Department of Veterinary Pathology, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan 48824
Received August 25, 2000; accepted November 21, 2000
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ABSTRACT |
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Key Words: ozone; endotoxin; mucous cell metaplasia; neutrophils; nasal epithelium.
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INTRODUCTION |
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Endotoxins are lipopolysaccharide-protein components of the cell wall of Gram-negative bacteria. Bacterial endotoxins are potent inflammagens, and are commonly found on airborne vectors such as organic dusts generated from agricultural or industrial sources (Rylander, 1995; Simpson et al., 1999
). Inhalation of endotoxin can elicit inflammatory cell recruitment and epithelial cell injury in humans and animals (Sandstrom et al., 1992
; van Helden et al., 1997
). We have previously described endotoxin-induced epithelial and inflammatory responses in the rodent nasal airways (Harkema and Hotchkiss, 1991
, 1993
). Intranasal instillation of endotoxin causes acute neutrophilic rhinitis that is accompanied by hypersecretion of mucus from secretory cells of the respiratory epithelium (RE), but engenders only acute inflammation (i.e., neutrophil influx) without MCM in the NTE. More recently, we have demonstrated that the presence of airway endotoxin enhances the development of ozone-induced MCM in the NTE (Cho et al., 1999a
; Fanucchi et al., 1998
). Endotoxin promotes ozone-induced metaplastic responses in the NTE where alone it causes only acute inflammation but not metaplasia. The cellular mechanisms of endotoxin's ability to augment ozone-induced epithelial cell responses in this tissue are unknown. We have shown in vitro that endotoxin can induce mucin gene expression in cultured nasal explants (Hotchkiss et al., 1998a
). In addition, several soluble mediators that are produced by endotoxin-activated inflammatory cells (e.g., tumor necrosis factor, interleukin-1, platelet-activating factor, and proteases) have been demonstrated to promote epithelial cell hyperplasia, MCM, and the expression of genes that code for mucin proteins (Borchers et al., 1999
; Lou et al., 1998
; Voynow et al., 1999
). Thus endotoxin might promote MCM by both direct and indirect pathways.
Because ozone-induced MCM is partially dependent on neutrophils, we hypothesized that enhancement by endotoxin of ozone-induced MCM is also dependent on the influx of neutrophils. To test this hypothesis, we used an antibody directed against rat polymorphonuclear leukocytes (PMNs) to deplete animals of circulating neutrophils and thereby block neutrophilic inflammation, caused by endotoxin, in nasal airways. In the present study we demonstrated, using histologic, morphometric, and immunohistochemical approaches, that the enhancement of ozone-induced MCM by endotoxin is neutrophil-dependent. Furthermore, we describe the nature of this dependence in relation to concurrent hyperplasia and mucin-specific gene expression that precedes the ozone-induced MCM in the NTE.
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MATERIALS AND METHODS |
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Ozone exposure (Days 1, 2, and 3).
Rats were exposed to filtered air (controls) or 0.5 ppm ozone for 3 days, 8 h/day (Fig. 1). At this concentration (0.5 ppm), ozone produces epithelial lesions in NTE of F344/N rats, but has little or no effect on adjacent nasal respiratory epithelium (Harkema and Hotchkiss, 1993
). 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 2 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 0.5 ± 0.011 ppm (mean ± SEM) for ozone chambers and less than 0.02 ppm for chambers receiving filtered air.
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Endotoxin instillations (Days 4 and 5).
Fourteen h after treatment with PMN antiserum (Day 4), animals were intranasally instilled with endotoxin by methods described in Harkema and Hotchkiss, 1991. Briefly, rats were anesthetized with 4% halothane in oxygen, and 50 µl of endotoxin (1 mg/ml, lipopolysaccharide from Pseudomonas aeruginosa Serotype 10; Sigma Chemical Co., St. Louis, MO) in pyrogen-free saline was instilled into each nasal passage of 48 rats (total dose of 100 µg). The other 48 rats were instilled with pyrogen-free saline. Instillation procedures were repeated 24 h later on Day 5.
Necropsy and tissue preparation (Days 5 and 8).
Forty-eight rats were killed 6 h after the second intranasal instillation of endotoxin. The remaining forty-eight rats in the study were killed 3 days after the last instillation. Rats were anesthetized with sodium pentobarbital (50 mg/kg), a midline laparotomy was performed, and 3 ml of blood was drawn from the abdominal vena cava into a Vacutainer containing EDTA as an anticoagulant. Total white blood cells in whole blood were enumerated with a Serono-Baker System 9000 automated cell counter (Serono-Baker Diagnostics, Allentown, PA). Total blood neutrophils were determined by their percent occurrence in at least 100 white blood cells counted in smears stained with Diff-Quik (Baxter, McGaw Park, IL). Rats were killed by exsanguination via the abdominal aorta. Immediately after death, the head of each rat was removed from the carcass and the lower jaw and skin were removed. The nasal airways were exposed by splitting the skull in a sagittal plane adjacent to the midline. One-half of the head was immersed in a large volume of zinc-formalin (Anatech, Kalamazoo, MI) for at least 48 h. After fixation, this half of the head was decalcified in 13% formic acid for 4 days, and then rinsed in distilled water for 4 h. A tissue block was removed from the anterior nasal cavity by making 2 cuts perpendicular to the hard palate, the first immediately posterior to the upper incisors, and the second at the level of the incisive papilla (Fig. 2A). The tissue blocks were embedded in paraffin, and 56 µm thick sections were cut from the anterior surface. Nasal sections were stained with hematoxylin and eosin (H&E) for routine histology or with Alcian Blue (pH 2.5)/Periodic Acid-Schiff (AB/PAS) to detect intraepithelial mucosubstances.
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Morphometry of stored intraepithelial mucosubstances.
To estimate the amount of the intraepithelial mucosubstances in NTE lining maxilloturbinates, 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 volume density (Vs) of intraepithelial mucosubstances is derived from the equation:
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and is expressed as nanoliters (nl) 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.
Morphometry of inflammatory cell densities.
The effect of endotoxin on neutrophil influx within the NTE of maxilloturbinates was determined in H&E-stained sections by counting the total number of neutrophils within the nasal mucosa (area between the turbinate bone and airway lumen) and dividing by the total length of the basal lamina. Neutrophils were identified by morphologic characteristics that included their size, darkly stained multilobed nuclei, and clear cytoplasm with dust-like granules.
RNA isolation.
Total RNA was isolated from microdissected, homogenized maxilloturbinates 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.
Steady state levels of rMuc-5AC mRNA were determined in rat maxilloturbinates from ozone-exposed rats using a quantitative RT-PCR technique. Muc5AC is a specific protein for secretory mucin glycoproteins, which is expressed in secretory epithelial cells and not in other cells of the maxilloturbinate. Therefore, RT-PCR of maxilloturbinate 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), which 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 (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 10-fold 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 (Ver. 4.0) 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 (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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Nasal Histopathology
Filtered air-exposed rats.
Both neutrophil-sufficient and neutrophil-depleted rats that were exposed to filtered air (filtered air-controls), and had also received intranasal instillations of saline, exhibited no exposure-related microscopic alterations in the examined nasal tissues at either 6 h or 3 days after the last instillation. In contrast, filtered air-exposed, neutrophil-sufficient rats that were instilled with endotoxin and killed 6 h post-instillation had a markedly diffuse neutrophilic rhinitis characterized by a conspicuous influx of neutrophils in the lamina propria and the overlying surface epithelium of the nasal mucosa. An associated neutrophilic exudate was often conspicuously present in the adjacent nasal airway lumens. The inflammatory cell infiltrate was most severe in the mucosal tissues that lined the lateral and middle meatus and contained NTE or respiratory epithelium. Only a mild inflammatory cell response was evident in the olfactory mucosa lining the dorsal meatus. In addition, there was no histologic evidence of degeneration or necrosis of the NTE, respiratory epithelium, or olfactory epithelium in these animals.
An endotoxin-induced neutrophilic rhinitis was predictably absent in the filtered air-exposed rats that were neutrophil-depleted and killed at 6-h post-instillation. In addition, there was no morphologic alteration in the NTE. There was a mild to moderate mucous cell hypertrophy, due to an increase in intracellularly stored mucosubstances in the respiratory epithelium lining the proximal aspect of the mid-nasal septum.
Filtered air-exposed and neutrophil-sufficient or -deficient rats that received endotoxin instillations and were killed 3 days after the end of the instillations had only a few mononuclear cells (lymphocytes and monocytes) in the lamina propria beneath the NTE and respiratory epithelium lining the lateral and middle meatus of the proximal nasal cavity. The only treatment-induced change in the NTE was a minimal to mild hyperplasia in neutrophil-sufficient rats exposed to endotoxin. In addition, moderate hypertrophy of mucous cells with increased amounts of mucosubstances was present in the respiratory epithelium lining the mid-nasal septum of both neutrophil-sufficient and -deficient rats exposed to endotoxin. No other microscopic alterations were present in either the respiratory or transitional epithelium.
Ozone-exposed rats.
The principal nasal lesion in the ozone-exposed and neutrophil-sufficient rats that received intranasal saline instillations and were sacrificed 6-h post-instillation was a marked epithelial hyperplasia of the NTE lining the lateral meatus and covering the mucosal surface of the proximal maxilloturbinates, lateral wall, and lateral aspects of the nasal turbinates. In addition, these rats had a minimal to mild influx of mononuclear cells with lesser numbers of eosinophils and only an occasional neutrophil in the lamina propria of the transitional mucosa lining the lateral meatus. Mild to moderate hypertrophy of mucous cells in the respiratory epithelium lining the mid-septum was also present in these rats.
Neutrophil-sufficient rats that were similarly exposed to ozone and intranasally instilled with saline, but were sacrificed 3 days after the last saline instillation, also had a markedly hyperplastic NTE. There was also a minimal to mild mucous cell metaplasia in the NTE of these ozone-exposed rats (Fig. 3). Only a minimal to mild increase in mononuclear cells and eosinophils was present in the lamina propria of the affected nasal mucosa.
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The principal nasal lesions in ozone-exposed, neutrophil-sufficient rats that were instilled with endotoxin and sacrificed 3 days post-instillation were moderate to marked mucous cell metaplasia and epithelial hyperplasia in the NTE that lines the lateral meatus of the proximal nasal airway (Fig. 5). A mild mononuclear-cell influx with a few eosinophils and neutrophils in the lamina propria accompanied the regionally restricted epithelial proliferation and differentiation. Interestingly, neutrophil-deficient animals that were similarly exposed and instilled with endotoxin had similar nasal lesions, with the noticeable difference that there was no or minimal mucous cell metaplasia in the NTE (Fig. 5
).
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Mucin (rMuc-5AC) mRNA Expression
Six h after instillation, endotoxin caused a significant increase in Muc-5AC mRNA expression compared to ozone, which was unaffected by depletion of circulating neutrophils (Fig. 8A). By 3 days after instillations, differences in mRNA levels between experimental groups were not statistically significant (Fig. 8B
).
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DISCUSSION |
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Early and transient inflammation, notably the presence of neutrophils in the NTE and the underlying submucosa, is required for metaplastic response to ozone exposure (Cho et al., 1999a). In the current study, endotoxin was given 24 h after 3 daily ozone exposures, at a time when the presence of ozone-induced inflammation in the NTE has subsided (Cho et al., 1999b
). Our results show that endotoxin's enhancement of MCM is accompanied by a significant influx of neutrophils that are not present with ozone exposure alone at this post-exposure time (i.e., 2 days after ozone exposures). Thus, the dosing protocol of ozone (3 days) followed by endotoxin (2 days) prolongs the presence of neutrophils in the NTE. By depleting animals of neutrophils after the ozone-induced inflammation had subsided, we were able to isolate the effects of endotoxin-induced neutrophilic influx on the ozone-induced MCM. Both the increase in mucosal neutrophils and MCM caused by endotoxin were blocked completely in neutrophil-depleted animals. This suggests that the neutrophilic influx caused by endotoxin instillation was responsible for enhancement of the MCM caused by ozone.
Airway endotoxin causes a more robust recruitment of neutrophils in upper airways than does exposure to ozone. Though not enumerated in the present study, we have observed previously that neutrophil accumulation in maxilloturbinates during acute ozone exposures are routinely 410-fold less than those elicited by endotoxin at the same time points (Cho et al., 1999b; Fanucchi et al., 1998
; Hotchkiss et al., 1998b
). In addition, the activation status of neutrophils after endotoxin or ozone treatments may be different. Endotoxin and ozone likely activate both similar and dissimilar inflammatory pathways that can affect neutrophil function. In this regard, the oxidant nature of ozone might induce oxidative stress in epithelial cells and lead to the activation of inflammatory cells, whereas endotoxin activates cells after receptor binding. We have shown that neutrophil recruitment into the NTE diminishes with repeated ozone exposures such that by the fourth or fifth day of repeated exposures, the numbers of infiltrated neutrophils are near control levels. It is notable that endotoxin can initiate a new round of neutrophilic infiltration at a time when ozone-induced signals for neutrophil recruitment have waned (i.e., at 4 and 5 days). This observation suggests that distinct chemotactic pathways (i.e., chemokines, cytokines) are invoked after treatment with endotoxin compared to repeated ozone exposure. Therefore, the activation status of endotoxin-elicited neutrophils, in addition to their cellular density, probably determines their contribution to MCM.
In addition to neutrophilic inflammation, ozone-induced MCM is always preceded by hyperplasia of epithelial cells in the NTE (Cho et al., 1999b; Harkema and Hotchkiss, 1994
; Harkema et al., 1997
). The hyperplastic response is maximal after 3 days of ozone, which is the same time, in the current study, when endotoxin and its accompanying inflammation were introduced. Endotoxin caused a decrease in epithelial cell density, which suggests cytotoxicity, in ozone-exposed animals 6 h after instillation with endotoxin. This cytotoxic effect appears to be selective for new epithelial cells within the multi-layered, hyperplastic NTE engendered by ozone exposure, because there was no change in the cell density (i.e., decrease) in air-exposed, monolayered NTE of animals instilled with endotoxin at this time point. These new cells may be more sensitive to the cytotoxic effects of endotoxin-induced inflammation. By 3 days after endotoxin instillation, however, the hyperplasia in ozone/endotoxin-treated animals is increased to levels even greater than that caused by ozone alone.
This sequence of decreased epithelial cellularity (i.e., cytotoxicity) followed by epithelial repair and hyperplasia is reminiscent of the response seen in NTE after ozone exposure. Specifically, after one exposure to ozone, a decrease in cell density occurs concomitantly with the appearance of epithelial neutrophils (Cho et al., 1999b). By 3 days, neutrophil numbers subside while epithelial cell density increases. In the present study, a similar sequence occurred when ozone-exposed animals were instilled with endotoxin. Specifically, cytotoxicity and neutrophil infiltration were evident by 6 h after endotoxin instillation, and both epithelial proliferation and MCM were significantly augmented compared to ozone-exposed, saline-instilled animals. Furthermore, all these endotoxin-induced events were inhibited in neutrophil-depleted animals. That endotoxin, in the absence of neutrophils, was incapable of promoting MCM in this model clearly implicates the neutrophil as a critical mediator of endotoxin's effects. However, our results do not rule out the possibility that, in addition to neutrophils, endotoxin itself or another mediator is required to promote MCM.
We have previously shown that upregulation of mucin gene transcription is an early event after ozone exposure (Cho et al., 1999b). Furthermore we have shown that neutrophils are not required for gene transcription after ozone exposure. Our present study shows that neutrophils are not required for the mucin gene transcription during endotoxin-enhanced MCM by ozone. Specifically, the increase in rMUC 5AC mRNA 6 h after endotoxin treatment in ozone-exposed animals was unaffected by neutrophil depletion. This finding is consistent with our work using nasal explants in vitro, in which endotoxin, in the absence of neutrophils, increases rMuc 5AC mRNA in pre-existing secretory cells (Hotchkiss et al., 1998a
). Taken together, these studies suggest that other mediators produced after ozone or endotoxin exposure are responsible for mucin gene expression, while neutrophils mediate post-transcriptional events that lead to the appearance of intraepithelial mucosubstances.
The protein product of Muc 5AC gene translation undergoes considerable modification by glycosyltransferases, which catalyze the addition of fucose and sialic acid among other saccharide groups, and sulfotransferases, which add sulfur-containing groups to the core mucin apoprotein. It is these sugar groups and sulfated residues within mucous cell globules that react histochemically with AB/PAS stains. Thus, unmodified, non-glycosylated and non-sulfated Muc5AC apoprotein may be present in epithelial cells in the NTE but is undetected by AB/PAS staining because it lacks reactive groups. One interpretation of these results is that neutrophils mediate the pathways responsible for the glycosylation and sulfation of mucin proteins. In the absence of neutrophils, the signal for processing mucin proteins might not be present. Alternatively, mucin protein may not be translated, despite the transcription of rMuc 5AC. Either possibility requires further study.
In summary, neutrophils mediate the ability of endotoxins to enhance ozone-induced MCM by a mechanism other than upregulation of mucin-gene expression. Our results suggest that neutrophils or neutrophil-derived products mediate an as yet undefined, post-transcriptional event that is necessary to complete metaplastic processes and cause the increased storage of intraepithelial mucosubstances. Although not examined directly in the present study, a number of neutrophil-derived products have been implicated in MCM in airway epithelium, including elastase (Voynow et al., 1999), TNF, and IL-1 (Borchers et al., 1999
). The role of these and other neutrophil products in endotoxin-enhanced MCM requires further investigation.
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ACKNOWLEDGMENTS |
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NOTES |
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