Division of Respiratory Medicine, City Hospital, University of Nottingham, Nottingham NG5 1PB, United Kingdom
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ABSTRACT |
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We have recently shown that endogenous prostanoids are critical in
bradykinin-stimulated interleukin (IL)-8 release from human airway
smooth muscle (ASM) cells. In this study, we tested the ability of
transforming growth factor (TGF)-1 to stimulate IL-8 release,
cyclooxygenase (COX)-2 expression and PGE2 generation in
cultured human ASM cells and explored the role of COX products and
COX-2 induction on IL-8 release. TGF-
1 stimulated IL-8 release, COX-2 induction, and PGE2 generation in a concentration-
and time-dependent manner. Maximal IL-8 release was achieved with 10 ng/ml of TGF-
1 after 16 h of incubation, which was inhibited by
the transcription inhibitor actinomycin D and the corticosteroid
dexamethasone but was not affected by the nonselective COX inhibitor
indomethacin and the selective COX-2 inhibitor NS-398 despite their
inhibition on TGF-
1-induced PGE2 release. These results
show for the first time that TGF-
1 stimulates IL-8 release, COX-2
induction, and PGE2 generation in human ASM cells and that
PGE2 generation is not critical for TGF-
1-induced IL-8
release. These findings suggest that TGF-
1 may play an important
role in the pathophysiology of asthma.
transforming growth factor-1; interleukin-8; cytokines; airway
inflammation; asthma; prostaglandin E2
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INTRODUCTION |
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ASTHMA IS A
CHRONIC airway inflammatory disease that is associated with
airway remodeling (30). Transforming growth factor (TGF)- is a multifunctional cytokine that contributes to the initiation and resolution of inflammatory events (36) and
may contribute to the pathophysiology of asthma. TGF-
1 gene
expression is upregulated in bronchial tissue from severe asthmatic
subjects (19), and increased immunostaining for TGF-
is
seen in bronchial biopsies from asthmatic and chronic bronchitic
subjects compared with biopsies from normal subjects (35).
Consistent with this, TGF-
concentrations are higher in
bronchoalveolar lavage fluid in subjects with atopic asthma compared
with those in normal control subjects (31). Furthermore,
the development of subepithelial fibrosis in asthmatic airways
correlates with the number of fibroblasts and the expression of
TGF-
1 (8). TGF-
is produced by a number of cells
including platelets, macrophages, bronchial epithelial cells,
(28) and airway smooth muscle (ASM) cells
(16). Several airway cells such as fibroblasts and
epithelial and endothelial cells serve as a target for the effects of
TGF-
(5, 29). TGF-
consists of a
superfamily of structurally related proteins, with TGF-
1, TGF-
2,
and TGF-
3 being expressed in mammals. TGF-
1 is the isoform
commonly implicated in inflammation and is upregulated in response to
tissue injury (5).
A previous study (4) has suggested that ASM cells
may serve as target cells for TGF-1 in asthma, whereas TGF-
1 has
been shown to enhance production of extracellular matrix components by
ASM. This is consistent with recent studies showing that ASM may be a
rich source of inflammatory mediators. For instance, ASM cells are able
to release neutrophil and eosinophil chemoattractants such as
interleukin (IL)-8 (24), regulated on activation
normal T cells expressed and secreted (RANTES) (10), and
granulocyte-macrophage colony-stimulating factor (32) in
response to cytokines. These cells also synthesize large quantities of
prostanoids via either the constitutive cyclooxygenase (COX)-1 or the
inducible COX-2 in response to cytokines (3,
22, 23) or inflammatory mediators (24).
TGF-1 has been shown to increase IL-8 release in human
endometrial cells and rat lung alveolar epithelial cells
(2, 7). Additionally, the COX-2 gene has also
been shown to have a TGF-
response element (38).
However, it is not known whether TGF-
1 can modify inflammation in
asthma by influencing the production of chemokines or prostanoids. Here
we tested the hypothesis that TGF-
1 might stimulate IL-8 release and
induce COX-2 expression and prostanoid release in human ASM cells.
Because a previous study by Pang and Knox (24)
with bradykinin has shown that PGE2 production was a
critical event in IL-8 release, we also investigated whether COX-2
induction and PGE2 release play a role in TGF-
1-induced IL-8 release.
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MATERIALS AND METHODS |
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Cell culture. Primary cultures of human ASM cells were prepared from explants of ASM according to methods previously reported by our group (22, 23). Human tracheae were obtained from two postmortem individuals within 12 h of death. The two patients were both male; one was an ex-smoker who died of a ruptured arterial aneurysm and hematoperitoneum at the age of 70, and the other was a nonsmoker who died of myeloma at the age of 82. There was no evidence of airway disease as determined by history and macroscopic examination of the trachea and lungs. Cells at passages 3 and 4 from the two donors were used for all experiments. Pang and Knox (22) have previously shown that cells grown in this manner depict the immunohistochemical and light-microscopic characteristics of typical ASM cells.
Experiment protocol.
The cells were plated at a density of 2 × 104
cells/well in 24-well culture plates and cultured to confluence in 10%
FCS (Seralab, Crownly Down, UK)-DMEM (Sigma, Poole, UK) in humidified
5% CO2-95% air at 37°C and growth arrested in FCS-free
DMEM for 24 h before the experiments. Immediately before each
experiment, fresh FCS-free DMEM containing TGF-1 (endotoxin tested;
Sigma) was added. In the time-course experiments, the cells were
incubated with TGF-
1 (10 ng/ml) for 30 min to 24 h. In the
concentration-response experiments, the cells were incubated for
16 h with 0.01-100 ng/ml of TGF-
1. The culture media were
harvested at the indicated times and stored at
20°C before analysis
with ELISA for IL-8 and with RIA for PGE2 content as a
representative of prostanoid generation. Protein extraction was
performed before Western blot analysis to measure COX-1 and COX-2
expression. Protein was extracted by incubating the cells for 5 min
with 50 µl/well of protein extraction buffer (0.9% NaCl, 20 mM
Tris · HCl, pH 7.6, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 0.01% leupeptin; all from Sigma) and shaking gently. The protein extraction buffer was then harvested and stored at
20°C for subsequent Western blot analysis. To test the mechanisms involved in the effect of TGF-
1, the nonselective COX
inhibitor indomethacin (Indo), the gene transcription inhibitor actinomycin D (Act D), the anti-inflammatory steroid dexamethasone (Dex; all from Sigma), the selective COX-2 inhibitor NS-398
{N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide; Cayman Chemical, Ann Arbor, MI}, or vehicle control (DMSO) was added
at various concentrations 1 h before the addition of TGF-
1.
IL-8 assay.
The concentration of IL-8 in the culture medium was determined by ELISA
(CLB, Amsterdam, The Netherlands) according to the manufacturer's instructions. Briefly, ELISA plates were coated overnight at room temperature with 200 µl of anti-human IL-8 coating antibody that had been diluted in 0.1 M carbonate-bicarbonate buffer
(pH 9.6). The plates were then washed five times with PBS (pH
7.2-7.4) containing 0.005% Tween 20 and blocked for 1 h at room temperature with 200 µl of blocking buffer. The plates were washed again, and 100 µl of samples containing standard amounts of
recombinant human IL-8 as well as study samples obtained from harvested culture medium (diluted 1:5 with dilution buffer) were added
in duplicate to individual wells and incubated at room temperature for
1 h. After five washes, 100 µl of biotinylated IL-8 antibody diluted in dilution buffer were added for 1 h. After another five washes, 100 µl of streptavidin-horseradish peroxidase (HRP) conjugate that had been diluted 1:10,000 in dilution buffer were added for 30 min. After a final wash, 100 µl of the substrate buffer containing the HRP substrate tetramethylbenzidine dihydrochloride and hydrogen peroxide in 0.05 M phosphate-citrate buffer (pH 5.0) were added for 30 min in the dark, and color developed in proportion to the amount of
IL-8 present. The reaction was stopped by adding 100 µl of stop
solution (1.8 M sulfuric acid). The degree of color that had been
generated was determined by measuring the optical density at 450 nm in
a Dynatech MR5000 microplate reader (Billinghurst, UK). The
standard curve was linearized and subjected to regression analysis. The
IL-8 concentration of unknown samples was extracted using the standard
curve. The results are expressed as picograms per milliliter. The
sensitivity of the ELISA kit in our study was at least 5 pg/ml, which
was consistent with the manufacturer's specifications. According to
the kit insert, the anti-IL-8 antibody does not cross-react with IL-1
through IL-7, IL-9 through IL-11, tumor necrosis factor,
interferon-, granulocyte-macrophage colony-stimulating factor, and
RANTES. All the reagents used were supplied by the ELISA kit
manufacturer with the exception of the HRP substrate tetramethylbenzidine dihydrochloride, which was obtained from Sigma.
Western blot analysis. COX-1 and COX-2 expression were assessed by Western blotting. The protein concentration of cell extracts was determined with the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hemel Hempstead, UK). Sufficient aliquots of sample (30 µg protein/track) were mixed 1:1 with sample buffer [20 mM Tris · HCl, pH 6.8, 20% glycerol, 2% sodium dodecyl sulfate (SDS), 5% 2-mercaptoethanol, and 0.025% bromphenol blue; all from Sigma] and boiled for 5 min before electrophoresis. Electrophoresis was performed on these samples on a 20 × 20-cm 7.5% SDS-polyacrylamide gel (45 mA, 5 h). The separated proteins were then electroblotted (150 V, 3 h) to pure nitrocellulose membrane (Gelman Sciences, Northampton, UK). The blot was blocked for 2 h at 4°C in blocking reagent [8% fat-free dried milk powder in PBS, pH 7.4, with 0.3% Tween 20 (PBS-T)], incubated with primary monoclonal anti-human COX-2 antibody (1:2,000 in blocking reagent; Cayman Chemical) for 2 h at room temperature before being washed with PBS-T and incubated with rabbit anti-mouse IgG coupled with HRP (1:2,000 in blocking reagent; Sigma) for 1 h at room temperature. Semiquantitative staining was achieved by using enhanced chemiluminescence detection. This detection was performed by washing the blot with PBS-T, incubating it with the SuperSignal CL-HRP substrate system (Pierce, Rockford, IL) for 1 min, and finally exposing it to Hyperfilm ECL (Amersham Life Science, Little Chalfont, UK). The positions and molecular masses of COX-2 and COX-1 were validated by reference to rainbow-colored molecular mass markers (Amersham Life Science). Reprobing of COX-1 was carried out by incubating the membrane in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris · HCl) at 50°C for 30 min with occasional agitation, washing the membrane in a large volume of PBS-T, blocking the membrane for 2 h in blocking reagent, and then following the steps described above to detect COX-1 with monoclonal anti-ovine COX-1 antibody (with cross-reactivity to human COX-1, 1:2,000 in blocking reagent; Cayman Chemical).
PGE2 assay.
Assays were performed in duplicate and are expressed as picograms per
milliliter. Doubling dilutions of the PGE2 standard (Sigma)
over the concentration range 1,000-7.5 pg/100 ml were made up in
assay buffer (1 g of gelatin, 6.1 g of NaCl, 10.8 g of
K2HPO4, 1.7 g of KH2PO4, and
0.7 g of NaN3 in 500 ml of distilled H2O)
and used to construct the standard curve. Prepared standards and
samples were then incubated overnight with
[3H]PGE2 (Amersham) and rabbit
PGE2 antiserum (1:8 with assay buffer; Sigma) in assay
buffer at 4°C. The bound labeled antibody complexes were then
separated with the use of dextran-coated charcoal. Samples and
standard were then centrifuged for 15 min at 3,500 rpm (4°C) to
precipitate the unbound labeled PGE2. The supernatants
were then transferred into scintillation vials, and 10 ml of
scintillation emulsifier cocktail (Packard, Pangbourne, UK) were added
to each vial. The samples were then counted with a -counter (5 min/vial). The anti-PGE2 antiserum (Sigma) in our hands had
negligible cross-reactivity (22).
Cell viability.
The toxicity of all chemicals used in this study [e.g.,
TGF-1, COX inhibitors, Act D, Dex, and drug vehicles DMSO and
ethanol (final concentration 1% vol/vol; Sigma)] to human ASM cells
was determined by the
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
assay. After a 24-h incubation with the chemicals, 20 µl of 5 mg/ml
of MTT were added to the culture medium in 96-well plates and incubated
for 1 h at 37°C. After the medium was removed, 200 µl of DMSO
were added to solubilize the blue-colored tetrazolium, the plates were
shaken for 5 min, and the optical density values at 550 nm were read in
a microplate reader. Viability was set as 100% in control cells.
Statistical analysis. Data are expressed as means ± SE from n determinations. The statistical analysis was performed with software from SPSS (34). A one-way ANOVA and/or an unpaired, two-tailed Student's t-test was used to determine the significant differences between the means. The results were adjusted for multiple testing with Bonferroni's correction. P values < 0.05 were accepted as significant.
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RESULTS |
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Effect of TGF-1 on IL-8 release.
Time-course and concentration-response experiments were conducted to
investigate the effect of TGF-
1 on IL-8 release. In the time-course
experiments (Fig. 1A), there
was a slight increase in IL-8 release from control cells over the 24-h
incubation (from 5.2 ± 2.1 pg/ml at 30 min to 9.9 ± 0.6 pg/ml at 24 h). In cells stimulated with TGF-
1, there was a
time-dependent increase in IL-8 release. A significant difference in
IL-8 release after stimulation with TGF-
1 was observed after 8 (P < 0.01), 16 (P < 0.001), and 24 (P < 0.01) h. Because the highest IL-8 release was
obtained at 16 h (133.7 ± 16.9 pg/ml), this time point was
used in subsequent experiments to evaluate the effect of TGF-
1. In
the concentration- response experiments, the cells were cultured with
TGF-
1 (0.01, 0.1, 1.0, and 10 ng/ml) for 16 h. A
concentration-dependent increase in IL-8 release, which became
significant at 1.0 (P < 0.01) and 10 (P < 0.001; Fig. 1B) ng/ml, was observed.
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Effect of Dex and Act D on TGF-1-induced IL-8 release.
The effect of the corticosteroid Dex and the gene transcription
inhibitor Act D on TGF-
1-induced IL-8 release was assessed. Pretreatment for 1 h with Act D and Dex (both 1.0 µM) before
incubation with TGF-
1 (10 ng/ml) for 16 h markedly inhibited
TGF-
1-induced IL-8 release (P < 0.001 for both;
Fig. 2).
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Effect of TGF-1 on PGE2 release.
TGF-
1 (10 ng/ml) caused a time-dependent accumulation of
PGE2 compared with that in the control cells (Fig.
3A). TGF-
1-induced PGE2 generation was significant after 2 h compared
with PGE2 release from control cells (P < 0.01), and the highest PGE2 concentration was achieved
after 16 h of stimulation (201.7 ± 30.4 pg/ml;
P < 0.01). In the concentration response experiments,
the cells were cultured with TGF-
1 (0.01, 0.1, 1.0, and 10 ng/ml)
for 16 h, and a concentration-dependent increase in
PGE2 release, which was significant at 0.1 (P < 0.05), 1.0 (P < 0.05), and 10 (P < 0.001) ng/ml compared with that in control cells
(Fig. 3B), was also observed.
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COX isoform induction in response to TGF-1.
COX-2 was undetectable in untreated human ASM cells. In the time-course
experiments, COX-2 protein bands appeared 2 h after treatment with
TGF-
1 (10 ng/ml), and a strong band was seen at 4, 8, and 16 h,
which then diminished at 24 h (Fig.
4). A concentration-dependent increase in
COX-2 induction was seen as TGF-
1 concentration was increased from
0.1 to 100 ng/ml (Fig. 4). Pretreatment of the cells with Dex (1.0 µM) abolished the TGF-
1-induced COX-2 expression (Fig. 4). COX-1
was expressed constitutively and did not alter after TGF-
1 treatment
(Fig. 4).
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Effect of COX inhibitors on TGF-1-induced IL-8 and
PGE2 release.
To determine whether COX products played a role in TGF-
1-induced
IL-8 release, we pretreated the cells with the nonselective COX
inhibitor Indo and the COX-2-selective inhibitor NS-398 for 1 h
before incubation with TGF-
1 (10 ng/ml) for 16 h. Neither Indo
(10 µM) nor NS-398 (10 µM) had any effect on TGF-
1-induced IL-8
release (Fig. 5A). However,
TGF-
1-induced PGE2 release was significantly inhibited
by both Indo (P < 0.01) and NS-398 (P < 0.05; Fig. 5B).
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Cell viability. Cell viability after 24 h of treatment with all of the chemicals used in this study was consistently >95% compared with vehicle-treated cells (data not shown).
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DISCUSSION |
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There are several novel findings in our study. This is the first
study to show that TGF-1 can cause release of IL-8, induction of
COX-2, and generation of PGE2 by human ASM cells. This is
consistent with the hypothesis that human ASM cells can actively modify
airway inflammation by expressing and secreting inflammatory products and cytokines (11). Unlike a previous study
(24) where prostanoid generation was a prerequisite
for bradykinin-induced IL-8 release, COX-2 isoenzyme induction and
PGE2 production were not critical for TGF-
1-induced IL-8
release. This suggests that TGF-
1 uses different signaling pathways
to release IL-8 compared with those for bradykinin.
We found that human ASM cells released IL-8 in a time- and
concentration-dependent manner in response to TGF-1. However, a
significant increase could only be seen after 8 h of treatment, suggesting that the induction of IL-8 is delayed. We have demonstrated that significant IL-8 release from human ASM cells can be observed after 2 h of treatment with bradykinin and that the effect is largely mediated by endogenous prostanoids. The present results suggest
that the effect of TGF-
1 may be due to the release of an as yet
unidentified mediator, but further work is needed to investigate this.
We also showed that the magnitude of IL-8 release by TGF-
1 was, in
general, similar to that seen with bradykinin (24). Our
findings are similar to the effects of TGF-
1 on IL-8 production in
human endometrial stromal cells and human proximal tubular epithelial
cells (2, 7). IL-8 has been implicated in
several airway diseases such as cystic fibrosis, bronchiectasis, and
chronic bronchitis (14). Increased levels of IL-8 have
also been demonstrated in bronchoalveolar lavage fluid
(37), by macrophages in bronchoalveolar lavage fluid
(6), in the blood and bronchial mucosa (33),
and by bronchial epithelial cells (17) in asthmatic patients. Overproduction of IL-8 has been implicated in inflammatory cell chemotaxis in asthma, and our results suggest that human ASM cells
may respond to TGF-
1 to act as a source of this cytokine in asthma.
Previous studies by Pang and Knox (24, 25)
with bradykinin and tumor necrosis factor-
show that they are also able to increase IL-8 release from human ASM cells.
We found that the corticosteroid Dex and Act D, a gene transcription
inhibitor, significantly inhibited TGF-1-induced IL-8 release,
suggesting that the increased release of IL-8 was transcriptionally regulated. The efficacy of glucococorticoids in asthma is known to be
due to their ability to disrupt cytokine networks in the lung tissue
(13). Several studies (reviewed in Ref. 26) showed that
there is a nuclear factor (NF)-
B site on the IL-8 promoter and that
Dex is thought to act by inhibiting NF-
B, thereby suppressing IL-8 transcription.
We also determined the effect of TGF-1 on COX isoform expression and
prostanoid generation from human ASM. We measured PGE2 as a
representative of other prostanoids because Pang and Knox (22) have previously shown that PGE2 is the
dominant prostanoid produced by these cells. Prostanoids are important
regulators in the inflammatory process of asthma. Their synthesis is
mediated by COX, which exists in two isoforms (18), the
constitutive COX-1 and the inducible COX-2. COX-1 is constitutively
expressed under physiological conditions in most cells in which it
maintains cellular homeostasis by producing physiological levels of
prostaglandin (18). Prostaglandins produced by COX-1
generally have a protective function, such as gastric mucus production
and renal blood flow maintenance, in non-airway cells. Similarly in the
airway, COX-1 is expressed constitutively in ASM cells, mast cells,
alveolar macrophages, and fibroblasts (3,
22). In contrast, COX-2 expression is regulated by
proinflammatory stimuli including cytokines (15,
22) and proinflammatory mediators such as bradykinin (23). As in previous studies (22, 23), we found
that COX-1 was the only isoform expressed under resting conditions,
consistent with its putative housekeeping role. We found that TGF-
1
stimulated PGE2 production through COX-2 induction in a
time- and concentration-dependent manner. No previous studies have
looked at the effect of TGF-
1 on COX-2 isoform expression in human
ASM cells. These findings are similar to the effect of TGF-
1 in
astrocytes (15), neurons (15), and lung
fibroblasts (9). TGF-
1 may induce COX-2 by activating
the TGF-
response element that is localized in a 166-bp region of
the COX-2 gene promoter near the transcriptional initiation site where
NF-I and NF-
B sites are also located (38). TGF-
may
also act on other response elements like jun B and serum response factor (38) to induce COX-2. We found that Dex inhibited
COX-2 induction. This was likely to be due to inhibition of NF-
B
because the human COX-2 promoter region has two NF-
B binding sites
(1). Our data on TGF-
1 complements previous studies
(18, 22-24) that have shown that COX-2
in ASM is induced by a number of cytokines and proinflammatory stimuli
such as bradykinin, suggesting that COX-2 expression may play a pivotal
role in the pathophysiological process of asthma.
However, it is unclear whether the increased expression of COX-2 and
PGE2 production in human ASM by TGF-1 would be
potentially beneficial or detrimental in the pathogenesis of airway
inflammation in asthma. Prostanoids have multiple functions in the
airways that include modulating airway tone, cell proliferation, and
mucus secretion. PGE2 is an important anti-inflammatory
mediator with considerable bronchoprotective effects (27),
and the exaggerated PGE2 production as a result of COX-2
induction may be part of a negative feedback mechanism to exert a
braking effect on the inflammatory process (13). There
are, however, some potential proinflammatory effects of COX-2 induction
because PGE2 at high concentrations can cause ASM
contraction due to agonism at the thromboxane receptor
(12). Induction of COX-2 in inflammatory cells may also
exaggerate airway inflammation by producing bronchoconstricter prostanoids such as PGD2 and PGF2
. Pang et
al. (20, 21) have also recently shown that
induction of COX-2 in response to IL-1
or bradykinin in human ASM
can cause heterologous desensitization of adenylyl cyclase in human
ASM, providing a possible explanation for the defective relaxation to
-adrenoceptor agonists demonstrated in human asthmatic bronchi in vitro.
Because our previous studies have shown that prostanoid
generation is required for IL-8 release by bradykinin (24), we also studied whether COX-2 induction and PGE2 generation were
essential for TGF-1-induced IL-8 release. In the present study, we
used the nonselective COX inhibitor Indo and the selective COX-2
inhibitor NS-398 to inhibit prostanoid generation. We found that
although both Indo and NS-398 markedly inhibited TGF-
1-induced
PGE2 generation, they did not inhibit TGF-
1-induced IL-8
release. These data suggest that COX products and COX-2 induction are
not directly involved in TGF-
1-induced IL-8 release, unlike
bradykinin-induced IL-8 release. This is consistent with results
obtained from our laboratory showing that IL-1
-induced IL-8 release
is also independent of COX-2 induction and PGE2 generation
(Pang and Knox, unpublished data). We have considered possible
explanations for these disparate findings. The time course and
mechanism of prostanoid generation with bradykinin differs from those
with TGF-
1 and IL-1
. Bradykinin causes a much earlier generation
of prostaglandins by release of arachidonic acid from phospholipase
A2 and subsequent generation of prostanoids from COX-1 as
well as from COX-2. It is thus possible that the critical signal in
IL-8 generation by bradykinin may be the early release of
prostaglandins via COX-1. Because both IL-1
and TGF-
lack the
early phase of PGE2 generation, this would explain the lack
of prostanoid dependence on IL-8 generation by these agents.
Alternatively, bradykinin-induced PGE2 generation may be
acting in concert with another signaling pathway that bradykinin but
not TGF-
1 or IL-1
activates.
In summary, we have shown that TGF-1 induces IL-8 release, COX-2
induction, and PGE2 generation from human ASM cells. Our data also suggest that COX-2 induction and prostanoid generation are
not essential for TGF-
1-induced IL-8 release. It is likely that
these properties of TGF-
1 play a role in the modification of airway
inflammation in asthma.
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ACKNOWLEDGEMENTS |
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We thank Colin Clelland for providing specimens of human trachea.
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FOOTNOTES |
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L. Pang was supported by the Wellcome Trust.
Address for reprint requests and other correspondence: A. J. Knox, Division of Respiratory Medicine, City Hospital, Univ. of Nottingham, Hucknall Rd., Nottingham NG5 1PB, UK (E-mail: alan.knox{at}nottingham.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 17 August 1999; accepted in final form 7 March 2000.
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