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Selective Functions in Human Eosinophils Activate2Secretory Phospholipases A

Vecchio and Gianni MaroneBalestrieri, Angelica Petraroli, Giulia Scalia, Luigi Del Massimo Triggiani, Francescopaolo Granata, Barbara

http://www.jimmunol.org/content/170/6/3279doi: 10.4049/jimmunol.170.6.3279

2003; 170:3279-3288; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2003 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Secretory Phospholipases A2 Activate Selective Functions inHuman Eosinophils1

Massimo Triggiani,2* Francescopaolo Granata,* Barbara Balestrieri,* Angelica Petraroli,*Giulia Scalia,† Luigi Del Vecchio,† and Gianni Marone*

Secretory phospholipases A2 (sPLA2s) are released in large amounts in the blood of patients with systemic inflammatory diseasesand accumulate at sites of chronic inflammation, such as the airways of patients with bronchial asthma. Blood eosinophils oreosinophils recruited in inflammatory areas therefore can be exposed in vivo to high concentrations of sPLA2. We have examinedthe effects of two structurally different sPLA2s (group IA and group IIA) on several functions of eosinophils isolated from normaldonors and patients with hypereosinophilia. Both group IA and IIA sPLA2 induced a concentration-dependent release of�-glu-curonidase, IL-6, and IL-8. Release of the two cytokines was associated with the accumulation of their specific mRNA. In addition,sPLA2s induced the surface expression of CD44 and CD69, two major activation markers of eosinophils. In contrast, none of thesPLA2s examined induced the production of IL-5, the de novo synthesis of leukotriene C4 and platelet-activating factor, orthe generation of superoxide anion from human eosinophils. Incubation of eosinophils with the major enzymatic products of thesPLA2s (arachidonic acid, lysophosphatidylcholine, or lysophosphatidic acid) did not reproduce any of the enzymes’ effects. Inaddition, inactivation of sPLA2 enzymatic activity by bromophenacyl bromide did not influence the release of�-glucuronidase orof cytokines. Stimulation of eosinophils by sPLA2s was associated with activation of extracellular signal-regulated kinases 1/2.These results indicate that sPLA2s selectively activate certain proinflammatory and immunoregulatory functions of human eo-sinophils through mechanism(s) independent from enzymatic activity and from the generation of arachidonic acid.The Journalof Immunology, 2003, 170: 3279–3288.

P hospholipases A2 (PLA2s)3 are a family of enzymes thatrelease fatty acids, including arachidonic acid (AA), fromthe sn-2-position of phospholipids (1, 2). Two major

classes of PLA2s have been identified and characterized: low-m.w.secretory PLA2s (sPLA2s) and high-m.w. cytosolic PLA2s (3).Within each class, there are several isoforms that have distinctprimary structures, Ca2� requirements, and cellular sources.sPLA2s were initially found in reptile (group IA) and bee venoms(group III) and in mammalian pancreatic fluid (group IB). It wasshown later that a variety of sPLA2s can be expressed and releasedin human tissues (3). Increased levels of extracellular sPLA2s havebeen detected in the plasma of patients affected by systemic in-flammatory diseases such as acute pancreatitis (4), septic shock(5), extensive burns (6), and autoimmune diseases (7). These mol-ecules also accumulate in inflammatory fluids such as the synovial

fluid of patients with rheumatoid arthritis (8–10), the bronchoal-veolar lavage of patients with bronchial asthma (11, 12), and thenasal secretions of patients with allergic rhinitis (13, 14).

sPLA2s released in plasma or within inflamed tissues are enzy-matically active molecules, and they participate in systemic or lo-cal inflammatory reactions by releasing AA from outer cell mem-brane phospholipids (2, 15). However, increasing evidencesuggests that at least some sPLA2s (groups IA, IB, and IIA) alsoactivate inflammatory cells by mechanisms unrelated to their en-zymatic activity (16–20). For example, group IA and IIA sPLA2sinduce exocytosis and cytokine production from human lung mac-rophages by interacting with binding sites expressed on these cells(18). Group IB sPLA2 induces bronchoconstriction and fibroblastproliferation via interaction with a 180-kDa receptor (M-type) (16,17). In addition, group IB and IIA sPLA2s activate mitogen-acti-vated protein kinases and induce phosphorylation of cytosolicPLA2 in murine mast cells expressing the M-type receptor forsPLA2 (19). These effects of sPLA2s are reproduced by catalyti-cally inactive sPLA2s and, therefore, are thought to be independentfrom the enzymatic activity of these molecules.

Several groups have shown that sPLA2s are released at sites ofallergic reactions, such as the airways of patients with bronchialasthma and the nasal mucosa of patients with allergic rhinitis (11–14). These tissues are the preferential areas of accumulation andactivation of eosinophils. Activated eosinophils release a variety ofproinflammatory molecules, including preformed mediators (e.g.,cationic proteins and lytic enzymes), lipid mediators (e.g., leuko-triene C4 (LTC4) and platelet-activating factor (PAF)), and severalcytokines (e.g., IL-5 and IL-6) and chemokines (e.g., IL-8) (21).These molecules concur to promote further recruitment of inflam-matory cells and to induce tissue injury.

In this study, we have examined the capability of two structurallydifferent sPLA2s (groups IA and IIA) to activate several immunolog-ical and biochemical functions of human eosinophils isolated from

*Division of Clinical Immunology and Allergy, University of Naples Federico II,Naples, Italy; and †Division of Hematology, A. Cardarelli Hospital, Naples, Italy

Received for publication September 10, 2002. Accepted for publication January9, 2003.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by grants from the Ministero dell’Istruzione,dell’Universita e della Ricerca, the Ministero della Salute “Alzheimer Project,” theConsiglio Nazionale delle Ricerche (Target Project Biotechnology Grants01.00191.PF31 and 01.00295.PF49), and the Istituto Superiore di Sanita (Rome, Italy;AIDS Project 40B.64). G.M. is the recipient of the 2002 Esculapio Award (Acca-demia Tiberina, Rome, Italy).2 Address correspondence and reprint requests to Dr. Massimo Triggiani, Division ofClinical Immunology and Allergy, University of Naples Federico II, Via Pansini 5,80131 Naples, Italy. E-mail address: triggian@unina.it3 Abbreviations used in this paper: PLA2, phospholipase A2; AA, arachidonic acid;sPLA2, secretory PLA2; LTC4, leukotriene C4; PAF, platelet-activating factor; ERK,extracellular signal-regulated kinase; lyso-PC, lysophosphatidylcholine; lyso-PA, ly-sophosphatidic acid; BPB, bromophenacyl bromide; MFI, mean fluorescence inten-sity; LDH, lactate dehydrogenase.

The Journal of Immunology

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normal donors and from patients with hypereosinophilia. Our resultsindicate that sPLA2s induce the release of �-glucuronidase and thesynthesis of IL-6 and IL-8. In addition, sPLA2s promote the expres-sion of CD44 and CD69, two activation markers of eosinophils. In-terestingly, the sPLA2s examined in this study have no effect on IL-5,cysteinyl LTC4, PAF, or superoxide anion production. Stimulation ofselected functions in human eosinophils by sPLA2s is mediated bymechanism(s) largely independent from the enzymatic activity and isassociated with activation of extracellular signal-regulated kinases 1/2(ERK1/2).

Materials and MethodsReagents and buffers

Group IA sPLA2 (from Naja mossambica mossambica venom), fatty acid-free human serum albumin, BSA, Histopaque-1077, PIPES, L-glutamine,antibiotic-antimycotic solution (10,000 U/ml penicillin, 10 mg/ml strepto-mycin, and 25 �g/ml amphotericin B), Triton X-100, phenolphthalein gluc-uronide, lysophosphatidylcholine (lyso-PC; 1-palmitoyl-sn-glycero-3-phosphocholine), lysophosphatidic acid (lyso-PA; 1-oleoyl-sn-glycero-3-phosphate), cytochrome c (from horse heart type VI), and superoxidedismutase (from bovine erythrocytes) were purchased from Sigma-Aldrich(St. Louis, MO). RPMI 1640, FCS, and eosin B were purchased from ICNPharmaceuticals (Costa Mesa, CA). The Ca2� ionophore A23187 andFMLP were purchased from Calbiochem (La Jolla, CA). AA, LTC4, andPAF were purchased from Biomol (Plymouth Meeting, PA). [3H]Aceticacid (Na salt; 1.9 Ci/mmol) was purchased from DuPont NEN (Boston,MA). GM-CSF was purchased from PeproTech (Rocky Hill, NJ). FITC-conjugated anti-CD44 and anti-CD69 and PerCP-conjugated anti-CD45mAbs were purchased from BD Biosciences (San Jose, CA). PE-conju-gated anti-CD45R0 mAb was obtained from DAKO (Glostrup, Denmark).Isotypic control Abs, FITC-conjugated mouse IgG1, and PE-conjugatedIgG2 were purchased from Immunotech (Marseille, France) and Caltag(Burlingame, CA), respectively. Rabbit anti-phospho-ERK1/2 Ab and rab-bit anti-ERK1/2 Ab were purchased from New England Biolabs (Beverly,MA). HRP-conjugated donkey anti-rabbit Ig Ab was purchased from Am-ersham Pharmacia Biotech (Buckinghamshire, U.K.).

Group IIA (recombinant human synovial) sPLA2 was a generous giftfrom J. Winkler (Smith Kline and Beecham, King of Prussia, PA). Bro-mophenacyl bromide (BPB)-inactivated group IA sPLA2, prepared as pre-viously described (20), was kindly provided by A. Fonteh (Wake ForestUniversity, Wiston-Salem, NC). BPB-inactivated group IIA sPLA2 wasprepared with the same protocol (20). IL-5, IL-6, IL-8, and �-actin primerswere designed by D. Essayan (Johns Hopkins University, Baltimore, MD)and were produced and purified by the Johns Hopkins DNA Core Facility.All other reagents were from Carlo Erba (Milan, Italy).

PIPES buffer was made of 25 mM PIPES, 110 mM NaCl, and 5 mMKCl. PCG buffer was made of PIPES buffer containing 1 mM CaCl2 and1g/L glucose (pH 7.4). Lysis buffer for Western blot of ERK1/2 was madeof 20 mM Tris (pH 7.5), 5 mM EDTA, 1 mM PMSF, 2 mM benzamidine,10 �g/ml aprotinin, 10 �g/ml leupeptin, 10 mM NaF, 150 mM NaCl, 1mM Na3VO4, 1% Nonidet P-40, and 5% glycerol.

Cell preparation

Eosinophils were isolated from peripheral blood of 19 healthy donors withnormal eosinophil count (�300/mm3) and 12 patients with primary hy-pereosinophilic syndrome (eosinophil count ranging from 500 to 2000/mm3). At the time of blood withdrawal, none of the patients was on or hadbeen previously on treatment with corticosteroids or other cytotoxic drugs.Informed consent was obtained from both normal donors and hypereosi-nophilic patients. Granulocytes were isolated by centrifugation over His-topaque-1077 and eosinophils were purified by negative immunomagneticselection (Miltenyi Biotec, Bergisch Gladbach, Germany), according to themanufacturer’s instructions. Briefly, granulocytes were resuspended (5 �107/50 �l) in PBS supplemented with 0.5% BSA and 2 mM EDTA andwere incubated (30 min, 4°C) with magnetic microbeads coated with amAb anti-CD16. At the end of incubation, the cells were washed andpassed through a magnetic field that retained CD16� cells. The free-flow-ing cell population consisted of eosinophils with a purity ranging from 95to 98%, as assessed by eosin B staining. The cells were then resuspended(2 � 106 cells/ml) in RPMI 1640 and incubated as described below.

The experiments on neutrophils were performed with cells isolated fromperipheral blood of healthy donors by centrifugation over Histopaque-1077. The preparations of neutrophils used in these experiments containeda percentage of eosinophils �2%.

Cell incubations

Eosinophils were incubated (37°C, 20 min to 18 h) in RPMI 1640 con-taining various concentrations of group IA (from N. mossambica mossam-bica) or IIA sPLA2 (recombinant human synovial PLA2). All sPLA2 prep-arations were repurified by size exclusion chromatography (19) before useand were routinely checked for LPS contamination (Limulus AmebocyteTest; ICN Pharmaceuticals). Preparations were discarded if LPS concen-tration was above the detection limit of the assay (0.125 endotoxin U/ml).In selected experiments, eosinophils were incubated with increasing con-centrations of AA, lysophospholipids, or BPB-inactivated sPLA2. In an-other group of experiments, eosinophils were incubated with group IIAsPLA2 (10 �g/ml) in the absence or in the presence of BSA (0.5–5 mg/ml).At the end of the incubation, the supernatants were removed, centrifugedtwice (1000 � g, 4°C, 5 min), and stored for up to 72 h at �80°C for thesubsequent determination of �-glucuronidase, IL-5, IL-6, IL-8, and LTC4

release. The cell pellets were lysed with 0.1% Triton X-100 to determinethe total cellular content of �-glucuronidase.

�-Glucuronidase assay

�-Glucuronidase activity in supernatants and cell pellets was measured bya colorimetric assay (22). �-Glucuronidase release was expressed as thepercentage of the total cellular content in cell aliquots lysed with 0.1%Triton X-100. All experiments were conducted in triplicate.

ELISA and RT-PCR for IL-5, IL-6, and IL-8

Cytokine release in the supernatant of eosinophils was measured in dupli-cate determinations using commercially available ELISA kits for IL-5(R&D Systems, Minneapolis, MN), IL-6, and IL-8 (Euro Clone, Devon,U.K.), according to the manufacturer’s instructions. The linearity range ofthe assay was between 8 and 500 pg/ml (IL-5), 6 and 200 pg/ml (IL-6), and62 and 2000 pg/ml (IL-8). The results were expressed as picograms ofcytokine per 106 cells.

In the experiments for RT-PCR, eosinophils (5 � 106/ml) were incu-bated in FCS-free medium alone or with group IIA sPLA2 (1 �g/ml). Atthe end of the incubation, RNA was isolated by the TRIzol technique(Invitrogen, Milan, Italy), according to the manufacturer’s instructions. Di-ethylpyrocarbonate-treated water without SDS was used for the final re-suspension step; RNA was stored at �80°C. Reverse transcription wasperformed with 5 mM MgCl2, oligo(dT)16 primer, and murine leukemiavirus reverse transcriptase according to the manufacturer’s instructions(Applied Biosystems, Norwalk, CT) on a thermocycler (GeneAmp PCRSystem 2400; Applied Biosystems). PCR was performed using Taq poly-merase (1–2.5 U/reaction) at the annealing temperature of 60°C, with tar-get-specific primers for IL-5 (5�-TCACCGAGCTCTGTTGACAA-3� and3�-GCGGTTTTCTCTTCACACC-5� at 0.2–1 �M/primer), IL-6 (5�-ATGAACTCCTTCTCCACAAGCGC-3� and 3�-GTCAGGTCGGACTCCCGAGAAG-5� at 0.2–1 �M/primer), and IL-8 (5�-GCATCTGGCAACCCTACAA-3� and 3�-CTATTTAAACCCCACCTTTCC-5� at 0.2–1 �M/primer). Number of cycles were 30 for IL-8 and 35 for IL-6 and IL-5. RNAwas normalized by RT-PCR for the constitutive marker gene �-actin (30cycles). All PCR products, together with a DNA ladder as a size standard,were separated on 2.5% agarose gel, stained with ethidium bromide, andphotographed. A semiquantitative measurement of relative cytokinemRNA expression was obtained by digital scanning and densitometricanalysis (Scion Image, Frederick, MD).

Flow cytometry analysis of CD44 and CD69 expression

Eosinophils were suspended in RPMI 1640 supplemented with 2.5% FCS,1% L-glutamine, and 1% antibiotic-antimycotic solution. Cells were cul-tured (106/ml) for up to 48 h with and without group IIA sPLA2 (0.1–10�g/ml) or with 10 ng/ml GM-CSF used as positive control. The cultureswere done in 12-well, sterile, flat-bottom plates (BD Biosciences) previ-ously coated with 1% BSA. At the end of incubation, the cells were har-vested and washed twice with PBS. Expression of CD44 and CD69 wasexamined by direct immunofluorescence and flow cytometry (FACSCali-bur; BD Biosciences) according to the following protocol. Cells were sus-pended at a concentration of 2 � 106/ml; 50 �l of cell suspension wereincubated (4°C, 30 min) with saturating amounts of CD44-FITC, CD45R0-PE, and CD45-PerCP (tube 1) and CD69-FITC, CD45R0-PE, and CD45-PerCP (tube 2). The pellets were then washed twice with PBS and wereresuspended in 300 �l of PBS for analysis. CellQuest software (BD Bio-sciences) was used for acquisition and Paint-a-Gate software (BD Bio-sciences) was used for the analysis according to a three-parameter proce-dure. Although the samples contained almost exclusively eosinophils, thesecells were identified according their light scatter properties and their typicalexpression of CD45 and CD45R0. The mean fluorescence intensity for the

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green channel (mean fluorescence intensity (MFI)) was recorded. Back-ground fluorescence was assessed by analyzing unstimulated and sPLA2-or GM-CSF-activated eosinophils incubated (4°C, 30 min) with isotypiccontrol Abs. Because background values under all experimental conditionswere comparable, they were not subtracted (background MFI at 24 h: un-stimulated � 13.67 � 2.33, group IIA sPLA2 � 14.00 � 1.67, GM-CSF �15.33 � 1.76; background MFI at 48 h: unstimulated � 18.33 � 1.67,group IIA sPLA2 � 19.00 � 1.76, GM-CSF � 19.67 � 2.00).

Phosphorylation of ERKs

Purified eosinophils were suspended in PCG buffer. The cells (2.0 � 106/sample) were incubated (37°C, 1–60 min) with 10 �g/ml group IA and IIAsPLA2s. At the end of incubation, the reactions were stopped by addingice-cold PIPES buffer, and the samples were microfuged for 15 s. Cellpellets were immediately lysed in lysis buffer. Cell lysates were kept on icefor 20 min and then microfuged for 10 min at 4°C. Supernatant was col-lected as a protein extract containing lysed cell components without nucleiand diluted in an equal volume of 2� loading buffer (125 mM Tris-HCl(pH 6.8), 4% SDS, 0.005% bromophenol blue, and 20% glycerol) contain-ing 5% 2-ME. Proteins were resolved by 10% SDS-PAGE and transferredto a nitrocellulose membrane (Bio-Rad, Hercules, CA). After immersionovernight in TBST (50 mM Tris (pH 7.5), 150 mM NaCl, and 0.05%Tween 20) containing 4% BSA, membranes were washed three times (10min each) with TBST and then blotted (22°C, 2 h) with anti-phospho-ERK1/2 Ab. After washing, the membranes were incubated (22°C, 1 h)with HRP-conjugated anti-rabbit Ab. Membranes were washed four times

and membrane-bound anti-rabbit Ig Ab was visualized with the ECL West-ern blotting detection reagent (Amersham Pharmacia Biotech) and Hy-perECL luminescence detection film (Amersham Pharmacia Biotech). Al-though comparisons were made on the basis of an equal number of cells,membranes were stripped with stripping buffer (7 M guanidine hydrochlo-ride in distilled water) and then reblotted with anti-ERK1/2 Ab to verifyequal protein content of each sample.

Production of LTC4 and PAF

LTC4 release was measured by RIA, as previously described (23). Thelinearity range of the assay was between 80 and 5000 pg/ml. The resultswere expressed as nanograms of LTC4 per 106 cells. The production ofPAF was determined in both supernatants and cell pellets as the incorpo-ration of [3H]acetate (22). The results were expressed as cpm of [3H]ac-etate incorporated into PAF/106 cells.

Generation of superoxide anion

Superoxide anion production was measured as superoxide dismutase-in-hibitable reduction of cytochrome c according to the method of Pick andMizel (24). The results were expressed as nanomoles of superoxide anionper 106 cells.

Lactate dehydrogenase (LDH) assay

LDH release at the end of the incubations was determined as an index ofcytotoxicity. LDH was measured in cell-free supernatants using a com-mercially available kit (Sigma-Aldrich).

Statistical analysis

The data are expressed as the mean � SE of the indicated number of exper-iments. The p values were determined with t test for unpaired samples (25).

ResultsEffect of sPLA2s on �-glucuronidase release from humaneosinophils

Initial experiments were performed to determine whether group IA(from cobra venom) and IIA (recombinant human synovial) sPLA2

induced the release of the lysosomal enzyme �-glucuronidase fromhuman eosinophils isolated from normal donors. The cells wereincubated (37°C, 2 h) with various concentrations of group IA orIIA sPLA2. Fig. 1 shows that group IA and IIA sPLA2s induce therelease of �-glucuronidase from purified human eosinophils in aconcentration-dependent fashion. The effect of sPLA2s was signif-icant at a concentration of 1 �g/ml, whereas concentrations �10�g/ml did not induce further release of �-glucuronidase. None ofthe sPLA2s used in these experiments significantly influenced cellviability, as assessed by trypan blue exclusion (data not shown). Inaddition, the release of LDH, used as an indicator of cytotoxicity,in sPLA2-treated eosinophil supernatants was always �4%.

Additional experiments were performed to compare sPLA2-in-duced �-glucuronidase release from eosinophils isolated from hy-pereosinophilic patients with eosinophils or neutrophils isolatedfrom normal donors (Table I). There was no significant difference

FIGURE 1. Effect of increasing concentrations of group IA and IIAsPLA2s on the release of �-glucuronidase from eosinophils. The cells wereincubated (37°C, 2 h) with the indicated concentrations of group IA andgroup IIA sPLA2s. At the end of the incubation, the supernatant was col-lected and centrifuged (1000 � g, 4°C, 5 min). �-Glucuronidase releasewas determined by a colorimetric technique (22). The values are expressedas the percentage of the total cellular content determined in cell aliquotslysed with 0.1% Triton X-100. The data are the mean � SE of six exper-iments. �, p � 0.01 vs control.

Table I. sPLA2-induced �-glucuronidase release from human eosinophils and neutrophilsa

Group IA sPLA2

�-Glucuronidase Release (% of total cellular content)

Eosinophils(normal donors; n � 8)

Eosinophils(hypereosinophilic patients; n � 8)

Neutrophils(normal donors; n � 8)

Unstimulated 6.7 � 1.1 8.4 � 1.2 10.6 � 0.50.1 �g/ml 8.5 � 0.6 9.6 � 1.0 12.4 � 1.01 �g/ml 10.4 � 0.5** 14.3 � 1.4** 13.1 � 0.7*10 �g/ml 16.3 � 1.3** 17.0 � 0.9** 16.7 � 1.8**

a The cells were incubated (37°C, 2 h) with the indicated concentrations of group IA sPLA2. At the end of the incubation,the supernatants were collected and centrifuged (1000 � g, 4°C, 5 min). �-Glucuronidase release was determined by a color-imetric technique (22). The values are expressed as the percentage of the total cellular content determined in cell aliquots lysedwith 0.1% Triton X-100.

�, p � 0.05 vs respective unstimulated.��, p � 0.01 vs respective unstimulated.

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in �-glucuronidase release induced by group IA sPLA2 betweeneosinophils from normal donors (blood eosinophils �300/mm3)and those from patients with hypereosinophilia (blood eosinophils�500/mm3). In addition, Table I shows that group IA sPLA2 alsoinduced the release of �-glucuronidase from human neutrophils(preparations containing �2% eosinophils) in a fashion quantita-tively similar to that induced from eosinophils. These data indicatethat sPLA2s induce a noncytotoxic release of �-glucuronidase thatis comparable in eosinophils (from normal donors and hypereosi-nophilic patients) and neutrophils.

Effect of sPLA2s on IL-5, IL-6, and IL-8 production fromeosinophils

In the next group of experiments, we determined whether, in ad-dition to a preformed mediator such as �-glucuronidase, group IAand IIA sPLA2s induced the production of IL-5, IL-6, and IL-8,three major cytokines produced by human eosinophils (26–28). Inthese experiments, the cells were incubated with increasing con-centrations of sPLA2s for 18 h (IL-5 and IL-6) or for 4 h (IL-8).Kinetic experiments indicated that these incubation times were op-timal for sPLA2-induced cytokine release (data not shown). Fig. 2shows that both group IA (upper panel) and IIA (lower panel)sPLA2s concentration dependently increase the basal secretion ofIL-6 and IL-8 from eosinophils. In contrast, none of the sPLA2sinduced IL-5 release from cells incubated up to 24 h. The effect ofsPLA2s on the production of IL-6 and IL-8 was significant at 1�g/ml and maximal at 10 �g/ml. There was no significant differ-

ence in sPLA2-induced IL-6 and IL-8 production between eosin-ophils from normal donors and eosinophils from hypereosinophilicpatients (data not shown).

We tested the hypothesis that sPLA2s induce cytokine gene ex-pression by evaluating IL-5, IL-6, and IL-8 mRNA expression byRT-PCR in eosinophils incubated with group IIA sPLA2. Fig. 3(upper panel) depicts the �-actin- and IL-6-specific RT-PCR am-plification products from an experiment representative of three inwhich eosinophils were cultured for 12 or 18 h with medium aloneor with group IIA sPLA2. Fig. 3 (lower panel) depicts the �-actin-and IL-8-specific RT-PCR amplification products from an exper-iment representative of three in which eosinophils were culturedfor 1 or 3 h with medium alone or with group IIA sPLA2. We useddifferent incubation times for IL-6 and IL-8 mRNA experimentsbecause kinetics data showed that IL-6 release was delayed (16–24h) compared with IL-8 release (2–6 h). Eosinophils constitutivelyexpress IL-6 and IL-8 mRNA; incubation with group IIA sPLA2

increased the amount of mRNA specific for both IL-6 and IL-8.There was no basal expression of IL-5 mRNA in eosinophil prep-arations and no sPLA2-induced increase in our experimental con-ditions (data not shown). Densitometric analysis of the signal in-tensity of IL-6 and IL-8 mRNA amplification products, obtained inthree experiments and expressed as the ratio with the correspond-ing signal intensity of �-actin, revealed that group IIA sPLA2sinduced a significant accumulation of IL-6 and IL-8 mRNA after12 h and 3 h of incubation, respectively (Table II). These resultsindicate that sPLA2-induced cytokine production is associatedwith the accumulation of their specific mRNA.

FIGURE 2. Effect of increasing concentrations of group IA and IIAsPLA2s on the release of cytokines from eosinophils. The cells were in-cubated (37°C, 18 h for IL-5 and IL-6 and 4 h for IL-8) with the indicatedconcentrations of group IA (upper panel) and group IIA (lower panel)sPLA2s. At the end of the incubations, the supernatant was collected andcentrifuged (1000 � g, 4°C, 5 min). IL-5, IL-6, and IL-8 release weredetermined by ELISA. The data are the mean � SE of six experiments. �,p � 0.05 vs control; ��, p � 0.01 vs control.

FIGURE 3. RT-PCR of IL-6 and IL-8 in sPLA2-stimulated eosinophils.Upper panel, �-Actin- (first row) and IL-6-specific RT-PCR amplificationproducts from a representative experiment in which eosinophils were cul-tured for 12 and 18 h with RPMI 1640 alone (unstimulated) or group IIAsPLA2 (1 �g/ml). The sizes for �-actin and IL-6 were 673 and 658 bp,respectively. A 100-bp DNA ladder was used as standard. Lower panel,�-Actin- (first row) and IL-8-specific RT-PCR amplification products froma representative experiment in which eosinophils were cultured for 1 and3 h with RPMI 1640 alone (unstimulated) or group IIA sPLA2 (1 �g/ml).The sizes for �-actin and IL-8 were 673 and 308, respectively. A 100-bpDNA ladder was used as standard.

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Effect of sPLA2 on CD44 and CD69 expression on humaneosinophilsCD44 and CD69 are distinct cell surface activation markers forhuman eosinophils (29). CD44 is constitutively expressed,whereas little if any CD69 is expressed on quiescent eosinophils.However, both markers are significantly increased on eosinophilsactivated in vivo or in vitro (29). We analyzed the effects of groupIIA sPLA2 on the expression of these two eosinophil activation

Ags. Fig. 4, A and B, shows that sPLA2 enhanced the expressionof CD44 in a concentration- and time-dependent fashion. In par-ticular, the effect of sPLA2 appeared after 24 h of incubation andreached maximal level after 48 h of incubation. As a control inthese experiments, we used GM-CSF (10 ng/ml) that inducesCD44 expression (29). Group IIA sPLA2 also induced the expres-sion of CD69 in a concentration- and time-dependent manner (Fig.5, A and B). As previously shown (30), because CD69 is expressed

FIGURE 4. FACS analysis of CD44 expression on human eosinophils. A,Eosinophils were cultured (37°C, 48 h) with medium alone (unstimulated;dotted line) or with group IIA sPLA2 (1 �g/ml; continuous line). At the end ofthe incubation, the cells were analyzed as described in Materials and Methods.The figure displays the results of one representative experiment of three. B,Eosinophils were incubated (37°C, 24–48 h) with medium alone (unstimu-lated), with increasing concentrations (0.1–10 �g/ml) of group IIA sPLA2, andwith GM-CSF (10 ng/ml) used as a positive control. At the end of the incu-bation, the cells were analyzed as described in Materials and Methods. Pair-matched isotypic control results of all experimental conditions were compa-rable as reported in Materials and Methods. The figure shows the mean � SEof three experiments. �, p � 0.05 vs unstimulated.

FIGURE 5. FACS analysis of CD69 expression on human eosinophils. A,Eosinophils were cultured (37°C, 24 h) with medium alone (unstimulated;dotted line) or with group IIA sPLA2 (1 �g/ml; continuous line). At the end ofthe incubation, the cells were analyzed as described in Materials and Methods.The figure displays the results of one representative experiment of three. B,Eosinophils were incubated (37°C, 24–48 h) with medium alone (unstimu-lated), with increasing concentrations (0.1–10 �g/ml) of group IIA sPLA2, andwith GM-CSF (10 ng/ml) used as a positive control. At the end of the incu-bation, the cells were analyzed as described in Materials and Methods. Pair-matched isotypic control results of all experimental conditions were compa-rable as reported in Materials and Methods. The figure shows the mean � SEof three experiments. �, p � 0.05 vs unstimulated.

Table II. Densitometric analysis of sPLA2-induced mRNA expression of IL-6 and IL-8 in humaneosinophilsa

Stimulus

IL-6(Ratio of IL-6:�-Actin)

IL-8(Ratio of IL-8:�-Actin)

12 h 18 h 1 h 3 h

Unstimulated 0.18 � 0.03 0.20 � 0.04 0.36 � 0.04 0.38 � 0.03Group IIA sPLA2 (1 �g/ml) 0.48 � 0.08* 0.28 � 0.05 0.56 � 0.07 1.05 � 0.09*

a Eosinophils were incubated for different times (1 or 3 h and 12 or 18 h for IL-8 and IL-6, respectively) with RPMI 1640alone (unstimulated) or with group IIA sPLA2. The results are expressed as the signal intensity ratio cytokine:�-actin. Data fromthree separate experiments are expressed as the mean � SE.

�, p � 0.05 vs unstimulated.

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at a low level in freshly isolated eosinophils, the effect of sPLA2 isalready near maximal after 24 h with only a small increase after48 h of incubation. The maximal response induced by sPLA2 (10�g/ml) was comparable to that induced by 10 ng/ml of GM-CSF.These data confirm and extend previous observations (30) demon-strating that sPLA2s induce the expression of two major activationmarkers on human eosinophils.

Effect of sPLA2s on superoxide anion generation from humaneosinophils

Eosinophils are a major source of oxygen radicals in hypereosi-nophilic diseases (31). In these experiments, we explored whethersPLA2s induced superoxide anion generation from eosinophils.Fig. 6 shows that neither group IA nor group IIA sPLA2 activatedthe production of superoxide anion from eosinophils isolated fromnormal donors or from patients with hypereosinophilia (data notshown). In the same experimental conditions, 5.9 � 1.4 nmoles ofsuperoxide anion/106 cells were produced by eosinophils stimu-lated with the the Ca2� ionophore A23187.

Effect of sPLA2s on LTC4 and PAF synthesis from humaneosinophils

Extracellular sPLA2s mobilize AA that is available for conversionto eicosanoids (15, 32). The major AA metabolite produced byhuman eosinophils is LTC4, a potent bronchoconstrictor and proin-flammatory mediator (33–35). In this group of experiments weevaluated the effect of sPLA2s on the de novo synthesis of LTC4.Group IA and group IIA sPLA2s did not induce release of LTC4

from eosinophils of either normal donors or hypereosinophilic pa-tients (Fig. 7). Under the same experimental conditions, A23187and FMLP, used as positive controls, induced the release of 21.3 �1.8 and 4.2 � 0.5 ng of LTC4/106 cells, respectively (Fig. 7).

Eosinophils are a major source of PAF (36), a lipid mediatorwhose synthesis is biochemically related to AA metabolism (37).In three experiments, incubation of eosinophils with group IIAsPLA2 up to 10 �g/ml did not induce the production of PAF asassessed by [3H]acetate incorporation (control, 22 � 7; group IIAsPLA2, 24 � 8 cpm/106 cells). In the same experiments, A23187

(10�6 M) induced the incorporation of 842 � 67 cpm of [3H]ac-etate into PAF/106 cells.

Role of sPLA2 enzymatic activity on the activation of humaneosinophils

The major enzymatic products of sPLA2s, AA and lysophospho-lipids, modulate several biochemical functions of inflammatorycells, including eosinophils (38). In particular, sPLA2-induced ex-pression of CD69 has been related to the generation of lysophos-pholipids (30). To understand whether the effects of sPLA2s weredue to the enzymatic activity, we designed two sets of experi-ments. We initially tested the hypothesis that enzymatic productsof sPLA2s could be involved in sPLA2-induced activation of eo-sinophils by evaluating the effect of AA, lyso-PC, or lyso-PA onthe release of �-glucuronidase and IL-8 and on the expression ofCD44 and CD69. Table III shows that AA, lyso-PC, and lyso-PA,at concentrations up to 10�5 M, did not affect any of the functionsactivated by group IIA sPLA2. We also performed experiments torule out that endogenous lipid mediators could have affectedsPLA2-induced activation of eosinophils. In these experiments, thecells were stimulated with group IIA sPLA2 in the presence ofBSA. At concentrations up to 5 mg/ml, BSA did not modify thesPLA2-induced release of �-glucuronidase and IL-8 (data notshown). These results indicate that the major enzymatic productsof sPLA2s are not involved in sPLA2-induced activation ofeosinophils.

In another group of experiments, eosinophils were incubatedwith group IA or IIA sPLA2, in which catalytic activity wasinactivated by treatment with BPB for 4 h at 37°C (20). Thisprocedure resulted in the irreversible inhibition of �98% ofenzymatic activity (data not shown). Fig. 8 shows that both BPB-inactivated sPLA2s (group IA and group IIA) retain their ability toinduce the release of �-glucuronidase (upper panel) and IL-8 (low-er panel) from eosinophils. However, although not statistically sig-nificant, the release of �-glucuronidase and IL-8 induced by en-zymatically inactive sPLA2s tended to be lower than that inducedby the active molecule. Taken together, the results of these groupsof experiments indicate that the enzymatic activity of sPLA2, ofboth snake and human origin, is not primarily responsible for in-ducing degranulation and cytokine release.

FIGURE 6. Effect of increasing concentrations of group IA and IIAsPLA2s on superoxide anion generation from eosinophils. The cells wereincubated (37°C, 30 min) with the indicated concentrations of group IA orIIA sPLA2s or with A23187 (10�6 M), used as a positive control. At theend of the incubation, the supernatant was collected and centrifuged(1000 � g, 4°C, 5 min). Superoxide anion generation was determined bythe superoxide dismutase-inhibitable reduction of cytochrome c. The dataare the mean � SE of four experiments. �, p � 0.01 vs control.

FIGURE 7. Effect of increasing concentrations of group IA and IIAsPLA2s on LTC4 production from eosinophils. The cells were incubated(37°C, 1 h) with the indicated concentrations of group IA or IIA sPLA2s orwith A23187 (10�6 M) and FMLP (10�6 M), used as positive controls. Atthe end of the incubation, the supernatant was collected and centrifuged(1000 � g, 4°C, 5 min). LTC4 production was determined by RIA. Thedata are the mean � SE of four experiments. �, p � 0.01 vs control.

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Activation of ERK1/2 by sPLA2 in human eosinophils

The aforementioned results indicated that the enzymatic activity ofsPLA2s was not mainly responsible for eosinophil activation and

suggested that the effects of sPLA2s could be mediated by alter-native mechanisms. Binding of sPLA2s to the M-type receptorexpressed on mast cells and macrophages has been shown to ac-tivate intracellular signaling through the ERK1/2 pathway (19, 39).To verify this mechanism in human eosinophils, cells were stim-ulated with group IA sPLA2 (10 �g/ml). The reaction was stoppedat various time points and cytosolic extracts were subjected toWestern blot with anti-phospho-ERK1/2 Ab. Group IA sPLA2 in-duced phosphorylation of both ERK1 and ERK2 (Fig. 9, uppergel). Phosphorylation became evident after 30 s and persisted up to15 min. Reprobing the membrane with the anti-ERK1/2 Ab (non-phosphorylated forms) confirmed equal protein loading (Fig. 9,lower gel). Similar results were obtained when eosinophils werestimulated with group IIA sPLA2 (data not shown). Phosphoryla-tion of ERK1/2 was not detected under our experimental condi-tions when the cells were incubated with AA or lysophospholipidsup to 10�5 M (data not shown).

DiscussionExtracellular PLA2s are attracting increasing attention as media-tors of local and systemic inflammation. Several studies haveshown that sPLA2s can activate inflammatory cells by variousmechanisms either related or unrelated to their enzymatic activity

FIGURE 8. Effect of enzymatically active and BPB-inactivated groupIA and IIA sPLA2s on �-glucuronidase (upper panel) and IL-8 (lowerpanel) release from eosinophils. The cells were incubated (37°C, 2 h for�-glucuronidase and 4 h for IL-8) with the indicated concentrations ofenzymatically active or BPB-inactivated group IA and IIA sPLA2. At theend of the incubation, the supernatant was collected and centrifuged(1000 � g, 4°C, 5 min). �-Glucuronidase release (upper panel) was de-termined by a colorimetric technique (22). The values are expressed as thepercentage of the total cellular content determined in cell aliquots lysedwith 0.1% Triton X-100. IL-8 release (lower panel) was determined byELISA. The data are the mean � SE of four experiments.

FIGURE 9. Kinetics of phosphorylation of ERKs induced by sPLA2.Esinophils were stimulated with group IA sPLA2 (10 �g/ml) for the timesindicated. Reactions were stopped with the addition of ice-cold PIPESbuffer. The cells were microfuged, lysed, and subjected to Western blotanalysis with anti-phosphorylated ERK1/2 Ab (upper gel) as described inMaterials and Methods. The membrane was then stripped and reblottedwith anti-ERK1/2 Ab (lower gel) to confirm equal protein content of eachsample. The Western blot shown is representative of two separateexperiments.

Table III. Effect of sPLA2 enzymatic products on human eosinophilsa

Stimulus�-Glucuronidase

(% of total cellular content)IL-8

(pg/106 cells)CD44(MFI)

CD69(MFI)

None 6.32 � 0.68 340 � 57 70.3 � 6.4 24.7 � 3.1AA, 10�7 M 5.93 � 0.35 313 � 71 69.0 � 4.4 24.3 � 2.6AA, 10�6 M 5.66 � 0.41 308 � 72 71.7 � 5.1 26.7 � 4.0AA, 10�5 M 6.03 � 0.37 325 � 67 67.0 � 6.5 26.3 � 3.3Lyso-PC, 10�7 M 6.57 � 0.73 332 � 50 69.7 � 5.2 24.3 � 3.9Lyso-PC, 10�6 M 6.92 � 0.75 350 � 47 68.7 � 3.9 23.0 � 3.7Lyso-PC, 10�5 M 6.96 � 0.58 352 � 63 71.3 � 4.9 25.3 � 2.9Lyso-PA, 10�7 M 6.01 � 0.69 362 � 61 73.3 � 8.4 25.7 � 3.5Lyso-PA, 10�6 M 6.10 � 0.57 328 � 58 69.0 � 7.5 26.0 � 2.8Lyso-PA, 10�5 M 6.20 � 0.74 344 � 64 69.3 � 4.3 23.3 � 4.3Group IIA sPLA2, 10 �g/ml 11.06 � 0.95** 713 � 97** 109.3 � 10.0* 55.3 � 6.3*

a The cells were incubated (37°C, 2 h for �-glucuronidase release, 4 h for IL-8 release, 48 h for CD44 and CD69 expression)with the indicated concentrations of AA, lyso-PC, lyso-PA, and group IIA sPLA2. At the end of the incubation, the supernatantwas collected and centrifuged (1000 � g, 4°C, 5 min). �-Glucuronidase release was determined by a colorimetric technique (22).IL-8 release was measured by ELISA. CD44 and CD69 expression were analyzed as described in Materials and Methods andexpressed as the MFI. The values of �-glucuronidase are expressed as the percentage of the total cellular content determined incell aliquots lysed with 0.1% Triton X-100. The values represents the mean � SE of three experiments.

�, p � 0.05 vs respective controls (none).��, p � 0.01 vs respective controls (none).

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(17–20, 39–41). We have examined the effects of two structurallydifferent sPLA2s on several functions of human eosinophils. Bothgroup IA and IIA sPLA2s induce the release of the specific granuleenzyme �-glucuronidase, the production of IL-6 and IL-8, and theexpression of activation markers (CD44 and CD69) on human eo-sinophils. In contrast, sPLA2s have no effect on IL-5, LTC4, PAF,and superoxide anion production.

Large quantities of sPLA2 are released in the blood of patientswith systemic inflammatory diseases. For example, concentrationsup to 4 �g/ml sPLA2 have been detected in the plasma of patientswith septic shock (5). In addition, calculation of the specific ac-tivity of human group IIA sPLA2 (42) and dilution of alveolar fluiddue to the bronchoalveolar lavage (43) indicate local concentra-tions of sPLA2 in the airways of patients with bronchial asthmaranging between 2 and 10 �g/ml (12). Therefore, eosinophils maybe exposed in vivo to concentrations of sPLA2s that efficientlyactivate selected biochemical and immunological functions inthese cells. Furthermore, eosinophils are a major cellular source ofgroup IIA sPLA2 (44). This observation suggests that sPLA2s mayfunction as an autocrine stimulus for eosinophil activation.

Interestingly, both group IA and IIA sPLA2s are more potentstimuli for IL-8 production than are other eosinophil agonists likePAF, RANTES, and PMA, and they do not require cell priming(28, 45). These data support the concept that sPLA2s may be rel-evant stimuli for systemic and/or local activation of eosinophils inallergic or hypereosinophilic disorders usually associated withhigh circulating levels of IL-6 and IL-8 (28, 46–48).

Surprisingly, sPLA2s do not induce, at least in vitro, IL-5 pro-duction. Previous data suggest that the production of cytokines isdifferentially regulated in normal human eosinophils. For example,secretory IgA induces the release of IL-8 but not of IL-5 (45). Inaddition, the production of IL-8, but not of other cytokines, inhuman eosinophils is closely associated with degranulation (45).Our data support the hypothesis of differential regulation of cyto-kine transcription in eosinophils by showing that sPLA2s selec-tively activate intracellular signals leading to IL-8 and IL-6, butnot IL-5, expression. Furthermore, sPLA2s induce the release of apreformed mediator (�-glucuronidase), but not of de novo synthe-sized mediators (LTC4, PAF, and superoxide anion). These obser-vations suggest that activation by sPLA2 is restricted to selectedsignaling pathways within human eosinophils. Our results indicatethat incubation of eosinophils with sPLA2s induces a rapid acti-vation of ERK1/2. This signaling pathway is crucial for degranu-lation and cytokine production, but not for other functions in hu-man eosinophils (49). Although these data clearly indicate thatsPLA2s generate intracellular signals in eosinophils, additional ex-periments are needed to understand the mechanism(s) underlyingthe selectivity of effect of sPLA2s.

Our results extend the previous observation that sPLA2 inducesthe expression of surface activation markers on eosinophils (30).CD44 and CD69, although not specific of eosinophils, are over-expressed on cells activated in vivo, such as the eosinophils re-trieved from the bronchoalveolar lavage, or in vitro by incubationwith cytokines (29, 50). The function of these markers in asthmaand in hypereosinophilic disease is unknown. Interestingly, how-ever, CD44 is highly expressed on leukocytes from patients withrheumatoid arthritis (51), a disease in which blood levels of sPLA2

are significantly increased. This observation supports the hypoth-esis that sPLA2s activate a broad spectrum of inflammatory cells.In line with this hypothesis is our observation that sPLA2 induceddegranulation of neutrophils in a fashion comparable to that ofeosinophils.

The activation of inflammatory cells induced by sPLA2 couldoccur through at least three mechanisms: 1) generation of AA and

lysophospholipids by direct enzymatic activity on membrane phos-pholipids with the concurrent generation of AA metabolites, 2)interaction of sPLA2s with membrane peptidoglycans throughtheir heparin-binding sites, and 3) activation of specific receptors,i.e., the M-type or N-type.

The enzymatic activity of sPLA2s generates AA and lysophos-pholipids, two intracellular messengers capable of inducing suchcellular responses as exocytosis and cytokine expression (38, 52).In addition, LTC4, the main AA metabolite synthesized by eosin-ophils, may function as an autocrine stimulus for these cells (53,54). Therefore, the enzymatic activity may be, at least in part,responsible for the eosinophil activation induced by sPLA2s. How-ever, a number of observations exclude that catalytic activity playsa primary role in the induction of degranulation and IL production.First, sPLA2-induced activation of eosinophils is not associatedwith significant LTC4 generation. Second, incubation of eosino-phils with the major enzymatic products of sPLA2, AA, lyso-PC,or lyso-PA, did not affect �-glucuronidase or IL-8 secretion. Fi-nally, inactivation of sPLA2 enzymatic activity by BPB did notabolish its capacity to induce eosinophil activation. These dataindicate that, as in other inflammatory cells (18–20), the catalyticactivity of sPLA2s is not mainly responsible for their ability toinduce the release of lysosomal enzymes and the de novo synthesisof cytokines and chemokines. However, inactivation of PLA2 ac-tivity tends to reduce the effect of sPLA2 on �-glucuronidase andIL-8 production, suggesting that one or more of the enzymaticproducts of sPLA2s may enhance the effects exerted by sPLA2s oneosinophils.

Several sPLA2s bind to peptidoglycans on cell membranesthrough their heparin-binding site (55, 56). This interaction resultsin the internalization of sPLA2, which is then capable of generatingintracellular signals leading to gene expression (57). Both groupIA and IIA sPLA2s possess a heparin-binding site (58). Therefore,a potential mechanism by which these molecules may activate eo-sinophils is through interaction with membrane peptidoglycans.Moreover, it is conceivable that sPLA2s, at least at high concen-trations, could induce membrane perturbations, which in turn maytrigger eosinophil activation.

Finally, sPLA2s may activate eosinophils by interacting withone or more specific receptors, i.e., the M-type or the N-type (59).These receptors are expressed in a variety of tissues and inflam-matory cells, including macrophages, monocytes, and mast cells,and they are functionally coupled to different cellular responses(17–19, 42, 59, 60). The lack of specific Abs or ligands for thesereceptors in human cells currently precludes their characterizationon eosinophils. However, we provide evidence that both group IAand IIA sPLA2s induce activation of ERK1/2, a signaling pathwaythat has been coupled to activation of the M-type receptor in othercell types (19, 39). These data support the hypothesis that sPLA2smay activate human eosinophils by interacting with the M-typereceptor. However, our data do not exclude the possibility thatactivation of N-type receptor or of other as yet uncharacterizedreceptors may be involved in sPLA2-induced activation ofeosinophils.

We were unable to confirm the observation by Urasaki et al. (30)that enzymatically generated lysophospholipids are responsible forthe expression of CD69 on eosinophils. This discrepancy may beexplained by differences in the procedure for purification of eo-sinophils, i.e., density gradients vs immunomagnetic selection. Inany event, our results do not exclude the possibility that more thanone mechanism underlies the effect of sPLA2s on eosinophils.

In conclusion, our results indicate that sPLA2s selectively acti-vate certain proinflammatory and immunomodulatory functions ineosinophils with mechanism(s) that appear mainly independent

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from their enzymatic activity. Activation of human eosinophils bysPLA2s may concur with the eicosanoid-forming capacity of thesemolecules to induce systemic and/or local activation of these cellsin inflammatory and allergic disorders.

AcknowledgmentsWe are indebted to Dr. Giuseppe Spadaro (Division of Clinical Immunol-ogy and Allergy, University of Naples Federico II, Naples, Italy) and Dr.Gennaro Liccardi (Division of Pneumology and Allergy, A. CardarelliHospital, Naples, Italy) for providing access to patients with hypereosino-philia. We are grateful to Jean Ann Gilder for text editing.

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