Division of Respiratory Medicine, City Hospital, Nottingham NG5 1PB, United Kingdom
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ABSTRACT |
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Interleukin (IL)-8, the C-X-C chemokine,
is a potent neutrophil chemoattractant that has been implicated in a
number of inflammatory airway diseases such as cystic fibrosis. Here we
tested the hypothesis that bradykinin, an inflammatory mediator and
chloride secretagogue, would increase IL-8 generation in airway
epithelial cells through autocrine generation of endogenous
prostanoids. Bradykinin increased IL-8 generation in both a non-cystic
fibrosis (A549) and cystic fibrosis epithelial cell line
(CFTE29
interleukin-8; lung; chemokines; inflammation; cystic fibrosis; cyclooxygenase-2
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INTRODUCTION |
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INTERLEUKIN
(IL)-8, the C-X-C chemokine, is a potent chemotractant for neutrophils
(37) that has been implicated in a number of inflammatory
diseases, such as cystic fibrosis (CF) (21), adult
respiratory distress syndrome (8), chronic obstructive pulmonary disease (COPD), and asthma (19). There are a
number of sources of IL-8 in the airway, including structural cells
such as airway epithelial cells (32). As airway epithelial
cells form a barrier against invading microorganisms, production of IL-8 by airway epithelial cells is likely to contribute to host defense
by promoting neutrophil chemotaxis and airway inflammation. Although
this is a useful response in acute inflammatory responses, an
exaggerated inflammatory response can contribute to pathogenesis in
chronic disease. Neutrophil-driven lung destruction is particularly important in CF, where the inflammatory response occurs early in the
disease (1, 4, 14) and is severe and sustained. Several
agents increase IL-8 release by airway epithelial cells, particularly
cytokines [IL-1, tumor necrosis factor (TNF)-
, interferon
(IFN)-
] and bacterial products (6, 10, 20). No
information is available on whether proinflammatory mediators such as
bradykinin (BK) also increase IL-8 and whether there are differences
between CF epithelial cells and non-CF epithelial cells in the
regulatory mechanisms.
BK is a nine-amino acid peptide that is formed locally in body fluids and tissues from the plasma precursor kininogen during inflammatory processes (25, 31, 33). Increased kallikrein levels have been reported in saliva in CF (18). BK is also a chloride secretagogue in several epithelial systems, including the airways (9, 26, 29). This may be of relevance in CF, where epithelial chloride secretion is impaired (28). BK increases IL-8 production in human lung fibroblasts (11), cultured human decidua-derived cells (5), and human airway smooth muscle cells (23), but not corneal epithelial cells (35), suggesting that the response to BK is tissue specific. The effect of BK on IL-8 release has not been studied in airway epithelial cells.
Previously, we reported that BK-induced IL-8 release is critically
dependent on endogenous prostanoids in airway smooth muscle (23), although the cyclooxygenase (COX) isoform
responsible was not defined, as airway smooth muscle expresses both
COX-1 and COX-2 after BK treatment. It is not known whether BK-induced IL-8 release is COX dependent in other cell types. This might be
particularly important in airway epithelial cells, which form a barrier
to protect against microorganisms and generate several neutrophil
chemoattractants. Prostanoid synthesis is mediated by two COX isoforms
(36), constitutive COX-1 (24) and COX-2, which is inducible in most cells but is constitutively expressed in
epithelial cells (2, 27). BK can release prostanoids in most cell systems by mobilizing arachidonic acid (AA) via phospholipase A2 (3, 34) and in some via induction of COX-2
(22). Here we tested the hypothesis that BK would induce
IL-8 release by airway epithelial cells and determined the prostanoid
dependence. Furthermore, we compared a non-CF airway epithelial cell
line (A549) with a F508-expressing human CF airway epithelial cell line (CFTE29
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MATERIALS AND METHODS |
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Cell culture. A549 cells were purchased from the European Collection of Animal Cell Cultures (ECAAC no.: 86012804; Salisbury, Wiltshire, UK). Aliquots of cells frozen in 10% dimethyl sulfoxide (dimethyl sulfoxide)-90% fetal calf serum (FCS) were thawed and suspended in culture medium comprising 90% DMEM-10% FCS containing 2 mM L-glutamine, 100 µg/ml; 10,000 U/ml penicillin G; 100 µg/ml streptomycin; and 2.5 µg/ml amphotericin.
CFTEExperiment protocols.
Aliquots of cells frozen in 10% DMSO-90% FCS were thawed and
centrifuged at 100 g. Cells were resuspended in 10% FCS
(Seralab, Crowly Down, UK)-DMEM (A549 cells) or MEM
(CFTE29
IL-8 assay. The concentration of the IL-8 in the culture medium was determined by ELISA (CLB, Amsterdam, The Netherlands) according to the manufacturer's instructions. We have described this in detail elsewhere (23).
PGE2 assay. PGE2 concentration was determined by RIA as previously described (22). We have previously validated this assay and shown it to have a low cross-reactivity with other metabolites of AA. Each sample analyzed for PGE2 was assayed in duplicate.
Western blot analysis. COX-1 and COX-2 expression were assessed by Western blotting (22). 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 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 horseradish peroxidase (HRP, 1:2,000 in blocking reagent; Sigma) for 1 h at room temperature. Semiquantitative staining was achieved by using enhanced chemiluminescence detection. We performed this detection 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). We carried out reprobing of COX-1 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).Statistical analysis. Data were expressed as means ± SE from n determinations. The statistical analysis was performed with the software program PRISM (Graphpad, San Diego, CA). A one-way ANOVA and/or an unpaired two-tailed Student's t-test were used to determine the significant differences between means. P values <0.05 were accepted as statistically significant.
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RESULTS |
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BK stimulates IL-8 release in A549 cells.
To investigate the time course of IL-8 production, we cultured A549
cells in the presence or absence of BK (10 µM) as shown in Fig.
1A. There was a time-dependent
increase in IL-8 production after stimulation with BK (88.9 ± 15.3 pg/ml at 1 h, 1,018 ± 104 at 20 h, 1,249 ± 60 at 24 h), which was significant compared with controls at all
time points, showing a maximal twofold increase at 20-24 h (20 h,
P < 0.01; 24 h, P < 0.001). A549
cells cultured with BK (0.001-100 µM) showed a
concentration-dependent rise in IL-8 (Fig. 1B) compared with
control after 8 h of incubation (1 µM, P < 0.001; 10 µM, P < 0.001; 100 µM, P < 0.001).
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BK stimulates PGE2 release in A549 cells.
Treatment of A549 cells with BK (10 µM) caused a time-dependent
accumulation of PGE2 (Fig.
2A). The increase was
significant at 1 h (P < 0.01). There was a
threefold increase in PGE2 production, reaching a peak
between 2 and 4 h (2 h, P < 0.001; 4 h,
P < 0.01), which declined at 8 h
(P < 0.05) and was not significant at 16 h. The
effect was concentration dependent as seen in Fig. 2B, with
a significant effect seen at a concentration of 10 µM
(P < 0.001).
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COX isoform responsible for PGE2 release in A549 cells.
Western blot showed that COX-2 was expressed under resting conditions
but that COX-1 was not (Fig. 3). There
was no effect of BK on COX-2 protein expression (Fig. 3), suggesting
that the increase in PGE2 produced by BK is likely to be
mediated by AA release and prostanoid production from existing COX-2
rather than further COX-2 induction.
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Effect of COX inhibitors on BK-induced IL-8 and PGE2
production in A549 cells.
To investigate the role of endogenous PGs in BK-induced IL-8 and
PGE2 generation, we studied the effect of the nonselective COX inhibitor IND and the selective COX-2 inhibitor NS-398 (both at
concentrations of 1 µM) on BK-induced IL-8 and PGE2
release. BK-induced IL-8 production was significantly inhibited by both NS-398 (P < 0.01) and IND (P < 0.001), as shown in Fig. 4A.
BK-induced PGE2 release was also inhibited by IND and
NS-398 (Fig. 4B; P < 0.001 for IND,
P < 0.01 for NS-398). These findings suggest that
BK-induced IL-8 production is partly mediated by endogenous COX
products.
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Effect of exogenous COX substrate AA or PGE2 on IL-8
production in A549 cells.
Furthermore, we examined whether exogenously applied COX substrate AA
or PGE2 would increase IL-8 release. A549 cells were cultured in the presence of AA (0.1-10 µM) or PGE2
(0.1-10 µM), and cell culture supernatants were collected at
16 h. Both AA and PGE2 significantly increased IL-8
release in a concentration-dependent manner, with values increasing
from 95 ± 5 pg/ml in control cells to 168 ± 10 pg/ml after
10 µM PGE2 and 128 ± 1 pg/ml after 10 µM AA (both
P < 0.001, Fig. 5).
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BK stimulates PGE2 release in
CFTE29
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COX isoform responsible for PGE2 generation in
CFTE29
Effect of COX inhibitors on BK-induced PGE2 and IL-8
production in CFTE29
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DISCUSSION |
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The aim of this study was to test the hypothesis that BK would
induce IL-8 release in airway epithelial cells and to determine the
prostanoid dependence of the effect. There are several novel findings
in our study. It is the first to show that BK increases IL-8 production
in A549 and CFTE29
We found that BK caused IL-8 release in both cell lines. Previous studies have shown that BK induces the release of neutrophil chemotactic activity in A549 cells and bronchial epithelial cells (15, 16), although the factors responsible have not been identified. Our results suggest that IL-8 may be a component of the neutrophil chemotractant activity produced by BK in airway epithelial cells. The IL-8 response of epithelial cells to BK appears to be tissue specific, as BK did not increase IL-8 release by corneal epithelial cells despite the presence of BK receptors (35).
CF is characterized by neutrophilic inflammation in the airways driven
by IL-8 (12). IL-8 production is increased in
bronchoalveolar lavage fluid and in cultured airway epithelial cells by
a number of stimulants, including cytokines (IL-1, TNF-
,
IFN-
), Pseudomonas aeruginosa, and respiratory syncytial
virus (6, 10, 20). We found no evidence of increased IL-8
production by CF cells compared with A549 cells. This is consistent
with the results of another study showing increased RANTES but not IL-8
production by CF epithelial cells (30). These findings do,
however, contrast with a study by DiMango and colleagues
(7), who show increased IL-8 production in CF IB3 cells, a
F508-expressing cell line, compared with wild-type cells due to
increased activation of nuclear factor-
B. In our studies there was
also no evidence of greater PGE2 production by
CFTE29
F508 compared with control cells (17). Subsequent
studies suggested the increase in AA production in response to BK was
due to increased phospholipase A2 activity (34). The disparity between our results and theirs is
likely to reflect differences in the cell lines studied or the
experimental protocols involved. CF is a heterogeneous condition with
over 800 genotypes, and there may be differences in AA metabolism
between genotypes. We used A549 cells because we and others have shown they are a good model system for the study of airway prostanoid production (2, 27).
Both A549 and CFTE29
We found that BK-induced IL-8 production was inhibited by the nonselective COX inhibitor IND, suggesting a role for endogenous prostanoids in IL-8 generation. Furthermore, the COX-2 selective inhibitor NS-398 had a similar effect, suggesting that endogenous prostanoids generated by constitutively expressed COX-2 are responsible for BK-induced IL-8 generation by airway epithelial cells. Because these agents are structurally dissimilar, this effect is likely to be a COX-related effect. The results with NS-398 are consistent with our Western blot results showing that COX-2 is the only COX isoform expressed by these cells and the fact that exogenous PGE2 stimulated IL-8 release. Unfortunately, there are no highly selective COX-1 inhibitors that can be used in whole cell systems. We have previously shown that both IND and NS-398 reduce COX-2-mediated PGE2 production by >90% at these concentrations and that NS-398 is COX-2 selective (27).
Our assertion that BK increases IL-8 release via prostanoid generation
is further strengthened by experiments with exogenous AA and
PGE2, both of which significantly increased IL-8 release in
A549 cells. The effect of exogenous AA was relatively small, which
probably reflects the fact that COX-2 is located at a perinuclear site;
therefore, endogenous AA release is likely to be more effective in
generating prostanoids, which can regulate gene transcription. We
performed selected experiments in CFTE29
We have considered the possible therapeutic implication of our studies. The fact that COX inhibitors can significantly reduce BK-induced IL-8 release in CF cells could provide an explanation for the beneficial effects reported with high doses of ibuprofen, a nonselective COX inhibitor on the rate of decline of lung function in children with CF (13). Further studies looking at the effect of selective COX-2 inhibitors in CF would therefore be of interest. COX-2 inhibitors could also have a role in other airway diseases characterized by neutrophilic inflammation, such as COPD or adult respiratory distress syndrome. It could be argued that effects of BK on IL-8 are relatively small and studies to determine whether such changes could alter neutrophil trafficking is vivo are required. BK does stimulate neutrophil chemotactic activity in epithelial cells in vitro, however, and it is likely that IL-8 is contributing to this. It would also be useful to determine whether COX inhibitors alter the IL-8 response to cytokines and bacterial agents.
In conclusion, we have shown that BK releases IL-8 by airway epithelial cells through generations of prostanoids from constitutively expressed COX-2. This suggests that there may be a role for selective COX-2 inhibitors in the treatment of lung diseases characterized by neutrophilic inflammation.
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ACKNOWLEDGEMENTS |
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This study was supported by the United Kingdom Cystic Fibrosis Trust.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. J. Knox, Respiratory Medicine Unit, Clinical Sciences Bldg., City Hospital, 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. Section 1734 solely to indicate this fact.
April 12, 2002;10.1152/ajplung.00483.2001
Received 17 December 2001; accepted in final form 9 April 2002.
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