* Department of Clinical Bacteriology, Sahlgrenska University Hospital, Göteborg, Sweden; and
Department of Rheumatology and Inflammation Research, Göteborg University, Sweden
Correspondence: Christine Wennerås, Department of Clinical Bacteriology, Sahlgrenska University Hospital, Guldhedsgatan 10, S-413 46 Göteborg, Sweden. E-mail: christine.wenneras{at}microbio.gu.se
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Key Words: degranulation • chemotaxis • respiratory burst • eosinophil cationic protein • eosinophil peroxidase
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Eosinophils are mainly tissue-dwelling cells, found in the lung, gastrointestinal, and lower genitourinary tracts [1 , 2 ]. During allergic inflammatory reactions, they are recruited in large numbers from the peripheral blood to the inflammatory site. Different substances, e.g., chemokines (i.e., regulated on expression, normal T expressed and secreted; eotaxin), the phospholipid platelet-activating factor (PAF), the cytokine interleukin (IL)-5, and complement factor 5a (C5a), induce extravasation of eosinophilic leukocytes by binding to specific receptors on the cell surface [3 4 5 ]. The downstream signaling effector enzymes that are activated by the occupied receptors promote intracellular calcium mobilization, alterations in the metabolism of phosphoinositides, and protein phosphorylation [6 ]. The integration by the eosinophil of the different intracellular signals results in chemotactic cell migration, release of granule constituents, and production of superoxide (O2-) by the eosinophil reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [7 ]. The type of cellular response depends on the identity of the agonist and to what extent the receptor involved in the activation process is expressed and desensitized [8 ].
By screening a peptide library, Seo and coworkers [9 ] identified a D-methionine-containing hexapeptide, Trp-Lys-Tyr-Met-Val-D-Met-NH2 (WKYMVm), with the capacity to activate neutrophils. The peptide has also been shown to bind to eosinophils [10 ]. Whereas prokaryotes use L- and D-isomers of amino acids in their protein synthesis, eukaryotic proteins as a rule incorporate only L-isomers [11 ]. Hence, D-isomers may constitute danger signals to the innate immune system.
As mentioned, WKYMVm has been shown to also bind to eosinophils [10 ]. In this study, we directly compare the responsiveness of eosinophils with that of neutrophils to WKYMVm. Our findings indicate that the peptide uses different members of the formyl peptide receptor family and partly elicits different patterns of activation in the two classes of granulocytes. Hence, the peptide activates eosinophils preferentially, although not exclusively via the N-formyl peptide receptor (FPR), thus eliciting chemotactic movement and the release of reactive oxygen species. In neutrophils, the peptide engages a structural homologue of the FPR, FPR-like 1 (FPRL1) [12 ], and gives rise to similar cell activation with the addition of release of the contents of secretory vesicles, measured as an up-regulation of complement receptor 3 (CR3) on the neutrophil cell surface.
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Purification of human eosinophils
Peripheral blood eosinophils were purified from fresh buffy coats obtained from healthy adult blood donors at Sahlgrenska University Hospital (Göteborg, Sweden). The cell fraction was diluted 1:1 (v/v) in physiological saline and was subjected to dextran sedimentation for removal of erythrocytes. The supernatant was centrifuged on a Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradient for 15 min at 450 g and room temperature to remove the mononuclear cell fraction. Remaining erythrocytes were removed by repeated hypotonic lysis using distilled water. The polynuclear cell fraction (neutrophils as well as eosinophils) was resuspended in KRG without Ca2+, and the contaminating neutrophils were removed by magnetic beads (MACS, Miltenyi Biotec Inc., Auborn, CA), coated with anti-CD16 monoclonal antibody (mAb; Miltenyi Biotec) [13
]. The eosinophils were washed, resuspended in KRG, and stored on melting ice until use. The purity of the eosinophils was routinely >95%, which was determined by Diff-quik stain (Dade Behring AG, Düdingen, Switzerland) of an aliquot of cytospinned cells, and the viability was >99%, assessed by Trypan blue stain.
Release of reactive oxygen species
An isoluminol-amplified chemiluminescence system was used to determine superoxide anion production. The chemiluminescence activity was measured in a six-channel Biolumat LB 9505 (Berthold Co., Wildbad, Germany) using disposable, 4 mL polypropylene tubes (Sarstedt, Nürnbrecht, Germany), each containing 5 x 104 eosinophils resuspended in KRG. The cells were equilibrated for 5 min at 37°C in the Biolumat before the addition of the various peptide agonists together with the chemiluminescence amplifiers isoluminol (a cell-impermeable isoform of luminol; 2x10-5 M) and horseradish peroxidase (4 U); the latter was added to provide the system with an excess of peroxidase. The system is designed to measure extracellular release of reactive oxygen species and continuously records light emission, indicated as counts per minute. Further details about the technique are given by Dahlgren and Karlsson [14
].
Degranulation of eosinophils
Eosinophils (105) suspended in KRG were put in 96-well low-binding polystyrene plates (NUNC AS, Roskilde, Denmark) and were activated with various peptides for different lengths of time (30 or 45 min at 37°C), whereafter the cell-free supernatants were collected and analyzed for release of eosinophil peroxidase (EPO) and eosinophil cationic protein (ECP). Peroxidase activity in supernatants of stimulated eosinophils or cell lysates was measured by the addition of H2O2 (4 µL 30% H2O2) and o-phenylenediamine (Sigma Chemical Co.; 20 mg), dissolved in 30 mL 100 mM sodium acetate, 20 mM EDTA, and 1% hexadecyl trimethyl ammonium bromide (HETAB) buffer (pH 6.0), followed by incubation for 10 min in the dark at room temperature. The reaction was stopped with 2 M H2SO4, and the absorbance was determined at 492 nm in a spectrophotometer (Multiscan MS, Labsystems, Helsinki, Finland). The detection limit of the EPO assay was 0.8%; i.e., the lowest amount of EPO detectable in a well containing 50,000 eosinophils was that corresponding to the total amount of EPO in 400 lysed cells (400/50,000). Similar readings were done to assess the release of myeloperoxidase from neutrophils. Release of ECP into supernatants was determined using a semiautomated enzyme immunoassay with fluorochrome-labeled antibodies (UniCAP 100, Pharmacia, Södertälje, Sweden). The secretagogues PAF (16 carbon atoms; Sigma Chemical Co.) and phorbol myristate acetate (PMA; Sigma Chemical Co.) were used as positive controls of degranulation.
Cytokine production
The levels of granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-5 in supernatants of eosinophils stimulated for 30 min or 18 h with the various peptide agonists or with the secretagogues PMA and PAF were measured by commercial sandwich enzyme-linked immunosorbent assay kits according to the manufacturer’s instructions (GM-CSF; MAB 615 and BAM 215 and Quantikine human IL-5, R&D Systems, Abingdon, UK). Immunoglobulin (Ig)A complexes were used to elicit de novo IL-5 synthesis in eosinophils as previously described by Dubucquoi et al. [15
]. Briefly, eosinophils were resuspended in serum-free culture medium X-vivo 15 (BioWhittaker, Göteborgs termometerfabrik, Göteborg, Sweden; 105 cells per microwell) and were incubated with monoclonal serum IgA at a final concentration of 15 µg/mL for 1 h at 4°C, followed by incubation with rabbit anti-human 
-chains of IgA (A0262, Dakopatts, Älvsjö, Sweden) at a final concentration of 20 µg/mL for 18 h at 37°C. Supernatants were collected and frozen at -20°C before analysis of IL-5 contents. The monoclonal IgA Igs were serum-derived myeloma proteins, monomeric IgA1 (Eve) and polymeric IgA2 (Fel), both a kind gift from Dr. Jiri Mestecky (University of Alabama at Birmingham).
Chemotaxis
Eosinophil migration was determined using 30 µL 96-well microplate-disposable chemotaxis/cell migration chambers with hydrophobic filters of pore size 3 µm (ChemoTx, Neuro Probe Inc., Gaithersburg, MD). Different chemoattractants (fMLF, WKYMVm, WKYMVM) were added to the lower chambers. Next, eosinophilic cell suspensions consisting of 30,000 cells/mL in KRG were added on top of the filters and incubated for 90 min at 37°C. The plate was next centrifuged at 400 g for 10 min, the filter was removed, and the plate was incubated for an additional 10 min at 37°C. Finally, supernatants were removed from the bottom chambers and any transmigrated cells were lysed by the addition of 1% Triton in phosphate-buffered saline (PBS) for 15 min at room temperature. The cell lysate was then transferred to a 96-well plate, and EPO was measured as described earlier.
Fluorescein-activated cell sorter (FACS)
Measurement of the surface expression of CR3 and C–C chemokine receptor 3 (CCR3) on eosinophils was done using identical protocols but different antibodies. Eosinophils (105 cells in 90 µL KRG) were incubated in 96-well plates for 5 min at 37°C, after which various concentrations of fMLF or WKYMVm were added (10-7 and 10-9 M), and the plates were reincubated for 30 min at 37°C. The plates were washed once with FACS buffer (0.1 mM EDTA, 0.02% NaN3 in PBS) and then incubated with purified human gammaglobulin (1 µg/105 cells; Pharmacia) for 15 min at room temperature to block Fc receptors. The cells were next incubated for 30 min at 4°C with either of the following phycoerythrin (PE)-labeled mAb: rat anti-human CCR3 (clone 61828.111, R&D Systems) or mouse anti-human CD11b moiety of CR3 (clone 12, Becton Dickinson Immunocytometry System, San Jose, CA). The following isotype-matched, PE-labeled control antibodies were used: rat IgG2a (clone 54447, R&D Systems) and mouse IgG2a (Becton Dickinson Immunocytometry System). Afterwards, the cells were fixated in ice-cold 3.7% paraformaldehyde-PBS and were left overnight at 4°C. The next day, the eosinophils were washed once in FACS buffer and resuspended in 80 µL PBS before FACS analysis using a FACScan (Becton Dickinson, Mountainview, CA).
Statistics
Statistical analyses were performed using GraphPad Prism 3.0 software (GraphPad, San Diego, CA). The unpaired two-tailed Student’s t-test was used, and a P value of <0.05 was used to indicate statistical significance.
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Secretion and chemotaxis
Recruitment of granulocytes from peripheral blood into the tissues is in part regulated through mobilization of intracellular organelles storing adhesion molecules and the concomitant release of enzymes/cytokines required for firm leukocyte adhesion to the vessel wall and subsequent endothelial transmigration [16
]. To investigate if WKYMVm could induce mobilization of organelles, we measured eosinophil surface expression of CR3 and CCR3, whose natural ligands are C3bi [17
] and eotaxin [18
], respectively. Using FACS analysis, we could not detect any modification of cell surface expression of CR3 (Fig. 1A
) or CCR3 (data not shown) on eosinophils incubated for up to 45 min with WKYMVm. In contrast, CR3 expression was up-regulated on neutrophils exposed to the peptide (Fig. 1B)
, in agreement with previously published results [19
].
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Figure 1. CR3 up-regulation on the granulocyte cell surface. Eosinophils (A) and neutrophils (B) were exposed to control diluent (- - -) or WKYMVm (10-7 M; –-) for 30 min at 37°C, and surface expression of CR3 was assessed by FACS. The binding of an isotype-matched control antibody to granulocytes exposed to control diluent (· · ·) or WKYMVm (10-7 M; ···) for 30 min at 37°C is also shown. Abscissa, intensity of fluorescence; ordinate, number of cells. The plot is derived from a representative experiment.
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Figure 2. Lack of degranulation of eosinophils exposed to WKYMVm. Open bars indicate the amounts of ECP found in supernatants of unstimulated eosinophils, and eosinophils stimulated for 30 min with WKYMVm (10-7 M), PAF (10-7 M), and PMA (5x10-8 M), respectively. Solid bars indicate levels of EPO. All data represent the arithmetic mean ± SE (n=5 different blood donors). *, P < 0.01; **, P < 0.001 versus medium.
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The ability of WKYMVm to elicit eosinophil migration was subsequently investigated. We found that the peptide promoted eosinophil chemotaxis (Fig. 3 ). The migratory dose-response curve and the maximal migration elicited in eosinophils after exposure to WKYMVm were very similar to the chemotactic responses seen after stimulation of eosinophils with fMLF (Fig. 3) . When the D-isomer of methionine at the C-terminus of WKYMVm was replaced by L-methionine (WKYMVM), the chemotactic activity in terms of maximal migration was increased, although a higher concentration (10-6 M vs. 10-8 M) of the modified peptide was needed to induce chemotactic movement (Fig. 3) .
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Figure 3. Chemotactic movement of eosinophils exposed to WKYMVm (with a D-methionine;  ), fMLF ( ), or WKYMVM (with an L-methionine; •), assayed in a 96-well microchamber system. The dashed line indicates the spontaneously transmigrated percentage of eosinophils (=5%). All data represent the mean ± SE (n=6 different blood donors). *, P < 0.01; **, P < 0.001; and ***, P < 0.0001 versus spontaneous migration.
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Figure 4. Time course of oxidative burst induced by PMA ( ; 5x10-8 M), fMLF (; 10-7 M), and WKYMVm (; 10-7 M) in eosinophils and neutrophils (inset), respectively. The oxidative burst was assessed by chemiluminescence. The plot is derived from a representative experiment.
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Figure 5. Capacity of cyclosporin H (A) and boc-MLF (10-5 M; B) to block the oxidative burst in neutrophils (open bars) and eosinophils (solid bars) elicited by fMLF, WKYMVm (with a D-methionine), and WKYMVM (with an L-methionine), respectively. The oxidative burst was assessed by measuring chemiluminescence. All data represent the mean ± SE of three to six experiments. **, P < 0.001; ***, P < 0.0001 versus eosinophils.
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Table 1. Heterologous Desensitization of Oxidative Burst
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Figure 6. (A) Desensitization of oxidative burst in eosinophils measured by chemiluminescence. Eosinophils were exposed to WKYMVm (10-7 M) at time zero (t=0) and restimulated 10 min later with fMLF (10-7 M). The inset graph indicates the oxidative burst in eosinophils that received no stimulus at t= 0 but were exposed to fMLF (10-7 M) after 10 min. (B) Eosinophils were first exposed to fMLF (10-7 M) and 10 min later were restimulated with WKYMVm (10-7 M). The inset graph indicates the oxidative burst in eosinophils that received no stimulus at t = 0 but were exposed to WKYMVm (10-7 M) after 10 min. All plots are derived from a representative experiment.
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In our earlier study, we demonstrated that although radioactively labeled WKYMVm bound to the receptors FPR and FPRL1 on neutrophils, cell activation occurred exclusively through FPRL1 [19 ]. This contrasts with the present findings, which indicate that WKYMVm-mediated activation of eosinophils is mainly transduced via the FPR. The FPR antagonists cyclosporin H and boc-MLF abrogated the greater part of the respiratory burst elicited by WKYMVm in eosinophils. However, as some activation remained after treatment with the FPR antagonists, we searched for another receptor candidate.
As FPRL1 is the preferred receptor for WKYMVm in neutrophils, we evaluated the capacity of this receptor to transmit activating signals in WKYMVm-stimulated eosinophils. To examine this hypothesis, we used the L-methionine-containing peptide WKYMVM, which has recently been found to be a ligand of FPRL1 and FPRL2 in transfected neutrophil-like HL-60 cells [26 ]. Here, we show that neither of the FPR antagonists cyclosporin H nor boc-MLF inhibited the oxidative response engendered in eosinophils by WKYMVM, and WKYMVM could desensitize WKYMVm-mediated superoxide release. As FPRL2 has only been identified in cells of the monocytic lineage, it is likely that WKYMVm (and WKYMVM) acts via FPRL1 in eosinophils.
WKYMVM stimulation of eosinophils not only transduced signals generating a modest oxidative burst but also elicited cell migration. Surprisingly, a larger fraction of eosinophils were found to respond to the L-conformer WKYMVM than to fMLF or WKYMVm. The bell-shaped dose-response curves to the studied chemoattractants reflect a well-known phenomenon; i.e., high concentrations of chemoattractants inhibit cell migration by a yet-to-be-defined mechanism [27 ]. We were able to attain the inhibitory concentration for WKYMVm but not for WKYMVM. Several possibilities may be imagined to explain why a higher proportion of eosinophils migrated toward the FPRL1 agonist WKYMVM than to WKYMVm, although we claim the latter peptide engages FPRL1 and FPR. FPRL1 might require a threshold degree of receptor occupancy, which once attained, would render it even more efficient than the FPR in eliciting chemotaxis. Alternately, a high degree of occupancy of the FPR might inhibit chemotactic movement transduced not only through itself but also through FPRL1. Finally, the high concentration (10-6 M) of WKYMVM required for it to act as a chemoattractant may be unphysiological and hence of limited biological relevance.
FPRL1, the putative receptor of WKYMVm and WKYMVM in eosinophils, is also referred to as the lipoxin A4-receptor (LXA4R), as it has been shown to bind this lipoxin [28 ] in addition to a large number of unrelated peptides [19 , 26 , 29 30 31 ]. LXA4 belongs to a relatively new class of arachidonate products generated, among other places, in the bronchoalveolar lavage fluid of patients with asthma and nasal polyps, e.g., eosinophil-associated pathologies [32 , 33 ].
Whereas the interaction of the peptide/protein ligands with FPRL1 elicits a G-protein-mediated signaling cascade resulting in chemotactic movement of neutrophilic granulocytes as well as mobilization of granules and release of reactive oxygen species [19 , 26 , 29 30 31 ], binding of LXA4 to the same receptor acts as a stop signal, abrogating cell trafficking, respiratory burst, and cytokine production in neutrophils [26 , 34 ]. An in vitro study has demonstrated the capacity of LXA4 to reduce eosinophil migration [35 ]. Notably, LXA4 has been shown to block allergen-induced eosinophil trafficking [36 ]. Although the mode of interaction of LXA4 and the other peptide ligands with FPRL1 is largely unknown, it has been proposed that they have separate binding sites on the receptor [26 ]. Removal of N-linked carbohydrates from FPRL1 drastically diminished the binding of the peptide ligands but not of LXA4 [26 ]. This could explain how signaling mediated through one and the same receptor could result in anti-inflammatory or proinflammatory cellular responses dependent on the identity of the ligand.
In summary, this study demonstrates that the hexapeptide WKYMVm activates eosinophils through the FPR and through another receptor, presumably the promiscuous and homologous chemoattractant receptor FPRL1. The high degree of amino acid homology shared by the FPR and FPRL1 in the cytoplasmic domain suggests that these two receptors share many signal transduction steps and have similar functions in host defense [37 ].
It is unknown whether homologues of the synthetic peptide WKYMVm exist in nature. Historically, the formylated peptides such as fMLF were synthesized in laboratories and were found to be potent leukocyte activators prior to their discovery as by-products of bacterial protein synthesis [38 ]. One of the major roles of neutrophils is to eliminate invading bacteria; hence, it is logical that this cell type should be equipped with receptors capable of recognizing bacterial compounds. However, eosinophils are relatively inefficient at phagocytosis and killing of bacteria [39 ], making it less obvious as to wherein the advantage lies for these cells to express this type of receptors. Possibly, parasites, the natural prey of eosinophils, may release related peptides [40 ]. Another alternative is that these receptors alert eosinophils to the presence of mitchondrially derived peptides, which are believed to signal tissue destruction [41 ].
Received November 4, 2001; revised May 10, 2002; accepted May 11, 2002.
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