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(Journal of Leukocyte Biology. 2002;72:769-779.)
© 2002 by Society for Leukocyte Biology

Human eosinophils express and release IL-13 following CD28-dependent activation

Gaetane Woerly*, Paige Lacy{dagger}, Amena Ben Younes*, Nadine Roger*,{ddagger}, Sylvie Loiseau*, Redwan Moqbel{dagger} and Monique Capron*,{ddagger}

* Centre d’Immunologie et Biologie Parasitaire, Unité INSERM U547 and IFR17, Institut Pasteur, Lille, France;
{dagger} Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Canada; and
{ddagger} Faculté de Pharmacie, Université de Lille 2, France

Correspondence: Dr. Monique Capron, Unité INSERM U547, Institut Pasteur de Lille, 1 rue du Prof. Calmette, BP 245, 59019 Lille Cedex, France. E-mail: monique.capron{at}pasteur-lille.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human eosinophils produce a large number of cytokines, including immunoregulatory cytokines. Given that eosinophils store and release interleukin (IL)-4, a key cytokine in the pathogenesis of allergic inflammation, and that IL-4 and IL-13 share common biological functions, we investigated the possibility that IL-13 may be synthesized by these cells. Using flow cytometry and immunocytochemistry, we show that eosinophils synthesize and store IL-13. Granule localization was demonstrated after subcellular fractionation, and IL-13 immunoreactivity was localized to crystalloid, granule-enriched fractions. Furthermore, electron microscopic analyses specifically localized IL-13 to the dense cores of bicompartmental secondary granules. Upon CD28 ligation, IL-13 was released by eosinophils, whereas a combination of CD28 and immunoglobulin A complexes resulted in decreased IL-13 secretion. Furthermore, eosinophil-derived IL-13 exerts a biological effect, inducing CD23 expression on B cells. By having the capacity to synthesize and release IL-13, eosinophils may participate in the development and maintenance of the T helper cell type 2 response, a prominent feature of allergic diseases.

Key Words: granulocytes • cytokines • flow cytometry • electron microscopy • secretion • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A significant feature of a number of human atopic diseases is elevation of T cell-derived type 2 (Th2) cytokines [interleukin (IL)-4, IL-5, IL-10, IL-13] and reduction of Th1 cytokines [IL-2, interferon-{gamma} (IFN-{gamma})]. The type 2-cytokine response is characterized by increased Th2 cell development, immunoglobulin E (IgE) production, and eosinophilia. Although eosinophils are classically involved in parasitic infections or allergic manifestations, they are in fact associated with most inflammatory or infectious processes. Their prominent role in allergic disorders was at first believed to be restricted to degranulation and release of highly charged cationic proteins. In the last decade, it has been shown that eosinophils also have the capacity to synthesize and release up to 28 cytokines [1 ], including the immunoregulatory cytokines type-1 (IL-2, IFN-{gamma}) [2 , 3 ], type-2 (IL-4 and IL-5) [4 5 6 7 8 ], and IL-10 [2 , 6 ]. Among them, IL-4 is recognized to play a central role in allergic and parasitic diseases. Recently, IL-13 was shown to be a key cytokine in certain Th2 cytokine-associated conditions, such as asthma and parasitic infections [9 10 11 12 ].

IL-13 shares many biological properties with IL-4 (reviewed in ref. [13 ]). These properties are shared as a result of the common {alpha} chain associated with receptors for IL-4 and IL-13 [14 ]. The effects of IL-4 and IL-13 stimulation include B cell growth and Ig class switching to IgE [15 16 17 ]; CD23 and human leukocyte antigen class II up-regulation on B lymphocytes [18 ]; down-regulation of monocyte function [e.g., IL-1, IL-12, tumor necrosis factor {alpha} production; refs. 19 20 ]; and increased expression of vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells [21 ]. However, in contrast to IL-4, IL-13 does not directly affect T cell functions, because of the lack of IL-13 receptors on these cells [14 ]. By inhibiting monocyte/macrophage Th1-inducing IL-12 production, IL-13 may favor Th2 differentiation. IL-13 is principally produced by T cells and in lower quantities by Epstein-Barr virus-transformed B cell lines, B cell lymphomas, keratinocytes, mast cells [22 , 23 ], basophils [24 , 25 ], and dendritic cells [26 ].

The demonstration that eosinophils synthesize and release IL-4 and that IL-4 and IL-13 share biological functions in common led us to investigate the possibility of synthesis of IL-13 by these cells. In this report, we show for the first time that human eosinophils purified from hypereosinophilic patients and healthy donors express IL-13 and that this expression exhibits donor variation. Furthermore, as already demonstrated for other cytokines, IL-13 was specifically localized to the core compartment of crystalloid granules. We also demonstrate that after CD28 ligation, eosinophils release significant amounts of bioactive IL-13. These findings have significant implications for a role in enhancement of the Th2 cytokine response by eosinophils in atopic and parasitic diseases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Heparinized peripheral blood was obtained from healthy donors and hypereosinophilic patients. The hypereosinophilia was associated with allergy and asthma, skin diseases (including eczema or pemphigoid), hypereosinophilic syndromes, haematological disorders (including tumors, myeloproliferative lymphoma, and eosinophilic leukaemia), and drug hypersensitivity. All patients had an eosinophilia above 1000/mm3.

Reagents
Anti-human CD16- and CD3-conjugated magnetic beads and the magnetic cell separation system (MACS) were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Percoll was obtained from Pharmacia (Uppsala, Sweden). RPMI-1640 medium, glutamine, penicillin, streptomycin, fetal calf serum (FCS), Hepes, and Nycodenz were from Gibco-BRL Life Technologies (Paisley, UK). Paraformaldehyde, saponin, N{alpha}-p-tosyl-L-arginine methyl ester (TAME), 4-methylumbelliferyl N-acetyl-ß-D-glucosaminide, adenosine 5'-triphosphate (ATP), aprotinin, phenylmethylsulfonyl fluoride (PMSF), leupeptin, tetramethylbenzidine (TMB), human secretory IgA (sIgA), and anti-mouse IgG F(ab')2 were purchased from Sigma Chemical Co. (St. Louis, MO). Fluorescein isothiocyanate (FITC)-conjugated and nonconjugated anti-IL-13 and anti-CD28 mouse monoclonal antibodies (mAb) were from Diaclone (Besançon, France). Phycoerythrin (PE)-conjugated anti-IL-4 and anti-CD23 mAb and the unlabeled mouse IgG1 antibody were obtained from Pharmingen (San Diego, CA). Anti-human IgA and PE-conjugated anti-CD19 mAb were from Immunotech (Coulter Corp., Miami, FL). The anti-Fc receptor for IgE (Fc{varepsilon}RI){alpha} (15.1, mIgG1) mAb was a kind gift from Dr. J-P. Kinet (Harvard Medical School, Boston, MA). The mouse alkaline phosphatase antialkaline phosphatase (APAAP) detection system, New Fuchsin kit, and FITC- and PE-conjugated mouse isotype controls were from Dako (Glostrup, Denmark). The recombinant human (rh) IL-13 and IL-4 were purchased from Peprotech (Rocky Hill, NJ), and the rh-granulocyte macrophage-colony stimulating factor (GM-CSF) was obtained from Novartis (Basel, Switzerland).

Eosinophil purification
Eosinophils were isolated by immunomagnetic separation technique using the MACS system, as previously described [2 ]. After density centrifugation on Percoll of diluted whole blood, the mononuclear cells at the interface [peripheral blood mononuclear cells (PBMC)] and the granulocyte pellet were collected. After hypotonic saline lysis, the pellet was incubated with anti-CD16- and anti-CD3-conjugated immunomagnetic beads to remove neutrophils and contaminating lymphocytes, respectively. Eosinophils were eluted by passage of the cells through the field of a permanent magnet. After isolation, eosinophil preparations were cytocentrifuged, and cytospins were stained with May-Grünwald Giemsa (RAL 555, Rieux, France). The purity of eosinophil preparations was normally above 98%, and contaminating cells were usually neutrophils and lymphocytes.

Reverse transcriptase-polymerase chain reaction (RT-PCR) of IL-13 mRNA
Total RNA was isolated from highly purified (>99%) human eosinophils and PBMC using RNAplus extraction reagent (Qbiogene, Carlsbad, CA). RT was performed using SuperScriptTMRT (Gibco-BRL). cDNA was amplified with primers for detection of IL-13 mRNA, based on those reported in an earlier publication [27 ]. The sense primer for IL-13 was 5'-CTGCCCGTCTTCAGCCTAGCCG-3', and the antisense was 5'-CGAGGCCCCAGGACCCCAG-3'. PCR amplifications were performed with the following settings: 40 cycles at 95°C for 1 min, 66°C for 1 min, and 72°C for 1 min. Primers for the housekeeping gene, ß2-microglobulin, were used as positive control: 5'-CAGCGTACTCCAAAGATTCAGGT-3' (sense) and 5'-TGGAGACAGCACTCAAAGTAGAA-3' (antisense). The primers were obtained from Invitrogen (Carlsbad, CA), and Taq polymerase was from Qbiogene. Amplified products were electrophoresed on a 1% agarose gel stained with ethidium bromide and were photographed under ultraviolet light.

Flow cytometric analysis of intracellular IL-4 and IL-13
Analysis of intracellular IL-13 expression by flow cytometry was performed as previously described [2 ]. Briefly, after fixation and permeabilization, eosinophils were labeled with FITC-conjugated anti-IL-13 or PE-conjugated anti-IL-4 mAb or FITC- or PE-conjugated isotype control antibodies at a final concentration of 5 µg/ml. Samples were analyzed on a FACSCaliburTM using the CellQuestTM software (Becton Dickinson, Mountain View, CA). Ten thousand events were usually acquired per sample. Thresholds were set on the control-isotype label. Results are expressed as mean fluorescence intensity (MFI), calculated by subtracting the MFI of the control from the MFI of the sample.

To control for specificity of intracellular staining, FITC- or PE-conjugated anticytokine mAb were preincubated for 15 min with an excess of rhIL-13, IL-4, or rhGM-CSF (50 µg/ml) before cell staining.

Immunocytochemistry
Eosinophil cytospins were fixed in cold acetone/methanol, rehydrated in 0.05 M Tris-buffered saline (TBS), pH 7.4, for 10 min. The APAAP method was used for immunoenzymatic staining [2 ]. After each incubation step, cytospins were washed for 3 x 10 min in TBS containing 0.1% bovine serum albumin (BSA). Briefly, after blocking with 3% BSA in TBS for 30 min, cytospins were incubated with unlabeled anti-IL-13 or isotype control mAb (40 µg/ml) in TBS-3% BSA overnight at 4°C. Slides were then incubated with rabbit anti-mouse Ig (1:25) in TBS-3% BSA for 1 h at room temperature, followed by incubation with the APAAP complex (1:40) for 1 h. After an additional wash for 2 x 10 min in TBS, the reaction was developed with New Fuchsin substrate. Slides were counterstained with Mayer’s hematoxylin and mounted with Immu-mount (Shandon, Pittsburgh, PA).

Indirect immunofluorescent staining was also performed. After cytospin rehydration as before, endogenous fluorescence was inhibited by 15 min incubation with 50 mM NH4Cl, pH 7.4. Slides were then washed in TBS for 10 min, and nonspecific binding sites were blocked with 3% BSA in TBS for 30 min. Samples were incubated overnight at 4°C with anti-IL-13 mAb or mouse IgG1 isotype control antibody (30 µg/ml final concentration) in TBS-3% BSA supplemented with 5% normal human serum. After washing as before, samples were further incubated with FITC-conjugated anti-mouse IgG F(ab)'2 (dilution 1:200) for 1 h. Slides were washed and mounted with Fluoromount G (Southern Biotechnology Assoc., Birmingham, AL).

Electron microscopy and immunogold staining
Cells suspended in phosphate-buffered saline (PBS) were fixed with an equal volume of paraformaldehyde and glutaraldehyde (2% and 0.1% final concentration, respectively) for 10 min and were further diluted (1:1) with 0.1 M phosphate buffer before fixation was prolonged for 90 min at room temperature. After centrifugation at 300 g for 10 min, the cell pellet was resuspended in PBS supplemented with 10% FCS and was centrifuged again at 300 g for 10 min. To obtain a consistent pellet, cells were resuspended in PBS-10% porcine gelatin, centrifuged at 2000 g for 2 min, and stored overnight at 4°C. Samples were then immersed overnight in PBS containing 2.3 M sucrose and 10% polyvinyl pyrrolidone before rapid freezing in liquid nitrogen.

Ultrathin sections (85 nm) were prepared using an ultracryomicrotome (Leica EM FCS, Austria) and mounted on nickel grids (Electron Microscopy Sciences, Fort Washington, PA). Sections were blocked in TBS-3% BSA and incubated overnight at 4°C with 50 µl anti-IL-13 mAb or isotype control (40 µg/ml final concentration). After rinsing in TBS-1% BSA, samples were incubated for 2 h with donkey anti-mouse IgG conjugated to 18 nm colloidal gold (Jackson Immunoresearch Laboratories, West Grove, PA). Sections were thoroughly rinsed in TBS, followed by water, and were stained with 2% methylcellulose containing 0.4% uranyl acetate. After air-drying, sections were examined by transmission electron microscopy (Hitachi 7500-2, Japan).

Subcellular fractionation
Purified peripheral blood eosinophils were homogenized by repeated passages through a ball-bearing cell homogenizer, and resulting organelles were separated by linear density gradient as described in earlier reports [28 , 29 ]. Briefly, at least 5 x 107 purified eosinophils were suspended in ice-cold 0.25 M Hepes-buffered sucrose (containing 10 mM Hepes, 1 mM EGTA, pH 7.4, supplemented with 100 µg/ml PMSF and 5 µg/ml each leupeptin, aprotinin, and TAME, 2 mM MgCl2, and 1 mM ATP) before homogenization through a 12 µm clearance in a ball-bearing cell homogenizer (EMBL, Heidelberg, Germany). The postnuclear supernatant from this was layered onto an 8-ml linear Nycodenz gradient (0–45% Nycodenz dissolved in Hepes-buffered sucrose) in a Beckman 14 x 89 mm Ultra-ClearTM centrifuge tube (Beckman, Palo Alto, CA). The gradient was subjected to equilibrium density centrifugation at 100,000 g for 1 h at 4°C, and fractions (16x0.8 ml) were collected from each preparation and stored at -80°C until used.

Marker enzyme assays
Marker enzyme assays were used to detect intracellular compartments in subcellular fractions. These included eosinophil peroxidase (crystalloid granule), measured by reactivity with TMB substrate, ß-hexosaminidase (crystalloid granule and secretory vesicles), measured by cleavage of the fluorescent substrate 4-methylumbelliferyl N-acetyl-ß-D-glucosaminide, and lactate dehydrogenase (LDH; cytosol) using an endpoint assay, as previously described [28 ]. Plasma membrane activity was determined by dot blot analysis with mAb to CD9 as previously described [29 ]. Enzyme activities were expressed as a percentage of the total sum of enzyme activity across all fractions as previously reported [28 , 29 ].

Cell culture
Culture medium consisted of RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cross-linking experiments were performed in 24-well culture plates, as previously described [2 ]. Wells were first coated with 40 µg/ml anti-mouse IgG F(ab')2 for 2 h at 37°C in 5% CO2. After washing, wells were incubated with 10 µg/ml mouse anti-CD28 (B-T3 mAb) or isotype-matched control mAb for 2 h at 37°C in 5% CO2. The wells were then washed twice with PBS, and 2 x 106 eosinophils in 1 ml culture medium were added per well. For stimulation with IgA and anti-IgA immune complexes, highly purified eosinophils were first incubated with sIgA at a final concentration of 15 µg/ml. After 1 h incubation at 37°C, cells were transferred in 24-well plates or anti-CD28-coated plates and were stimulated with 20µg/ml anti-IgA mAb at 37°C in 5% CO2. For high-affinity Fc{varepsilon}RI activation, eosinophils were stimulated with 10 µg/ml 15.1 mAb followed by the addition of 10 µg/ml anti-mIgG F(ab')2. After 18 h of culture, supernatants were collected and analyzed for IL-13 secretion.

Cytokine measurements
After subcellular fractionation, fractions were diluted 1:2 in assay diluent before measurement of immunoreactivity using a human IL-13 OptEIA enzyme-linked immunosorbent assay (ELISA) set (BD Pharmingen Canada, Mississauga, Ontario). The sensitivity of this immunoassay was 3.1 pg/ml, which was the lowest concentration of standard used. Results were expressed as an average value of IL-13 immunoreactivity (pg/ml) in each fraction.

IL-13 was also assayed in eosinophil supernatants using a specific ELISA kit (Diaclone), according to the manufacturer’s instructions. The lower detection limit of the assay was 1.5 pg/ml.

Biological activity of IL-13
Mononuclear cells, isolated by centrifugation over Percoll, were enriched in B cells by negative sorting using anti-CD3 immunomagnetic beads. Flow cytometric analysis of the cell preparation indicated that 32–63% CD19+ B lymphocytes were present in the gated lymphocytes. Enriched B cells were cultured in round-bottomed 96-well plates (1.6x105 cells/well) in the presence of increasing concentrations of rhIL-13 (10–1000 pg/ml) or eosinophil supernatants. Specificity was controlled by the addition of a neutralizing anti-IL-13 mAb (5 µg/ml). After 48 h of culture, cells were stained with a PE-conjugated anti-CD23 mAb and analyzed by flow cytometry.

Statistical analysis of data
Statistical significance was determined using the Mann-Whitney U-test for the biological assay. ANOVA was used to compare cytokine expression among the groups of patients and normal donors. P values <0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of IL-13 in eosinophils
To determine whether eosinophils were expressing IL-13 mRNA, total RNA was extracted from highly purified eosinophils from eosinophilic donors, and RT-PCR was performed with specific primers. As shown in Figure 1 , IL-13 product was detected in all eosinophil preparations. To rule out the possibility that the IL-13-specific amplicon obtained using RNA from purified eosinophils was a result of the minor PBMC contaminant present in the preparation, we performed RT-PCR using, as template, the amount of RNA corresponding to twice (2%) the highest percentage of PBMC observed in our entire set of eosinophil preparations. As a result of the high amplification of the RT-PCR technique, even a low amount of contaminating lymphocytes could result in the detection of a weak IL-13 signal (Fig. 1) . However, the signal obtained for potentially contaminating cells was not overlapping the one for eosinophils. These results clearly indicated that eosinophils are expressing IL-13 mRNA.



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  		Figure 1. Expression of IL-13 mRNA by eosinophils. RNA extracted from highly purified human eosinophils (in the order: ND, ND, HES) was subjected to RT and amplified by PCR by using primers specific for IL-13 and ß2-microglobulin (housekeeping gene). As a positive control, total RNA from human PBMC (100%) was used. A sample corresponding to 2% PBMC was also subjected to RT-PCR to determine that the IL-13 signal observed in eosinophil extracts was not resulting from contaminating cells. The products were electrophoresed on a 1% agarose gel and were stained with ethidium bromide.

 
The intracellular expression of IL-13 was then examined in freshly isolated eosinophils from hypereosinophilic patients or from normal healthy donors by cytofluorometric analysis. After fixation and permeabilization, cells were stained with FITC-conjugated anti-IL-13 mAb or an isotype-related control antibody. Results presented in Figure 2 clearly showed that all circulating eosinophils expressed IL-13, as demonstrated by the obtention of a unimodal pattern of fluorescence. The specificity of the intracellular staining was assessed by cell staining in the presence of excess recombinant cytokine. A tenfold excess of rhIL-13 was able to significantly reduce the intracellular staining of IL-13 in eosinophils (Fig. 2A) , whereas an equivalent excess of rhGM-CSF was without effect (Fig. 2B) .



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  		Figure 2. Flow cytometric analysis of intracellular IL-13. After fixation and permeabilization, freshly purified eosinophils (allergic donor) were stained with FITC-conjugated anti-IL-13 (solid line) or isotype-matched Ab (dotted line), as described in Materials and Methods. Competition assay (dashed line) was performed by cell staining in the presence of an excess (50 µg/ml) rhIL-13 (A) or rhGM-CSF (B). Cell fluorescence was measured using a FACSCaliburTM equipped with CellQuestTM software (Becton Dickinson). Thresholds were set according to the isotype-matched control. A total of 104 cells was usually acquired. Results are representative of one experiment.

 
To confirm the presence of IL-13 detected by flow cytometry, we used another detection method. Cytospin preparations of purified eosinophils were processed for immunohistochemical analysis using the APAAP method (Fig. 3A and 3B ) or immunofluorescence detection (Fig. 3C and 3D) as described in Materials and Methods. As shown in Figure 3B , eosinophils exhibited positive staining for IL-13, localized in the cytoplasm and which seemed associated with the granules. An additional cell membrane-like staining was also observed. Such a membrane-staining pattern has already been described for IL-2 [30 ] and stem cell factor [31 ]. No staining could be observed when cells were incubated with an irrelevant isotype control antibody (Fig. 3A) . Similar results were obtained using immunofluorescence detection. A positive granular pattern of fluorescent staining was observed when cells were incubated with anti-IL-13 mAb followed by FITC-conjugated anti-mouse IgG (Fig. 3D) . No fluorescence was detected with isotype control (Fig. 3C) . The fraction of cells stained for IL-13 was giving 15–25% positive cells, and the membrane-like staining was detected in only one eosinophil preparation, suggesting that the presence of IL-13 at the cell periphery was depending on specific physiological conditions.



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  		Figure 3. Immunoenzymatic (A, B) and immunofluorescence (C, D) detection of IL-13. Cytospin of freshly purified eosinophils (allergic donor) were incubated with anti-IL-13 (B, D) or an isotype-matched Ab (A, C). Cell staining was revealed using the APAAP detection system and New Fuchsin coloration (A, B) or fluorescence detection (C, D). Cells were counterstained with Mayer’s hematoxylin. Original magnification: x100.

 
The results obtained by immunohistochemistry on cytospin preparations confirmed the presence of IL-13 within eosinophils. Moreover, the staining pattern detected by immunofluorescence would implicate a preferential localization of this cytokine within the granules.

Intracellular distribution of IL-13
The intracellular distribution of IL-13 in eosinophils was determined after subcellular fractionation. Eosinophils (5x107) were homogenized using a specialized ball-bearing cell homogenizer, and their resulting organelles were separated down a 0–45% Nycodenz gradient. As previously determined [28 , 29 ], marker enzyme assays for eosinophil peroxidase and ß-hexosaminidase indicated the presence of crystalloid granules in high-density regions of the gradient (fractions 3–8), and dot blot analysis of CD9 demonstrated the position of plasma membrane fractions at low-density regions (fractions 9–12, Fig. 4A ). Cytosolic fractions, which do not enter the Nycodenz solution, remain afloat on the gradient as indicated by LDH activity (fractions 13–18). Immunoassay results for IL-13, shown in Figure 4B , indicated that most of the IL-13 (90% of the total immunoreactivity) coeluted with fractions enriched in crystalloid granules (fractions 3–5), suggesting that the major intracellular site of storage of IL-13 is the crystalloid granule, similar to other cytokines shown to be expressed in these cells. It is interesting that a small percentage of IL-13 immunoreactivity was also detected in low-density fractions (fractions 9 and 10) partially overlapping with CD9+ plasma membrane fractions. These fractions are known to be enriched in the chemokine RANTES, as determined in an earlier report [29 ]. These results suggest that IL-13 is stored as a preformed mediator in at least two separate intracellular locations in eosinophils; the first, which contains the majority of IL-13, is the crystalloid granule and the second, in a population of small secretory vesicles.



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  		Figure 4. IL-13 immunoreactivity in subcellular fractions of eosinophils. Subcellular fractionation was carried out using unstimulated peripheral blood eosinophils (5x107) obtained from an asthmatic donor. Fractions were collected from a 0–45% linear Nycodenz gradient and were analyzed for marker enzyme activities to obtain profiles of subcellular compartments. Marker assays used were eosinophil peroxidase (secretory granules), ß-hexosaminidase (secretory granules and lysosomal granules), CD9 (plasma membrane), and LDH (cytosol) and are expressed as a percentage of the maximal value. Quantification of IL-13 was carried out by ELISA for each fraction and is expressed as pg/ml. The total IL-13 content across all fractions was equivalent to 53 pg/5 x 107 eosinophils.

 
Localization of IL-13 by electron microscopy
To obtain a more precise localization of IL-13 in eosinophils, immunocytochemical staining with anti-IL-13 mAb, followed by colloidal gold-labeled anti-mouse IgG antibodies, was performed on cryo-ultrathin sections and analyzed by electron microscopy. As shown in Figure 5 , colloidal gold-detected immunoreactivity was essentially associated with the cores of bicompartmental secondary granules when samples were incubated with anti-IL-13 mAb (Fig. 5B and 5D , solid arrows), whereas no labeling was seen with isotype control antibody (Fig. 5A) , suggesting a specific localization of this cytokine in eosinophil crystalloid granules, although not all granules were positively stained (15–20% positive cores). IL-13 labeling was also detected in some unicompartmental secondary granules (Fig. 5C) , which were observed in eosinophils isolated from a hypereosinophilic syndrome (HES) donor. This type of granule, not common in blood patients, has been described in mature eosinophils derived in vitro from cord blood cells [32 ]. Furthermore, in one eosinophil preparation [skin disease (SK) donor], gold particles were present in small vesicles (Fig. 5D and inset, arrowheads) and at the cell membrane (Fig. 5D , open arrow), suggesting a possible transport of this cytokine to the cell membrane through small secretory vesicles and its release.



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  		Figure 5. Immunogold staining of IL-13. Cryo-ultrathin section of eosinophils were stained with anti-IL-13 (B–D) or an isotype-matched Ab (A), followed by gold (18 nm)-labeled anti-mouse IgG. Analysis by electron microscopy of eosinophils isolated from a patient with eczema (A, B, D) showed a preferential localization of IL-13 within the core compartment of secondary granules (B and D, solid arrows). IL-13 was also detected in some unicompartmental secondary granules present in eosinophils from an HES donor (C). Gold particles were also present in small vesicles (D and inset, arrowheads) and at the cell membrane (open arrow).

 
These observations not only confirmed that eosinophil crystalloid granules are storage sites for IL-13 but further localized IL-13 to the core compartment of these granules. In addition, IL-13 was found associated with small vesicular membrane structures, in agreement with the detection of IL-13 immunoreactivity in subcellular fractionation.

IL-13 expression in eosinophils in comparison with IL-4
We then compared eosinophil-associated IL-13 with the pathological status of respective donors. Patients were divided into five groups according to the etiology of diseases: HES, drug hypersensitivity (DHS), haematological disorders (HD), allergy and asthma (A), or SK, as well as healthy (normal) donors (ND). As shown in Figure 6A , eosinophils from all donors expressed IL-13, although individual variations in the IL-13 intracellular content were observed in patients within each group. Two groups of patients, HES and DHS, were highly heterogeneous in their content of IL-13. Furthermore, the intensity of staining, represented by the MFI, was significantly increased in almost all groups of patients compared with ND.



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  		Figure 6. Intracellular expression of IL-13 (A) and IL-4 (B) in eosinophils according to the etiology of eosinophilia. The donors were classified as follow: HES, hypereosinophilic syndrome (n=6); DHS, drug hypersensitivity (n=6); HD, haematological disorders (n=5); A, allergy and asthma (n=7); SK, skin diseases (n=8); ND, normal donors (n=4). After fixation and permeabilization, eosinophils were incubated with FITC-conjugated anti-IL-13, PE-conjugated anti-IL-4, or a FITC- or PE-conjugated isotype control Ab. Samples were analyzed by flow cytometry using a FACSCaliburTM equipped with CellQuestTM software (Becton Dickinson). Results are expressed as MFI, calculated by subtracting the MFI of the control from the MFI of the sample. Statistical analysis was performed using ANOVA, and P < 0.05 was considered significant.

 
Because IL-13 and IL-4 are defined as Th2-type cytokines and share many functions, we analyzed IL-4 intracellular content in eosinophil preparations in parallel with IL-13 expression. The specificity of IL-4 staining in human eosinophils was verified in the same manner used for IL-13 labeling. Only a tenfold excess of rhIL-4 could inhibit the labeling of anti-IL-4, whereas an equal excess of rhGM-CSF was ineffective (data not shown). In contrast to results with IL-13, IL-4 expression was less heterogeneous among individual patients, except for four patients, who showed very strong staining (Fig. 6B) . Whereas all groups of patients were significantly higher than the group of normal donors for IL-13 expression, only patients with allergies (A) exhibited statistically higher intracellular IL-4 content than ND.

Taking into account that these eosinophil samples were purified from patients with varying immunological profiles, where previous in vivo cytokine secretion may have already occurred, we investigated the intracellular content of IL-13 and IL-4 after cell culture in the presence of brefeldin A. This compound inhibits intracellular protein transport at the level of the trans-Golgi network and thus prevents secretion of newly synthesized protein. Purified eosinophils were cultured for 18 h in the presence or absence of an optimal concentration of brefeldin A (10 µg/ml) and were analyzed for IL-13 and IL-4 intracellular staining by flow cytometry. No change in IL-13 expression was detected in the presence of brefeldin A (Fig. 7A ), whereas a strong increase in IL-4 immunoreactivity was observed (Fig. 7B) . These results suggest that IL-4 and IL-13 may be differentially synthesized and released in the eosinophil using separate trafficking mechanisms.



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  		Figure 7. Detection of intracellular IL-13 (A) and IL-4 (B) in cultured eosinophils. Purified cells (obtained from a patient with eczema) were cultured (2x106/ml) for 18 h in the presence (dotted line) or absence (solid line) of brefeldin A (10 µg/ml). Cell staining was performed as before, and samples were analyzed by flow cytometry. The isotype-matched Ab, represented by the dashed line, was identical for both culture conditions.

 
IL-13 release by purified eosinophils
Our findings led us to investigate whether IL-13 may be released following immunological activation. Previous studies have shown that sIgA immune complexes could induce IL-4, IL-5, and IL-10 secretion by human eosinophils, suggesting that Th2-type cytokine secretion was induced after IgA-dependent activation [2 ]. Therefore, highly purified eosinophils were activated with sIgA immune complexes for 18 h, and culture supernatants were analyzed for IL-13 release. No IL-13 was detected in sIgA-treated eosinophil supernatants compared with control (Fig. 8 ). Lymphocytes have been shown to produce high levels of IL-13 and IL-13 mRNA when activated by phorbol 12-myristate 13-acetate (PMA) and anti-CD28 [33 ]. In our previous work, we demonstrated that the costimulatory molecule CD28 was expressed on eosinophils and that CD28 activation resulted in Th1-type cytokine release by these cells [2 ]. As CD28 stimulation potently induced IL-2 and IFN-{gamma} release by eosinophils, we investigated whether CD28 ligation could also induce IL-13 secretion. Purified eosinophils were added to plates coated with anti-CD28 or isotype control mAb and were incubated for 18 h. As shown in Figure 8 , CD28 ligation resulted in substantial IL-13 secretion from eosinophils (mean: 20.6±4.7 pg/ml; range: 5–60 pg/ml).



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  		Figure 8. Release of IL-13 by eosinophils after stimulation by CD28 ligation. Purified eosinophils (2x106/ml) were stimulated with sIgA-anti-IgA (n=5), immobilized anti-CD28 mAb (n=13), sIgA-anti-IgA together with immobilized anti-CD28 (n=5), cross-linked anti-Fc{varepsilon}RI mAb (n=3), or an isotype-matched Ab (n=13), as described in Materials and Methods. Eosinophils were obtained from normal donors or donors with various pathologies: A, HES, DHS, and SK. After 18 h, supernatants were harvested and analyzed by ELISA for IL-13 content. Data are presented as mean ± SEM.

 
Furthermore, as previously demonstrated for IL-2 and IFN-{gamma} [2 ], we observed that CD28-induced IL-13 release was strongly inhibited following coincubation with sIgA-IgA complexes. Recently, we have shown that human eosinophils express the high-affinity receptor for IgE Fc{varepsilon}RI and that ligation of this receptor results in cell activation and IL-10 release [34 ]. Therefore, we investigated whether this activation process could also lead to IL-13 secretion. Stimulation of eosinophils with anti-Fc{varepsilon}RI{alpha} (15.1) mAb followed by anti-mouse IgG F(ab')2 did not induce IL-13 release by eosinophils (Fig. 8) , although secretion of IL-10 was detected (119±60 pg/ml). These results indicate that only activation by CD28 pathway induced IL-13 production by eosinophils. Furthermore, this finding indicates that although preformed cytokines are stored in granules, the pathways leading to their secretion are different.

Biological activity of eosinophil-derived IL-13
To investigate the biological relevance of these findings, we evaluated the role of IL-13 on CD23 expression by B cells. Enriched fractions of B lymphocytes were incubated with rhIL-13 or CD28-activated eosinophil supernatants for 48 h, and CD23 expression was determined by flow cytometric analysis. Supernatants from eosinophils activated on CD28 ligation induced a significant increase in CD23 expression on B lymphocytes (Fig. 9B ). Furthermore, given the quantity of IL-13 released by these cells, this effect was larger than the effect of rIL-13 (Fig. 9A) . IL-4 and IL-13 are known to exert such an effect on B cells. As previously shown [2 ], we were unable to detect IL-4 in supernatants from CD28-activated eosinophils (data not shown), supporting a direct effect of IL-13 on B lymphocytes. To confirm these results, a neutralizing anti-IL-13 mAb was added to eosinophil supernatants before incubation with B lymphocytes, leading to the absence of induction of CD23 expression. These results indicate that eosinophil-derived IL-13 is biologically active and suggest that this cytokine is the major factor contributing to CD23 regulation by eosinophils.



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  		Figure 9. Biological activity of eosinophil-derived IL-13. (A) Enriched B cells were cultured in round-bottomed 96-well plates (1.6x105 cells/well) in the presence of variable concentrations of rhIL-13 (solid bars), and specificity was controlled by the addition of a neutralizing anti-IL-13 mAb (open bars). (B) Enriched B cells were cultured with supernatants from eosinophils (1:2 dilution) stimulated with immobilized anti-CD28 (S) or an isotype-matched Ab (NS). Specificity was controlled by incubation of cell supernatant with a neutralizing anti-IL-13 Ab. After 48 h of culture, cells were stained with a PE-conjugated anti-CD23 mAb and analyzed by flow cytometry. Data are expressed as % CD23+ cells above basal level and are presented as mean ± SEM from two replicate experiments. Statistical analysis was performed using Mann-Whitney, and P < 0.05 was considered as significant.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first demonstration that IL-13 is synthesized and released from CD28-stimulated eosinophils and that eosinophil-derived IL-13 is bioactive. By intracellular flow cytometry analysis, we detected IL-13 within eosinophils isolated from normal donors and hypereosinophilic patients. Inhibition of the intracellular staining after preincubation with rhIL-13 but not with rhGM-CSF confirmed the specificity of our IL-13 staining. The presence of IL-13 in eosinophils was further supported by immunocytochemistry on cytospin preparations, showing a positive, granular staining pattern for IL-13. Previous work has demonstrated that in human basophils, IL-13 is localized in the granules [24 ]. In human eosinophils, the intracellular distribution of IL-13 after subcellular fractionation was found to overlap essentially with fractions corresponding to the crystalloid granule compartment (eosinophil peroxidase and ß-hexosaminidase-positive). These results indicate that eosinophil-crystalloid granules constitute a major intracellular site for IL-13 storage. Immunofluorescence and immunogold staining further revealed a preferential localization of IL-13 in the crystalloid granules. Immunogold staining has previously been used to determine whether cytokines are localized in the core compartment or matrix of bicompartmental secondary granules (see review in ref. [35 ]). IL-13 was in fact found in the core compartment of secondary granules, as well as in some unicompartmental secondary granules that were present in eosinophils from a HES patient. It is now recognized that eosinophils store many cytokines in their crystalloid granules to provide a means for them, unlike lymphocytes, to rapidly release a preformed pool of these cytokines. Based on our results, IL-13 may now be added to this intracellular cytokine pool. In addition to this major storage site, some IL-13 was found associated with small vesicular membrane structures. This was evidenced by the association of IL-13 immunoreactivity with plasma membrane fractions (CD9+) and detection of IL-13 immunogold particles in small secretory vesicles and cell membrane. Until now, only RANTES and transforming growth factor-{alpha} have been detected in small secretory vesicles of the cytoplasm [29 , 36 ]. RANTES and IL-4 have been shown to be released in a piecemeal manner through translocation of small secretory vesicles [29 , 37 ]. This interesting observation suggests that following activation, IL-13 may also be transported from the crystalloid granule to the cell membrane through small secretory vesicles and released by piecemeal degranulation.

Besides activated T cells, which produce the highest concentrations of IL-13, other cell populations have been shown to produce IL-13 upon stimulation. After engagement of Fc{varepsilon}RI using anti-IgE or anti-Fc{varepsilon}RI Ab, human basophils release IL-13 in vitro [24 , 25 ]. Similar results were found for human mast cells [23 ]. In contrast to these cell populations, which produce IL-13 only after cell activation, human eosinophils appear to store this cytokine in crystalloid granules. Even cells from normal donors were positive for intracellular IL-13 staining. In our hands, stimulation of eosinophils by Fc{varepsilon}RI engagement did not induce detectable IL-13 secretion. This was not a result of a lack of expression of Fc{varepsilon}RI at the cell surface, as we have shown in another study that all eosinophil preparations expressed the high-affinity receptor for IgE, although individual variations existed among donors [34 ]. In their work on IL-13 release by basophils, Li et al. [24 ] also looked at IL-13 production by activated eosinophils. Similar to our results, no IL-13 could be released during cell culture with anti-Fc{varepsilon}RI Ab alone. However, in their system, the failure to activate eosinophils with unaggregated anti-Fc{varepsilon}RI Ab alone is likely a result of the lack of receptor ligation at the cell surface, as we have already shown for IL-10 secretion [34 ]. The authors also went on to show that human neutrophils do not appear to produce IL-13 after cell activation. In contrast to this observation, we have evidence for the detection of IL-13 in freshly purified neutrophils by intracellular flow cytometry (unpublished data), suggesting that neutrophils also have the potential to release IL-13 under appropriate conditions. In T cells activated by CD28 ligation in the presence of PMA, large concentrations of IL-13 were detected in supernatants [33 ]. In our hands, activation of eosinophils by CD28 ligation alone induced IL-13 release, similar to our observation for IL-2 and IFN-{gamma} secretion [2 ]. It is interesting that these results indicate that different cytokines stored in eosinophil granules are not released under the same processes of activation.

Because IL-13 shares many biological functions in common with IL-4, and the latter is produced by human eosinophils [4 , 5 ], we examined the intracellular content of both cytokines in freshly purified eosinophils according to disease etiology. IL-13 was detected in eosinophils from all donors, indicating that cells are able to store this cytokine as a preformed mediator regardless of pathophysiology. The intracellular content of IL-13 was significantly higher in patients with disease than in normal donors, suggesting a possible role for IL-13 in these conditions. Analysis of the intracellular expression of IL-4 in the same eosinophil preparations revealed that its level was low and less heterogeneous than IL-13 expression. This suggests that IL-4 has already been released in vivo or that IL-4 is not a major storage product in these cells. To answer this question, we compared IL-13 and IL-4 expression after cell culture in the presence of brefeldin A. This compound interferes with protein secretion by blocking their exit from the Golgi apparatus. Therefore, cytokine production is expected to accumulate within the cell for detection by flow cytometry. In the presence of brefeldin A, IL-4 immunoreactivity was increased in eosinophils, indicating that cells were engaged in de novo synthesis of this cytokine. Similar results have already been shown for human neutrophils [38 ]. In contrast, no detectable increase in intracellular IL-13 was produced during culture with brefeldin A, as the levels of IL-13 immunoreactivity were similar before and after treatment. These results suggest that IL-4 is constitutively released during culture or has been released in vivo and that upon culture with brefeldin A, eosinophils accumulated a larger intracellular pool of IL-4. Favoring this hypothesis is the recent work of Bandeira-Melo et al. [37 ], showing that all circulating eosinophils contained preformed IL-4. The fact that the levels of IL-13 expression were unchanged would rather indicate that the intracellular pool of IL-13 was unmodified, suggesting that spontaneous release of IL-13 had not occurred in vivo in the case of the eosinophilic patients under study. These results also indicate that in eosinophils, IL-4 and IL-13 production is differentially regulated. This conclusion is supported by the demonstration that IL-13 is released after anti-CD28 activation, and IL-4 is secreted in response to activation by IgA complexes. Taken all together, these results suggest that IL-4 and IL-13 are released from eosinophils by independent processes and that eosinophils might participate in asthma or other inflammatory reactions in a different context of stimulation [10 ].

One major function of IL-13 is its ability to regulate B cell functions and to switch B cells to the production of IgE in the absence of IL-4 [39 ]. The low-affinity receptor for IgE, CD23, is required for enhancement of IgE-mediated allergic responses because of its capacity to focus allergen/IgE complexes to allergen-specific T cells [40 ]. CD23 expression on B cells is up-regulated by IL-4 and IL-13. Therefore, it was of interest to examine whether eosinophil-derived IL-13 could regulate CD23 expression on B lymphocytes. Our experiments indicated that supernatants from anti-CD28-stimulated eosinophils significantly increased CD23 expression on B cells. The higher stimulatory effect on CD23 expression of supernatants from anti-CD28-stimulated eosinophils compared with recombinant IL-13 is likely a result of the presence of other factors in eosinophil supernatants, which would synergize the effect of cell-derived IL-13 on B cells. Taking the opposite view, one would also conceive that the recombinant IL-13 would not be endowed with the same stimulatory ability than the natural molecule, as it may not be post-translationally modified in the same manner as immune cell-derived IL-13. However, the demonstration that eosinophil-derived IL-13 was inducing CD23 expression on B cells suggests a possible role for eosinophils in regulating the transfer and amplification of signals from antigen-presenting cells (APC) to B cells, potentially in the lymph nodes where eosinophils have been shown to traffic during atopic responses [41 ].

Other functions of IL-13 have been reported, including its capacity to induce VCAM-1 and P-selectin expression on endothelial cells [21 , 42 ] and to activate eotaxin production by airway epithelial cells [43 ] and human nasal fibroblasts [44 ]. These findings suggest that through the release of IL-13, eosinophils could induce the influx of inflammatory cells, thereby sustaining its own recruitment. Eosinophil-derived IL-13 may also activate eosinophils in an autocrine or paracrine manner, resulting in an up-regulated expression of CD69 and increased survival [45 ].

At the present time, it is not known whether eosinophils release IL-13 in vivo and what role eosinophil-derived IL-13 may play in regulation of the immune response. The only evidence in favor of a functional role is the detection of IL-13 mRNA in thymic eosinophils, potentially leading to IL-13 synthesis and release in vivo [46 ]. A massive eosinophil infiltration is observed in lung inflammation during asthma and in granuloma formation during Schistosoma mansoni infection. The presence at the inflammation sites of APC such as macrophages expressing B7 molecules would therefore allow a rapid interaction of CD28-positive eosinophils with these cells and the release of IL-13.

Many reports in the literature suggest that eosinophil-derived cytokines may have an important role in allergy. First, in eosinophilic inflammation, eosinophils outnumber T cells in the tissues by as much as 100-fold. As such, the magnitude of the presence of eosinophils may be a determining factor in regulating immune responses at a local level. Secondly, eosinophils have the potential to release IL-13 locally to influence the function of other cells, such as T helper cells and endothelial cells, in a juxtacrine manner. The release of eosinophil IL-13 takes place within a much shorter period than T cell-released IL-13, as this cytokine is stored as a preformed mediator in crystalloid granules, which may be secreted in response to stimuli in a matter of minutes. Thus, eosinophil-derived IL-13 is predicted to further enhance T cell-initiated eosinophilic inflammation at a local level and within a short time. Thirdly, eosinophils have been shown to traffic to paratracheal-draining lymph nodes (in a mouse model of asthma), where they were shown to function as APC expressing major histocompatibility complex class II and costimulatory CD80 and CD86 to stimulate CD4+ T cells [41 ]. In this case, IL-13 would not be expected to be required in abundance to carry out important immunomodulatory events, such as enhanced switching of T cells to Th2 phenotype and increased IgE synthesis, both of which are hallmarks of allergic disorders.

Bearing in mind the prominent role of IL-13 demonstrated recently in murine experimental asthma [9 , 10 ], as well as in S. mansoni infection [11 , 12 ], characterized by a massive eosinophil infiltrate, the finding that eosinophils have the capacity to elaborate functional IL-13 supports an important role for this cell in type 2 immune responses.


   ACKNOWLEDGEMENTS
 
This work was supported by the Institut National de la Santé et de la Recherche Médicale U547, Institut Pasteur de Lille, and Université de Lille II. It was also supported by the MRC/Canadian Institutes of Health Research. M. C. is a member of the Institut Universitaire de France, P. L. is a Canadian Lung Association/Canadian Institutes of Health Research Scholar, and R. M. is an Alberta Heritage Senior Medical Scholar. The authors thank Prof. E. Delaporte, D. Staumont, and the Centre de Médecine Préventive de l’Institut Pasteur de Lille for access to patients. We are also grateful to A-S. Roumier and V. Angeli for helpful discussions, to M. Loyens and M. Steward for technical assistance, to J. L. Neyrinck for computer help, and to J.M. Merchez for image analysis.

Received April 24, 2002; revised July 2, 2002; accepted July 3, 2002.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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A. P Rogerio, C. R Cardoso, C. Fontanari, M. A Souza, S. R Afonso-Cardoso, E. V. Silva, N. S Koyama, F. L Basei, E. G Soares, J. B Calixto, et al.
Anti-asthmatic potential of a D-galactose-binding lectin from Synadenium carinatum latex
Glycobiology, August 1, 2007; 17(8): 795 - 804.
[Abstract] [Full Text] [PDF]

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 Proc. Natl. Acad. Sci. USAHome page
L. A. Spencer, R. C. N. Melo, S. A. C. Perez, S. P. Bafford, A. M. Dvorak, and P. F. Weller
Cytokine receptor-mediated trafficking of preformed IL-4 in eosinophils identifies an innate immune mechanism of cytokine secretion
PNAS, February 28, 2006; 103(9): 3333 - 3338.
[Abstract] [Full Text] [PDF]

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 J. Immunol.Home page
E. R. Machado, M. T. Ueta, E. V. Lourenco, F. F. Anibal, C. A. Sorgi, E. G. Soares, M. C. Roque-Barreira, A. I. Medeiros, and L. H. Faccioli
Leukotrienes Play a Role in the Control of Parasite Burden in Murine Strongyloidiasis
J. Immunol., September 15, 2005; 175(6): 3892 - 3899.
[Abstract] [Full Text] [PDF]

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 J. Immunol.Home page
S. L. Kimzey, P. Liu, and J. M. Green
Requirement for CD28 in the Effector Phase of Allergic Airway Inflammation
J. Immunol., July 1, 2004; 173(1): 632 - 640.
[Abstract] [Full Text] [PDF]