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Two Distinct Pathways of Interleukin-5 Synthesis in Allergen-Specific
Human T-Cell Clones Are Suppressed by Glucocorticoids
By Akio Mori, Osamu Kaminuma, Matsunobu Suko, Satoshi Inoue, Takeo Ohmura, Akihiko Hoshino,
Yumiko Asakura, Keiji Miyazawa, Toyokazu Yokota, Yasushi Okumura, Koji Ito, and Hirokazu Okudaira
Glucocorticoids (GC) have long been used as the most effective agents for the treatment of allergic diseases accompanied by eosinophilia such as chronic asthma and atopic
dermatitis. The development of chronic eosinophilic inflammation is dependent on interleukin-5 (IL-5), a selective
eosinophil-activating factor, produced by helper T cells. To
delineate the regulatory mechanisms of human IL-5 synthesis, we established allergen-specific CD4" T-cell clones from
asthmatic patients. GC efficiently suppressed IL-5 synthesis
of T-cell clones activated via either T-cell receptor (TCR) or
IL-2 receptor (IL-2R). Induction of IL-5 mRNA upon TCR and
IL-2R stimulation was totally inhibited by dexamethasone.
Human IL-5 promoter/enhancer-luciferase gene construct
transfected to T-cell clones was transcribed on either TCR
or IL-2R stimulation and was clearly downregulated by dexamethasone, indicating that the approximately 500-bp human
IL-5 gene segment located 5* upstream of the coding region
contains activation-inducible enhancer elements responsible for the regulation by GC. Electrophoretic mobility shift
assay analysis suggested that AP-1 and NF-kB are among
the possible targets of GC actions on TCR-stimulated T cells.
NF-AT and NF-kB were not significantly induced by IL-2 stimulation. Our results showing that GC suppressed IL-5 production by human CD4" T cells activated by two distinct
stimuli, TCR and IL-2R stimulation, underscore the efficacy
of GC in the treatment of allergic diseases via suppression
of T-cell IL-5 synthesis.
q 1997 by The American Society of Hematology.
I
inducing IL-5 production by T cells. IL-2 exerts its effects
after binding with its receptor, which is composed of three
subunits, IL-2 receptor (IL-2R) a, b, and g chain.30,31 The
IL-2R signal activates multiple signal transduction pathways,
eg, protein tyrosine kinases,32,33 PI-3 kinase,34 and protooncogenes.35-37 The signal transduced by IL-2R is reported to
be very distinct from the signal transduced by T-cell receptor
(TCR) in that (1) TCR signal induces Ca2/ influx but IL-2R
signal does not,38,39 (2) TCR signal induces PI turnover but
IL-2R signal does not,40 (3) TCR signal induces protein kinase C (PKC) activation but IL-2R signal does not,41,42 and
(4) TCR signal is sensitive to FK506 and cyclosporin, but IL2R signal is not.43-45 It was also reported that TCR-induced
cytokine production was inhibited by GC at quite low concentrations, but, in contrast, the proliferation of murine cytotoxic T cells induced by IL-2 was affected only marginally
by GC,46 suggesting that the TCR signal is mediated by quite
distinct pathways from the IL-2R signal. So it is now an
intriguing question as to whether IL-2–induced IL-5 synthesis is inhibited by GC as effectively as TCR-stimulated IL5 synthesis. We showed here for the first time that GC inhibited IL-2–induced cytokine synthesis and proliferation of
helper T cells. Our results further supported the efficacy of
GC for the treatment of atopic diseases by acting as IL-5
synthesis inhibitors.
NTERLEUKIN-5 (IL-5) is a potent selective growth factor, differentiation factor, activating factor, and chemotactic factor for human eosinophils.1-3 Several animal studies
have shown that tissue infiltration of eosinophils is dependent on IL-5 produced by T cells.4,5 Persistent eosinophilic
inflammation of the bronchial mucosa is a characteristic
pathologic feature of bronchial asthma.6-8 The number of
CD4/ T cells expressing IL-5 mRNA is increased in the
bronchial mucosa of symptomatic asthmatics,9 is correlated
with the number of activated eosinophils, and is further increased upon allergen inhalation challenge.10,11 L-5 is the
predominant eosinophil-active cytokine present in the broncho-alveolar lavage fluids obtained during allergen-induced
late-phase reaction.12 Oral prednisolone therapy reduced serum IL-5 concentration13 and the number of IL-5 mRNAexpressing cells in broncho-alveolar lavage fluid,14 in parallel
with clinical improvement. Control of T-cell IL-5 production
seems to be an effective strategy for the management of
allergic diseases characterized by eosinophilic inflammation.
Glucocorticoids (GC) have long been considered the most
effective treatment for eosinophilic disorders, including
chronic asthma.15 The efficacy of GC is ascribed to their
multiple pharmacologic actions, one of which is the suppression of inflammatory cytokine production.16,17 We and others
reported that IL-5 synthesis by peripheral blood mononuclear
cells was inhibited by GC in vitro.18,19 Because of the mixed
cell preparations used in those studies, the question of
whether GC act directly on T cells or not still remains to be
answered. It is well known that GC induce non-T cells to
produce anti-inflammatory mediators, eg, lipocortins, which
exhibit immunosuppressive effects on T cells.20-24 To address
this issue, we used allergen-specific human T-cell clones in
the present study and showed that GC directly act on T cells
to suppress IL-5 synthesis at the level of gene transcription.
We have recently reported that human helper T-cell clones
produce IL-5 in response to IL-2 as well as to antigenic
stimulation.25 Several investigators have reported that the
production of not only Th2 cytokines but also IL-2 (a Th1
cytokine) by infiltrating T cells is enhanced at the sites of
allergic inflammation.26-29 Locally produced IL-2 may facilitate the development of eosinophilic inflammation through
From the Departments of Medicine and Physical Therapy and
Geriatrics, Faculty of Medicine, University of Tokyo, Bunkyo-ku,
Tokyo, Japan; and the Bioscience Research & Development Laboratory, Asahi Breweries, Ltd, Ohta-ku, Tokyo, Japan.
Submitted September 9, 1996; accepted December 2, 1996.
Address reprint requests to Hirokazu Okudaira, MD, Phd, Associate Professor, Department of Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
q 1997 by The American Society of Hematology.
0006-4971/97/8908-0023$3.00/0
Blood, Vol 89, No 8 (April 15), 1997: pp 2891-2900
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MATERIALS AND METHODS
Reagents. Hydrocortisone, prednisolone, and dexamethasone
were purchased from Sigma Chemicals (St Louis, MO) and dissolved
in ethanol at a concentration of 2 mmol/L as stock solutions. AntiCD3 (Leu 4), anti-CD4 (Leu 2), and anti-CD8 (Leu 3) monoclonal
antibodies (MoAbs) were purchased from Becton Dickinson (San
Jose, CA). Anti-CD3 MoAb (OKT3) was from Ortho (Raritan, NJ).
Phorbol 12-myristate 13-acetate was from Sigma and ionomycin was
from Calbiochem (La Jolla, CA). Recombinant human IL-2 (rIL-2)
was kindly provided by Shionogi Pharmaceutical Co (Osaka, Japan).
The specific activity of the recombinant material for IL-2 was approximately 1 U/ng protein. Recombinant Der f II (rDer f II) was
directly expressed in Escherichia coli using the Der f II cDNA clone
and prepared as previously described.47 AIM-V medium (GIBCO
BRL, Gaitherburg, MD) was used for T-cell cultures. RPMI 1640
medium (GIBCO) supplemented with 10% fetal bovine serum, 100
mg/mL streptomycin, and 100 U/mL penicillin was used for the
transient transfection experiments.
Establishment of antigen-specific T-cell clones. Der f II-specific
human T-cell clones were derived from peripheral blood mononuclear cells (PBMC) of atopic asthmatic patients by antigen stimulation followed by the limiting dilution method, as described previously.48
Briefly, PBMC (2 1 106/mL) were cultured with rDer f II protein
(1 mg/mL) for 10 days in 24-well culture plates and nonadherent
cells were recovered. Then,102 to 104 live cells were cultured in 96well round-bottom culture plates (Nunc, Roskilde, Denmark) with
antigen and 2,500-rad–irradiated autologous PBMC (5 1 104 cells).
Fresh medium containing 10 U/mL rIL-2 was added once weekly.
When less than 1 of 10 wells contained proliferating cells, the resulting cell lines were considered to have originated from a single
clone. To ensure their clonality, these T-cell clones were further
subcloned by limiting dilution using irradiated autologous PBMC
and antigen. After 10 to 14 days, expanding cultures were transferred
to 24-well culture plates (Becton Dickinson). T-cell clones were
maintained by antigenic stimulation with irradiated autologous
PBMC (2 1 106/well) and rDer f II protein every 2 to 3 weeks.
Stimulation of T-cell clones. T cells were harvested at least 10
days after the last antigenic stimulation, layered onto Ficoll-Paque,
and centrifuged. The interface was recovered, washed twice, and
resuspended in fresh medium. The resulting preparation usually consisted of more than 98% CD3/ cells, as determined by flow cytometry. Cells (105/well) were cultured in triplicate with various stimuli
in 96-well round-bottom culture plates for 24 hours, and then supernatants were harvested and kept frozen at 0707C until use. In some
cultures, wells were preincubated with 10 mg/mL anti-CD3 MoAb
(OKT3) in 0.05 mol/L carbonate-bicarbonate buffer (pH 9.6) at 47C
overnight. For proliferation analysis, cells were cultured for 72
hours. 3H-Thymidine (0.5 mCi/well) was pulsed for the last 16 hours.
To obtain cytoplasmic RNA, T cells (4 1 106/well) were cultured
in 24-well culture plates with various stimulants for the designated
time periods.
Quantitation of cytokines. IL-5 was measured by a sandwich
enzyme-linked immunosorbent assay (ELISA) using monoclonal antihuman IL-5 (D138) as the capture antibody and biotinylated purified rabbit antihuman IL-5 as the second antibody, as described
previously.49 The linear portion of the standard curve was between
3.9 and 500 pg/mL. IL-2 and IL-4 were measured by specific ELISA
(Quantikine ELISA kits; R&D Systems, Minneapolis, MN) according to the manufacturer’s directions.
Reverse transcription-polymerase chain reaction (RT-PCR) assay.
IL-5 gene expression was analyzed using the RT-PCR method as
reported previously.19 Briefly, RNA was extracted from the pelleted
cells essentially following the one-step acid guanidinium isothiocya-
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nate/phenol-chloroform extraction method of Chomczynski and Sacchi50 using Isogene (Nippongene, Tokyo, Japan). cDNA was synthesized from 1 mg of cytoplasmic RNA using random primers and
murine Moloney leukemia virus reverse transcriptase (GIBCO BRL).
PCRs were performed using the following primers (Clontech, Palo
Alto, CA): IL-5: sense strand primer, 5*-GCTTCTGCATTTGAGTTTGCTAGCT-3*, and antisense strand primer, 5*-TGGCCGTCAATGTATTTCTTTATTAAG-3*; b-actin: sense strand primer, 5*ATGGATGATGATATCGCCGCG-3*, and antisense strand primer,
5*-CTAGAAGCATTTGCGGTGGACGATGGGGGCC-3*. To 50
mL (final volume) of amplification solution (50 mmol/L KCl, 10
mmol/L Tris-HCl, pH 8.3, 2 mmol/L MgCl2 , 0.01% [wt/vol] gelatin,
0.2 mmol/L of each deoxynucleotide triphosphate; Toyobo, Osaka,
Japan), 2 mL of cDNA (corresponding to about 250 ng of starting
RNA material), 0.4 mmol/L of each primer, and 2 U of Taq DNA
polymerase (AmpliTaq; Perkin Elmer Cetus, Norwalk, CT) were
added. The mixture was heated at 957C for 5 minutes, followed by
25 cycles, each consisting of incubations for 1 minute at 957C, 2
minutes at 607C, and 3 minutes at 727C. The PCR products were
analyzed by agarose gel electrophoresis in the presence of ethidium
bromide.
Plasmid constructs. Human genomic DNA isolated from HeLa
cells was used as a template. IL-5 promoter/enhancer gene (0511
to /4 relative to the transcription initiation site) was PCR amplified
using a sense strand primer (5*-ATACTCGAGGGATCCTAATCAAGACCC-3*) and an antisense strand primer (5*-TGCAAGCTTTGCATAGTACAAGACTGC-3*) according to the DNA sequence
reported by Tanabe et al.51 This 515-bp PCR product was inserted
into the Xho I-HindIII site of the pGL2 basic vector (Promega,
Madison, WI) to construct pIL-5(-511)Luc. Identity of the promoter/
enhancer sequence with the originally reported DNA sequence was
confirmed by chain termination sequencing using Sequenase 2.0
(Strategene, La Jolla, CA). pCMV-b-gal control vector (Riken DNA
Bank, Tokyo, Japan) was used as a transfection control.
Transient transfection, luciferase assay, and b-galactosidase
assay. T-cell clones were transfected by electroporation using a
Gene Pulser (Bio-Rad, Richmond, CA). Briefly, 2.5 1 107 T-cell
clones in 500 mL RPMI 1640 medium supplemented with 10% fetal
bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin, and
10 U/mL rIL-2 in a 0.4-cm electroporation cuvette were incubated
with 50 mg of pIL-5(-511)Luc and 10 mg of pCMV-b-gal and electroporated at 270 V, 960 mF with a mean time constant of 19 milliseconds. After transfected cells were cultured in a fresh medium with
or without stimulation for 24 hours, protein extracts were prepared
and luciferase activity was assayed using the Luciferase Assay System (Promega). Relative luciferase unit (RLU) was calculated by
the following formula: RLU Å Luciferase Activity in Cell Lysate/
Protein Content (in milligrams per milliliter). b-Galactosidase activity was assayed using chlorophenol red-b-D-galactopyranoside
(CPRG) as the substrate. First, 150 mL of cell lysates diluted with
Z buffer (0.1 mol/L Na2HPO4 -NaH2PO4 , pH 7.5, 10 mmol/L KCl,
1 mmol/L MgSO4 , 50 mmol/L 2-mercaptoethanol) and 30 mL of 15
mmol/L CPRG were incubated at 377C for 30 minutes. The reaction
was then stopped by the addition of 75 mL of 1 mol/L Na2CO3 and
OD574 was measured.
Preparation of nuclear extracts. Crude nuclear and cytoplasmic
extracts were prepared from unstimulated or stimulated cells as described by Schreiber et al,52 with modifications. Cells were washed
in ice-cold phosphate-buffered saline, suspended at 5 1 107cells/mL
in ice-cold buffer A (10 mmol/L HEPES-KOH, pH 7.9, 10 mmol/
L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L dithiothreitol [DTT], and 0.5 mmol/L phenylmethyl sulfonyl fluoride [PMSF])
and kept on ice for 15 minutes. Then 1/16 vol of 10% Nonidet
P-40 was added and the mixture was vigorously vortexed. After
centrifugation at 12,000 rpm for 30 seconds at 47C, the cytoplasmic
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GC EFFECT ON T-CELL IL-5 GENE TRANSCRIPTION
supernatant was retained on ice and the nuclear pellet was washed
with the same buffer (buffer A containing NP-40). The pellet was
next incubated with 3 vol of ice-cold buffer C (10 mmol/L HEPESKOH, pH 7.9, 400 mmol/L NaCl, 10 mmol/L EDTA, 1 mmol/L
EGTA, 1 mmol/L DTT, 1 mmol/L PMSF) at 5 1 108 nuclei/mL
for 15 minutes and centrifuged at 15,000 rpm for 15 minutes at
47C. The nuclear and cytoplasmic supernatants were kept frozen in
aliquots at 0707C. For some experiments, they were dialyzed against
500 vol of dialysis buffer (10 mmol/L HEPES-KOH, pH 7.9, 50
mmol/L NaCl, 50% glycerol [vol/vol], 1 mmol/L DTT, and 1 mmol/
L MgCl2 ) for 12 hours at 47C.
Electrophoretic mobility shift assay (EMSA). The oligonucleotides used for AP-1, NF-kB and Oct-1 mobility shift assays were
purchased from Promega: AP-1 (5*-CGCTTGATGAGTCAGCCGGAA-3*),53 NF-kB (5*-AGTTGAGGGGACTTTCCCAGGC-3*),54
and Oct-1 (5*-TGTCGAATGCAAATCACTA GAA-3*).55 NF-AT
oligonucleotides were from Sawady Technology (Tokyo, Japan);
NF-AT (5*-GGAGGAAAAACTGTTTCATACAGAAGGCGT-3*)
from distal NF-AT site of human IL-2 gene.56 Pairs of synthetic
high-performance liquid chromatography-purified oligonucleotides
containing complementary sequences were annealed by boiling equimolar concentrations of each strand for 10 minutes and allowing the
mixture to slowly cool in a water bath to room temperature. Then,
3.5 pmol of annealed oligonucleotides was incubated in a 10-mL
reaction mixture containing 70 mmol/L Tris-HCl, pH 7.6, 10 mmol/
L MgCl2 , 5 mmol/L DTT, and 10 mCi of [g-32P]ATP (3,000 Ci/
mmol; Amersham, Arlington Heights, IL) with 10 U of T4 polynucleotide kinase (Takara, Otsu, Japan) for 30 minutes at 377C. The
reaction was stopped by adding 1 mL of 0.5 mol/L EDTA and 89
mL TE buffer (10 mmol/L Tris-HCl, pH 8.0, 1 mmol/L EDTA).
Gel-shift analysis was performed according to the manufacturer’s
protocol (Gel Shift Assay Systems; Promega) with slight modifications. Thirty-five femtomoles of 32P end-labeled oligonucleotides
was incubated in a 10-mL reaction mixture containing 10 mmol/L
Tris-HCl, pH 7.5, 0.5 mmol/L EDTA, 0.5 mmol/L DTT, 4% glycerol, 50 mmol/L NaCl, 1 mmol/L MgCl2 , and 0.5 mg poly (dIdC)rpoly (dI-dC) with 4 mg of nuclear extracts for 30 minutes at
room temperature. In competition experiments, a 20 to 200 molar
excess of unlabeled oligonucleotides, relative to the probe, was
added to the binding reaction. After incubation, bromophenol blue
and xylene cyanol were added to 0.02% and the resulting complexes
were resolved on 4% polyacrylamide gel (acrylamide:bisacrylamide,
30:1 wt/wt) by electrophoresis at 100 V in 0.51 TBE buffer (11
TBE: 89 mmol/L Tris-HCl, pH 8.0, 89 mmol/L boric acid, and 2
mmol/L EDTA) at room temperature. The gel was subsequently
dried and exposed to RX film (Fuji Photo Film, Tokyo, Japan) at
0707C.
Protein assay. Protein concentrations were determined using bicinchoninic acid protein assay reagent (Pierce, Rockford, IL) according to the manufacturer’s directions.
Statistical analysis. Statistical analysis was performed using the
Student’s t-test. A value of P õ .05 was considered to be statistically
significant. Responses are presented as the mean { standard error
of the mean (SEM).
RESULTS
GC suppressed cytokine production of human T-cell
clones stimulated via T-cell receptor complex. The first
experiment was performed to test whether IL-5 production
of human helper T-cell clones is suppressed by GC. T-cell
clones were used for experiments at least 10 days after the
last antigenic stimulation. As described in the Materials and
Methods, T cells obtained from the interface of Ficoll-Paque
density gradient consisted of more than 98% pure CD3/
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CD4/ cells. They were washed three times, resuspended
in fresh medium, and stimulated via T-cell receptor using
immobilized anti-CD3 MoAb. Culture supernatants were
harvested after 24 hours. As shown in Fig 1, all of the three
agents, hydrocortisone (A), prednisolone (B), and dexamethasone (C), suppressed the production of IL-5 by activated
T-cell clones in a dose-dependent manner. The concentrations of each agent required for half maximal inhibition
(IC50 ) were Ç100 nmol/L, Ç20 nmol/L, and Ç1 nmol/L,
respectively. All three agents did not alter the viability of
cells after 24 hours of incubation at the concentrations used
in these experiments, thereby excluding nonspecific toxicity
of the agents (data not shown). The results clearly indicate
that GC directly act on T cells to suppress IL-5 synthesis.
Dexamethasone suppressed IL-5 production induced by
IL-2. We have recently reported that T-cell clones that produce IL-5 upon antigenic stimulation also produce IL-5 in
response to IL-2 and that IL-5 production of peripheral T
cells is dependent on IL-2.25 PI turnover, PKC activation,
and Ca2/ influx were not observed in T cells stimulated with
IL-2,38-42 although these early biochemical events are the
hallmarks of TCR-mediated T-cell activation.57 TCR-induced proliferation of T cells is inhibited by FK506, but IL2–induced proliferation is not.43-45 Therefore, the effects of
GC on IL-2–induced IL-5 production were next examined.
T-cell clones HK5, YA5, HK2, and YA8 were stimulated
by recombinant human IL-2 (100 U/mL) for 24 hours in the
presence or absence of dexamethasone. As shown in Fig 2,
dexamethasone clearly suppressed IL-5 production (A) and
proliferation (B) of these T-cell clones in a dose-dependent
manner, indicating that IL-2 signals leading to cytokine production and proliferation of helper T cells are both sensitive
to GC. Essentially the same results were obtained in five
other T-cell clones, indicating that this observation was not
restricted to these particular clones (data not shown).
IL-2–induced proliferation of CTLL-2 cells is not affected
by dexamethasone. Because dexamethasone-resistant proliferation of murine CD8/ T cells stimulated with IL-2 was
reported by previous investigators,46 we confirmed the inability of dexamethasone to suppress IL-2–induced proliferation
employing a murine cytotoxic T-cell line, CTLL-2, which
is a well-characterized IL-2–dependent cell line commonly
used for the bioassay of human IL-2, murine IL-2, and murine IL-4.58 As shown in Fig 3, CTLL-2 cells proliferated in
response to exogenous rIL-2 in a dose-dependent manner.
Dexamethasone at concentrations of 100 nmol/L and 1 mmol/
L did not significantly affect the proliferative response of
CTLL-2 cells, suggesting that the IL-2R signal leading to
the proliferation of CD8/ (cytotoxic) T cells is transduced
by GC-resistant signaling pathway(s) that is distinct from
the IL-2R signal of CD4/ T cells.
Dexamethasone suppressed IL-5 gene expressions. We
next examined the effects of dexamethasone on IL-5 gene
expression using RT-PCR analysis. T-cell clone (YA5) was
stimulated with immobilized anti-CD3 MoAb or rIL-2 (100
U/mL) for the designated time periods and total cytoplasmic
RNA was extracted. RNA was then reverse transcribed and
amplified by PCR using primer sets specific for human IL5 and b-actin. The number of cycles was titrated before the
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MORI ET AL
Fig 1. TCR-induced IL-5 production was suppressed by glucocorticoids. A total of 105 T-cell clones ([s] HK5; [n] YA5; [h] HK2) were
cultured in 96-well round-bottom culture plates pretreated with
OKT3 MoAb (10 mg/mL). Designated concentrations of hydrocortisone (A), prednisolone (B), and dexamethasone (C) were included
from the start of some cultures. Culture supernatants were harvested
after 24 hours and assayed for IL-5 by a specific ELISA. Data are
expressed as the mean of triplicate cultures Ô SEM. IL-5 production
in the unstimulated cultures was always below the detection limit
of the ELISA system (Ú1 pg/mL).
experiment and reactions were repeated for 25 cycles, because the PCR products were amplified exponentially between 23 and 30 cycles. IL-5 mRNA was induced in T-cell
clones stimulated with either immobilized anti-CD3 antibody or rIL-2 and reached maximum after 6 to 9 hours, as
reported previously.25,48 A representative result is shown in
Fig 4. IL-5 mRNA induced by either stimulus was totally
downregulated by dexamethasone. Essentially the same results were obtained when PCR was performed for 23 and
27 cycles, because the PCR reaction was linear between 23
and 30 cycles (data not shown), indicating that the amounts
of PCR products at 25 cycles well reflect the relative quantity
of mRNA in the original RNA preparations.
Dexamethasone suppressed the transcriptional activity
mediated by Ç500-bp human IL-5 promoter/enhancer. It
has been shown that dexamethasone inhibits the induction
of IL-2 gene transcription mainly by interfering with AP-1
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activity.59,60 We used a transient transfection system to analyze whether dexamethasone suppresses human IL-5 transcriptional activity. The 511-bp human IL-5 promoter/enhancer-luciferase gene construct, pIL-5(-511)Luc, was
transiently transfected to T-cell clones by electroporation.
The transfected cells were then stimulated with immobilized
anti-CD3 MoAb or rIL-2 for 24 hours. As shown in Table
1, luciferase activity was induced upon stimulation and was
clearly downregulated by dexamethasone, indicating that the
effect of GC on IL-5 synthesis is exerted at the level of gene
transcription. b-Galactosidase activity derived from pCMVb-gal, a transfection control, did not differ significantly either between stimulated and unstimulated cells or between
GC-treated and GC-nontreated cells. GC-sensitive transcription of pIL-5Luc was confirmed in two other T-cell clones
(HK2 and HK5).
EMSA analysis of dexamethasone-treated T-cell clones.
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GC EFFECT ON T-CELL IL-5 GENE TRANSCRIPTION
2895
The findings given above that both activation signals mediated through TCR- and IL-2R–induced GC-sensitive gene
transcription and protein synthesis of IL-5 prompted us to
examine whether AP-1 and NF-kB, GC-sensitive transcription factors, were induced by either TCR or IL-2R stimula-
Fig 3. IL-2–induced proliferation of CTLL-2 cells was not affected
by dexamethasone. CTLL-2 cells (104/well) were incubated with the
designated concentrations of rIL-2 for 24 hours. Either 100 nmol/L or
1 mmol/L dexamethasone was included from the start of some cultures. 3H-Thymidine (0.5 mCi/well) was pulsed for the last 6 hours.
Data are expressed as the mean of triplicate cultures Ô SEM.
Fig 2. IL-2–induced IL-5 production and proliferation of T-cell
clones were suppressed by dexamethasone. (A) A total of 105 T-cell
clones ([s] HK5; [n] YA5; [h] HK2; [●] YA8) were cultured in 96-well
round-bottom culture plates with human rIL-2 (100 U/mL). Designated concentrations of dexamethasone were included from the start
of some cultures. Culture supernatants were harvested after 24 hours
and assayed for IL-5 by a specific ELISA. Data are expressed as the
mean of triplicate cultures Ô SEM. IL-5 production in the unstimulated cultures was always below the detection limit of the ELISA
system (Ú1 pg/mL). (B) For proliferation assays, cells were cultured
for 72 hours. 3H-Thymidine (0.5 mCi/well) was pulsed for the last 16
hours. Data are expressed as the mean of triplicate cultures Ô SEM.
3
H-Thymidine incorporation of unstimulated HK5, YA5, HK2, and YA8
cells was 362 Ô 154, 526 Ô 193, 759 Ô 227, and 668 Ô 149, respectively.
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tion and were downregulated by dexamethasone. Representative results are shown in Fig 5. Each band indicated by the
arrowhead shows the specific binding that was diminished by
the addition of excess amounts of unlabeled probes in the
preliminary experiments (data not shown). NF-AT, AP-1,
and NF-kB binding activities were clearly upregulated in the
nuclear protein preparations upon stimulation with immobilized anti-CD3 MoAb. Oct-1 binding activities were constitutively present in the stimulated and unstimulated nuclear
proteins and were not affected by dexamethasone. AP-1 and
NF-kB binding activities were clearly downregulated by
dexamethasone, suggesting that these transcription factors
are the targets of GC action on IL-5 synthesis. NF-AT binding was not affected by dexamethasone. In contrast to the
TCR stimulation, neither NF-AT nor NF-kB was significantly induced in the nuclear extracts of IL-2–stimulated T
cells. Essentially the same results were obtained in three
other T-cell clones (data not shown). Although AP-1 and
NF-AT are critical transcription factors for the induction of
IL-2 gene,57 these findings suggest that IL-5 gene transcription using human helper T cells may occur in the absence
of apparent induction of AP-1 and NF-AT and still in a
manner sensitive to GC.
DISCUSSION
In our present study, allergen-specific human T-cell clones
established from atopic asthmatic donors were used to delineate the action of GC on IL-5 synthesis. It was clearly shown
that IL-5 synthesis of human helper T cells induced by two
distinct stimuli mediated through TCR and IL-2R was completely suppressed by GC (Figs 1 and 2). IL-5 mRNA expression induced by either stimulus was clearly downregulated
by dexamethasone (Fig 4). Dexamethasone suppressed the
transcriptional activity mediated through the Ç500-bp hu-
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Fig 4. Dexamethasone suppressed IL-5 gene expression of
human T-cell clones stimulated
via TCR and IL-2R. (A) T-cell
clone (YA5, 2 Ì 106/well) was
cultured in 24-well culture plates
for the designated time periods.
Some wells were pretreated
with OKT3 MoAb (10 mg/mL), as
indicated. rIL-2 (100 U/mL) was
added to the designated wells.
Dexamethasone (100 nmol/L)
was included throughout the
culture period in some wells, as
indicated. Cells were harvested
after 0, 4, 8, and 16 hours. Total
RNA was then extracted, reverse
transcribed, and amplified by
PCR. The 279- and 1,128-bp products correspond with the expected size of IL-5 and b-actin
amplification products, respectively. (B) T-cell clone (HK2 and
HK5) was stimulated for 8 hours.
man IL-5 promoter/enhancer gene segment located 5* upstream of the coding region (Table 1), indicating that the
inhibition of IL-5 synthesis by GC was exerted at the level
of gene transcription.
It has been shown that GC inhibit the production of various
cytokines, such as IL-2, IL-3, IL-4, IL-6, IL-8, interferon-g, and
granulocyte-macrophage colony-stimulating factor.46,61-66 The
mechanisms of action of GC have been elucidated since the
Table 1. Dexamethasone Suppressed Human IL-5 Promoter/Enhancer Activity Induced by Either TCR or IL-2R Stimulation
Clone*
Stimulation†
Dex‡
YA5
None
Anti-CD3
0
0
/
0
/
10.9
192.4
21.0
118.2
17.2
IL-2
Luciferase Activity§ (per mg protein)
{
{
{
{
{
b-Galactosidase Activity§ (per mg protein)
1.0
12.2
2.0
10.0
1.2
0.67
0.59
0.62
0.66
0.69
{
{
{
{
{
0.07
0.08
0.07
0.08
0.06
HK5
None
Anti-CD3
0
0
/
4.2 { 1.0
479.2 { 30.9
12.3 { 1.0
0.92 { 0.08
0.87 { 0.10
0.97 { 0.13
HK2
None
IL-2
0
0
/
8.2 { 1.0
269.2 { 20.1
9.2 { 2.9
0.72 { 0.08
0.67 { 0.11
0.77 { 0.10
* IL-5–producing T-cell clones (2.5 1 107 cells in 500 mL RPMI1640 medium supplemented with 10% FBS and 10 U/mL rIL-2) were transiently
transfected with pIL-5Luc (50 mg) / pCMV-b-gal (10 mg) by electroporation (270 V, 960 mF) using a 0.4-cm cuvette. Transfected cells were
suspended in a fresh medium and cultured in triplicate in 24-well culture plates.
† Cells were either stimulated with rIL-2 (100 U/mL) or immobilized anti-CD3 MoAb or kept unstimulated for 24 hours.
‡ Dexamethasone (1 mmol/L) was included from the start of some cultures, as indicated..
§ After 24 hours, cell lysates were prepared and tested for luciferase activity using Luciferase Assay System and b-galactosidase activity using
CPRG as a substrate. Protein concentrations were determined with bicinchoninic acid protein assay reagent. Data are presented as the mean
of triplicate cultures { SEM.
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GC EFFECT ON T-CELL IL-5 GENE TRANSCRIPTION
Fig 5. Effects of dexamethasone on NF-AT, AP-1, NF-kB, and Oct1 binding activities. T-cell clones (YA5) were stimulated with immobilized anti-CD3 MoAb or rIL-2 (100 U/mL) for 2 hours. Dexamethasone
(1 mmol/L) was added to some cultures throughout the culture period, as indicated. Nuclear proteins were then extracted and DNA
binding activity was measured by EMSA. Arrows indicate specific
binding to NF-AT (A), AP-1 (B), NF-kB (C), and Oct-1 (D) oligonucleotides.
molecular identification of the GC receptor (GR).67,68 GC exerts
its effects through binding with its receptor existing in the cytoplasm. GC-GR complex can translocate into the nucleus and bind
with the specific enhancer gene segment called glucocorticoid
responsive element (GRE; GGTACANNNTGTTCT), either to
enhance or suppress the target gene transcription. In some instances, GC-GR complex interferes with the transcriptional activity of AP-1, a ubiquitous transcription factor that is composed
of Jun and Fos families (c-Jun, Jun-B, Jun-D, c-Fos, FosB,
Fra1, and Fra2) through direct protein-protein interaction.69-71
The inhibition of IL-2 enhancer by GC maps to the locus containing AP-1 element.59,60 It is also reported that GC interfere
with NF-kB activity through induction of IkB synthesis.72 Our
present results are consistent with the previous reports that AP1 and NF-kB are the targets of GC actions. The possible location
of AP-1 and NF-kB elements responsible for the GC sensitivity
of human IL-5 gene warrants further investigation.
Because the CD3 molecule is a component of the T-cell
receptor complex, the activation signal transduced by the
cross-linkage of CD3 molecules is considered to mimic antigenic stimuli delivered through TCR under physiologic con-
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ditions.57 We and others reported that IL-5 synthesis of human PBMC was suppressed by GC.18,19 Because GC induce
anti-inflammatory second messengers, including lipocortins,
in various cell populations,20-24 it is still unclear whether the
suppressive effect of GC on IL-5 production is a direct action
on T cells or an indirect one. Our present findings indicate
that GC can exert their effects directly on T cells, because
the T-cell preparations used in our study consisted of more
than 98% pure T-cell populations.
In addition to the activating signal through T-cell receptor
complex, exogenous IL-2 is sufficient to induce IL-5 synthesis by human allergen-specific T-cell clones.25 Although the
roles of Th2 cytokines in atopic diseases have been emphasized,11,73 it has been reported that local IL-2 production was
also enhanced during the late phase reaction after allergen
exposure,26-29 suggesting the involvement of IL-2 (Th1 cytokine) in addition to Th2 cytokines in the pathogenesis of
atopic diseases. So it is an intriguing question as to whether
IL-2–induced IL-5 production is also suppressed by GC. IL2 induces PTK activation and PI-3 kinase activation, but
neither Ca2/ mobilization, PI turnover, nor PKC activation
occurs in T cells in response to IL-2 stimulation,32-34,38-42
whereas these early biochemical events are the hallmarks of
TCR-mediated T-cell activation.57 TCR signal is sensitive
to FK506 and cyclosporin A, but IL-2R signal is not,43-45
indicating that cytokine synthesis induced by TCR signal is
transduced by a quite different pathway from that induced by
IL-2R signal. It was also reported that TCR-induced cytokine
production was inhibited by quite low concentrations of GC,
but, in contrast, the proliferation of murine cytotoxic T cells
induced by IL-2 was affected only marginally by GC,46 suggesting that the signals mediating cytokine synthesis use
different pathways from those mediating IL-2–induced cell
proliferation in T cells. It was clearly shown here for the first
time that not only the proliferation but also IL-5 synthesis of
human helper T cells induced by IL-2 is totally suppressed
by GC. These findings further support the efficacy of GC in
the treatment of eosinophilic inflammation acting as IL-5
synthesis inhibitors. GC suppressed the expression of IL-5
mRNA induced by IL-2 (Fig 4) and at the same time suppressed the gene transcription mediated by the human IL-5
promoter/enhancer gene located 5* upstream of the coding
region (Table 1), suggesting that GC exert their action on T
cells at the transcriptional level.
EMSA analysis indicated that AP-1 and NF-kB complex
were among the possible targets of GC action on TCR-stimulated helper T cells (Fig 5), which is consistent with previous
reports.59,60,72 Whether the effect of GC on IL-5 gene specifically maps to AP-1 or NF-kB element within human IL-5
promoter/enhancer gene should further be determined by the
transfection analysis using mutant promoter/enhancer plasmids as performed in IL-2 gene.60 AP-1, NF-kB, and NF-AT
binding was not detectable after IL-2R stimulation, in contrast
to TCR stimulation, despite the fact that endogenous IL-5
gene expression (Fig 4) and transcriptional activity as determined by pIL-5(-511)Luc (Table 1) induced by either TCR
or IL-2R stimulation were quite comparable, suggesting that
AP-1 and NF-kB activities seem to be important for IL-5
synthesis induced by TCR stimulation, but are not essential
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2898
MORI ET AL
for IL-5 synthesis induced by IL-2. Because the induction of
NF-AT, AP-1, and NF-kB complexes in IL-2–stimulated T
cells may follow different kinetics than in TCR-stimulated
cells, we examined NF-AT, AP-1, and NF-kB binding activities at different time points after IL-2 and TCR stimulation.
No significant binding activities of NF-AT, AP-1, and NFkB were detected at 0.5, 1, and 4 hours after IL-2 stimulation,
in accordance with the results obtained at 2 hours (shown in
Fig 5), although those binding activities were clearly induced
by TCR stimulation (data not shown). The present findings
suggest that GC may exert their effects through affecting a
still unknown transcription factor(s) other than AP-1, NF-AT,
NF-kB, and Oct-1, and a GC-sensitive unique transcription
factor(s) might regulate human IL-5 gene activation in allergic
diseases associated with eosinophilic inflammation such as
asthma and atopic dermatitis.
To date, several transcription elements, including AP-1,
NF-kB, NF-AT, Oct-1, GATA, and conserved lymphokine
element 0 (CLE0), have been indicated by the EMSA and
homology search analyses.74-78 NF-AT binding activity was
induced in an activated murine mast cell line and the human
IL-5 sequence between 0120 and 098 corresponded to the
activity.76 Both GATA binding element located 082 to 062
and CLE0 element located 056 to 042 are essential for the
spontaneous, not inducible, transcriptional activity expressed
in an adult T-cell leukemia virus-transformed T-cell line.77
Two octamer binding sites located 0244 to 0237 and somewhere between 067 and 030 were also identified.78 Our
results showing that human IL-5 promoter/enhancer-luciferase gene construct transfected to T-cell clones was transcribed upon stimulation and was downregulated by dexamethasone (Table 1) indicate that the approximately 500-bp
human IL-5 gene segment located 5* upstream of the coding
region contains an activation-inducible enhancer element(s)
regulated by GC. Because the function of transcription factors is context-dependent, it is essential to determine (1)
whether those possible elements homologous to the consensus binding sequences of known transcription factors such
as NF-AT, AP-1, and NF-kB really bind their corresponding
transcription factors and (2) whether those elements and transcription factors are functionally active in human helper T
cells and regulated by GC. The studies necessary to answer
these questions are currently being undertaken using transient transfection systems, EMSA, and footprint analyses
using genuine human IL-5 sequences. The further identification of IL-2–responsive and GC-sensitive transcriptional elements in the human IL-5 promoter/enhancer gene will improve understanding of the molecular mechanisms of human
T-cell IL-5 synthesis and may facilitate future therapeutic
intervention for severe atopic diseases by the development
of selective IL-5 synthesis inhibitors.
ACKNOWLEDGMENT
The authors thank Dr S. Morimura for helpful discussion, Dr
W.A. Gray for reviewing this manuscript, and M. Komatsu and Y.
Shirai for technical assistance.
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1997 89: 2891-2900
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