Regulation of MUC5AC mucin secretion and airway surface liquid metabolism by IL-1{beta} in human bronchial epithelia

Thomas Gray,1,* Ray Coakley,1,* Andrew Hirsh,2 David Thornton,3 S. Kirkham,3 Ja-Seok Koo,4 Lauranell Burch,2 Richard Boucher,2 and Paul Nettesheim1

1Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park 27709; 2Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599-7248; 4Department of Thoracic/Head & Neck Medical Oncology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030; and 3Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester M13 9PT, United Kingdom

Submitted 19 December 2002 ; accepted in final form 26 September 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mucociliary transport in the airways significantly depends on the liquid and mucin components of the airway surface liquid (ASL). The regulation of ASL water and mucin content during pathological conditions is not well understood. We hypothesized that airway epithelial mucin production and liquid transport are regulated in response to inflammatory stimuli and tested this hypothesis by investigating the effects of the pleiotropic, early-response cytokine, IL-1{beta}, on cultured primary human bronchial epithelial and second-passage, normal human tracheo-bronchial epithelial (NHTBE) cell cultures. Fully differentiated NHTBE cultures secreted two major airway mucins, MUC5AC and MUC5B. IL-1{beta}, in a dose- and time-dependent manner, increased the secretion of MUC5AC, but not MUC5B. MUC5AC mRNA levels were only transiently increased at 1 and 4 h after the start of IL-1{beta} treatment and returned to control levels thereafter, even though MUC5AC mucin production remained elevated for at least 72 h. Synchronous with elevated MUC5AC secretion, ASL volume increased, its percentage of solid was reduced, and the pH/[] of the ASL was elevated. ASL volume changes reflected altered ion transport, including an upregulation of Cl- secretory currents (via CFTR and Ca2+-activated Cl- conductance) and an inhibition of epithelial sodium channel (ENaC)-mediated absorptive Na+ currents. IL-1{beta} increased CFTR mRNA levels without affecting those for ENaC subunits. The synchronous regulation of ASL mucin and liquid metabolism triggered by IL-1{beta} may be an important defense mechanism of the airway epithelium to enhance mucociliary clearance during airway inflammation.

normal human tracheobronchial epithelial; air-liquid interface; cystic fibrosis transmembrane conductance regulator


AIRWAY SURFACE LIQUID (ASL) is composed of a thin, periciliary liquid layer and an overlying mucus layer. It traps inhaled particles, microorganisms, and environmental toxicants and transports them out of the airways. Despite the importance of this mucociliary clearance mechanism, its regulation under normal conditions and particularly during airway disease is poorly understood. Because inflammatory stimuli may affect ion transport at many levels, it is possible that transepithelial water flux is actively modulated during inflammation. The ability to hydrate ASL is important, because an increase in mucin production alone in response to an inflammatory challenge might impair mucociliary clearance, allowing mucins to accumulate in a dehydrated ASL. We speculated that airway epithelia might exhibit adaptive responses that lead to modulation of mucin content and ASL volume.

Mucins are highly glycosylated, large-molecular-weight glycoproteins that are the major macromolecular components of mucus. Mucins are responsible for the viscoelastic properties and hydrophilicity of the complex mucus layer that provide lubrication and protection for epithelia (39). Thirteen mucin genes have been reported (26, 39, 53), and MUC2, MUC5AC, and MUC5B encode for polymeric mucins that are expressed by airway epithelium and are found in airway secretions. MUC5AC and MUC5B were shown to be the major components of airway mucus; however, all three have been shown to be increased during episodes of airway inflammation (11, 17, 49, 52). The biochemical and molecular mechanisms involved in the regulation of mucin synthesis under normal and pathological conditions are the subjects of intensive investigation (3, 11, 12, 28-30, 51).

The surface clearance properties of ASL are determined not only by its mucin content, but also by its hydration. Vectorial ion transport by ciliated epithelial cells regulates ASL volume (4). Basal transepithelial water absorption is mediated via electrogenic Na+ absorption through apical epithelial Na+ channels (ENaC, 4). However, airway epithelia are also capable of Ca2+- and cyclic AMP-activated Cl-/liquid secretion mediated by Ca2+-activated Cl- channels and CFTR, respectively (22).

ASL pH may also affect airway defense (1, 2, 44, 50). Inflammation may acidify ASL, and, consequently, alkalinization of ASL by the epithelium may be an important component of airway defense. Thus synchronous regulation of mucin production, ASL volume, and ASL pH are likely essential components of the integrated airway's defensive response to inflammation.

Inflammatory mediators that regulate mucin production and ASL volume and pH have not been reported. IL-1{beta} is a pleiotropic and an early-response cytokine in the pulmonary inflammatory cascade that is produced by many cell types; however, resident and migratory macrophages are considered to be the principal source during inflammatory episodes (10). Because increased levels of IL-1{beta} have been reported in inflammatory airway diseases associated with hypersecretion (34, 37, 42), we speculated that it might be responsible for concerted regulation of both liquid and mucin metabolism. Accordingly, we designed studies to measure the effects of IL-1{beta} on mucin production and secretion, ASL hydration, ion transport, and pH regulation by human airway epithelium. For these studies, we used primary and passage 2 human airway epithelial cell cultures that mimic many of the properties of bronchial epithelium in vivo (14). Using this system we were able to assess the direct effects of IL-1{beta} on the regulation of mucin secretion and fluid metabolism in pure populations of fully differentiated airway epithelial cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture, Cell Counts, and Immunocytochemical Detection of MUC5AC Mucin

In experiments in which mucin production and mucin gene expression were investigated, passage 2 normal human tracheobronchial epithelial (NHTBE) cells (two strains from different donors purchased from Clonetics, La Jolla, CA) were seeded onto 24-mm uncoated, semipermeable, Transwell clear membranes (Corning Costar, Cambridge, MA) at 2 x 104 cells per cm2 (14). The complete development of the mucociliary phenotype in these air liquid interface (ALI) cultures was observed in random day 35 cultures after fixation with neutral buffered formalin, paraffin embedding, sectioning, and staining with Alcian blue-periodic acid Schiff (see Fig. 1).



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Fig. 1. Histomorphology of well-differentiated, human bronchial epithelial cultures. Normal human tracheobronchial epithelial (NHTBE) cells were grown for 35 days in air-liquid interface cultures. The cultures were formalin fixed, paraffin embedded, sectioned, and stained with Alcian blue-periodic acid Schiff (x600 magnification). Goblet cells (large arrowheads) and ciliated cells (small arrowheads).

 

Completely differentiated cultures were exposed on the basolateral surface to different concentrations of IL-1{beta} (R&D Systems, Minneapolis, MN) dissolved in sterile PBS. After treatment, the number of cells per culture was determined by visual cell counts of cell suspensions obtained by enzymatic dissociation of the cultures (triplicate cultures per group). Cytospin slides, which were prepared from the cell suspensions, were fixed in methanol-acetone (1:1 volume), and cells containing MUC5AC protein were detected with the anti-MUC5AC antibody, 45M1 (1:250 dilution; NeoMarkers, Freemont, CA; for more details on antibody specificity see immunodetection methods in the next paragraph), using an immunostaining kit (Mouse Immuno Cruz Staining System, Santa Cruz, CA). Control slides were stained with either an irrelevant antibody, with the primary antibody omitted or with purified mouse IgG. The data are expressed as the means ± SD from triplicate cultures. We determined the mean number of positive cells by scoring 500 cells per slide, and statistical comparisons were made by Student's t-test with a P value of <0.05 indicating statistical significance.

Immunodetection, Quantitation, and Characterization of Secreted and Intracellular Mucins

Quantitation of MUC5AC and MUC5B produced by NHTBE cultures and the immunoblotting methods used to detect them have been previously reported (14, 48). The apical secretions (triplicate cultures per group) were collected to measure secreted MUC5AC and MUC5B mucins. To measure cell-associated mucins, following removal of the apical secretions, we lysed and collected the cultured cells with 2x Laemmli buffer. To measure total mucin, i.e., extracellular and intracellular mucin per culture, we collected the entire contents of the culture in Laemmli buffer. Diluted apical secretions, cell lysates, or whole culture lysates were applied to nitrocellulose membranes, which were incubated with either anti-human MUC5AC (45M1, Neomarkers) or anti-MUC5B antibodies (49), both diluted 1:250. After primary antibody exposure, the blots were treated with horseradish peroxidase conjugated goat anti-mouse or anti-rabbit IgG, and the signal was detected by chemiluminescence (ECL kit; Amersham, Buckinghamshire, UK). The levels of MUC5AC and MUC5B reactivity were measured densitometrically. The data are presented either as mucin levels per culture (i.e., densitometric units ± SD, calculated by optical density x dilution factor of the sample) or as fold increase (i.e., mean ± SD mucin levels in IL-1{beta}-treated cultures divided by values from vehicle controls).

MUC5AC mucin produced by the cultures was detected by use of the mouse monoclonal 45M1 antibody (Neomarkers), which has been shown to react with gastric MUC5AC (35) and MUC5AC produced by NCI-H292 cells (24). We confirmed the specificity of the Neomarkers' antibody by comparing its reactivity with that of the MAN-5AC-1, produced and characterized by Thornton et al. (47) against human airway MUC5AC mucin (40). We found that the Neomarker and MAN-5AC-1 antibodies have quantitatively very similar reactivity with purified MUC5AC mucin as well as with secretions from control and IL-1{beta}-stimulated NHTBE cultures.

Methods to assess the size distribution and charge density of mucins produced by NHTBE cultures have been previously reported in detail (48). Briefly, to characterize the secreted MUC5AC mucin from 24-h IL-1{beta}-treated and vehicle control cultures, we collected the apical secretions in PBS (0.5 ml/well) and diluted them 1:1 with 8 M guanidine hydrochloride (GuHCl) and the mucin purified by two stages of CsCl density gradient centrifugation (48). The size distribution of the mucin was determined by rate zonal centrifugation on 6-8 M GuHCl gradients and detection with the anti-MUC5AC antibody MAN-5AC-1 (41). To determine the effect of IL-1{beta} treatment on the charge density of secreted MUC5AC, we subjected apical secretions to anion exchange chromatography on a Mono Q HR 5/5 column (Pharmacia, Buckinghamshire, UK) and eluted them with a linear (0-0.4 M) lithium perchlorate gradient.

Quantitative Real-Time RT-PCR

Total RNA was isolated from triplicate, individual cultures per condition. Real-time RT-PCR was performed with a Perkin-Elmer ABI 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) and the SYBR Green DNA PCR Core Reagent Kit (Perkin-Elmer) in accordance with the manufacturer's instructions. The sequences for primers for MUC5AC were based on those described by Ordonez et al. (36) and are forward, 5'-TGTGGCGGGAAAGACAGC-3'; reverse, 5'-CCTTCCTATGGCTTAGCTTCAGC-3'; and those for the control gene, transferrin receptor (Trfr, 36), are forward, 5'-CAGGAACCGAGTCTCCAGTGA-3'; reverse, 5'-GGTGAAGTCTGTGCTGTCCAGT-3'. All reactions were performed in triplicate. RT-PCRs were run twice. Reverse transcription reactions and PCRs were performed in the same tube according to methods described by Hamadeh et al. (16). MgCl2 (4 mM), dNTPs with dUTP (0.8 mM), SYBR Green Buffer (1x), total RNA (50 ng in 2.5 µl of water), primers (20 µM), RNasin (0.4 U/µl), AmpliTaq Gold DNA polymerase (0.025 U/l) and RT (0.25/µl, Superscript; Invitrogen) were added to the tube, and the volume was brought to 50 µl with water. The RT reactions were carried out for 30 min at 48°C followed by 10 min at 95°C to activate AmpliTaq Gold, and the PCRs at 40 cycles of denaturation for 15 s at 95°C, and annealing/extension for 60 s at 60°C. PCR products were resolved by gel electrophoresis and yielded the predicted sizes of 116 bp for MUC5AC and 96 bp for Trfr (36). The specificity of the amplified products was monitored by its melting curve as well as by DNA sequencing. Fluorescence emission was detected for each PCR cycle, and the threshold cycle (Ct) values and the average Ct of the triplicate reactions were determined for MUC5AC and Trfr. The Ct value was defined as the actual PCR cycle when the fluorescence signal increased above the background threshold, and the {Delta}Ct was determined for each sample by subtracting the Ct for Trfr from Ct for MUC5AC, and the mean Ct of the triplicate samples was determined. The mean level of induction of MUC5AC (±SD) was calculated as follows: fold increase = e-[{Delta}Ct (IL-1{beta} treated)-{Delta}Ct (control)], and the results were compared by Student's t-test. P < 0.05 represented statistical significance.

Cell Culture Model for ASL Volume Flow/Regulation Experiments

For experiments addressing transepithelial volume and ionic flux as well as CFTR and ENaC expression, human bronchial epithelial (HBE) cells were obtained at the time of lung transplantation from main stem or lobar bronchi under the auspices of protocols approved by the Institutional Committee on the Protection of the Rights of Human Subjects (University of North Carolina). Disaggregated airway epithelial cells from 16 normal subjects were seeded onto collagen-coated Snapwell porous filters (1.13 cm2, Corning Costar) and cultured as previously described for at least 35 days at 37°C, 5% CO2 in a humidified atmosphere (33). Once confluent, cell preparations were grown at ALI. Function was assessed by transepithelial resistance (Rt) and potential difference (PD), which were measured with an STX-2 electrode connected to an epithelial voltohmeter (World Precision Instruments, Sarasota, FL). We examined how IL-1{beta} affected ion transport and liquid metabolism by NHTBE cultures and identified similar changes in amiloride-sensitive current (decreased), forskolin response (increased), and response to ATP (increased), as well as finding increased apical liquid on treated preparations (unpublished observations). However, since we have defined the characteristics of HBE cells in numerous prior publications and demonstrated how this model's bioelectric properties recapitulate those of airway epithelium in vivo, we preferred to report results from this culture preparation.

Measurement of the Percentage of Solids in the Apical Liquid and Transepithelial Volume Flux

Percentage of solids in ASL. After cultures were IL-1{beta} or control vehicle-treated for 48 h, fixed volumes of apical liquid were aspirated at a time designated time zero (t0). Samples were immediately transferred to preweighed aluminum weighing pans on an electronic balance (model no. 27; Cahn Electronic Balance, Cerritos, CA). The first weight measurement was made 20 s following t0, and serial measurements were made at fixed intervals thereafter. Recorded weights decreased over time due to evaporative water loss. A regression line was fitted from the recorded values and extrapolated back to t0 to estimate the actual weight of the sample at t0. The sample, on its aluminum pan, was then placed in a plastic vial, thoroughly desiccated to dryness in an oven overnight, and then reweighed. The percentage change in weight was a measure of percentage water content at t0, and this value was used to calculate the percentage solids (23).

Transepithelial volume flux. Two experimental protocols were used to measure the effect of IL-1{beta} on ASL volume regulation. In both protocols, epithelia were cultured as previously described (32) in a specially humidified incubator to prevent evaporative water loss from the culture surface.

The first protocol addressed the effect of IL-1{beta} on the capacity of epithelia to modulate absorption of a luminal liquid challenge. Cultures were pretreated with IL-1{beta} for 24 h, following which the ASL was aspirated, and 100 µl of Krebs bicarbonate Ringer (KBR) solution containing 2% blue dextran (BD), a cell-impermeant ASL volume marker dye, were added. After an additional 24 h, microaliquots (2-5 µl) of apical liquid were sampled.

The second protocol tested for ASL secretion. At the initiation of the experiment, all liquid possible was aspirated from the apical surface of the cultures, and the volume remaining was measured and compared with the apical volume after 24 and 48 h of IL-1{beta} or vehicle control treatment. The baseline apical volume at 0 h (after aspiration of surface liquid) and apical volume after 24 and 48 h were measured by adding 3 µl of KBR solution containing 3% BD to the apical surface, gently mixing for 5 s, reaspirating the same volume (3 µl) of ASL, and measuring the dilution of BD in these samples. The BD was diluted in the resident liquid on the apical surface, and the magnitude of dilution was used to calculate ASL volume at t0, t24h, and t48h.

In both protocols, sampling was performed by insertion of a constant-bore, nitric acid-washed, microcapillary pipette into the apical liquid via a small-bore hole drilled in the culture dish lid. Water-saturated mineral oil was introduced into the microcapillary pipette on either end of the sample (though not in contact with it), and each end was plugged with clay. Samples were stored at -20°C until analyzed. BD concentration was measured optically (32).

Measurement of Bioelectric Properties of Airway Epithelia

Primary, bronchial epithelial cultures, grown at ALI on Snapwell (1.13 cm2 surface area) permeable supports (Costar) coated with human placental collagen (50 µg, type VI; Sigma), were mounted in modified Ussing chambers (Physiological Instruments, San Diego, CA). The epithelium was bathed on both sides with 5 ml of warmed (37°C) KBR solution circulated by gas lifts with 95% O2-5% CO2. Solution pH was maintained at 7.4. The epithelial culture was voltage clamped, i.e., short-circuit current (Isc), and Rt was measured with an automatic voltage clamp (Physiological Instruments). Data were acquired and analyzed with Acquire and Analysis (version 1.2) software. After 15-min equilibration, amiloride (10 µM) was added to the luminal bath, followed sequentially by bilateral isoproterenol (10 µM), luminal UTP (100 µM), and finally serosal bumetanide (50 µM).

Ribonuclease Protection Assay for CFTR and ENaC

Methods for the ribonuclease protection assay (RPA) with sodium channel subunit probes were as previously described (5). A 465-bp probe corresponding to part of the NH2 terminus of the human CFTR sequence (accession no. M28668 [GenBank] ) was prepared by PCR from a plasmid template containing a full-length CFTR cDNA using the following primers: CFTR 75F 5'-TGGCATTAGGAGCTTGAGCCCAGACGGCCC-3', and CFTR 539R 5'-AGTGTCCTCACAATAAAGAGAAGGCATAAG-3'. The PCR product was gel purified and ligated into the pCRII vector (Invitrogen, Carlsbad, CA). To determine orientation and verify wild-type sequence, we submitted individual clones for automatic sequencing. A template for in vitro transcription was prepared by PCR from the CFTR pCRII plasmid with PCRII vector primers: PCRII SP6 5'-ATGATTACGCCAAGCTATTTAGGTGACACT-3', and PCRII T7 5'-GACGGCCAGTGAATTGTAATACGACTCACT-3'. The gel-purified PCR product was transcribed incorporating [32P]UTP with the Maxiscript Kit (Ambion, Austin, TX), and the RPA was performed with Ambion's RPA III kit. Protected fragments were quantified by phosphorimage analysis with ImageQuant software (Molecular Dynamics, San Leandro, CA).

Measurement of ASL Ionic Composition and pH

The culture surface was washed twice with PBS; a third wash was carried out with the apical study solution. This liquid was carefully aspirated, and a fixed volume of apical test liquid was added to the culture surface. Aliquots (1-5 µl) were obtained from the apical compartment after designated intervals and stored as described above. For measurements of [Na+], [K+], and [Cl-], sample volumes were measured from the length of the liquid column in the capillary tube, the contents were diluted into ultrapure 0.1 N nitric acid, and measurements of [Na+] and [K+] were made by flame emission spectroscopy and of [Cl-] by amperometric titration, as previously described (23). [] was measured by coupled enzymatic reactions and spectrophotometric analysis of NAD (Sigma-Aldridge, St. Louis, MO).

For measurement of ASL pH, microaliquots (0.5-1.0 µl) were aspirated directly from microcapillary tubes (see above) into the tip of a short (0.5 cm) section of CO2-permeable silicone tubing (0.025 mm inner diameter and 0.047 mm outer diameter, Helix Medical). We inserted the tip of the pH-sensitive microelectrode (Microelectrodes, Bedford, NH) into the sample by stretching the end of the silicone tubing containing the sample over the microelectrode. The tight fit of the electrode in the tubing trapped a thin layer of liquid between the wall of the tubing and the electrode, so that the reference and pH electrodes made contact with the sample. The microelectrode and silicone tubing were placed in a water bath that was continually gassed and equilibrated with 5% CO2. A column of air in the tubing, distal to the electrode tip, prevented water from reaching the sample. CO2 equilibration was usually complete within 2 min, as evidenced by a stable pH. A two-point calibration with standard buffers was repeated at regular intervals to test for drift in the electrode output. Measurements were highly accurate and reproducible within ±0.01 pH units.

Data Presentation and Analysis

Unless otherwise stated, data are presented as means ± SE. A Student's t-test was employed to compare two groups. Where more than one group was analyzed, a one-way analysis of variance was performed with a Student-Newman-Keuls posttest to isolate differences between groups. Where the response of a variable in two groups over time was compared, two-way analysis of variance was employed with a Bonferroni posttest. A P value of <0.05 was chosen to indicate statistical significance. Computational analysis was performed with the GraphPad Prism software on a personal computer.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of IL-1{beta} on MUC5AC and MUC5B Mucin Secretion

Increased levels of IL-1{beta} have been reported to be present in bronchoalveolar lavage from patients with ongoing airway inflammatory disease (34). To determine the effect of IL-1{beta} on mucin secretion, we exposed well-differentiated, NHTBE cultures (Fig. 1) to IL-1{beta} (2.5 ng/ml) for 24 h. As seen in Fig. 2A, IL-1{beta}-treated cultures secreted two- to threefold more MUC5AC mucin than vehicle-treated controls. MUC5B mucin secretion was not significantly altered (Fig. 2A), which suggests differential regulation of MUC5AC and MUC5B mucins in airway epithelial cells by IL-1{beta}. Because MUC5B mucin secretion was not affected by IL-1{beta} treatment, we focused on MUC5AC mucin in all subsequent experiments.



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Fig. 2. Characterization of mucins from IL-1{beta}-treated NHTBE cultures. Apical secretions were collected from 35-day-old cultures treated for 24 h with IL-1{beta} (2.5 ng/ml) or vehicle control (PBS), and the levels of mucin present in the apical secretions were determined by dot-blot analysis and densitometry. A: data are expressed as the mean ± SD increases of MUC5AC or MUC5B mucin in IL-1{beta}-treated cultures relative to vehicle control cultures. Results are based on 3 independent experiments, 3 cultures each per IL-1{beta} and vehicle control treatment group (P < 0.01). On the basis of densitometric scanning, the levels of MUC5AC mucin in vehicle control and IL-1{beta}-treated cultures were 95 ± 18 and 220 ± 23 units, whereas the levels of MUC5B mucin were 140 ± 16 and 129 ± 22 units, respectively. B: size distribution of the MUC5AC mucin from IL-1{beta}-treated ({triangledown}) and vehicle control ({bullet}) cultures were determined following rate zonal centrifugation and density gradient separation. C: charge density of MUC5AC mucin from IL-1{beta} ({triangledown})- and vehicle control ({bullet})-treated cultures was determined by anion exchange chromatography.

 

The physicochemical properties of airway mucin were reported altered in airway inflammatory disease, including status asthmaticus where viscous plugs of mucus obstruct the conducting airways (19). To determine whether IL-1{beta} could affect the physical-chemical properties of secreted MUC5AC mucin, we treated cultures for 24 h with IL-1{beta}. Apical secretions were collected and purified by CsCl gradient centrifugation. We found that the MUC5AC exhibited similar buoyant densities regardless of IL-1{beta} treatment (data not shown). The size distribution of the mucins was analyzed by rate zonal centrifugation on 6-8 M GuHCl. As seen in Fig. 2B, the MUC5AC mucin isolated from the secretions from IL-1{beta}- and control vehicle-treated cultures exhibited similar profiles, characterized by a broad range of sedimentation rates, consistent with polydisperse, high-molecular-mass, oligomeric mucins. To determine whether IL-1{beta} treatment caused differences in charge densities of secreted MUC5AC mucin, the mucin was reduced and carboxymethylated and subjected to anion-exchange chromatography on Mono Q columns (48). These studies showed that the charge density for MUC5AC mucin in IL-1{beta}-treated cultures was similar to that in vehicle control cultures (Fig. 2C).

Dose- and Time-Dependent Stimulation of MUC5AC Secretion by IL-1{beta}

Twenty-four hour treatment of NHTBE cultures with IL-1{beta} increased the levels of secreted MUC5AC in a dose-dependent manner (see Fig. 3A). Concentrations of IL-1{beta} >2.5 ng/ml had no increased effect. To determine how rapidly IL-1{beta} induced MUC5AC hypersecretion, we treated cultures with IL-1{beta} (2.5 ng/ml) for up to 72 h. As seen in Fig. 3B, secreted MUC5AC was significantly increased (approximately twofold) within 8 h, reaching a peak (approximately threefold) at 24 h. MUC5AC secretion remained elevated 48 and 72 h after the start of IL-1{beta} treatment.



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Fig. 3. Concentration- and time-dependent effects of IL-1{beta} on MUC5AC mucin secretion. A: cultures were treated for 24 h with various concentrations of IL-1{beta}, apical secretions were collected from triplicate cultures, and the levels of MUC5AC mucin were determined. The data are expressed as the increase in the mean ± SD levels of MUC5AC mucin in IL-1{beta}-treated cultures relative to vehicle controls. B: cultures were treated with IL-1{beta} (2.5 ng/ml, open bars) or vehicle control (solid bars) for various time periods. After treatment, apical secretions were collected from triplicate cultures per condition at 6, 8, 12, and 24 h. At the 48- and 72-h time points, secretions were allowed to accumulate for 24 h, i.e., from 24 to 48 h after the start of treatment and from 48 to 72 h after treatment, respectively. The levels of secreted MUC5AC mucin were determined at each time point, and the data are reported as arbitrary densitometric units (means ± SD) of secreted MUC5AC per culture. The results are representative of data obtained from 2 separate experiments. *P < 0.05; **P < 0.01 vs. control.

 

IL-1{beta} Stimulates MUC5AC Production and Secretion

Three mechanisms could explain the increased MUC5AC secretion in response to IL-1{beta}: 1) IL-1{beta} might increase the number of mucin-producing cells; 2) IL-1{beta} might act as a secretagogue, stimulating mucin exocytosis; or 3) IL-1{beta} might simultaneously stimulate production and secretion of MUC5AC.

To determine whether IL-1{beta} increased the number of MUC5AC mucin-producing cells, we treated cultures for 24 h and then enzymatically dissociated them. We found that the total number of cells and the number of MUC5AC positive cells were not significantly altered by IL-1{beta} treatment (4.8 ± 0.4 x 106 vs. 5.0 ± 0.8 x 106 cells per culture and 10.8 ± 1.2 vs. 9.9 ± 1.9% MUC5AC positive cells in vehicle control vs. IL-1{beta}-treated cultures).

To determine whether IL-1{beta} was acting as a secretagogue selectively via exocytosis, we treated cultures with IL-1{beta} (0 or 2.5 ng/ml) for 24 h, after which time apical secretions and cell lysates were collected separately from individual wells and the amount of MUC5AC mucin was determined. As seen in Fig. 4A, IL-1{beta} treatment increased secreted (extracellular) MUC5AC approximately twofold. In contrast, the intracellular levels of MUC5AC were the same in control and IL-1{beta}-treated cultures. This experiment was repeated three times, consistently showing that following IL-1{beta} treatment, the secreted, but not the cell-associated, MUC5AC was increased, indicating that IL-1{beta} caused a significant increase in MUC5AC secretion without depletion of intracellular mucin levels. Eight-hour treatment of the cultures with puromycin (25 µg/ml) prevented the IL-1{beta}-induced increase in mucin production (data not shown).



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Fig. 4. The effect of IL-1{beta} treatment on cell-associated, secreted, and total MUC5AC mucin. Cultures were IL-1{beta} (2.5 ng/ml, open bars) or vehicle control (solid bars) treated for 24 h (quadruplicate cultures per treatment). A: cell lysates and apical secretions were collected, and the mean ± SD levels of cell-associated and secreted MUC5AC mucin were determined for each and reported as arbitrary densitometric units. The results are representative of data obtained from 3 independent experiments; *P < 0.01. B: in 2 additional experiments the total amount of MUC5AC mucin per culture was determined following 24 h of IL-1{beta} treatment (0 or 2.5 ng/ml). The entire contents of the culture were collected in lysis buffer, and the levels of MUC5AC were determined and reported as arbitrary densitometric units. **P < 0.01.

 

In two additional experiments, the contents of the entire cultures (cells and extracellular material) were collected and assayed for MUC5AC mucin. As seen in Fig. 4B, following 24 h of IL-1{beta} treatment, the amount of MUC5AC mucin was increased approximately threefold over sham-treated controls.

Effect of IL-{beta} Treatment on MUC5AC mRNA Levels

To determine whether IL-1{beta} stimulation of MUC5AC mucin production was regulated at the level of mRNA, we collected total RNA from triplicate, individual cultures treated for 24 h with IL-1{beta} (0 and 2.5 ng/ml), and the levels of MUC5AC mRNA were determined by quantitative real-time RT-PCR. As seen in Fig. 5, MUC5AC mRNA levels were increased only at 1 and 4 h following IL-1{beta} treatment and returned to control levels by 8 h. However, the increases were not statistically significant.



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Fig. 5. The effect of IL-1{beta} on MUC5AC mRNA levels. Cultures were treated with IL-1{beta} (0 or 2.5 ng/ml) for 24 h, and at timed intervals total RNA from triplicate, individual cultures was isolated, and the levels of MUC5AC mRNA were determined by quantitative, real-time RT-PCR. The data are expressed as the increase in the mean ± SD levels of MUC5AC mRNA in IL-1{beta}-treated cultures relative to vehicle controls.

 

Effect of IL-1{beta} on the Percentage of Solids in Surface Liquid

To determine whether the increased MUC5AC secretion led to increased mucin concentration within ASL, we measured ASL percentage of solids in IL-1{beta}- or control vehicle-treated cultures. Figure 6A demonstrates that the percentage of solids in ASL fell significantly in response to IL-1{beta} treatment. This observation suggests that increased mucin production was accompanied by proportionately more liquid secretion into ASL.



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Fig. 6. The effect of IL-1{beta} on the percentage of solids in airway surface liquid (ASL) and on ASL volume. A: ASL was sampled 48 h after IL-1{beta} (2.5 ng/ml, solid bars) or vehicle control treatment (open bars). The percentage of solids in ASL from IL-1{beta}-vs. control vehicle-treated cultures was calculated by ratio of wet-dry weight. *P < 0.05 IL-1{beta} treated vs. control. B: effect of IL-1{beta} on volume absorptive capacity. After 24 h of IL-1{beta} (solid bars) or vehicle control (open bars) treatment, 100 µl of Krebs bicarbonate Ringer solution containing blue dextran (BD) was applied to the luminal surface. Twenty-four hours later the remaining apical volume was calculated by concentration of BD. *P < 0.05. C: effect of IL-1{beta} on volume secretory capacity. Cultures were vehicle control ({circ}) or IL-1{beta} (2.5 ng/ml, {bullet}) treated for 48 h (quadruplicate cultures per treatment, from 8 different subjects). Change in resident ASL volume over time in IL-1{beta}- and vehicle-treated cultures was estimated by dilution of BD (see MATERIALS AND METHODS) vs. time zero. *P < 0.05, **P < 0.05 IL-1{beta} vs. control vehicle-treated cultures.

 

Effect of IL-1{beta} on the Transepithelial Volume Flux

To test the notion that IL-1{beta} increased ASL volume, we assessed the effect of IL-1{beta} exposure on transepithelial volume flux in bronchial cultures.

To confirm that liquid absorption in response to an apical liquid challenge was Na+ dependent and unaffected by evaporative losses, we performed ion-substitution experiments, where Na+ in the apical solution was isotonically replaced with N-methyl-D-glucamine. Apical liquid (100 µl) was added to the cultures (triplicate cultures from three subjects) at the beginning of the experiment. After 24 h of incubation, we found that the absence of Na+ in the apical liquid almost completely abrogated liquid absorption (apical volume after 24 h: Na+-free 97.2 ± 3.56 vs. Na+-containing 27.8 ± 1.08; P < 0.0001).

To test for an IL-1{beta} effect on volume absorption, we exposed cultures to an apical liquid volume (100 µl) challenge. IL-1{beta}- and vehicle-treated cultures both absorbed liquid, but the rate of absorption in IL-1{beta}-treated cultures was significantly slowed (Fig. 6B). To test for IL-1{beta} induction of liquid secretion, we aspirated all possible liquid from culture surfaces, leaving a residual liquid volume of 3 µl. Forty-eight hours following aspiration of the apical surface, the volume of apical liquid increased following IL-1{beta} treatment but had decreased, i.e., was absorbed, in the control vehicle-treated preparations (Fig. 6C). These data are consistent with IL-1{beta}-induced secretion.

Effect of IL-1{beta} on Ion Transport

Because transepithelial water flux is determined by active ion transport, we evaluated the effects of IL-1{beta} on the bioelectric properties of bronchial cultures. IL-1{beta} did not affect Rt or PD acutely within 30 min (data not shown). Detailed studies of ion transport were undertaken in cultures treated with IL-1{beta} or vehicle for 48 h. A representative tracing is shown in Fig. 7A. Compared with controls, IL-1{beta}-treated cultures had an increased basal Isc. The raised basal Isc was absolutely and proportionately less amiloride sensitive, suggesting both a reduction in Na+ absorption and an increase in Cl- secretion. Analysis of mean data confirmed a raised basal Isc, a reduction in amiloride-sensitive Isc, and an increased amiloride-insensitive Isc in IL-1{beta}-treated cultures (Fig. 7B).



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Fig. 7. The effect of IL-1{beta} on short circuit current (Isc). A: representative Isc tracing from triplicate IL-1{beta} ({bullet}, 2.5 ng/ml, 48 h)- and control vehicle ({circ})-treated preparations from a single subject. Cells grown on Snapwell inserts were mounted in modified Ussing chambers. Isc was measured basally and in response to amiloride (10-5 M), isoproterenol (10 µM), UTP (100 µM), and bumetanide (50 µM). P < 0.0005 IL-1{beta} treated vs. control, 2-way ANOVA. B: basal, amiloride-sensitive, and amiloride-insensitive Isc. Mean data from triplicate IL-1{beta} (solid bars)- and vehicle control (open bars)-treated cultures from 6 subjects. *P < 0.01 IL-1{beta} treated vs. control. **P < 0.05 IL-1{beta} treated < control, by ANOVA. C: peak {Delta}Isc in response to isoproterenol and UTP. Mean data from triplicate control (open bars) and IL-1{beta} (solid bars)-treated bronchial epithelial cultures from 6 subjects. *P < 0.001 IL-1{beta} treated vs. vehicle control.

 

To test whether IL-1{beta} modulated CFTR-dependent Cl- secretion, we stimulated cells with isoproterenol. The increase in Isc in response to isoproterenol was enhanced in IL-1{beta}-treated preparations, suggesting an increase in CFTR-dependent Cl- conductance (Fig. 7, A and C). To test whether a Ca2+-activated Cl- conductance was also increased by IL-1{beta}, we exposed cultures to apical UTP. The increase in Isc was somewhat greater in IL-1{beta}-treated vs. control preparations (Fig. 7, A and C). Bumetanide sensitivity was used to test for the requirement of basolateral Cl- entry via the sodium, potassium, two-chloride, cotransporter (NKCC1) to support Cl- secretion. Bumetanide-sensitive Isc (IL-1{beta}, 20 ± 3.4 µA/cm2 vs. vehicle; control, 11 ± 3.1 µA/cm2; P < 0.05) was increased following IL-1{beta} treatment (Fig. 7A).

Effect of IL-1{beta} on CFTR and ENaC mRNA levels

IL-1{beta} effects on isoproterenol-stimulated and amiloride-sensitive currents could reflect transcriptional upregulation of CFTR and downregulation of ENaC, respectively. Therefore, we quantified mRNA levels for CFTR and the three subunits of ENaC in vehicle control and IL-1{beta}-treated cultures. IL-1{beta} significantly increased CFTR mRNA levels but did not affect ENaC mRNA levels. (Fig. 8, A and B).



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Fig. 8. The effect of IL-1{beta} on CFTR and epithelial sodium channel (ENaC) mRNA levels. RNA was isolated from triplicate bronchial epithelial cultures following 48 h of IL-1{beta} (2.5 ng/ml) or vehicle control treatment (from 4 normal subjects). Levels of mRNA for CFTR and ENaC mRNA were quantitated by ribonuclease protection assay. A: CFTR mRNA in IL-1{beta}-treated samples (solid bars) and vehicle control (open bars), *P < 0.05 IL-1{beta} vs. control. B: {alpha}-, {beta}-, and {gamma}-subunit ENaC mRNA in IL-1{beta}-treated (solid bars) and vehicle control (open bars) samples.

 

Effect of IL-1{beta} on pH and Ion Composition of ASL

We measured pH and ionic composition of apical liquid from IL-1{beta}- and control vehicle-treated cultures. Time-dependent depletion and ASL acidification were detected in control vehicle-exposed cultures, as previously reported (7, 41). In contrast, pH remained at 7.4 in IL-1{beta}-treated cultures, reflecting a constant [] (Fig. 9, A and B). Although the apical liquid remained isotonic in both control and IL-1{beta}-treated cultures following 24 h of treatment (data not shown), ASL Cl- was significantly elevated in control compared with IL-1{beta}-treated preparations (Fig. 9C). IL-1{beta} did not affect ASL K+ depletion (vehicle control = 0.63 ± 1.2 meq/l vs. IL-1{beta} = 0.52 ± 1.1 meq/l) or steady-state Na+ (vehicle control = 129.3 ± 1.2 meq/l vs. IL-1{beta} = 130.3 ± 1.6 meq/l) after 48 h.



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Fig. 9. The effect of IL-1{beta} on ASL pH and ionic composition. Bronchial epithelial cultures were IL-1{beta} (2.5 ng/ml, {bullet}) or vehicle control ({circ}) treated for 48 h (quadruplicate cultures per treatment, from 8 different subjects). At the beginning of the experiment, 200 µl of Krebs bicarbonate Ringer were applied to the apical surface of the cultures. After 24 and 48 h, pH (A), [] (B), and [Cl-] (C) were determined. *P < 0.001 IL-1{beta} treated vs. vehicle control.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The airways are continually exposed to inhaled pathogens, particulates, and environmental toxicants. Mucociliary clearance is a major innate defense mechanism to prevent airway damage from these exposures (21). The efficacy of mucociliary clearance depends on trapping particles and bacteria in the mucus layer and transporting this material from the lung via the actions of ciliary beat.

Dysregulation of any component of ASL can degrade the efficacy of mucociliary clearance and promote airway damage. For example, mucus hypersecretion is typically associated with airway inflammation and is often considered to have adverse consequences for airway defense. However, it is not yet clear what specific inflammatory processes trigger mucus hypersecretion, nor is it clear that hypersecretion per se must have adverse consequences for mucociliary clearance. For example, in vivo studies suggest that many cellular products released during inflammation from inflammatory cells, as well as the epithelium, play important roles in altering the regulation of mucin secretion. Among the products implicated in causing mucin hypersecretion are cytokines, growth factors, eicosanoids, eosinophil products, free radicals, reactive oxygen species, and neutrophil elastase (3, 6, 11, 12, 15, 18, 28-30, 54).

IL-1{beta} is a prototypical inflammatory cytokine implicated in many inflammatory diseases and, consequently, is a useful probe to test whether, in response to inflammation, airway epithelia 1) secrete mucins in a manner that may have adverse consequences for airway defense, e.g., selective mucin secretion would render ASL more viscous and less clearable, or 2) coordinate secretion of mucins with addition of adequate liquid to promote entrapment and efficient clearance of inhaled inflammatory particles from the lung (8).

Previous studies using explant cultures of gastrointestinal lining show that IL-1{beta} stimulated mucin exocytosis (8). Here we report that IL-1{beta} treatment increased MUC5AC mucin production and secretion by human airway epithelia in a time- and dose-dependent manner. Interestingly, MUC5B mucin secretion was not increased by IL-1{beta} treatment. MUC5AC mucin production was stimulated by as little as 0.25 ng/ml of IL-1{beta} and was increased within 8-12 h of IL-1{beta} exposure (Fig. 3). Although there are no reports that cytokines alter the physicochemical properties of mucins, there have been a number of reports indicating that in airway inflammatory diseases some of these properties are altered (41). In the present studies, no evidence was found to indicate that IL-1{beta} altered the physicochemical properties of MUC5AC mucin.

In vivo, airway inflammation is commonly associated with goblet cell hyperplasia, increased MUC5AC mRNA levels, and mucin overproduction. In the studies reported here using normal human bronchial cells, no increase in the number of MUC5AC mucin-producing cells occurred following treatment with IL-1{beta}. However, the cytokine did increase the total (intracellular plus extracellular) amount of MUC5AC mucin per culture. With respect to mechanism of induction of MUC5AC production, IL-1{beta} treatment did not deplete cell-associated MUC5AC, as might be expected if the cytokine was acting selectively as a secretagogue (Fig. 4A). Because IL-1{beta} treatment resulted in an approximately threefold increase in the total amount (intracellular plus secreted levels) per culture of MUC5AC mucin (Fig. 4B) and because the protein synthesis inhibitor puromycin inhibited the levels of MUC5AC mucin, we conclude that in NHTBE cell cultures, the main IL-1{beta} effect was to increase production of mucin rather than to act as a secretagogue.

Several investigators have reported that diverse inflammatory mediators increase MUC5AC mRNA levels in airway cell lines by transcriptional or posttranscriptional mechanisms (3, 4, 11, 12, 27-30). We also found this to be true using NCIH292 cells and reported that IL-1{beta}, TNF-{alpha}, lipopolysaccharide, or neutrophil elastase increases MUC5AC expression (24). In the present study using normal, well-differentiated, HBE cells, IL-1{beta} only slightly and transiently increased MUC5AC mRNA expression, namely at 1 and 4 h posttreatment, whereas MUC5AC mucin production was elevated twoto threefold for at least 72 h. This raises the question whether the transient elevation of MUC5AC mRNA alone is sufficient to explain the protracted increase in mucin production or whether the cytokine triggers other mechanisms resulting in sustained, elevated MUC5AC translation. The mechanisms of translational control of a variety of proteins are the subject of intensive investigations (for review see Refs. 20, 25).

A key aspect of this study was to determine whether the increased production of mucin was accompanied by an increase in surface liquid. We have recently shown that normal airway epithelia regulate ion transport so that they absorb liquid in times of volume excess on airway surfaces and secrete liquid when there is volume depletion (45). Consequently, we evaluated functional consequences of IL-1{beta} on ASL volume metabolism by two approaches. First, we asked whether ASL volume could be increased by inhibition of volume absorption. This question is probably most relevant clinically because in vivo ASL normally is transported cephalad along airway surfaces, imposing volume loads on the proximal airways (43). IL-1{beta} did indeed inhibit volume absorption, suggesting that IL-1{beta} can effectively add liquid to airway surfaces by this mechanism (Fig. 6B). Second, we asked whether airway epithelia could add liquid to "dry" airway surfaces in response to IL-1{beta}. Our data suggest that this was also possible (Fig. 6C).

We explored the ion transport mechanisms regulated by IL-1{beta}. We found that IL-1{beta} appeared to expand ASL volume by altering the balance between Na+ absorption, which was inhibited, and anion secretion, which was stimulated (Figs. 6, 7). IL-1{beta} also appeared to prime airway epithelia to respond to secretagogues, since the anion secretory responses to isoproterenol (CFTR) and UTP (Ca2+-activated chloride conductance) were also augmented (Fig. 7). Increased CFTR-dependent secretion may, at least in part, reflect an increase in CFTR gene expression (Fig. 8). The inhibition of amiloride-sensitive current did not reflect reduced ENaC gene expression but could reflect increased CFTR expression, as CFTR interacts with ENaC to inhibit Na+ absorption in airway epithelia (38), or it could reflect activation of other mechanisms that directly inhibit ENaC.

Together, our data indicate that airway epithelia coordinate mucin and ASL metabolism. Importantly, the expansion of ASL volume (secretion) exceeded that of mucin secretion, as indicated by the drop in the percentage of solids (Fig. 6). We have recently shown that increased ASL volume, particularly if the percentage of solids decreases, increases mucus transport rates (46). Thus the paralleled ASL volume and mucin secretory responses to IL-1{beta} would be predicted to be favorable with respect to lung defense.

Airway inflammation also is predicted to acidify the luminal environment, with adverse consequences on a variety of processes. In the control vehicle-treated cultures, ASL pH and [K+] fell, likely due to the activity of a K-+H+-ATPase, which we have previously identified in the apical membrane of airway epithelium (7). IL-1{beta} treatment prevented the acidification of ASL. Because IL-1{beta} treatment did not inhibit K+ removal from the ASL, lack of ASL acidification in IL-1{beta}-treated cultures probably did not reflect inhibition of K+-H+-ATPase. CFTR, however, has also been suggested to play a role by increasing the pH of ASL by mediating an apical conductance (38). In this context, prevention of ASL acidifi-cation by IL-1{beta} treatment might be a consequence of the increased CFTR activity. Thus the more alkaline ASL in IL-1{beta}-treated cultures might be an important defense mechanism by the epithelium anticipating an acidic environment that occurs during airway inflammation.

These data may have implications for the pathogenesis of many inflammatory airway diseases associated with increased IL-1{beta} levels. IL-1{beta}-mediated airway epithelial liquid secretion was, at least in part, CFTR dependent. By virtue of absent CFTR function, CF airways lack a critical compensatory mechanism for adding liquid to the airway surface. The prediction is thus that inflammation (i.e., IL-1{beta})-stimulated mucin secretion, in the absence of liquid secretion, would worsen mucus viscosity and clearance from CF airways (46). It has also been reported that IL-1{beta} expression is induced by rhinoviral infection of normal human airway epithelia (37). Our findings of increased ASL and mucin production may explain in part the copious production of secretions that typically occur with this infection; however, we should note that other cytokines might have contrasting effects on airway epithelial ion (and thus liquid) transport. Further studies of the effects of these cytokines on mucin secretion and ASL volume transport will be required.

Although it is poorly understood if, and how, inflammation elicits adaptive responses in airway epithelial ion transport and ASL composition, recent evidence suggests a role for inflammatory mediators in the regulation of these vital processes. A hypersecretory phenotype (inhibition of amiloride-sensitive currents and activation of UTP- and forskolin-dependent responses) has also been induced in airway epithelial cells by IL-13 and IL-4 (9). In contrast, IL-4 and IL-13 have been reported to have differential effects on epithelial ion transport in T84 cells. INF-{gamma}, but not TNF-{alpha}, has been reported to inhibit both Na+- and CFTR-dependent Cl- currents while upregulating a Ca2+-activated Cl- conductance in airway epithelia (13, 55). Our results demonstrate that IL-1{beta} regulates both airway epithelial ion transport and, as consequence, ASL volume. The differential affects of inflammatory mediators on ion transport might be an important facet of lung host defense.

In summary, IL-1{beta}, a cytokine involved in the early component of the inflammatory response, triggers airway epithelia to mount an adaptive response to increase mucin production, ASL volume, and ASL alkalinization. The net functional, i.e., key "defense," effect of this coordinated secretion is to provide a solution that will "flush" noxious agents from airway surfaces, much as has been proposed as a mechanism to flush noxious agents from the crypts of gut epithelia (31).


    ACKNOWLEDGMENTS
 
Current address for L. Burch: Dept. of Pulmonary Med., Duke University Medical Center, Durham, NC 27710.


    FOOTNOTES
 

Address for reprint requests and other correspondence: T. Gray, MD C4-09, PO Box 12233, National Inst. of Environmental Health Sciences, Research Triangle Park, NC 27709 (E-mail: grayt{at}niehs.nih.gov).

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.

* T. Gray and R. Coakley contributed equally to this work. Back


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