Early life exposure to allergen and ozone results in altered development in adolescent rhesus macaque lungs.

In rhesus macaques, previous studies have shown that episodic exposure to allergen alone or combined with ozone inhalation during the first 6 months of life results in a condition with many of the hallmarks of asthma. This exposure regimen results in altered development of the distal airways and parenchyma (Avdalovic et al., 2012). We hypothesized that the observed alterations in the lung parenchyma would be permanent following a long-term recovery in filtered air (FA) housing. Forty-eight infant rhesus macaques (30 days old) sensitized to house dust mite (HDM) were treated with two week cycles of FA, house dust mite allergen (HDMA), ozone (O3) or HDMA/ozone (HDMA+O3) for five months. At the end of the five months, six animals from each group were necropsied. The other six animals in each group were allowed to recover in FA for 30 more months at which time they were necropsied. Design-based stereology was used to estimate volumes of lung components, number of alveoli, size of alveoli, distribution of alveolar volumes, interalveolar capillary density. After 30 months of recovery, monkeys exposed to HDMA, in either group, had significantly more alveoli than filtered air. These alveoli also had higher capillary densities as compared with FA controls. These results indicate that early life exposure to HDMA alone or HDMA+O3 alters the development process in the lung alveoli.

Lung effects of inhaled corticosteroids in a rhesus monkey model of childhood asthma.

BACKGROUND: The risks for infants and young children receiving inhaled corticosteroid (ICS) therapy are largely unknown. Recent clinical studies indicate that ICS therapy in pre-school children with symptoms of asthma result in decreased symptoms without influencing the clinical disease course, but potentially affect postnatal growth and development. The current study employs a primate experimental model to identify the risks posed by ICS therapy. OBJECTIVE: To (1) establish whether ICS therapy in developing primate lungs reverses pulmonary pathobiology associated with allergic airway disease (AAD) and (2) define the impact of ICS on postnatal lung growth and development in primates. METHODS: Infant rhesus monkeys were exposed, from 1 through 6 months, to filtered air (FA) with house dust mite allergen and ozone using a protocol that produces AAD (AAD monkeys), or to FA alone (Control monkeys). From three through 6 months, the monkeys were treated daily with ICS (budesonide) or saline. RESULTS: Several AAD manifestations (airflow restrictions, lavage eosinophilia, basement membrane zone thickening, epithelial mucin composition) were reduced with ICS treatment, without adverse effects on body growth or adrenal function; however, airway branching abnormalities and intraepithelial innervation were not reduced. In addition, several indicators of postnatal lung growth and differentiation: vital capacity, inspiratory capacity, compliance, non-parenchymal lung volume and alveolarization, were increased in both AAD and Control monkeys that received ICS treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Incomplete prevention of pathobiological changes in the airways and disruption of postnatal growth and differentiation of airways and lung parenchyma in response to ICS pose risks for developing primate lungs. These responses also represent two mechanisms that could compromise ICS therapy's ability to alter clinical disease course in young children.

Airway generation-specific differences in the spatial distribution of immune cells and cytokines in allergen-challenged rhesus monkeys.

BACKGROUND: Accumulation of immune cell populations and their cytokine products within tracheobronchial airways contributes to the pathogenesis of allergic asthma. It has been postulated that peripheral regions of the lung play a more significant role than proximal airways with regard to inflammatory events and airflow obstruction. OBJECTIVE: To determine whether immune cell populations and associated cytokines are uniformly distributed throughout the conducting airway tree in a non-human primate model of allergic asthma. METHODS: We used a stereologic approach with a stratified sampling scheme to measure the volume density of immune cells within the epithelium and interstitium of trachea and 4-5 intrapulmonary airway generations from house dust mite (HDM) (Dermatophagoides farinae)-challenged adult monkeys. In conjunction with immune cell distribution profiles, mRNA levels for 21 cytokines/chemokines and three chemokine receptors were evaluated at four different airway generations from microdissected lungs. RESULTS: In HDM-challenged monkeys, the volume of CD1a+ dendritic cells, CD4+ T helper lymphocytes, CD25+ cells, IgE+ cells, eosinophils, and proliferating cells were significantly increased within airways. All five immune cell types accumulated within airways in unique patterns of distribution, suggesting compartmentalized responses with regard to trafficking. Although cytokine mRNA levels were elevated throughout the conducting airway tree of HDM-challenged animals, the distal airways (terminal and respiratory bronchioles) exhibited the most pronounced up-regulation. CONCLUSION: These findings demonstrate that key effector immune cell populations and cytokines associated with asthma differentially accumulate within distinct regions and compartments of tracheobronchial airways from allergen-challenged primates.

Smooth muscle hypertrophy in distal airways of sensitized infant rhesus monkeys exposed to house dust mite allergen.

BACKGROUND: Airway smooth muscle hypertrophy is closely associated with the pathophysiology of hyper-reactive airways in allergic asthma. OBJECTIVE: To determine whether repeated exposure to allergens during postnatal lung development promotes remodelling of airway smooth muscle. METHODS: Infant, male rhesus monkeys (30-day-old) were sensitized to house dust mite allergen (HDMA) and then exposed to HDMA aerosol periodically over 5 months. Smooth muscle mass and bundle size and abundance in conducting airways were measured and compared with age-matched control (filtered air-exposed) monkeys. RESULTS: Total smooth muscle mass and average bundle size were significantly greater in the conducting airways of monkeys exposed to HDMA. Smooth muscle bundle abundance was not affected by exposure to HDMA. CONCLUSION: Repeated cycles of allergen exposure alter postnatal morphogenesis of smooth muscle, affecting both total mass and bundle size, in conducting airways of infant monkeys.

The remodelled tracheal basement membrane zone of infant rhesus monkeys after 6 months of recovery.

BACKGROUND: In previous studies, we showed that repeated exposure to (1) house dust mite allergen (HDMA) (Dermatophagoides farinae) caused thickening of the basement membrane zone (BMZ) and (2) HDMA+ozone (O3) caused depletion of BMZ perlecan and atypical development of BMZ collagen (irregular thin areas<2.0 microm in width). OBJECTIVE: The purpose of this study was to determine if these remodelling changes were reversible after 6 months of recovery. METHODS: Rhesus monkeys were exposed to a regimen of HDMA and or O3 or filtered air (FA) for 6 months. After the exposure protocol was completed FA and O3 groups were allowed to recover in FA for 6 months. The HDMA and HDMA+O3 exposure groups recovered in a modified environment. They were re-exposed to HDMA aerosol for 2 h at monthly intervals during recovery in order to maintain sensitization for pulmonary function testing. To detect structural changes in the BMZ, collagen I and perlecan immunoreactivity were measured and compared to data from the previous papers. RESULTS: The remodelled HDMA group had a significantly thicker BMZ and after 6 months of recovery the width had not regressed. In the remodelled BMZ of the HDMA+O3 group, perlecan had returned to the BMZ after 6 months of the recovery protocol, and the thin, irregular, collagen BMZ had been resolved. CONCLUSION: In summary, this study has shown that: (1) The width of the remodelled HDMA BMZ did not regress during a recovery protocol that included a sensitizing dose of HDMA. (2) The atypical collagen BMZ in the HDMA+O3 BMZ was resolved in the absence of O3. (3) Depletion of perlecan from the BMZ by O3 was reversed by recovery in the absence of O3.

Total number and mean size of alveoli in mammalian lung estimated using fractionator sampling and unbiased estimates of the Euler characteristic of alveolar openings.

Estimation of alveolar number in the lung has traditionally been done by assuming a geometric shape and counting alveolar profiles in single, independent sections. In this study, we used the unbiased disector principle to estimate the Euler characteristic (and thereby the number) of alveolar openings in rat lungs and rhesus monkey lung lobes and to obtain robust estimates of average alveolar volume. The estimator of total alveolar number was based on systematic, uniformly random sampling using the fractionator sampling design. The number of alveoli in the rat lung ranged from 17.3 x 10(6) to 24.6 x 10(6), with a mean of 20.1 x 10(6). The average number of alveoli in the two left lung lobes in the monkey ranged from 48.8 x 10(6) to 67.1 x 10(6) with a mean of 57.7 x 10(6). The coefficient of error due to stereological sampling was of the order of 0.06 in both rats and monkeys and the biological variation (coefficient of variance between individuals) was 0.15 in rat and 0.13 in monkey (left lobe, only). Between subdivisions (left/right in rat and cranial/caudal in monkey) there was an increase in variation, most markedly in the rat. With age (2-13 years) the alveolar volume increased 3-fold (as did parenchymal volume) in monkeys, but the alveolar number was unchanged. This study illustrates that use of the Euler characteristic and fractionator sampling is a robust and efficient, unbiased principle for the estimation of total alveolar number in the lung or in well-defined parts of it.

Immune and airway effects of house dust mite aeroallergen exposures during postnatal development of the infant rhesus monkey.

BACKGROUND: The effect of chronic environmental aeroallergen exposure on the immune system and airways has not been experimentally defined in very young children. OBJECTIVE: The purpose of this study was to determine the immunophenotype of peripheral blood and airway leucocytes in the newborn rhesus macaque monkey, following recurrent aerosol exposure to house dust mite (HDM) (Dermatophagoides farinae). METHODS: A regimen of HDM aerosolization was initiated for 2 h per day, three times per week, starting when rhesus macaque monkeys were 1 week of age. All monkeys were inoculated with diptheria, tetanus, and acellular pertussis vaccine at 5 weeks of age to simulate human infant vaccination schedules. RESULTS: Following 8 weeks of HDM aeroallergen exposure, infant monkeys exhibited a significant reduction in the total peripheral blood lymphocyte numbers and a decreased frequency of peripheral blood CD4+ T lymphocytes with a CD45RA-'memory' immunophenotype. Lavage CD4+ T lymphocytes from HDM-exposed monkeys showed elevated expression of CD25, as well as an increase in CD45RA-/CD62L-/CD11ahigh immunophenotype. Eosinophils were more abundant within airways of HDM-exposed monkeys, accumulating maximally within the trachea. CONCLUSION: These data demonstrate the development of immunological responses following chronic inhalation of a common environmental allergen during postnatal maturation in the non-human primate.

Allergen-specific IgG and IgA in serum and bronchoalveolar lavage fluid in a model of experimental feline asthma.

Allergic asthma, a Th2 cell driven response to inhaled allergens, has classically been thought of as predominantly mediated by IgE antibodies. To investigate the role of other immunoglobulin classes (e.g., IgG and IgA) in the immunopathogenesis of allergic asthma, levels of these allergen-specific immunoglobulins were measured in serum and mucosal fluids. Bermuda grass allergen (BGA)-specific IgG and IgA ELISAs in serum and bronchoalveolar lavage fluid (BALF) were developed and optimized in an experimental model of BGA-induced feline asthma. Levels of BGA-specific IgG and IgA significantly increased over time in serum and BALF after allergen sensitization. Additionally, these elevated levels of BGA-specific IgG and IgA were seen in conjunction with the development of an asthmatic phenotype indicated by positive intradermal skin tests, enhanced airways hyperreactivity, and increased eosinophil percentages in the BALF.

Production of polyclonal antisera against feline immunoglobulin E and its use in an ELISA in cats with experimentally induced asthma.

Serum samples from six cats with experimentally induced asthma were used to purify feline IgE using gel filtration and affinity chromatography. The resultant IgE, evaluated for purity by immunoelectrophoresis (IEP) and reactivity by Prausnitz-Kustner (PK) testing, was used to develop polyclonal rabbit anti-feline IgE antisera. Using reverse cutaneous anaphylaxis (RCA), the antisera were determined to be specific for feline IgE. The polyclonal rabbit anti-feline IgE antiserum was then validated in an allergen-specific ELISA. Serum samples from an additional five asthmatic cats sensitized with Bermuda grass allergen (BGA) were evaluated prior to sensitization, after parenteral sensitization, and after aerosol sensitization and challenge. A significant increase in serum BGA-specific IgE was noted over time.

Abrogation of bleomycin-induced lung fibrosis by nitric oxide synthase inhibitor, aminoguanidine in mice.

The effects of aminoguanidine (AG), a specific inhibitor of inducible nitric oxide synthase, on the bleomycin (BL)-induced lung fibrosis was evaluated in mice. The animals were placed into five groups: saline (SA)-instilled drinking water (SA+H(2)O), saline-instilled drinking water containing 0.5%AG (SA+0.5%AG), BL-instilled drinking water (BL+H(2)O), BL-instilled drinking water containing 0.2%AG (BL+0.2%AG), and BL-instilled drinking water containing 0.5%AG (BL+0.5%AG). The mice had free access to H(2)O or H(2)O containing AG and lab chow ad lib 2 days prior to intratracheal (IT) instillation of BL (0.07U/mouse/100 microL) or an equivalent volume of sterile isotonic saline. The mice in the SA+0.5%AG group consumed the greatest amount of AG without any ill effects than the mice in any other group. There were no differences in any of the measured biochemical determinants between the SA+H(2)O and SA+0.5%AG control groups. The IT instillation of BL in the BL+H(2)O group caused significant increases in the lipid peroxidation, hydroxyproline content, and prolyl hydroxylase activity of lungs and influx of inflammatory cells in the broncheoalveolar lavage fluid (BALF) as compared to both control groups. The intake of aminoguanidine by mice in the BL+0.5%AG group caused significant reductions in the BL-induced increases in all measured biochemical indices of lung fibrosis without any effects on the influx of inflammatory cells in the BALF. In fact, AG in both BL-treated groups additionally increased the total cell counts in the BALF from mice in the BL+0.2%AG and BL+0.5%AG groups as compared to the BL+H(2)O group. Histopathological evaluation of the lungs revealed that the mice in the BL+0.5%AG group had markedly fewer fibrotic lesions than mice in the BL+H(2)O group. These results demonstrate that aminoguanidine minimizes the BL-induced lung fibrosis at both the biochemical and the morphological level and support our earlier hypothesis that the production of nitric oxide plays a significant role in the pathogenesis lung fibrosis caused by BL.

Effect of C-fiber-mediated, ozone-induced rapid shallow breathing on airway epithelial injury in rats.

We examined the relationship between C-fiber-mediated, ozone-induced rapid shallow breathing and airway epithelial cell injury at different airway sites within the lower respiratory tract of conscious Wistar rats (n = 24). We combined an acute 8-h ozone inhalation with vagal perineural capsaicin treatment, a selective C-fiber conduction block, and 5-bromo-2'-deoxyuridine (BrdU) labeling as an index of epithelial injury. Vehicle-treated rats that inhaled ozone developed a rapid shallow breathing pattern during ozone inhalation, whereas the capsaicin-treated rats that inhaled ozone showed no changes in respiratory frequency. In vehicle-treated, ozone-exposed rats that developed rapid shallow breathing, a progressive increase in BrdU-labeling density (no. of BrdU-labeled cells/mm(2) airway) was observed starting at the bifurcation of the left main stem bronchi (central airway) and going down either a short or long airway path. In vehicle-treated, ozone-exposed rats, terminal bronchioles supplied by short and long airway paths had a similar degree of BrdU-labeling density that was significantly (P < 0.05) greater than the BrdU-labeling density of the proximal airways that supply them. In contrast, the attenuation of rapid shallow breathing produced by capsaicin treatment resulted in a significantly reduced BrdU-labeling density in the terminal bronchioles supplied by short airway paths compared with the terminal bronchioles supplied by long airway paths. Our data indicate that ozone-induced rapid shallow breathing protects large conducting airways while producing a more even distribution of injury to terminal bronchioles.

Mild and moderate asthma is associated with airway goblet cell hyperplasia and abnormalities in mucin gene expression.

Excessive airway mucus is an important cause of morbidity and mortality in asthma, but the relationship between accumulation of mucus and goblet cell size, number, and function is incompletely understood. To address these questions, stored mucin in the epithelium and goblet cell size and number were measured morphometrically, and mucin gene expression was measured by polymerase chain reaction and immunohistochemistry in endobronchial biopsies from 13 subjects with mild and moderate asthma and from 12 healthy control subjects. Secreted mucin was measured in induced sputum. We found that stored mucin in the airway epithelium was three times higher than normal in the subjects with asthma (p < 0.005). Goblet cell size was similar in both groups, but goblet cell number was significantly higher in the subjects with asthma (93,043 +/- 15,824 versus 41,959 +/- 9,230/mm3, p < 0.05). In mild asthma (FEV1 > or = 80% pred, n = 7), the level of stored mucin was as high as in moderate asthma (FEV1 < 80% pred, n = 6), but the level of secreted mucin was significantly lower (28.4 +/- 6.3 versus 73.5 +/- 47.5 microg/ml, p < 0.05). Secreted mucin was inversely correlated with stored mucin for the whole asthma group (rs = -0.78, p = 0.007). MUC5AC was the predominant mucin gene expressed in healthy subjects and subjects with asthma, and MUC5AC protein was increased in the subjects with asthma. We conclude that even mild asthma is associated with goblet cell hyperplasia and increased stored mucin in the airway epithelium, whereas moderate asthma is associated with increased stored mucin and secreted mucin. These findings suggest that acute degranulation of hyperplastic goblet cells may represent a mechanism for asthma exacerbations in mild and moderate asthma and that chronic degranulation of goblet cells may contribute to chronic airway narrowing in moderate asthma.

Mechanism of dexamethasone-mediated interleukin-8 gene suppression in cultured airway epithelial cells.

The effects of dexamethasone, a glucocorticoid analog, on interleukin 8 (IL-8) gene expression were studied in cultures of primary human tracheobronchial epithelial cells and an immortalized human bronchial epithelial cell line, HBE1 cells. Dexamethasone inhibited IL-8 mRNA and protein expression in a concentration- and time-dependent manner. The inhibition did not occur at the transcriptional level since both nuclear run-on activity and IL-8 promoter-reporter gene expression assay revealed no significant effect. Instead, there was a change in IL-8 mRNA stability in dexamethasone-treated cultures. Under actinomycin D treatment, IL-8 mRNA was quite stable in dexamethasone-depleted cultures, while in dexamethasone-pretreated cultures, IL-8 message was rapidly degraded within the first hour, then leveled off. When dexamethasone and actinomycin D were added simultaneously to dexamethasone-depleted cultures, IL-8 mRNA remained rather stable. When cycloheximide was used to inhibit new protein synthesis, dexamethasone-dependent inhibition was not observed. These results suggest that a posttranscriptional mechanism, which requires dexamethasone-dependent new protein synthesis, is involved in the regulation of IL-8 mRNA by dexamethasone in airway epithelial cells.

Allergic asthma induced in rhesus monkeys by house dust mite (Dermatophagoides farinae).

To establish whether allergic asthma could be induced experimentally in a nonhuman primate using a common human allergen, three female rhesus monkeys (Macaca mulatta) were sensitized with house dust mite (Dermatophagoides farinae) allergen (HDMA) by subcutaneous injection, followed by four intranasal sensitizations, and exposure to allergen aerosol 3 hours per day, 3 days per week for up to 13 weeks. Before aerosol challenge, all three monkeys skin-tested positive for HDMA. During aerosol challenge with HDMA, sensitized monkeys exhibited cough and rapid shallow breathing and increased airway resistance, which was reversed by albuterol aerosol treatment. Compared to nonsensitized monkeys, there was a fourfold reduction in the dose of histamine aerosol necessary to produce a 150% increase in airway resistance in sensitized monkeys. After aerosol challenge, serum levels of histamine were elevated in sensitized monkeys. Sensitized monkeys exhibited increased levels of HDMA-specific IgE in serum, numbers of eosinophils and exfoliated cells within lavage, and elevated CD25 expression on circulating CD4(+) lymphocytes. Intrapulmonary bronchi of sensitized monkeys had focal mucus cell hyperplasia, interstitial infiltrates of eosinophils, and thickening of the basement membrane zone. We conclude that a model of allergic asthma can be induced in rhesus monkeys using a protocol consisting of subcutaneous injection, intranasal instillation, and aerosol challenge with HDMA.

Expression of the beta6 integrin subunit is associated with sites of neutrophil influx in lung epithelium.

Inhalation of ozone by Rhesus monkeys results in epithelial injury and granulocyte influx in both conducting airways and respiratory bronchioles. We have reported that ozone-induced neutrophil recruitment and subsequent epithelial repair can be inhibited in vivo with a CD18 antibody. The antibody-mediated effect is abrogated by local instillation of C5a (a CD18-independent neutrophil chemoattractant), thereby demonstrating a role for neutrophils in lung epithelial repair processes. As an extension of this study, we examined the effect of ozone and neutrophil influx on epithelial expression of the beta6 integrin, an adhesion molecule associated with proliferation and repair. Expression of beta6 integrin was determined by immunohistochemistry for ozone-exposed monkeys treated with either control immunoglobulins or a CD18 antibody. The tracheal epithelium of ozone-exposed monkeys treated with control immunglobulins expressed the beta6 integrin. In contrast, the tracheal epithelium of ozone-exposed monkeys treated with CD18 antibody exhibited very low to undetectable expression of beta6 integrin. In association with C5a instillation and neutrophil influx, beta6 integrin was also observed in respiratory bronchiolar epithelium from both control and ozone-exposed animals. These findings cumulatively suggest that lung epithelial cell expression of beta6 integrin is associated with sites of neutrophil recruitment.

Effect of antibody against integrin alpha4 on bleomycin-induced pulmonary fibrosis in mice.

Integrins are a family of transmembrane glycoproteins that can interact with components of the extracellular matrix. The alpha4beta1 and alpha4beta7 integrins are heterodimeric leukocyte cell surface molecules critical to their cell and matrix adhesive interactions. Evidence for a central role for the alpha4 integrins in leukocyte pathophysiology in the lung is well documented. In this study, we tested the hypothesis that neutralizing antibody for integrin alpha4 (PS2) may reduce bleomycin (BL)-induced lung fibrosis in vivo. Male C57BL/6 mice were injected intratracheally with saline (SA) or BL (0.08 U/mouse) followed by intraperitoneal injection of SA, isotype control antibody (1E6), or PS2 (100 microg) three times a week. Twenty-one days after the intratracheal instillation, mice were killed for bronchoalveolar lavage (BAL), biochemical, histopathological, and immunohistological analyses. Treatment with PS2 significantly reduced BL-induced increases in lung lipid peroxidation and hydroxyproline content. Lung histopathology also showed reduced fibrotic lesions in the BL-treated lungs by treatment with PS2. BL-treated mouse lungs also showed induction of cells with the myofibroblast phenotype, as indicated by the increased expression of alpha-smooth muscle actin (alphaSMA), whereas treatment with PS2 minimized the BL-induced alphaSMA expression. Furthermore, treatment with PS2 reduced the BL-induced increase in the BAL total cell number, and attenuated the BL-induced increase in the BAL protein level. It is concluded that integrin alpha4 may play an important role in BL-induced pulmonary fibrosis, and the use of anti-alpha4 antibody offers therapeutic antifibrotic potential in vivo.

Epithelial desquamation in asthma: artifact or pathology?

To determine whether the denudation of the bronchial epithelium observed in endobronchial biopsies from asthmatic subjects is a true pathologic feature or an artifact of tissue sampling, we analyzed epithelial integrity in bronchial biopsies from 14 subjects with mild and moderate asthma and 12 healthy subjects. In each subject, 4 to 8 bronchial biopsies were taken from large airways during bronchoscopy, fixed in 4% paraformaldehyde, embedded in glycomethacrylate, cut into 2-microM sections, and stained with toluidine blue. A x4 image of each biopsy was copied to a computer file using a video camera, and lines were drawn and measured along the basement membrane underlying areas completely denuded of overlying epithelium, areas covered by a single layer of basal cells, and areas of intact epithelium. We found that the percentage of basement membrane that was denuded of epithelium was similar in the healthy and asthmatic subjects (14.8 +/- 11.8 versus 11.4 +/- 9.8% respectively, p = 0.38); the percentage of basement membrane that was covered by a single layer of basal cells was also similar in the two groups (46.4 +/- 11.0 versus 54.5 +/- 9.8%, respectively, p = 0. 11). In the asthmatic subjects, we found no significant correlation between the percentage of basement membrane covered by denuded epithelium or by a single layer of basal cells and the FEV(1) percentage of predicted or the PC(20) methacholine. We conclude that denudation of bronchial epithelium in endobronchial biopsies from asthmatic subjects with stable mild and moderate disease is an artifact of tissue sampling and is not a true pathologic feature of the disease, and that the extent of airway epithelial denudation is not correlated with the severity of airway narrowing or the severity of bronchial hyperresponsiveness.

Effect of respiratory pattern on ozone injury to the airways of isolated rat lungs.

Ozone stimulates the "defensive" C-fibers in the lungs, changing breathing pattern to rapid and shallow. We hypothesized that when ozone is administered to the isolated lung with a rapid shallow breathing pattern rather than a slow deep pattern, relatively less airway epithelial damage would occur. Four groups of isolated buffer perfused rat lungs were exposed to ozone (1 ppm) or to filtered air for 90 min with either a slow deep (SDB, tidal volume 2.4 ml, frequency 40 breaths/min) or a rapid shallow breathing pattern (RSB, tidal volume 1.2 ml, frequency 80 breaths/min), resulting in an equivalent inspired dose. The absorbed dose of ozone did not differ between the exposed groups. Ethidium homodimer-1 was then instilled into the trachea to identify injured airway epithelial cells. The lungs were fixed, the airways were microdissected, and the airway epithelial cells were counterstained with YPRO-1 prior to evaluation with confocal microscopy. Ozone-induced airway epithelial cell injury occurred to a lesser overall degree when lungs were exposed by the RSB pattern (p = 0.003). The relative reduction in injury was greater (p < 0.05) in the proximal axial airway than in its adjacent airway branch and terminal bronchioles. Ozone induced an increase in pulmonary resistance with the SDB pattern but not with the RSB pattern. Thus, at an equivalent dose of inspired ozone, a RSB pattern resulted in less total damage than a SDB pattern and the distribution of protection was heterogeneous with proximal axial airways displaying the greatest relative reductions in epithelial damage.

Ozone-induced epithelial injury in the ferret is similar to nonhuman primates.

Extrapolation to humans from rodent ozone exposure is limited by the anatomic differences between the species. Ferrets have similar pulmonary structures to humans, with well developed respiratory bronchioles and submucosal glands. We exposed adult ferrets, monkeys, and rats to 1 ppm ozone (O(3)) or filtered air for 8 h followed by 1 h in filtered air. Bronchoalveolar lavage (BAL) analysis, histopathology, and confocal microscopy were used to evaluate ozone-induced epithelial injury and inflammation. BAL showed significantly increased numbers of neutrophils in ozone-exposed as compared with filtered air ferrets, monkeys, and rats. However, there were 3- to 4-fold more neutrophils in monkeys and ferrets per milliliter of BAL than in rats. Ozone-exposed lungs showed a severe, acute infiltration of neutrophils in regions with necrotic epithelial cells, especially in the centriacinar region that was more severe in ferrets and monkeys than rats. We conclude that acute ozone exposure in ferrets induce severe epithelial necrosis and inflammation, results in similar epithelial injury compared with monkeys, and represents a better model of humans than rodents.