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Clinical and Diagnostic Laboratory Immunology, January 1998, p. 18-23, Vol. 5, No. 1
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Induction of Interleukin-4 and Interleukin-5
Expression in Mast Cells Is Inhibited by Glucocorticoids
William A.
Sewell,*
Lyndee L.
Scurr,
Helen
Orphanides,
Simon
Kinder, and
Russell I.
Ludowyke
Centre for Immunology, University of New
South Wales, and St. Vincent's Hospital, Sydney, New South Wales,
Australia
Received 28 April 1997/Returned for modification 9 July
1997/Accepted 22 September 1997
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ABSTRACT |
Inflammation in asthma and other allergic diseases is characterized
by excessive production of immunoglobulin E (IgE) and the influx of
leukocytes, especially eosinophils. Interleukin 4 (IL-4) and IL-5 are
essential for IgE production and eosinophilia, respectively, and are
produced by mast cells in allergic conditions, for which
glucocorticoids are widely used therapeutically. We assessed the effect
of glucocorticoids on IL-4 and IL-5 mRNA production by the RBL-2H3 cell
line, an analog of mucosal mast cells. IL-4 and IL-5 mRNAs were induced
by an antigen that is used to cross-link receptor bound IgE, by calcium
ionophore, or by ionophore with phorbol ester and were markedly
inhibited by dexamethasone. In cells activated with ionophore and
phorbol ester, 10
6 M dexamethasone reduced the IL-4 and
IL-5 mRNA levels to only 12.8 and 5.7%, respectively, of those in
cells without dexamethasone, and 109 M dexamethasone
caused reductions to 27 and 56%, respectively. Hydrocortisone at
106 and 107 M almost completely inhibited
IL-4 and IL-5 mRNA production. Dexamethasone was markedly inhibitory
even if it was added after the cells were activated, provided that it
was present in the cultures for at least 1.5 h. These studies
indicate that the expression of IL-4 and IL-5 mRNAs by mast cells is
highly sensitive to glucocorticoids. The data suggest that these
inhibitory effects may contribute to the clinical efficacy of
glucocorticoids in the therapy of allergic diseases.
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INTRODUCTION |
Cellular inflammation in asthma and
other allergic diseases is characterized by the influx of leukocytes,
especially eosinophils. Such inflammation is critical in the airway
obstruction in asthma, and secreted products of eosinophils have been
shown to be toxic to respiratory mucosa (2). Cytokines are
considered to have a major role in the coordination of these cellular
inflammatory conditions. In particular, interleukin 4 (IL-4) and IL-5
have prominent roles in allergic reactions. In IL-4 knockout mice
immunoglobulin E (IgE) production is totally abolished (17).
IL-4 is also important in attracting eosinophils to sites of
inflammation (35). This may be achieved by upregulating
local endothelial expression of the cell surface adhesion molecule
VCAM-1 (33) or inducing the production of the eosinophil
chemotactic factor eotaxin (30). IL-5 is a very important
cytokine for eosinophils; it accelerates their production and has
several actions on mature eosinophils, including priming for activation
and prolongation of their life span (31). Antigen-induced
eosinophil infiltration into the lungs was absent in IL-5 knockout mice
(12). Cells containing mRNA for IL-5, detected by in situ
hybridization, have been found at increased frequency in the airways of
patients with asthma (15). Thus, production of IL-5 is
particularly important in the development of the cellular infiltrate in
asthma and other conditions with prominent eosinophilia.
Mast cells have the capacity to produce cytokines in response to
cross-linking of receptor-bound IgE by specific allergen (13). Evidence for the mast cell as a source of cytokines in vivo has been obtained by immunohistochemical analysis of bronchial mucosal biopsy specimens from patients with asthma. IL-4, IL-5, IL-6,
and tumor necrosis factor alpha were predominantly found in mast cells,
although some IL-5 was found in eosinophils (6). In studies
in which mRNA was detected in bronchoalveolar lavage fluid and
bronchial biopsy specimens from patients with asthma by in situ
hybridization, the number of cells expressing mRNA for IL-4 and IL-5
was greatly increased, and expression was found in T cells, mast cells,
and eosinophils (43).
Glucocorticoids are widely regarded as the most effective available
treatment for asthma, and they are particularly able to suppress
cellular inflammation. Glucocorticoids can inhibit the production of
most cytokines, and this may be an important general mechanism of their
clinical efficacy (2). Glucocorticoids inhibit the
expression of IL-4 (42) and IL-5 (28) by T cells.
In the present study we have assessed the effects of glucocorticoids on
the expression of IL-4 and IL-5 in the RBL-2H3 cell line, an analog of
rat mucosal mast cells (34). IL-4 and IL-5 mRNAs were readily induced in these cells after activation by a variety of stimuli. Glucocorticoids markedly inhibited the expression of IL-4 and
IL-5 mRNAs, even when the glucocorticoids were added well after the
cells were activated. These effects could account, at least in part,
for the therapeutic efficacy of glucocorticoids in the management of
clinical allergic conditions.
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MATERIALS AND METHODS |
Cells.
RBL-2H3 cells were obtained from M. A. Beaven,
National Institutes of Health, Bethesda, Md., and were cultured and
activated as described previously (20, 21). For each aliquot
taken from liquid nitrogen, the time course and dose-response of
release of granule contents in response to antigen used to cross-link receptor-bound IgE were tested. Aliquots were used for no more than 20 passages. Cells were maintained as monolayers in minimal essential
medium (MEM) without CaCl2 (Gibco BRL, Life Technologies, Gaithersburg, Md.) and with 2 mM glutamine (Gibco BRL) and 10% (vol/vol) fetal calf serum (P.A. Biologicals, Sydney, New South Wales,
Australia) at 37°C in 5% CO2. There is sufficient
calcium in the serum for normal cell growth and secretion (data not
shown). Cells were harvested by trypsin treatment, and 0.8 × 106 cells were seeded into each well of 12-well culture
dishes and were allowed to form monolayers overnight. Glucocorticoids
were then added to the monolayers, and the mixture was incubated
overnight prior to activation unless otherwise stated. Overnight
glucocorticoid treatment did not affect cell number, cell viability, or
total 
-hexosaminidase content. Dexamethasone and hydrocortisone were obtained from Sigma (St. Louis, Mo.); the water-soluble form of dexamethasone was used. For experiments involving antigen activation, a
2,4-dinitrophenol (DNP)-specific monoclonal IgE antibody (75 ng/ml;
Sigma) was added at the time that the cells were seeded.
Cells were washed twice with MEM (Gibco BRL) containing 200 mg of
CaCl2 per liter but without fetal calf serum. All of the following activators were diluted in MEM at the indicated
concentrations unless stated otherwise: 100 ng of DNP-bovine serum
albumin (BSA) per ml (24 molecules of DNP conjugated with 1 molecule of
BSA, kindly donated by H. Metzger, National Institutes of Health,
Bethesda, Md.), 1,000 nM A23187 (Sigma), and 50 nM phorbol myristate
acetate PMA (Sigma). In preliminary experiments, IL-4 mRNA was elicited to a similar extent by DNP-BSA concentrations of 10 to 1,000 ng/ml (data not shown). The cells were incubated in the solutions for 30 min,
and the supernatants were collected for analysis of -hexosaminidase secretion. The activated cells were then incubated for the remaining time in MEM without CaCl2 and were then lysed for RNA
extraction. For samples treated with glucocorticoids overnight, prior
to activation, cells were maintained in glucocorticoids throughout the
time between activation and RNA extraction.
Analysis of IL-4 and IL-5 mRNAs.
The cells were harvested
4 h after activation unless otherwise stated. Total cellular RNA
was extracted as described previously (8), the RNA
concentration was estimated by measuring the optical density at 260 nm,
and cDNA was prepared as described previously (23) from 1 µg of RNA in 50-µl volumes with oligo(dT) (4 ng/µl) and 4 U of
avian myelobastosis virus reverse transcriptase (Promega, Madison,
Wis.). PCR was performed with cDNA derived from 0.1 µg of total RNA
with 1 U of Taq polymerase (Boehringer Mannheim, Mannheim,
Germany) and 250 ng of each amplification primer as described
previously (23). PCR was performed in a Gene Machine (Innovonics, Melbourne, Victoria, Australia) for 26 cycles for IL-4, 35 cycles for IL-5, and 22 cycles for -actin. Denaturation, annealing,
and extension conditions were 95°C for 60 s, 58°C for 30 s, and 75°C for 30 s, respectively. The primers, based on
published sequences (22, 25, 36), were
5'-ACCTTGCTGTCACCCTGTTC-3' and
5'-TTGTGAGCGTGGACTCATTC-3' for IL-4,
5'-CTCTGTTGACGAGCAATGAG-3' and
5'-CTCTTGCAGGTAATCCAGGA-3' for IL-5, and
5'-TAACCAACTGGGACGATATG-3' and
5'-ATACAGGGACAGCACAGCCT-3' for -actin. The expected
product sizes were 351 bp for IL-4, 239 bp for IL-5, and 202 bp for
-actin. The primers were designed to anneal to different exons, so
that any contaminating genomic DNA in the cDNA samples would contain one or more introns and would yield a product larger than that derived
from cDNA.
PCR products were electrophoresed in 2% agarose gels and were stained
with ethidium bromide. The sizes of the PCR products
were determined
with reference to molecular size markers (
X174
cleaved with
HaeIII; Boehringer Mannheim). In some experiments,
the gels
were photographed with reversed-image film (Polaroid),
and band
intensities were measured by laser densitometry (Molecular
Dynamics,
Sunnyvale, Calif.). In other experiments, the specificities
of the
products were confirmed by Southern blotting. The gels
were transferred
to a nylon membrane (Hybond N
+; Amersham, Amersham,
Buckinghamshire, England) under vacuum,
and the membranes were
hybridized to oligonucleotide probes designed
to anneal to the PCR
product but not to the amplification primers.
The hybridization probes
were 5'-TACCTCCGTGCTTGAAGAAC-3' for IL-4,
5'-TCAGTATGTCTAGCCCCTGA-3' for IL-5, and
5'-CAGCCATGTACGTAGCCATC-3'
for
-actin. These were
end-labelled with [
32P]ATP and T4 polynucleotide kinase
(Pharmacia, Uppsala, Sweden).
The blots were washed and exposed to
X-ray film (DuPont, Wilmington,
Del.). After development of the films,
band intensities were determined
by laser densitometry as described
above.
Reverse transcription-PCR (RT-PCR) for
-actin was performed with all
samples. Fewer than 5% of the samples had markedly reduced
or absent
-actin signals; these samples were excluded from further
analysis.
Semiquantitative PCR data from time course studies was
obtained by
performing RT-PCR with all samples and identifying
the sample with the
strongest signal. Threefold dilutions of this
sample were prepared, and
PCR was repeated with all samples and
these dilutions. The dilutions
were used to prepare a standard
curve against which the other samples
were read. The undiluted
specimen of the sample with the strongest
signal was defined as
having a signal of 100%, and the other signal
data were expressed
as a percentage of the signal for this sample.
Similar procedures
were used to obtain semiquantitative data in the
experiments with
glucocorticoids, except that the signals for the
samples which
were stimulated and not treated with glucocorticoids were
defined
as being 100% and were used to prepare the dilutions.
Assessment of release of granule contents.
Secretion of
granule contents by RBL-2H3 cells was determined by measuring the
release of the granule marker -hexosaminidase into the medium
(26). Culture supernatants were collected 30 min after the
cells had been activated. Supernatant (20 µl) was incubated with 20 µl of 5 mM
p-nitrophenyl-N-acetyl-D-glucosamide (Sigma) in 0.05 M sodium citrate buffer (pH 4.5) in triplicate in a
96-well plate at 37°C for 2 h. At the end of the incubation, 200 µl of 0.1 M sodium carbonate-sodium bicarbonate buffer (pH 11.0) was
added, and the absorbance at 405 nm was read in an enzyme-linked immunosorbent assay plate reader (Diagnostics Pasteur). The release of
-hexosaminidase was expressed as a percentage of the total -hexosaminidase present in unactivated cells. Any effect of phenol red in the medium was accounted for by the inclusion of medium as a
control. The cell lysate was obtained by incubating cells with 1 ml of
0.1% Triton X-100 for 10 min. Spontaneous release in the absence of
stimuli was in the range of 2 to 6% of the total -hexosaminidase
and was subtracted from the values given above. Release from cells
stimulated with antigen or with the combination of PMA and A23187
(PMA-A23187) was typically in the range of 40 to 50% of total
-hexosaminidase. The means for triplicate samples were determined,
and these were used to calculate the means and standard errors of the
means (SEMs) for replicate experiments. Data were then expressed as a
percentage of the release from stimulated cells not treated with
glucocorticoids.
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RESULTS |
Cytokine expression by RBL-2H3 cells.
The capacity of RBL-2H3
cells to express cytokines was assessed in response to antigen
cross-linking of receptor-bound IgE, to PMA, and to the calcium
ionophore A23187. At 4 h after activation, cells were lysed and
RT-PCR was performed for IL-4, IL-5, and -actin mRNA. The sizes of
the principal products determined by gel electrophoresis were within 10 bp of their predicted sizes, and their identities were confirmed by
Southern transfer and hybridization to radiolabelled oligonucleotide
probes (Fig. 1). In the Southern blots,
in addition to the principal bands, some samples contained other bands
which hybridized specifically. The bands with slower electrophoretic
mobilities represent single-stranded PCR products (37); the
bands with faster electrophoretic mobilities probably arise from
mispriming of specific cDNA.
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FIG. 1.
Inducible expression of IL-4 and IL-5 by RBL-2H3 cells.
(a) Cells were exposed to no stimulus (lane 1), antigen (lane 2), or
PMA-A23187 (lane 3) for 4 h, and RNA was then extracted. RT-PCR
was performed, and products were detected by electrophoresis and
Southern hybridization. The control RNA, -actin RNA, was readily
detected in all samples, attesting to the integrity of the RNA.
Negative controls were omission of reverse transcriptase enzyme from
the RT (lane 4, which was otherwise identical to lane 3) and omission
of cDNA from the PCR (lane 5). (b) Cells were exposed to no stimulus
(lane 6), PMA (lane 7), A23187 (lane 8), or PMA-A23187 (lane 9), and
mRNA was detected as described above. Percent -hexosaminidase
releases in this experiment were as follows: no stimulus, 0.5%; PMA
stimulus, 2.4%; A23187 stimulus, 43.8%; PMA-A23187 stimulus,
36.7%.
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mRNAs for IL-4 and IL-5 were expressed at low levels in unstimulated
cells. After stimulation with antigen, mRNA levels for
IL-4 and IL-5
were markedly increased. Stimulation with the combination
of PMA and
A23187 had similar effects (Fig.
1a). IL-4 mRNA was
very readily
detected, with bands in ethidium bromide-stained
gels being visible
after only 26 cycles of PCR amplification of
cDNA derived from 0.1 µg
of total RNA. Stimulation of cells with
A23187 alone was almost as
effective as PMA-A23187 in inducing
IL-4 and IL-5 mRNAs. However, PMA
alone had little or no effect
(Fig.
1b). The expression of IL-4 and
IL-5 mRNAs in response to
the different activators correlated with
granular secretion, measured
by the release of
-hexosaminidase (see
the legend to Fig.
1).
To define the peak time of mRNA production, cells were harvested from
15 min to 8 h after activation. There was differential
induction
of IL-4 and IL-5 (Fig.
2). IL-4 mRNA was
induced earlier,
with markedly increased production detectable within
1 h. IL-4
mRNA levels peaked at 2 h and returned to baseline
levels at 8
h. IL-5 had a slower induction rate, with little
change within
the first hour, peak expression 4 h after
stimulation, and a slower
decay of mRNA levels than those for IL-4
mRNA. In these experiments,
IL-5 mRNA was detectable in unstimulated
cells and IL-5 mRNA levels
were not significantly increased by PMA
alone. On the basis of
these findings, subsequent mRNA harvest was
performed 4 h after
activation.
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FIG. 2.
Time course of IL-4 and IL-5 mRNA production. Cells were
activated with PMA-A23187 and were incubated for various times before
lysis for RNA extraction. IL-4 and IL-5 mRNAs were detected by RT-PCR,
and band intensity was determined by laser densitometry. Each value
represents the mean and SEM for four (IL-4) or three (IL-5) separate
experiments.
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Effects of glucocorticoids.
The effect of the synthetic
glucocorticoid dexamethasone on the production of IL-4 and IL-5 mRNAs
in RBL-2H3 cells was determined. Concentrations of dexamethasone
ranging from 106 to 1011 M were tested in
the presence of PMA-A23187 (Fig. 3).
Dexamethasone at 1010 M reduced the level of IL-4 mRNA
production significantly and at 106 to 108
M almost abolished it (Fig. 3a), with a 50% inhibitory concentration (IC50) of 1010.3 M. Dexamethasone at
109 M reduced IL-5 mRNA levels significantly and at
107 and 106 M reduced the mRNA to very low
levels (Fig. 3b), with an IC50 of 108.8 M. At
the higher doses tested, dexamethasone was moderately inhibitory to
granule secretion, as measured by -hexosaminidase release. However,
even at doses as high as 106 M, only 40 to 50%
inhibition of granule release was observed (Fig. 3c).
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FIG. 3.
Effects of dexamethasone on cells stimulated with
PMA-A23187. Cells were incubated overnight in the presence of various
concentrations of dexamethasone and were stimulated with PMA-A23187.
Cells were harvested 4 h later for assessment of IL-4 (a) and IL-5
(b) mRNA levels. Supernatants were collected 30 min after activation
for determination of -hexosaminidase secretion (c). Data are
expressed as a percentage of the values for the samples from cells
given no dexamethasone (dex). Each value represents the mean and SEM
for three (a) or two (b and c) separate experiments.
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The naturally occurring glucocorticoid hormone hydrocortisone had
effects similar to those of dexamethasone. There was a dramatic
concentration-dependent decrease in the levels of expression of
both
IL-4 and IL-5, with the mRNAs of both cytokines essentially
being
abolished at hydrocortisone concentrations of 10
6 M (Fig.
4a and b). As with dexamethasone, IL-4
mRNA production
was more sensitive than IL-5 mRNA production to
hydrocortisone.
The IC
50s were 10
8.9 M for
IL-4 and 10
8.0 M for IL-5. Granule secretion was only
moderately inhibited by
the highest concentrations of hydrocortisone
(Fig.
4c).
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FIG. 4.
Effects of hydrocortisone. IL-4 mRNA (a), IL-5 mRNA (b),
and secretion (c) were determined as described in the legend to Fig. 3,
but with hydrocortisone instead of dexamethasone. Data are expressed as
a percentage of the values for the samples from cells given no
hydrocortisone (HC). Each value represents the mean and SEM for two (a
and b) or three (c) separate experiments.
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Studies were then performed to determine the effect of dexamethasone on
cells activated by antigen-induced cross-linking of
IgE receptors.
These experiments were performed because this is
a more physiological
method of mast cell activation than PMA-A23187
stimulation. Cells were
incubated with IgE antibody overnight
and were exposed to specific
antigen. Production of IL-4 and IL-5
mRNAs was markedly inhibited by
dexamethasone concentrations from
10
8 to
10
6 M (Fig.
5). The
IC
50s were 10
8.6 M for IL-4 and
10
8.8 M for IL-5. These effects were similar to those
described in
Fig.
3 for cells stimulated with PMA-A23187. However, the
inhibitory
effect of dexamethasone on the secretion of
-hexosaminidase was
much greater for cells stimulated with antigen
(Fig.
5c) than
for cells stimulated with PMA-A23187 (Fig.
3c).
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FIG. 5.
Effect of dexamethasone on cells stimulated with
antigen. Cells were activated with DNA-BSA to cross-link receptor-bound
IgE. IL-4 mRNA (a), IL-5 mRNA (b), and secretion (c) were determined as
described in the legend to Fig. 3. Data are expressed as a percentage
of the values for the samples from cells given no dexamethasone (dex).
Each value represents the mean and SEM for three (a and b) or five (c)
separate experiments.
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In the experiments described above, dexamethasone was added overnight,
prior to activation of the cells. The effects of adding
it at a range
of times prior to and after activation were assessed
(Fig.
6). Cells were activated at time zero and
4 h later were
harvested for RNA extraction. Dexamethasone was
added at different
times, from 16 h before activation to 4 h
after activation, i.e.,
immediately prior to cell lysis for RNA
extraction. Dexamethasone
markedly inhibited IL-4 and IL-5 mRNA
production when it was added
16 and 2 h prior to activation. It
was also markedly inhibitory
when it was added at the time of
activation and at later times,
as late as 2.5 h after activation,
which was only 1.5 h before
the cells were harvested for RNA
extraction (Fig.
6). The effects
were variable when dexamethasone was
added only 1 h prior to harvest
(3 h after activation), and there
was no effect when it was added
30 min prior to harvest (3.5 h after
activation).
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FIG. 6.
Effect of time of addition of dexamethasone. Cells were
activated at time zero and 4 h later were harvested for RNA
extraction. Dexamethasone was added at times ranging from 16 h
prior to activation ( 16) to immediately before cell harvest, i.e.,
4 h after activation. RT-PCR was performed for IL-4 and IL-5.
Quantitative data were determined as described in the legend to Fig. 3
and were expressed as a percentage of the values for the samples to
which dexamethasone (dex) was added 4 h after activation. The data
are the means and SEMs of two separate experiments.
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DISCUSSION |
In this paper we demonstrate that expression of IL-4 and IL-5 can
be induced in the RBL-2H3 cell line, an analog of mucosal mast cells,
by cross-linking of receptor-bound IgE or by a calcium ionophore.
Induction of IL-4 and IL-5 mRNAs was strongly inhibited by the
glucocorticoids dexamethasone and hydrocortisone. These findings are
consistent with reports that glucocorticoids inhibit the expression or
production of a number of cytokines by mast cells, including tumor
necrosis factor alpha, IL-1, IL-3, IL-6, and IL-8 (18, 19, 38,
39, 41). Inhibition of IL-4 mRNA by dexamethasone was recently
reported in the human mast cell line HMC-1 (38). In T cells,
by contrast, there are conflicting reports on the effect of
glucocorticoids on IL-4 production. High concentrations of
glucocorticoids were reported to inhibit IL-4 production by human blood
T lymphocytes or mononuclear cells (7, 42) and by
CD4+ T cells from rat lymph nodes (27). In a
study with mice, however, low concentrations of dexamethasone from
1011 to 108 M were reported to enhance IL-4
production by lymph node or spleen cells (10). In the
present studies, dexamethasone and hydrocortisone doses from
1011 to 106 M were tested, and there was no
evidence of the enhancement of IL-4 or IL-5 mRNA production at any
concentration, but there was marked inhibition at higher concentrations
(Fig. 3 and 4).
The present findings confirm and extend other recent observations on
the effects of glucocorticoids on the production of IL-5 mRNA by mast
cells. Dexamethasone inhibits the expression of IL-5 in rat peritoneal
mast cells (41) and in human lung explants stimulated with
IgE (14). In these studies with cells prepared from tissue,
the possibility that glucocorticoids might be acting on another cell
population to induce a factor that could inhibit IL-5 mRNA production
by mast cells cannot be completely excluded. The use of the RBL-2H3
mast cell line in the present study demonstrates unequivocally that
these inhibitory effects of glucocorticoids on IL-5 expression are
directly on the mast cells. In cells activated with PMA-A23187, lower
concentrations of either dexamethasone or hydrocortisone were required
to inhibit IL-4 mRNA compared with the concentrations required to
inhibit IL-5 mRNA (Fig. 3 and 4). This effect suggests that the cells
may use different signaling pathways for IL-4 and IL-5 mRNA expression.
However, differences in glucocorticoid concentrations were not observed in cells activated by antigen (Fig. 5).
These findings raise the possibility that the beneficial therapeutic
effects of glucocorticoids in allergic diseases may be mediated, at
least in part, by inhibition of production of IL-4 and IL-5 by mast
cells. An intriguing and novel observation was that dexamethasone may
be added after activation but still markedly reduce the abundance of
cytokine mRNA. When dexamethasone was added 2.5 h after activation
and the cells were harvested 4 h after activation, the inhibition
of IL-4 and IL-5 expression was as marked as when dexamethasone was
added prior to activation (Fig. 6). These findings also suggest that in
a therapeutic setting, IL-4 and IL-5 production can be inhibited by
glucocorticoids after mast cells have been activated by an allergen.
This effect may contribute to the clinical efficacy of therapy with
glucocorticoids when they are introduced after the onset of an asthma
attack.
It is interesting to speculate whether the glucocorticoid
concentrations used in the present experiments are similar to those achieved therapeutically. In a pharmacokinetic study with humans, a
single oral dose of 20 mg of hydrocortisone, a modest glucocorticoid dose, was followed by a peak concentration in plasma of 0.8 × 106 M (11). In the present experiments, IL-4
and IL-5 expression was almost completely abolished by
107 and 106 M hydrocortisone (Fig. 4). It
is difficult to make exact comparisons between concentrations in vivo
and in vitro because of differences in protein binding, the more rapid
elimination of glucocorticoid in vivo, and different kinetics of
receptor occupation during changing extracellular concentrations.
Nevertheless, it is reasonable to propose that the concentrations of
glucocorticoids achieved in the therapy of allergic conditions are
sufficient to inhibit IL-4 and IL-5 mRNA expression.
Dexamethasone was approximately 1 order of magnitude more potent than
hydrocortisone (Fig. 3 and 4), a difference consistent with previous
observations (5).
In T cells, IL-4 and IL-5 mRNA abundance is principally regulated by
control of the rate of gene transcription rather than by alterations in
mRNA stability (24, 29). From the data presented in Fig. 6,
the levels of IL-4 mRNA 4 h after activation were very low in
cells given dexamethasone 2.5 h after activation, by which time
IL-4 mRNA levels have already peaked (Fig. 2). In activated T cells,
the half-life of IL-4 mRNA was 60 min (3). If IL-4 has a
similar half-life in mast cells, the findings in Fig. 6 could not be
fully accounted for by effects on the rate of gene transcription, and
acceleration of mRNA decay might also be involved. In the case of IL-5,
peak mRNA expression was not reached until 4 h after activation.
In mitogen-stimulated T cells, dexamethasone reduced total mRNA levels
by inhibition of IL-5 gene transcription, without affecting mRNA
stability (29a). The data therefore suggest that the effects
of glucocorticoids on IL-5 in mast cells are based on inhibition of the
rate of gene transcription.
The stimuli required for IL-4 and IL-5 gene expression are similar to
those required for granule release (Fig. 1b). Thus, cross-linking of
IgE receptors or calcium ionophore alone, but not PMA alone, is
sufficient for both responses. IgE receptor cross-linking initiates a
sequence of intracellular events leading to an increased intracellular
calcium ion concentration. These elements of the signaling pathways may
be required for both granule release and cytokine mRNA production. In
cells stimulated with PMA-A23187, secretion was only partially
inhibited by glucocorticoids (Fig. 3 and 4), whereas in cells
stimulated with antigen, secretion of granule contents was markedly
inhibited (Fig. 5), as has been reported previously (9, 40).
The data in Fig. 3 and 4 suggest that the effects of glucocorticoids on
cytokine mRNA production may involve components of the signaling
pathway not required for granule release. A number of possible
mechanisms whereby glucocorticoids might inhibit expression of cytokine
genes have been described. Glucocorticoids inhibit the effects of
transcription factors AP-1 and NF-
B, which are involved in the
expression of cytokine genes. The glucocorticoid receptor-hormone
complex interacts directly with AP-1 to prevent it from binding to its
DNA motifs in promoter regions (16). Glucocorticoids
stimulate production of I-B, which inhibits the translocation of
NF-B from the cytoplasm to the nucleus (1, 32).
Glucocorticoids can also accelerate mRNA degradation, as has been
described in the reduction of IL-2 mRNA levels induced with
dexamethasone in T cells (4). The effects of glucocorticoids
on IL-4 and IL-5 mRNA production in mast cells may be mediated by these
and/or other mechanisms.
The relative importance of mast cells as producers of cytokines in
allergic conditions is controversial. Mast cells are the most numerous
cells that contain IL-4 and IL-5 proteins in immunohistochemical studies with biopsy specimens from asthmatic patients (6). By contrast, in in situ hybridization studies, IL-4 and IL-5 mRNAs were
found more frequently in T cells than in mast cells (43). Mast cells may be more effective early in allergic reactions because they are able to be activated rapidly after the arrival of antigen, which cross-links surface-bound IgE. By contrast, before T cells can be
activated, antigen must be processed and presented by other cells. The
rapid induction of cytokine mRNA described in Fig. 2 is consistent with
a role for mast cells in the initiation of clinical allergic reactions.
| |
ACKNOWLEDGMENTS |
This project was supported by the Asthma Foundation of New South
Wales. R.I.L. was supported by the E. Sternberg Research Fellowship.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centre for
Immunology, St. Vincent's Hospital Sydney, Darlinghurst, NSW 2010, Australia. Phone: 61 2 9361 7700. Fax: 61 2 9361 2391. E-mail:
w.sewell{at}cfi.unsw.edu.au.
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Clinical and Diagnostic Laboratory Immunology, January 1998, p. 18-23, Vol. 5, No. 1
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
