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Blood, Vol. 95 No. 5 (March 1), 2000:
pp. 1600-1607
HEMATOPOIESIS
Interleukin 5 regulates the isoform expression of its own receptor
 -subunit
Jan Tavernier,
José Van der Heyden,
Annick Verhee,
Guy Brusselle,
Xaveer Van Ostade,
Joël Vandekerckhove,
Janet North,
Sara
M. Rankin,
A. Barry Kay, and
Douglas S. Robinson
From the Flanders Interuniversity Institute for Biotechnology and
Department of Respiratory Diseases, University of Ghent, Belgium;
Allergy and Clinical Immunology, National Heart and Lung Institute and
Leukocyte Biology, BioMedical Sciences Division, Imperial College
School of Medicine, London, UK.
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Abstract |
The receptor for interleukin 5 (IL-5) consists of a
cytokine-specific  chain (IL-5R) and a signaling  chain, which
is shared with interleukin 3 (IL-3) and granulocyte-macrophage
colony-stimulating factor (GM-CSF). These 3 cytokines can act in
eosinophil development and activation in vitro, but gene deletion or
antibody blocking of IL-5 largely ablates eosinophilic responses in
models of allergic disease or helminth infection. We investigated
factors acting in differential IL-5R gene splicing to generate
either the membrane-anchored isoform (TM-IL-5R) which
associates with the common chain to allow IL-5 responsiveness, or
a secreted, antagonist variant (SOL-IL-5R). In a murine myeloid cell
line (FDC-P1), transfected with minigenes allowing expression of either
IL-5R variant, IL-5 itself, but not IL-3 or GM-CSF, stimulated a
reversible switch toward expression of TM-IL-5R. A switch from
predominantly soluble isoform to TM-IL-5R messenger RNA (mRNA)
expression was also seen during IL-5-driven eosinophil development from
human umbilical cord blood-derived CD34+ cells; this was
accompanied by surface expression of IL-5R and acquisition of
functional responses to IL-5. IL-3 and GM-CSF also supported eosinophil
development and up-regulation of TM-IL-5R mRNA in this system, but
this was preceded by expression of IL-5 mRNA and was inhibited by
monoclonal antibody to IL-5. These data suggest IL-5-specific
signaling, not shared by IL-3 and GM-CSF, leading to a switch toward
up-regulation of functional IL-5R and, furthermore, that IL-3 and
GM-CSF-driven eosinophil development is dependent on IL-5, providing an
explanation for the selective requirement of IL-5 for expansion of the
eosinophil lineage.
(Blood. 2000;95:1600-1607)
© 2000 by The American Society of Hematology.
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Introduction |
Selective accumulation of eosinophils is a feature of
allergic inflammation, as observed in atopic asthma, where cells are implicated in tissue damage contributing to bronchial
hyperresponsiveness.1,2 Interleukin 5 (IL-5) acts in
development, activation, and tissue survival of
eosinophils.3-5 Increased expression of IL-5 at sites of
allergic disease,6-8 which correlates with clinical disease activity,9,10 and data from blocking antibody or gene
deletion experiments in animal models11-13 suggest that
IL-5 is the major determinant of eosinophilic inflammation.
High-affinity receptors for human IL-5 are formed from
cytokine-specific  chain associated with a common signaling receptor chain shared with interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF).14,15 Responsiveness of cells to IL-5 appears to be controlled at the level of expression of
IL-5R.16 Differential splicing of the IL-5R gene can
generate different messenger RNA (mRNA) isoforms: a membrane-anchored
isoform (TM-IL-5), which can associate with the common chain to
form the active receptor complex, or at least 2 secreted variants
(SOL-IL-5R), which lack the membrane anchor and show receptor
antagonist activity.17 We hypothesize that control of
this splicing event will determine IL-5 receptor expression, IL-5
responsiveness, and thus eosinophil expansion and activation.
We have previously examined expression of IL-5R isoforms in
bronchial mucosal biopsies from asthma subjects and found increased relative expression of mRNA for TM-IL-5R and decreased SOL-IL-5R in those subjects with more active disease.18 Thus,
increased IL-5 responsiveness of eosinophils might result in more
pronounced inflammation and airway responsiveness. In addition, we have
recently detected TM-IL-5R expression by CD34+ cells in
the airway in asthmatic subjects.19 Together with a report
of increased numbers of bone marrow
CD34+/IL-5R+ cells after allergen inhalation
challenge of asthmatic subjects,20 this raises the
possibility that IL-5 could act early in eosinophil development from progenitors.
Here, minigene constructs and human primary CD34+ cells
were used to examine cytokine regulation of IL-5R isoform
expression. Our data suggest that IL-5, but not IL-3 or GM-CSF, can
switch toward expression of the active TM-IL-5R and that eosinophil development, even that driven by IL-3 or GM-CSF, may be, at least in
part, IL-5 dependent.
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Materials and methods |
Materials
Vectors pSV-SPORT (Life Technologies, Rockville, MD) and
pBluescriptII (Stratagene, La Jolla, CA) were used to construct
minigenes. Restriction enzymes HindIII, BglII, Xho1, Sac1, EcoR1, Not1,
and BamH1 were from Gibco BRL. Dulbecco's modified Eagle's medium (DMEM) and RPMI medium were from Gibco BRL, DEAE-dextran from Pharmacia, and chloroquine, puromycin, TPA, and PKH2-GL were from Sigma
Chemicals (St Louis, MO). Poly-A+ RNA was extracted using
the FastTrack system (Invitrogen Inc, San Diego, CA), and total RNA was
extracted for reverse transcriptase-polymerase chain reaction (RT-PCR)
by lysis in 4 mol/L guanidine thiocyanate, 25 mM sodium citrate, 0.5%
n-lauryl sulfate, and 100 mM mercaptoethanol (all from Sigma). Cell
selection and IL-5R isoform switching experiments were performed
using rmIL-3 (Nippon Roche, Kamakura, Japan), rhIL-5 (purified using a
5A5 immunoaffinity column as described previously22),
rmGM-CSF (Biogen SA, Geneva, Switzerland), or rmIL-4 (conditioned
medium from transfected CHO cells, a gift from Dr W. Muller). In
certain experiments, conditioned medium from phorbol myristate acetate
(PMA)-stimulated EL-4 cells was used as the source of cytokines. In all
cases, saturating concentrations supporting growth of FDC-P1 cells were used.
Human umbilical cord blood was collected into McCoy's 5A medium
(Sigma), with 15% fetal calf serum (FCS; Sigma), and antibiotic, antimycotic (penicillin, streptomycin, and amphotericin mix; Life Technologies, Paisley, Scotland) and 50µM -mercaptoethanol (Life Technologies), and cultured in Iscove's modified Dulbecco's medium (Life Technologies), in 12-well plates (Nunc, Roskilde, Denmark). Histopaque was from Sigma, and MACS-CD34+ immunomagnetic
columns from Miltenyi Biotech, (Bisley, UK). Monoclonal antibodies were
anti-CD34 (HPCA2, Becton Dickinson, Oxford, UK), antimajor basic
protein (BMK 13, Pharmacia Upjohn, Milton Keynes, UK), anti-CD9
(Serotec, Oxford, UK), anti-hIL-3R,6H6 anti-hGM-CSF
8G6, and antihuman common chain (IC1) (all kind gifts
from Prof. Angel Lopez, Hanson Institute, Adelaide, Australia) and
anti-hIL-5R (16, Van der Heyden et al, unpublished observations).
For CD34+ cultures rhIL-3 (Genzyme Corporation, West
Malling, UK), rhIL-5 (Pharmingen), or rhGM-CSF (Genzyme) was used.
Chromotrope 2R and May Grunwald Giemsa stains, used to identify
eosinophils in cultures, were from BDH, Lutterworth, UK.
Generation of minigenes
pSV-SPORT was used as vector frame for the minigene constructions. A
Xho1-Sac1 cDNA fragment from pCDM8-hIL-5RSOL14 was combined with a Sac1-EcoR1 fragment containing exon 11 from
 ghIL5R-623 in pBluescript II. Likewise, a
HindIII-BglII fragment containing exon 12 from
ghIL5R-1123 was combined with a BglII-Xba1 cDNA segment from pCDM8-hIL-5RTM.17 pSV-MINI-1 was assembled
by combining Xho1-Not1 (BamH1 linkered) and HindIII (BamH1
linkered)-Not1 from the previous constructs. pSV-MINI-2 contains an
additional SV40 polyadenylation site at the BamH1 fusion point. The
minigene constructs are shown in Figure 1C.
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| Fig 1.
Structure of the hIL-5R minigene and PCR primer
design.
(A) hIL-5R gene structure. The alternative splicing choice leading
to the transcripts encoding the secreted or membrane-anchored isoforms
is indicated by dashed lines. Exons are shown as boxes and are numbered
on top, with exon 11 corresponding to the "soluble"-specific exon
and exon 12 encoding the transmembrane region. Polyadenylation sites
are shown below. (B) The 2 major transcripts from the hIL-5R gene.
SOL encodes the major secreted variant; TM encodes the
membrane-associated receptor.8 Boxes are translated
regions, with protein domains shown: S indicates signal peptide; EC,
extracellular domain; M, membrane anchor; C, cytoplasmic tail. (C) The
structure of the minigenes. Boxes are translated regions, either from
cDNA segments or from exons. (A)n is the polyadenylation site present
in the "soluble"-specific exon; SV(A)n comes from SV40 and is
artificially added. The position of the extra SV(A)n in pSV-MINI-2 is
shown by a dashed line. The arrow represents the SV40 early promoter.
On top, arrows indicate the positions of the primers used in the
(competitive) RT-PCR experiments. Sizes of possible reaction products
are indicated. Throughout the figure, black, gray, and hatched boxes
correspond to exons, protein domains, or coding regions specifying the
signal peptide, secreted isoform, or membrane-anchored isoform,
respectively.
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Analysis of IL-5R isoform expression and switching
Cos-1 cells maintained in DMEM with 10% FCS were transfected
following established procedures using DEAE-dextran and chloroquine. Cells were harvested and mRNA was prepared approximately 72 hours after
transfection. FDC-P1 cells were grown in RPMI with 10% FCS and 10%
conditioned medium from TPA-stimulated EL4 cells (as a source of murine
IL-3), and were transfected by electroporation using settings at 300 V
and 1500 µF (Easyject plus electroporator, EquiBio, Kent, UK). A
stable transfectant FDC-P1-MINI-1.1 was obtained by
cotransfection with a vector conferring puromycin resistance and
selection in 1µg/mL puromycin. PolyA+ RNA was prepared using the
FastTrack procedure (Invitrogen). Total RNA samples were prepared from
isothiocyanate lysed cells essentially as described.24
Expression of mRNA for either soluble or membrane-associated IL-5R
isoforms was detected by Northern analysis or RT-PCR. For Northern
blots, Zeta-probe GT membranes (Biorad, Hercules, CA), and probes
corresponding to the extracellular domains, thus hybridizing with both
mRNA isoforms, or corresponding to the cytosolic domain only, were used
as specified, and products were identified on size criteria (see Figure
1). Competitive RT-PCR was optimized allowing rapid estimation of the
ratios of expression of both hIL-5R isoforms. RT-PCR was performed
using a cDNA synthesis kit with RnaseH MMLV Reverse
Transcriptase according to the supplier's instructions, Taq polymerase
was from Gibco-BRL. A forward primer was designed on the extracellular
part of the receptor at position 1033 to 105617 and 2 reverse primers, specific for the transcripts encoding either the
secreted (position 1279-1298, 5'-TCAGATACGGTGTGGGGCAG)22 or membrane-anchored isoform (position 1539-1561, 5'-TGAGGCACTGAGGCATGTGTGAG)22 (referred to as
SOL-transcripts and TM-transcripts, respectively). Predicted PCR
reaction products are 245bp and 527bp for the SOL- and TM-transcripts,
respectively. Pilot experiments, using various ratios of plasmids
containing the cloned cDNAs for either isoform, indicated an
approximately 5-fold bias toward amplification of the SOL-transcript
(data not shown). A likely explanation for such bias is the different
sizes of the amplified reaction products. In cultured human umbilical
cord blood-derived CD34+ cells, IL-5 mRNA expression was
also analyzed by RT-PCR (forward: 5'-CAAACGCAGAACGTTTCAAGA;
reverse: 5'-CAGGTACCCCCTTGCACAGTT). In certain experiments,
fainter bands migrating close to the predicted bands could be observed,
but these were found to be artefacts by cloning and sequencing.
Surface expression of IL-5R protein
Scatchard analysis was used to examine transfected cell lines for
high-affinity IL-5 binding, as described.21,25 Membrane expression of IL-5R on developing eosinophils was analyzed by flow
cytometry using the monoclonal antibody 16.
Cell labeling and analysis of clonal expansion in selection of
IL-5R isoform expression
To determine whether IL-5R isoform switching represented
selective outgrowth of a subpopulation from the cell line, or a switch
of the whole population, a number of techniques were used. To avoid
rhIL-5 exerting selection pressure in cell expansion, FDC-P1 cells were
grown in rmIL-4 together with rhIL-5, or were cotransfected with the
pSVZeo-mIL-5R vector, which directs the expression of the murine
IL-5R subunit. Here, a cDNA encoding the mIL-5R chain is
expressed under control of an SV40 early promoter. These cells were
then cultured in murine IL-5.
To measure the plating efficiency of cells growing in either
conditioned medium or rhIL-5, FDC-P1-MINI-1.1 cells (FDC-P1 cells transfected with the IL-5R minigene) were plated in microtiter plates at 1 or 3 cells per well in medium containing either
EL4 conditioned medium alone or in medium containing IL-5. Colonies were counted at day 7 (EL4 CM) or day 12 (hIL-5).
To label cells, the lipophilic chromophore PKH2-GL was purchased from
Sigma and used according to the manufacturer's instructions and
reference 26. Briefly, 107 FDC-P1-MINI 1.1 cells were
collected in 1 mL and were incubated for 7 minutes at room temperature
with 4 µM PKH2-GL. Medium with 10% FCS was added and cells were
separated from free label by centrifugation through a serum cushion.
These cells were then cultured in conditioned medium or rhIL-5 as above
and analyzed at 14 days by flow cytometry to examine distribution of
the fluorescent dye among daughter cells.
Umbilical cord blood CD34+ cell culture
Human umbilical cord blood was collected from the Maternity Unit,
Chelsea and Westminster Hospital (with approval of the local Ethics
Committee). Mononuclear cells were isolated by centrifugation over
Histopaque (Sigma) followed by red cell lysis in sterile water and
adherence at 1 to 2 hours at 37°C. CD34+ cells were
isolated from nonadherent cells using MACS-CD34+
immunomagnetic columns (Miltenyi Biotech), and checked for purity by
staining with anti-CD34 monoclonal antibody HPCA2 (Becton Dickinson). For CD34+ cultures rhIL-3, rhIL-5, or rhGM-CSF was added at
1 ng/mL and cells were grown in Iscove's modified Dulbecco's medium,
with 20% FCS, 1% antibiotic/antimycotics, and 50 µM mercaptoethanol in 12-well plates. Cells were counted and fed with fresh medium at days
7 and 14 of culture.
Eosinophil phenotype was identified by chromotrope 2R and May Grunwald
Giemsa staining, and by immunostaining with antibodies directed against
eosinophil major basic protein (BMK13). Positive cells were enumerated
and expressed as a percentage after counting at least 200 total cells.
RNA was extracted from cells at days 0, 3, 5, 7, and 14 of culture as
above, and flow cytometry was performed at days 0, 3, 5, 7, and 14 for
cytokine receptor expression and phenotypic markers, and cells were
used for chemokinesis and shape change assays at days 0, 4, 7, and 14 of culture. In some experiments, to determine IL-5 dependence of
eosinophil development in IL-3 and GM-CSF, the blocking antibody to
IL-5, 5A5,22 was added to cultures at a concentration of 10 µg/mL.
Functional responses to IL-5
For chemokinesis experiments Transwell filters (3 µm pore size)
were purchased from Millipore (Watford, UK), and the rhIL-5 used was a
kind gift of Dr Tim Wells, Serono, Geneva, Switzerland. Umbilical cord
blood CD34+ cells cultured for various periods in cytokines
were added to the top well, then exposed to medium alone or IL-5 at 0.1 nM or 1 nM added for 1 hour to both chambers. Cells migrating to the bottom chamber were counted using a flow cytometer (FACScan, Becton Dickinson, San Jose, CA), with relative cell counts obtained by acquiring events for 60 seconds.27 Chemokinetic index was
expressed as the number of cells in the bottom well at 1 hour with
stimulus divided by that with medium alone.
Polarization in response to medium alone or 0.1 nM and 1 nM IL-5 was
assessed by change in forward scatter (FSC) signal on flow
cytometry.28,29 Mature peripheral blood eosinophils showed a change of 100 to 150 units to a similar stimulus (data not shown).
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Results |
Minigene constructs allow differential expression of membrane
associated (TM-IL-5R) or soluble (SOL-IL-5R) IL-5R mRNA
isoforms
The signaling competent transmembrane or the soluble
antagonist isoforms of IL-5R are generated by differential gene
switching to exclude exon 11 and skip to include exons 12, 13, and 14 (TM-IL-5R) or to include and terminate at exon 11 (SOL-IL-5R)14,17 (Figure 1A and B). We constructed
minigenes combining cDNA and genomic DNA segments of these IL-5R
isoforms to allow examination of factors regulating relative expression
of these 2 isoforms (Figure 1C). Transient transfection of Cos-1 cells
led to expression of the membrane-associated IL-5R only
(TM-transcript, Figure 2A). In contrast, in
FDC-P1-MINI-1.1 cells, a stable subclone derived from the murine
promyelocytic FDC-P1 cell line, predominant expression of the secreted
variant (SOL-transcript, Figure 2B and C, CM lanes) was seen, and the
TM-transcript could only be detected by RT-PCR (data not shown).
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| Fig 2.
Transcription patterns from hIL-5R gene and minigenes.
(A) Northern blot hybridization experiments were performed using a
probe that detects both membrane associated (TM) and soluble (SOL)
IL-5R mRNA isoforms, using RNA from Cos-1 cells transfected with the
pSV-MINI-1 or -2 constructs. In both cases, only IL-5R-TM was
detected. Construct pSV-MINI-2 includes an SV40 polyadenylation
sequence downstream of exon 11, ruling out the possibility that the
absence of the SOL mRNA is due to a deficiency in recognizing the SOL
transcript-specific polyadenylation signal. The size and location of
the TM-specific transcript is indicated. (B and C) Competitive RT-PCR
analysis and Northern analysis of IL-5R transcripts from
FDC-P1-MINI-1.1 cells grown in EL4-conditioned medium (lanes CM, a
source of murine IL-3), or rhIL-5 (lanes IL-5) for 2 weeks, or in
rhIL-5 for 2 weeks followed by EL-4-conditioned medium for a further 7 days (lanes IL-5/CM). Representative of 3 independent experiments. (D)
Culture in rhIL-5 induces high-affinity IL-5 binding sites on
FDC-P1-MINI-1.1 cells. Human IL-5 was radiolabeled using
125I by the iodogen procedure and Scatchard plot analysis
was performed. No binding was detected to cells grown in EL-4
conditioned medium alone.
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IL-5, but not IL-3 or GM-CSF, up-regulates membrane expression of
IL-5R
To examine factors regulating relative expression of mRNA for
transmembrane and soluble IL-5R isoforms, FDC-P1-MINI-1.1cells were
cultured with cytokines for 2 weeks, then examined by RT-PCR and
Northern analysis. As shown in Figure 2A and B, when the
FDC-P1-MINI-1.1 cells were cultured with recombinant human IL-5 there
was up-regulation of the TM-IL-5R mRNA, which was not seen when
cells were cultured with rmIL-3, rmGM-CSF, rmIL-4, or conditioned
medium from PMA-stimulated EL-4 cells (Figure
3). This IL-5-dependent switch to
TM-IL-5R mRNA expression was accompanied by acquisition of
high-affinity IL-5 binding at the cell surface, detected by binding of
radiolabeled IL-5 (Figure 2D). There was no detectable IL-5 binding to
FDC-P1 transfectants before culture with IL-5, nor to cells cultured in
conditioned medium alone.
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| Fig 3.
Expression of membrane-associated IL-5R.
IL-5 but not IL-3 or GM-CSF induces a switch toward membrane-associated
IL-5R isoform expression. Northern analysis using a probe that
detects the membrane-associated (TM) IL-5R mRNA isoform using RNA
extracted from FDC-P1-MINI-1.1 cells grown in conditioned medium (CM),
rmIL-3, rmIL-4, rhIL-5, or rmGM-CSF for 2 weeks.
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IL-5 switching to membrane-associated IL-5R expression in FDC-P1
transfectants is an induced event
The kinetics of switching to hIL-5R TM-transcripts in the
FDC-P1-MINI-1.1 system were slow, with a maximal effect after
approximately 2 weeks (data not shown). Although TM-IL-5R expression
by FDC-P1 transfectants at baseline was only detectable by RT-PCR, it
remained possible that IL-5 selectively expanded a minority
TM-IL-5R+ population rather than inducing a switch to
TM-IL-5R expression. To address this issue we performed a number of
experiments. First, as shown in Figure 2A and B, the IL-5-induced
switch to TM-IL-5R mRNA expression was fully reversible if cells
were cultured for a further week in conditioned medium alone, in the
absence of IL-5. Second, we labeled FDC-P1-MINI-1.1 cells with the
green fluorescent lipophilic chromophore PKH2-GL, which incorporates into the membrane with an elution half-life of 5 to 8 days. On every
cell division the fluorescent dye is equally partitioned into the
daughter cells, resulting in halving of the fluorescence as measured by
flow cytometry. We reasoned that if clonal selection and expansion
occurred, a small subpopulation of cells with decreased fluorescence
intensity would become apparent. As a control, we took advantage of the
fact that FDC-P1-MINI-1.1 cells become factor independent when grown in
the absence of any growth factor. Figure 4A illustrates that cells
grown in human IL-5 (top panel) behaved as a single cell population,
and this growth paralleled that seen in medium alone (middle panel).
The same conclusion was reached when we measured the plating efficiency
of cells growing in either conditioned medium or IL-5. Plating
efficiencies were above 70% in both cases, arguing against selection
of a minority of the starting cells by IL-5 (data not shown). Third, we
examined whether the change in hIL-5R isoform expression could also
be observed when FDC-P1-MINI-1.1 cells were grown in the presence of
murine IL-4 together with human IL-5. Because this cell line can use mIL-4 as a growth factor, no selection pressure on cell survival and
proliferation depending on the presence of functional hIL-5R subunits can exist under these circumstances. Results are shown in
Figure 4B. Panels show results from RT-PCR experiments when cells were
grown in medium containing conditioned medium, IL-5 alone, IL-4 alone,
or IL-4 and IL-5 combined as indicated. The upper part of each panel
shows the products of a competitive PCR; the lower part represents PCR
data using primers for the TM-transcript only. TM-transcripts are
detected in the presence of IL-5, but also, although at a lower level
and with slower kinetics, in the presence of IL-4 plus IL-5. Building
on this observation, we transfected the FDC-P1-MINI-1.1 cell-line with
the pSVZeo-mIL-5R vector, which directs the expression of the murine
IL-5R subunit. Here, a cDNA encoding the mIL-5R chain is
expressed under control of an SV40 early promoter. Such cells are now
capable of growing in the presence of murine IL-5. Under those
conditions, we could again observe the isoform expression switch of
hIL-5R transcripts from the minigene (Figure 4C). This again
underscores the inducible character of the switching, because IL-5 will
not exert any growth selection in these conditions.
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| Fig 4.
Induced up-regulation of the membrane-bound hIL-5R
isoform by IL-5.
(A) Flow cytometry plots of FDC-P1-MINI-1.1 cells labeled with the
lipophilic chromophore PKH2-GL. Cell growth in human IL-5 (bottom
panel) showed a single population, which parallels spontaneous
autocrine outgrowth (middle panel). If a fast proliferating
subpopulation were present, dilution of the fluorescent dye would be
much more pronounced and would approach fluorescence intensities as
shown for cells grown in IL-4-containing medium (top panel).
Representative of 2 independent experiments. (B) RT-PCR analysis of
IL-5R isoform mRNA expression by FDC-P1-MINI-1.1 cells grown in
medium containing EL4 CM, IL-5 alone, IL-4 alone, or IL-4 and IL-5
combined. The upper part of each panel shows the products of a
competitive PCR; the lower part represents PCR data using primers for
the TM-transcript only. TM-transcripts are detected in the presence of
IL-5, but also, although at a lower level, and with slower kinetics in
the presence of IL-4 plus IL-5. Representative of 2 independent
experiments. (C) FDC-P1-MINI-1.1 cells transfected with the
pSVZeo-mIL-5R vector, which directs the expression of the murine
IL-5R subunit, will grow in the presence of murine IL-5,
independent of hIL-5. Under these conditions, switch of isoform
expression of hIL-5R transcripts from the minigene was also observed
(arrow). Sampling time points are shown. Representative of 2 independent experiments.
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Membrane associated IL-5R is up-regulated during development of
human eosinophils from umbilical cord blood-derived CD34+
cells
Although the data from FDC-P1-MINI 1.1 cells suggested that IL-5
could switch to TM-IL-5R isoform expression, it was possible that
this was a function of the cell line or transfection system. To examine
control of IL-5R isoform expression during primary human eosinophil
development, cultures of umbilical cord blood-derived CD34+
cells were used. When these cells were cultured in IL-3 and IL-5, 84.5% (range 75-88%, n = 10 independent cultures) had eosinophilic granules identified by chromotrope 2R staining and immunocytochemistry for major basic protein (MBP) by day 14 of culture. Eosinophil lineage
was confirmed by flow cytometric staining for phenotypic markers,
which showed loss of CD34 by day 3 of culture, with acquisition of
surface CD9, IL-3R, GM-CSFR, and common chain increasing to
day 14 (data not shown).
The RT-PCR of cells cultured in IL-3 and IL-5 showed up-regulation of
TM-IL-5R mRNA relative to that of the soluble isoform at days 5 to 7 of culture (Figure 5A). Expression of mRNA
for the TM isoform diminished by day 14 of culture, to a pattern of predominant SOL-IL-5R mRNA expression, as seen in mature blood eosinophils. Presumably this reflects stage-specific regulation of
relative expression of mRNA for IL-5R SOL and TM isoforms rather
than simple consumption of cytokines, because cytokines were added to
cultures at days 7 and 14.
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| Fig 5.
IL-5R expression by developing primary human
eosinophils.
(A) The effect of cytokine treatment on IL-5R isoform mRNA
expression by human cord blood CD34+ cell-derived
eosinophil progenitors. RT-PCR using primers specific for the
TM-transcript, both TM and SOL transcripts, and actin mRNA was
performed on RNA extracted from cord blood CD34+ cells
cultured in IL-3+IL-5 for 0, 3, 5, 7, or 14 days. Results are
representative of 5 separate experiments. (B) Interleukin 5R surface
expression is detected after 7 days of culture of CD34+
progenitors in IL-3 and IL-5. Flow cytometry plot shows surface
staining with 16 (an IgG1 mouse monoclonal antibody to human
IL-5R chain) or isotype control IgG1 antibody. CD34+
cord blood-derived progenitors were stained at the time of isolation
(day 0) and after 7 days of culture in IL-3 and IL-5. The plot shown is
typical of 10 independent experiments. (C) CD34+ umbilical
cord blood progenitors acquire biologic responsiveness to IL-5 by day 7 of culture in IL-3 and IL-5. Chemokinetic and polarization responses of
cord blood-derived CD34+ cells cultured in IL-3 and IL-5
for 4, 7, and 14 days. Cells added to the top well were exposed to
medium alone (open bars) or IL-5 at 0.1 nM (hatched bars) or 1 nM
(black bars). Chemokinetic index is the number of cells in the bottom
well at 1 hour with stimulus divided by that with medium alone.
Polarization in response to medium alone or 0.1 nM and 1 nM IL-5 was
assessed by change in forward scatter (FSC) signal on flow cytometry
(mature peripheral blood eosinophils showed a mean change in forward
scatter of 120 units to 1 nM IL-5, n = 3, data not shown). The figure
shows a typical response at days 7 and 14. No response to IL-5 was seen
at day 3 to 4 in 7 independent experiments. Responsiveness of cells
cultured for 7 days to IL-5 was variable and may reflect different
rates of eosinophil development in different cultures; however, similar
responses to those shown were seen from cells cultured for 7 days in
IL-3 and IL-5 in 4 independent experiments.
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Up-regulation of relative expression of mRNA for the TM-IL-5R
isoform at day 7 of culture of CD34+ cells in IL-3 and IL-5
was accompanied by the acquisition of IL-5R expression at the cell
surface (Figure 5B).
To determine the functional responsiveness of these developing cells to
IL-5 we examined chemokinetic responses and cellular activation by IL-5
as judged by shape change. Chemokinetic responses to IL-5 were not seen
at day 4 of culture, but were maximal by day 7 of culture (Figure 5C).
In addition, cellular activation in response to IL-5 was maximal by
day 7 (see Figure 5C).
Eosinophil development from human CD34+ cells cultured
in IL-3 and GM-CSF is preceded by autologous expression of IL-5 mRNA
and can be inhibited by a monoclonal antibody blocking IL-5
Results from the FDC-P1 cell line suggested that the activity of
IL-5 in switching to predominant expression of the TM-IL-5R is not
shared by IL-3 and GM-CSF. However, previous reports had documented
that IL-3 and GM-CSF can support eosinophil development. To examine
this further, we cultured human umbilical cord blood-derived CD34+ cells in IL-3 alone, or IL-3+GM-CSF, without addition
of exogenous IL-5. In these cultures eosinophil development was also
observed, and this was accompanied by up-regulation of mRNA for
TM-IL-5R also seen at days 5 to 7, as seen when cells were cultured
with IL-5 (Figure 6A). To evaluate whether
endogenous IL-5 might play a role in this system, we examined IL-5 mRNA
expression by RT-PCR, and added a blocking antibody to IL-5. As shown
in Figure 6A, IL-5 mRNA was detected in cells grown in IL-3+GM-CSF or
IL-3 alone at days 3 to 5 of culture, before the switch to predominant
TM-IL-5R expression. Furthermore, addition of the blocking anti-IL-5
monoclonal antibody, 5A5, to CD34+ cells cultured in IL-3
or IL-3+GM-CSF inhibited eosinophil development (Figure 6B).
View larger version (4716K):
[in this window]
[in a new window]
| Fig 6.
Up-regulation of TM-IL-5R.
Up-regulation of TM-IL-5R mRNA expression in umbilical cord blood
CD34+ cells grown in IL-3 and GM-CSF is preceded by IL-5
mRNA expression, and anti-IL-5 inhibits eosinophil development from
cord blood-derived CD34+ cells grown in IL-3 and GM-CSF.
(A) RT-PCR for TM and SOL IL-5R mRNA and IL-5 mRNA expression by
umbilical cord blood CD34+ cells grown in IL-3 or IL-3 and
GM-CSF for 0, 3, 5, 7, and 14 days. Results are representative of 3 independent experiments. (B) A neutralizing anti-hIL-5 monoclonal
antibody (5A5, 10 µg/mL) was added to CD34+ cord blood
cells cultured at 2 × 104 cells per 100µL in
96-well plates with IL-3 and GM-CSF, as above. Three wells were set up
for each time point and cells were harvested at 3, 5, 7, and 14 days of
culture for cell counts after May Grunwald Giemsa or major basic
protein staining. Data shown are means and standard error for 3 separate experiments.
|
|
| |
Discussion |
Expression of IL-5 is increased in atopic allergic disease and
helminth parasite infections and is closely implicated in the control
of tissue eosinophilia.3,7,11 Control of the relative expression of the signaling, membrane-associated (TM) or soluble, antagonist (SOL) IL-5 receptor -chain isoforms appears to be at the
level of gene splicing, and may determine IL-5 responsiveness of both
developing and mature eosinophils.16,17 The present data
show that IL-5 itself switches to predominant expression of the
TM-IL-5R in a minigene reporter system and during primary human
eosinophil development, suggesting ligand control of receptor expression at the gene level. Furthermore, this property of IL-5 was
not shared by IL-3 and GM-CSF, which share common receptor chains,
suggesting that IL-5 can signal through cytokine-specific pathways not
dependent on the common chain.
One explanation for our findings would be specific signaling via the
IL-5R chain, not involving the chain, and thus not shared with
IL-3 and GM-CSF. IL-5 signaling has been defined via both IL-5R and
c, with activation of STAT1 and 5 via Jaks 1 and 2, the
Ras/mitogen activated protein kinase, phosphatidyl inositol
3kinase, JNK/ERK, and SHP-2 pathways.30-33 Whether
differential activation of these pathways by different receptor chains could result in cytokine-specific downstream events remains to
be established. To date, evidence for chain-specific signaling
within this cytokine subfamily is limited to selective CREB
phosphorylation and subsequent Egr-1 activation in TF-1
cells34 and selective FAK up-regulation in differentiating
myeloid cells35 by GM-CSF but not IL-3. Differential phosphorylation effects between IL-3 or GM-CSF and IL-5 have also been
described in TF-1 cells.36 Evidence for specific
differentiation signals is also suggested from work showing that
transfection of FDCP-Mix cells with either the GM-CSFR or IL-3R
leads to either macrophage differentiation or expansion of blast cells respectively (Dr T. Whetton, personal communication, 1999). IL-5 specific signaling may also be explained by direct DNA binding of IL-5,
which has been shown to possess a nuclear localization sequence and to
have the capability of direct nuclear binding, at least in in vitro
systems.37 Linear accumulation of translocated IL-5 instead
of a receptor-mediated amplification process would also explain the
slow kinetics observed in isoform switching. Alternatively, the very
low IL-5R expression levels in untreated FDC-P1-MINI-1.1 cells may
retard a full response. Signaling competence via such low expression
levels has been shown to occur for IL-5-induced differentiation
of B cells.38
The observed switch to TM-IL-5R expression by IL-5 in FDC-P1 cells
was shown to result from an induced event in the whole cell population,
rather than selective outgrowth of a minority population of cells
expressing TM-IL-5R at baseline. We have not conclusively
demonstrated whether this results from a switch at the level of gene
transcription, but detected a similar switch in immature transcripts
(data not shown), suggesting that this is indeed a switch in gene
splicing. The minigene transfection system used here resulted in
predominant SOL-IL-5R in FDC-P1 cells, in which IL-5 was shown to
induce a switch to TM-IL-5R expression, but led to constitutive
TM-IL-5R mRNA expression in Cos-1 cells. The IL-5-induced
up-regulation of TM-IL-5R during development of eosinophils from
CD34+ cells occurred at around day 7 of culture, but did
not persist. Despite continuing exposure of the cells to IL-5, by day
14 cells expressed predominant SOL-IL-5R mRNA, a pattern also
detected in mature peripheral blood eosinophils. Taken together, the
present data suggest cell- and stage-specific signaling events and
factors acting in control of IL-5R gene splicing. Two IL-5R
promoter sites have been defined,39,40 although the
transcription factors acting to control gene splicing remain to be established.
It is of note that recent data suggest that in mature peripheral blood
eosinophils predominant expression of TM-IL-5R at the mRNA level is
rapidly down-regulated by IL-5, IL-3, and GM-CSF at the level of
transcription, although the effects of longer term cultures were not
examined.41 The relative expression of IL-5R isoforms
and its regulation in mature eosinophils remains to defined. It also
needs to be mentioned that the SOL-IL-5R protein has
antagonistic properties in vitro, which may indicate a subtle balance
in the IL-5-induced control of its receptor isoform expression.
However, it remains unclear which signals are required for
production and secretion of this SOL-IL-5R isoform by eosinophils.
The data from umbilical cord blood-derived CD34+ cells
suggest that IL-5-induced up-regulation of TM-IL-5R expression is
also relevant in primary human cells, and, in particular, may play an
important role in eosinophil development. The demonstration that even
when eosinophils develop in culture with IL-3 and GM-CSF, this is
dependent on endogenously produced IL-5, may go some way to explaining
the IL-5 dependence of eosinophilia seen in vivo when compared to in
vitro culture systems. Thus, although IL-3 and GM-CSF could support
development of eosinophils from bone marrow cultures,42
anti-IL-5 monoclonal antibody treatment or gene deletion of IL-5
largely abrogated eosinophilic responses in both helminth parasitized
and inhaled allergen challenged mice.11,13,43 This is
encouraging for therapeutic approaches based on selective inhibition of
IL-5.12 Transgenic mice expressing IL-5 under the
T-cell-dependent CD2 promoter had marked eosinophilia.44 It
is of note that mice deficient in IL-5 do have bone marrow and blood
eosinophils, albeit in reduced numbers, and that the eosinophil
phenotype is similar to that seen in mice lacking both c
and IL-3, where IL-3 and GM-CSF could not substitute for IL-5 in
driving eosinophil development.43,45 This suggests that factors other than IL-3, IL-5, or GM-CSF can induce some eosinophil development, but that IL-5 is crucial to eosinophil expansion and
mobilization in allergic and parasitic disease.7,46
The current results suggest that the very low expression level of the
hIL-5R subunit is the initial rate-limiting step in isoform
switching. The slow kinetics may represent a built-in protection
mechanism against premature eosinophil expansion. Progenitor cells at
this stage may be dependent on IL-3 or GM-CSF or both for survival and
expansion, explaining the requirement for these cytokines in eosinophil
differentiation assays. Indeed, in our CD34+ culture system
eosinophil expansion was not seen in IL-5 alone (data not shown).
Proliferation of eosinophilic progenitor cells may then first require
the up-regulation of the IL-5R-subunit, which is only obtained on a
prolonged exposure to IL-5. Such a situation is obtained in persistent
helminth parasite infections in vivo or in allergic
inflammation.1,7,11 It is of note that FDCP-MIX cell lines
transfected with IL-5R can proliferate to IL-5, but do not show
increased differentiation to eosinophils.47 This suggests
that either additional signaling machinery via IL-5R or additional
factors are required for eosinophil differentiation and that IL-5 may
act as a growth and survival factor for progenitor cells developing
toward the eosinophil lineage via stochastic or programmed mechanisms.
Although the switch to TM-IL-5R was slow in transfected cell lines,
taking 2 weeks, the kinetics in the CD34+ cord blood system
were more rapid, with maximal expression by day 7. This may reflect
differences in the cells studied or culture systems, but it is of note
that increased numbers of CD34+ cells expressing
TM-IL-5R were detected in the bone marrow as early as 24 hours after
allergen inhalation challenge of asthmatics.20 In addition,
CD34+/TM-IL-5R mRNA+ cells were recently
detected in airway biopsies from atopic asthmatic subjects.19 Taken together, this suggests that switching to TM-IL-5R expression may be accelerated in vivo when compared to the
culture systems studied and that IL-5 may act with other factors in
induction of IL-5R on CD34 cells, which may be a key step in
eosinophil development. The ability of CD34+ progenitors
themselves to produce IL-5 is supported by the recent finding of IL-5
mRNA+ CD34+ cells in bone marrow of sensitized
Balb/c mice after allergen challenge,48 and suggests that
autocrine (and paracrine) production of IL-5 by CD34+ cells
may contribute to IL-5R isoform switching.
The specific role of IL-5 in switching mRNA isoforms for its own
receptor may have broader significance for many other cytokine receptor
systems. It may also represent a specific target for therapeutic
intervention in diseases such as asthma.
| |
Acknowledgments |
We wish to thank Dr Philippe Lassalle and Tania Tuypens for help in
setting up the competitive RT-PCR procedure and generating the minigene
constructions, respectively; Dr Geert Plaetinck, Professor Colin
Sanderson, and Professor Tim Williams for helpful discussions;
Professor Tony Whetton for sharing unpublished data; Professor Angel
Lopez for the gift of monoclonal antibodies; and Wim Drijvers for artwork.
| |
Footnotes |
Submitted August 23, 1999; accepted October 27, 1999.
X.V.O. and A.V. were supported by a grant from the Verkennend Europees
Onderzoek (VEO-011VO797) and the Geconcerteerde Onderzoeks Acties (GOA
96012), respectively. Part of this work was supported by the Wellcome
Trust, UK, by the Medical Research Council, UK, and by GlaxoWellcome, UK.
Reprints: Jan Tavernier, Flanders Interuniversity Institute for
Biotechnology, University of Ghent, Faculty of Medicine, Department of
Medical Protein Research, K.L. Ledeganckstraat 35, B-9000 Ghent,
Belgium; e-mail: jan.tavernier{at}rug.ac.be.
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.
| |
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Top
Abstract
Introduction