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Published ahead of print on January 27, 2005, doi:10.1165/rcmb.2004-0302OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 462-469, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0302OC

Regulated Hydrogen Peroxide Production by Duox in Human Airway Epithelial Cells

Radia Forteza, Matthias Salathe, Françoise Miot, Rosanna Forteza and Gregory E. Conner

Department of Cell Biology and Anatomy, and Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Miami School of Medicine, Miami, Florida; and IRIBHM, Faculté de Médecine, Université Libre de Bruxelles, Brussels, Belgium

Correspondence and requests for reprints should be addressed to Gregory E. Conner, Ph.D., University of Miami School of Medicine, P.O. Box 016960 (R124), Miami, FL 33101. E-mail: gconner{at}miami.edu


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Hydrogen peroxide (H2O2) is found in exhaled breath and is produced by airway epithelia. In addition, H2O2 is a necessary substrate for the airway lactoperoxidase (LPO) anti-infection system. To investigate the source of H2O2 produced by airway epithelia, PCR was used to screen nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression in human airway epithelia redifferentiated at the air–liquid interface (ALI) and demonstrated the presence of Duox1 and 2. Western blots of culture extracts indicated strong expression of Duox, and immunohistochemistry of human tracheal sections localized the protein to the apical portion of epithelial cells. Apical H2O2 production was stimulated by 100 µM ATP or 1 µM thapsigargin, but not 100 µM ADP. Diphenyleneiodonium, an NADPH oxidase inhibitor, and dimethylthiourea, a reactive oxygen species scavenger, both inhibited this stimulation. ATP did not stimulate the basolateral H2O2 production by ALI cultures. ATP and thapsigargin increased intracellular Ca2+ with kinetics similar to increasing H2O2 production, and thus consistent with the expected Ca2+ sensitivity of Duox. These data suggest that Duox is the major NADPH oxidase expressed in airway epithelia and therefore a contributor of H2O2 production in the airway lumen. In addition, the data suggest that extracellular H2O2 production may be regulated by stimuli that raise intracellular Ca2+.

Key Words: Duox • hydrogen peroxide • calcium • purinergic


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Hydrogen peroxide (H2O2) is present in exhaled breath condensate and airway secretions and is produced by airway epithelial cells (1–4). We have shown that lactoperoxidase (LPO) is present and active in airway secretions (5). This host defense enzyme protects epithelial surfaces from colonization and infection by using H2O2 to oxidize thiocyanate anions (SCN–) to hypothiocyanite, a strong antimicrobial agent (6). Thus, LPO requires H2O2 to make this antibacterial substance in airway secretions. The enzymatic source for H2O2 production in human airway, however, remains unclear.

Phagocytes (neutrophils, eosinophils, monocytes, and macrophages) contain an equivalent host defense system (i.e., myeloperoxidase and eosinophil peroxidase) that uses H2O2 to oxidize Cl– or SCN– to form HOCl and HOSCN, powerful anti-infection agents (7). In phagocytes, the enzymatic generation of H2O2 is achieved by the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. This enzyme is a multicomponent electron transport system anchored in the plasma membrane, where the catalytic subunit, gp91phox, reduces the molecular oxygen in an NADPH-dependent manner to form superoxide, which dismutates to H2O2 (8, 9). Several homologs of the gp91phox NADPH oxidase subunit of phagocytes have been identified and form the Nox gene family (10). The Nox messenger RNAs are present in a variety of human tissues including kidney (11), colon (12), vascular smooth muscle cells (13), osteoclast (14), and thyroid (15), and their orthologs have been found in rat and Caenorhabditis elegans (16). An evolutionary tree has been constructed in which three subgroups have been proposed to describe the Nox family members (17). One group comprises Nox 1, Nox 2 (gp91phox), Nox 3, and Nox 4, all having in common a similar molecular mass (~ 65 kD). Another subgroup consists of Dual oxidase 1 and 2 (Duox1, Duox2), the high molecular weight homologs (~ 180 kD) characterized by an additional N-terminal peroxidase domain and two EF-hand Ca2+-binding domains. Finally, Nox 5 is divergent from the other members.

Although the predicted domain structure of Nox family members is well established, their functions in all tissues remain to be elucidated. Different functions have been proposed in which Nox proteins may participate in biological processes including control of cell growth (18, 19), bone remodeling (14), control of vascular tone (20), regulation of thyroid hormone biosynthesis (21, 22), and acid secretion (23). Because airway LPO requires H2O2 for activity, we investigated NADPH oxidase in human airway epithelia that perhaps could provide a regulated substrate source for LPO. Previous reports by us and others (16, 23–25) have indicated the presence of Duox in the human respiratory tract. Our data, described here and earlier in abstract form (25), confirm that Duox 1 and 2 are the major NADPH oxidases expressed in airway epithelia. The data described here also show the production of extracellular H2O2 by airway epithelia that is enhanced by stimuli that increase [Ca2+]i, consistent with the EF hand domains present in Duox1 and 2.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Source of Materials
TRIzol reagent, SuperScript First Strand Synthesis System for reverse transcriptase–polymerase chain reaction (RT-PCR), and PCR reagent System were purchased from Invitrogen Corp. (Carlsbad, CA). BCA protein assay kit was purchased from Pierce (Rockford, IL); dithiothreitol (DTT) from Boehringer Mannheim Biochemicals (Indianapolis, IN); 7.5% Ready Gel Precast SDS-polyacrylamide gels and gelatin from Bio-Rad (Hercules, CA); polyvinylidene fluoride (PVDF) Immobilon-P transfer membrane from Millipore (Billerica, MA). Horseradish peroxidase (HRP)-conjugated Streptavidin, alkaline phosphatase–coupled goat anti-rabbit IgG, rabbit nonimmune serum, and the affinity-purified biotin labeled goat anti-rabbit IgG were purchased from KPL (Gaithersburg, MD). Normal goat serum was purchased from Chemicon (Tamecula, CA); Endogenous Biotin Blocking kit, 10-acetyl-3, 7 dihydroxyphenoxazine (Amplex Red), pluronic acid F-127, and fura-2 AM from Molecular Probes (Eugene, OR); and thapsigargin from Calbiochem (San Diego, CA). All other materials were purchased from Sigma Chemical Co. (St.Louis, MO).

Cell Culture
Human airways were obtained from organ donors whose lungs were not to be used for transplant from the Life Alliance Organ Recovery Agency of the University of Miami. All tissues were obtained after appropriate consent and according to local IRB-approved protocols. Airway epithelial cells were isolated, grown, and redifferentiated at the air–liquid interface (ALI) on 24- or 6.5-mm Transwell-clear culture inserts from Corning Costar Corporation (Cambridge, MA) and coated with human placenta collagen Type IV as previously described (26, 27). Fischer rat thyroid epithelial cells (FRTL-5) were a generous gift from Dr. Robert B. Levy (Department of Microbiology and Immunology, University of Miami School of Medicine). These cells were maintained as previously described (28). The cells were trypsinized after 5–7 d of culture and used for Western blot analyses.

RT-PCR
RNA was extracted from ALI epithelial cells using TRIzol reagent. RNA (2 µg) was reverse transcribed using the SuperScript First Strand Synthesis System for RT-PCR. The final reaction mixture (0.025 µg/µl oligodT, 0.5 mM dNTPs, 1x RT buffer, 5 mM MgCl2, 0.01 M DTT, and RNase inhibitor) was incubated with SuperScript II reverse transcriptase (2.5 U/µl) for 50 min at 42°C. The reaction was terminated at 37°C for 20 min with RNase H. To control for genomic DNA contamination, additional RNA samples were processed without reverse transcriptase. First-strand cDNAs were amplified by PCR using Taq polymerase (2.5 U/µl) and the oligonucleotide primers described in Table 1. Annealing temperatures were: primer sets 1 and 5 (Table 1), 55°C; set 2, 48°C; and set 3, 51°C. Amplification with primer sets 1–3 and set 5 were 30 cycles of 45 s denaturation at 94°C, 45 s annealing, and 1 min extension at 72°C, followed by a final extended elongation of 8 min at 72°C. The amplification using set 4 primers was performed for 27 cycles of 15 s at 94°C, 30 s at 60°C, and 4 min at 68°C; followed by an extended elongation of 10 min at 68°C using a thermal cycler (Brinkmann Instruments Inc., Westbury, NY).


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  		TABLE 1. Oligonucleotide primers

 
Western Blotting
Protein extracts from ALI cultures, FRTL-5 cells, and CHO cells were obtained by lysis at 100°C in 1% SDS, 10 mM Tris pH 8.5, 0.1 M EDTA, followed by sonication, and centrifugation at 14,000 x g. Total protein was determined using the BCA protein assay. For each cell type, 20 µg of protein extract were solubilized with 100 mM Tris pH 6.8, 4% SDS, 20% glycerol, and 200 mM DTT, separated by a 7.5% SDS/polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically to polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked at room temperature for 2 h with 1% gelatin prepared in 20 mM Tris Cl, pH 7.5, 140 mM NaCl (TBS), and containing 0.05% Tween-20 (TTBS). The membranes were incubated overnight at 4°C with either a rabbit nonimmune serum prepared in 1% gelatin or a rabbit polyclonal antibody raised against the Arg618-His1044 intracellular fragment of human Duox1/ThOx1 (15, 29) that recognizes both Duox1 and 2. The membranes were then incubated for 1 h with an alkaline phosphatase–coupled goat anti-rabbit IgG. The immune complex was detected using 0.16 mg/ml 5-bromo-4-chloro-3-indoyl phosphate (BCIP) plus 0.32 mg/ml nitroblue tetrazolium (NBT) in 100 mM Tris pH 9.5, 100 mM NaCl, and 5 mM MgCl2.

Immunohistochemistry
Normal human tracheal rings were fixed with 4% paraformaldehyde and prepared for embedding in paraffin in a microwave Tissue Processor (Microwave Materials Technologies Inc., Knoxville, TN) according to the manufacturer's protocol using a nonxylene method. Embedding and sectioning were performed by the Histology Laboratory at University of Miami Hospital and Clinic-Sylvester Comprehensive Cancer Center.

The sections were deparaffinized, rehydrated, and quenched for endogenous peroxidase activity with 3% H2O2 in methanol for 45 min at room temperature. Phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBS-T) was used for washes. Sections were treated for antigen retrieval using 10 mM citrate buffer (pH 6.0) for 15 min at 80°C and blocked with 5% normal goat serum diluted in PBS-T for 1 h at room temperature. Biotin background binding was reduced using the Endogenous Biotin Blocking kit according to manufacturer's instructions. Sections were incubated overnight at 4°C with either anti-Duox1/ThOx1 antibody (1:200 dilution) or a rabbit nonimmune serum as a negative control, both prepared in 1% normal goat serum/PBS-T. Sections were then incubated for 45 min with an affinity-purified biotin-labeled goat anti-rabbit IgG (2.5 µg/ml), followed by an incubation for 30 min with HRP-conjugated streptavidin in 1% normal goat serum/PBS-T (0.3 µg/ml). Immunoreactivity was visualized using the AEC Chromogen kit (2.5 M acetate buffer, pH 5.0, 3-amino-9-ethylcarbazole [AEC] in N,N-dimethylformamide and 3% H2O2) according to the manufacturer's instruction.

H2O2 Measurements
H2O2 was measured by two different approaches. First, ALI epithelial cells were incubated with 500 µl Dulbecco's PBS in the apical chamber and culture media either alone or with 1 µM thapsigargin, 1 µM diphenyleneiodonium (DPI), or 1 µM of both thapsigargin and DPI in the basolateral chamber. After 60 min of incubation at 37°C, samples were collected from the apical chamber. Triplicates of each sample were loaded in a 96-well microplate and mixed with 50 µM of 10-acetyl-3,7 dihydroxyphenoxazine (Amplex Red) combined with 0.1 U/ml of HRP (30). For each experiment, the amount of apical H2O2 production was determined using a H2O2 standard curve from 0–2 µM. The fluorescence was read at Ex 530 nm/Em 590nm (9 nm bandwidth) using a SpectraMax Gemini EM fluorometer (Molecular Devices, Sunnyvale, CA).

Alternatively, triplicates of ALI epithelial cells in 6.5-mm Transwell-clear culture inserts were directly placed onto the SpectraMax Gemini EM fluorometer (Molecular Devices). Instead of collecting samples, the reaction mixture containing Amplex Red (50 µM) and HRP (0.1 U/ml) in Dulbecco's PBS was applied to the apical surface of the ALI cultures, and 500 µl of Hanks' Balanced Salt Solution (HBSS) was added to the basolateral chamber. ALI cultures subjected to DPI inhibition were preincubated for 30 min at 37°C with 20 µM DPI. Apical fluorescence measurements (Ex 530 nm/Em 590 nm) were performed at 37°C every 20 s for 5 min to establish a baseline. ALI cultures were then removed from the fluorometer and treated at the apical surface with PBS alone or PBS containing: 100 µM ATP, 100 µM ATP plus either 20 µM DPI or 40 mM dimethylthiourea (DMTU), 100 µM ADP, or 1 µM thapsigargin. The fluorescence was read at 20-s intervals for an additional 30 min at 37°C. Fluorescence was normalized to the last recorded baseline value and plotted as Fx/F0 (NRFU). For basolateral H2O2 production by ALI cultures, 50 µl of reaction mixture (sufficient to fill the space between the membrane and the culture dish) containing Amplex Red (50 µM) with HRP (0.1 U/mL) in HBSS supplemented with Hepes (pH 7.4) was applied to the basolateral compartment and, to the apical surface, 100 µM ATP in PBS or PBS alone was added. Moreover, in combination with Amplex red/HRP solution in the basolateral compartment, we added either 100 µM ATP, 1 µM thapsigargin or PBS. The basolateral fluorescence measurement was performed as described above except the SpectraMax plate reader was set to bottom read.

[Ca2+]i Measurements
Culture inserts (6.5 mm) of ALI epithelial cells were incubated with the calcium indicator fura-2 AM (5 µM) supplemented with 0.1% pluronic acid (vol/vol) in the basolateral and apical chambers for 30 min at 37°C followed by 30 min at room temperature. The filters were then washed three times with HBSS supplemented with 10 mM Hepes (pH 7.4) in the basolateral and Dulbecco's PBS in the apical compartments over a period of 30 min, after which filters were loaded onto the SpectraMax Gemini EM fluorometer. Fura-2 fluorescence was recorded at 510 nm using sequential excitation at 340 and 380 nm. Measurements were repeated at 30-s intervals for the duration of the experiment. After the baseline was established (5 min), cultures were stimulated with 100 µM ATP or 1 µM thapsigargin, and readings continued every 30 s for another 30 min. Background fluorescence, determined in parallel cultures not incubated with fura-2, was subtracted from measured values before calculating ratios.

Statistical Analysis
Data from the H2O2 production measurements were expressed as mean ± SE. The statistical significance of differences between means was determined by ANOVA followed by the Tukey-Kramer honestly significantly difference test using JMP software from SAS (Cary, NC). P values < 0.05 were considered significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Duox mRNA in Human Airway Epithelial Cells
We previously demonstrated that LPO is a functional airway host defense system using an in vivo sheep model (31) and that LPO in human airway secretions actively kills bacteria in vitro (5). LPO requires H2O2 as a substrate and our preliminary investigations showed that Duox homologs of the phagocytic NADPH oxidase were expressed in human airway epithelial cells (25). To confirm these data and the recent results of others (23, 24), oligonucleotides were designed to be complementary to: (1) gp91phox, Nox1, and Duox/ThOx 1 and 2; (2) Nox 3 and 4; and (3) Nox 5 based on available published sequences (Table 1). Set 1 and 2 primers were degenerate. RT-PCR with set 1 primers yielded a cDNA fragment of 248 bp as described in a previous abstract (25), whereas the other oligonucleotide primers only gave faint, nonspecific PCR products (Figure 1). When sequenced and aligned, the 248-bp fragment was identical with human Duox1, a large molecular weight NADPH oxidase homolog. Sequence analysis of six different cDNAs that were weakly amplified by the Nox 3 and 4 and the Nox 5 primer sets showed that none was related to other NADPH oxidases. To further confirm these results, gene-specific primers chosen within the 5'UTR and 3'UTR of human Duox1 (Genbank accession No. NM_017434) were used for RT-PCR. This procedure generated a single 5.5-kb cDNA sequence (Figure 2a), and its sequence analysis showed that it was identical with human Duox1, previously identified in human thyroid cells under the name of ThOx1 (15). Since Schwarzer and colleagues (23) have recently shown that both Duox1 and 2 were present in airway epithelia, we also designed specific primers for Duox2 and detected this mRNA by RT-PCR as well (Figure 2b).



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  		Figure 1. RT-PCR of NADPH oxidase homolog expression. RT-PCR of mRNA of human ALI epithelial cells was performed using oligonucleotide primers described in Table 1: primer set 1 (lanes 1 and 2), set 2 (lanes 3 and 4), and set 3 (lanes 5 and 6). RT-PCR using hGAPDH primers gave a 250-bp band and served as a positive control (lanes 7 and 8). Reactions without reverse transcriptase (lanes 2, 4, 6, and 8) served as controls for DNA contamination. This broad screen of NADPH oxidase expression generated a 248-bp cDNA fragment with set 1 primers, whereas two other primer sets yielded nonspecific products that were unrelated by sequence to the Nox gene family. The 248-bp RT-PCR product was dependent on the presence of reverse transcriptase (lane 2).

 


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  		Figure 2. RT-PCR of human Duox mRNA. Two sets of gene specific primers (Table 1) designed to complement human Dual oxidase 1 (a) and human Dual oxidase 2 (b) were used to perform RT-PCR with total mRNA of ALI epithelial cells. (a) A cDNA sequence of 5.5 kb corresponding to Duox1 was detected (lane 1), while no band was amplified in the absence of RT (lane 2). (b) A 181-bp cDNA fragment was identical with Duox2 (lane 1), which was not detected in absence of reverse transcriptase (lane 2). In both panels, hGAPDH was also amplified in the presence (lane 3, 250 bp), but not in the absence (lane 4), of RT.

 
Duox Protein Expression in Airways
To investigate whether Duox protein is expressed in human airway epithelial cells, fully redifferentiated ALI cultures were solubilized in SDS and Western blot analysis was performed using a rabbit polyclonal antibody raised against the intracellular domain of Duox1, although this antibody recognizes both Duox1 and 2 and cannot differentiate between the two. An immune complex was detected with an apparent molecular weight of 185 kD (Figure 3a). This was consistent with previous analyses of thyroid tissue in which Duox proteins appeared between 164 and 190 kD, depending on the degree of glycosylation (15, 32). No labeling was detected with a rabbit nonimmune serum (Figure 3b). FRTL-5 cells, previously used to detect Duox proteins (15, 33), served as a positive control, but showed a weaker signal for Duox consistent with reduced crossreactivity of the antibody to rat Duox. Chinese hamster ovary (CHO) cells were used as a negative control because none of the Duox proteins are expressed in them (29); no significant labeling was detected. These data suggest that Duox1 and/or 2 were major NADPH oxidase homologs expressed at the mRNA and protein level in human airway epithelial cells.



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  		Figure 3. Western blot analysis of Duox protein expression. Twenty micrograms of each extract from human ALI cultures, and FRTL-5 and CHO cells were separated by 7.5% SDS-PAGE. Immunoblot analyses were performed using a rabbit polyclonal antibody raised against the Arg618-His1044 fragment of Duox1 (a) or a rabbit nonimmune serum (b). (a) A single band of protein was detected in ALI epithelial cells (lane 1) with an apparent molecular weight of 185 kD corresponding to Duox1 and 2; only a fainter expression was seen in FRTL-5 cells (lane 2), and no expression was observed in CHO cell line (lane 3). (b) No labeling was detected using the rabbit nonimmune serum to blot the membrane.

 
Cellular Localization of Duox Protein in Human Airway Tissues
To localize Duox protein expression, we examined tissue sections of human trachea by immunohistochemistry with the rabbit polyclonal anti-Duox antibody used for Western blot analysis. Positive labeling by the anti-Duox antibody was seen in the airway epithelium (Figure 4a). The majority of the staining was observed both in the apical plasma membrane and in the apical portion of epithelial cells, the greater labeling being present in the latter. No labeling was obtained when the tracheal tissues were incubated with a rabbit nonimmune serum (Figure 4b).



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  		Figure 4. Localization of Duox protein in human airway tissues by immunohistochemistry. Immunostaining of human tracheal sections was performed using a rabbit polyclonal anti-Duox1 antibody (a) or a rabbit nonimmune serum (b), followed by incubation with an affinity-purified biotin-labeled goat anti-rabbit IgG. The immune complex was then identified using HRP-conjugated Streptavidin followed by AEC chromogen: Duox protein was localized both in the apical plasma membrane and in the apical cytoplasm of epithelial cells (a); no staining was detected using the rabbit nonimmune serum (b). Arrows indicate the apical side of the epithelium and arrowheads indicate the basement membrane. Bar = 20 µm.

 
H2O2 Production at the Apical Surface of Airway Epithelial Cells
The catalytic subunit of the phagocytic NADPH oxidase generates superoxide that rapidly dismutates to H2O2 (9). To test if the Duox 1 and 2 being expressed in epithelial cells were the source of the H2O2 generated by these cells, we investigated the function of Duox protein in human ALI epithelial cells using a quantitative fluorometric micro-assay based on HRP-catalyzed H2O2 oxidation of Amplex Red (30). The assays examined the release of H2O2 into the apical extracellular compartment during a 1-h period by sampling PBS applied to the surface of the culture.

Because previous studies on porcine and human thyroid plasma membrane preparations demonstrated the existence of a Ca2+-dependent NADPH oxidase activity (34–37) and the predicted molecular structure of Duox1 and 2 showed two EF-hand motifs forming the calcium-binding domain (38), we assessed basal and Ca2+-stimulated H2O2 production in airway epithelia. ALI cultures were stimulated for 1 h with thapsigargin (1 µM) that has previously been shown to increase [Ca2+]i in our cultured airway epithelial cells (39). After thapsigargin treatment, the apical H2O2 increased from a baseline value of 14.0 ± 3.75 pmoles/h/cm2 to 30.4 ± 4.10 pmoles/h/cm2 (mean ± SE, n = 18, P < 0.05) (Figure 5). To confirm that this H2O2 production was due to an NADPH oxidase–like activity, ALI cultures were treated with thapsigargin in the presence of 1 µM of diphenyleneiodonium (DPI), an NADPH oxidase inhibitor. DPI treatment blocked the thapsigargin-induced increase in apical H2O2 production (14.1 ± 4.27 pmoles/h/cm2, n = 10, P < 0.05 compared with thapsigargin treatment). However, 1 µM DPI alone did not significantly reduce the baseline H2O2 production of unstimulated cultures (10.6 ± 4.48 pmoles/h/cm2, n = 13, P > 0.05 compared with untreated control), suggesting that thapsigargin-stimulated H2O2 production was due to NADPH oxidase activity. Cultures grown and differentiated under identical conditions contained 2–4 x 106 cells/cm2.



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  		Figure 5. H2O2 production activity at the surface of airway epithelial cells during a 1-h incubation. ALI epithelial cells were incubated for 1 h at 37°C with PBS at the apical surface and either PBS, 1 µM thapsigargin (T), 1 µM DPI, or 1 µM of both thapsigargin and DPI (T/DPI) in the basolateral medium. After 1 h, samples were collected from the upper chamber and the amount of apical H2O2 determined using a quantitative fluorometric microassay based on HRP-catalyzed H2O2 oxidation of Amplex Red. H2O2 values were obtained by interpolation of the standard curve and are expressed as mean ± SE, n = 10–18.

 
Continuous Measurement of H2O2 Production by Human Airway Epithelia
The previous measurements were of samples from apically applied PBS after 1 h incubation, and thus any increase in [H2O2] over PBS control could only be due to stimulated appearance in the apical compartment. To further investigate the calcium-regulated apical H2O2 production, H2O2 was measured continuously by loading Amplex Red and HRP directly to the surface of ALI cultures maintained at 37°C, and fluorescence was measured at 20-s intervals.

Our previous studies have shown that apically applied extracellular ATP was able to cause a rapid, large, and transient increase in [Ca2+]i in these human airway epithelial cells via activation of P2Y receptors (40, 41). Therefore, we tested the effect of ATP, alone or in combination with DPI, on apical H2O2-dependent and HRP-mediated oxidation of Amplex Red. Fluorescence was measured before stimulation to establish baseline H2O2 production, and post-stimulation values were normalized to the last recorded baseline value (NRFU) after addition of either ATP alone or ATP with DPI, ADP, thapsigargin, or PBS control to the apical surface (Figure 6). Within the first 200 s after addition of ATP, H2O2 production showed a rapid and significant increase (0.0096 ± 0.0036 NRFU/s, n = 39, compared with the control of 0.0022 ± 0.0009 NRFU/s, n = 39, P < 0.05). DPI inhibited this ATP-induced fluorescence increase (0.0036 ± 0.0009 NRFU/s, n = 33, P < 0.05). Moreover, the ATP-induced increase in H2O2 production was specific, as ADP did not induce a significant increase over baseline production (0.0032 ± 0.0003 NRFU/s, n = 27, P > 0.05). Thapsigargin induced a slower increase in NRFU compared with ATP, seen 400–600 s after addition (0.0046 ± 0.0003 NRFU/s, n = 24, compared with the control 0.0022 ± 0.0002 NRFU/s, n = 39, P < 0.05). DPI treatment did not lower baseline production (0.0025 ± 0.0002 NRFU/s, n = 33, P > 0.05).



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  		Figure 6. Continuous measurement of Duox activity. A reaction mixture of Amplex red (50 µM) and HRP (0.1 U/ml) was loaded on the apical surface of ALI cultures, which were then placed directly into the fluorometer at 37°C. Measurements were performed at 20-s intervals for 5 min to establish a baseline. Cultures were stimulated with 100 µM ATP (filled squares), 100 µM ATP combined with 20 µM DPI (triangles), 100 µM ADP (open squares), 1 µM thapsigargin (open circles), or PBS (filled circles), and the fluorescence was read for an additional 30 min at 20-s intervals and normalized to the last prestimulation value. Cultures were returned to the fluorometer at t = 0 s. Data were expressed as mean values of Fx/F0 (NRFU) ± SE, n = 24–39.

 
In addition, ALI epithelial cell cultures were apically stimulated with either ATP alone or ATP plus dimethylthiourea (DMTU) (Figure 7A), a permeable chemical scavenger of reactive oxygen species (ROS) including H2O2, hydroxyl radicals, and other hydroxylated products (42–44). DMTU significantly reduced the ATP-induced increase in apical ROS production within the first 200 s after addition (0.0067 ± 0.0009 NRFU/s, n = 9, compared with ATP alone 0.0115 ± 0.0015 NRFU/s, n = 9, P < 0.05) and ROS production remained lower than the ATP-stimulated one throughout the experiment. However, DMTU did not lower ROS production to control values (0.0038 ± 0.0007 NRFU/s, n = 9, compared with ATP + DMTU, n = 9, P < 0.05). The scavenging activity of DMTU on ATP-stimulated ROS production was dose-dependent (data not shown).



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  		Figure 7. H2O2 dependent oxidation of Amplex Red. (A) Triplicates of ALI epithelial cell cultures were incubated at 37°C with a reaction mixture of Amplex Red (50 µM) and HRP (0.1 U/ml) in the apical chamber. Fluorescence measurements were performed at 20-s intervals for 5 min to obtain a baseline. At t = 0, cultures were stimulated with 100 µM apical ATP (filled squares), 100 µM ATP plus 40 mM DMTU (triangles), or PBS (filled circles) and the fluorescence was read for an additional 30 min at 20-s intervals. (B) Apical fluorescence measurement of oxidized Amplex Red was performed from ALI cultures stimulated at t = 0 with 100 µM ATP in the presence (filled squares) or in the absence (open squares) of HRP (0.1 U/ml). Control cultures were incubated with PBS in the reaction mixture of Amplex Red/HRP (filled circles). The data were expressed as mean values of Fx/F0 (NRFU) ± SE, n = 9 for each experimental condition.

 
To further confirm that the measured fluorescence was due to H2O2 produced at the apical surface of ALI cultures, we performed the assay by omitting HRP during the ATP stimulation (Figure 7B). The results showed a significantly smaller increase in NRFU within the first 200 s after addition of ATP in absence of HRP (0.0054 ± 0.0009 NRFU/s, n = 9, compared with ATP in the presence of HRP 0.0226 ± 0.0053 NRFU/s, n = 9, P < 0.05). There was no difference seen between the NRFU values after ATP in absence of HRP and the control values where the cells were unstimulated (0.0058 ± 0.0005 NRFU/s, n = 9, P > 0.05).

To assess H2O2 production at the basolateral (BL) compartment of the ALI cultures, the fluorescence from Amplex Red oxidation by H2O2 in the presence of HRP was measured at 20-s intervals from the bottom side of the cultures as described above for the apical side. Addition of ATP at the apical surface did not increase the BL H2O2 production (0.0017 ± 0.0009 NRFU/s, n = 9, compared with the control 0.0016 ± 0.0007 NRFU/s, n = 9, P > 0.05) (Figure 8A). Moreover, when ATP (Figure 8B) or thapsigargin (Figure 8C) was added to the BL compartment along with Amplex Red/HRP, there was no increase of NRFU compared with the control (ATP: 0.0016 ± 0.0003 NRFU/s, n = 9, compared with the control 0.0019 ± 0.0004 NRFU/s, n = 9, P > 0.05; thapsigargin: 0.0018 ± 0.0004 NRFU/s, n = 9, compared with the control 0.0019 ± 0.0003 NRFU/s, n = 9, P > 0.05). These data suggest that the Ca2+-regulated H2O2 production measured by Amplex Red/HRP is restricted to the apical compartment of ALI epithelial cells.



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  		Figure 8. Basolateral H2O2 production by human ALI epithelial cells. ALI cultures were incubated at 37°C with 50 µl of Amplex Red (50 µM) plus HRP (0.1 U/ml) in the BL compartment. Fluorescence measurements were made from the BL side of ALI cultures for 5 min to establish a baseline. At t = 0, cultures were stimulated for 30 min and fluorescence was read every 20 s after addition of: 100 µM ATP (filled squares) or PBS (open circles) to the apical surface (A); 100 µM ATP (filled squares) or PBS (open circles) to the basolateral compartment (B); 1 µM thapsigargin (triangles) or PBS (open circles) to the basolateral compartment (C). Data were expressed as mean values of Fx/F0 (NRFU) ± SE, n = 9 for each experimental condition.

 
To confirm that both ATP and thapsigargin raise intracellular calcium concentration in ALI cultures under these conditions, changes in [Ca2+]i were estimated using the calcium indicator fura-2 after stimulation with ATP or thapsigargin (Figure 9). A similar time course of changes in the fura-2 ratio and H2O2 production was observed upon addition of either ATP or thapsigargin. Addition of ATP induced a rapid transient peak increase of the fura-2 ratio immediately after addition before decreasing back toward baseline (Figure 9B). Thapsigargin induced a slower increase of the fura-2 ratio during the first 200 s after its addition, but maintained an increased fura-2 ratio (Figure 9C). DPI did not inhibit ATP-mediated increases in the fura-2 ratio. Thus, DPI does not block ATP-stimulated H2O2 production by blocking increases in [Ca2+]i (data not shown).



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  		Figure 9. Estimation of [Ca2+]i using fura-2 ratio measurements. ALI cultures were loaded with fura-2 AM. Changes in [Ca2+]i were followed by measuring ratios of fura-2 fluorescence emission at 340 and 380 nm excitations. After measuring fura2 ratios for prestimulation values, cultures were removed from the fluorometer, stimulants were added and the cultures returned to the instrument at t = 0 s. All measurements were at 37°C. (A) PBS control; (B) 100 µM ATP; (C) 1 µM thapsigargin. Plotted ratios are means ± SE, n = 5.

 
The demonstration of DPI inhibitable apical H2O2 generation under conditions that increase [Ca2+]i is consistent with the notion that Duox in airway epithelia catalyzes the formation and release of H2O2 in the airway lumen.


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Hydrogen peroxide is an essential substrate for the LPO antibacterial system found in human airways. The studies presented here were undertaken to identify the enzymatic source of H2O2 in the airway lumen that might provide this substrate for LPO. The data showed that human airway epithelial cells express Duox mRNA, confirming reports by us and others (16, 23–25), and providing evidence of Duox protein expression in native airway epithelial sections. Our immunohistochemistry studies showed an apical localization of Duox in the tracheal surface epithelia in agreement with previous studies of thyroid gland (29) and isolated cultured airway epithelial cells (23). The assays, used here, measured H2O2 released to the apical, extracellular space, also suggesting that Duox is located on or close to the surface of the epithelial cells. The data also show that extracellular, apical H2O2 production, likely by Duox, is stimulated by ATP and thapsigargin, both agents that increased [Ca2+]i. Because Duox1 and 2 are highly related and of similar molecular size, Western blot analysis does not adequately distinguish the two forms; thus, we were not able to distinguish protein expression and activity differences between these two forms.

Two independent lines of evidence suggested that Duox is the major NADPH oxidase found in airway epithelia. First, RT-PCR studies suggested that Duox mRNA is the most abundant homolog. Second, airway epithelia increased H2O2 production after stimulation with thapsigargin or ATP, which both increased [Ca2+]i as shown by fura-2 fluorescence (40, 41). This stimulated H2O2 production was DPI-sensitive. These data are consistent with the known presence of EF-hand motifs in a Duox domain predicted to be on the cytosolic face of the membrane (15). Thus, it is probable, although not directly demonstrated, that the observed DPI-sensitive H2O2 production in airway epithelia reflects Duox activity. Significant oxidation of Amplex red was not observed in the absence of HRP in the assay, suggesting that Duox was not capable of oxidizing significant quantities of the substrate and also suggesting that the assay measured H2O2.

Previous studies have suggested that H2O2 production inside airway epithelia did not rise in response to thapsigargin-mediated [Ca2+]i increases (45). These latter studies used changes in fluorescence of Rhodamine 123-loaded cells to assess increases in intracellular reactive oxygen species. In contrast, the studies reported here show that extracellular H2O2 production by airway epithelial cells rises in response to thapsigargin. Our studies measured changes in H2O2 released at the apical surface of epithelia that would largely be undetected by the Rhodamine 123 method.

The presence of Ca2+-regulated H2O2 release at the apical surface is consistent with the immunolocalization data showing that Duox activity is on or near the surface of the cells. In fact, Duox topology predicts the release of superoxide into the extracellular compartment. Why Duox is not active inside thyroid and airway epithelia is not known. Other membrane proteins have been reported to coexist on the surface and in internal structures (46) and to be regulated in part by altering the relative amounts found on the surface.

Since ADP did not stimulate H2O2 production, it appears that ATP stimulation of H2O2 production may be mediated by a P2Y receptor. Pseudomonas aeruginosa flagellin binding to bronchial epithelial cells has been shown to release ATP from cells, and it has also been shown that the released ATP mediates increased mucin transcription via P2Y receptors (47). P2Y receptors also mediate a variety of other epithelial host defense responses, e.g., increases in ciliary beating (27, 39, 48) and secretion of mucus (49–51). P2Y receptor activation may also stimulate H2O2 production thus complementing other aspects of airway host defense. It is interesting that Duox also contributes to acidification of airway surface liquid (23). Because airway LPO activity increases with decreasing pH (48), stimulation of Duox activity may increase LPO activity through multiple mechanisms.

LPO appears to be constitutively expressed and secreted by human airways (5). Because it requires H2O2 for its activity, the previously reported presence of H2O2 in airways (2–4) may reflect the normal production of substrate for LPO. LPO is not expressed at measurable levels in these culture conditions, and thus it was not possible to show a direct link between Duox-mediated H2O2 production and LPO activity in vitro. However, increased airway H2O2 seen during stimulation by irritants or allergens may serve in part to increase LPO-mediated host defense. Because Duox is the major NADPH oxidase in airway epithelia and appears to respond to P2Y stimulation to release H2O2 to the apical surface, it is an excellent candidate source for LPO substrate needed for airway host defense.


   Footnotes
 
Conflict of Interest Statement: R.F. has no declared conflicts of interest; M.S. has no declared conflicts of interest; F.M. has no declared conflicts of interest; R.F. has no declared conflicts of interest; and G.E.C. has no declared conflicts of interest.

Received in original form September 23, 2004

Received in final form January 21, 2005


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