Persistent rhinitis and epithelial remodeling induced by cyclic ozone exposure in the nasal airways of infant monkeys

Abstract

Children chronically exposed to high levels of ozone (O₃), the principal oxidant pollutant in photochemical smog, are more vulnerable to respiratory illness and infections. The specific factors underlying this differential susceptibility are unknown but may be related to air pollutant-induced nasal alterations during postnatal development that impair the normal physiological functions (e.g., filtration and mucociliary clearance) serving to protect the more distal airways from inhaled xenobiotics. In adult animal models, chronic ozone exposure is associated with adaptations leading to a decrease in airway injury. The purpose of our study was to determine whether cyclic ozone exposure induces persistent morphological and biochemical effects on the developing nasal airways of infant monkeys early in life. Infant (180-day-old) rhesus macaques were exposed to 5 consecutive days of O₃ [0.5 parts per million (ppm), 8 h/day; “1-cycle”] or filtered air (FA) or 11 biweekly cycles of O₃ (FA days 1–9; 0.5 ppm, 8 h/day on days 10–14; “11-cycle”). The left nasal passage was processed for light microscopy and morphometric analysis. Mucosal samples from the right nasal passage were processed for GSH, GSSG, ascorbate (AH₂), and uric acid (UA) concentration. Eleven-cycle O₃ induced persistent rhinitis, squamous metaplasia, and epithelial hyperplasia in the anterior nasal airways of infant monkeys, resulting in a 39% increase in the numeric density of epithelial cells. Eleven-cycle O₃ also induced a 65% increase in GSH concentrations at this site. The persistence of epithelial hyperplasia was positively correlated with changes in GSH. These results indicate that early life ozone exposure causes persistent nasal epithelial alterations in infant monkeys and provide a potential mechanism for the increased susceptibility to respiratory illness exhibited by children in polluted environments.

ozone, the principal oxidant air pollutant in photochemical smog, causes tissue injury to the nasal airways in adults and children (25, 48). Initial exposure to ambient concentrations of ozone induces acute, neutrophilic inflammation in the nasal airways of healthy adults (26) and children (47). Both adult and juvenile residents of southwest Mexico City, a region in which ambient concentrations of ozone frequently exceed the National Ambient Air Quality Standards (NAAQS), exhibit histological evidence of epithelial injury and remodeling in their nasal airways (6–9). These pollution-related nasal alterations may impair normal physiological functions (e.g., filtration and mucociliary clearance) that serve to protect the upper and lower respiratory tract from potentially harmful inhaled infectious agents and xenobiotics.

Recent evidence suggests that children may be more vulnerable to the respiratory health effects of ozone than adults (22, 40). This is of particular concern for several reasons. The U.S. Environmental Protection Agency estimates that the majority (53%) of children in the United States live in areas that exceed the NAAQS level for ozone (72). There also exists a strong association between childhood exposure to air pollution and the incidence of acute respiratory infections, a major cause of morbidity and mortality in children (16, 22, 24). Finally, children spend more time outdoors than adults (2), have higher minute ventilation relative to body size than adults (4, 61), and engage in more physical activity than adults, further increasing their potential exposure to airborne pollutants (40, 64).

Studies employing whole animal ozone exposure models are important for establishing exposure-dose-response relationships. Several studies have characterized the nature and persistence of ozone-induced nasal airway injury using adult laboratory animal models. Adult monkeys exposed short-term (6 days) to ambient concentrations [0.15 parts per million (ppm)] of ozone develop acute rhinitis, epithelial hyperplasia, and mucous cell metaplasia in the nonciliated transitional epithelium (NTE) and the ciliated respiratory epithelium (RE) lining the anterior nasal airways. Adult monkeys exposed to long-term ozone (0.15 or 0.30 ppm, 8 h/day for 90 days) exhibit an increase in intraepithelial mucus compared with short-term exposure along with resolution of nasal inflammation and epithelial hyperplasia (30, 31). The epithelial remodeling and attenuated inflammatory cell influx observed following long-term exposure in adult monkeys may represent an adaptive response that protects the nasal airways from ongoing ozone-induced injury. We have recently reported that infant monkeys exposed to repeating cycles of ozone for 2 mo develop persistent rhinitis and nasal epithelial necrosis and do not develop epithelial hyperplasia or mucous cell metaplasia (11), raising the possibility that this adaptive response in adult nasal airways may be absent in the developing nasal airways of infant monkeys.

Many host factors contribute to the magnitude, persistence, and anatomic site of injury following ozone exposure (13, 55, 71). For example, it is well-recognized that genetic factors influence the susceptibility to ozone-induced airway inflammation (45, 46). Variations in the anatomic structure of the nasal cavity affect airflow and the site of subsequent ozone deposition within the nasal airways (42). Homeostatic control of tissue antioxidants has been proposed as an important upstream factor modulating the responses to oxidant challenge. Alterations in the concentrations and regulation of airway tissue antioxidants may alter the capacity of that tissue to respond to oxidant challenges, including ozone exposure (18). Although many of these factors have been evaluated using adult animal models, additional controlled exposure studies using the infant monkey model, whose nasal airways are structurally and functionally similar to those of children, are needed to elucidate better the fundamental differences between the nasal responses to ozone exposure in adults and children as well as identify the potential mechanisms underlying the heightened susceptibility exhibited by children.

Fundamental differences may exist between infants undergoing postnatal respiratory development and adults in the nasal response to acute ozone exposure as well as the ability to adapt to chronic ozone exposure. The current study aimed to examine the morphological and biochemical effects of acute and chronic ozone exposure on the nasal airways of infants and the impact of ozone exposure during postnatal development on adaptive responses. To address this knowledge gap, we exposed infant rhesus monkeys to cyclic episodes of ozone for 5 days (1 cycle) or 5 mo (11 cycles). Using histochemical, image analysis, and rigorous morphometric techniques, we quantitatively characterized the morphological alterations induced by short-term and long-term cyclic ozone exposure in the nasal airways of infant rhesus monkeys. We determined the site, nature, magnitude, and persistence of ozone-induced nasal injury in infant monkeys and compared these with previous reports from adult monkeys to determine the influence of age-related differences in the nasal architecture, epithelial responses, and inflammatory responses to short-term and long-term ozone exposure in infants. Using site-matched biochemical and molecular analyses, we also compared these site-specific morphological changes in the nasal airways to local, ozone-induced fluctuations in the steady-state levels of intracellular antioxidants and their regulatory enzymes.

MATERIALS AND METHODS

Animals and ozone exposure.

Fourteen male infant rhesus monkeys (Macaca mulatta) were raised from birth in a filtered air (FA) environment. All monkeys were obtained from the breeding colony at the California National Primate Research Center at the University of California, Davis. Care and housing of the animals before, during, and after treatment complied with the provisions of the University of California, Davis, Institute of Laboratory Animal Resources and conformed to practices established by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). All animal protocols were reviewed and approved by the University of California, Davis, Institutional Animal Care and Use Committee. While undergoing exposures, animals were housed in stainless steel, open-mesh cages. Before exposure, animals were housed in small social groups within the exposure chambers to allow acclimation to the chamber environment. Five monkeys were exposed to 5 consecutive days of 0.5 ppm ozone for 8 h/day, ending at 6 mo of age (1-cycle O₃). Four monkeys were exposed to 9 consecutive days of FA followed by 5 consecutive days of 0.5 ppm ozone, 8 h/day, for a total of 11 cycles, with ozone exposures beginning at 32–37 days of age and ending at 6 mo of age (11-cycle O₃). Five control monkeys were exposed to FA for 5 mo (Fig. 1). All animals were 180 ± 5 days of age at the end of the experimental period. This exposure protocol was selected because it combined a high ambient ozone concentration (0.5 ppm) consistent with the level achieved in highly polluted areas (73), intermittent exposures to clean and polluted air, which better represent the nature of long-term exposure experienced by people in polluted regions (65), and nighttime exposures, during which our animal model is less active. Consequently, the dose of ozone delivered to target tissues by this exposure protocol is likely lower than would be experienced by active children outdoors in polluted, daytime air. All experimental tissues were harvested on the morning immediately following the end of exposure.

Fig. 1.

Schematic of the ozone exposure protocol. Animals in 1-cycle O₃ group were exposed to 0.5 parts per million (ppm) ozone for 5 consecutive days, 8 h/day, ending at 6 mo of age (top chart). Animals in 11-cycle O₃ group were exposed to 11 biweekly cycles of filtered air (FA; 9 days) and ozone (5 days, 8 h/day), starting at ∼30 days of age and ending at 6 mo of age (middle chart). Control (FA) animals were raised in FA until 6 mo of age (bottom chart). All animals were killed immediately following the end of the last exposure. Numbers above charts indicate age (in days) of animals during ozone exposures, with the number at the end of the chart indicating the age at the end of exposure and necropsy.

Ozone exposures were conducted in 4.2-m³ stainless steel and glass, whole body inhalation chambers. Ozone was produced from vaporized liquid, medical grade oxygen by electronic discharge ozonizers. Generated ozone was diluted with FA (24°C, 40–60% relative humidity) to the appropriate concentration and injected into the inlet airflow of the exposure chamber. FA was supplied to the exposure chambers at a rate of 2.1 m³/min, providing 30 air exchanges/h. Chamber ozone concentration was monitored throughout exposures with an ultraviolet ozone analyzer (Model 1003-AH; Dasibi, Glendale, CA).

Necropsy and tissue preservation.

Immediately at the end of the last exposure, monkeys were sedated using ketamine hydrochloride (10 mg/kg im) followed by induction of deep anesthesia using propofol (Diprivan; 0.1–0.2 mg/kg/min iv), with the dose adjusted as necessary by an attending veterinarian. The trachea, sternum, and abdominal cavity were exposed by a midline incision. Anesthetized animals were euthanized by exsanguination via the abdominal aorta. Immediately after death, the head was removed from the carcass, and the lower jaw, skin, brain, and musculature were removed from the head. The nasal cavity was exposed by splitting the skull sagittally, 1–2 mm to the right of midline, yielding a complete left nasal cavity and intact nasal septum and a free right lateral wall, as previously described (11). All nasal tissues were harvested and preserved within 45 min of exposure cessation. The left nasal cavity was prepared for histopathological and morphometric analyses. Briefly, the left nasal cavity was flushed retrograde through the nasopharyngeal duct with a solution of 1% paraformaldehyde and 0.1% glutaraldehyde (0.01 M phosphate buffer, pH 7.4), immersed in ∼100 ml of 1% paraformaldehyde and 0.1% glutaraldehyde, and fixed at 4°C for at least 24 h.

The right side of the nasal cavity was used to harvest site-specific mucosal samples for RNA isolation and biochemical antioxidant analyses corresponding to the sites examined by light microscopy in the left nasal cavity. Mucosal samples were dissected from the anterior maxilloturbinate (MT), posterior MT, and anterior ethmoturbinate (ET; Fig. 2A). Each mucosal sample was divided into two equal parts, each approximately 2 × 2 × 2 mm. One part from each region was stored in 0.5 ml of RNAlater (Ambion, Austin, TX), held at room temperature for 24 h, and then stored at −20°C. The samples from the anterior MT were further processed for RNA extraction and gene expression analyses (described below). The other part from each region was immediately placed into 300 μl of 10% metaphosphoric acid and snap-frozen in liquid nitrogen. Acidified, frozen nasal mucosal samples were later thawed, homogenized for 30 s using a Polytron homogenizer, refrozen, and stored at −80°C until further processing for low molecular weight antioxidant analysis via HPLC.

Fig. 2.

Anatomic location of nasal tissues selected for morphometric analysis. A: exposed right lateral wall of the nasal cavity of a 180-day-old rhesus monkey. Vertical lines indicate the locations of the 12 transverse sections processed for light microscopy. The 2 black lines indicate the sections through the anterior maxilloturbinate (MT) that were used for morphometric analyses. B and C: low magnification photomicrographs of hematoxylin and eosin (H&E)-stained transverse sections through the anterior MT of a FA-exposed monkey. The area highlighted in green represents the dorsomedial surface of the MT. The mucosa underlying this region was selected for morphometric analysis. n, Naris; i, incisor tooth; et, ethmoturbinate; hp, hard palate; np, nasopharyngeal meatus; s, nasal septum.

Tissue processing for light microscopy and morphometric analysis.

After fixation, the left nasal cavity specimen was decalcified in 200 ml of 13% formic acid for 14–16 days and then rinsed with distilled water for 2–4 h. After decalcification, the nasal cavity was transversely sectioned at 10–12 specific anatomic locations in a plane perpendicular to the hard palate and nasal septum using gross dental and palatine landmarks, as previously described (11). The tissue blocks were embedded in paraffin for light microscopic and morphometric analyses. Slides were prepared using 4- to 5-μm-thick sections from the anterior face of each of the 10–12 tissue blocks. One tissue section from each block was stained with hematoxylin and eosin (H&E) for routine histopathological examination. Another tissue section was stained with Alcian blue (pH 2.5)/periodic acid-Schiff (AB/PAS) for identification of acidic and neutral mucosubstances.

Our histological and morphometric investigations focused on the epithelium lining the anterior nasal cavity. This region of the nasal cavity has been reported to be the primary site for damage induced by inhaled toxicants in monkeys (27, 30, 31) and children (8, 9). The first two transverse tissue sections that included a complete profile of the MT (i.e., the anterior MT) were selected for morphometric analyses (Fig. 2). A tissue section from these blocks was stained with rabbit anti-human granulocytic myeloperoxidase polyclonal antibody (Lab Vision, Fremont, CA) for immunohistochemical identification of neutrophils.

Morphometry of neutrophilic inflammation and epithelial numeric cell density.

The epithelium lining the dorsomedial surface of the anterior MT was analyzed through standard morphometric techniques and computerized image analysis (29). Neutrophil numeric densities were determined by counting the number of nuclear profiles of neutrophils within the surface epithelium, in the lamina propria, and within the subepithelial vasculature of the mucosa lining the dorsomedial MT and dividing this number by the total length of the basal lamina underlying the epithelium in this region. Neutrophils were identified on the basis of characteristic, multilobed nuclear morphology and cytoplasmic myeloperoxidase immunoreactivity. The basal lamina length was calculated from the contour length of the basal lamina on a digitized image using image analysis software (Scion Image; Scion, Frederick, MD). The epithelial cell numeric density was determined by counting the total number of epithelial cell nuclear profiles in the epithelium lining the dorsomedial MT and dividing this number by the length of the basal lamina. The mucous cell numeric density was determined by counting the total number of AB/PAS-positive epithelial cells with nuclear profiles and dividing this number by the basal lamina length.

Morphometry of epithelial metaplasia.

Metaplastic changes in the epithelium lining the dorsomedial, anterior MT were quantified using stereological methods. The volume density of nuclei, cilia, and intraepithelial mucus within this region was morphometrically evaluated as previously described for airway epithelium (11, 35, 36). All measurements were obtained at a final magnification of ×1,710 using a light microscope (Olympus BX40; Olympus America, Melville, NY) coupled to a 3.3-megapixel digital color camera (Q-Color3 Camera; Quantitative Imaging, Burnaby, British Columbia, Canada) and a personal computer (Dimension 8200; Dell, Austin, TX). The morphometric analyses were performed using a 135-point cycloid grid overlay with an automated software package for counting points and intercepts within the grid (Stereology Toolbox; Morphometrix, Davis, CA) (35, 36). The percent volume density (the proportion of the total epithelial volume), V_v, of the epithelial constituents was determined by point counting and calculated using the following formula:

$$V_v=P_p=P_n/P_t$$ (1)

where P_p is the point fraction of P_n, the number of test points hitting the structure of interest (e.g., nuclei), divided by P_t, the total number of points hitting the reference space (i.e., epithelium). The volume of each epithelial component of interest per unit basal lamina length (cubic micrometers per square micrometer; S_v) was determined by point- (epithelial component) and intercept- (basal lamina) counting and was calculated using the following formula:

$$S_v=2I_o{L}_{\gamma }$$ (2)

where I_o is the number of cycloid intercepts with the object (basal lamina), and L_γ is the length of the test line in the reference volume (epithelium within the test grid). To determine the thickness of the total epithelium, a volume per unit area of basal lamina (cubic micrometers per square micrometer) was calculated using the following formula for arithmetic mean thickness (τ):

$$\tau =V_v/S_{v\mathrm{.}}$$ (3)

Morphometry of stored intraepithelial mucosubstances.

The amount of stored mucosubstances in the surface epithelium of the dorsomedial MT was estimated by quantifying the volume of AB/PAS-stained mucosubstances per unit basal lamina (volume density, V_s) using computerized image analysis and standard morphometric techniques. The area of AB/PAS-stained mucosubstances was calculated by circumscribing the perimeter of the stained material using the Scion Image program. The length of the basal lamina underlying the surface epithelium was calculated as described above. The volume density was estimated using a previously described method (30) and was expressed in nanoliters per square millimeter basal lamina.

Determination of intracellular low molecular weight antioxidant concentrations in nasal mucosa.

Acid-stabilized nasal tissue homogenates were thawed and centrifuged at 12,500 g for 4 h. Protein pellets were resuspended in 500 μl of PBS, neutralized with 25 μl of 1 N NaOH, and sonicated for 1 h at 37°C. The protein content of resuspended pellets was measured using the Pierce BCA Protein Assay (Pierce Biotechnology, Rockford, IL). Supernatants were filtered through a 0.22-μm syringe filter, and samples were fractioned in triplicate on a Shimadzu LC-10Ai HPLC (Shimadzu Scientific Instruments, Columbia, MD) using a Phenomenex Luna C18(2) 250 × 4.6 mm, 5-μm reversed-phase column, preceded by a Phenomenex ODS 4 × 3 mm guard column (Phenomenex, Torrance, CA). The mobile phase consisted of an isocratic mixture of 50 mM phosphate buffer, pH 3.1, containing 50 μM octanesulfonic acid and methanol (95:5). Samples were fractioned at a mobile phase flow rate of 0.5 ml/min. GSH, GSSG, ascorbate (AH₂), and uric acid (UA) were simultaneously detected with an eight-channel ESA CoulArray Model 5600A electrochemical detector (ESA, Chelmsford, MA). GSH, GSSG, AH₂, and UA values were normalized to total protein content of centrifuge pellets.

Analysis for glutamate-cysteine ligase catalytic and modifier subunit mRNA in nasal tissues.

Tissue samples for RNA isolation were homogenized using a Polytron homogenizer. Total cellular RNA was isolated with TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions from samples stored in RNAlater (Ambion). To avoid DNA contamination, RNA pellets were resuspended in nuclease-free water and DNase-treated with DNA-free (Ambion) for 1 h at 37°C. cDNA was prepared using random hexamer primers (Amersham Pharmacia Biotech, Piscataway, NJ) and Moloney murine leukemia virus reverse transcriptase (Invitrogen).

Glutamate-cysteine ligase catalytic (GCL-C) and modifier (GCL-M) subunit mRNA levels were determined by real-time PCR as previously described (1, 69). Briefly, samples were tested in duplicate, and the PCR for the GAPDH housekeeping gene and the target gene from each sample were run in parallel on the same plate. The reaction was carried out in a 96-well optical plate (Applied Biosystems, Foster City, CA) in a 25-μl reaction volume containing 5 μl of cDNA and 20 μl of Master Mix (Applied Biosystems). Sequences were amplified using the 7900 default amplification program (2 min at 50°C, 10 min at 95°C, followed by 40–45 cycles of 15 s at 95°C and 1 min at 60°C). The results were analyzed with the SDS 7900 system software version 2.1 (Applied Biosystems). GCL-C and GCL-M mRNA expression levels were calculated from the normalized ΔC_T cycle threshold values. Fold increases were calculated using the ΔΔC_T method (62).

Statistical analyses.

All data were expressed as mean group values ± SE. The differences among groups were analyzed by one-way ANOVA. Pairwise comparisons were performed a priori using Student-Newman-Keuls multiple comparisons test. The relationship between epithelial cell numeric density and intracellular antioxidant concentration was analyzed by using post hoc correlation analysis (Pearson product moment correlation). The criterion for statistical significance was set to P ≤ 0.05 for all analyses. All statistical analyses were performed using a commercial statistical software package (SigmaStat; SPSS, Chicago, IL).

RESULTS

Nasal histopathology.

In FA-exposed (0 ppm) infant monkeys, the preturbinate region of the main chamber was lined with a combination of squamous epithelium, NTE, and RE (Fig. 3A). Most of the remainder of the main chamber of the nasal airways, including the entire surface of the MT, was lined with RE. A small region along the dorsal meatus and the dorsal half to third of the ET was lined with olfactory epithelium. This epithelial distribution is in accordance with previous reports on the nasal airways of monkeys (11, 27, 30, 31). Occasional intraepithelial and subepithelial neutrophils were present in the mucosa of the NTE and the RE lining the anterior nasal cavity in FA-exposed monkeys. This mild inflammation was primarily localized near the ducts and luminal openings of submucosal glands and near the junctions of two adjacent epithelial populations (e.g., the junction of RE and olfactory epithelium). No gross or microscopic morphological alterations were observed in the nasal mucosa of monkeys exposed to FA.

Fig. 3.

Light photomicrographs of the nasal mucosa lining the dorsomedial surface of the MT of 180-day-old monkeys exposed to 0 ppm O₃ (FA; A), 1-cycle O₃ (B), or 11-cycle O₃ (C). The epithelial surface in A is lined by an intact layer of cilia (long arrows). Marked epithelial necrosis and exfoliation (arrowheads) of the surface epithelium (e) are present throughout the nasal mucosa following 1-cycle O₃ exposure (B). Squamous metaplasia and epithelial hyperplasia are present in the surface epithelium following 11-cycle O₃ exposure (C). Note the loss of surface cilia and intracellular mucosubstances following 1-cycle (B) and 11-cycle (C) ozone exposure. Tissues were stained with H&E. Dotted line, basal lamina between epithelium and lamina propria; g, glands; bv, blood vessels.

The principal nasal injury in monkeys exposed to 1-cycle O₃ was acute, multifocal, necrotizing rhinitis. This response was restricted primarily to the NTE and ciliated RE lining the ventral half of the anterior main chamber. In all animals exposed to 1-cycle O₃, there were focal areas of epithelial necrosis and exfoliation, localized primarily to sites along the MT and ventral aspect of the nasal septum. Associated with these areas of necrosis was an influx of neutrophils in the affected epithelium and lamina propria and endothelial margination of neutrophils in the underlying capacitance vessels of the nasal mucosa.

The site of the most severe necrotizing rhinitis in 1-cycle O₃-exposed animals was consistently observed in the RE lining the dorsomedial surface of the anterior MT. This location was further characterized by a conspicuous attenuation or complete loss of cilia and a reduction in the amount of stored intraepithelial mucosubstances (Fig. 3B). No significant histological abnormalities were noted in the olfactory epithelium lining the dorsal nasal cavity or in the nasal mucosa of the posterior aspect of the nasal cavity in 1-cycle O₃-exposed animals.

The exposure-related injury in 11-cycle O₃-exposed animals was also restricted to the ventral part of the anterior main chamber, similar to the distribution observed in the 1-cycle O₃-exposed animals. The principal lesions in animals exposed to 11-cycle O₃ were epithelial hyperplasia, squamous metaplasia, and necrotizing rhinitis, located along the dorsomedial surface of the MT (Fig. 3C). This location is consistent with the site of maximal injury found in the 1-cycle O₃-exposed animals. However, in contrast to the pseudostratified ciliated RE described in 1-cycle O₃ animals, the surface epithelium at this site in the 11-cycle O₃-exposed animals was 3–4 cells thick and consisted primarily of layers of small, polygonal epithelial cells devoid of intracellular mucosubstances or surface cilia. There was an overall decrease in the amount of stored intraepithelial mucosubstances in the 11-cycle O₃-exposed animals. However, this overall loss of intraepithelial mucus was accompanied by the appearance of occasional clusters of small, mucus-containing epithelial cells forming intraepithelial mucous glands. Focal areas of acute rhinitis and epithelial necrosis with and without exfoliation and loss of cilia were also present along the ventral septum and ventral meatus and immediately adjacent to the area of squamous metaplasia along the MT.

Morphometry of nasal epithelial thickness.

Results of stereological analysis of the dorsomedial surface of the anterior MT are presented in Fig. 4. Eleven-cycle O₃ exposure caused a 30% increase in mean epithelial thickness compared with FA-exposed animals. This ozone-induced epithelial thickening observed in 11-cycle O₃-exposed animals was associated with a significant (2.5-fold) increase in the volume density of epithelial nuclei compared with FA-exposed animals, thus indicating that the increase in epithelial thickening is due to cellular hyperplasia rather than increases in extracellular matrix. Despite the necrosis and exfoliation described above, there was no significant difference in the mean epithelial height or nuclear volume density between 1-cycle O₃ and FA-exposed animals. Both 1- and 11-cycle O₃ exposure regimens caused a significant reduction in the volume density of cilia (56 and 91% reductions, respectively) from the surface of the anterior MT compared with FA exposure.

Fig. 4.

Morphometry of ozone-induced nasal epithelial injury in infant rhesus monkeys. Eleven-cycle O₃ exposure caused epithelial hyperplasia and squamous metaplasia, resulting in an increase in epithelial height (A) and in the volume density of epithelial cell nuclei (B). One- and eleven-cycle ozone exposure each resulted in a reduction in the volume density of cilia (C). Bars represent group means ± SE. *Significantly different from respective FA group (P ≤ 0.05); ^asignificantly different from respective 1-cycle ozone group (P ≤ 0.05).

Morphometry of intraepithelial mucosubstances.

The epithelial hyperplasia and squamous metaplasia observed in animals exposed to 11-cycle O₃ was also associated with a significant reduction in the amount of stored intraepithelial mucosubstances (88% decrease) and the numeric density of mucous cells (90% decrease) in the anterior MT compared with FA exposure (Fig. 5). Monkeys exposed to 1-cycle O₃ also exhibited a mild, but not statistically significant, decrease in stored intraepithelial mucosubstances (36% decrease) and mucous cells (7.6% decrease) compared with FA-exposed monkeys.

Fig. 5.

A and C: light photomicrographs of MT from monkeys exposed to FA (A) or 11-cycle O₃ (C). Tissues were stained with Alcian blue/periodic acid-Schiff (AB/PAS) to detect acidic and neutral mucosubstances. Note the presence of cilia (solid arrow) and intraepithelial AB/PAS-stained mucosubstances (dashed arrow) in the epithelium of the FA-exposed (A) monkey and the loss of these features following 11-cycle O₃ exposure (C). Dotted line, basal lamina between epithelium and lamina propria. B and D: morphometry of intraepithelial mucus. Episodic ozone exposure caused a decrease in the volume density of intraepithelial mucosubstances (B) and mucous cell numeric cell density (D). Bars represent group means ± SE. *Significantly different from respective FA group; ^asignificantly different from respective 1-cycle O₃ group (P ≤ 0.05). V_s, volume per unit surface area.

Morphometry of neutrophilic inflammation.

All ozone-exposed animals (1- or 11-cycle) exhibited neutrophilic rhinitis in the nasal airway mucosa lining the dorsomedial MT (Fig. 6). Both of the ozone-exposed groups had ∼4-fold more total neutrophils in the nasal mucosa (intraepithelial + interstitial + intravascular) than the FA-exposed group. There was no significant difference in the total or interstitial neutrophil numeric density between 1- and 11-cycle O₃ animals. However, the 11-cycle O₃ group had significantly more intraepithelial neutrophils (by 2.4-fold) and fewer intravascular neutrophils (75% fewer) along this site than the 1-cycle O₃ group. The numeric cell densities of neutrophils in the mucosa along the dorsomedial surface of the anterior MT are summarized in Fig. 7.

Fig. 6.

Light photomicrographs of MT from monkeys exposed to FA (A), 1-cycle O₃ (B), or 11-cycle O₃ (C). Tissues were stained with anti-granulocytic myeloperoxidase antibody for immunohistochemical identification of neutrophils (PMNs). Note the infiltration of PMNs (arrows) into the epithelium and lamina propria of monkeys exposed to 1-cycle O₃ (B) and the persistence of this inflammatory response in monkeys exposed to 11-cycle O₃ (C).

Fig. 7.

Effect of ozone exposure on the numeric cell density of PMNs in the nasal mucosa along the anterior MT of infant monkeys. A: total PMN numeric cell density (intraepithelial + interstitial + intravascular PMN). B: intraepithelial PMN. C: interstitial PMN. D: intravascular PMN. Bars represent group means ± SE. *Significantly different from respective FA group (P ≤ 0.05); ^asignificantly different from respective acute ozone group (P ≤ 0.05).

Intracellular concentrations of low molecular weight nasal antioxidants.

Baseline antioxidant concentrations of GSH, GSSG, AH₂, and UA in FA-exposed animals were similar in the anterior MT, posterior MT, and the anterior ET. The mucosal concentrations of GSH, GSSG, AH₂, and UA did not change significantly in any of the 3 nasal regions examined following 1-cycle O₃ exposure. However, there were slight decreases in the concentrations of GSH, AH₂, and UA in all regions following 1-cycle O₃ exposure compared with FA-exposed animals. In contrast, 11-cycle O₃ exposure caused an increase in mucosal GSH concentrations in the anterior MT (65% increase), posterior MT (140% increase), and anterior ET (75% increase) compared with FA exposure (Fig. 8A). There was no change in GSSG concentration in the 2 MT regions following 11-cycle O₃ exposure, however, the GSSG concentration in the anterior ET increased by ∼2.4-fold (Fig. 8B). No differences were observed in AH₂ or UA concentrations in any of the 3 nasal regions examined in 11-cycle O₃ ozone animals (Fig. 8, C and D).

Fig. 8.

Effect of 1- and 11-cycle O₃ exposure on the intracellular concentrations of GSH (A), GSSG (B), ascorbate (AH₂; C), and uric acid (UA; D) in the nasal mucosa from the anterior MT, posterior MT, and anterior ET of infant monkeys. Note the robust increase in intracellular GSH concentrations in all regions of the nasal mucosa following episodic ozone exposure. Bars represent group means ± SE. *Significantly different from respective FA group (P ≤ 0.05); ^asignificantly different from respective 1-cycle O₃ group (P ≤ 0.05).

GCL-C and GCL-M mRNA expression in the anterior MT.

Eleven-cycle O₃ exposure induced a significant increase (1.4-fold) in the steady-state level of GCL-C mRNA in the anterior MT compared with FA exposure (Fig. 9A). There was no change in GCL-C mRNA expression at this site following 1-cycle O₃ exposure. There were no changes in the expression of GCL-M mRNA following either 1- or 11-cycle O₃ exposure (Fig. 9B).

Fig. 9.

Effect of 1- and 11-cycle O₃ exposure on glutamate-cysteine ligase catalytic (GCL-C; A) and modifier (GCL-M; B) subunit gene expression in the nasal mucosa of the anterior MT. Bars represent group means ± SE. *Significantly different from respective FA group (P ≤ 0.05); ^asignificantly different from respective acute ozone group (P ≤ 0.05).

Correlation analysis for epithelial cell numeric density and antioxidant concentration along the anterior MT.

Correlation analysis was used to determine the association between epithelial cell numeric density and intracellular antioxidant concentration. There was a significant positive correlation between epithelial cell numeric density and intracellular GSH concentration at the anterior MT (r = 0.684; P = 0.007) across all exposure groups (Fig. 10A). There were no significant correlations found between epithelial cell numeric density and the intracellular concentrations of AH₂ (r = 0.0215; P = 0.942) or UA (r = 0.328; P = 0.252; Fig. 10, B and C).

Fig. 10.

Correlations between epithelial hyperplasia (numeric cell density; x-axes) and the intracellular concentrations of GSH (A), AH₂ (B), and UA (C) (y-axes) in the nasal mucosa lining the anterior MT across all exposure groups. The associations were described as the Pearson product moment correlation coefficients (r) and were considered significant if P ≤ 0.05.

DISCUSSION

In the present study, exposure of infant monkeys to cyclic ozone yielded 3 major nasal alterations that were distinct from the responses previously observed in ozone-exposed adult monkeys: persistent inflammation, the development of squamous metaplasia, and a lack of mucous cell metaplasia. The nasal responses to both acute and chronic, daily ozone exposure in adult monkeys have been extensively characterized. In previous reports from our laboratory (30, 31), acute ozone exposure (0.3 ppm, 8 h/day, 6 days) caused neutrophilic rhinitis, epithelial necrosis, and epithelial hyperplasia that were confined to the NTE and ciliated RE of the anterior nasal cavity in adult monkeys. Continued daily exposure of adult monkeys for 90 days resulted in a resolution of the inflammation and epithelial hyperplasia observed at 6 days (30). Both acute and chronic daily ozone exposures also resulted in the development of mucous cell metaplasia in the anterior nasal airways of adult monkeys. In the present study, we found that infant (180-day-old) monkeys exposed acutely to ozone (1-cycle O₃) also exhibited epithelial necrosis, ciliated cell loss, and neutrophilic inflammation in the anterior nasal cavity. Following 5 mo (11 cycles) of cyclic ozone exposure, infant monkeys exhibited rhinitis, epithelial hyperplasia, and squamous metaplasia in the anterior nasal airways. Furthermore, the magnitude of the inflammatory response was similar between infant monkeys exposed to 1 and 11 cycles. Our results indicate that cyclic ozone exposures cause inflammation and epithelial alterations in the nasal airways of infant monkeys. Importantly, this also suggests that unlike adults, infant monkeys remain susceptible to ozone-induced nasal airway injury following long-term exposure.

An important distinction between the present study in infant monkeys and our previous studies in adult monkeys is that the chronic studies in adults used daily exposures, whereas the chronic exposure in the present study employed a cyclic exposure protocol. It is possible that the differences observed in infant monkeys may actually reflect a response to repeated cycles of ozone-induced injury. However, it is also important to note that the infant monkeys in the present study also exhibited a different response to acute exposure than adult monkeys despite similar daily exposure conditions. Another important similarity is that the tissues in both studies were harvested immediately after the end of an ozone exposure, indicating that the morphological and biochemical alterations observed in both experiments reflected the effects of prior exposure on the response to a recent exposure. Finally, the exposure protocol used in the present study reflects intermittent exposures to clean and polluted air, which more accurately reflects the nature of exposure experienced by children in highly polluted regions (65).

The findings of persistent inflammation and the lack of adaptation in a model of the nasal airways of children has important potential biological implications. Preexisting rhinitis has been associated with the development or enhancement of allergen-mediated airway inflammation in human studies (52) and in experimental models (68) and has been shown to be a risk factor in the development of respiratory infections (63). Rhinitis has also been associated with the development or exacerbation of inflammatory lower airway conditions, including asthma. Furthermore, the resolution of nasal inflammation and the restoration of normal nasal function have been associated with a beneficial effect on the management of comorbid bronchial conditions (32). Indeed, epidemiologic studies have demonstrated associations with childhood exposure to ozone and the development of allergic (56) and infectious (16, 24) respiratory disorders. The results of the present study suggest that children living in highly polluted areas are at risk for the development of persistent nasal inflammation and epithelial injury and may also remain vulnerable to other nasal and pulmonary comorbidities.

Maintenance of neutrophilia at a site of inflammation can be due to persistent chemokine-mediated recruitment of neutrophils from circulation, cytokine-mediated enhancement of neutrophil survival (19), or a combination of these factors. Altered neutrophil survival has been demonstrated to play a role in the development of pulmonary tolerance to ozone (21). In the present study, we found a higher numeric density of interstitial and intraepithelial neutrophils and a lower numeric density of intravascular neutrophils following 11-cycle O₃ compared with 1-cycle O₃ exposure. This shift in the tissue localization of neutrophils could be consistent with a decrease in neutrophil recruitment and an increase in peripheral neutrophil survival. Additional studies are needed to more fully examine the mechanisms behind this persistent inflammatory response in the nasal airways of infant monkeys and the possible implications toward the response to ozone exposure exhibited by children.

The nasal airways of people (3) and nonhuman primates (11, 39) undergo significant postnatal growth and development. Age-related changes in the gross and microscopic structure of the nasal cavity may influence local tissue susceptibility, the distribution of susceptible epithelial populations, and the dose of inhaled toxicants delivered to susceptible sites. The ozone-induced inflammatory and epithelial alterations observed in infant monkeys in the present study were confined to specific epithelial types (NTE and ciliated RE) within the anterior nasal cavity. This distribution of ozone-induced injury observed in 180-day-old infant monkeys was similar to the pattern of nasal injury previously described in adult monkeys exposed to ozone (30, 31) and is also consistent with the distribution of nasal injury that we (11) have previously described in younger (90-day-old), infant monkeys similarly exposed to ozone. These results indicate that the site specificity of ozone-induced injury is consistent in infants and adults despite significant postnatal growth of the nasal cavity in nonhuman primates. This finding is also in accordance with a recent report comparing computational fluid dynamics simulations of reactive gas uptake in the nasal airways of children and adults (23) and suggests that age-related changes in nasal epithelial distribution, nasal airflow, and ozone deposition are not the sole mitigating factors in the persistent susceptibility to ozone-induced injury exhibited by infant monkeys.

Mucous cell metaplasia is a frequent morphological feature of both the nasal and pulmonary airway epithelium following inhaled pollutant challenge (15, 28, 33, 37, 60). The development of mucous cell metaplasia is generally considered a pathological response following airway epithelial injury (58, 67). However, airway mucus provides an effective barrier between the airway lumen and underlying epithelium, whereas intraepithelial mucus has been suggested to act as an antioxidant by trapping reactive oxygen species (14, 17). We (30) have previously found that adult monkeys develop mucous cell metaplasia in the anterior nasal airways following acute exposure (6 days, 8 h/day) that persists with chronic (90 days, 8 h/day) daily exposure to 0.3 ppm ozone. This early morphological alteration may contribute to the reduced susceptibility observed following chronic ozone exposure in adult monkeys. In the present study, neither 1- nor 11-cycle O₃ ozone exposure caused mucous cell metaplasia (or hyperplasia) in infant monkeys. In fact, both 1- and 11-cycle O₃ exposures caused a decrease in the amount of stored intraepithelial mucosubstances in the ciliated RE lining the anterior MT. The lack of this mucus barrier in the nasal airways of infant monkeys may allow ozone and/or its reaction products to penetrate more easily the epithelial lining fluid layer and cause persistent nasal epithelial injury. These morphological changes may represent inherent, age-related differences in the response of the infant nasal airways to oxidant pollutant exposure.

Squamous metaplasia and hyperplasia of the nasal epithelium have been reported as sequelae of chronic exposure to oxidant pollutants in children (8, 9). In the present study, monkeys exposed to 11-cycle O₃ exhibited focal areas of epithelial hyperplasia and squamous metaplasia of the ciliated RE lining the anterior MT. This metaplastic change resulted in an increase in epithelial thickness and was also associated with a loss of surface cilia and a reduction in the amount of stored intraepithelial mucosubstances. Although these morphological changes in the nasal airways may result in an epithelium that is locally more resistant to uptake of reactive, pollutant gases (38, 43, 44), they may also serve to disrupt filtration, reactive gas absorption, and mucociliary clearance functions in the anterior nasal airways. The loss of nasal filtering function could potentially result in delivery of xenobiotics and infectious agents to more distal sites in the respiratory tract, including the conducting airways and pulmonary parenchyma (5, 54). Furthermore, the disruption of mucociliary clearance could also lead to increased transit times for airborne xenobiotics and pathogens trapped in the nasal airways, resulting in prolonged contact with the nasal epithelium and enhanced upper airway toxicity or susceptibility to airway infection.

In the present study, we report that cyclic exposure to ozone causes squamous metaplasia, a decrease in intraepithelial mucosubstances, and loss of ciliated cells in the anterior nasal airways of infant monkeys. The nature of this nasal airway injury is morphologically similar to that described in the nasal biopsies of children chronically exposed to air pollutants in photochemical smog (8, 9). This comparison suggests that the nasal alterations experienced by children in highly polluted environments are due, at least in part, to exposure to ambient levels of ozone. The nasal biopsies in these studies were obtained from the posterior aspect of the inferior nasal turbinates in children. Interestingly, in the present study, we found that ozone-induced injury was consistently confined to the anterior part of the MT (the most inferior of the 2 turbinates in the monkey nose), whereas the posterior aspect remained morphologically normal. Several factors may contribute to the discrepancy in the sites of injury between these studies. Our experimental animals were exposed at night, during which time they are less active. In contrast, children are typically exposed to outdoor air pollutants during more active periods in the daytime. In addition, children exposed to ozone in photochemical smog are concurrently exposed to other air pollutants, including particulate matter and nitrogen dioxide (53). Finally, children raised from birth in polluted environments experience lifelong exposure to air pollutants, whereas our experimental model was exposed episodically for a period of only 5 mo. It is possible that these differences between experimental and natural exposure may alter the site specificity and distribution of pollutant-induced nasal airway injury.

Low molecular weight antioxidants are considered as the first line of defense against inhaled oxidant challenge (18). In the distal lung, the site specificity of injury and remodeling induced by inhaled xenobiotics are dependent on local changes in the regulation of intracellular low molecular weight antioxidants. Regional changes in GSH status have been correlated with toxicant-induced responses in the lungs of monkeys and rats exposed to ozone (20, 57). Similar relationships among antioxidant regulation, xenobiotic-induced epithelial injury, and epithelial repair have been documented in the lungs of laboratory rodents following exposure to hyperoxia (41) and naphthalene (70) and in the nasal airways of rats exposed to cigarette smoke (50). To investigate the potential role of intracellular low molecular weight antioxidants in ozone-induced nasal injury and repair in our study, we compared the baseline and postexposure tissue concentrations of AH₂, UA, GSH, and GSSG at an intranasal site of ozone-induced injury and repair (anterior MT) and at 2 sites at which no exposure-related morphological changes were observed (posterior MT and anterior ET). The only exposure-related change in antioxidant levels was an increase in GSH concentration following 11-cycle O₃ exposure. This increase was associated with an increase in the steady-state mRNA levels of the catalytic subunit of GCL, the rate-limiting enzyme in de novo GSH synthesis, in animals exposed to 11-cycle O₃. Although the increase in GCL-C expression may explain part of the increase in GSH, other regulators of GSH were also likely involved (e.g., GSH redox cycling via glutathione reductase and γ-glutamyl transpeptidase-mediated recycling of extracellular GSH). Importantly, in our study, the ozone-induced upregulation in GSH levels was not limited to the site of ozone-induced injury but was found in samples taken throughout the nasal cavity. This suggests that the upregulation in mucosal GSH concentration observed in our study was not a site-specific response to ozone-induced injury but rather a widespread nasal mucosal response to ozone exposure itself. One possible explanation for the discrepancy between ozone-induced injury and antioxidant response is heterogeneity in the dose of ozone delivered to different sites in the nose. Previous reports describing predictive models of ozone mass flux suggest that ozone flux is highest in the anterior nasal airways of monkeys (10) and rodents (34). It is possible that higher ozone mass transfer at these sites exceeds the local tissue defenses against oxidant challenge (e.g., antioxidant capacity), supporting the concept that airflow-driven ozone deposition also plays an important role in the observed pattern of injury.

Although the exact role of GSH in cell proliferation and cell survival remains unclear, a growing body of evidence supports a role for GSH in cell cycle regulation and cell proliferation. Previous reports indicate that upregulation and subcellular localization of GSH are important in the control of G₁-to-S transition (51) and DNA synthesis (66). The importance of GSH in oxidant-induced cell proliferation has been previously documented in cultured bronchial epithelial cells. Cigarette smoke condensate causes a dose-dependent increase in 5-bromo-2-deoxyuridine (BrdU) incorporation in primary bronchial epithelial cells in vitro. This effect is inhibited by glutathione depletion (49). Another recent investigation examined the role of GSH in nuclear factor-E2-related factor (Nrf2)-mediated cell proliferation. Using type II pneumocytes from Nrf2^−/− mice, these investigators demonstrated that Nrf2-deficiency leads to impaired cell proliferation in vitro and that GSH supplementation restores replicative capacity to Nrf2^−/− cells (59). In the present study, we found a positive correlation between the total numeric density of epithelial cells lining the anterior MT, a measure of epithelial hyperplasia, and the intracellular concentration of GSH at that site. This finding is consistent with our (12) previous report that younger (90-day-old) infant rhesus monkeys exposed to 1 or 5 cycles of ozone do not develop epithelial hyperplasia or squamous metaplasia and exhibit an associated decrease in intracellular GSH concentrations. Although these data do not establish a cause-and-effect relationship between GSH regulation and cell proliferation or survival, they do provide a possible mechanistic basis for the persistence of the epithelial alterations observed in the nasal airways of infant monkeys.

In the present study, we have shown that cyclic exposure to ozone induces persistent injury and morphological alterations in the nasal airways of infant monkeys, a relevant model of the developing nasal airways of children. Several of these responses in infant monkeys are distinct from those observed in adult monkeys and may provide a mechanistic basis for the heightened susceptibility to inhaled xenobiotics exhibited by children. The presence of ongoing inflammation following long-term exposure suggests that the adaptive response to cyclic ozone may be diminished in the developing nasal airways of infants. Furthermore, the long-lasting epithelial remodeling may cause decrements in normal nasal functions, including loss of barrier function and impairment of mucociliary clearance, providing several mechanisms by which the nasal airways of children are potentially rendered more vulnerable to the effects of inhaled infectious agents or xenobiotics. Ozone-induced alterations in antioxidant capacity may provide a biochemical basis for the persistence of these changes in the infant model; however, a data gap still exists in our understanding of the role of low molecular weight antioxidants in ozone-induced nasal injury and repair in both infants and adults. Taken together, our results illustrate the complexity of the relationships among ozone exposure, local tissue susceptibility, inflammatory responses, and antioxidant capacity in the nasal airways and highlight the importance of considering age-related differences in these risk factors when extrapolating experimental data from animal models for the assessment of risk of air pollutant exposures in children.

GRANTS

This work was supported by the National Institutes of Health, National Institute of Environmental Health Sciences Grant P01-ES-011617 (E. M. Postlethwait).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).