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The Journal of Immunology, 2003, 170: 3279-3288.
Copyright © 2003 by The American Association of Immunologists

Secretory Phospholipases A2 Activate Selective Functions in Human Eosinophils1

Massimo Triggiani2,*, Francescopaolo Granata*, Barbara Balestrieri*, Angelica Petraroli*, Giulia Scalia{dagger}, Luigi Del Vecchio{dagger} and Gianni Marone*

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Secretory phospholipases A2 (sPLA2s) are released in large amounts in the blood of patients with systemic inflammatory diseases and accumulate at sites of chronic inflammation, such as the airways of patients with bronchial asthma. Blood eosinophils or eosinophils recruited in inflammatory areas therefore can be exposed in vivo to high concentrations of sPLA2. We have examined the effects of two structurally different sPLA2s (group IA and group IIA) on several functions of eosinophils isolated from normal donors and patients with hypereosinophilia. Both group IA and IIA sPLA2 induced a concentration-dependent release of {beta}-glucuronidase, 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 the sPLA2s examined induced the production of IL-5, the de novo synthesis of leukotriene C4 and platelet-activating factor, or the generation of superoxide anion from human eosinophils. Incubation of eosinophils with the major enzymatic products of the sPLA2s (arachidonic acid, lysophosphatidylcholine, or lysophosphatidic acid) did not reproduce any of the enzymes’ effects. In addition, inactivation of sPLA2 enzymatic activity by bromophenacyl bromide did not influence the release of {beta}-glucuronidase or of 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 eosinophils through mechanism(s) independent from enzymatic activity and from the generation of arachidonic acid.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Phospholipases A2 (PLA2s)3 are a family of enzymes that release fatty acids, including arachidonic acid (AA), from the 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 distinct primary 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 was shown later that a variety of sPLA2s can be expressed and released in human tissues (3). Increased levels of extracellular sPLA2s have been detected in the plasma of patients affected by systemic inflammatory diseases such as acute pancreatitis (4), septic shock (5), extensive burns (6), and autoimmune diseases (7). These molecules also accumulate in inflammatory fluids such as the synovial fluid of patients with rheumatoid arthritis (8, 9, 10), the bronchoalveolar lavage of patients with bronchial asthma (11, 12), and the nasal secretions of patients with allergic rhinitis (13, 14).

sPLA2s released in plasma or within inflamed tissues are enzymatically active molecules, and they participate in systemic or local inflammatory reactions by releasing AA from outer cell membrane phospholipids (2, 15). However, increasing evidence suggests that at least some sPLA2s (groups IA, IB, and IIA) also activate inflammatory cells by mechanisms unrelated to their enzymatic activity (16, 17, 18, 19, 20). For example, group IA and IIA sPLA2s induce exocytosis and cytokine production from human lung macrophages by interacting with binding sites expressed on these cells (18). Group IB sPLA2 induces bronchoconstriction and fibroblast proliferation via interaction with a 180-kDa receptor (M-type) (16, 17). In addition, group IB and IIA sPLA2s activate mitogen-activated protein kinases and induce phosphorylation of cytosolic PLA2 in murine mast cells expressing the M-type receptor for sPLA2 (19). These effects of sPLA2s are reproduced by catalytically inactive sPLA2s and, therefore, are thought to be independent from the enzymatic activity of these molecules.

Several groups have shown that sPLA2s are released at sites of allergic reactions, such as the airways of patients with bronchial asthma and the nasal mucosa of patients with allergic rhinitis (11, 12, 13, 14). These tissues are the preferential areas of accumulation and activation of eosinophils. Activated eosinophils release a variety of proinflammatory molecules, including preformed mediators (e.g., cationic proteins and lytic enzymes), lipid mediators (e.g., leukotriene C4 (LTC4) and platelet-activating factor (PAF)), and several cytokines (e.g., IL-5 and IL-6) and chemokines (e.g., IL-8) (21). These molecules concur to promote further recruitment of inflammatory cells and to induce tissue injury.

In this study, we have examined the capability of two structurally different sPLA2s (groups IA and IIA) to activate several immunological and biochemical functions of human eosinophils isolated from normal donors and from patients with hypereosinophilia. Our results indicate that sPLA2s induce the release of {beta}-glucuronidase and the synthesis of IL-6 and IL-8. In addition, sPLA2s promote the expression of CD44 and CD69, two activation markers of eosinophils. Interestingly, the sPLA2s examined in this study have no effect on IL-5, cysteinyl LTC4, PAF, or superoxide anion production. Stimulation of selected functions in human eosinophils by sPLA2s is mediated by mechanism(s) largely independent from the enzymatic activity and is associated with activation of extracellular signal-regulated kinases 1/2 (ERK1/2).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents 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 streptomycin, and 25 µg/ml amphotericin B), Triton X-100, phenolphthalein glucuronide, 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 superoxide dismutase (from bovine erythrocytes) were purchased from Sigma-Aldrich (St. Louis, MO). RPMI 1640, FCS, and eosin B were purchased from ICN Pharmaceuticals (Costa Mesa, CA). The Ca2+ ionophore A23187 and FMLP were purchased from Calbiochem (La Jolla, CA). AA, LTC4, and PAF were purchased from Biomol (Plymouth Meeting, PA). [3H]Acetic acid (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-CD45 mAbs were purchased from BD Biosciences (San Jose, CA). PE-conjugated anti-CD45R0 mAb was obtained from DAKO (Glostrup, Denmark). Isotypic control Abs, FITC-conjugated mouse IgG1, and PE-conjugated IgG2 were purchased from Immunotech (Marseille, France) and Caltag (Burlingame, CA), respectively. Rabbit anti-phospho-ERK1/2 Ab and rabbit anti-ERK1/2 Ab were purchased from New England Biolabs (Beverly, MA). HRP-conjugated donkey anti-rabbit Ig Ab was purchased from Amersham Pharmacia Biotech (Buckinghamshire, U.K.).

Group IIA (recombinant human synovial) sPLA2 was a generous gift from J. Winkler (Smith Kline and Beecham, King of Prussia, PA). Bromophenacyl bromide (BPB)-inactivated group IA sPLA2, prepared as previously described (20), was kindly provided by A. Fonteh (Wake Forest University, Wiston-Salem, NC). BPB-inactivated group IIA sPLA2 was prepared with the same protocol (20). IL-5, IL-6, IL-8, and {beta}-actin primers were 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 mM KCl. PCG buffer was made of PIPES buffer containing 1 mM CaCl2 and 1g/L glucose (pH 7.4). Lysis buffer for Western blot of ERK1/2 was made of 20 mM Tris (pH 7.5), 5 mM EDTA, 1 mM PMSF, 2 mM benzamidine, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 mM NaF, 150 mM NaCl, 1 mM Na3VO4, 1% Nonidet P-40, and 5% glycerol.

Cell preparation

Eosinophils were isolated from peripheral blood of 19 healthy donors with normal eosinophil count (<300/mm3) and 12 patients with primary hypereosinophilic syndrome (eosinophil count ranging from 500 to 2000/mm3). At the time of blood withdrawal, none of the patients was on or had been previously on treatment with corticosteroids or other cytotoxic drugs. Informed consent was obtained from both normal donors and hypereosinophilic patients. Granulocytes were isolated by centrifugation over Histopaque-1077 and eosinophils were purified by negative immunomagnetic selection (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. Briefly, granulocytes were resuspended (5 x 107/50 µl) in PBS supplemented with 0.5% BSA and 2 mM EDTA and were incubated (30 min, 4°C) with magnetic microbeads coated with a mAb anti-CD16. At the end of incubation, the cells were washed and passed through a magnetic field that retained CD16+ cells. The free-flowing cell population consisted of eosinophils with a purity ranging from 95 to 98%, as assessed by eosin B staining. The cells were then resuspended (2 x 106 cells/ml) in RPMI 1640 and incubated as described below.

The experiments on neutrophils were performed with cells isolated from peripheral blood of healthy donors by centrifugation over Histopaque-1077. The preparations of neutrophils used in these experiments contained a percentage of eosinophils <2%.

Cell incubations

Eosinophils were incubated (37°C, 20 min to 18 h) in RPMI 1640 containing various concentrations of group IA (from N. mossambica mossambica) or IIA sPLA2 (recombinant human synovial PLA2). All sPLA2 preparations were repurified by size exclusion chromatography (19) before use and were routinely checked for LPS contamination (Limulus Amebocyte Test; ICN Pharmaceuticals). Preparations were discarded if LPS concentration was above the detection limit of the assay (0.125 endotoxin U/ml). In selected experiments, eosinophils were incubated with increasing concentrations of AA, lysophospholipids, or BPB-inactivated sPLA2. In another group of experiments, eosinophils were incubated with group IIA sPLA2 (10 µg/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, centrifuged twice (1000 x g, 4°C, 5 min), and stored for up to 72 h at -80°C for the subsequent determination of {beta}-glucuronidase, IL-5, IL-6, IL-8, and LTC4 release. The cell pellets were lysed with 0.1% Triton X-100 to determine the total cellular content of {beta}-glucuronidase.

{beta}-Glucuronidase assay

{beta}-Glucuronidase activity in supernatants and cell pellets was measured by a colorimetric assay (22). {beta}-Glucuronidase release was expressed as the percentage 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 duplicate 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 of the assay was between 8 and 500 pg/ml (IL-5), 6 and 200 pg/ml (IL-6), and 62 and 2000 pg/ml (IL-8). The results were expressed as picograms of cytokine per 106 cells.

In the experiments for RT-PCR, eosinophils (5 x 106/ml) were incubated in FCS-free medium alone or with group IIA sPLA2 (1 µg/ml). At the end of the incubation, RNA was isolated by the TRIzol technique (Invitrogen, Milan, Italy), according to the manufacturer’s instructions. Diethylpyrocarbonate-treated water without SDS was used for the final resuspension step; RNA was stored at -80°C. Reverse transcription was performed with 5 mM MgCl2, oligo(dT)16 primer, and murine leukemia virus reverse transcriptase according to the manufacturer’s instructions (Applied Biosystems, Norwalk, CT) on a thermocycler (GeneAmp PCR System 2400; Applied Biosystems). PCR was performed using Taq polymerase (1–2.5 U/reaction) at the annealing temperature of 60°C, with target-specific primers for IL-5 (5'-TCACCGAGCTCTGTTGACAA-3' and 3'-GCGGTTTTCTCTTCACACC-5' at 0.2–1 µM/primer), IL-6 (5'-ATGAACTCCTTCTCCACAAGCGC-3' and 3'-GTCAGGTCGGACTCCCGAGAAG-5' at 0.2–1 µM/primer), and IL-8 (5'-GCATCTGGCAACCCTACAA-3' and 3'-CTATTTAAACCCCACCTTTCC-5' at 0.2–1 µM/primer). Number of cycles were 30 for IL-8 and 35 for IL-6 and IL-5. RNA was normalized by RT-PCR for the constitutive marker gene {beta}-actin (30 cycles). All PCR products, together with a DNA ladder as a size standard, were separated on 2.5% agarose gel, stained with ethidium bromide, and photographed. A semiquantitative measurement of relative cytokine mRNA expression was obtained by digital scanning and densitometric analysis (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 cultured (106/ml) for up to 48 h with and without group IIA sPLA2 (0.1–10 µg/ml) or with 10 ng/ml GM-CSF used as positive control. The cultures were done in 12-well, sterile, flat-bottom plates (BD Biosciences) previously coated with 1% BSA. At the end of incubation, the cells were harvested and washed twice with PBS. Expression of CD44 and CD69 was examined by direct immunofluorescence and flow cytometry (FACSCalibur; BD Biosciences) according to the following protocol. Cells were suspended at a concentration of 2 x 106/ml; 50 µl of cell suspension were incubated (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 were resuspended in 300 µl of PBS for analysis. CellQuest software (BD Biosciences) was used for acquisition and Paint-a-Gate software (BD Biosciences) was used for the analysis according to a three-parameter procedure. Although the samples contained almost exclusively eosinophils, these cells were identified according their light scatter properties and their typical expression of CD45 and CD45R0. The mean fluorescence intensity for the green channel (mean fluorescence intensity (MFI)) was recorded. Background fluorescence was assessed by analyzing unstimulated and sPLA2- or GM-CSF-activated eosinophils incubated (4°C, 30 min) with isotypic control Abs. Because background values under all experimental conditions were comparable, they were not subtracted (background MFI at 24 h: unstimulated = 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 x 106/sample) were incubated (37°C, 1–60 min) with 10 µg/ml group IA and IIA sPLA2s. At the end of incubation, the reactions were stopped by adding ice-cold PIPES buffer, and the samples were microfuged for 15 s. Cell pellets were immediately lysed in lysis buffer. Cell lysates were kept on ice for 20 min and then microfuged for 10 min at 4°C. Supernatant was collected as a protein extract containing lysed cell components without nuclei and diluted in an equal volume of 2x loading buffer (125 mM Tris-HCl (pH 6.8), 4% SDS, 0.005% bromophenol blue, and 20% glycerol) containing 5% 2-ME. Proteins were resolved by 10% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). After immersion overnight in TBST (50 mM Tris (pH 7.5), 150 mM NaCl, and 0.05% Tween 20) containing 4% BSA, membranes were washed three times (10 min 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 Western blotting detection reagent (Amersham Pharmacia Biotech) and HyperECL luminescence detection film (Amersham Pharmacia Biotech). Although comparisons were made on the basis of an equal number of cells, membranes were stripped with stripping buffer (7 M guanidine hydrochloride in distilled water) and then reblotted with anti-ERK1/2 Ab to verify equal protein content of each sample.

Production of LTC4 and PAF

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

Generation of superoxide anion

Superoxide anion production was measured as superoxide dismutase-inhibitable reduction of cytochrome c according to the method of Pick and Mizel (24). The results were expressed as nanomoles of superoxide anion per 106 cells.

Lactate dehydrogenase (LDH) assay

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

Statistical analysis

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


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of sPLA2s on {beta}-glucuronidase release from human eosinophils

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 {beta}-glucuronidase from human eosinophils isolated from normal donors. The cells were incubated (37°C, 2 h) with various concentrations of group IA or IIA sPLA2. Fig. 1 shows that group IA and IIA sPLA2s induce the release of {beta}-glucuronidase from purified human eosinophils in a concentration-dependent fashion. The effect of sPLA2s was significant at a concentration of 1 µg/ml, whereas concentrations >10 µg/ml did not induce further release of {beta}-glucuronidase. None of the sPLA2s used in these experiments significantly influenced cell viability, as assessed by trypan blue exclusion (data not shown). In addition, the release of LDH, used as an indicator of cytotoxicity, in sPLA2-treated eosinophil supernatants was always <4%.



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  		FIGURE 1. Effect of increasing concentrations of group IA and IIA sPLA2s on the release of {beta}-glucuronidase from eosinophils. The cells were incubated (37°C, 2 h) with the indicated concentrations of group IA and group IIA sPLA2s. At the end of the incubation, the supernatant was collected and centrifuged (1000 x g, 4°C, 5 min). {beta}-Glucuronidase release was determined by a colorimetric technique (22 ). The values are expressed as the percentage of the total cellular content determined in cell aliquots lysed with 0.1% Triton X-100. The data are the mean ± SE of six experiments. *, p < 0.01 vs control.

 
Additional experiments were performed to compare sPLA2-induced {beta}-glucuronidase release from eosinophils isolated from hypereosinophilic patients with eosinophils or neutrophils isolated from normal donors (Table I). There was no significant difference in {beta}-glucuronidase release induced by group IA sPLA2 between eosinophils 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 also induced the release of {beta}-glucuronidase from human neutrophils (preparations containing <2% eosinophils) in a fashion quantitatively similar to that induced from eosinophils. These data indicate that sPLA2s induce a noncytotoxic release of {beta}-glucuronidase that is comparable in eosinophils (from normal donors and hypereosinophilic patients) and neutrophils.


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  		Table I. sPLA2-induced {beta}-glucuronidase release from human eosinophils and neutrophilsa

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

In the next group of experiments, we determined whether, in addition to a preformed mediator such as {beta}-glucuronidase, group IA and IIA sPLA2s induced the production of IL-5, IL-6, and IL-8, three major cytokines produced by human eosinophils (26, 27, 28). In these experiments, the cells were incubated with increasing concentrations of sPLA2s for 18 h (IL-5 and IL-6) or for 4 h (IL-8). Kinetic experiments indicated that these incubation times were optimal for sPLA2-induced cytokine release (data not shown). Fig. 2 shows that both group IA (upper panel) and IIA (lower panel) sPLA2s concentration dependently increase the basal secretion of IL-6 and IL-8 from eosinophils. In contrast, none of the sPLA2s induced IL-5 release from cells incubated up to 24 h. The effect of sPLA2s on the production of IL-6 and IL-8 was significant at 1 µg/ml and maximal at 10 µg/ml. There was no significant difference in sPLA2-induced IL-6 and IL-8 production between eosinophils from normal donors and eosinophils from hypereosinophilic patients (data not shown).



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

 
We tested the hypothesis that sPLA2s induce cytokine gene expression by evaluating IL-5, IL-6, and IL-8 mRNA expression by RT-PCR in eosinophils incubated with group IIA sPLA2. Fig. 3 (upper panel) depicts the {beta}-actin- and IL-6-specific RT-PCR amplification products from an experiment representative of three in which eosinophils were cultured for 12 or 18 h with medium alone or with group IIA sPLA2. Fig. 3 (lower panel) depicts the {beta}-actin- and IL-8-specific RT-PCR amplification products from an experiment representative of three in which eosinophils were cultured for 1 or 3 h with medium alone or with group IIA sPLA2. We used different incubation times for IL-6 and IL-8 mRNA experiments because kinetics data showed that IL-6 release was delayed (16–24 h) compared with IL-8 release (2–6 h). Eosinophils constitutively express 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 preparations and no sPLA2-induced increase in our experimental conditions (data not shown). Densitometric analysis of the signal intensity of IL-6 and IL-8 mRNA amplification products, obtained in three experiments and expressed as the ratio with the corresponding signal intensity of {beta}-actin, revealed that group IIA sPLA2s induced a significant accumulation of IL-6 and IL-8 mRNA after 12 h and 3 h of incubation, respectively (Table II). These results indicate that sPLA2-induced cytokine production is associated with the accumulation of their specific mRNA.



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

 

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

 
Effect of sPLA2 on CD44 and CD69 expression on human eosinophils

CD44 and CD69 are distinct cell surface activation markers for human eosinophils (29). CD44 is constitutively expressed, whereas little if any CD69 is expressed on quiescent eosinophils. However, both markers are significantly increased on eosinophils activated in vivo or in vitro (29). We analyzed the effects of group IIA sPLA2 on the expression of these two eosinophil activation Ags. Fig. 4, A and B, shows that sPLA2 enhanced the expression of CD44 in a concentration- and time-dependent fashion. In particular, the effect of sPLA2 appeared after 24 h of incubation and reached maximal level after 48 h of incubation. As a control in these experiments, we used GM-CSF (10 ng/ml) that induces CD44 expression (29). Group IIA sPLA2 also induced the expression of CD69 in a concentration- and time-dependent manner (Fig. 5, A and B). As previously shown (30), because CD69 is expressed at a low level in freshly isolated eosinophils, the effect of sPLA2 is already near maximal after 24 h with only a small increase after 48 h of incubation. The maximal response induced by sPLA2 (10 µg/ml) was comparable to that induced by 10 ng/ml of GM-CSF. These data confirm and extend previous observations (30) demonstrating that sPLA2s induce the expression of two major activation markers on human eosinophils.



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  		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 µg/ml; continuous line). At the end of the 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 (unstimulated), with increasing concentrations (0.1–10 µg/ml) of group IIA sPLA2, and with GM-CSF (10 ng/ml) used as a positive control. At the end of the incubation, the cells were analyzed as described in Materials and Methods. Pair-matched isotypic control results of all experimental conditions were comparable as reported in Materials and Methods. The figure shows the mean ± SE of three experiments. *, p < 0.05 vs unstimulated.

 


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  		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 µg/ml; continuous line). At the end of the 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 (unstimulated), with increasing concentrations (0.1–10 µg/ml) of group IIA sPLA2, and with GM-CSF (10 ng/ml) used as a positive control. At the end of the incubation, the cells were analyzed as described in Materials and Methods. Pair-matched isotypic control results of all experimental conditions were comparable as reported in Materials and Methods. The figure shows the mean ± SE of three experiments. *, p < 0.05 vs unstimulated.

 
Effect of sPLA2s on superoxide anion generation from human eosinophils

Eosinophils are a major source of oxygen radicals in hypereosinophilic diseases (31). In these experiments, we explored whether sPLA2s induced superoxide anion generation from eosinophils. Fig. 6 shows that neither group IA nor group IIA sPLA2 activated the production of superoxide anion from eosinophils isolated from normal donors or from patients with hypereosinophilia (data not shown). In the same experimental conditions, 5.9 ± 1.4 nmoles of superoxide anion/106 cells were produced by eosinophils stimulated with the the Ca2+ ionophore A23187.



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  		FIGURE 6. Effect of increasing concentrations of group IA and IIA sPLA2s on superoxide anion generation from eosinophils. The cells were incubated (37°C, 30 min) with the indicated concentrations of group IA or IIA sPLA2s or with A23187 (10-6 M), used as a positive control. At the end of the incubation, the supernatant was collected and centrifuged (1000 x g, 4°C, 5 min). Superoxide anion generation was determined by the superoxide dismutase-inhibitable reduction of cytochrome c. The data are the mean ± SE of four experiments. *, p < 0.01 vs control.

 
Effect of sPLA2s on LTC4 and PAF synthesis from human eosinophils

Extracellular sPLA2s mobilize AA that is available for conversion to eicosanoids (15, 32). The major AA metabolite produced by human eosinophils is LTC4, a potent bronchoconstrictor and proinflammatory mediator (33, 34, 35). In this group of experiments we evaluated 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 patients (Fig. 7). Under the same experimental conditions, A23187 and 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).



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

 
Eosinophils are a major source of PAF (36), a lipid mediator whose synthesis is biochemically related to AA metabolism (37). In three experiments, incubation of eosinophils with group IIA sPLA2 up to 10 µg/ml did not induce the production of PAF as assessed by [3H]acetate incorporation (control, 22 ± 7; group IIA sPLA2, 24 ± 8 cpm/106 cells). In the same experiments, A23187 (10-6 M) induced the incorporation of 842 ± 67 cpm of [3H]acetate into PAF/106 cells.

Role of sPLA2 enzymatic activity on the activation of human eosinophils

The major enzymatic products of sPLA2s, AA and lysophospholipids, modulate several biochemical functions of inflammatory cells, including eosinophils (38). In particular, sPLA2-induced expression of CD69 has been related to the generation of lysophospholipids (30). To understand whether the effects of sPLA2s were due to the enzymatic activity, we designed two sets of experiments. We initially tested the hypothesis that enzymatic products of sPLA2s could be involved in sPLA2-induced activation of eosinophils by evaluating the effect of AA, lyso-PC, or lyso-PA on the release of {beta}-glucuronidase and IL-8 and on the expression of CD44 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 functions activated by group IIA sPLA2. We also performed experiments to rule out that endogenous lipid mediators could have affected sPLA2-induced activation of eosinophils. In these experiments, the cells were stimulated with group IIA sPLA2 in the presence of BSA. At concentrations up to 5 mg/ml, BSA did not modify the sPLA2-induced release of {beta}-glucuronidase and IL-8 (data not shown). These results indicate that the major enzymatic products of sPLA2s are not involved in sPLA2-induced activation of eosinophils.


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  		Table III. Effect of sPLA2 enzymatic products on human eosinophilsa

 
In another group of experiments, eosinophils were incubated with group IA or IIA sPLA2, in which catalytic activity was inactivated by treatment with BPB for 4 h at 37°C (20). This procedure resulted in the irreversible inhibition of >98% of enzymatic activity (data not shown). Fig. 8 shows that both BPB-inactivated sPLA2s (group IA and group IIA) retain their ability to induce the release of {beta}-glucuronidase (upper panel) and IL-8 (lower panel) from eosinophils. However, although not statistically significant, the release of {beta}-glucuronidase and IL-8 induced by enzymatically inactive sPLA2s tended to be lower than that induced by the active molecule. Taken together, the results of these groups of experiments indicate that the enzymatic activity of sPLA2, of both snake and human origin, is not primarily responsible for inducing degranulation and cytokine release.



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

 
Activation of ERK1/2 by sPLA2 in human eosinophils

The aforementioned results indicated that the enzymatic activity of sPLA2s was not mainly responsible for eosinophil activation and suggested that the effects of sPLA2s could be mediated by alternative mechanisms. Binding of sPLA2s to the M-type receptor expressed on mast cells and macrophages has been shown to activate intracellular signaling through the ERK1/2 pathway (19, 39). To verify this mechanism in human eosinophils, cells were stimulated with group IA sPLA2 (10 µg/ml). The reaction was stopped at various time points and cytosolic extracts were subjected to Western blot with anti-phospho-ERK1/2 Ab. Group IA sPLA2 induced phosphorylation of both ERK1 and ERK2 (Fig. 9, upper gel). Phosphorylation became evident after 30 s and persisted up to 15 min. Reprobing the membrane with the anti-ERK1/2 Ab (nonphosphorylated forms) confirmed equal protein loading (Fig. 9, lower gel). Similar results were obtained when eosinophils were stimulated with group IIA sPLA2 (data not shown). Phosphorylation of ERK1/2 was not detected under our experimental conditions when the cells were incubated with AA or lysophospholipids up to 10-5 M (data not shown).



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  		FIGURE 9. Kinetics of phosphorylation of ERKs induced by sPLA2. Esinophils were stimulated with group IA sPLA2 (10 µg/ml) for the times indicated. Reactions were stopped with the addition of ice-cold PIPES buffer. The cells were microfuged, lysed, and subjected to Western blot analysis with anti-phosphorylated ERK1/2 Ab (upper gel) as described in Materials and Methods. The membrane was then stripped and reblotted with anti-ERK1/2 Ab (lower gel) to confirm equal protein content of each sample. The Western blot shown is representative of two separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Extracellular PLA2s are attracting increasing attention as mediators of local and systemic inflammation. Several studies have shown that sPLA2s can activate inflammatory cells by various mechanisms either related or unrelated to their enzymatic activity (17, 18, 19, 20, 39, 40, 41). We have examined the effects of two structurally different sPLA2s on several functions of human eosinophils. Both group IA and IIA sPLA2s induce the release of the specific granule enzyme {beta}-glucuronidase, the production of IL-6 and IL-8, and the expression of activation markers (CD44 and CD69) on human eosinophils. 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 patients with systemic inflammatory diseases. For example, concentrations up to 4 µg/ml sPLA2 have been detected in the plasma of patients with septic shock (5). In addition, calculation of the specific activity of human group IIA sPLA2 (42) and dilution of alveolar fluid due to the bronchoalveolar lavage (43) indicate local concentrations of sPLA2 in the airways of patients with bronchial asthma ranging between 2 and 10 µg/ml (12). Therefore, eosinophils may be exposed in vivo to concentrations of sPLA2s that efficiently activate selected biochemical and immunological functions in these cells. Furthermore, eosinophils are a major cellular source of group IIA sPLA2 (44). This observation suggests that sPLA2s may function as an autocrine stimulus for eosinophil activation.

Interestingly, both group IA and IIA sPLA2s are more potent stimuli for IL-8 production than are other eosinophil agonists like PAF, RANTES, and PMA, and they do not require cell priming (28, 45). These data support the concept that sPLA2s may be relevant stimuli for systemic and/or local activation of eosinophils in allergic or hypereosinophilic disorders usually associated with high circulating levels of IL-6 and IL-8 (28, 46, 47, 48).

Surprisingly, sPLA2s do not induce, at least in vitro, IL-5 production. Previous data suggest that the production of cytokines is differentially regulated in normal human eosinophils. For example, secretory IgA induces the release of IL-8 but not of IL-5 (45). In addition, the production of IL-8, but not of other cytokines, in human eosinophils is closely associated with degranulation (45). Our data support the hypothesis of differential regulation of cytokine transcription in eosinophils by showing that sPLA2s selectively activate intracellular signals leading to IL-8 and IL-6, but not IL-5, expression. Furthermore, sPLA2s induce the release of a preformed mediator ({beta}-glucuronidase), but not of de novo synthesized mediators (LTC4, PAF, and superoxide anion). These observations suggest that activation by sPLA2 is restricted to selected signaling pathways within human eosinophils. Our results indicate that incubation of eosinophils with sPLA2s induces a rapid activation of ERK1/2. This signaling pathway is crucial for degranulation and cytokine production, but not for other functions in human eosinophils (49). Although these data clearly indicate that sPLA2s generate intracellular signals in eosinophils, additional experiments are needed to understand the mechanism(s) underlying the selectivity of effect of sPLA2s.

Our results extend the previous observation that sPLA2 induces the expression of surface activation markers on eosinophils (30). CD44 and CD69, although not specific of eosinophils, are overexpressed on cells activated in vivo, such as the eosinophils retrieved from the bronchoalveolar lavage, or in vitro by incubation with cytokines (29, 50). The function of these markers in asthma and in hypereosinophilic disease is unknown. Interestingly, however, CD44 is highly expressed on leukocytes from patients with rheumatoid arthritis (51), a disease in which blood levels of sPLA2 are significantly increased. This observation supports the hypothesis that sPLA2s activate a broad spectrum of inflammatory cells. In line with this hypothesis is our observation that sPLA2 induced degranulation of neutrophils in a fashion comparable to that of eosinophils.

The activation of inflammatory cells induced by sPLA2 could occur through at least three mechanisms: 1) generation of AA and lysophospholipids by direct enzymatic activity on membrane phospholipids with the concurrent generation of AA metabolites, 2) interaction of sPLA2s with membrane peptidoglycans through their 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 lysophospholipids, two intracellular messengers capable of inducing such cellular responses as exocytosis and cytokine expression (38, 52). In addition, LTC4, the main AA metabolite synthesized by eosinophils, 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. However, a number of observations exclude that catalytic activity plays a primary role in the induction of degranulation and IL production. First, sPLA2-induced activation of eosinophils is not associated with significant LTC4 generation. Second, incubation of eosinophils with the major enzymatic products of sPLA2, AA, lyso-PC, or lyso-PA, did not affect {beta}-glucuronidase or IL-8 secretion. Finally, inactivation of sPLA2 enzymatic activity by BPB did not abolish its capacity to induce eosinophil activation. These data indicate that, as in other inflammatory cells (18, 19, 20), the catalytic activity of sPLA2s is not mainly responsible for their ability to induce the release of lysosomal enzymes and the de novo synthesis of cytokines and chemokines. However, inactivation of PLA2 activity tends to reduce the effect of sPLA2 on {beta}-glucuronidase and IL-8 production, suggesting that one or more of the enzymatic products of sPLA2s may enhance the effects exerted by sPLA2s on eosinophils.

Several sPLA2s bind to peptidoglycans on cell membranes through their heparin-binding site (55, 56). This interaction results in the internalization of sPLA2, which is then capable of generating intracellular signals leading to gene expression (57). Both group IA and IIA sPLA2s possess a heparin-binding site (58). Therefore, a potential mechanism by which these molecules may activate eosinophils is through interaction with membrane peptidoglycans. Moreover, it is conceivable that sPLA2s, at least at high concentrations, could induce membrane perturbations, which in turn may trigger eosinophil activation.

Finally, sPLA2s may activate eosinophils by interacting with one or more specific receptors, i.e., the M-type or the N-type (59). These receptors are expressed in a variety of tissues and inflammatory cells, including macrophages, monocytes, and mast cells, and they are functionally coupled to different cellular responses (17, 18, 19, 42, 59, 60). The lack of specific Abs or ligands for these receptors in human cells currently precludes their characterization on eosinophils. However, we provide evidence that both group IA and IIA sPLA2s induce activation of ERK1/2, a signaling pathway that has been coupled to activation of the M-type receptor in other cell types (19, 39). These data support the hypothesis that sPLA2s may activate human eosinophils by interacting with the M-type receptor. However, our data do not exclude the possibility that activation of N-type receptor or of other as yet uncharacterized receptors may be involved in sPLA2-induced activation of eosinophils.

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

In conclusion, our results indicate that sPLA2s selectively activate certain proinflammatory and immunomodulatory functions in eosinophils with mechanism(s) that appear mainly independent from their enzymatic activity. Activation of human eosinophils by sPLA2s may concur with the eicosanoid-forming capacity of these molecules to induce systemic and/or local activation of these cells in inflammatory and allergic disorders.


   Acknowledgments
 
We are indebted to Dr. Giuseppe Spadaro (Division of Clinical Immunology and Allergy, University of Naples Federico II, Naples, Italy) and Dr. Gennaro Liccardi (Division of Pneumology and Allergy, A. Cardarelli Hospital, Naples, Italy) for providing access to patients with hypereosinophilia. We are grateful to Jean Ann Gilder for text editing.


   Footnotes
 
1 This work was supported in part by grants from the Ministero dell’Istruzione, dell’Università e della Ricerca, the Ministero della Salute "Alzheimer Project," the Consiglio Nazionale delle Ricerche (Target Project Biotechnology Grants 01.00191.PF31 and 01.00295.PF49), and the Istituto Superiore di Sanità (Rome, Italy; AIDS Project 40B.64). G.M. is the recipient of the 2002 Esculapio Award (Accademia Tiberina, Rome, Italy). Back

2 Address correspondence and reprint requests to Dr. Massimo Triggiani, Division of Clinical Immunology and Allergy, University of Naples Federico II, Via Pansini 5, 80131 Naples, Italy. E-mail address: triggian{at}unina.it Back

3 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, lysophosphatidic acid; BPB, bromophenacyl bromide; MFI, mean fluorescence intensity; LDH, lactate dehydrogenase. Back

Received for publication September 10, 2002. Accepted for publication January 9, 2003.


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