Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan 48824
Received February 10, 2003; accepted April 17, 2003
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ABSTRACT |
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Key Words: ozone; lipopolysaccharide; mucous cell metaplasia; mucin; neutrophil; inflammation.
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INTRODUCTION |
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Exposure of people to ozone, the primary oxidant gas in photochemical smog, is associated with altered pulmonary function and airway reactivity (Lippmann and Schlesinger, 2000), airway inflammation (Graham and Koren, 1990
), and increased hospital admissions in people with preexisting airway diseases (Thurston et al., 1992
; Wong et al., 2002
). We have documented ozone-induced epithelial lesions and mucous cell metaplasia in the nasal epithelium of rats and primates (Harkema et al., 1987
; Hotchkiss et al., 1991
). However unlike endotoxin, ozone exposure has no effect on the mucous apparatus in axial, pulmonary airways in laboratory rodents (Harkema and Hotchkiss, 1993
, Postlethwait et al., 2000
). In these animals, ozone-induced pulmonary lesions are limited to neutrophilic inflammation and minor epithelial injury in centriacinar regions of the lung. Despite these relatively minor responses, ozone exacerbates the severity of toxic responses of pulmonary airways to such airborne pollutants as nitrogen dioxide and particulate matter (Bouthillier et al., 1998
; Farman et al., 1999
; Madden et al., 2000
). It is not known if ozone would similarly worsen the toxic responses to endotoxin in these airways.
Recent studies in our laboratory describe the toxic interaction of endotoxin and ozone to produce enhanced alterations in the nasal mucous apparatus (Fanucchi et al., 1998; Wagner et al., 2001a
). In pulmonary airways, ozone elicits neutrophilic inflammation similar to endotoxin (Hotchkiss et al., 1989
), and we have previously shown that endotoxin-induced mucous cell metaplasia in axial pulmonary airways of rats is dependent in part on neutrophilic inflammation (Hotchkiss and Harkema, 1994
). Using an endotoxin/ozone coexposure model, we hypothesized that ozone exposure would exacerbate endotoxin-induced mucous cell metaplasia in axial pulmonary airways of rats. In the present study, we demonstrated, using histological, morphological, biochemical, and molecular approaches, that ozone exposure enhances endotoxin-induced alterations in the mucus apparatus in rat lungs. Specifically, ozone enhanced endotoxin-induced neutrophilic inflammation, mucin gene expression, and production and hypersecretion of mucin glycoproteins in rat airways.
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MATERIALS AND METHODS |
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Ozone exposure.
Rats were exposed to 1 ppm ozone for all studies. National Ambient Air Quality Standards for ambient ozone are 120 ppb for 1 h and 80 ppb for 8 h, which is exceeded during summer months in some areas of the country where levels reach greater than 300 ppb (EPA, 2000) Dosimetry studies suggest that rats require four- to fivefold higher doses of ozone than humans to create an equal deposition and pulmonary inflammatory response (Hatch et al., 1994
). Therefore, 1 ppm is a reasonable exposure level from which to make comparisons with humans.
Ozone was generated with an OREC model O3V1-O ozonizer (Ozone Research and Equipment Corp., Phoenix, AZ) using compressed air as a source of oxygen. Total airflow through the exposure chambers was 250 l/min (15 chamber air changes/h). The concentration of ozone within the chambers was monitored throughout the exposure using two Dasibi 1003 AH ambient air ozone monitors (Dasibi Environmental Corp., Glendale, CA). Sampling probes were placed in the breathing zone of rats within the middle of the cage racks. The concentration of ozone during exposures was 1.0 ± 0.11 ppm (mean ± SEM) for ozone chambers and less than 0.02 ppm for chambers receiving filtered air.
Endotoxin instillation.
Rats were anesthetized with 4% halothane in oxygen, and 150 µl of endotoxin (lipopolysaccharide from Pseudomonas aeruginosa, serotype 10), in pyrogen-free saline was instilled into each nasal passage (total doses of 0, 2, or 20 µg). The highest dose of 20 µg elicits neutrophilic inflammation and mucous cell metaplasia that is resolved by seven days (Harkema and Hotchkiss, 1992; Steiger et al., 1995
). Clinical human studies use doses of 20100 µg of inhaled endotoxin to elicit the same degrees of pulmonary inflammation (Michel et al., 1997
, 2000
).
Coexposure protocol (days 1 and 2).
Rats were first instilled with saline or endotoxin, and 6 h later they were exposed to air or 1 ppm ozone for 8 h. This dosing-exposure regimen was chosen so that endotoxin-elicited airway neutrophils (which peak between 6 and 12 h), were present at the time of ozone exposure. One day later, endotoxin instillation and ozone exposures were repeated (Fig. 1).
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After lavage, the right lung lobes were ligated and removed. The axial conducting airway from the right caudal lobe was removed by microdissection and homogenized in 0.5 ml Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH) using a post-mounted homogenizer with a 5-mm generator (Model 250, Pro-Scientific, Inc., Monroe, CT). Samples were kept at 80°C until further processing for RNA isolation.
The left lung was processed for histological analysis as follows. The clamp was removed from the left bronchus, and the left lobe was inflated under constant pressure (30 cm H2O) with zinc formalin (Anatech, Kalamazoo, MI) for 2 h. The bronchial airway was ligated and the inflated lobe was stored in a large volume of the same fixative for at least 24 h until further processing.
The intrapulmonary airways of the fixed left lung lobe from each rodent was microdissected according to a modified version of the technique of Plopper et al.(1983) and fully described in one of our previous publications (Harkema and Hotchkiss, 1992
). Beginning at the lobar bronchus, airways will be split down the long axis of the largest daughter branches (i.e., main axial airway; large diameter conducting airway) through the twelfth airway generation. Tissue blocks that transverse the entire lung lobe at the level of the fifth and eleventh airway generation of the main axial airway were excised and processed for light microscopy and morphometric analyses. The tissue blocks were embedded in paraffin, and 56 µm thick sections were cut from the anterior surface. Lung sections were stained with hematoxylin and eosin (H&E) for routine histopathology or with Alcian Blue (pH 2.5)/Periodic Acid-Schiff (AB/PAS) to detect intraepithelial mucosubstances.
Bronchoalveolar Lavage
Cellularity.
Total leukocytes in BALF were enumerated with a hemocytometer, and fractions of neutrophils, macrophages, and lymphocytes were determined in a cytospin sample stained with Diff-Quick (Dade Behring, Newark, DE).
Secreted mucosubstances.
Secreted mucosubstances recovered in BALF fluid was determined by an ELISA for mucin glycoprotein 5AC using a mouse monoclonal antibody to the human MUC5AC protein (Mucin 5AC Ab-1, Neomarkers, Fremont, CA) that has reactivity to the rat rMuc5AC core protein. Fifty microliter aliquots of BALF were applied to a 96-well microtiter plate (Microfluor 2 Black, Dynex Technologies, Chantilly, VA) and dried overnight at 40°C. Plates were blocked with a solution of 1.5% horse serum and 2% rat serum in Automation Buffer Solution (ABS, pH 7.5; Biomeda Corp., Foster City, CA) for 30 min at 37°C. Plates were then incubated with anti-rMuc5AC antibody (1:400 in ABS containing 1.5% horse serum) for 1 h at 37°C and then washed three times with ABS. Bound primary antibody was detected with a biotinylated rabbit anti-mouse secondary antibody and quantitated using horseradish-peroxidase-conjugated avidin/biotin complex (ABC Reagent; Vector Laboratories, Burlingame, CA) and a fluorescent substrate (QuantaBlue; Pierce Chemical, Rockford, IL) using a fluorescence microplate reader (SpectraMax Gemini; Molecular Devices; 318 nm excitation/410 nm emission). Readings were taken at 3 min intervals for 24 min. Duplicate samples were averaged and the group data is represented as mean Vmax units/s.
Lavaged elastase.
Airway elastase recovered in BALF was determined by an ELISA for elastase using a rabbit monoclonal antibody to the human elastase (Calbiochem, La Jolla, CA). Fifty microliter aliquots of BALF were applied to a 96-well microtiter plate (Microfluor 2 Black, Dynex Technologies, Chantilly, VA) and dried overnight at 40°C. Plates were blocked with a solution of 1.5% goat serum in Automation Buffer Solution (ABS, pH 7.5; Biomeda Corp., Foster City, CA) for 30 min at 37°C. Plates were then incubated with anti-elastase antibody (1:400 in ABS containing 1.5% goat serum) for 1 h at 37°C and then washed three times with ABS. Bound primary antibody was detected with a biotinylated goat anti-rabbit secondary antibody and quantitated using horseradish-peroxidase-conjugated avidin/biotin complex (ABC Reagent; Vector Laboratories, Burlingame, CA) and a fluorescent substrate (QuantaBlue; Pierce Chemical, Rockford, IL) using a fluorescence microplate reader (SpectraMax Gemini; Molecular Devices; 318 nm excitation/410 nm emission). Readings were taken at 3 min intervals for 24 min. Duplicate samples were averaged and the group data is represented as mean Vmax units/s.
Morphometry of stored intraepithelial mucosubstances.
To estimate the amount of the intraepithelial mucosubstances (IM) in respiratory epithelium lining axial airways, the volume density (Vs) of AB/PAS-stained mucosubstances was quantified using computerized image analysis and standard morphometric techniques. The area of AB/PAS stained mucosubstance was calculated from the automatically circumscribed perimeter of stained material using a Power Macintosh 7100/66 computer and the public domain NIH Image program (written by Wayne Rasband, U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). The length of the basal lamina underlying the surface epithelium was calculated from the contour length of the digitized image of the basal lamina. The volume of stored mucosubstances per unit of surface area of epithelial basal lamina was estimated using the method described in detail by Harkema et al.(1987). The Vs of intraepithelial mucosubstances is expressed as nanoliters of intraepithelial mucosubstances per mm2 of basal lamina.
Morphometry of epithelial cell numeric density.
The numeric epithelial cell density was determined by counting the number of epithelial cell nuclear profiles in the surface epithelium and dividing by the length of the underlying basal lamina. The length of the basal lamina was calculated from its contour length in a digitized image using the NIH image system described above.
RNA isolation.
Total RNA was isolated from microdissected, homogenized axial airways by following the method of Chomczynski and coworkers (Chomczynski and Mackey, 1995; Chomczynski and Sacchi, 1987). Isolated RNA pellets were resuspended in nuclease-free water and incubated with DNase solution (100 units rRNasin [Promega, Madison, WI], 100 mM DTT [Life Sciences Technology Inc., Grand Island, NY], and 10 units DNase I [Boehringer Mannheim, Indianapolis, IN] in 5X transcription buffer [Promega]) for 45 min at 37°C. The RNA was extracted sequentially with equal volumes of phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:1), and precipitated with 10 M ammonium acetate and isopropanol. The pellet was washed with 75% ethanol, air dried, and resuspended in nuclease-free water containing rRNasin (40 units/100 µl). RNA concentrations were determined with a fluorescent RNA-binding assay (RiboGreen; Molecular Probes, Eugene, OR), using a SpectraMax GEMINI spectrofluorometer (Molecular Devices Corp., Sunnyvale, CA).
Quantitative RT-PCR (reverse transcriptase polymerase chain reaction).
Steady state levels of rMuc-5AC mRNA were determined in rat airway from ozone-exposed rats using a quantitative RT-PCR technique. Muc5AC is a specific protein for secretory mucin glycoproteins that is expressed in secretory epithelial cells and not in other cells of the airway. As such, RT-PCR of airway RNA was used to estimate the rMuc-5AC mRNA that is present in epithelium. The quantitative RT-PCR technique employs a recombinant competitor RNA (rcRNA), used as an internal standard (IS), that is reverse transcribed and amplified in the same tubes as the target sequence (rMuc-5AC). The IS rcRNA was synthesized as described previously (Fanucchi et al., 1999). The IS contains the same sequences recognized by the amplification primers for rMuc-5AC, but has a different-sized intervening sequence and therefore yields a different-sized RT-PCR product. The concentration of rMuc-5AC mRNA was estimated by adding increasing, known amounts of IS (in numbers of molecules) to the RT-PCR mixtures that contain a constant, unknown amount of sample RNA. Because both the IS and sample RNA are amplified at the same rate, this procedure results in an absolute experimental readout (molecules of target gene mRNA per unit sample).
RT-PCR for rMuc-5AC was performed as outlined by Gilliland and coworkers (Gilliland et al., 1990a,b
), except that known amounts of the IS rcRNA were reverse-transcribed into complementary DNA (cDNA) in a volume of 20 µl containing PCR buffer plus 5 mM MgCl2, 1 mM each dNTP, 10 units rRNasin, 125 ng oligo(dT)1218 (Becton Dickinson, Bedford, MD), 100 ng total RNA from maxilloturbinates, and 40 units of MMLV reverse transcriptase (Promega). For each RNA sample from individual animals, a known concentration of IS rcRNA molecules was added that was similar in concentration to the RNA samples. This was determined in a preliminary range-finding experiment using pooled samples of each experimental group to be between 106107 molecules per sample. A standard curve was also prepared by adding tenfold serial dilutions of the IS (104109 molecules per tube) to a constant amount of RNA (pooled from all samples). All RNA samples were then incubated at 42°C for 15 min, followed by an incubation at 95°C for 4 min. A PCR master-mix consisting of PCR buffer, 4 mM MgCl2, 6 pmol each of rMuc-5AC forward (5'-CATCATTCCTGTAGCAGTAGTGAGG-3') and reverse (5'-GGTACCCAGGTCTACACCTACTCCG-3') primers, and 1.25 units Taq DNA polymerase were added to the cDNA samples, for a final volume of 50 µl (Taq polymerase was added to the PCR master-mix after it had been heated to 85°C for 5 min). Samples were then immediately heated to 95°C for 4 min and then cycled 36 times at 94°C for 30 s, 56°C for 30 s, and 72°C for 30 s, after which an additional final extension step at 72°C for 10 min was included.
PCR products (10 µl) were electrophoresed on a 3% agarose gel (NuSieve 3:1; FMC Bioproducts, Rockland, ME) and visualized by ethidium bromide staining. Densitometry was carried out using a Bio-Rad ChemiDoc image acquisition system and Quantity One (v4.0) quantitation software (Bio-Rad, Hercules, CA), running on a Dell OptiPlex GX1 computer. The density ratio of the rMuc-5AC PCR product band to the corresponding IS PCR product band present in each sample was determined as described by Gilliland and colleagues (Gilliland et al., 1990a,b
). A standard curve was constructed by plotting the log of the density ratio (i.e., rMuc-5AC PCR product band/IS PCR product band) versus the log of IS serial dilution concentrations added to the standards (i.e., 104109 molecules/tube). Linear regression was performed on the standard curve to determine slope and y-intercept, which yielded the amount of rMuc-5AC mRNA (molecules) present in the pooled RNA standard sample when mRNA/IS = 1.
The rMuc-5AC mRNA value was then divided by each IS serial dilution concentration to arrive at an "actual" ratio. A transformed standard curve was then calculated by plotting the original density ratio versus the actual ratio, and linear regression was performed. The transformed standard curve was used to calculate the single point measurements of the experimental samples, which were obtained with the following equation:
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Data are expressed as the number of rMuc-5AC mRNA molecules per ng of total sample RNA that was added to the RT-PCR reaction.
Statistical analysis.
Data are expressed as mean ± standard error of the mean (SEM). Data were analyzed using a completely randomized analysis of variance. Multiple comparisons were made by Student-Newman-Keuls post hoc test. Criterion for significance was taken to be p 0.05.
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RESULTS |
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Microscopically, the affected areas of lung consisted of a mild-moderate acute bronchopneumonia. The principal features in the alveolar parenchyma included mild congestion of alveolar capillaries, interstitial edema within alveolar septa, type II cell hyperplasia and a mixed inflammatory cell infiltrate of mononuclear cells (monocytes/macrophages and lymphocytes) and neutrophils. Aggregates of large vacuolated alveolar macrophages containing phagocytized cellular debris were widely scattered throughout the affected areas of the alveolar parenchyma along with modest amounts of eosinophilic proteineous material in the alveolar lumens (pulmonary edema). In addition, the preterminal and terminal bronchioles in the areas of pneumonia often had hypertrophic surface epithelium. Intersititial edema and accumulation of mononuclear cells, neutrophils, and some eosinophils also surrounded these distal airways and adjacent pulmonary arteries.
The principal lesion in the main intrapulmonary axial airways, and several of the other large diameter, preterminal bronchioles branching off of the axial airways, was a conspicuously thickened, columnar surface epithelium with numerous mucous (goblet) cells containing copious amounts of AB/PAS-stained mucosubstances (mucous cell metaplasia; Fig. 2). This endotoxin-induced mucous cell metaplasia occasionally extended into the surface epithelium lining more distal preterminal and terminal airways.
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Rats instilled with endotoxin and exposed to ozone.
The lungs of animals that were instilled with endotoxin and exposed to ozone had gross and microscopic lung lesions that were characteristic of both toxicants, described above. However, the mucous cell metaplasia in the axial airways of these coexposed rats was more severe (see morphometric analyses below) than that observed in the rats instilled with endotoxin but exposed only to filtered air (0 ppm ozone).
Rats instilled with saline and exposed to filtered air.
No exposure-related histologic lesions were present in the lungs of control rats exposed only to filtered air and intranasally instilled with saline.
Bronchoalveolar Lavage
Cellularity.
Endotoxin instillation caused a dose-dependent increase of neutrophils and lymphocytes recovered in BALF from rats (Figs. 3 and 4). By comparison, ozone exposure alone caused significant accumulations of BALF macrophages but not of neutrophils or lymphocytes. Exposure of rats to ozone enhanced by twofold the numbers of BALF neutrophils elicited by instillation with 20 µg of endotoxin, and of BALF lymphocytes elicited by 2 µg endotoxin.
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DISCUSSION |
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The mechanism by which endotoxin promotes mucus production in airway epithelium is unknown. In cultured airway cells and nasal explants, endotoxin administered in vitro directly induces mucin gene expression, apparently in the absence of secondary mediator(s) (Hotchkiss et al., 1998; Li et al., 1997
). In animal studies, endotoxin elicits production in airways of several mediators known to induce mucin gene expression and mucous cell metaplasia, including TNF-
, IL-1, platelet activating factor, and neutrophil-derived elastase. Thus, in addition to direct effects of endotoxin on airway epithelium, many soluble mediators may contribute to endotoxin-induced mucous cell metaplasia in vivo.
We have demonstrated in F344 rats that endotoxin-induced mucous cell metaplasia is partially dependent on neutrophils. In endotoxin instilled rats depletion of circulating neutrophils blocks neutrophilic inflammation completely, and significantly inhibits mucous cell metaplasia in both nasal and pulmonary airways by approximately 60% (Hotchkiss and Harkema, 1994; Wagner et al., 2001a
). In the present study, ozone enhanced both airway neutrophil accumulation and increases in mucus storage and secretion that was elicited by the instillation of 20 µg endotoxin. These data suggest that ozones effects may be mediated by neutrophils or neutrophil-derived products. Because endotoxin-induced mucous cell metaplasia is driven by neutrophilic inflammation, enhancement by ozone may be due simply to increased numbers or the activation of neutrophils. Data was collected at a single timepoint, 72 h after the last endotoxin instillation and ozone exposure. Thus, is it unknown if ozone caused a higher peak in numbers of neutrophils elicited early after endotoxin instillation, or if it caused their persistence in airways beyond that invoked by endotoxin alone.
We began ozone exposures 6 h after instillation with endotoxin, at a time when airway neutrophil recruitment induced by endotoxin is near maximal. Significant numbers of airway neutrophils were therefore exposed directly to ozone. Neutrophil response to ozone exposure has not been adequately characterized in vitro or in vivo, but ozone exposure of monocytic and epithelial cells in culture systems in vitro elicits their production of inflammatory mediators (Jaspers et al., 1997; Samet et al., 1992
). Similarly, ozone may directly stimulate neutrophils to undergo degranulation or oxidative burst within airways. In the present study, ozone significantly increased airway elastase concentrations induced by instillation with 2 µg endotoxin without increasing neutrophil numbers. These data suggest that airway neutrophils in endotoxin-instilled, ozone-exposed rats were more activated to produce inflammatory mediators than airway neutrophils in endotoxin-instilled rats breathing filtered air. Using a specific elastase inhibitor, we have recently demonstrated that endotoxin-induced mucous cell metaplasia in F344 rats is elastase dependent (Wagner et al., 2002b
). Elastase has been implicated in ozone-induced airway reactivity and mucus hypersecretion (Matsumoto et al., 1999
; Nogami et al., 2000
). Thus, although elastase was not detected in BALF from saline-instilled and ozone-exposed rats, ozone may be enhancing endotoxin-induced mucous cell metaplasia by the same, elastase-dependent mechanism.
It is notable that ozones effects are not additive with those induced by endotoxin. Indeed, ozone exposure alone did not engender significant responses of neutrophilic inflammation, BALF elastase accumulation, mucus secretion and storage, or gene expression. Ozone served only to enhance or act synergistically with endotoxin. Endotoxins can initiate cellular responses by binding to the CD14 receptor on inflammatory cells, or to the soluble form of CD14 to interact with epithelial and endothelial cells (Heumann and Roger, 2002). A significant number of recent studies show that endotoxin also binds and activates Toll-like receptors (TLR), a previously described class of cell surface receptors that are linked intercellularly to NF-
B pathways and are important in innate and adaptive immune responses (Modlin, 2002
). TLRs are pattern recognition receptors that are activated by lipids and lipid moieties on lipopolysaccharides, lipoproteins, and peptidoglycans, and thereby recognize a variety of bacterial, viral, and fungal products. However, at least one animal model suggests that TLRs are also important in ozone-induced lung injury. Using mice that are deficient in TLR-4, a specific Toll-like receptor, Kleeberger and coworkers (2000, 2001) showed that TLR-4 is necessary to fully develop ozone-induced lung permeability. It was also demonstrated that induction of pulmonary Tlr4 gene expression by ozone was required for injury in this model. In these studies, mice were exposed to 0.3 ppm ozone, whereas in the present study we exposed rats to 1 ppm. In our model, we hypothesize that ozones ability to upregulate TLR4, and thereby provide more available receptors to transduce cellular responses to endotoxin, may serve to prolong and magnify the effects of endotoxin on epithelial and inflammatory cells. Other studies suggest that upregulation of Tlr genes in both inflammatory and epithelial cells is an important for prolonging the inflammatory processes (Modlin, 2002
). Extending our exposure regimen to employ more and lower doses of ozone, and assessing gene induction (i.e., Tlr4) at earlier timepoints may support the hypothesis that TLR-4 mediates the ozone-induced enhancement we observe in this animal model.
Our results illustrate a unique interaction between two airborne toxicants to alter airway epithelium that would not have been predicted from the known toxicological profile of either pollutant given alone. Ozone enhanced the toxicity of endotoxin, a ubiquitous biogenic substance, at ozone exposure concentrations that alone are nontoxic to rat airways. Because safety standards of air pollutants are primarily based on the toxicological effects of a single pollutant (e.g., ozone), it is possible that the health risk of breathing a mixture of pollutants is underestimated. Our demonstration of ozone enhancement of endotoxin-induced mucous cell metaplasia supports this premise. It is notable that the doses of endotoxin and ozone used in this study are reasonable models for human exposures. Humans living in highly polluted areas and that have high domestic or occupational exposure to endotoxin may be at an increased risk to adverse health effects. Conversely, individuals with a polymorphism in TLR4 and have altered response to inhaled endotoxin may exhibit less sensitivity to coexposures (Schwartz, 2001). Controlled, human exposures are required to test this hypothesis. Furthermore, our results with ozone and endotoxin might be extended to predict the potential airway responses to exposures to other oxidant gases and biogenic substances. Indeed we have recently demonstrated ozone enhancement of airway lesions induced in allergic airways (Wagner et al., 2002a
) and by instillation with vanadium (Wagner et al., 2001b
). Further research is needed to elucidate the mechanism of these toxic interactions and the role played by inflammatory cells and their soluble mediators.
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ACKNOWLEDGMENTS |
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NOTES |
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