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Am J Physiol Lung Cell Mol Physiol 292: L1289-L1296, 2007. First published January 12, 2007; doi:10.1152/ajplung.00356.2006
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Regulation of interleukin-8 via an airway epithelial signaling cascade

Takashi Nakanaga, Jay A. Nadel, Iris F. Ueki, Jonathan L. Koff, and Matt X. G. Shao

Cardiovascular Research Institute and Departments of Medicine and Physiology, University of California, San Francisco, California

Submitted 12 September 2006 ; accepted in final form 5 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Airways function as an innate immune organ against airborne bacteria that are inhaled and deposited in airways. One of the mechanisms of host defense is to recruit neutrophils into airways to clear the invaders. Airway epithelial cells produce neutrophil chemoattractant interleukin (IL)-8 in response to invading bacteria. In this study we show a signaling pathway on the plasma surface of human airway epithelial NCI-H292 cells that regulate IL-8 production in response to a model inflammatory stimulus, phorbol 12-myristate 13-acetate, and a pathophysiological stimulus, gram-negative bacterial lipopolysaccharide. First, we show that EGF receptor (EGFR) and MAP kinase ERK1/2 are involved in IL-8 expression by these stimuli. Second, we show that EGFR ligand transforming growth factor (TGF)-{alpha} mediates IL-8 production. Third, we show that tumor necrosis factor-{alpha}-converting enzyme (TACE) is required for IL-8 production by cleaving EGFR proligand proTGF-{alpha} into soluble TGF-{alpha}, activating EGFR. Last, we show that dual oxidase 1 (Duox1), a homolog of NADPH oxidase in airways, mediates TACE activation and IL-8 expression via generation of reactive oxygen species. In summary, we describe a signaling pathway, Duox1-TACE-TGF-{alpha}-EGFR, on the surface of airway epithelial (NCI-H292) cells that mediates airway epithelial defense against bacterial infection by producing IL-8. This pathway, which also regulates mucin production in human airways, provides mechanisms for killing foreign organisms and for their clearance.

chronic inflammatory airway disease; innate immunity; bacterial infection

WHEN FISH GAVE UP THEIR GILLS and came ashore, lungs developed as an improved apparatus for gas exchange at the alveolar-capillary interface; airways developed as conduits between the alveoli and the environment. However, inhaled air allows environmental microbes (e.g., bacteria) to deposit in airways, threatening the organism (lungs). Thus the airway epithelium has become the "battleground" where the body confronts the invading bacteria. In response to bacterial invasion, signals are generated at the airway epithelial surface to defend the organism. One of the mechanisms defending the host against invaders is the recruitment of inflammatory cells (e.g., neutrophils) into airways, which kill the invaders. Airway epithelial cells are a source of interleukin (IL)-8, which recruits neutrophils into the infected airways. Appropriate neutrophil recruitment results in bacterial killing and prevents bacterial invasion. However, excessive neutrophil influx into airways contributes to the development of chronic inflammatory airway diseases (e.g., severe asthma, cystic fibrosis, and chronic obstructive pulmonary disease) (8). Thus better understanding of the molecular mechanisms regulating IL-8 production by the airway epithelial cells can be useful in the development of effective therapies for these diseases.

Human airway epithelial cells express EGF receptor (EGFR) (26), which is implicated in both maintenance and repair of normal airway epithelial tissues and in the pathogenesis of chronic inflammatory airway diseases (3). The expression of EGFR is increased in chronic airway diseases including severe asthma (15, 26), cystic fibrosis (12), and chronic obstructive pulmonary disease (COPD) (12), in which neutrophil infiltration is a prominent feature, suggesting a causal relationship between the upregulation of EGFR and the neutrophil influx into the airways. In support of this speculation, EGFR has been shown to mediate IL-8 production in normal human bronchial epithelial cells (16), epithelial cells from severe asthmatic (27) and COPD subjects (16), and in human pulmonary mucoepidermoid carcinoma cell lines [e.g., BEAS-2B cells (24), NCI-H292 cells (7, 16)]. EGFR phosphorylation leads to the activation of NF-{kappa}B (1, 6), which moves into the nucleus and induces the upregulation of IL-8 gene expression (17).

Recently, our laboratory discovered a novel signaling pathway on the plasma surface of human airway epithelial cells that regulates MUC5AC mucin expression in response to multiple stimuli including phorbol 12-myristate 13-acetate (PMA), cigarette smoke, and neutrophil elastase (19, 20, 22, 23). These stimuli activate dual oxidase 1 (Duox1), which generates reactive oxygen species (ROS), resulting in activation of TNF-{alpha}-converting enzyme (TACE), cleaving EGFR proligand pro-transforming growth factor (TGF)-{alpha} into soluble TGF-{alpha}, which binds to and activates EGFR, leading to activation of MAP kinases and subsequent upregulation of MUC5AC mucin gene expression. Because both mucins and neutrophils are implicated in airway innate immunity and in the pathogenesis of chronic inflammatory airway diseases, we hypothesize that human airway epithelial cells use a similar signaling pathway to upregulate IL-8 gene expression and protein production in response to inflammatory stimuli.

To examine this hypothesis, we stimulated human airway epithelial (NCI-H292) cells with a model inflammatory stimulus, PMA, and a pathophysiological stimulus, Pseudomonas aeruginosa lipopolysaccharide (LPS). We used selective inhibitors, neutralizing antibodies, and small interfering RNA (siRNA) to prevent the function of each molecule involved in the pathway, and we analyzed IL-8 expression at both mRNA and protein levels. We found that a Duox1-ROS-TACE-TGF-{alpha}-EGFR cascade is involved in IL-8 upregulation in human airway epithelial NCI-H292 cells.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Amplex UltraRed reagent was purchased from Molecular Probes (Eugene, OR). AG1478, diphenyleneiodonium chloride (DPI), an EGFR blocking antibody, GM6001, negative control of GM6001 [GM(–)], neutralizing EGF antibody, neutralizing TGF-{alpha} antibody, PD-98059, and TNF-{alpha} proteinase inhibitor (TAPI)-1 were purchased from Calbiochem (La Jolla, CA). BIBX1522 was generously provided by Boehringer Ingelheim Pharma (Ingelheim, Germany). Horseradish peroxidase (HRP), PMA, n-propyl gallete (nPG), and P. aeruginosa LPS serotype 10 were purchased from Sigma (St. Louis, MO).

Cell culture. NCI-H292 cells, a human pulmonary mucoepidermoid carcinoma cell line, were plated at 4–5 x 105 cells in 2 ml in each well of a 6-well plate or at 1–2 x 105 cells in 1 ml in each well of a 24-well plate (both 6-well and 24-well plates were purchased from BD Falcon, Bedford, MA) and were grown in RPMI 1640 medium containing 10% fetal bovine serum, penicillin (10 U/ml), streptomycin (100 µg/ml), and HEPES (25 mM) at 37°C in a humidified, 5% CO2-95%, water-jacketed incubator. After the cells reached confluence, they were further cultured for 5–10 days. Cells were serum-starved for 24 h before experiments.

Culture conditions of cells with stimuli and inhibitors. After 24 h of serum starvation, cells were treated with stimuli as indicated in each experiment. For inhibitor studies, serum-starved cells were pretreated with inhibitors for 30 min before exposure to stimuli. In studies of PMA, cells were treated with PMA (10 ng/ml) for various times. The cells were then washed three times with serum-free medium (SFM) and cultured for 10 or 24 h in SFM with the same concentrations of inhibitors as in the pretreatment period. In studies of LPS, cells were incubated with LPS (10 µg/ml) for 10 or 24 h. Cell culture supernatants and cell lysates were then collected to measure Il-8 protein production.

Cytotoxicity detection and measurement of total protein. Lactate dehydrogenase (LDH) activity in supernatants of cell cultures treated with or without inhibitors was measured with the cytotoxicity detection kit (Roche Diagnostic, Indianapolis, IN). Total protein in cell lysates of cell cultures treated with or without inhibitors was measured with the BCA protein assay kit (Pierce, Rockford, IL). None of the measurements showed significant cytotoxicity for the inhibitors at the concentrations used in the present studies.

siRNA preparation and transfection of cells. Predesigned human TACE siRNAs (nos. 104029, 104030, 104031) were purchased from Ambion. In our preliminary studies, siRNA no. 104029 (100 nM) had the greatest inhibition of TACE gene expression examined by RT-PCR. Therefore, this siRNA was selected for subsequent studies. The 21-nt sequences for TACE siRNA (no. 104029) are (sense) GGUUUUAAAGGCUAUGGAAtt and (antisense) UUCCAUAGCCUUUAAAACCtg. Duox1 siRNA no. 24969 (Ambion), which inhibits Duox1 expression (19), was used in the present studies. The 21-nt sequences of Duox1 siRNA (no. 24969) were (sense) GGACUUAUCCUGGCUAGAGtt and (antisense) CUCUAGCCAGGAUAAGUCCtg. Silencer negative control siRNA no. 1 (Ambion) was used as a nonspecific siRNA. SiRNA transfection into NCI-H292 cells was carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Specific silencing of TACE and Duox1 was confirmed by RT-PCR 72 h after transfection.

H2O2 detection and quantification. The cell-impermeable reagent Amplex UltraRed (A36006 [GenBank] ; Molecular Probes), which is converted into a fluorescent compound in the presence of H2O2 and HRP, was used to detect extracellular H2O2. The fluorescence intensity was monitored in a CytoFluor 2350 multiwell fluorescent plate reader (Applied Biosystems, Foster City, CA) by using wavelength ratios of 530/25 and 620/40 for excitation and emission, respectively. H2O2 reaction mixture contained 50 µM Amplex UltraRed reagent and 1 U/ml HRP diluted in warm PBS. For the H2O2 standard curves, 10 µl/well of different dilutions of H2O2 in PBS were added to 100 µl of the reaction mixture in 96-well plates.

NCI-H292 cells were grown in 24-well plates and then serum starved for 24 h before H2O2 analysis was performed. For stimulation studies, the cells were washed with 1 ml of PBS (prewarmed in a 37°C incubator) twice and then incubated with 0.5 ml of H2O2 reaction mixture with or without LPS (10 µg/ml, final concentration) or PMA (10 ng/ml, final concentration) for various times as indicated in each experiment. For inhibition studies, cells were pretreated with inhibitors diluted in serum-free RPMI 1640 medium for 30 min, washed twice with warm PBS, and then incubated with 0.5 ml of H2O2 reaction mixture (containing the same concentrations of inhibitors) with or without LPS (10 µg/ml, final) or PMA (10 ng/ml, final) for various times. At the end of experiments, reaction mixtures were transferred into 1.5-ml microcentrifuge tubes and incubated at room temperature for 15 min. One hundred microliters of each reaction mixture were transferred into 96-well plates, and the fluorescence intensity was measured. H2O2 production from each well was calculated with a standard curve. Cells in each well were counted, and H2O2 production was expressed as picomoles per 1 x 106 cells.

RNA isolation and RT-PCR. Total RNA was isolated, and reverse transcription was performed as described previously (23). One microliter of the RT reaction was PCR-amplified in a 50-µl reaction with SuperTaq DNA polymerase (Invitrogen). The PCR protocol was as follows: 94°C for 4 min, 72°C for 10 min, followed by 25–40 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 45 s, and extension at 72°C for 45 s. IL-8 primer sets were purchased from R&D Systems (Minneapolis, MN). Primers directed against ribosomal RNA (Rig/S15) were used as the endogenous control. Primers were as follows: TACE, (forward) 5'-ACCTGAAGAGCTTGTTCATCGAG-3' and (reverse) 5'-CCATGAAGTGTTCCGATAGATGTC-3'; Duox1 (forward) 5'-GCCCTGTACAACCAGGACTT-3' and (reverse) 5'-AGGTGGTATTTCGGATTTCT-3'; and Rig/S15 (forward) 5'-TTCCGCAAGTTCACCTACC-3' and (reverse) 5'-CGGGCCGGCCATAGCTTTACG-3'. After PCR, 5- to 10-µl aliquots were subjected to 2.0% agarose gel electrophoresis and stained with ethidium bromide.

Analysis of IL-8 protein production. Cells were grown in 24-well plates. IL-8 in cell lysates and in cell culture supernatants was measured using an IL-8 ELISA kit (R&D Systems) according to the manufacturer's instructions.

Immunocytochemical staining of IL-8. NCI-H292 cells were grown in eight-chamber slides. The cells were treated with PMA for various times. After treatments, the cells were fixed with 4% paraformaldehyde for 30 min. Cells were treated with 0.3% H2O2-methanol for 30 min to quench endogenous peroxidase, incubated with 2% BSA in PBS for 1 h, and then incubated with anti-IL-8 polyclonal antibody (BioSource, Carlsbad, CA) for 1 h. After removing excess antibody by washing with PBS, we incubated cells with biotinylated goat anti-rabbit immunoglobulin G (1:200 dilution; Vector Laboratories, Burlingame, CA) for 1 h at room temperature. Bound antibody was visualized according to standard procedure for the avidin-biotin-peroxidase complex method (Elite ABC kit; Vector Laboratories).

Statistical analysis. Data are presented as means ± SE (n ≥ 3). ANOVA (Newman-Keuls procedure) was used to examine statistical differences among (between) groups. A P value < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PMA upregulates IL-8 gene expression and increases IL-8 protein production. First, we examined IL-8 gene expression and protein production in response to increasing concentrations of PMA: PMA upregulated IL-8 gene expression (Fig. 1A) and increased IL-8 protein production (Fig. 1B) dose dependently. PMA (10 ng/ml) induced ~20 times higher IL-8 protein production than control. Next, we examined the timing of IL-8 gene expression and protein production by PMA (10 ng/ml). IL-8 gene expression and protein production started to increase after 2 h of incubation with PMA and increased significantly after 4 h of incubation (Fig. 1, C and D). Approximately 95% of the IL-8 protein induced by PMA at 24 h was secreted into the supernatant (Fig. 1, B and D); therefore, we measured only the secreted IL-8 in the subsequent experiments. Immunocytochemical studies confirmed the ELISA measurements: IL-8 staining was weak in the untreated cells [Fig. 1E, left (0 h)], but PMA increased IL-8 staining on the cell surface conspicuously at 6 h (Fig. 1E, middle), an effect that was even greater at 12 h (Fig. 1E, right). These results show that PMA induces rapid and marked upregulation of IL-8 gene expression and protein production in human airway epithelial NCI-H292 cells.


Figure 1
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  		Fig. 1. Interleukin (IL)-8 gene expression and protein production in response to phorbol 12-myristate 13-acetate (PMA). NCI-H292 cells were exposed to PMA at various dilutions and times. Total RNA was prepared and subjected to RT-PCR; Rig/S15 was used as an internal marker. A and B: effect of various dilutions of PMA when given for 12 h on RT-PCR-amplified IL-8 gene expression (A) and when given for 24 h on total IL-8 protein production by ELISA (B). C–E: effect of PMA (10 ng/ml) given for various times on RT-PCR-amplified IL-8 gene expression (C) and on IL-8 protein production by ELISA (D) or by immunocytochemical staining at different times following PMA administration (E). Results shown are typical of the results in 3 separate experiments. IL-8 proteins were measured in the cell lysate (dark areas) and in the supernatant (light areas). Data in B and D are expressed as means ± SE (n = 3). *P < 0.05 compared with control.

 
PMA induces IL-8 via EGFR-MEK signaling pathway. Our previous studies showed that EGFR-MEK cascade controls MUC5AC mucin expression (23). We examined whether this cascade also is involved in IL-8 induction. Pretreatment with selective EGFR inhibitors AG1478 or BIBX1522 or the MEK inhibitor PD-98059 significantly reduced PMA-induced IL-8 gene expression (Fig. 2A) and protein production (Fig. 2B). These results show that an EGFR-MEK cascade controls IL-8 gene expression and protein production by PMA.


Figure 2
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  		Fig. 2. Effect of EGF receptor (EGFR) and MEK inhibitors on IL-8 expression by PMA. NCI-H292 cells were treated with vehicle alone (–) or PMA (10 ng/ml) alone or pretreated with the selective EGFR phosphorylation inhibitors AG1478 (AG; 10 µM) or BIBX1522 (BIBX; 5 µg/ml) or with the MEK inhibitor PD-98059 (PD; 10 µM) for 30 min and then treated with PMA (10 ng/ml) for 8 h for the analysis of IL-8 gene expression by RT-PCR (A) or treated for 24 h for the measurement of IL-8 protein in the supernatant by ELISA (B). Data in B are expressed as means ± SE (n = 3). *P < 0.05 compared with PMA alone.

 
PMA induces IL-8 via TGF-{alpha}-dependent EGFR activation. Having shown the role of EGFR activation in IL-8 production, we next examined whether an EGFR ligand is involved. We preincubated cells with an EGFR blocking antibody (4 µg/ml) that binds to and occupies the ligand-binding sites on EGFR. This pretreatment reduced IL-8 gene expression (Fig. 3A) and protein production (Fig. 3B) by PMA significantly (~75% reduction of IL-8 protein production), indicating that an EGFR ligand plays a major role in IL-8 production by PMA. Because PMA has been shown to induce the cleavage of pro-TGF-{alpha} into mature soluble TGF-{alpha} in NCI-H292 cells (23), we examined the role of TGF-{alpha} in IL-8 production. Pretreatment with a TGF-{alpha} neutralizing antibody (4 µg/ml) significantly inhibited IL-8 gene expression (Fig. 3A) and protein production (Fig. 3B), whereas preincubation with an EGF neutralizing antibody (4 µg/ml) was without effect. From these results we conclude that TGF-{alpha} is a major EGFR ligand involved in PMA-induced IL-8 production.


Figure 3
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  		Fig. 3. Effect of EGFR- and ligand-neutralizing antibodies on IL-8 expression. Cells were treated with vehicle alone (–) or PMA alone or pretreated with neutralizing antibody (Ab; 4 µg/ml) for EGFR or transforming growth factor (TGF)-{alpha} or EGF for 30 min and then treated with PMA (10 ng/ml) for 8 h to examine IL-8 mRNA expression by RT-PCR (A) or with PMA for 24 h to examine IL-8 protein production in the supernatant by ELISA (B). Data are expressed as means ± SE (n = 3). *P < 0.05 compared with PMA alone and with PMA + EGF Ab.

 
TACE is involved in IL-8 production. The metalloprotease TACE has been shown to cleave membrane-bound EGFR proligand (e.g., pro-TGF-{alpha}) into a mature soluble ligand, which induces mucin production in human airway epithelial cells (19, 20, 22, 23), but the role of TACE in IL-8 expression has not been examined. We used both metalloprotease inhibitors and siRNA to investigate the role of TACE in IL-8 induction by PMA. Pretreatment with a broad-spectrum metalloprotease inhibitor, GM6001 (10 µM), significantly reduced IL-8 gene expression (Fig. 4A) and protein production (Fig. 4B); GM(–) (10 µM), a negative control of GM6001, was without effect. Pretreatment with TAPI-1 (10 µM), a relatively selective TACE inhibitor, significantly reduced IL-8 gene expression and protein production (Fig. 4, A and B). To confirm the role of TACE in the response, we knocked down TACE expression using siRNA (Fig. 4C). TACE siRNA significantly reduced IL-8 gene expression (Fig. 4D) and protein production (Fig. 4E). From these results we conclude that TACE is involved in IL-8 induction by PMA.


Figure 4
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  		Fig. 4. Effect of metalloprotease inhibitors and knockdown of tumor necrosis factor-{alpha}-converting enzyme (TACE) on IL-8 expression. NCI-H292 cells were treated with vehicle alone (–) or PMA alone or pretreated with metalloprotease inhibitors GM6001 [GM(+); 10 µM] or the negative control of GM6001 [GM(–); 10 µM], or TNF-{alpha} proteinase inhibitor (TAPI; 10 µM) for 30 min and then stimulated with PMA for 8 h to examine IL-8 mRNA expression by RT-PCR (A) or for 24 h to measure IL-8 protein production in the supernatant (B). Data are expressed as means ± SE (n = 3). *P < 0.05 compared with PMA alone and with PMA + GM(–). C: cells were transfected with or without TACE small interfering RNA (siRNA) (TACE; 100 nM) or negative control siRNA (NC; 100 nM), cultured for 72 h, and then analyzed for TACE mRNA expression by RT-PCR. D and E: cells were treated with vehicle or with transfection agent Lipofectamine 2000 (Lipo) or transfected with TACE siRNA (100 nM) or NC siRNA (100 nM). After culture for 72 h, the cells were treated with PMA (10 ng/ml) for 8 h to examine IL-8 mRNA expression by RT-PCR (D) or for 24 h to measure IL-8 protein production in the supernatant (E). Data are expressed as means ± SE (n = 3). *P < 0.05 compared with Lipo + PMA and with NC + PMA treatment.

 
Duox1 generates ROS that mediate IL-8 expression. Airway epithelial cells express NADPH oxidase homologue Duox1, which generates ROS in response to a variety of stimuli (19, 20). Duox1 and ROS have been shown to be involved in TACE activation and mucin expression (19). We hypothesized that Duox1 and ROS are involved in PMA-induced IL-8 production. To test this hypothesis, we first examined PMA-induced ROS (e.g., H2O2) generation. PMA (10 ng/ml) induced H2O2 generation time dependently (Fig. 5A). We then examined the role of ROS in IL-8 production. We preincubated cells with a ROS scavenger, nPG (100 µM); this preincubation significantly reduced PMA-induced IL-8 gene expression (Fig. 5B) and protein production (Fig. 5C), implicating ROS. Last, we examined the involvement of Duox1 in IL-8 production, using both pharmacological and genetic approaches. 1) We pretreated cells with the NADPH oxidase inhibitor DPI. DPI significantly reduced PMA-induced H2O2 generation (Fig. 5A), IL-8 gene expression (Fig. 5B), and protein production (Fig. 5C), implicating NADPH oxidase (most likely Duox1) in PMA-induced IL-8 production. 2) We transfected cells with Duox1 siRNA to knock down the gene expression of Duox1 (Fig. 5D). Knockdown of Duox1 significantly reduced PMA-induced H2O2 generation (Fig. 5E), IL-8 gene expression (Fig. 5F), and protein production (Fig. 5G). These results suggest that PMA induces IL-8 production via Duox1 activation and ROS generation in human airway epithelial NCI-H292 cells.


Figure 5
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  		Fig. 5. Effect of inhibition of NADPH oxidase and reactive oxygen species (ROS) generation and knockdown of dual oxidase 1 (Duox1) on IL-8 production. A: NCI-H292 cells were preincubated with or without NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI; 3 µM) for 30 min and then treated with or without PMA (10 ng/ml) for various times (5, 15, and 30 min). H2O2 detection was performed as described in MATERIALS AND METHODS. Data are expressed as means ± SE (n = 4). *P < 0.05 compared with PMA treatment. #P < 0.05 compared with control. B and C: NCI-H292 cells were treated with vehicle alone (–) or pretreated with ROS scavenger n-propyl gallete (nPG; 100 µM) or DPI (3 µM) for 30 min and then were either treated with PMA (10 ng/ml) for 8 h and IL-8 gene expression was analyzed by RT-PCR (B) or treated with PMA for 24 h and IL-8 protein production was analyzed by ELISA (C). Data are expressed as means ± SE (n = 3). *P < 0.05 compared with PMA alone. D: cells were transfected with or without Duox1 siRNA (100 nM) or with NC siRNA (100 nM), cultured for 72 h, and then analyzed for Duox1 mRNA expression by RT-PCR. E–G: cells were incubated with vehicle or transfection agent Lipo or transfected with Duox1 siRNA (100 nM) or NC siRNA (100 nM). After culture for 72 h, the cells were treated with or without PMA (10 ng/ml) for 30 min to detect H2O2 production as described in MATERIALS AND METHODS (E), for 8 h to examine IL-8 mRNA expression by RT-PCR (F), or for 24 h to measure IL-8 protein production in the supernatant (G). Data in E are means ± SE (n = 4); data in G are means ± SE (n = 3). *P < 0.05 compared with Lipo + PMA and with NC + PMA treatment.

 
Duox1-ROS-TACE-EGFR cascade contributes to LPS-induced IL-8 production. Having determined the involvement of Duox1-ROS-TACE-EGFR cascade in PMA-induced IL-8 production, we examined whether this cascade plays a role in LPS-induced IL-8 production in NCI-H292 cells. Blockade of EGFR with the selective inhibitors AG1478 or BIBX1522 or blockade of MEK with the selective inhibitor PD-98059 significantly inhibited LPS-induced IL-8 gene expression (Fig. 6A) and protein production (Fig. 6B), implicating an EGFR-MEK signaling pathway. Blockade of TACE with TAPI-1 or knockdown of TACE using TACE siRNA significantly reduced LPS-induced IL-8 gene expression (Fig. 6, C and E) and protein production (Fig. 6, D and F), implicating TACE. To examine the role of ROS in IL-8 production by LPS, we measured LPS-induced H2O2 generation. LPS (10 µg/ml) induced H2O2 generation time dependently (Fig. 6G). We pretreated the cells with ROS scavenger nPG; this treatment significantly reduced LPS-induced IL-8 gene expression (Fig. 6C) and protein production (Fig. 6D), implicating ROS. Pretreatment with NADPH oxidase inhibitor DPI or knockdown of Duox1 using siRNA significantly reduced LPS-induced H2O2 generation (Fig. 6, G and H), IL-8 gene expression (Fig. 6, C and E), and protein production (Fig. 6, D and F), implicating Duox1. Together, these results suggest that the Duox1-ROS-TACE-EGFR cascade is involved in LPS-induced IL-8 production in human airway epithelial NCI-H292 cells.


Figure 6
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  		Fig. 6. Effect of inhibition of Duox1-ROS-TACE-EGFR-MEK1/2 signaling pathway on Pseudomonas aeruginosa lipopolysaccharide (LPS)-induced IL-8 production. A and B: NCI-H292 cells were incubated with vehicle alone (–) or pretreated with EGFR inhibitors AG (10 µM) or BIBX (5 µg/ml) or MEK1/2 inhibitor PD (10 µM) for 30 min and then treated with LPS (10 µg/ml) for 8 h to examine IL-8 gene expression by RT-PCR (A) or treated with LPS (10 µg/ml) for 24 h to measure IL-8 protein production by ELISA (B). Data are means ± SE (n = 3). *P < 0.05 compared with LPS treatment alone. C and D: cells were incubated with vehicle alone (–) or pretreated with TACE inhibitor TAPI-1 (10 µM), ROS scavenger nPG (100 µM), or NADPH oxidase inhibitor DPI (3 µM) for 30 min and then treated with LPS (10 µg/ml) for 8 h to examine IL-8 gene expression by RT-PCR (C) or for 24 h to measure IL-8 protein production by ELISA (D). Data are means ± SE (n = 3). *P < 0.05 compared with LPS alone. E and F: cells were incubated with vehicle, treated with transfection agent Lipo, or transfected with TACE siRNA (100 nM), Duox1 siRNA (100 nM), or NC siRNA (100 nM). After 72 h, the cells were treated with LPS (10 µg/ml) for 8 h to examine IL-8 mRNA expression by RT-PCR (E) or for 24 h to measure IL-8 protein production in the supernatant (F). Data are means ± SE (n = 3). *P < 0.05 compared with Lipo + LPS and with NC + LPS. G: NCI-H292 cells were preincubated with or without DPI (3 µM) for 30 min and then treated with or without LPS (10 µg/ml) for various times (5, 15, and 30 min) to detect H2O2 production as described in MATERIALS AND METHODS. Data are means ± SE (n = 4). *P < 0.05 compared with LPS alone. #P < 0.05 compared with control. H: cells were incubated with vehicle, treated with Lipo, or transfected with Duox1 siRNA (100 nM) or NC siRNA (100 nM). After 72 h, the cells were treated with or without LPS (10 µg/ml) for 30 min to detect H2O2 production as described in MATERIALS AND METHODS. Data are means ± SE (n = 4). *P < 0.05 compared with Lipo + LPS and with NC + LPS.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
During evolution from gills to lungs, airways have developed not only as conduits connecting lungs and environment but also as an innate immune organ against airborne toxins and microbes that deposit in airways. For instance, airways produce mucus to trap foreign toxins and microbes and remove these harmful materials from the respiratory tract by mucociliary clearance. Airway epithelial cells produce IL-8 to recruit neutrophils in response to these environmental stimuli. Because the first contact site of the stimuli is the apical surface of the airways, we hypothesized that airway epithelial cells have signaling cascades that allow the host to mount innate immune defense quickly. In the present studies, we took advantage of our prior findings showing the sequence of a Duox1-ROS-TACE-TGF-{alpha}-EGFR cascade that is involved in the upregulation of MUC5AC mucin expression in response to multiple pathophysiological stimuli in both normal human bronchial epithelial cells and a human pulmonary mucoepidermoid carcinoma cell line (NCI-H292 airway epithelial cells). We have shown that IL-8 production by NCI-H292 cells is dependent at least in part on a Duox1-ROS-TACE-TGF-{alpha}-EGFR-MEK cascade in response to a model inflammatory stimulus PMA and a pathophysiological stimulus, the gram-negative bacterial product LPS.

Human airway epithelial cells express EGFR, which is implicated in airway innate immunity. EGFR has been shown to be involved in IL-8 production in human airway epithelial cells in chronic inflammatory airway diseases (4, 5). Richter and colleagues (7, 16) showed that cigarette smoke induces IL-8 production in bronchial epithelial BEAS-2B cells, an effect that was inhibited by a selective EGFR tyrosine kinase inhibitor, implicating EGFR activation in IL-8 production. They also showed that ~50% of the secreted IL-8 was blocked by an EGFR neutralizing antibody, implicating the EGFR ligand release in the response. However, previous studies have not examined the nature and sequence of events that result in the release of EGFR ligand by the stimulated airway epithelial cells. In our studies examining MUC5AC mucin production in normal human airway epithelial cells and NCI-H292 cells, we showed that TACE, a member of the "disintegrin and metalloprotease" (ADAM) family, cleaves pro-TGF-{alpha} into mature soluble TGF-{alpha} (2, 14), which binds to and activates EGFR, resulting in MUC5AC mucin gene expression and mucin protein production (23). We hypothesized that airway epithelial cells use a similar signaling pathway to activate EGFR, leading to IL-8 production. To examine the role of TACE-TGF-{alpha} cascade in IL-8 production, we used multiple independent approaches to block the cascade, including siRNA, protease inhibitors, and blocking antibodies for EGFR and for the ligands TGF-{alpha} and EGF. We found that blockade of this pathway significantly inhibited IL-8 gene upregulation and protein production in response to PMA and LPS, implicating TACE-TGF-{alpha} cascade in IL-8 production in human airway epithelial NCI-H292 cells.

TACE can be activated by ROS (28). ROS disrupt a cysteine-zinc bond between a cysteine residue in the inhibitory domain and zinc in the catalytic domain of TACE, resulting in a conformational change and exposure of the catalytic domain, leading to TACE activation (23). Recently, we showed that Duox1, a homolog of NADPH oxidase in human airway epithelial cells (4, 5), generates hydrogen peroxide in response to PMA and to neutrophil elastase (19). Knockdown of Duox1 expression using siRNA, or depletion of ROS using ROS scavengers, prevents TACE activation and mucin expression, implicating a Duox1-ROS-TACE cascade. We used similar strategies to examine the role of this cascade in IL-8 production. We found that both blockade of Duox1 activation with a NADPH oxidase inhibitor and knockdown of Duox1 expression significantly inhibited H2O2 generation, IL-8 gene expression, and protein production, implicating Duox1. The ROS scavenger nPG inhibited IL-8 production, supporting the involvement of Duox1 in IL-8 response. Thus we have shown that a linear signaling pathway Duox1-ROS-TACE-TGF-{alpha}-EGFR-MEK on the cell surface is not only involved in MUC5AC mucin expression (19, 21) but also plays an important role in mediating the IL-8 response to inflammatory stimuli such as PMA and LPS.

Duox1 can be activated by PMA via a PKC-dependent mechanism in human airway epithelial cells (19), but the mechanism of Duox1 activation by LPS is unknown. LPS induces cell responses via binding to Toll-like receptor 4 (TLR4) on cell surfaces (25). In phagocytes, LPS binding to TLR4 induces the activation of NADPH oxidase (10). After TLR4 activation, MyD88 is recruited to the Toll/IL-1 receptor (TIR) domain, resulting in phosphorylation of p47phox, a critical cytosolic component of NADPH oxidase. Phosphorylated p47phox translocates to the plasma membrane to join p91phox and other components, leading to the assembly of functional NADPH oxidase in phagocytes, producing ROS (10). Because Duox1 shares structural homology with phagocytic NADPH oxidase and because airway epithelial cells express p47phox (18), it is speculated that LPS activates Duox1 via a mechanism similar to NADPH oxidase. In fact, McNamara and Basbaum (11) reported that dominant negative mutant MyD88 inhibited mucin expression by LPS in NCI-H292 cells, supporting the speculation that MyD88 may be involved in Duox1 activation and subsequent mucin expression in NCI-H292 cells. Studies investigating whether MyD88 induces p47phox phosphorylation by LPS are ongoing in this laboratory.

This work was presented in abstract form in September 2005 (13). In May 2006, during the preparation of the present article for publication, neutrophil elastase-induced IL-8 upregulation in A549 human lung carcinoma cells was reported via an EGFR cascade (9).

In summary, we have shown that stimulation of NCI-H292 cells with PMA and LPS activates the signaling pathway Duox1-ROS-TACE-TGF-{alpha}-EGFR on the epithelial surface, resulting in IL-8 production (and mucin production). Inhibition of this pathway suggests novel mechanisms for the treatment of chronic inflammatory airway diseases such as COPD, cystic fibrosis, and acute asthma, where mucus hypersecretion and neutrophilic inflammation predominate.


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This work was supported by private funding (to J. A. Nadel) and by the Alpha One Foundation (to M. X. G. Shao).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Nadel and M. X. G. Shao, Cardiovascular Research Institute and Depts. of Medicine and Physiology, Univ. of California, San Francisco, CA 94143-0130 (e-mail: jay.nadel{at}ucsf.edu and matt.shao{at}ucsf.edu)

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.


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