Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan 48824
Received October 5, 2001; accepted January 25, 2002
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ABSTRACT |
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Key Words: ozone; allergen; ovalbumin; mucous cell metaplasia; eosinophil; neutrophil; inflammation.
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INTRODUCTION |
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Among major air pollutants, ozone is currently considered one of the most pervasive problems in regard to the attainment of the National Ambient Air Quality Standards (NAAQS). The U.S. Environmental Protection Agency (U.S. EPA) estimates that almost 50% of the United States population lives in areas where ambient ozone levels exceed NAASQ limits (U.S. EPA, 2000). Approaches to establishing safe ozone exposure limits for humans involve consideration for the most susceptible populations. In this regard, people with preexisting respiratory conditions such as asthma and chronic obstructive pulmonary diseases are considered at risk for adverse health effects from exposure to high ambient ozone concentrations that occur during summer months (Bascom et al., 1995; Cody et al., 1992
; White et al., 1994
). Results from both human and animal studies show that ozone exacerbates airway hyperresponsiveness and immune and inflammatory responses in lower airways of asthmatic subjects (Gilmour, 1995
; Hiltermann et al., 1998
; Jenkins et al., 1999
; Vagaggini et al., 1999
).
Rhinitis is frequently associated with asthma, and similar mechanisms are postulated to underlie the inflammatory and physiologic responses of each condition (Casale, 1999; Simons, 1999
; Vignola et al., 1998
). As such, upper airway responses to ozone might be exacerbated in individuals with allergic rhinitis by mechanisms similar to those that occur in lower airways of asthmatics after ozone exposure. Both healthy and asthmatic individuals respond to ozone exposure with nasal inflammation as indicated by the presence of neutrophils, eosinophils, and inflammatory mediators in nasal lavage fluid (Graham and Koren, 1990
; Hiltermann et al., 1997
). However, these lavage markers of ozone-induced inflammation are greater in asthmatics than in nonasthmatics (McBride et al., 1994
). Furthermore, ozone enhances some antigen-specific allergic nasal responses. Concentrations of inflammatory cells and soluble mediators in nasal lavage are increased after allergen challenge when allergic subjects are first exposed to ozone (Bascom et al., 1990
; Peden et al., 1995
). Thus, in addition to the sensitive populations mentioned above (i.e., asthmatics, children, etc.), people with allergic rhinitis might also be included as a susceptible target group when assessing the risks to human health of ozone exposure. Indeed, recent epidemiological studies in the United Kingdom demonstrate a 37% increase in allergic rhinitis patients on days following high ambient concentrations of ozone (Hajat et al., 2001
).
The mechanism or mechanisms by which ozone enhances allergic inflammatory responses in the nasal airways of humans are unknown. Similar studies in laboratory animals have not been performed. Apart from inflammatory cell infiltration, alteration in nasal epithelial cells, and the production and secretion of mucus are common symptoms in humans with allergic rhinitis (Varney et al., 1992; Watanabe and Kiuna, 1998
). Although allergic asthma has been well characterized in animal models, there are few animal models of allergic rhinitis that describe the alterations in epithelial and mucous cells (Shimizu et al., 2000
). Conversely, we have developed sensitive techniques to examine the inflammatory, histologic, and morphologic changes in rat nasal epithelium after acute and chronic exposures to ozone (Harkema et al., 1989
, 1997
).
Because ozone exacerbates responses to allergens in the lower airway and promotes mucous cell changes by itself in upper airways, we hypothesized that exposure to ozone will enhance nasal epithelial responses in a rat model of allergic rhinitis. In the present study we exposed sensitized rats to ozone and allergen, and used histochemical, image analysis, and morphometric techniques to characterize the development of inflammation and morphologic alterations in the nasal epithelium. Specifically, we examined two types of nasal epithelium that line specific sites in proximal nasal airways: the respiratory epithelium (RE), a pseudostratified, ciliated epithelium that lines the mid-nasal septum and contains several mucous (goblet) cells, and the nasal transitional epithelium (NTE), a nonciliated, cuboidal epithelium that lines the maxilloturbinates and contains few, if any, mucous cells (Harkema et al., 1991). In general, allergen elicits mucous cell responses in the RE (Varney et al., 1992
), and ozone causes mucous cell metaplasia in the NTE (Cho et al., 1999
, 2000
; Harkema et al., 1989
; Hotchkiss et al., 1997
). In this manner, we were able to compare the responses of distinct nasal tissues to these agents and determine the potential interactions of ozone-and allergen-engendered responses. Using morphometry and histologic descriptions of epithelial cell remodeling, we extended the findings of human studies that used nasal lavage to indicate ozone enhancement of allergic rhinitis. Moreover, our results provide the first evidence in animals or humans that ozone exposure enhances the nasal inflammatory and epithelial alterations caused by exposure to allergens.
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MATERIALS AND METHODS |
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Ozone exposure.
Rats were exposed to filtered air or 0.5 ppm ozone for either 1 or 3 days for 8 h/day. The ozone concentration used produces minimal lesions in the NTE of Brown Norway rats in acute studies, and has little or no effect on respiratory epithelium (Harkema et al., 1999). By comparison, ozone induces a pronounced MCM in the NTE of F344/N rats, but these rats are not highly responsive to allergen sensitization and intranasal challenge (Harkema et al., 1989
, 1997
; Hotchkiss et al., 1999
). Thus, the Brown Norway rat provides a good animal model of allergic rhinitis in which to test the effects of ozone exposure.
Ozone was generated with an OREC model 03VI-0 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 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 cage racks. The concentration of ozone during exposures was 0.536 ± 0.008 ppm (mean ± SEM) for ozone chambers and less than 0.02 ppm for chambers receiving filtered air.
Allergen exposure.
Immediately after each inhalation exposure, rats were removed from the chambers, anesthetized with 4% halothane in oxygen, and 50 µl of either pyrogen-free saline or an ovalbumin solution (1%, prepared in pyrogen-free saline) was instilled into each nasal airway passage of rats. Rats were exposed to ozone for 8 h to elicit inflammatory cell infiltration, which would be present at the time of intranasal ovalbumin instillation. Animals were returned to the inhalation chambers after IN challenges.
Necropsy and tissue preparation.
Twenty-four rats were killed at 24 h after a single inhalation and intranasal challenge, and twenty-four were killed 24 h after three daily inhalation and intranasal treatments. Two h before necropsies, rats were injected intraperitoneally with bromodeoxyuridine (BrdU; 50 mg/kg body weight) to label epithelial cells undergoing DNA synthesis (s-phase of cell cycle). Rats were killed via exsanguination by cutting the abdominal aorta, after being deeply anesthetized with sodium pentobarbital (50 mg/kg, ip). Immediately after death, the head of each animal was removed from the carcass. After the lower jaw and skin were removed, the head was placed in a large volume of zinc-formalin (Anatech, Kalamazoo, MI) for at least 48 h. After fixation, the head was decalcified in 13% formic acid for 4 days, and then rinsed in tap distilled water for 4 h. A tissue block was removed from the anterior nasal cavity by making two cuts perpendicular to the hard palate: (1) immediately posterior to the upper incisors, and (2) at the level of the incisive papilla (Fig. 1A). The tissue blocks were embedded in paraffin, and 5 to 6 µm-thick sections were cut from the anterior surface. Nasal sections were stained with hematoxylin and eosin (H&E) for routine histology, Alcian blue (pH 2.5)/periodic acid-Schiff AB/PAS) to detect intraepithelial mucosubstances, May-Grunwald stain and hematoxylin to detect eosinophils, or immunohistochemically to detect BrdU-labeled cells. Neutrophils were identified by morphologic characteristics that include their size, their darkly stained, multilobed nuclei, and their clear cytoplasm with dust-like granules. In contrast, eosinophils were slightly larger in size than neutrophils, had a bilobed nucleus, and contained numerous intracytoplasmic eosinophilic granules.
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Morphometry of stored intraepithelial mucosubstances.
To estimate the amount of the intraepithelial mucosubstances in NTE and RE, 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 on a Power Macintosh 7100/66 computer using the public domain NIH Image program (written by Wayne Rasband, U.S. National Institutes of Health and available on the Internet at http://rsb.infb.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 length of surface area of epithelial basal lamina was determined as described previously (Harkema et al., 1987). It was expressed as nl of intraepithelial mucosubstances per mm2 of basal lamina (i.e., volume density).
Morphometry of epithelial DNA synthesis and cell density.
The number of BrdU-labeled nuclei divided by the total epithelial nuclei x 100 (percentage of BrdU-labeled epithelial cells) was used as an estimate of DNA synthesis in the NTE and RE. 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 ozone and ovalbumin exposures on neutrophil and eosinophil influx was determined in May-Grunwald stained sections by counting the total number of each cell type within the mucosa of the septum (area between the airway and septal cartilage) or mucosa of the maxilloturbinate (area between the airway and the turbinate bone). Granulocytes with a bilobed nucleus and large bright pink staining granular cytoplasm were counted as eosinophils, whereas smaller polymorphonuclear cells with clear cytoplasm that excluded May-Grunwald stain were identified as neutrophils.
Statistical analysis.
Morphometric data are expressed as the mean ± the standard error of the mean (SEM) and were statistically 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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After three consecutive days of intranasal instillations of saline and 8-h inhalation exposures to ozone, the multiply exposed rats had a mild rhinitis with an associated regenerative hyperplasia of NTE and RE lining the lateral meatus in both the proximal and distal sections examined. The inflammatory cell influx consisted of eosinophils with lesser numbers of mononuclear cells (lymphocytes and plasma cells) and neutrophils. Though the intranasal distribution of the surface epithelial lesions was similar in the single and multiple exposures, the character of the epithelial lesions was markedly different.
The ozone-induced epithelial degeneration and necrosis observed after a single 8-h inhalation exposure was not a consistent feature in the nasal passages of the multiply exposed rats. Instead, the affected NTE and RE were distinctively hyperplastic, consisting of increased numbers of hypertrophic cells with basophilic cytoplasm and enlarged nuclei with prominent nucleoli (Fig. 3B). Attenuation and loss of cilia also was a characteristic feature of the regenerative RE lining the lateral wall in the distal nasal passage. RE lining the nasal septum was free of ozone-induced degeneration or necrosis, but there was a mild MCM in the proximal nasal passage.
Rats exposed to both ovalbumin and ozone.
Rodents exposed to both a single intranasal instillation of ovalbumin and a single 8-h inhalation exposure to ozone had both ovalbumin-and ozone-related nasal lesions. These rats had a marked rhinitis characterized by a mixed inflammatory cell influx of numerous eosinophils and neutrophils with lesser numbers of mononuclear cells (lymphocytes and plasma cells). Like the other rodents that were exposed to either ozone or ovalbumin, the nasal inflammation was restricted to the nasal mucosa lined by NTE or RE and not in areas lined by olfactory or squamous epithelium. The rhinitis appeared to be most severe in the mucosa lining the lateral meatus in both the proximal and distal sections. The inflammation was also conspicuous in septal mucosa lining the middle meatus.
Associated with the rhinitis were prominent alterations in the NTE and RE of the affected mucosa. The NTE on the lateral wall, maxilloturbinates, and lateral surfaces of the nasoturbinates were markedly hyperplastic, with focal areas of mild to moderate MCM and nonkeratinizing squamous metaplasia (SM; Fig. 3D). The latter lesion was usually present on the dorsolateral surfaces of the maxilloturbinates and the lateral ridge and scroll of the nasoturbinate. Both the MCM and the SM were metaplastic lesions of the NTE that were not present in rats exposed only to ovalbumin or ozone.
The principal lesion of the RE lining the mid-septum in both the proximal and distal nasal passage was a marked MCM (Fig. 4). The RE lining the lateral wall in the distal nasal passage was markedly hyperplastic with attenuation and loss of cilia. The epithelial alterations caused by ozone, ovalbumin, and the coexposure to ozone and ovalbumin, are summarized in Table 1
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DISCUSSION |
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Exposure to ozone or allergen elicits distinct yet overlapping responses in the nose. For example, both agents cause a marked infiltration of inflammatory cells, epithelial cell injury, mucus secretion, and increased storage of epithelial mucosubstances (Harkema et al., 1997; Hotchkiss et al., 1997
; Miadonna et al., 1999
; Varney et al., 1992
; Watanabe and Kiuna, 1998
). Agent-specific differences exist in the nature of nasal alterations and inflammatory responses. In general, nasal responses to allergens involve the RE, which consists of ciliated cells and preexisting secretory cells and is marked by recruitment and activation of eosinophils. Conversely, ozone elicits neutrophilic inflammation and mucous cell metaplasia in the epithelium of the lateral meatus, lined by NTE in the proximal nasal airways.
In the present study, ovalbumin challenge caused increases in DNA synthesis and stored mucosubstances in the RE lining the septum but not in the NTE of the maxilloturbinates. The increase in stored mucus in the RE was not due to increases in cell density. This is consistent with observations in nasal RE of humans with allergic rhinitis, where goblet cell density is unaltered after acute allergen challenge (Berger et al., 1997; Karlsson and Pipkorn, 1989
). Our data suggest that preexisting secretory cells in the septum produce and store more mucosubstances in response to allergen instillation.
We also found that the combination of ozone exposure and ovalbumin challenge resulted in increased stored mucosubstances in the septum that were significantly greater than those induced by ovalbumin challenge alone. Again, cell density was not altered by these treatments, and therefore preexisting secretory cells contain increased volumes of stored mucus. These results demonstrate that ozone can potentiate the ovalbumin-induced alterations in the existing mucous apparatus in regions of the nose (i.e., in the RE) where ozone alone normally has no effect.
We have recently described in detail how repeated daily exposures to ozone cause inflammation, hyperplasia, and mucous cell metaplasia in the NTE of maxilloturbinates in Fisher F344 rats (Cho et al., 1999, 2000
). Specifically, increased cell density is evident after one day of exposure and mucous cells appear by 24 days after repeated daily ozone exposures. We have also reported that 4 days of ozone exposure of Brown Norway rats result in hyperplasia but not in MCM in the NTE (Hotchkiss et al., 1999
). We extended these findings in the present study by evaluating inflammatory and epithelial changes after 1 day of ozone exposure, at a time when ozone-initiated, premetaplastic events occur (i.e., inflammation, epithelial cell density). In the present study we found that intranasal challenge with allergen enhanced ozone-induced hyperplastic responses (Fig. 7
), and that the combination of allergen and ozone caused MCM (Fig. 5
) in the NTE of Brown Norway rats. These results suggest that the allergen acts either to increase ozone-initiated pathways or to initiate distinct pathways that act synergistically with ozone. Similar hypotheses can be proposed for the effect of ozone to augment allergen responses in the RE of the septum. The results of the present study suggest that each agent enhances the site-specific epithelial responses of the other.
As a first step in elucidating the mechanism(s) for these responses, we evaluated inflammatory cell influx at times that precede (i.e., after a single instillation or exposure) and that are concurrent (i.e., after 3 such treatments) with epithelial cell changes. Ozone exposure alone had no effect on neutrophil recruitment into the septum, and in fact, partially blocked the accumulation of neutrophils elicited by allergen challenge (Fig. 9B). Thus, the ability of ozone to enhance production of mucus in preexisting secretory cells is not due to a numeric increase in infiltrated neutrophils.
Associations exist between the presence of eosinophils in airway mucosa and allergen-induced epithelial cell responses and hypersensitivity (Blyth et al., 1996; Durham et al., 1992
; Elwood et al., 1992
). We report here a relationship between eosinophil accumulation and increased mucosubstances in the RE of the septum. Although ozone exposure enhances the increase in intraepithelial mucosubstances in the septum, it has no effect on the eosinophil accumulation in these tissues. Taken together, if the presence of either eosinophils or neutrophils underlies the mechanism of ozone-induced enhancement of mucous cell responses in the RE, our data suggests their presence may be qualitative rather than quantitative. That is, ozone or a mediator of ozone might activate these inflammatory cells or alter their function such that they contribute to mucous cell responses.
Repeated coexposures to ozone and ovalbumin induced epithelial hyperplasia and mucous cell metaplasia in the NTE of maxilloturbinates where there are normally no mucus-containing cells (Figs. 5 and 7). The only inflammatory cell response after coexposure that differed from a single agent exposure was a synergistic increase in eosinophils after a single coexposure (Fig. 8A
). One exposure to ozone alone also caused increases in eosinophilic inflammation, but did not lead to significant increases in stored mucosubstances. Repeated ozone exposures did result in hyperplasia, albeit less than that produced by coexposure to ozone and allergen. It is possible that the magnitude of eosinophilic inflammation early after treatments dictates the rate and degree of epithelial cell changes in NTE (i.e., hyperplasia and metaplasia). In the present study, the rate of DNA synthesis after a single coexposure and the cell density achieved after multiple coexposures both correlate with the early eosinophilic response. Thus, the stronger eosinophilic response induced by ozone and allergen may portend a more robust hyperplastic and metaplastic response in the NTE.
Our work in Fisher F344 rats suggests that either neutrophil-derived products or cellcell interactions of neutrophils and epithelial cells mediate, in part, the ozone-induced MCM (Cho et al., 1999). In this regard, neutrophil products such as platelet activating factor (PAF), elastase, and reactive oxygen species have been implicated by others in the activation of mucin protein coding genes in airway epithelial cells (Lou et al., 1998
; Voynow et al., 1999
). MCM or the induction of mucin genes in airway cells can be mediated by factors such as interleukin-4 (IL-4), IL-9, IL-13, prostaglandin E2, tumor necrosis factor, 15-HETE, and members of the epidermal growth factor family (Borchers et al., 1999
; Dabbagh et al., 1999
; Takeyama et al., 1999
). It is notable that all of these factors can be secreted or produced by eosinophils. As such, the eosinophil may secrete mediators that are responsible in part for MCM in nasal epithelium.
Recent reports, however, suggest that eosinophils are not required for allergen-induced MCM in the lower airways of mice (Cohn et al., 1999; Haile et al., 1999
). Likewise, it is possible that eosinophils in the nasal airways of exposed rats in the present study are not necessary for the concurrent epithelial cell changes. Mast cells and T lymphocytes also produce mediators which promote MCM in airways (e.g., IL-4, IL-9) during allergic inflammation. Furthermore, ozone alone is capable of activating mast cells and T lymphocytes to release putative mediators of metaplasia (Chen et al., 1995
; Schierhorn et al., 1999
). Thus, redundant signals for epithelial cell differentiation are likely present after ozone and allergen exposures. Future studies in which eosinophils, neutrophils, or mast cells are depleted or their activation inhibited before ozone and allergen challenge are needed to understand the contribution of these inflammatory cells to the epithelial changes we have described here.
Previous descriptions of upper airway inflammation after ozone and allergen challenge have been limited to the analysis of nasal lavage. The present study provided a morphologic approach to understanding the temporal involvement of inflammatory cells with epithelial changes induced by coexposure of an oxidant pollutant and an inhaled allergen. Specifically, we have documented increased mucus storage, hyperplasia, MCM, and the relationship of these epithelial changes with eosinophilic and neutrophilic inflammation. Based on the changes in the nasal mucous apparatus in different nasal airway epithelium populations, our results suggest that each stimulus (i.e., ozone and allergen) enhances the effects of the other. As such, people with allergic rhinitis may be more sensitive to the health risks of ozone exposure than are normal, noncompromised individuals. Further studies are required to identify the cellular and molecular mechanisms responsible for the enhanced mucous cell responses after ozone and allergen coexposure.
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ACKNOWLEDGMENTS |
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NOTES |
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