115t5.59 330t7.89 161t6.70 132t5.96 Genistein 10 mM 106t15.0 321t58.1 116t15.348.8t2.79

2+ Data are mean tSEM from three experiments. Baseline = mean [Ca ]i in the 60 seconds immediately preceding addition of 1 mM PAF; peak = 2+ 2+ maximum value of [Ca ]i after addition of PAF; `plateau'=[Ca ]i ) 60 seconds after addition of PAF; RT = time taken for [Ca2+] to return M 90 i

(n from peak to within 10% of baseline. i ] 2+

[Ca 2+ Effects of genistein on [Ca ]i The possibility of suppression of early cell signalling events by PTK inhibition was addressed by studying the effect of genistein on PAF-induced Ca2+ mobilization. Pre-incubation of eosinophils with 10 mM genistein for 10 min caused a small, 2+ non-signi®cant reduction in baseline [Ca ]i and had no effect 2+ on peak PAF-induced [Ca ]i elevations (Fig. 3). The return of 2+ [Ca ]i to baseline levels after peaking, however, was accelerated in the presence of genistein (Fig. 3), with both 2+ plateau [Ca ]i (level 60 seconds after addition of PAF) and time taken for [Ca2+] to return to 10% of peak rise (90% Figure 3. Effects of genistein on PAF-induced calcium mobilization in i 2+ recovery time: RT90) being reduced signi®cantly (Table 1). human eosinophils. Changes in [Ca ]i induced by PAF are shown in the absence and presence of 10 mM genistein. Data are mean from three experiments. Additional details are given in Table 1. Effects of genistein on PAF-induced protein tyrosine phosphorylation that was consistently signi®cantly higher than basal production PAF (1 mM) caused a time-dependent phosphorylation of . (P<0 05 in all sets of experiments). Both basal and PAF- tyrosine residues on a number of eosinophil proteins (Fig. 4a). Å. induced O2 generation were inhibited in a concentration- There was notable, reversible tyrosine phosphorylation of dependent manner by the non-selective PTK inhibitor, proteins with apparent molecular masses of 30 000 and below, genistein, with a median inhibitory concentration (IC50)in and of 63 000 and 109 000. Genistein caused a concentration- . PAF-stimulated cells of 0 93 mM (geometric mean, 95% CI dependent inhibition of the PAF-induced phosphorylation of . . 0 18±4 8 mM; Fig. 1a). all of these proteins with similar potency (Fig. 4b,c). Within the To determine which PTK(s) might participate in this range of concentrations of genistein that were effective in response, two drugs with greater selectivity were studied. suppressing eosinophil functions, the degree of inhibition of Tyrphostin AG126 had no signi®cant effect on either basal or PAF-induced tyrosine phosphorylation was less than 50% Å. PAF-induced O2 generation (Fig. 1b). In contrast, tyrphostin (Fig. 4c). B42 (AG490) caused signi®cant suppression of PAF-induced Å. . O2 generation at concentrations in the range 0 1±10 mM (Fig. 1c). DISCUSSION The objective of this study was to identify the role of PTK in mediating responses of human eosinophils to the in¯ammatory mediator, PAF. The broad-spectrum PTK inhibitor, genistein, Effects of PTK inhibitors on PAF-induced LTC release 4 was used to demonstrate PTK involvement in respiratory 2+ PAF induced a concentration-dependent release of LTC4 from burst, LTC4 production and Ca mobilization responses, human eosinophils in the range 20 nMx20 mM that declined at while two more selective inhibitors ± AG126, which inhibits MAPK 13 higher concentrations (not shown). In the presence of 10 mM tyrosine phosphorylation of p42 , and AG490, which 14,15 genistein, PAF-induced LTC4 release was abolished (Fig. 2a). inhibits the activity of Janus kinase 2 (Jak2) ± were studied A second broad-spectrum PTK inhibitor, lavendustin A (as the to determine which PTKs might be involved in eosinophil cell-permeant methyl ester), also abolished PAF-induced LTC4 activation. release at 10 mM and inhibited substantially at 1 mM (Fig. 2b). PAF stimulated respiratory burst and LTC4 release in Lavendustin A methyl ester could not be studied for effects on eosinophils and also induced increases in cytoplasmic free Å. 2+ O2 production owing to its deep orange colour, which calcium concentration [Ca ]i, which may be involved in the interfered profoundly with the cytochrome c reduction assay. activation of one or both of these responses.16,17 PAF also Å. Similarly to O2 production, PAF-induced LTC4 release induced rapid, transient phosphorylation of tyrosine residues was unaffected by AG126 (Fig. 2c) but was inhibited by on multiple eosinophil proteins, indicating the activation of one approximately 50% by 10 mM AG490 (Fig. 2d). or more tyrosine kinase enzyme(s). Genistein suppressed both

# 2000 Blackwell Science Ltd, Immunology, 100, 231±237 Eosinophil activation by platelet-activating factor 235

(a)

PAF 1 µM Control MW (kDa) 2 5 10 20 min

220

97·4

(b) (c) Control PAF 1 µM

1 2 3 4 5 100 pp54 pp57 pp63 220 pp108 80

97·4

66 Optical density (% control) 46 40

0 0·1 1·0 10 µM 0·1 1·0 10 µM

Genistein Genistein (µM)

Figure 4. PAF-induced tyrosine phosphorylation of eosinophil proteins. (a) Western blot of phosphotyrosine-containing proteins in unstimulated human eosinophils (left lane) and eosinophils activated with 1 mM PAF for 2, 5, 10 and 20 min. (b) Western blot of phosphotyrosine-containing proteins in unstimulated eosinophils (lane 1), and eosinophils activated with 1 mM PAF in the absence (lane 2) or presence of genistein (lanes 3±5). (c) Effects of genistein on tyrosine phosphorylation of selected eosinophil proteins, quanti®ed by laser densitometry of Western blots. Data are mean of two experiments.

Å. O2 generation and LTC4 release in human eosinophils, in eosinophils by PKC inhibitors is accompanied by an increase 2+ 12 demonstrating that tyrosine kinase activation is required for in both peak and post-peak [Ca ]i. Since this indicates induction of both of these responses. A second broad-spectrum possible involvement of intracellular calcium mobilization in

PTK inhibitor, lavendustin A, also potently inhibited PAF- PAF-induced LTC4 production, we investigated the effect of induced LTC4 release, con®rming the dependence of this genistein, at a concentration that inhibits this response, on the 2+ response on PTK. [Ca ]i signal following PAF stimulation of eosinophils. While 2+ We have demonstrated previously that an augmentation of peak [Ca ]i was unaffected by 10 mM genistein, the post-peak PAF-induced eicosanoid (thromboxane and LTC4) production return to baseline was accelerated signi®cantly, indicating that

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2+ the prolonged [Ca ]i elevation depends upon PTK activation, concentration (5 mM) that causes only partial inhibition of 2+ 14 while the induction of the initial [Ca ]i `spike' is apparently protein tyrosine phosphorylation. It would appear that a high independent of PTK. The ®nding that a PKC inhibitor and a level of tyrosine phosphorylation is required to effect some cell PTK inhibitor ± drugs that have opposite actions on PAF- functions and that a 50% inhibition of PTK activity can have induced LTC4 release ± also have opposite effects on PAF- profound effects on PTK-dependent functions. induced Ca2+ mobilization supports the hypothesis that the In conclusion, PAF activates one or more PTK in human generation of arachidonic acid metabolites in eosinophils is eosinophils and this activation is involved in the maintenance 2+ 2+ Å. dependent upon [Ca ]i elevation. of intracellular Ca elevations and in the generation of O2 To identify which PTK(s) may be involved in transduction and of LTC4 by these cells. The speci®c PTK enzymes of PAF signals in eosinophils, we studied two more selective responsible for evoking these responses remain to be identi®ed PTK inhibitors: tyrphostins AG126 and AG490. AG126 had but may include an AG490-sensitive Jak2. no effect on either respiratory burst or LTC4 release induced by PAF in eosinophils at a concentration close to that which abolishes lipopolysaccharide-induced tumour necrosis factor-a ACKNOWLEDGMENTS production in murine macrophages, suggesting that mitogen- This work was supported by grant 4001-01 KE 9301 from the activated protein kinase (MAPK) activation is unlikely to be Bundesministerium fuÈr Forschung und Technologie, Germany, grant required for transduction of PAF signals in human eosinophils. HL-46368 from the Division of Lung Disease, National Heart, Lung & PAF has been demonstrated to induce transient activation of Blood Institute, National Institutes of Health, Bethesda, MD, USA, MAPK 18 p42 (Erk2); it might therefore seem surprising that and a grant from GlaxoWellcome Medicines Research, UK. AG126, which prevents Erk2 activation,13 was without effect on PAF-induced eosinophil responses. In fact, suppression of Erk2 activation by an inhibitor of MAPK/Erk kinase (Mek) NOTE ADDED IN PROOF causes no inhibition of respiratory burst responses in PAF- primed eosinophils.18 This ®nding indicates a similarity with While no role is apparent for PAF-activated MAP kinase in the leukotriene B -stimulated guinea-pig eosinophils, in which p42/ induction of respiratory burst or eicosanoid generation in eosinophils, 4 Miike et al. have recently demonstrated that PAF-induced human p44 MAPK (Erk1/2) are activated but play no part in eosinophil chemotaxis is dependent upon activation of MAP kinase mediating either respiratory burst or arachidonic acid meta- [Miike S., Kurasawa K., Saito S. & Iwamoto I. (2000) Platelet- 19 bolism. It seems likely that PAF activates Erk2 in human activating factor activates mitogen-activated protein kinases through 2+ eosinophils but that Ca mobilization, respiratory burst and the activation of phosphatidylinositol 3-kinase and tyrosine kinase in LTC4 secretion are not dependent upon activation of this human eosinophils. J Leukoc Biol 67, 117]. enzyme. AG490, on the other hand, caused roughly 50% inhibition of both responses, indicating that both responses involve a REFERENCES PTK that is sensitive to inhibition by this tyrphostin. While AG490 has been demonstrated to exert inhibitory actions in 1. SPRY C.J.F. (1988) Eosinophils: a Comprehensive Review and Guide leukaemic B lymphocytes through Jak2 inhibition,14 it also to the Scienti®c and Medical Literature Oxford University Press, Oxford. inhibits receptor PTKs such as epidermal growth factor 2. H K.J. (1996) In¯ammatory cells in airways. 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