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IMMUNOBIOLOGY
From Meakins-Christie Laboratories,
McGill University, Montreal, Quebec, Canada; the Department of
Allergy and Clinical Immunology, Imperial College School of Medicine,
National Heart and Lung Institute, London, England; the Department
of Medical Protein Chemistry, Flanders InterUniversity Institute
for Biotechnology, Ghent, Belgium; and Magainin Pharmaceuticals,
Plymouth Meeting, PA.
Interleukin-9 (IL-9) has been implicated in the pathogenesis
of allergic disorders. To examine the interaction between IL-9 and
eosinophils, we evaluated mature peripheral blood eosinophils for their
expression of the specific Airway eosinophilia have been recognized as a
predominant feature of allergic asthma, and elevated numbers within the
inflammatory infiltrate are often associated with disease
severity.1-3 These cells are believed to contribute to the
pathophysiology of asthma through release of cationic granule proteins,
reactive oxygen metabolites, and proinflammatory and profibrotic
cytokines.4 In addition to eliciting tissue damage,
eosinophil-derived proinflammatory mediators can perpetuate the
inflammatory reaction and lead to chronic changes in airway
function.5 Asthmatic airways also play host to an
increased number of CD4+ T cells, which appear to
orchestrate the specific immune response occurring within the lungs.
These cells produce a range of Th2-type cytokines, particularly
interleukin-5 (IL-5), with regulatory effects on eosinophil growth,
differentiation, and activation.6-8
IL-9 is a Th2-type cytokine first described in the mouse as a T-cell
and mast-cell growth factor.9-11 This regulatory cytokine inhibits lymphokine production by interferon- There is extensive literature on the complex interactions between
the eosinophil and the CD4+ T lymphocyte, which are
fundamental to the development of allergic inflammation. While it
has been shown that IL-9 promotes eosinophilia in
vivo,22-24 the mechanism remains obscure. To further
investigate the role of IL-9 on eosinophils, we examined the expression
of functional IL-9R on the surface of human eosinophils and whether IL-9 could influence eosinophil function, survival, and/or
development. In this report we show that eosinophils from human
peripheral blood express IL-9R on their surface. Furthermore,
stimulation of eosinophils with IL-9 up-regulates the cell surface
expression of the IL-5R- Isolation of eosinophils from peripheral human blood
Reverse transcriptase-polymerase chain reaction and Southern
blot analysis
The oligonucleotide primers were synthesized on the basis of the entire
coding region of the IL-9R- Cytofluorographic analysis of surface expression of IL-9R on human eosinophils Samples of 2 × 105 cells were incubated with saturating concentrations of the primary antibodies (5 µg/mL purified mouse mAb anti-IL-9R- chain or isotype control IgG1) in the
presence of 2 mg/mL affinity-purified human IgG in phosphate-buffered
saline (PBS) and 5% fetal calf serum (FCS) for 30 minutes on ice. The cells were washed twice with PBS/2%FCS and incubated in the dark for
30 minutes with fluorescein isothiocyanate (FITC) goat antimouse IgG at
a final dilution of 1:200. The cells were then washed and resuspended
in 300 µL PBS and analyzed on a fluorescence-activated cell sorter
(FACS) (FACScan; Becton Dickinson, Oxnard, CA). The results
were analyzed using Cell Quest software (Becton Dickinson).
Cytospin preparations Cytospin slides were prepared from peripheral blood eosinophils or bronchoalveolar lavage (BAL),32 fixed in 4% paraformaldehyde for 20 minutes at room temperature, and washed with 0.05 mol/L Tris-HCl (tris[hydroxymethyl] aminomethane-hydrochloride)-buffered isotonic saline (TBS) (pH.7.6). After drying, the slides were stored at 20°C before immunocytochemistry.
Immunocytochemistry The cytopreparations of purified eosinophils or BAL slides32 were washed with TBS. After saturation for 20 minutes with TBS containing 10% normal human serum and 5% normal goat serum, the cells were incubated with 1 µg/mL rabbit polyclonal antibody (pAb) anti-IL-9R-, which is specific to the C-terminus
intracellular domain of the IL-9R-, in antibody dilution buffer
(Dako SA, Glostrup, DK) overnight at 4°C. As a negative control, a
dilution of 1:500 normal rabbit serum was used as a primary antibody.
After washing, a concentration of 1:200 biotinylated swine antirabbit
IgG was added for 30 minutes at 37°C followed by 1:200
streptavidin-conjugated alkaline phosphatase for 1 hour at room
temperature. The slides were developed using Fast Red and
counterstained with Mayer's hematoxylin.
Determination of eosinophil apoptosis Morphological assessment of nuclei alteration. Freshly isolated human eosinophils were incubated in complete Roswell Park Memorial Institute medium (RPMI 1640) with 10 ng/mL recombinant human (rh)IL-9, rhIL-5, or medium alone for 18 hours. Apoptosis was determined by morphological assessment.33 In this system, cytospin preparations were made and stained by Diff Quick (American Scientific Products, McGray, IL), and cells exhibiting apoptotic nuclei were enumerated in different fields in a blinded fashion using a random coded order. A minimum of 500 total cells was counted. This was achieved using a Nikon light microscope (Nikon, Japan) at the original magnification × 400. The number of apoptotic cells was calculated as a percentage of the total cell count. The results are reported as the mean percentage of apoptotic cells plus or minus SD. Determination of eosinophil viability. The percentage of necrotic eosinophils was determined by counting trypan blue positive cells. DNA fragmentation assay Apoptosis was also assessed by analysis of the staining characteristics of fixed permeabilized cells exposed to the DNA-binding dye, propidium iodide (PI), as described previously.34 Briefly, after 18 or 36 hours of culture, 0.3 × 106 eosinophils were washed with PBS and fixed with 70% ethanol for 30 minutes at 4°C. The fixed cells were then washed twice with 2 mL cold PBS and resuspended in 200 µL PBS containing 200 U/mL ribonuclease A (RNase A) for 30 minutes at 37°C. PI was then added to the suspension and analyzed using cell cycle parameters on the FACScan machine.Competitive RT-PCR Competitive PCR was optimized to allow rapid estimation of the ratios of soluble and membrane-associated IL-5R- isoforms. A
forward primer was designed for the extracellular portion of the
receptor (position 1033-1056) and 2 reverse primers for a soluble isoform (position 1279-1298) and a membrane-anchored isoform (position 1539-1561), as previously described.35
Cell line and culture conditions The human cell line HL-60 (clone 15; American Type Culture Collection, Rockville, MD) was used in the study. The cells were cultured at 37°C in humidified 5% carbon dioxide (CO2) in RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Eosinophil differentiation was induced as described previously.36 Briefly, the cells were grown in RPMI 1640 and 10% FCS in the presence of 0.3 mmol/L butyric acid for 7 days to generate eosinophils.Effect of IL-9 on IL-5R- chain was then performed by flow
cytometry analysis using 5 µg/mL mouse mAb anti-IL-5- chain as
described above.
Eosinophil differentiation of human umbilical cord blood progenitors and HL-60 cells Human umbilical cord blood was collected from the maternity unit of Chelsea and Westminster Hospital (London, UK) with approval of the local ethics committee. Mononuclear cells were isolated after centrifugation over Histopaque (Sigmal Chemical Co, Oakville, Ontario, Canada) followed by red cell lysis in sterile water and 1-hour adherence to plastic. The CD34+ cells were isolated from nonadherent cells by MACS immunomagnetic cell separation using anti-CD34 beads. The cells were then cultured in Iscove's modified Dulbecco's medium (IMDM) with 15% FCS, penicillin, streptomycin, amphotericin mix, and 50 µmol/L -mercaptoethanol in 24-well plates
(Nunc, Roskilde, Denmark) at 1 × 105 cells per well. The
cytokines were added at 1 ng/mL for IL-3 and IL-5 and at 1, 5, or 10 ng/mL for IL-9. The cells were counted, and the medium and cytokines
were replenished at 7 and 14 days of culture, when flow cytometry was
performed. To detect the presence of soluble and membrane-bound forms
of IL-5R- messenger RNA (mRNA) in these cells over time, RNA was
extracted at days 0, 3, 5, 7, and 14 for RT-PCR.
To assess the ability of IL-9 to influence the eosinophilic
differentiation of the HL-60 cell line, the cells were taken on day 6, washed twice with RPMI 1640, and incubated with 10 ng/mL IL-9 protein
in complete RPMI 1640 or with the vehicle control (medium alone). The
cells were then harvested at 4, 6, 8, 18, or 24 hours after
stimulation. For umbilical cord progenitors and HL-60 cells, the
eosinophilic phenotype of the cells was confirmed by
May-Grünwald-Giemsa and chromotrope 2R staining and by
immunocytochemistry to detect a major basic protein with mAb BMK-13.
Surface expression of IL-5R- Quantification and statistics The differences between groups were analyzed using the Student t test, and P < .05 was considered statistically significant.Reagents and antibodies The following was used in the study: rabbit pAb affinity-purified anti-IL-9R- directed to C-terminal intracellular
domain specific and FITC-conjugated goat antimouse IgG (Santa Cruz
Biotechnology, Santa Cruz, CA); mAb antihuman IL-5R- chain (-16,
prepared by J.T.); rhIL-9 (Calbiochem, La Jolla, CA); mouse mAb
affinity-purified antihuman IL-9R-, which was directed to
N-terminal extracellular domain specific and was used for cord blood
culture IL-3 (Genzyme Corporation, West Malling, England); IL-5
(PharMingen, San Diego, CA); mAb antimajor basic protein (BMK-13)
(Sanbio b.v., Uden, The Netherlands); normal rabbit serum and PI
(Cedarlane, Toronto, Ontario, Canada); affinity-purified human IgG and
IgG1 isotype control (clone MOPC1), chromotrope 2R, and Histopaque
(Sigma); alkaline phosphatase antialkaline phosphatase (APAAP),
biotinylated and unconjugated Fab'-2 swine antirabbit IgG, Fast Red,
and streptavidin-conjugated alkaline phosphatase (Dako, Glostrup,
Denmark); anti-CD16, anti-CD34, and anti-CD3 immunomagnetic beads
(Miltenyi Biotech); FCS (Hyclone Laboratories, Logan, UT); and
-mercaptoethanol (Life Technologies, Inc, Grand Island, NY)
Detection of mRNA encoding IL-9R in human peripheral blood eosinophils To determine whether freshly isolated peripheral blood eosinophils express the IL-9R- chain transcript, mRNA preparation from highly purified eosinophils was analyzed by RT-PCR. As shown in
Figure 1, a specific band at the expected
size (325 base pair [bp]) corresponding to the IL-9R- chain mRNA
was detected in eosinophils from both nonasthmatic controls (lanes 3 and 4) and asthmatic patients (lanes 5 and 6), and the
eosinophil-differentiated HL-60 cell line, used as a positive control
(lane 2). There was no specific band seen in the absence of cDNA (lane
1). Amplification products specific for -actin were of similar
intensity between all samples, which suggests the equality of the RNA
preparations.
Cell surface expression of IL-9R- chain is expressed on the
cell surface of freshly isolated peripheral blood eosinophils, we performed flow cytometry analyses of human eosinophils from different subjects stained with mouse mAb to the human IL-9R- chain. Purified human eosinophils from an asthmatic patient and the
eosinophil-differentiated HL-60 cell line showed positive reactivity
using the mouse mAb anti-IL-9R-, with a mean percentage positivity
of 23% and 65%, respectively (Figure 2A,B). We also confirmed the
expression of the IL-9R- chain in eosinophils from healthy
controls, but there was no significant difference in the level of the
IL-9R- expression eosinophils from 5 asthmatics
(10.0% ± 12.7%) and 5 nonasthmatic healthy control subjects
(8.9% ± 8.2%) (P > .05), as depicted in Figure
2C.
Detection of IL-9R protein in human peripheral blood and BAL eosinophils by immunocytochemistry To further investigate the protein expression of the IL-9R- by human eosinophils, immunocytochemistry was performed with rabbit pAb anti-IL-9R- chain on human peripheral blood eosinophils from 12 donors. Specific staining with anti-IL-9R- antibodies was
observed in peripheral blood (Figure 3A)
and BAL eosinophils from an asthmatic subject (Figure 3C). Substitution
of the primary antibody with normal rabbit serum eliminated the
positive immunoreactivity, demonstrating the specificity of the
analysis (Figure 3B,D). Our immunohistochemical studies demonstrated
the presence of IL-9R- on cells that showed the eosinophilic
phenotype. Furthermore, there was no significant difference observed
between the percentage of IL-9R-+ eosinophils from 8 asthmatics (68.0% ± 24.7%) and 5 nonasthmatic controls
(55.0% ± 18.9%) (P > .05), as seen in Figure 3E.
However, in both groups of subjects it was noted that higher numbers of IL-9R-+ eosinophils were detected by
immunocytochemistry compared to flow cytometry analysis. This suggests
that the surface expression of IL-9R- is under regulatory control,
and it can be speculated that factors, such as cytokines, can increase
the surface expression of IL-9R, which may potentiate the function of
recruited eosinophils during an inflammatory
reaction.2
Effect of IL-9 on eosinophil survival As demonstrated in thymic lymphoma cell lines37 and T cell lines,38 the major activity of IL-9 on T cells has been proposed to be protection from apoptosis. To date, there have been no studies that have examined the effect of this cytokine on human eosinophil survival. To test whether stimulation of the IL-9R influenced eosinophil apoptosis, human purified blood eosinophils were treated with increased concentration of either rhIL-9, rhIL-5, or medium alone for 18 hours. The extent of cellular apoptosis was then evaluated by morphological assessment (Figure 4).33 As observed in Figure 4A, treatment with IL-9 significantly decreased, in a concentration-dependent manner, the percentage of eosinophils undergoing apoptosis compared to untreated cells. The minimal effective concentration of IL-9 was 0.1 ng/mL, and the maximum effect was observed at 10 ng/mL. The mean percentage of apoptotic cells was 20.8% ± 2.5% vs 44.5% ± 6.2% with medium (P < .01). Similarly, as expected, the percentage of eosinophils undergoing apoptosis decreased significantly in the presence of IL-5 (P < .01) (Figure 4A,B).
The antiapoptotic effect of IL-9 was then confirmed in additional
subjects, and significant inhibition of eosinophil apoptosis was
obtained when the cells were stimulated with IL-5 or IL-9 for 18 hours
(Figure 5A,C). No difference in the
percentage of necrotic cells could be detected between different
samples at 18 hours (Figure 5B), which suggests that the untreated
cells are at the first stage of the apoptosis process.33
Using a DNA fragmentation assay, we demonstrated that IL-9 inhibited
apoptotic cell death. We found that treatment of eosinophils with IL-9, as well as IL-5, significantly inhibited DNA fragmentation compared to
untreated cells at 18 hours. The mean percentage of inhibition was
34.7% ± 6.3% and 36.4% ± 10.4%, respectively (Figure 5D). Furthermore, comparable results were observed when apoptotic cells were
assessed by morphology analysis (Figure 5D).
We next investigated whether IL-9 could increase human eosinophil
survival at 36 hours. In this system fewer apoptotic eosinophils were
detected in preparations cultured with IL-9 or IL-5 compared to
untreated cells, as showed by morphology assessment and DNA fragmentation (P < .05) (Figure
6A,C). A significant difference in the
percentage of necrotic cells could be observed between IL-9 or IL-5
compared to untreated cells at 36 hours (Figure 6B). Altogether these
results demonstrate that IL-9 increases human eosinophil survival.
Effect of IL-9 stimulation on human eosinophil IL-5R- following IL-9 incubation. Compared to
unstimulated cells, stimulation for 18 hours with IL-9 of the
peripheral blood human eosinophils increased the expression of the
IL-5R- chain by 2.4 ± 0.6-fold compared with baseline cells
(Figure 7A,B). Similarly, the expression
of IL-5R- increased the baseline level by 4-fold when
differentiated HL-60 cells (harvested on day 7) were stimulated with
IL-9 for 18 hours (data not shown). Therefore, IL-9 augmented the
expression of IL-5R- on human eosinophils. Taken together, these
results suggest that the antiapoptotic effect exerted by IL-9 on human
eosinophils may be accounted, at least partially, by the increased
surface expression of the IL-5R- chain.
Effect of IL-9 on IL-5R- -subunit and common -subunit.6 We next tested
whether IL-9 has an effect on IL-5R- expression. As previously
reported, human umbilical cord blood CD34+ cells cultured
in IL-3 and IL-5 developed into eosinophils by day 14, as determined by
the presence of eosinophilic granules using the
May-Grünwald-Giemsa stain (mean, 84.5%; range, 75%-88%; n = 10 independent cultures). This showed a switch to the
mRNA expression for the transmembrane isoform of the IL-5R-
accompanied by surface expression of the IL-5R-.39
Culture of CD34+ cells in IL-9 alone lead to surface
IL-5R- expression at day 7 and day 14 (Figure
8A). The specific mean fluorescence
intensity (SMFI) for IL-5R- staining at day 7 for cells
cultured in IL-9 alone was 0.122 (SE, 0.06), and the SMFI for cells
cultured in IL-3 and IL-5 was 0.135 (SE, 0.09) (n = 5). This was
confirmed by RT-PCR, which showed mRNA for both soluble and
membrane-associated IL-5R- isoforms at days 7 and 14 of culture in
IL-9 alone (Figure 8B). IL-9 supported modest cell survival, with a
2.18-fold mean expansion in cell number by day 14 (n = 3).
IL-9 increases IL-5R- . We could not detect
surface expression of the IL-5R- chain on eosinophil-differentiated HL-60 cells before day 6. A small percentage of cells expressed the
membrane-bound IL-5R- after 6 days of culture with the vehicle control for IL-9 (SMFI, 3.4 ± 1.8). Kinetic experiments revealed a
significant and progressive increase in the surface expression of the
IL-5R- chain at 4, 6, 8, and 18 hours, which reached a maximum
expression after 18 hours of culture (SMFI, 58 ± 6.5). As suggested
for eosinophil-derived CD34+ cells,60
this showed that surface expression of the IL-5R- is a
feature of the differentiation and maturation of HL-60 cells. The
introduction of 10 ng/mL IL-9 in the culture on day 6 induced a
6.2-fold increase in the IL-5R- chain surface expression on the
eosinophil-differentiated HL-60 cell line by 6 hours (SMFI,
119 ± 13.5), which then decreased with time to reach the baseline
expression by 18 hours (Figure 9A,B).
These results suggest that IL-9 may potentiate the terminal differentiation and maturation of eosinophils by increasing the surface
expression of the IL-5R-.
This study set out to examine the expression of the IL-9R on
eosinophils and the functional implications of its stimulation with
respect to eosinophil viability. Using flow cytometry and immunohistochemistry, our results demonstrate that human peripheral blood eosinophils express the specific Although originally described as a T-cell growth factor with very narrow specificity for certain T cells, IL-9-mediated activities have been described on erythroid progenitors; B cells; mast-cell fetal thymocytes9-13; and most recently, airway epithelia.24 Recently, IL-9 has received considerable attention by virtue of the genetic studies linking the IL-9 and IL-9R gene loci to indices of airway hyper-responsiveness and asthma.15-17,27 Moreover, IL-9 has been shown to be produced by activated CD4+ T lymphocytes of the Th2-like phenotype11 and is implicated in the Ig isotype switching to favor IgE production.13 The finding that human peripheral blood eosinophils express a functional receptor for IL-9 suggests that IL-9 may influence the differentiation and activation of effector cells linked to the pathogenesis of allergic disease. In our initial experiments, the expression of the IL-9R- In this study, we provided evidence of increased survival of
human peripheral blood eosinophils stimulated with IL-9. Using different criteria to determine apoptotic cell death, we found that
IL-9 has a consistent and significant effect on human eosinophil survival compared to medium alone, both at 18 and 36 hours.
Furthermore, the percentage of necrotic eosinophils differs
significantly between IL-5 or IL-9 and medium-treated cells at 36 hours
but not at 18 hours. At 18 hours the cells are at the early stage of
the apoptotic process, in which membrane integrity is still
conserved.33 However, at 36 hours cells undergoing
apoptosis reach secondary necrosis, in which the integrity of the cell
membrane is compromised. These results not only confirm the presence
and the functional integrity of the IL-9R on human eosinophils, but
they also provide a novel mechanism to explain the continued presence
of eosinophils within inflamed tissues such as asthmatic airways. In
agreement with our findings, airway eosinophilia were observed in
transgenic mice that overexpress IL-9 selectively within the
lungs22-23 and in naive mice administered with recombinant
IL-9 intratracheally.21 These previous studies suggested a
mechanism where IL-9 could influence IL-5 activity on eosinophils. To
further address this issue, we determined whether IL-9 was able to
influence the expression of the specific Previous studies have suggested that IL-5 is the most important
cytokine for the terminal differentiation of the committed eosinophil
precursors and is a potent inducer of eosinophil
survival.39-42 These actions are mediated via a
membrane-bound IL-5R that is composed of a ligand-specific From in vitro studies little is known about the factors regulating the
expression of the IL-5R- However IL-9 alone did up-regulate the IL-5R- Taken together, our results show for the first time the presence of a
functional receptor for IL-9 on human eosinophils and suggest that IL-9
may induce airway eosinophilia both by promoting eosinophil
differentiation and inhibiting cellular apoptosis. These actions may be
attributed to its ability to up-regulate the IL-5R-
The authors would like to acknowledge Ms Elsa Schotman for technical assistance.
Supported by grant MT13273 from the Medical Research Council of Canada, Canada; the Wellcome Trust, Glaxo-Wellcome plc; and Magainin Pharmaceuticals Inc., Philadelphia, PA.
Submitted November 8, 1999; accepted May 12, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Qutayba Hamid, Meakins-Christie Laboratories, 3626 St Urbain Street, Montreal, Quebec, Canada H2X 2P2; e-mail: hamid{at}meakins.lan.mcgill.ca.
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Top
Abstract
Introduction
(IFN-
eosinophils were highly
purified in the fraction that flowed through the column. Contaminating
T lymphocytes were further removed by incubating the CD16 fraction for
another 15 minutes with saturating concentrations of anti-CD3 magnetic
beads. Cytocentrifugation slides of the eosinophils stained with
Wright-Giemsa showed that the purity of the isolated eosinophils was
greater than 97%.
,10 ng/mL) were used as a
positive control (*P < .05). Freshly purified peripheral
blood eosinophils stimulated for 18 hours with a graded amount of IL-9
(
) showed fewer apoptotic cells compared to medium alone (
,
P < .01 at 10 ng/mL of IL-9). (B) Data from panel A are
presented as the percentage of inhibition compared to the control
(medium alone). The assays were performed in duplicate on eosinophils
from the same donor. These results are representative of 3 independent
experiments performed under the same conditions.
