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-induced eotaxin release from cultured human airway smooth muscle cells by ß2-agonists and corticosteroids
Division of Respiratory Medicine, City Hospital, University of Nottingham, Nottingham, U.K.
1Correspondence: Division of Respiratory Medicine, Clinical Sciences Building, City Hospital, Hucknall Road, Nottingham NG5 1PB, U.K. E-mail: mfzlp{at}unix.ccc.nottingham.ac.uk or alan.knox{at}nottingham.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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stimulated eotaxin release (assayed by ELISA)
from HASM cells and that the release was partially inhibited by
salbutamol and salmeterol. The effect of ß2-agonists was
mimicked by forskolin and 8-bromo-cAMP and potentiated by the
cAMP-dependent phosphodiesterase inhibitor rolipram, suggesting that it
is cAMP dependent. We also found that the cAMP inhibition was likely at
the transcription stage, although experiments with the PKA inhibitors
H-89 and Rp-cAMP or the PKG inhibitor KT5823 suggested that none of
these kinases was involved. Partial inhibition of eotaxin release was
also seen with the corticosteroids dexamethasone and fluticasone. The
combined use of ß2-agonists, rolipram, and steroids
abolished TNF--induced eotaxin release. These results suggest that
the combination of a ß2-agonist, PDE inhibitor, and a
corticosteroid may have additive beneficial effects in the treatment of
the eosinophilia associated with asthma and other allergic
diseases—Pang, L., Knox, A. J. Regulation of TNF--induced
eotaxin release from cultured human airway smooth muscle cells by
ß2-agonists and corticosteroids.
Key Words: eosinophil • cyclic AMP • asthma • phosphodiesterase • cytokine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
and was later demonstrated to be a unique member of the chemokine
family that is specifically and directly chemotactic for eosinophils
in vitro and in vivo (1
2
3
4
5)
. Recent
studies suggest that it also attracts and activates basophils (6
, 7)
and the Th2-like lymphocytes (8)
through its
specific receptor CCR3. Since several reports have shown up-regulated
eotaxin gene expression and increased eotaxin protein production in
bronchial mucosa and BALF of asthmatic patients compared with
nonasthmatic subjects (9
10
11
12
13)
, and the up-regulation of
eotaxin expression correlates with the number of airway eosinophils
(9
, 11
12
13)
, the clinical parameters of disease severity
(11)
, and levels of airway hyperresponsiveness (11
, 12)
, eotaxin is believed to be critically involved in the
pathogenesis of airway eosinophilic inflammatory diseases.
A variety of cell types have been identified as sources of eotaxin
production. These include not only various airway inflammatory cells,
such as monocyte-macrophages, T lymphocytes, and eosinophils themselves
(9
, 11
, 12
, 14)
, but also several types of airway
structural cells such as epithelial cells and smooth muscle cells
(9
10
11
12
, 14)
. Furthermore, in vitro studies with
tumor necrosis factor (TNF-) and interleukin 1ß (IL-1ß),
whose levels are significantly increased in BALF from asthmatic
subjects (15
, 16)
, have shown that eotaxin expression and
release in human lung epithelial cells (17)
and human
airway smooth muscle (HASM) cells (10
, 18)
can be markedly
enhanced after stimulation with these cytokines, suggesting that airway
structural cells may also actively contribute to airway eosinophilia
via eotaxin generation.
Although the human eotaxin gene has been cloned and several regulatory
promoter elements potentially regulating eotaxin gene expression and/or
mediating the effects of anti-inflammatory drugs have been identified
(3
, 19
, 20)
, the mechanisms on the regulation of eotaxin
expression still remain largely unknown. Glucocorticoids have been
generally regarded as potent inhibitors of the expression of several
proinflammatory cytokines. However, it appears from studies so far that
the regulation of eotaxin expression by glucocorticoids may differ from
one type of cells to another. For instance, cytokine-induced eotaxin
release from human lung epithelial cells is suppressed by
glucocorticoids (17)
, whereas the release from HASM cells
is not affected (18)
. cAMP has been considered a
ubiquitous regulator of inflammatory and immunological reactions.
Increases of intracellular cAMP have been demonstrated to inhibit the
release of proinflammatory cytokines such as IL-1ß and TNF-
(21
, 22)
and the induction of inflammatory gene products
such as inducible nitric oxide synthase (iNOS) (23
, 24)
and cyclooxygenase-2 (COX-2) (23)
. However, the regulation
of eotaxin expression by cAMP has not been explored. Since steroids and
cAMP stimulants, i.e., ß2-agonists, through
their anti-inflammatory and bronchodilator effects, provide the
mainstay of asthma management and HASM has been shown to be a rich
source of biologically active chemokines and mediators
(25)
, the present study was designed to investigate the
regulation of TNF--stimulated eotaxin release from HASM cells by
ß2-agonists and steroids, explore the possible
additive/synergistic inhibition when both types of drugs were combined,
and determine the cAMP dependence of the
ß2-agonist effects.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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27
28)
. Human tracheas were obtained
from four postmortem individuals (two males aged 44 and 70 years and
two females aged 52 and 70 years, respectively) within 12 h of
death. The donors had no history of respiratory diseases and no
evidence of airway abnormalities as determined by history and
pathological examination of the trachea and lungs. Cells at passage
4–6 were used for all experiments. We have previously shown that the
cells grown in this manner depict the immunohistochemical and light
microscopic characteristics of typical HASM cells (26)
.
Experiment protocol
The cells were cultured to confluence in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, the
antibiotics penicillin (100 U/ml) and streptomycin (100 µg/ml), the
antifungal amphotericin B (2.5 µg/ml), and L-glutamine (4 mM) in
humidified 5% CO2/95% air at 37°C in 24-well
culture plates and growth-arrested in serum-free medium for 24 h
prior to experiments. Immediately before each experiment, fresh
serum-free medium containing TNF- was added. In the time course
experiments, the cells were incubated with TNF- (10 ng/ml) for 1–24
h; in the concentration response experiments, the cells were incubated
for 16 h with 0.1–100 ng/ml TNF-. In most experiments
thereafter the cells were incubated with 10 ng/ml TNF- for 16 h. At the indicated times, the culture media were harvested and stored
at -20°C until the enzyme-linked immunosorbent assay (ELISA) for
eotaxin. To test the inhibition by various drugs on the effect of
TNF-, the ß2-agonists salbutamol (Salbu) and
salmeterol (Salme), the direct adenylyl cyclase activator forskolin
(FSK), the membrane-permeable cAMP analog 8-bromoadenosine 3':5'-cyclic
monophosphate (8-Br-cAMP), and the corticosteroids dexamethasone (Dex)
and fluticasone propionate (Flut) were added 1 h prior to the
incubation with TNF- unless otherwise specified in figure legends.
To analyze the mechanism of the inhibition on TNF--induced eotaxin
release by steroids and ß2-agonists, the
specific ß2-receptor antagonist ICI-118,551
(ICI), the type IV cAMP-dependent phosphodiesterase inhibitor rolipram
(Roli), the cAMP-dependent protein kinase A (PKA) inhibitors H89
(Ki = 48 nM) and adenosine 3':5'-cyclic
monophosphothioate Rp-isomer (Rp-cAMP, Ki = 11
µM), and the cGMP-dependent protein kinase G (PKG) inhibitor KT5823
(Ki = 234 nM) were added 1 h prior to the
addition of steroids and cAMP stimulants and analog. All the agents
were dissolved in dimethyl sulfoxide (DMSO, final concentration 
0.6% v/v). In all the experiments, a group of control cells was
incubated with the drug vehicle for the same period of time as the
experimental cells. The effect of some of the above agents on eotaxin
release by their own was examined with increasing concentrations and
various time points.
Eotaxin assay
The concentration of eotaxin in the culture medium was measured
by a sandwich enzyme immunoassay kit according to the manufacturer’s
instructions. Briefly, 50 µl of standards or samples were pipetted
into the 96-well plates and the eotaxin present was bound by the
precoated monoclonal antibody specific for eotaxin. After incubation
and washing away any unbound substances, an enzyme-linked polyclonal
antibody specific for eotaxin was added to the wells. After incubation
and a wash to remove any unbound antibody-enzyme reagent, a substrate
solution was added and the color developed in proportion to the amount
of eotaxin bound in the initial step. The reaction was then stopped and
the intensity of the color was measured by a microplate reader. The
standard curve was linearized and subjected to regression analysis. The
eotaxin concentration of unknown samples was extracted by using the
standard curve. The results were expressed as picograms/milliliter of
culture medium. The sensitivity of the ELISA kit at our hands was at
least 5 pg/ml, which was consistent with the manufacturer’s
specifications. According to the kit insert, no significant
cross-reactivity or interference with other human cytokine and
chemokines was observed.
Cell viability
The toxicity of all the chemicals used in this study and their
vehicle DMSO to HASM cells was determined by MTT assay in a separate
series of experiments in 96-well plates, as described previously
(26)
. Viability was set as 100% in control cells.
MATERIALS
Fetal calf serum was purchased from Seralab (Crowly Down, U.K.).
Recombinant human TNF- was obtained from Genzyme (West Malling,
Kent, U.K.). Fluticasone propionate was kindly provided by Dr. Malcolm
Johnson (GlaxoWellcome Research and Development, Uxbridge, Middlesex,
U.K.). ICI-118,551, H89, adenosine 3':5'-cyclic monophosphothioate
Rp-isomer, and KT5823 were from Calbiochem-Novabiochem (Nottingham,
U.K.). The human eotaxin ELISA kit was purchased from R & D Systems
(Abingdon, Oxon, U.K.). DMEM, the antibiotics, amphotericin B,
L-glutamine, salbutamol, salmeterol, forskolin, 8-bromoadenosine
3':5'-cyclic monophosphate, rolipram, dexamethasone, dimethyl
sulfoxide, and all other chemicals were purchased from Sigma (Poole,
Dorset, U.K.).
Statistical analysis
Results were expressed as the mean ± SE of
n determinations from HASM cells obtained from two donors.
One-way analysis of variance (ANOVA) and/or unpaired two-tailed
t test were used to determine the significant differences
between the means. P values of less than 0.05 were accepted
as statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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on eotaxin release, HASM
cells were cultured in the presence or absence of TNF- (10 ng/ml)
for 0.5–24 h. Eotaxin release from control cells was low even though
there was a slight increase over the incubation period. There was a
marked and time-dependent increase in eotaxin release after stimulation
with TNF-; significant difference was observed after 2 h of
stimulation as compared with eotaxin release from control cells
(P<0.05) and the highest eotaxin concentration was observed
after 16 h stimulation (P<0.001) (Fig. 1A
). When the cells were cultured with TNF- at
concentrations of 0.1, 1.0, 10, and 100 ng/ml for 16 h, a
concentration-dependent increase in eotaxin release was also observed
that was significant from 0.1 ng/ml (P<0.001) and peaked at
10 ng/ml (Fig. 1B
). Ten nanograms/milliliter of TNF- with
an incubation time of 16 h was chosen for the ensuing experiments.
|
Effect of cAMP stimulants on TNF--induced eotaxin release
To investigate whether increases in cAMP had an effect on
cytokine-induced eotaxin release, we studied the effect of the
ß2-agonists Salbu and Salme and the direct
adenylyl cyclase activator FSK on TNF--induced eotaxin generation
from HASM cells. As shown in Fig. 2
, TNF- strongly stimulated eotaxin release, but pretreatment of the
cells with the cAMP stimulants all significantly inhibited the effect
of TNF-. The effect of Salbu, Salme, and FSK on basal eotaxin
release from human ASM cell was also assessed. Salbu (1 and 10 µM),
Salme (0.1 and 1 µM), and FSK (1 and 10 µM) did not alter eotaxin
accumulation at any time point studied as compared with the control
(data not shown).
|
Effect of steroids on TNF--induced eotaxin release
Pretreatment of the cells with steroids Dex (0.1–10 µM) and
Flut (0.01–1 µM) prior to TNF- stimulation
concentration-dependently inhibited but did not abolish TNF--induced
eotaxin release (Fig. 3
).
|
Combined inhibition by steroids and salbutamol on TNF--induced
eotaxin release and the cAMP dependence of the inhibition.
We then went on to study whether the combined use of steroids and
Salbu could further reduce TNF--induced eotaxin release. As shown
before, pretreatment of the cells with either 1 µM Dex or 0.1 µM
Flut strongly inhibited but did not abolish TNF--induced eotaxin
release (Fig. 4A
, B
, P<0.001); Salbu (10 µM) alone also
exhibited an inhibitory effect (Fig. 4A
, B
,
P<0.001, P<0.01, respectively). When Dex and
Flut were used in combination with Salbu, a significant further
inhibition over the effect of steroids or Salbu alone on eotaxin
release was observed (Fig. 4A
, B
, P<0.01). To
clarify the role of cAMP in this combined inhibition on eotaxin
release, we examined whether ICI, a potent
ß2-receptor antagonist, could reverse the
inhibition and if rolipram (Roli), a specific inhibitor of the
cAMP-dependent phosphodiesterase, could further enhance the inhibition.
As shown in Fig. 4
, the combined inhibition was indeed significantly
reversed by ICI (1 µM) (Fig. 4A
, B
, P<0.01,
P<0.05, respectively) and further enhanced by Roli (1
µM) (Fig. 4A
, B
, P<0.01, P<0.001,
respectively). The effect of ICI and Roli on their own on
TNF--induced eotaxin release was also assessed, and the results did
not show any significant change on the effect of TNF- (data not
shown).
|
Experiments with the combination of Salme and steroids resulted in a
response similar to that of Salbu and steroids. TNF--induced eotaxin
release was markedly inhibited by Salme (1 µM) alone
(P<0.01), significantly further inhibited when Salme was
used in combination with either Dex or Flut (P<0.01), and
abolished with the addition of Roli. FSK also mimicked the effect of
ß2-agonists by exerting further inhibition with
Dex on TNF--induced eotaxin release (Fig. 5
, P<0.01). The combined inhibition was further enhanced by
Roli (Fig. 5
, P <0.01). ICI had no effect on the FSK-induced
reduction of TNF--induced eotaxin release.
|
To pursue the role of cAMP in the effect of Salbu and Salme, we
determined whether the effect of cAMP stimulants could be mimicked by
the membrane-permeable cAMP analog 8-Br-cAMP. We found that 8-Br-cAMP
(10 and 100 µM), like the cAMP stimulants tested, had no effect on
basal levels of eotaxin release (data not shown), but that pretreatment
with 8-Br-cAMP (100 µM) significantly inhibited TNF--induced
eotaxin release (Fig. 6A
, B
, P<0.05, P<0.01, respectively);
when 8-Br-cAMP was used in combination with Dex (1 µM) or Flut (0.1
µM), a significant additional inhibition over the effect of 8-Br-cAMP
or the steroid alone was achieved (Fig. 6A
, B
,
P<0.05, P<0.001, respectively).
|
The results suggest that the inhibition of eotaxin release by ß2-agonists alone or in combination with steroids is via a cAMP-dependent mechanism.
PKA and PKG involvement in the combined inhibition
To clarify the role of cAMP in the above combined inhibition of
eotaxin release, we examined whether H-89 and Rp-cAMP, potent and
selective inhibitors of the cAMP-dependent PKA, and KT5823, a potent
and selective inhibitor of the cGMP-dependent PKG, could reverse the
combined inhibition. As shown in Fig. 7A
, H-89 (0.1 and 1 µM), Rp-cAMP (10 and 100 µM), and
KT5823 (0.1 and µM) on their own did not significantly alter
TNF--induced eotaxin release. The combined inhibition on
TNF--induced eotaxin release by Salbu and Dex was not reversed but
was further enhanced by both H-89 (1 µM, P<0.001) and
KT5823 (1 µM, P<0.001) but not by Rp-cAMP (100 µM)
(Fig. 7B
). The results suggest that neither PKA nor PKG is
involved in the inhibition on TNF--induced eotaxin release by the
combination of ß2-agonists and steroids.
|
Comparison of the effects of Salbu and Dex on TNF--induced
eotaxin release at various time points
To compare different mechanisms involved in the inhibition of
TNF--induced eotaxin release by the
ß2-agonists and steroids, Salbu and Dex were
added at different time points against TNF- stimulation. Maximum
inhibition by Salbu was observed when it was added 1 h before
TNF- (Fig. 8
, P<0.001). The inhibition was thereafter time-dependently
reduced when Salbu was added at the same time as TNF-
(P<0.01) or 1 h (P<0.001), 2 h
(P<0.001), and 4 h (P<0.001) after
TNF-. Marked inhibition by Dex was also achieved when it was added
1 h before TNF- (Fig. 8
, P <0.001). However, no
significant changes were observed when Dex was added 1 h, 2 h, and 4 h after TNF- (Fig. 8)
.
|
Cell viability
Cell viability after 16 h treatment with the chemicals used
in this study was consistently >95% compared with cells treated with
the vehicle.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We demonstrated that TNF- is a potent inducer of eotaxin release by
HASM cells as has been described previously (18)
. Elevated
levels of proinflammatory cytokines, such as IL-1ß and TNF-, in
BALF from asthmatic subjects have been reported (15
, 16)
,
indicating they may play an important role in the development of airway
inflammation in asthma. We used TNF- in this study rather than
IL-1ß, as we have shown that IL-1ß, induces COX-2, the inducible
form of cyclooxygenase in HASM cells, causing substantial prostanoid
release and resulting in impairment of ß-agonist-induced cAMP
generation (26
, 27)
. In contrast, TNF- neither induced
COX-2 nor impaired adenylyl cyclase function over the time course used
in our experiments (26
, 27)
, thus providing a good model
to study cAMP manipulation of cytokine-induced eotaxin release.
We showed that when given alone, the short- and long-acting
ß2-agonists Salbu and Salme did not alter the
basal level eotaxin release but, when given before TNF- stimulation,
markedly inhibited TNF--induced eotaxin release. We also found from
this study that TNF--stimulated eotaxin release was
concentration-dependently but partially inhibited by both Dex and Flut.
This is in consistent with the fact that steroids are strong inhibitors
of the expression of a large number of inflammatory genes, including
chemokines such as IL-8 (28
, 29)
, RANTES
(30)
, and eotaxin (17)
, but does not agree
with a recent study showing that Dex (1 µM) does not inhibit both
IL-1ß- and TNF--induced eotaxin release and mRNA expression
(18)
.
We then studied the possible synergistic or additive inhibitory effects
when ß2-agonists and other cAMP stimulants were
used in combination with corticosteroids. We found that an additive but
not synergistic inhibition on TNF--induced eotaxin release was
achieved when the two types of drugs were used together. In subsequent
experiments to explore the cAMP dependence of the effect of
ß2-agonists, we found that the effects of
ß2-agonists on TNF--stimulated eotaxin
release and on steroid-mediated suppression of the release were
mimicked by both FSK and 8-Br-cAMP, reversed by the specific
ß2-receptor antagonist ICI 118,551, and
potentiated by the type IV phosphodiesterase inhibitor rolipram. As
would be expected, ICI 118,551 had no effect on the additive inhibition
produced by Dex and FSK as FSK activates adenylyl cyclase directly.
Neither FKS, the direct activator of adenylyl cyclase, nor the
membrane-permeable cAMP analog 8-Br-cAMP altered the basal level of
eotaxin release. The results suggest that the inhibition of eotaxin
release by ß2-agonists alone or in combination
with steroids is mediated by ß2-receptors via a
cAMP-dependent mechanism.
The precise mechanism of the effect of cAMP on TNF--induced eotaxin
release is not known. We found that the inhibition of TNF--induced
eotaxin release was greater when Salbu was added 1 h before
TNF- than when added at the same time or 1–4 h after TNF-. In
contrast, the inhibition by Dex remains virtually unchanged whether
added 1 h before, the same time as, or 1–4 h after TNF-. The
results suggest that the mechanisms by which cAMP and steroids regulate
TNF--induced eotaxin release are likely to be different. The cAMP
regulation is largely at the early stage of eotaxin release (i.e., at
transcription stage), whereas the effect of Dex may involve the late
stage of eotaxin release (i.e., at post-transcription stage). Our
results with Dex may explain why it did not inhibit cytokine-induced
eotaxin mRNA expression (18)
but cannot explain why it did
not inhibit cytokine-induced eotaxin release in one previous study
(18)
. However, the study did show a 35% inhibition,
although not statistically significant, of TNF--induced eotaxin
release from HASM cells by Dex; if larger samples had been studied, a
significant inhibition by Dex might have been achieved
(18)
.
Since NF
B, AP-1, and glucocorticoid response element, but not cAMP
response element (CRE), are present on the eotaxin gene promoter
(19
, 20)
, transcriptional elements other than CRE are
likely to play a major role in the regulation of eotaxin gene
expression by cAMP. Some reports suggest that cAMP-dependent PKA may
stabilize IB, impair nuclear transport of NFB p65, and
consequently inhibit certain gene expression (31)
. If this
is the mechanism for the effect of cAMP in our present study, the
inhibition by Dex and Salbu on TNF--induced eotaxin release would be
significantly reversed by PKA inhibitors. In fact, we found that the
inhibition was further enhanced by the specific PKA inhibitor H-89.
Similar results were also obtained with another specific PKA inhibitor,
KT5720 (data not shown), but Rp-cAMP was without effect. Although it is
possible that high levels of PKA stimulation may be difficult to be
antagonized by PKA inhibitors and higher concentrations of PKA
inhibitors may result in nonspecific inhibition of other protein
kinases, the results nevertheless suggest that the inhibitory effect of
cAMP stimulants on eotaxin release may not be mediated by PKA. Indeed,
PKA may potentiate TNF--stimulated eotaxin release from HASM cells.
There may be cross talk between cAMP and other signaling pathways that
is independent of PKA. This is supported by a recent report
demonstrating that both PKA-dependent and -independent pathways
contribute to cAMP-mediated mitogenesis and that phosphatidylinositol
3-kinase is involved in the PKA-independent signaling pathways
(32)
. Some transcription factors may also be involved in
the cAMP-mediated but PKA-independent inhibition of TNF--induced
eotaxin release. For instance, CRE binding protein 2 (CREB-2) is an
unconventional member of the activation transcription factors
(ATF)/CREB family because it lacks a potential PKA phosphorylation site
and has been demonstrated to negatively regulate CRE-dependent
transcription (33)
. How it regulates the transcription of
genes that lack CRE in their promoters, however, requires further
investigation. Our results showed that the inhibition by Dex and Salbu
on TNF--induced eotaxin release was also further enhanced by a
PKG-specific inhibitor, KT5823, suggesting that PKG may antagonize the
cAMP-mediated inhibition of eotaxin release, although the mechanisms
are not known.
The family of transcription factors includes nuclear receptors for
steroids, which are generally activated by binding of specific ligands.
A variety of nuclear receptors appear to respond to elevations of cAMP,
but whether the response is due to phosphorylation of the receptor by
PKA is not known. For some nuclear receptors, stimulation by PKA may
activate the receptor in the absence of ligand. Such an effect has been
demonstrated for the androgen receptor (34)
.
ß2-Agonists have also been recently shown to
increase the nuclear translocation of the glucocorticoid receptor (GR)
in primary lung fibroblasts and vascular smooth muscle cells
(35)
. To examine whether the mechanism could provide an
explanation for the effect of cAMP seen in our studies, we conducted
experiments to compare the effects of Dex and Salbu, Salme, and FSK on
the nuclear translocation of the GR in HASM cells. After 30 min to
4 h incubation, Dex stimulated the nuclear translocation of the
GR, whereas none of the cAMP stimulants had any effect and no further
translocation was observed when Dex was used together with the cAMP
stimulants (data not shown). The results clearly indicate that the
inhibitory effect of cAMP on eotaxin release differs from that of
steroids and is not mediated by the ligand-independent activation of
GR. In general, the mechanisms of cAMP-dependent inhibition on
TNF--induced eotaxin release from HASM cells are complex and largely
remain to be explored. Future studies to determine the roles of PKA and
PKG, the interaction between PKA/PKG and transcription factors and the
cross talk between cAMP and other signal transduction systems are
likely to provide some answers.
We have also performed studies looking at the effects of
ß2-agonists and steroids on TNF--stimulated
IL-8 release (unpublished observations). The results of the IL-8
studies differ slightly in that ß2-agonists
stimulate IL-8 release on their own, but when given in combination with
steroids they synergistically inhibit TNF--induced IL-8 release.
This suggests that interactions between
ß2-agonists and steroids are chemokine
specific.
The additive inhibition of ß2-agonists and
steroids on TNF--stimulated eotaxin release seen in our study is
interesting. Recent studies, particularly those with long-acting
ß2-agonists, have shown that these agents
potentiate the effects of corticosteroids on airflow and asthma
symptoms and cause a reduction in asthma exacerbations (36
, 37)
. Our data may be relevant particularly to the reduction in
asthma exacerbations that occurred in these studies and may suggest a
possible mechanism whereby ß2-agonists
potentiate the reduction in cytokine-stimulated eotaxin release
produced by corticosteroids. The cAMP-specific type IV PDE inhibitors
have been extensively studied for the development of new PDE inhibitors
for asthma therapy. Most evidence so far suggests that they possess
bronchodilator activity and may exert an effect against the
inflammatory aspects of asthma. Our results showed that the combined
use of a ß2-agonist, a steroid, and a PDE
inhibitor abolished TNF--induced eotaxin release, suggesting that
the anti-inflammatory aspect of PDE inhibitors may be an important
mechanism for their use in asthma therapy.
In conclusion, we found that TNF- stimulated eotaxin release in HASM
cells and the release was partially inhibited by
ß2-agonists and corticosteroids. An additive
inhibition was achieved when both types of drugs were used together.
FSK and 8-bromo-cAMP mimicked and rolipram enhanced the effect of
ß2-agonists, indicating that it is cAMP
dependent. Although the inhibition by cAMP was likely to be at the
transcription stage, experiments with PKA and PKG inhibitors suggested
that neither of these kinases is involved in the process. Our findings
may be relevant to the mechanisms of action of these drugs in asthma
and may provide a mechanistic explanation for the findings that the
combined use of a long-acting ß2-agonist and an
inhaled corticosteroid produces complementary benefits on symptoms and
airflow and potentiates the ability of steroids to reduce asthma
exacerbations.
|
ACKNOWLEDGMENTS |
|---|
Received for publication April 20, 2000.
Revision received July 17, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
: effects of IL-10 and corticosteroids. Br. J. Pharmacol. 127,1145-1150[Medline]
and interferon-
on the induction of cyclo-oxygenase-2 in cultured human airway smooth muscle cells. Br. J. Pharmacol. 121,579-587[Medline]
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 O. Tliba, Y. Amrani, and R. A. Panettieri Jr Is Airway Smooth Muscle the "Missing Link" Modulating Airway Inflammation in Asthma? Chest, January 1, 2008; 133(1): 236 - 242. [Abstract] [Full Text] [PDF]
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 O. Tliba and Y. Amrani Airway Smooth Muscle Cell as an Inflammatory Cell: Lessons Learned from Interferon Signaling Pathways Proceedings of the ATS, January 1, 2008; 5(1): 106 - 112. [Abstract] [Full Text] [PDF]
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 M. Kaur, J. E. Chivers, M. A. Giembycz, and R. Newton Long-Acting 2-Adrenoceptor Agonists Synergistically Enhance Glucocorticoid-Dependent Transcription in Human Airway Epithelial and Smooth Muscle Cells Mol. Pharmacol., January 1, 2008; 73(1): 203 - 214. [Abstract] [Full Text] [PDF]
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 M. R. Edwards, J. Haas, R. A. Panettieri Jr., M. Johnson, and S. L. Johnston Corticosteroids and beta2 Agonists Differentially Regulate Rhinovirus-induced Interleukin-6 via Distinct Cis-acting Elements J. Biol. Chem., May 25, 2007; 282(21): 15366 - 15375. [Abstract] [Full Text] [PDF]
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S. Keslacy, O. Tliba, H. Baidouri, and Y. Amrani Inhibition of Tumor Necrosis Factor-{alpha}-Inducible Inflammatory Genes by Interferon-{gamma} Is Associated with Altered Nuclear Factor-{kappa}B Transactivation and Enhanced Histone Deacetylase Activity Mol. Pharmacol., February 1, 2007; 71(2): 609 - 618. [Abstract] [Full Text] [PDF]
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M. R. Edwards, M. W. Johnson, and S. L. Johnston Combination Therapy: Synergistic Suppression of Virus-Induced Chemokines in Airway Epithelial Cells Am. J. Respir. Cell Mol. Biol., May 1, 2006; 34(5): 616 - 624. [Abstract] [Full Text] [PDF]
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 L. Pang, M. Nie, L. Corbett, A. Sutcliffe, and A. J. Knox Mast Cell beta-Tryptase Selectively Cleaves Eotaxin and RANTES and Abrogates Their Eosinophil Chemotactic Activities J. Immunol., March 15, 2006; 176(6): 3788 - 3795. [Abstract] [Full Text] [PDF]
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O. Tliba, J. A. Cidlowski, and Y. Amrani CD38 Expression Is Insensitive to Steroid Action in Cells Treated with Tumor Necrosis Factor-{alpha} and Interferon-{gamma} by a Mechanism Involving the Up-Regulation of the Glucocorticoid Receptor beta Isoform Mol. Pharmacol., February 1, 2006; 69(2): 588 - 596. [Abstract] [Full Text] [PDF]
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 S. Baouz, J. Giron-Michel, B. Azzarone, M. Giuliani, F. Cagnoni, S. Olsson, R. Testi, G. Gabbiani, and G. W. Canonica Lung myofibroblasts as targets of salmeterol and fluticasone propionate: inhibition of {alpha}-SMA and NF-{kappa}B Int. Immunol., November 1, 2005; 17(11): 1473 - 1481. [Abstract] [Full Text] [PDF]
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 O. S. Usmani, K. Ito, K. Maneechotesuwan, M. Ito, M. Johnson, P. J. Barnes, and I. M. Adcock Glucocorticoid Receptor Nuclear Translocation in Airway Cells after Inhaled Combination Therapy Am. J. Respir. Crit. Care Med., September 15, 2005; 172(6): 704 - 712. [Abstract] [Full Text] [PDF]
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S. E. Overbeek, P. G. Mulder, S. M. Baelemans, H. C. Hoogsteden, and J.-B. Prins Formoterol Added to Low-Dose Budesonide Has No Additional Antiinflammatory Effect in Asthmatic Patients Chest, September 1, 2005; 128(3): 1121 - 1127. [Abstract] [Full Text] [PDF]
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