Introduction

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Mucus obstruction of the airways causes significant morbidity in chronic inflammatory airway diseases, including cystic fibrosis (CF), chronic bronchitis, and bronchiectasis. Inflammation in these airway diseases is characterized by numerous neutrophils in the bronchoalveolar lavage fluid of these patients (1, 2). The presence of neutrophils in bronchoalveolar lavage fluid directly correlates with impaired pulmonary function (3). These clinical studies suggest that neutrophilic inflammation and airway mucus obstruction are closely linked.

Neutrophils release elastase (EC 3.4.21.37), a serine protease that is found in high concentrations in airway surface fluids of patients with chronic airway diseases (1, 2, 4, 5). Neutrophil elastase (NE) impairs mucociliary clearance by several mechanisms, leading to mucus obstruction of the airways: NE injures cilia and impairs ciliary function (6), and stimulates mucin production, secretion, and release (7). In addition, NE causes secretory granule accumulation and secretory cell metaplasia/hyperplasia in the airways (10, 11). We reported that NE treatment increases MUC5AC mucin gene expression and glycoprotein production in respiratory epithelial cells. The increase in MUC5AC gene expression was attributed to NE enhancement of mRNA stability (12).

In this report, we sought to determine NE-induced intracellular signaling events required for MUC5AC gene regulation. NE activates several epithelial intracellular signals such as kinases (e.g., interleukin-1 receptor-associated kinase [13] and mitogen activated protein kinase [14]) and reactive oxygen species (ROS) (15). Using in vitro models of respiratory epithelial cells, we demonstrate that NE triggers generation of ROS and that antioxidants inhibit NE-induced MUC5AC expression. These results indicate that NE induces MUC5AC gene expression in airway epithelial cells via an oxidant-dependent mechanism.

	Materials and Methods

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Reagents

A549 cells were obtained from ATCC (CCL-185, Rockville, MD). Ham's F-12K medium, Dulbecco's modified essential medium, fetal bovine serum, penicillin, streptomycin, trypan blue, and TRIZOL were from GIBCO-BRL (Rockville, MD). Normal human bronchial epithelial cells, bronchial epithelial basic medium, and Singlequot supplements were from Clonetics (San Diego, CA). Rat tail collagen type I was purchased from Collaborative Biochemical (Bedford, MA). Transwell filters were purchased from Corning Costar (Cambridge, MA). Epidermal growth factor and bovine serum albumin were from Intergen (Purchase, NY). Neutrophil elastase (875 U/mg protein) and N-Suc-Al-Al-Al-pNitroanalide were from Elastin Products, Co. (Owensville, MO). Nylon filter (Nytran plus) was from Schleicher and Schuell (Keene, NH). X-Omat AR film was purchased from Kodak (Rochester, NY). [-³²P]dCTP and [-³²P]ATP were from Amersham Corp. (Arlington Heights, IL). Biospin columns and sodium dodecyl sulfate were purchased from Bio-Rad Laboratories (Hercules, CA) and cesium chloride from ICN (Costa Mesa, CA). Dichlorodihydrofluorescein diacetate was purchased from Molecular Probes (Eugene, OR) and anhydrous dimethyl sulfoxide from Aldrich, (Milwaukee, WI). Glass and Permanox plastic chamber slides were purchased from Nalge NUNC (Naperville, IL). Cytotox 96 Non-radioactive cytotoxicity assay kit was purchased from Promega (Madison, WI). Urea was purchased from Mallinckrodt Baker, Inc. (Paris, KY). Retinoic acid, 1,3-dimethyl-2-thiourea, desferrioxamine mesylate, guanidine thiocyanate, methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (AAPV- CMK), and all other chemicals were from Sigma Chemical Co. (St. Louis, MO).

Cell Culture

A549 Cells. A549, a lung carcinoma cell line, was cultured in Ham's F-12K medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml). Cells were grown at 37°C in a humidified 5% CO₂ atmosphere.

Differentiated primary normal human bronchial epithelial cells. Normal human bronchial epithelial cells (NHBE) were seeded, as previously described, on rat tail collagen type I-coated microporous membranes (Transwell filters) in a serum-free 1:1 mixture of bronchial epithelial cell basic medium, and Dulbecco's Modified Essential Medium with SingleQuot supplements, bovine pituitary extract (13 mg/ml), epidermal growth factor (0.5 ng/ml), bovine serum albumin (1.5 µg/ml), and all-trans retinoic acid (5 × 10⁸ M) in place of SingleQuot retinoic acid. When cells were 70% confluent, culture conditions were changed to an air-liquid interface (12, 16); medium was removed from the apical surface and media in the basolateral chamber were changed daily. Cells were cultured for an additional 2 wk and then used for experiments.

Treatment Protocol

All studies were done in serum-free media for both A549 and NHBE cells. NHBE cells in air-liquid interface cultures were exposed on both apical and basolateral surfaces. For studies including antioxidants, A549 or NHBE cells were preincubated with the antioxidant for 30-60 min and then coincubated with NE. A549 cells were stimulated with 100 nM (2.6 U/ml) NE, and NHBE cells were treated with 500 nM (13 U/ml) NE, for the indicated time periods. Concentrations of the antioxidants were based on previously reported concentrations used for respiratory epithelial cells (17, 18) and were tested for cytotoxicity under our treatment conditions by trypan blue dye exclusion and lactate dehydrogenase (LDH) release. Dimethylthiourea (DMTU), urea, and desferrioxamine (DF) were solubilized in water. Controls included cells treated with NE dilution buffer (50 µM sodium acetate, pH 5.2, and 100 µM sodium chloride) or the antioxidants alone.

Northern Analysis

RNA was isolated from A549 cells cultures as previously described (12) by the guanidinium thiocyanate/cesium chloride method. For NHBE cells, RNA was isolated with TRIZOL according to the manufacturer's instructions. Total RNA (10 µg) was separated by 1.2% agarose-formaldehyde gel electrophoresis and transferred by capillary blot to a nylon filter (Nytran plus) in 1 M ammonium acetate. After UV crosslinking, the filters were hybridized at 62°C with ³²P-labeled probes (specific activity > 10⁸ cpm/µg) for MUC5AC (12) and 28 s rRNA (19). Filters were washed twice with 2× saline sodium citrate (SSC) and 0.1% sodium dodecyl sulfate (SDS) at room temperature for 30 min, and then with 0.1× SSC and 0.1% SDS at 62°C for 15 min. Filters cells were exposed for autoradiography at 80°C. Band density on autoradiographs was determined by digitalization with Fotolook and Photoshop softwares and quantitation using NIH image software.

Dichlorodihydrofluorescein Fluorescent Microscopy Assay for Intracellular Oxidation

Dichlorodihydrofluorescein (DCF) is an intracellular oxidation indicator. In the presence of reactive oxygen species (ROS), such as hydrogen peroxide, dichlorodihydrofluorescein is oxidized to the fluorescent compound dichlorofluorescein (20, 21). A549 cells were treated with NE or control vehicle, rinsed twice, and then loaded with DCF (2.5 µM) for 25 min. NHBE were loaded with DCF (10 µM) before NE exposure for 45-60 min, rinsed twice, and then treated with NE or control vehicle. At the conclusion of the experiments, cells were rinsed three times and evaluated for oxidation of DCF by fluorescent microscopy. Cells were also examined for changes in autofluorescence in response to NE or control vehicle in the absence of DCF.

Cytotoxicity Assessment

Cytotoxicity was assessed by two methods: trypan blue dye exclusion and LDH release. Cell counts were determined for adherent and nonadherent cells, and viability was assessed by trypan blue dye exclusion for all treatment conditions. Percent of LDH release was assessed using a commercially available colorimetric assay for LDH and performed according to manufacturer's instructions. A549 cells were exposed to NE and inhibitors separately as well as NE plus inhibitors. Both supernatants and cell lysates were collected and assessed for LDH content. Percent of LDH release was calculated by taking the ratio of LDH released into the supernatant to the total LDH in the supernatant and the cell lysate.

Elastase Activity Assay

To evaluate the effect of the antioxidants on NE activity in vitro, in the absence of cells, we performed an elastase enzyme activity assay provided by the manufacturer of the NE (22). In a TRIS-based buffer (0.1 M TRIS, pH 7.5, and 0.5 M NaCl), NE was incubated with the substrate N-Suc-Al-Al-Al-pNitroanalide and the change in absorbance at 410 nm was monitored over time. NE activity correlated to the change in absorbance per minute. The following conditions were tested: heat-inactivated NE, NE plus each antioxidant, and NE plus water (antioxidant solubilization vehicle). NE was preincubated with each buffer or inhibitor for 10 min before incubating with the substrate. Specific activity for NE plus water was compared with NE plus inhibitor to determine if NE enzyme activity was altered.

Statistical Analysis

Analysis of data was performed using the Kruskal-Wallis one-way nonparametric analysis of variance (ANOVA) and post-hoc comparisons by the Wilcoxon rank sum/Mann-Whitney rank sum test. Differences were considered significant at P < 0.05 (23).

	Results

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Antioxidants Inhibit NE-induced MUC5AC Expression

We hypothesized that ROS are second messengers required for NE-regulated MUC5AC expression. We approached this problem by using two different antioxidants, dimethylthiourea (DMTU) and desferrioxamine (DF), to evaluate their effect on NE-regulated MUC5AC mRNA expression. In both A549 and NHBE cells, DMTU (15 mM) significantly inhibited NE-induced MUC5AC expression (Figures 1 and 2). To control for the osmotic load associated with DMTU, urea was used at equimolar concentrations to DMTU in the experiments. Urea (15 mM) had no significant effect on control (unstimulated) or NE-induced MUC5AC expression (Figure 1). DF significantly inhibited NE-induced MUC5AC mRNA expression in A549 cells (Figure 3). Neither the antioxidants nor solvents themselves used in these studies significantly affected control levels of MUC5AC mRNA expression. There was no significant effect by NE, either antioxidant or the combination of NE and antioxidant on the expression of the control gene, 28 s rRNA (Figures 1B, 2B, and 3B). In addition, NE and each of the inhibitors, alone or in combination, at the concentrations used, were determined by both trypan blue dye exclusion and LDH release assays to be noncytotoxic (data not shown). The effect of DMTU and DF on NE regulation of MUC5AC expression was not due to inhibition of NE activity (data not shown).

View larger version (42K): [in this window] [in a new window]   View larger version (34K): [in this window] [in a new window]   Figure 1.   Dimethylthiourea (DMTU) inhibits the NE-induced increase in MUC5AC expression in A549 cells. A549 cells were treated with DMTU (0 or 15 mM) or urea (15 mM) 35-45 min before and during control vehicle or NE treatment (100 nM [2.6 U/ml], 6 h). RNA was isolated and evaluated by Northern analyses and autoradiography for MUC5AC expression (A) and 28 s rRNA (B). The graph summarizes the densitometry data from two separate experiments performed in triplicate (C). The line at 100% represents control level. Results are expressed as a percent of control of the ratio of MUC5AC:28 s rRNA (mean ± SEM; n = 5-6). *, significantly different from control, (P < 0.05); , significantly different from NE treatment alone (P < 0.05). CTL, control; D, 15 mM DMTU; U, 15 mM urea; ND, 100 nM NE plus 15 mM DMTU; NE, 100 nM neutrophil elastase; and NU, 100 nM NE plus 15 mM urea.

View larger version (60K): [in this window] [in a new window]   View larger version (28K): [in this window] [in a new window]   Figure 2.   Dimethylthiourea (DMTU) inhibits the NE-induced increase in MUC5AC expression in NHBE cells. NHBE cells were treated with DMTU (0 or 15 mM) 50 min before and during control vehicle or NE treatment (500 nM [13 U/ml], 1 h). RNA was isolated and evaluated by Northern analyses and autoradiography for MUC5AC expression (A) and 28 s rRNA (B). The graph summarizes the densitometry data from three separate experiments (C). The line at 100% represents control level. Results are expressed as a percent of control of the ratio of MUC5AC:28 s rRNA (mean ± SEM; n = 6-8). *, significantly different from control (P < 0.05); , significantly different from NE treatment alone (P < 0.05). CTL, control; D, 15 mM DMTU; ND, 500 nM NE plus 15 mM DMTU; and NE, 500 nM neutrophil elastase.

View larger version (51K): [in this window] [in a new window]   View larger version (27K): [in this window] [in a new window]   Figure 3.   Desferrioxamine (DF) blocks the NE-induced increase of MUC5AC in A549 cells. A549 cells were treated with DF (250-500 µM) 50 min before and during control vehicle or NE treatment (100 nM [2.6 U/ml], 6 h). RNA was isolated and evaluated by Northern analyses and autoradiography for MUC5AC expression (A), and 28 s rRNA (B). The graph summarizes the densitometry data from five separate experiments performed in duplicate (C). The line at 100% represents control level. Results are expressed as a percent of control of the ratio of MUC5AC:28 s rRNA (mean ± SEM, n = 8 - 10). *, significantly different from control (P < 0.05); , significantly different from NE treatment alone (P < 0.05).

NE Treatment Triggers the Generation of ROS in Respiratory Epithelial Cells

The effect of antioxidants on NE-induced MUC5AC expression suggested that NE may trigger an increase in ROS and ROS could mediate the regulation of MUC5AC mRNA expression. Therefore, to determine whether NE treatment generated increased ROS in respiratory epithelial cells, we used a fluorescent indicator of intracellular oxidation, dichlorodihydrofluorescein (DCF). Fluorescent photomicroscopy revealed that NE increased cellular fluorescence in A549 and NHBE cells (Figures 4 and 5). Importantly, the iron chelator, DF, blocked NE-triggered DCF fluorescence in A549 cells (Figure 4). In the absence of DCF, there was no autofluorescence of cells at rest (Figures 4 and 5), or after treatment with control vehicle or NE (data not shown). There was no fluorescence in cells treated with proteolytically inactivated NE, either due to boiling or due to binding with the synthetic NE inhibitor, methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone [AAPV-CMK] (data not shown). Therefore, oxidation of DCF and NE-induced MUC5AC gene expression (12) required proteolytically active NE.

View larger version (14K): [in this window] [in a new window]   Figure 4.   Desferrioxamine inhibits NE-induced oxidative stress in A549 cells. A549 cells were treated with DF (500 µM) 30-60 min before and during exposure to NE or control vehicle (100 nM [2.6 U/ml], 30 min). Cells were then labeled with dichlorodihydrofluorescein (DCF; 2.5 µM, 25 min) and evaluated by fluorescent microscopy. Photomicrographs for the following cell treatment conditions are shown: autofluorescence (A), control-vehicle treatment (B), NE treatment (C), DF 500 µM (D), and NE 100 nM plus DF 500 µM (E). These photographs are representative of results from three separate experiments performed in duplicate. NE-exposed cells showed increased fluorescence, reflecting enhanced generation of intracellular ROS.

View larger version (29K): [in this window] [in a new window]   Figure 5.   NE treatment triggers oxidative stress in NHBE cells. NHBE cells were loaded with DCF (10 µM, 50 min) then rinsed and treated with NE (500 nM [13 U/ml], 1 or 4 h) or control vehicle. Cells were then evaluated for oxidative stress by fluorescent microscopy. Photomicrographs for the following conditions are shown: autofluorescence (A), control cells (B), and NE treatment (C). These photographs are representative of results from three separate experiments. NE increases fluorescence, reflecting enhanced generation of intracellular ROS in NHBE cells.

	Discussion

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We have previously reported that NE upregulates MUC5AC gene expression in respiratory epithelial cells by enhancing mRNA stability (12). In this report, we demonstrate that NE appeared to induce MUC5AC gene expression by an oxidant-dependent mechanism. This conclusion was based on two lines of evidence: antioxidants attenuated NE-regulated MUC5AC gene expression, and NE increased generation of intracellular oxidants in respiratory epithelial cells. Two different antioxidants, DMTU and DF, were evaluated for their effect on NE-induced MUC5AC gene expression. DMTU is a broad-spectrum antioxidant that scavenges hydroxyl radical and associated hydroxylated products and peroxynitrite. The "antioxidant" capacity of DF is attributed to its iron-chelating properties. DF inhibits the formation of hydroxyl radical through iron-catalyzed ROS generation (e.g., Fenton reaction). Both antioxidants significantly inhibited NE-induced increase in MUC5AC gene expression. In conjunction with our previously reported results, DMTU inhibited NE-induced MUC5AC expression in a concentration-dependent manner (24). The results presented in this report suggest that ROS generated by NE treatment are required for NE-induced MUC5AC expression. To confirm that NE treatment triggers generation of ROS or alters the cellular oxidant balance, we demonstrated that NE treatment triggered DCF fluorescence in A549 and NHBE cells. Furthermore, DF inhibited NE-induced DCF fluorescence. Thus, both gene expression and DCF experiments implicate ROS as requisite mediators for NE-induced MUC5AC expression.

ROS-mediated regulation of gene expression can occur at either the transcriptional or post-transcriptional level. For a variety of genes, ROS, including superoxide and hydrogen peroxide, have been reported to mediate changes in mRNA stability. In human retinal pigment epithelial cells, superoxide, produced by the reaction of xanthine with xanthine oxidase, increases expression of vascular endothelial growth factor by increasing its mRNA stability (25). In HeLa cells, diethylmaleate-induced oxidative stress, which is attributed to glutathione depletion, increases p21^waf1/cip1 mRNA expression and mRNA stability (26). Hydrogen peroxide treatment of endothelial cells upregulates eNOS expression and the mRNA half-life (27). In addition, in the alveolar macrophage cell line NR8383, hydrogen peroxide exposure upregulates macrophage inflammatory protein-2 (MIP-2) gene expression, in part, as a result of enhanced mRNA stability (28). Vanadium treatment of the mouse macrophage cell line RAW 264.7 increases expression of MIP-2 mRNA by a mechanism involving ROS and enhanced mRNA stability (29). In lungs of rats exposed to hyperoxia, several antioxidant enzyme genes are post-transcriptionally regulated. In neonatal rat lungs, expression of Mn-superoxide dismutase, Cu-Zn-superoxide dismutase, glutathione peroxidase, and catalase mRNA are increased in response to ROS via post-transcriptional mechanisms (30, 31). The changes in catalase mRNA stability are dependent on the oxidation-reduction state of the cellular proteins (32). Iron, a transition metal important in normal cell processes as well as inflammatory and oxidant-mediated reactions and diseases, plays a pivotal role in transferrin receptor mRNA stability. This regulatory process is post-transcriptionally controlled by interaction between specific secondary structure(s) in the 3' untranslated region of the mRNA, iron-responsive elements (IRE), and a unique mRNA binding factor, iron-responsive element-binding protein (IRE-BP) (33). As iron levels decrease, there is increased binding of IRE-BP to the IRE. This interaction sterically hinders RNase, interferes with RNase activity, and thus prolongs the half-life of the transferrin receptor mRNA. In conjunction with our previous report that NE enhances MUC5AC mRNA stability (12), our results suggest that ROS may be important in the regulation of mucin mRNA stability.

There are several potential sources of NE-triggered ROS production. The mitochondrial electron transport system serves as one source of NE-induced ROS (15). NE may also reduce antioxidant function. For example, activated neutrophils or trypsin, a serine protease, inactivate extracellular superoxide dismutase (34). Alternatively, NE may activate a pro-oxidant enzyme. Caspases, a family of cysteine proteases, cleave and subsequently activate cytosolic phospholipase A₂ (35). This may result in arachidonic acid production and subsequent generation of cycloxygenase or lipoxygenase products. NE has been reported to increase xanthine oxidase activity in endothelial cells by catalyzing the conversion of xanthine dehydrogenase to xanthine oxidase (36).

As no specific NE receptor has been identified for respiratory epithelial cells, the targets for NE activity are currently unknown. Our laboratory and others have reported that proteolytic activity is required for NE-induced gene expression (12, 37). In studies with airway epithelial cells, NE has been immunolocalized to the extracellular space (38). Therefore, we predict that the primary event in NE-regulated gene expression is NE interaction with the epithelial cell membrane. We speculate that iron bound to phospholipids or associated with membrane proteins may be released in response to NE treatment and then taken up by the cell in the form of nontransferrin-bound iron (39). In the cell, depending on the iron storage proteins available (e.g., ferritin H-type versus L-type), iron may be available for ROS-generating reactions (40).

In summary, we report that NE regulates MUC5AC expression by a novel signaling cascade via the generation of ROS. This report suggests a new arena of potential therapeutic targets to attenuate the effect of NE on airway epithelial cells.