Department of Environmental Health, University of Washington, Seattle, Washington
Received July 21, 2000; accepted January 3, 2001
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ABSTRACT |
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Key Words: airway; cell culture; cytokine; electron spin resonance; epithelial cell, free radical; inflammation; nasal cell; ozone; transcription factor; tumor necrosis factor..
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INTRODUCTION |
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Free radical-forming oxidants are known to activate NF-B in airway epithelial cells (Barnes and Adcock, 1998
; Martin et al., 1998
). NF-
B is a ubiquitously expressed transcription factor present in most cell types, which plays an active role in the inflammatory response. The inactive protein exists as a heterodimer of 2 DNA-binding, Rel family subunits, p50 (NF-
B1) and p65 (RelA), bound in the cytosol by the inhibitory protein I
B (Barnes and Adcock, 1997
). Activation of NF-
B involves degradation of I
B (Baeurle et al., 1994; Schieven et al., 1993
) and subsequent translocation of active NF-
B to the nucleus, where it binds the
enhancer in the promoter regions of inducible genes (Schreck et al., 1992
). The genes of many inflammatory proteins, including TNF-
, IL-8, GM-CSF, ICAM-1, and inducible nitric oxide synthase, possess
B-binding sequences and are therefore inducible by NF-
B (Barnes et al., 1997). Studies have shown oxidant-induced increases in NF-
B activity in animal airway epithelium as well as human cell lines (Haddad et al., 1996
; Jany et al., 1995
). Jaspers and associates (1997) reported increased DNA binding of NF-
B in A549 cells following in vitro exposure to 100 ppb O3 for 5 h.
At this time, however, the relationship between O3 exposure and NF-B activation has not been adequately demonstrated in primary cultures of human airway epithelium. This is surprising, as the release of inflammatory mediators by these cells in vitro represents the most realistic model of the inflammatory response to O3 in vivo. Human bronchial epithelial cells release significantly elevated amounts of IL-8, GM-CSF, and soluble ICAM-1 following exposure to 50 ppb O3, and TNF-
at O3 concentrations as low as 10 ppb (Rusznak et al., 1996
). Beck et al. (1994) found that primary human nasal epithelial (HNE) cells exposed to 500 ppb O3 for 6 h significantly increased expression of surface ICAM-1, IL-1
, and IL-6. That study also reported a significant 89% increase in TNF-
release from HNE cells exposed to O3 versus cells exposed to air.
The principal objective of the present study was to demonstrate increased activation of NF-B in primary cultures of HNE cells following exposure to O3. Electron spin resonance (ESR) spectroscopy was employed to demonstrate free radical generation in cells prior to NF-
B activation, and increased TNF-
production confirmed an inflammatory response. Because O3 is a ubiquitous urban air pollutant, persistent exposure of large populations to O3 constitutes a significant public health concern. The present findings may contribute to development of strategies that might reduce the adverse respiratory effects associated with O3 exposure.
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MATERIALS AND METHODS |
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Reagents.
Lipopolysaccharide (LPS) from Escherichia coli Serotype O26:B6, dithiothreitol (DTT), phenylmethylsulfonyl fluoride (PMSF), N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), molecular biology-grade water, alcohols, and buffer salts were purchased from Sigma Chemical Co. (St. Louis, MO). Poly(dI-dC)-Poly(dI-dC) double-strand sodium salt [Poly(dI-dC)] was obtained from Pharmacia Biotech (Alameda, CA). Precast, 10-cm2, 6% polyacrylamide gels prepared with 0.5x Tris-borate-EDTA (TBE) buffer, 5x TBE running buffer stock, and 6x sample buffer stock (with bromophenyl blue and xylene cyanol dyes) were obtained from NOVEX (San Diego, CA). Calcium- and magnesium-free Dulbecco's phosphate-buffered saline (DPBS), Pen/Strep Fungizone Mix (PSF), trypsin-versene mixture (trypsin), 1:1 DMEM:Ham's F12 culture medium (DMEM:F12), and fetal bovine serum (FBS) were purchased from BioWhittaker (Walkersville, MD). Dispase II (Dispase), Nonidet P-40, and T4 kinase were acquired from Boehringer Mannheim (Indianapolis, IN). NuSerum was obtained from Collaborative Research (Lexington, MA). Aqueous solutions for ESR studies were chromatographed on chelex-100 resin to remove adventitious metal contaminants prior to use. DMPO was treated with activated charcoal to remove ESR-detectable impurities (Buettner and Oberley, 1978).
HNE cell culture.
HNE cell cultures were established according to the method described by Beck et al. (1994). In order to maintain an atmospheric interface with the apical surface of the monolayers, the HNE cells were plated on collagen-coated 12-mm Transwell® (0.4-µm pore polytetrafluoroethylene membrane) filter inserts (Costar, Cambridge, MA) in 12-well culture plates at a concentration of 106 cells/insert. Culture medium was removed by suction every 2 days and 1 ml fresh E-medium (consisting of DMEM:F12 culture medium supplemented with 10% NuSerum and 1% PSF) added under each insert to contact only the basal surface of the monolayers. All incubations were carried out at 37°C with 95% humidity and 5% CO2 in a water-jacketed incubator (Forma-Scientific, Marietta, OH) until the monolayers reached confluence, usually within one week.
Ozone exposure.
Just prior to exposure, half the cultures from each donor were randomized to 120-, 240-, or 500-ppb O3 treatment groups, while the remaining half were designated as controls. Cells were then placed in a flow-through exposure system that allowed simultaneous exposure of control groups to filtered air and exposure of treatment groups to O3 (detailed by McManus et al., 1989). Both chambers received 5% CO2 monitored with a medical gas analyzer (Beckman, Model LB-2, Irvine, CA). O3 was generated by ultraviolet irradiation of compressed oxygen (Ozone Research and Equipment Co., Model 03V1-0, Phoenix, AZ) and diluted in filtered air. O3 levels were maintained at 120, 240, or 500 ppb and monitored using an ultraviolet photometric analyzer (Dasibi, Model 1003-AH, Glendale, CA). Temperature and humidity were maintained at approximately 37°C and 95%, respectively. All exposures were carried out in duplicate for a duration of 3 h, and cells were returned to the incubator upon completion.
Electron spin resonance.
Following O3 or air exposure for 3 h, media was removed by aspiration, and cells were washed in PBS to remove any remaining O3-containing media. Cells were then gently scraped from their culture inserts and transferred in 1-ml DPBS to sterile microfuge tubes to which DMPO was then added, for a final concentration of 100 mM. Cell suspensions were immediately transferred to 50-µl capillaries (VWR 53432783) via capillary action, and were sealed on the bottom with critoseal (Fisher 0267620). Capillaries were placed in 4-mm ESR tubes (Wilmad, 707-SQ-250M), and samples were analyzed for free radical formation using a Bruker EMX-band (9 GHz) ESR spectrometer under the following conditions: 20 mW microwave power, 10 Hz modulation frequency, 1.25 G modulation amplitude, 100 G scan range, and 1024 points per scan. Readings were taken at 30, 60, 90,120, and 360 min after initiation of spectra acquisition.
Nuclear protein extraction.
HNE nuclear proteins were extracted and NF-B activity determined using the electrophoretic mobility shift assay (EMSA), as previously described (Woods et al., 1999
). For positive controls, LPS was added to a final concentration of 1 µg/ml to the basal media of randomly chosen, air-exposed cell inserts, and all inserts were returned to the incubator for 30 min. Basal media was then evacuated from beneath control and treated culture inserts. In order to extract HNE-cell nuclear proteins, the monolayers were first gently washed with two 0.5 ml aliquots of DPBS. These washes were performed with DPBS at 37°C, while all further procedures were carried out with buffers on ice. Using a glass rod, the HNE cells were gently scraped from each insert in 150 µl Buffer A (an aqueous stock containing 10-mM HEPES [pH 7.9], 1.5 mM MgCl2, and 10 mM KCl, which was supplemented with 0.5 mM DTT [with a stock prepared in 0.01 M sodium acetate buffer, pH 5.2], 0.5 mM PMSF [stock prepared in molecular biology grade isopropanol], and 10 µg/ml leupeptin hydrogen sulfate [Boehringer Mannheim]) and samples (pooled in groups of 3) removed to sterile microfuge tubes. Tubes were centrifuged at 12,000 rpm for 15 s at 4°C, the supernatant aspirated, and 20 µl Buffer A supplemented with 0.1% Nonidet P-40 was added to each tube. Tubes were then vortexed, incubated on alcohol ice for 10 min, re-vortexed, and centrifuged at 12,000 rpm for 10 min. After aspirating the supernatant, 20 µl Buffer C (an aqueous stock containing 20 mM HEPES, 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, and 25% glycerol, supplemented exactly as for Buffer A above) was added, and the tubes were once again vortexed, incubated in alcohol ice for 10 min, re-vortexed, and centrifuged at 12,000 rpm for 10 min. The supernatant containing the extracted nuclear proteins was removed and mixed with 30 µl Buffer D (a stock solution containing 20 mM HEPES [pH 7.9], 50 mM KCl, 0.2 mM EDTA, and 20% glycerol, supplemented to 0.5 mM DTT, 0.2 mM PMSF, and 10 µg/ml leupeptin), at which time 4 µl was removed and stored at 20°C for total protein determination using the Bio-Rad Total Protein Assay (Hercules, CA). Protein extracts were then split into primary and backup 23-µl samples and immediately stored at 80°C.
Oligonucleotide probe.
An oligonucleotide containing the B sense sequence (5'-AGT GTA GGG GAC TTT CCC AGG C-3') was ordered as an annealed probe from IDT (Coralville, IA). The double-stranded oligo probe was end-labeled with [
-32P]dATP using T4 kinase. The radiolabeled probe was separated from free nucleotides by using a Nuc Trap push column (Stratagene, La Jolla, CA). Competition experiments with either an excess of the unlabeled probe containing the
B sequence or with a mutant sequence (5'-AGT TGA GGC GAC TTT CCC AGG C-3'), also acquired from IDT, were used to confirm specificity. The experimental protocol employed to confirm that the specific proteinDNA complex observed was that of NF-
B has been previously reported (Woods et al., 1999
).
Electrophoretic mobility shift assay.
In order to analyze NF-B activation, nuclear protein extracts (12 µg in a volume equal to or less than the binding reaction volume [usually 12.5 µl]) were first incubated for 15 to 30 min at 37°C in a solution containing 2 µg poly (dl/dC), 12.8 mM Tris, pH 7.5, 64 mM NaCl, 1.28 mM EDTA, 1.28 mM DTT, 0.08% Nonidet P-40, and an NF-
B-binding 32P-end-labeled double-stranded oligonucleotide probe (equal to 4 x 104 cpm/5 µl). Following addition of 4 µl of sample buffer containing the running dyes bromophenol blue and xylene cyanol, 5 µl of each reaction mixture was loaded onto a pre-cast TBE 6% polyacrylamide DNA retardation gel. Samples were electrophoresed in 0.5x TBE running buffer containing 44.5 mM Tris base, 44.5 mM boric acid, and 1 mM EDTA, at 4°C for approximately 90 min at 90 V constant O/C on an Owl "Penguin" Model P8D8 unit (International Scientific Corp., Kaysville, UT) driven by a Model 250 electrophoresis power supply (GIBCO-BRL, Gaithersburg, MD). Gels were dried and exposed to Kodak X-OMAT AR X-ray film with intensifying screens for up to 24 h. Autoradiograms were analyzed using Bio-Rad Gel Doc 1000 and Bio-Rad Molecular Analyst Version 2.1.1 software.
TNF- assay.
In order to quantitate the release of TNF- from HNE cells, basal media was collected from underneath the Transwell® inserts following O3 or air exposure. Samples were collected at 0,1, 2, 4, 16, 20, and 22 h post-exposure to determine the time interval that revealed maximum TNF-
expression. TNF-
concentration in each sample was determined, in duplicate, using the QuantikineTM Human TNF-
Immunoassay (ELISA) from R&D Systems (Minneapolis, MN).
Statistical analysis.
Two-tailed, paired t-tests were used to compare mean TNF- values between groups of air- and O3-exposed samples collected 16 h post-exposure. Significance was defined at p < 0.05. All data are expressed as mean ± SE.
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RESULTS |
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DISCUSSION |
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To our knowledge, this is the first direct demonstration of O3-induced free radical generation, as well as of NF-B activation by LPS and O3, in primary human nasal epithelial cells. The identity of the specific free radicals that give rise to the spectra presented in Figure 1
remain to be defined, but most likely represent products of O3-induced lipid peroxidation. The nasal epithelium, a critical target for reaction with ozone, is rich in unsaturated phospholipids, such as palmitoleic and oleic acids, which readily react with O3 (Cueto et al, 1994
). Pryor et al (1981) have previously demonstrated the formation of alkyl (dienyl) and alkoxy radicals from the interaction of O3 with the polyunsaturated fatty acid, methyl linoleate, in vitro using DMPO as spin trap. The spectrum presented in Figure 1F
is reminiscent of that obtained from the reaction of O3 with linoleate (Pryor et al., 1981
) and may represent reaction products derived from the interaction of O3 with membrane unsaturated fatty acids in HNE cells. Further studies are required to identify the specific free radical species as well as the intermediate reaction products involved. On the other hand, it is known that O3 reacts directly with DMPO in vitro to produce ESR-detectable reaction products (Pryor et al., 1981
). Although cells were washed after O3 exposure to remove O3-containing media prior to addition of DMPO in the present studies, it is possible that unreacted O3 interacted with DMPO intracellularly to produce the spectra obtained. This possibility appears unlikely, however, inasmuch as no ESR signals were observed when DMPO was added to O3-exposed media alone.
The precise mechanism by which ozone promotes activation of NF-B in HNE cells remains to be defined, although evidence of free radical involvement is strongly suggested by the ESR data. Activation of NF-
B by reactive oxidants and consequent NF-
B-mediated inflammation and proliferation in airway epithelial cells have been described by numerous investigators (Haddad et al., 1996
; Jany et al., 1995
; Martin et al., 1998
; Rahman, 2000
). Notably, Chung and Adcock (2000) recently reported increased NF-
B-DNA binding associated with the oxidative effects of O3 exposure in rat lung cells. Additionally, Salmon et al (1998) reported prevention of O3-induced proliferation of bronchial and alveolar epithelium by the antioxidant, apocynin (Salmon et al, 1998
), suggestive of a free radical mechanism underlying O3-induced NF-
B activation in these related airway cell types. In the present studies, radical species were detected in HNE cells as early as 30 min after O3 exposure, consistent with the time of detection of NF-
B-DNA binding. Further studies are required, however, to define the specific role of O3-derived free radical species in NF-
B activation in HNE cells. On the other hand, the possibility remains that proinflammatory cytokines released during O3 exposure, including TNF-
, may induce NF-
B activation in HNE cells, as has been observed in other cell types (Morales et al., 1997
; Rahman et al., 1999
). This possibility seems unlikely however, inasmuch as maximal TNF-
release in the present studies occurred more than 16 h following O3 exposure, much later than the observed NF-
B activation.
The observed NF-B activation and increased TNF-
release also confirm previous research showing O3-induced activation of NF-
B and release of proinflammatory mediators in transformed airway cells. Jaspers et al. (1997) reported increased NF-
B activation and release of IL-8 from A549 cells into basal media collected 16 h after exposure to 100 ppb O3 for 5 h. The present study confirms the study by Jaspers and colleagues on a cell line and extends the observations to primary cultures of human upper airway epithelial cells.
Because TNF- is a marker of airway inflammation, TNF-
release from HNE cells into the basal media is an indicator of an inflammatory response to inhaled O3. Maximal TNF-
release occurred at longer than 16 h post-exposure to O3, which is the expected chronological order after free radical generation and NF-
B activation. This 16-h lag between NF-
B activation and maximal TNF-
release implies that there may be a cascade of signal transduction events that occur between inflammatory gene transcription and extracellular release. The unchanged TNF-
release from cells exposed to 120 ppb O3 for 3 h compared to controls implies that any effects induced by this exposure were below the level of detection. It should be noted, however, that only one marker of inflammation was analyzed in this study after an acute O3 exposure. It is plausible that screening for several cytokines under chronic exposure conditions might result in a different picture of the inflammatory response in HNE cells exposed to 120 ppb O3. Like NF-
B activation, TNF-
levels varied among individuals and was greatest at time points greater than or equal to 16 h after exposure to 500 ppb O3. This contrast to the relatively small variation in TNF-
levels seen at 0 to 4 h post-exposure might be explained by O3-induced cytotoxicity and cell death at this high dose. A greater proportion of necrotic cells, obviously unable to continue expressing inflammatory cytokines, would lead to greater variation in TNF-
levels seen between subjects, which would increase as long as the intact cells were allowed to continue mounting an inflammatory response.
A limitation of this study may be its small sample population. Also the cells were exposed for one 3-h period, whereas human exposures are often for more hours per day as well as throughout an entire summer ozone season.
In conclusion, the present study describes free radical generation, NF-B activation, and upregulation of TNF-
in primary cultures of HNE cells, following exposure to O3. Since the airway epithelium is the first cell layer to come into contact with inhaled O3 and the free radical products generated by ozonation of lipids and proteins in the airway lining fluid, these results support the hypothesis that the airway epithelium plays a role in directing the inflammatory response to inhaled O3 via free radical-mediated NF-
B activation.
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ACKNOWLEDGMENTS |
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NOTES |
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