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J Appl Physiol 90: 713-723, 2001;
8750-7587/01 $5.00
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Vol. 90, Issue 2, 713-723, February 2001

Airway responses to chronic ozone exposure are partially mediated through mast cells

Steven R. Kleeberger, Yoshinori Ohtsuka, Liu-Yi Zhang, and Malinda Longphre

Department of Environmental Health Sciences, Division of Physiology, The Johns Hopkins University, Baltimore, Maryland 21205


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Airways inflammation and epithelial injury induced by chronic ozone (O3) in genetically mast cell-deficient mice (KitW/KitW-v) were compared with those in mast cell-sufficient mice (+/+) and KitW/KitW-v mice repleted of mast cells (KitW/KitW-v-BMT). Mice were exposed to 0.26 ppm O3 8 h/day, 5 days/wk, for 1-90 days. Background was 0.06 ppm O3. Age-matched mice were exposed to filtered air for O3 controls. Reversibility of lesions was evaluated 35 days after exposure. Compared with KitW/KitW-v, O3 caused greater increases in lavageable macrophages, epithelial cells, and polymorphonuclear leukocytes in +/+ and KitW/KitW-v-BMT mice. O3 also caused lung hyperpermeability, but the genotypic groups were not different. Cells and permeability returned to air control levels after O3. O3 induced lung cell proliferation only in +/+ and KitW/KitW-v-BMT mice; proliferation remained elevated or increased in +/+ and KitW/KitW-v-BMT mice after O3. Greater O3-induced cell proliferation was found in nasal epithelium of +/+ and KitW/KitW-v-BMT mice compared with KitW/KitW-v mice. Results are consistent with the hypothesis that mast cells affect airway responses induced by chronic O3 exposure.

inflammation; proliferation; mast cell-deficient mouse; stem cell factor; air pollution


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE OXIDANT POTENTIAL OF OZONE (O3) is well recognized, and this highly reactive pollutant is still an important public health concern in urban environments of the United States and other industrialized countries. In healthy human subjects, acute (2-6 h) exposure to 0.08-0.75 ppm O3 causes decrements in pulmonary function (2, 45), elicits the release of bronchoactive chemical mediators (7, 26), and induces airway inflammation (1, 7, 26). Epidemiological studies also suggest that short-term O3 exposure increases bronchial reactivity, reduces lung function, and, in general, is a risk factor for childhood and adult respiratory health (21, 38, 46). Although these studies have provided insight into the potential mechanisms of acute O3-induced lung injury, it remains unclear whether acute responses are predictive of airway effects of chronic O3 exposures.

Investigations have examined the effects of simulated urban chronic intermittent O3 exposures on airways of animal models (6, 16, 27, 35). These studies have indicated that the most susceptible cell type of the upper (nasal) airways is the cuboidal or transitional epithelium, whereas injury of the lower airways occurs in the conducting and respiratory epithelium and in alveolar epithelium. The effects of O3 on nasal airways almost certainly compromise upper respiratory defense mechanisms and may also compromise "scrubbing" efficiency of these airways (13), thereby influencing the susceptibility of lung tissues. Although these studies provide useful insight into the time course and reversibility of chronic O3 exposure, there remain questions regarding the mechanisms that initiate and propagate pathogenesis of airway injury induced by chronic O3 exposure.

Although a number of cells of the respiratory tract, including macrophages, epithelial cells, neutrophils, and neurons, are likely involved in responding to environmental stress, there is increasing evidence that mast cells may be key to the stress response. Mast cells are found in large numbers in mammalian lungs, closely associated with pulmonary endothelium and nerve termini such as C fibers (4). When stimulated, mast cells may contribute significantly to bronchoconstriction, airway hyperreactivity, and inflammation. Proinflammatory mast cells have been implicated in a large number of disease processes, including sarcoidosis, chronic fibrosing alveolitis, asthma, and allergic rhinitis (for review, see Ref. 20). These cells may be stimulated to release numerous preformed, or de novo synthesized, mediators, e.g., tumor necrosis factor-alpha , that are critical to the sequence of events that leads to inflammatory cell recruitment and other events associated with tissue injury (9, 14). Mast cells also contribute to normal tissue repair and have been associated with the early overproduction of basement membrane components observed in a variety of fibrotic conditions (37, 44).

A number of studies suggest a role for mast cells in environmentally induced pulmonary disease, including observations that mast cell degranulation (22, 33, 41) and migration occur in the lungs of some animals after O3 exposure. In healthy human subjects, acute O3 exposures elicit the release of mast cell-specific tryptase into lavageable spaces of the upper and lower airways (42). Although each of these studies implicates the mast cell in acute responses to O3, the potential relationship between these processes and subsequent physiological responses or inflammatory events or chronic effects of O3 exposure have not been firmly established. The overall objective of this project was to test the hypothesis that mast cells contribute significantly to the initiation and propagation of inflammation and epithelial injury induced by chronic O3 exposure.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General

Male (6-8 wk, 20-25 g) mast cell-deficient (WBB6F1-KitW/KitW-v) and mast cell-sufficient (WBB6F1-+/+) congenic mice were purchased from Jackson Laboratories (Bar Harbor, ME). Hereafter, WBB6F1-KitW/KitW-v and WBB6F1-+/+ mice have been designated KitW/KitW-v and +/+, respectively. Mice were housed in microisolation cages in an antigen- and virus-free room. Water and mouse chow (Agway Pro-Lab RMH 1000) were provided ad libitum. Sentinel animals were examined periodically (titers and necropsy) to ensure that the animals remained free of infection. Animals were handled in accordance with the standards established by the U.S. Animal Welfare Acts set forth in National Institutes of Health guidelines and the Johns Hopkins Animal Care and Use Committee.

Characteristics of the Mast Cell-Deficient Mouse Model

Compared with normal congenic +/+ mice, the mast cell-deficient KitW/KitW-v (formerly designated WBB6F1-W/Wv) mouse is sterile and anemic, lacks melanocytes in the skin, and has <1% of normal densities of peripheral tissue mast cells (and none in the respiratory tract). However, the KitW/KitW-v mouse has normal levels of basophils and other granulocytes, T cells, and B cells (11, 30). The mast cell deficiency and associated other abnormalities are due to a mutation at the W locus on chromosome 5 that results in a defect in the lineage of stem cell factor receptor that is expressed on the surface of hematopoietic progenitor cells, mast cells, melanocytes, and germ cells. The mast cell deficiency in KitW/KitW-v mice can be repaired by adoptive transfer of bone marrow cells from +/+ donors, as described elsewhere (11, 25). This model system has been used to evaluate the role of mast cells in a number of immunologic and physiological processes, including immediate hypersensitivity reactions, host defense, bleomycin-induced pulmonary fibrosis, sepsis, and silica-induced inflammation (8, 9, 43).

Bone Marrow Transplantation

Bone marrow transplantation was performed to replete KitW/KitW-v mice with mast cells as described previously (25, 29). Briefly, +/+ mice were killed by cervical dislocation, and femurs were isolated. Bone marrow was obtained by flushing sterile saline through the bones into a sterile glass tube, and the cells were suspended in saline. Marrow cell suspension (1.5 ml; 2.5 × 107 cells) was injected intraperitoneally into each KitW/KitW-v animal. Age-matched +/+ and KitW/KitW-v animals were sham transplanted intraperitoneally with sterile saline (vehicle). Animals were housed for an additional 16 wk before experimentation because full mast cell reconstitution in the airways has been shown to require at least 15 wk (25).

To determine the success of mast cell repletion, the trachea and lung were removed en bloc from separate groups of 25-wk-old +/+, KitW/KitW-v, and KitW/KitW-v-BMT mice and were fixed in Carnoy's fixative (60:30:10, ethyl alcohol-chloroform-glacial acetic acid) at a constant pressure of 25 cmH2O for 2 h. The tissues were embedded in paraffin, sectioned (5 µm), and stained with toluidine blue. Sections were examined at ×400 for mast cells within or in juxtaposition to the tracheobronchiolar epithelium (one cell layer beneath). Airway lumen diameter of the trachea and main stem bronchi were ~1.2 and 1.0 mm, respectively. These tissues were chosen for verification of mast cell repletion because previous studies have indicated that these tissues are normally relatively densely populated with mast cells and have been good indicators of mast cell repletion throughout the lung (25, 29). Sections were imaged on a digitizing tablet by camera lucida. The area was calculated from the contour tracing using Sigma Scan (Jandel Scientific, Corte Madera, CA). Longitudinal sections along the entire trachea or bronchus were examined for mast cells. The data for each group were expressed as the mean ± SE number of mast cells per square millimeter. The trachea and main stem bronchi were easily identified in the sagittal plane and were therefore the focus of study. To determine the effect of bone marrow transplantation on blood cell counts, hemoglobin, and hematocrit, blood samples were taken from the three groups of mice by retroorbital bleeding. Samples were analyzed with a Cell-dyne 3500 Hematology Analyzer (Abbott Diagnostics, Abbott Park, IL).

O3 Exposures

Mice were placed individually in stainless steel wire cages with free access to food and water during the exposures. Cages were set inside 700-liter laminar flow inhalation chambers that were equipped with a charcoal- and high-efficiency particle acceptor-filtered air supply. Chamber air was renewed at ~20 changes/h with 50-65% relative humidity and an ambient temperature of 20-25°C. O3 was generated by directing dried, filtered air through an ultraviolet light O3 generator (Orec, Phoenix, AZ) located upstream of the exposure chamber. The O3-air mixture was metered into the inlet air stream with computer-operated stainless steel mass flow controllers. Simultaneous exposures to filtered air were done in a separate chamber for age- and treatment-matched groups to serve as O3 controls. Intermittent O3 exposures were performed automatically by using a control program and microcomputer that were interfaced with the O3-generating system. O3 concentrations were monitored regularly at different levels within the chamber with a Dasibi 1003-AH (Dasibi Environmental, Glendale, CA) and recorded on a strip-chart recorder. The Dasibi 1003-AH was calibrated regularly against a standard source Dasibi 1008-PC. Standard 8-h exposures to 0.26 ppm O3 (peak of profile pattern) were done Monday through Friday between 0830 and 1630; mice were exposed to 0.06 ppm O3 for the remaining 16 h of each day. Weekend exposures were continuous 0.06 ppm O3. Daily chamber cleaning and food and water replacement took 30 min.

Tissue Preparation and Histological Analyses

Lungs. The lungs were excised and fixed by perfusion through the trachea with Carnoy's fixative at a constant pressure of 25 cmH2O. After 2 h of perfusion and fixation, the lungs were immersed in a large volume of the same fixative. The right caudal lobes were then removed and cut sagittally into three pieces. Tissues were embedded in paraffin, sectioned (5 µm), and stained with hematoxylin and eosin.

Noses. The head of each mouse was removed from the carcass, depelted, and fixed in Carnoy's fixative. After fixation, the heads were decalcified in 13% formic acid for 2 days. A tissue block (2-3 mm thick) from the anterior nasal cavity of each mouse was excised by making two transverse cuts, perpendicular to the hard palate, at specific gross anatomic locations previously described for the rat (47): 1) immediately posterior to the upper incisor teeth and 2) at the incisor papillae of the nasal palate. Tissue blocks were embedded in paraffin. Sections (5 µm) from the anterior surface of the nasal tissue block for each mouse were cut and stained with hematoxylin and eosin.

Serial sections from both lung and nasal tissues were immunohistochemically stained for bromodeoxyuridine (BrdU) as previously described (19) and counterstained with hematoxylin and lithium carbonate. Sections were imaged on a digitizing tablet by camera lucida. Using ×400 magnification, all BrdU-labeled and nonlabeled epithelial cells were counted throughout each section. The linear length of the basal lamina was calculated from the contour tracing by using Sigma Scan. The data for each group were expressed as the mean number of BrdU-labeled nuclei divided by total cells ± SE. BrdU labeling, when expressed as the number of BrdU-labeled nuclei per millimeter of basal lamina (data not shown), was also measured, and the findings led to similar conclusions. In the nasal cavity, the transitional epithelium lining the medial and lateral surfaces of the maxilloturbinates and the lateral wall was the focus of study because it has previously been shown to have the most significant epithelial damage with O3 exposure (18). Likewise, terminal bronchioles were the focus of study in the lungs because O3 exposure causes histologically evident epithelial lesions in these areas (18). The airway diameters of the terminal bronchioles averaged ~100 µm. The bronchiolar airway is bounded by a continuous wall that is lined by a cuboidal epithelium. Only terminal bronchioles that were continuous with the proximal alveolar ducts in the section were used to determine the labeling index. The epithelium lining the luminal surface of the bronchioles was identified on the basis of the presence of a basal lamina supporting cuboidal cells. No distinction was made between nonciliated and type 2 pneumocytes, and luminal macrophages and interstitial fibroblasts were not included in the BrdU counts. At least 10 profiles/lung section and at least 3 sections per mouse were counted. The sample size for these studies was five per genotypic group per exposure per sample date.

BAL, Total Protein, and Cell Preparation

Mice were killed by cervical dislocation, and the lungs were lavaged in situ four times with Hanks' balanced salt solution (HBSS; 35 ml/kg; pH 7.2-7.4). Recovered bronchoalveolar lavage (BAL) fluid was immediately cooled at 4°C. The HBSS contains (g/l): 8.0 NaCl, 0.4 KCl, 0.06 KH2PO4, 0.05 NaHPO4, 0.35 NaHCO3, 1.0 dextrose. For each mouse, the BAL returns were centrifuged (500 g at 4°C), and the supernatant from the first lavage return was decanted. The total protein concentration in the supernatant was measured and used as an indicator of lung permeability. A BSA protein assay kit (Pierce, Rockford, IL) was used to measure BAL protein. The assay follows the method of Bradford (5) and is accurate from 10 to 2,000 µg/ml.

The cell pellets from all four lavages were combined and resuspended in 1 ml HBSS. Cells were counted with a hemocytometer, and results were expressed as the number of cells recovered in the total BAL return. Aliquots (10 µl) were cytocentrifuged (Shandon Southern Instruments, Pittsburgh, PA), and the cells were stained with Diff-Quik (AHS del Caribe) for differential cell analysis. Differential cell counts were done by identifying 300 cells according to standard cytological techniques. Epithelial cells in particular were identified by the presence of cilia. It is therefore likely that epithelial cell loss after exposure is underestimated because nonciliated cells were not counted. Sample sizes for these studies ranged from four to six per genotypic group per exposure per sample date.

Experimental Protocol

Bone marrow cells were transplanted from +/+ donor mice to KitW/KitW-v recipients as described above. Sham transplantation was done in age- and gender-matched +/+ and KitW/KitW-v mice. When peripheral tissues of KitW/KitW-v BMT mice were reconstituted with mast cells (16 wk after transplantation), mice from all three age-matched genotypic groups were exposed intermittently to O3 or filtered air for 1, 3, 14, 30, and 90 days (i.e., animals entered the exposure chamber at the age of 22-24 wk). Immediately after O3 or air exposure, lung inflammatory cell infiltration, epithelial sloughing, and lung hyperpermeability were evaluated. Epithelial proliferation in the nose and lung were also quantitated, except on day 1 when 8 h was insufficient time for epithelial proliferation to be measured by BrdU technique. To determine whether the effects of chronic O3 exposure were reversible, sets of mice were exposed for 90 days, and nasal and lung responses were assessed 35 days postexposure. The high frequency of sampling early in the exposure regimen reflected the expectation that the peak of the inflammatory response would occur over the first 3 days.

Statistical Analyses

The effects of exposure (O3, air), strain or genotype (+/+, KitW/KitW-v, KitW/KitW-v-BMT), and time course (1, 3, 14, 30, and 90 days exposure, and 90 days exposure plus 35 days recovery) on dependent variables were assessed by three-way ANOVA. The dependent variables were indicators of inflammatory response (lavageable cells and total protein) and epithelial injury (epithelial proliferation in lung and nasal airways). All ANOVA were done by using SuperANOVA software package (Abacus Concepts, Berkeley, CA). Group sizes (repeated measures) were 4-6 animals/group. The effects of bone marrow cell transplantation on tissue mast cell densities were assessed by comparing transplant animals with age-matched controls by one-factor ANOVA. Data sets were tested for homoscedasticity as required for parametric analyses, and data that did not meet this requirement (that is, heteroscedastic) were transformed (ln or arcsine). Fisher's protected least significant difference analyses were used for a posteriori comparisons of means. Statistical significance was accepted at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the experiments reported below, the O3 concentration in the exposure chambers did not vary (+/-) from the target concentration by more than 10% during the peak and background exposures, and the standard deviations of the O3 concentrations were <2% of the means. At entry and exit from the exposure chambers, there were no differences in body weights between genotypic groups. There were no deaths recorded during the exposures.

BAL Cell Responses to Filtered Air and O3

Macrophages. For illustration purposes and because there was no statistically significant effect of time, air controls for each genotypic group were pooled, and the means ± SE are shown in Fig. 1 (ANOVA was done without pooling). The mean number of macrophages recovered from O3-exposed mice of all genotypes was greater than those from air-exposed animals (ANOVA, P < 0.05). The mean numbers of macrophages from +/+ and KitW/KitW-v-BMT mice were not different from each other but were significantly (P < 0.05) greater than those from KitW/KitW-v mice (Fig. 1). Exposure time also significantly affected the macrophage response, because there were increases (P < 0.05) in BAL macrophages up to 14 days (Fig. 1). Macrophages remained elevated over air controls in +/+ and KitW/KitW-v-BMT mice through 90 days. However, in mast cell-deficient KitW/KitW-v mice, mean macrophage numbers increased relative to air controls only after 14 and 30 days of O3. After 90 days, macrophage numbers were not statistically different from air controls (Fig. 1). After 35 days recovery from O3 and air, the mean numbers of macrophages in all three groups of mice returned toward air controls. Among O3-exposed animals, there was a slightly but significantly greater number of macrophages in +/+ and KitW/KitW-v-BMT animals compared with KitW/KitW-v mice (Fig. 1).


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  		Fig. 1.   Mean numbers of macrophages recovered by bronchoalveolar lavage (BAL) from +/+ (mast cell-sufficient), KitW/KitW-v (mast cell-deficient), and KitW/KitW-v-BMT (KitW/KitW-v mice repleted of mast cells) mice after exposure to filtered air or ozone (O3) for up to 90 days and after 35 days recovery. Values are means ± SE. Means from air-exposed animals (controls) were not significantly different from each other at any time point and were thus pooled. Sample sizes = 4-6 animals/group. Statistical comparison of air vs. O3: *P < 0.05; comparison of +/+ or KitW/KitW-v-BMT with KitW/KitW-v: +P < 0.05.

PMNs. The numbers of recovered polymorphonuclear leukocytes (PMNs) were ln transformed to correct heterogeneity (heteroscedasticity) of the group variances. Significant exposure and genotype effects on the mean number of BAL PMNs were detected in the three groups of animals (ANOVA, P < 0.05). Relative to air controls, O3 caused significant infiltration of PMNs into the airways of +/+ and KitW/KitW-v-BMT mice after 3, 14, and 30 days (Fig. 2). The peak of the response in both genotypes occurred after 3 days. No significant effect of O3 on PMN infiltration in KitW/KitW-v mice was detected (Fig. 2). After 90 days of O3 and 35 days of recovery, mean numbers of PMNs in all three groups were not different from respective air controls (Fig. 2). However, among O3-exposed mice, the mean numbers of PMNs in +/+ and KitW/KitW-v-BMT mice were greater than those in KitW/KitW-v mice.


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  		Fig. 2.   Mean numbers of polymorphonuclear leukocytes (PMNs) recovered by BAL from +/+, KitW/KitW-v, and KitW/KitW-v-BMT mice after exposure to filtered air or O3 for up to 90 days and after 35 days recovery. Statistical comparison of air vs. O3: *P < 0.05; comparison of +/+ or KitW/KitW-v-BMT with KitW/KitW-v: +P < 0.05.

The mean numbers of PMNs recovered from +/+ and KitW/KitW-v-BMT mice were not significantly different from each other, and both were significantly greater than the mean number of PMNs from KitW/KitW-v mice (P < 0.05).

Epithelial cells. Genotype, exposure, and time significantly affected the mean numbers of epithelial cells recovered by BAL (ANOVA, P < 0.05). Relative to air controls, O3 significantly increased the mean number of BAL epithelial cells (P < 0.05) (Fig. 3). The number of epithelial cells recovered from +/+ and KitW/KitW-v-BMT mice were not significantly different from each other, and both were significantly greater than the number of epithelial cells from KitW/KitW-v mice (P < 0.05). Within each of the three genotypic groups, the greatest number of epithelial cells was recovered after 3 days exposure (Fig. 3). Numbers declined slightly after 14 days and remained significantly elevated relative to respective air controls up to 90 days in +/+ and KitW/ KitW-v-BMT mice. In KitW/KitW-v mice, epithelial cell numbers returned to air control levels after 30 days and remained at these levels throughout the remainder of the exposure. In all genotypic groups, mean epithelial cell numbers were not different from respective air controls after 35 days recovery (Fig. 3).


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  		Fig. 3.   Mean numbers of epithelial cells recovered by BAL from +/+, KitW/KitW-v, and KitW/KitW-v-BMT mice after exposure to filtered air or O3 for up to 90 days and after 35 days recovery. Statistical comparison of air vs. O3: *P < 0.05; comparison of +/+ or KitW/KitW-v-BMT with KitW/KitW-v: +P < 0.05.

Total BAL protein. Statistically significant exposure and time effects on the total BAL protein concentration were detected (ANOVA, P < 0.05). Compared with air-exposed animals, O3 elicited significant increases in BAL protein that changed over time (Fig. 4). The maximum increases in protein occurred after 3 days exposure and returned toward air control levels thereafter. Mean BAL protein concentrations in O3- and air-exposed animals were not significantly different between groups after 90 days exposure or after 35 days recovery. In contrast to the cellular responses to O3, genotype had no effect on BAL protein (P > 0.05).


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  		Fig. 4.   Mean concentration of total protein in bronchoalveolar lavage from +/+, KitW/KitW-v, and KitW/KitW-v-BMT mice after exposure to filtered air or O3 for up to 90 days and after 35 days recovery. Sample sizes = 4-6 animals/group. Statistical comparison of air vs. O3: *P < 0.05; comparison of +/+ or KitW/KitW-v-BMT with KitW/KitW-v: +P < 0.05.

Epithelial Cell Proliferation in Response to Filtered Air and O3

Lung. Exposure, genotype, and time significantly (P < 0.05) affected the mean numbers of BrdU-labeled cells per millimeter basal lamina. In contrast with BAL cells and protein reported above, exposure time significantly affected the mean number of labeled cells in air-exposed mice (P < 0.05). The peak of the air effect occurred after 90 days exposure (Fig. 5). Genotype-specific effects of air exposure on BrdU-labeled cells were not detected. O3 caused significantly greater numbers of BrdU-labeled cells compared with mice exposed to filtered air (P < 0.05). Furthermore, the mean numbers of BrdU-labeled cells were significantly greater in +/+ and KitW/KitW-v-BMT mice compared with KitW/KitW-v, and there were no statistically significant differences between labeled cells in +/+ and KitW/KitW-v-BMT mice (genotype effect, P < 0.05).


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  		Fig. 5.   Mean numbers of bromodeoxyuridine (BrdU)-labeled cells/mm basal lamina in lung tissue of +/+ (A), KitW/KitW-v (B), and KitW/KitW-v-BMT (C) mice after exposure to filtered air or O3 for up to 90 days and after 35 days recovery. Values are means ± SE. Sample sizes = 4-6 animals/group. Statistical comparison of air vs. O3: *P < 0.05.

Relative to respective air controls, O3 caused significantly greater increases in BrdU incorporation in +/+ and KitW/KitW-v-BMT mice at all time points (Fig. 5). There was an O3 effect in KitW/KitW-v mice only after 14 days exposure (Fig. 5). Interestingly, compared with the respective 90-day exposure groups, the mean numbers of BrdU-labeled cells remained significantly elevated (+/+) or increased (KitW/KitW-v-BMT) 35 days after O3 (Fig. 5). BrdU incorporation in O3-exposed KitW/KitW-v mice was not significantly different from that in air controls after 35 days recovery. Occasional mild accumulations of alveolar macrophages were found in the airway lumens of some centriacinar regions of the O3-exposed +/+ and KitW/KitW-v-BMT mice after recovery, but no obvious tissue necrosis was detected by light microscopy. No inflammatory cell accumulation or epithelial lesions were evident in tissue distal to the centriacini.

Nose. Compared with the proliferative responses observed in the lung, the responses to O3 in the nose were slight, although statistically significant. Exposure and time effects on the mean number of BrdU-labeled cells were detected (ANOVA; P < 0.05), but the effect of genotype was not statistically significant. There were no obvious inflammatory lesions or epithelial damage in the nasal airways of any of the genotypic groups at any time point.

The mean number of labeled epithelial cells in the nasal airways increased significantly in the air-exposed animals of each genotype up to 90 days exposure and then declined after 35 days recovery (P < 0.05; Table 1). No statistically significant differences between genotypes at any time point after air exposure were detected. Relative to respective air controls, a significant increase in the number of labeled cells was found in +/+ and KitW/KitW-v-BMT, but not KitW/KitW-v, mice after 35 days recovery from O3 (Table 1).

                              
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  		Table 1.   Time course of changes in the mean number of BrdU-labeled cells/mm basal lamina in nasal tissue of +/+, KitW/KitW-v, and KitW/KitW-v-BMT mice after chronic exposure to filtered air or 0.26 ppm O3

Peripheral Tissue Mast Cell Densities and Blood Parameters

A statistically significant effect of genotype on the mean number of mast cells was found in trachea and main stem bronchi of the three groups of mice (ANOVA; P < 0.05). In both tissue types, the density of mast cells was significantly greater in +/+ mice compared with age-matched KitW/KitW-v mice, in which no mast cells were found (P < 0.05) (Table 2). Bone marrow transplant restored mast cell densities in main stem bronchi, and there were no significant differences between +/+ and KitW/KitW-v-BMT mice (Table 2). Mast cell densities were also significantly increased in tracheae of KitW/KitW-v-BMT mice compared those with KitW/KitW-v animals, although the densities were still less than those in the +/+ group (Table 2). Our laboratory and others have shown that the mast cell-deficient mouse is anemic compared with wild-type control mice, and the anemia is reversed by bone-marrow transplantation (see, e.g., Ref. 30). In the present study, red blood cell (RBC) counts (6.9 ± 0.2 × 106/ml), hematocrit (36.9 ± 0.6%), and hemoglobin (12.0 ± 0.1 meq/l) were significantly lower in KitW/KitW-v mice compared with +/+ mice (9.8 ± 0.6 × 106/ml; 47.8 ± 0.2%; 14.2 ± 0.2 meq/l). Bone marrow transplantation reversed the deficiency (10.8 ± 0.4 × 106/ml; 46.3 ± 0.9%; 14.1 ± 0.2 meq/l). Platelet counts were also significantly different between KitW/KitW-v and KitW/KitW-v-BMT mice, but bone marrow transplantation did not reverse the deficiency (data not shown). The changes in O3-induced cellular responses correlated with RBCs, hematocrit, and hemoglobin. However, we do not believe that the changes in RBCs contributed to the differences seen between groups because we are unaware of a demonstrated role of RBCs in modulating oxidant-induced inflammation and epithelial cell injury in the lungs. Bone marrow transplantation also corrected a deficiency in total white blood cells (+/+: 9.9 ± 1.0 × 106/ml; KitW/KitW-v: 6.6 ± 0.9 × 106/ml; KitW/KitW-v-BMT: 9.0 ± 0.1 × 106/ml). Although the numbers of white blood cells were lower in KitW/KitW-v mice, they were within the range of numbers normally found in inbred strains of mice (39). Furthermore, others have demonstrated that leukocyte infiltration in non-mast cell-mediated inflammation models is the same in KitW/KitW-v mice and their +/+ littermates (12). It is therefore unlikely that differences in numbers of white blood cells between the two genotypic groups account for their differential responses to O3 exposure.

                              
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  		Table 2.   Mast cells in tracheal and mainstem bronchiolar tissues for 25-wk-old +/+, sham-transplanted mast cell-deficient KitW/KitW-v, and KitW/KitW-v-BMT mice


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The overall objective of this study was to determine the role of proinflammatory mast cells in orchestrating the pathological responses to ozone exposure. Table 3 was generated to summarize the major findings and assist the reader in the interpretation of the results.

                              
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  		Table 3.   Summary of effects of chronic exposure to O3 on BrdU uptake (epithelial proliferation) and lavageable cells and protein in +/+, KitW/KitW-v, and KitW/KitW-v-BMT mice

A profile pattern of O3 exposure that peaked at 0.26 ppm O3 and declined to 0.06 ppm background was utilized in this study. It was designed to mimic the pattern of O3 production that may occur in heavily populated urban environments where O3 concentrations gradually increase during morning hours and peak concentrations may exceed 0.26 ppm before decreasing later in the afternoon (28). It is important to note that the actual delivered dose of O3 to the lung of rodents may be considerably less than may be expected by concentration × time considerations in human subjects, because rodents are obligate nose breathers. Accordingly, the nasal airways are very efficient at removing toxicants from inhaled air. Support for the enhanced scrubbing efficiency of upper airways of rodents compared with humans comes from Hatch et al. (17). Using 18O-labeled O3, these investigators found that the amount of O3 that reaches bronchoalveolar regions of resting rats exposed to 2.0 ppm O3 for 2 h was less than the amount of 18O incorporated in homologous regions of human subjects exposed to 0.4 ppm O3 for 2 h with exercise. Therefore, the dose of O3 delivered to the lungs of rodents with the protocol used in the present study is considerably less than what would occur in human subjects with similar exposure.

Responses to Chronic O3 Exposure in +/+ Mice

Inflammation. The kinetics of the inflammatory cell responses to chronic O3 exposure in +/+ mice suggested that, although adaptation to the exposure occurred for some cells, macrophages and PMNs remained elevated, and epithelial sloughing continued to occur. After 35 days recovery from exposure, all inflammatory response parameters returned to levels that were not different from air controls.

The mechanisms of adaptation are not certain, but a number of possibilities exist. For example, the upper airways may have become more efficient scrubbers of the inhaled O3. Although we detected no obvious characteristics of O3-exposed upper airways that would enhance scrubbing (e.g., increased mucus production or epithelial surface area), it is possible that antioxidant capacity of the epithelial cells lining the upper airways may have been upregulated. Acute and subacute exposures of rats to O3 have caused increased activities of whole lung antioxidant enzymes such as glucose-6-phosphate dehydrogenase, glutathione reductase, and glutathione peroxidase (see, e.g., Ref. 32). It is possible that similar enhanced production of these and other antioxidant enzymes may have contributed to the selective adaptation observed in the present study.

Cell proliferation. The proliferative response of the centriacinar epithelium to chronic O3 exposure in +/+ mice tracked the epithelial sloughing detected by BAL. Interestingly, DNA synthesis remained elevated after 35 days recovery in both nasal and lung tissues. That is, even in the absence of O3 and the subsequent decline in inflammatory response parameters (macrophages, PMNs, total protein), upregulation of epithelial cell proliferation was maintained compared with air-exposed controls. These results may imply that the signal(s) for epithelial proliferation are distinct from those that signal the infiltration of inflammatory cells, although the mast cell likely mediates both events (see below). It is important to note that, although a concerted effort was made to identify only epithelial cells in this study, it is possible that some basal cells were also counted. Although we believe it is unlikely that cell types other than epithelial cells contributed significantly to the reported proliferation indexes, it is possible that some were included.

Role of Mast Cells in Mediating Airway Responses to Chronic O3 Exposure

Inflammation. The role of mast cells in mediating inflammatory responses to chronic O3 was determined by comparing inflammation and proliferation in mast cell-deficient KitW/KitW-v mice with those in age-matched +/+ animals. Although O3 caused an increase in the mean number of lavageable macrophages (14 and 30 days) and epithelial cells (3-30 days), but not PMNs, in KitW/KitW-v animals, the magnitudes of response were significantly less than those of the congenic +/+ animals. Furthermore, there was apparent adaptation in these animals after 90 days exposure that was not observed in the +/+ mice. These results, therefore, suggest that mast cells contribute significantly to the pathogenesis of cellular responses to O3 in this model. To further test this hypothesis, we determined whether the inflammatory responses to O3 in KitW/KitW-v mice could be reversed with reconstitution of peripheral tissue mast cells. The kinetics of response in the reconstituted animals closely resembled +/+ mice, given that there were no statistically significant differences in lavageable cells and protein recovered in the two groups of animals after O3 exposure. However, the magnitude of change in macrophages, PMNs, and epithelial cells was significantly greater in KitW/KitW-v-BMT mice than in those of KitW/KitW-v animals. There were no differences in O3-induced lung hyperpermeability between the two groups. These results, therefore, provide further support for the hypothesis that mast cells mediate the infiltration of macrophages and PMNs as well as epithelial cell sloughing responses to O3 exposure in the lung. The effect of O3 on lung permeability appears to be regulated independent of mast cell activity. It should be noted that the experimental design does not rule out the contribution of other bone marrow-dependent factors, but it is unlikely that these factors contributed significantly to O3 response differences between genotypic groups (see RESULTS).

These observations are largely consistent with previous studies that demonstrated a role for mast cells in lung cellular responses to short-term (3-4 h) exposure to 1-3 ppm O3 (25, 29, 33). In the acute O3 exposure models, short-term reversible inflammation and epithelial cell loss, as well as permeability induced by high concentrations of O3 were significantly less in the lungs and nasal passages of mast cell-deficient mice compared with mast cell-sufficient animals. The present study has demonstrated that, in addition to the role in modulating O3-induced acute inflammation, the mast cell is also important in propagating and maintaining the inflammatory cell infiltration and epithelial disruption induced by chronic O3 exposure. The lack of a detectable effect of mast cells on the permeability response to chronic O3 may be a consequence of the nature of the two exposure protocols. It is speculated that, although the acute exposure to high O3 concentrations leads to degranulation and secretion by the mast cells, the chronic exposure to lower O3 concentrations may lead to "selective" stimulation of the mast cells and induce qualitatively and quantitatively different secretory profiles.

Compared with acute and subacute O3 exposure models, the magnitude of inflammation and epithelial cell loss induced by chronic exposure in this model was somewhat smaller. The numbers of PMNs recovered 6 h after acute (3 h) exposure to 2 ppm O3 were 11.6 ± 1.7 × 103/ml in +/+ mice and 3.0 ± 1.2 × 103/ml in KitW/KitW-v animals (25), which were ~3.1- and 2.0-fold greater, respectively, than the maximum numbers of cells recovered in the present study. In a subacute O3 exposure study (23), the numbers of PMNs and epithelial cells recovered from C57BL/6J mice (with O3 susceptibility similar to +/+ mice) after 48-72 h continuous exposure to 0.3 ppm O3 were 2.7- and 1.5-fold greater than those recovered in the present study. The disparity in magnitudes of inflammation and epithelial cell loss between the models may be due to a number of factors, including the obvious differences in the exposure regimen. Another important factor is the age of the mice. Because 16 wk were required to restore mast cells to peripheral tissues of KitW/KitW-v mice after bone marrow transplantation, the mice were ~24 wk of age before exposures were initiated. In a previous study with this model, our laboratory demonstrated that the inflammatory responses induced in 6-8 wk +/+ and KitW/KitW-v mice were significantly greater than those found in 22- to 24-wk-old animals (25). Interestingly, the numbers of lavageable PMNs recovered after acute O3 exposure of 22- to 24-wk-old animals were similar to those in the present study. The importance of age as a susceptibility factor in pulmonary responses to O3 has been demonstrated previously in other species, including humans (31) and rats (15).

Because O3 does not penetrate epithelial membranes and murine pulmonary mast cells are located beneath the epithelium, mast cells likely modulate inflammatory responses secondary to the initial O3-epithelium/macrophage interaction. There is evidence from in vitro experiments that normal epithelial cell-mast cell interaction may have important consequences on basal function of mast cells (34). These investigators found that medium conditioned by nonstimulated epithelial cells inhibits immunoglobulin E- and ionophore-induced mast cell degranulation, although the identity of the inhibitory factor(s) has not been identified. It is not known whether factors released by O3-stimulated epithelial cells or macrophages could induce mast cell degranulation and secretion. It has been hypothesized that a cascade of events initiated by the interaction of O3 with cell membranes leads to the release of a number of cell signals such as lipid ozonation products, which may act directly or indirectly to cause the release of other activating mediators (36). These signals may, in turn, induce mast cells to release a number of preformed and de novo synthesized mediators that may lead to inflammatory cell influx and epithelial damage. Mast cells contain a number of proteases that may contribute to epithelial disruption. Mast cells are also an important source of proinflammatory cytokines, which may contribute significantly to inflammatory processes (10). These include the pleiotropic tumor necrosis factor-alpha (14), which may have an important role in the pathogenesis of O3-induced inflammation (24).

Cell proliferation. The proliferative response to O3 in mast cell-deficient KitW/KitW-v animals was considerably smaller than the response in +/+ animals. In O3-exposed KitW/KitW-v-BMT mice, the time course of the proliferative response in these mice was largely intermediate to those of the +/+ and KitW/KitW-v animals. However, as in the +/+ mice, epithelial cell proliferation in the centriacinar region of the lung and in the nasal airways did not return to baseline after 35 days recovery from O3. Surprisingly, the number of labeled cells actually increased significantly after the animals were removed from the O3 chamber. We therefore conclude that, as in the inflammatory cell response, mast cells contributed to the initiation and maintenance of epithelial cell proliferation in the lung and nose of mice after chronic O3 exposure. It is not certain whether this effect was directly mediated by the mast cells or indirectly mediated via infiltrating inflammatory cells. It may be speculated that, inasmuch as the proliferating epithelium may have different phenotypic properties than normal nonproliferating cells, it may also have different susceptibility to exogenous stimuli. It would be of interest to characterize the functional properties of the centriacinar epithelium in conditions of nonproliferation and O3-induced proliferation.

Results of these experiments are consistent with the hypothesis that mast cells significantly contribute to the initiation and maintenance of bronchiolar inflammation and epithelial responses induced by chronic O3 exposure in this model. A potentially important implication of these studies is that individuals with increased densities of bronchiolar mast cells may be at increased risk to O3. Studies that have compared O3-induced airway inflammatory responses in asthmatic and normal subjects indicate that such responses are more severe in asthmatic subjects, a group with preexisting allergic inflammation of the airways of which mast cells are a central component (3, 40). This may account, in part, for greater O3-induced inflammation in allergic asthmatic compared with healthy nonasthmatic subjects.


    ACKNOWLEDGEMENTS

This work was supported by grants from the Health Effects Institute (HEI 95-5) and the National Institute of Environmental Health Sciences (ES-03519).


    FOOTNOTES

Address for reprint requests and other correspondence: S. R. Kleeberger, Division of Physiology, Rm. 7006, School of Public Health, Johns Hopkins Univ., 615 N. Wolfe St., Baltimore, MD 21205 (E-mail: skleeber{at}jhsph.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2 June 2000; accepted in final form 15 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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