Introduction

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Ozone (O₃), the principal oxidant gas of photochemical smog, represents a major health concern in urban areas because of its potential toxic effects. O₃ induces a variety of morphologic, functional, biochemical, and immunologic alterations in the respiratory tract of humans and experimental animals at near-ambient and higher concentrations. However, the exact mechanisms that account for O₃-induced injury are not well characterized. The morphologic response to acute O₃ inhalation involves epithelial cell injury, which results in cell necrosis, loss, and replacement. In particular, type I alveolar and ciliated epithelial cells of the centriacinar region appear to be the most sensitive.

O₃-induced epithelial cell damage is accompanied by a predominantly neutrophilic inflammatory infiltrate (1). Neutrophil emigration to the sites of epithelial injury is a fairly rapid process, with maximal migration occurring between 8 and 12 h after initiation of O₃ exposure in the rat (3). Although their exact role in the pathophysiology of O₃-induced injury is not entirely clear, neutrophils have been shown to play a key role in the pathogenesis of both experimental and naturally occurring types of lung injury. Previous work has demonstrated that the products of activated neutrophils, including oxidants, proteases, and defensins, are capable of disrupting epithelial integrity as well as causing direct cytotoxicity (4).

Although previous data imply that neutrophils exacerbate or directly cause O₃-induced epithelial damage, recent evidence has demonstrated a possible beneficial role for the neutrophil. A prior study in our laboratory in monkeys exposed to O₃ demonstrated a strong relationship between epithelial necrosis and repair and the emigration and retention of neutrophils in the epithelium and interstitium at all levels of the tracheobronchial tree (2). Morphometric techniques showed that the degree of airway epithelial repair following exposure was directly associated with an increase of neutrophils adjacent to necrotic epithelial cells. A subsequent study in our laboratory showed that rats, depleted of circulating neutrophils prior to O₃ exposure, had no significant difference in the amount of epithelial degeneration and necrosis compared with normal rats exposed to O₃. However, the neutrophil-depleted group showed a marked delay in the onset of epithelial repair during the postexposure recovery period (5). No data were available to determine whether the response profile for neutrophils corresponded to epithelial cell mitogenesis. A recent study in our laboratory demonstrated that neutrophils facilitate the removal of injured alveolar epithelial cells after exposure to low concentrations of O₃ in vitro (6). Such in vivo and in vitro data suggest that neutrophils facilitate the repair process by assisting with the destruction and/or removal of O₃-injured airway epithelial cells.

In addition to inducing epithelial necrosis and inflammation, acute O₃ inhalation is known to result in numerous physiologic responses including the lung C-fiber-initiated reflex of rapid, shallow breathing (7, 8). This reflex is thought to be the result of the stimulation of vagal airway C-fiber afferent nerves by eicosanoic inflammatory mediators released from injured airways (9). The specific cell type that produces these eicosanoic inflammatory mediators and the role neutrophils play in their release during exposure and subsequent recovery remain uncertain. Rapid, shallow breathing is thought to effect the distribution of the inhaled dose of O₃ (10) and influence the distribution and severity of O₃-induced injury and inflammation in the airways. Therefore, the effects of rapid, shallow breathing and its effect on local tissue dose must be considered when assessing O₃-induced injury and inflammation at a specific region of the respiratory tract.

The present study was undertaken to examine the effect of O₃ on nasal and airway epithelial injury and repair in rats depleted of circulating neutrophils compared with neutrophil-sufficient rats. The design of the experiments enabled us to measure changes in breathing pattern to assess the role of neutrophils in the physiologic response to O₃ inhalation. We also evaluated the severity and distribution of O₃-induced epithelial injury (vacuolation, necrosis, and sloughing) throughout the respiratory tract using a semiquantitative scoring system. Finally, morphometric techniques measuring the incorporation of 5-bromo-2-deoxyuridine (BrdU) into replicating epithelial cells within terminal bronchioles enabled us to assess the repair process in neutrophil-sufficient rats compared with neutrophil-depleted rats.

	Materials and Methods

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Animals and Neutrophil Depletion

We obtained male Wistar rats, approximately 43 d old, from a specific pathogen-free colony (Charles River Laboratories, Wilmington, MA) and housed them in polycarbonate cages with fir wood chips. Animals were fed Purina Rodent Laboratory Chow 5001 (Purina Mills Inc., St. Louis, MO) and water ad libitum and maintained on a 12-h light/dark photoperiod. Rats were allowed to acclimate in the cages for at least 1 wk prior to the O₃ exposure. We studied 37 animals between 6 and 9 wk old (181 to 340 g).

We randomly divided rats into two groups: neutrophil-depleted animals and normal rabbit serum (NRS) controls. Rats in the first group were given a single, 1-ml intraperitoneal injection of rabbit antirat neutrophil serum (ANS) (Accurate Chemical Inc., Buffalo, NY) 12 h prior to O₃ or filtered air (FA) exposure. A pilot study using a single intraperitoneal injection of ANS in rats showed that there was a marked reduction in circulating neutrophils by 12 h and neutrophil numbers remained depressed through 48 h, which was ample time to complete our experiments. The second group was given a single, 1-ml intraperitoneal injection of NRS 12 h prior to exposure. The experimental design and exposure regimens were as follows: (1) NRS-treated rats exposed to FA for 16 h (n = 10), (2) NRS-treated rats exposed to 1 ppm O₃ for 8 h followed by 8 h of FA (n = 9), (3) ANS-treated rats exposed to FA for 16 h (n = 9), and (4) ANS-treated rats exposed to 1 ppm O₃ for 8 h followed by 8 h of FA (n = 9). Therefore, the experimental time for each rat was 16 h. Rats from both the ANS- and NRS-treated groups were killed at the end of the 16-h experiment time for collection of lungs and nasal cavities using the methods described below (see INTRAVASCULAR PERFUSION FIXATION AND TISSUE PREPARATION section). We made blood smears from all rats using blood obtained by clipping the tail tip while the rats were still anesthetized. The blood smears were stained with a modified Wright's stain (LeukoStat; Fisher Scientific, Pittsburgh, PA), and differential cell counts (200 cells counted per smear) were performed to assess the effectiveness of the ANS in causing neutrophil depletion.

Anesthesia and O₃ Exposure

Rats were initially anesthetized with a solution of 2% -chloralose, 25% urethane, and 5% sodium tetraborate (Sigma, St. Louis, MO), 0.3 ml/100 g body weight intraperitoneally. We then aseptically placed a catheter into the femoral vein of each rat. We attached the catheter to an infusion pump and maintained anesthesia with a constant infusion of 2% -chloralose, 25% urethane, and 5% sodium tetraborate. Body temperature was maintained at 37 ± 0.5°C by placing the rats on a water-filled heating pad. We monitored body temperature via a rectal probe and adjusted the thermostat on the heating pad as needed.

We used a head-only exposure system to expose rats to O₃ (1 ppm) or FA (Figure 1). Briefly, FA and medical-grade oxygen flowed through a calibrated flow-control panel at known rates. Oxygen was passed through a Sanders Model 25 ozonizer (Germany) to produce O₃. The O₃ was then mixed with FA (3 liters/min) and analyzed by a Dasibi O₃ analyzer (Model 1003H; Dasibi Environmental Corp., Glendale, CA) before passing to the glass exposure hood. The hood consisted of a glass cylinder (5 cm inside diameter, 7 cm high, 140 ml volume) with an inlet port and an exhaust port. We used an O₃-resistant silicone rubber diaphragm with a hole large enough to allow a rat's head to pass through it to cover the open end of the hood. We applied liberal amounts of a high-viscosity silicone lubricant (7 Release Compound; Dow Corning, Midland, MI) to the neck of each rat to prevent leakage of gas between the rubber diaphragm and the animal. Any gas (1 liter/ min) passing through the hood then passed through a pneumotachograph (Model Series 8300; Hans Rudolph, Inc., Kansas City, KS) attached to the exhaust port. We vented the exhaust gas from the laboratory directly to the outside. We used a second similar system delivering only FA (1 liter/min) to enable us to study two rats simultaneously. A control breathing period was obtained by allowing the rats to breathe FA for 10 min. One rat received only FA for 16 h, and the other rat received 1 ppm O₃ for 8 h, followed by an 8-h FA postexposure period.

View larger version (26K): [in this window] [in a new window]   Figure 1.   Schematic of system used to expose two anesthetized rats simultaneously. One rat received FA for 16 h, while the other rat received 1 ppm ozone for 8 h followed by an 8-h FA postexposure period. Anesthesia was maintained with a constant infusion of 2% -chloralose, 25% urethane, and 5% sodium tetraborate. A flow rate of 1 liter/min into each exposure hood was controlled with a calibrated flow panel.

Physiologic Measurements

We used the pneumotachographs attached to the outlet ports of the exposure hoods to measure tidal volume (VT), breathing frequency (f), and minute ventilation (E). We balanced the pneumotachographs to zero voltage with the full flow (1 liter/min) going through them. The rats were subsequently attached to the system, and their VT were measured by integrating the periodic change in flow passing through the pneumotachographs using the PO-NE-MAH digital data acquisition and analysis system (Gould Instrument Systems, View Valley, OH). VT, f, and E were measured during the entire 16-h experimental period and recorded for 120 s every 20 min and then averaged to give a mean value for VT, f, and E. An hourly time point was obtained by averaging three consecutive means. The flow panel controls were manually adjusted as needed to maintain an O₃ concentration of 1.0 ± 0.05 ppm over the 8-h exposure period without altering the flow rate of 1 liter/min through both hoods.

In Vivo Cumulative Cell Labeling

We placed one osmotic minipump (ALZET pump Model 2ML1, nominal pumping rate 10 µl/h; ALZA Corp., Palo Alto, CA) subcutaneously between the shoulder blades of the anesthetized rats after insertion of the femoral vein catheter. The pump was loaded with BrdU (Sigma, St. Louis, MO) dissolved in phosphate-buffered saline (PBS) (20 mg/ml). To ensure immediate release of BrdU, the pumps were primed by immersion in 0.9% saline at 37°C 8 h prior to insertion. BrdU, a thymidine analog incorporated into DNA by cells undergoing DNA replication, is indicative of cell proliferation and a marker of O₃-induced airway epithelial injury (11). The duodenum served as a positive labeling control for each rat. The incorporated BrdU was detected using a monoclonal antibody and immunocytochemical techniques as outlined by Hsu and coworkers (12, 13). Briefly, 30-µm-thick paraffin sections were deparaffinized in three changes of xylene (5 min each) and rehydrated in a graded series of ethyl alcohol (100%-95%-75% distilled water, 5 min each). The tissue sections were then incubated in 2.5 N HCl for 20 min at 37°C to denature the DNA. We neutralized the acid by two borate buffer washes (5 min each) and a PBS wash (5 min). We incubated the sections in 3% hydrogen peroxide for 30 min to block the endogenous peroxidase activity, washed the sections for 10 min in PBS, and then incubated the sections in 5% NRS with 5% powdered milk for 1 h to block nonspecific binding. After blotting excess fluid, we incubated the sections with the primary monoclonal mouse antiBrdU (Dako Corp., Carpenteria, CA) diluted 1:50 in PBS with 5% powdered milk for 2 h. Tissue sections incubated with 5% powdered milk in PBS were used as negative controls. After washing with PBS, the sections were incubated for 30 min in biotinylated, rabbit antimouse immunoglobulin (Dako) diluted 1:200 in PBS with 5% NRS. We followed this procedure with two PBS washes and incubation of the sections in Vectastain ABC reagent (Vector Laboratories, Burlingame, CA) for 30 min. After two washes in PBS (5 min each), the sections were incubated in peroxidase substrate solution (7.5 µl 30% hydrogen peroxide in 1.0 mg/ml diaminobenzidine in 0.1 M Tris-HCl buffer at pH 7.6) for 3 to 5 min. Finally, we rinsed the sections with PBS and then distilled water. Labeled nuclei appeared reddish-brown by light microscopy, as no counterstain was used.

Intravascular Perfusion Fixation and Tissue Preparation

We used intravascular perfusion to fix the lungs and nasal cavity via the pulmonary artery and aorta, respectively. This fixation method was chosen to avoid the redistribution of intraluminal inflammatory cells and sloughed epithelial cells in airways and alveoli (14). Briefly, a tracheostomy was performed on the anesthetized rats and the lungs were ventilated using a small animal respirator (60 breaths/min). The chest was opened and 0.15 ml of heparin (1,000 U/ml) was injected into the right and left ventricles. We then cannulated the pulmonary artery and aorta. We flushed the pulmonary vasculature with PBS until the lungs were white (approximately 30 s). Prior to flushing, both atria were removed to allow the escape of fluid from the pulmonary and systemic circulation. We then inflated the lungs with air via the tracheal cannula to a pressure of 30 cm water, slowly deflated them to 12 cm water, and held the pressure there. Finally, we used a buffered formalin fixative (Z-Fix; Anatech Ltd., Battle Creek, MI) to perfuse the pulmonary and systemic vasculature. Perfusion was performed in a nonrecirculating fashion at a pressure of approximately 18 mm Hg for the lungs and 40 mm Hg for the nasal cavity for 15 to 25 min. Lungs, heads, and duodenums (BrdU-positive control tissue) were then stored in fixative for at least 48 h before processing.

Beginning at the level of the lobar bronchus, we used a dissecting microscope and a cool fiberoptics illuminator to microdissect the fixed right cranial lung lobe along its long axis. This dissection plane through the lobe exposed the majority of small side branches. The tissues were then routinely processed and embedded in paraffin for light microscopy. We sagittally cut a 7-µm-thick section from the embedded tissue along the axial pathway and stained it with hematoxylin and eosin (H&E). A second serial 30-µm-thick section was also cut and immunohistochemically stained to identify cells labeled with BrdU as described previously. After fixation and removal of the musculature, skin, lower jaw, and eyes, we decalcified the heads in 13% formic acid for at least 4 d and then rinsed them in tap water for at least 4 h. We transversely sectioned the nasal cavity of each rat at or immediately posterior to the upper incisor teeth as previously described (15). The nasal cavity was then routinely processed and embedded in paraffin for light microscopy. Six-micron-thick sections were cut from the anterior surfaces of the paraffin-embedded blocks and stained with H&E.

Airway Morphology and Semiquantitative Scoring System

We used a semiquantitative scoring system to assess the H&E sections of nasal cavities and lungs by light microscopy without knowledge of exposure group (Table 1). Scoring systems have been used to provide a simple but relatively reproducible method to assess pathologic changes in the lung (16, 17). We evaluated 17 histopathologic features within the nasal cavity, bronchi, terminal bronchioles, and proximal alveolar ducts that were divided into two broad categoriesinflammatory/exudative changes and epithelial changesand scored each feature in a semiquantitative fashion. Scores were based on a zero to 5 grading scheme where 1 = absent, 2 = occasional, 3 = less than 25% tissue involvement, 4 = 25 to 50% tissue involvement, and 5 = greater than 50% tissue involvement.

Morphometry of Lungs

Under low magnification, we examined the 7-µm-thick H&E sections and numbered the terminal bronchioles in a stratified manner on the slide, using NIH Image software.¹ Four terminal bronchioles were systematically sampled from the 10 to 20 terminal bronchioles sectioned per slide using a random start. We used an optical disector to count the BrdU-positive epithelial cells (18). We chose to assess BrdU labeling in the terminal bronchioles because the primary lesion of acute O₃ toxicity in the lung is epithelial cell necrosis, especially in the centriacinar regions (3).

Stereologic techniques were used to determine the number of BrdU-positive cells per epithelial basement membrane surface area (18). Briefly, we estimated the number of cells per volume of epithelium using the following equation:

where N_{V BrdU/epi} is the number of BrdU-positive cells per volume of epithelium; N_BrdU is the number of BrdU-positive cells counted in the 30-µm sections, excluding those that intersected the top of the section and two sides of the counting frame; A_epi is the area of epithelium in the counting frame; and H_epi is the height of the optical disector (usually 30 µm). Using Stereology Toolbox (Morphometrix, Davis, CA) software and the adjacent 7-µm H&E vertical section, we estimated the surface area of the basement membrane to the epithelial volume (S_{V bm/epi}) by point and intersection counting as follows:

where I_bm are intersections of the test line of a cycloid grid with the epithelial basement membrane and L_epi is the test line length over the epithelium. Finally, we normalized the number of BrdU-labeled epithelial cells by the epithelial basement membrane surface area to allow us to compare epithelial cells between terminal bronchioles as shown in the following equation:

where the epithelial volumes are the same and divide to one.

Statistical Analysis

We analyzed breathing pattern data using a three-way analysis of variance (ANOVA) with repeated measures, where treatment (NRS-treated, ANS-treated) and exposure (FA, O₃) were the grouping factors and time was the repeated measure to check for significant interactions. Differences between the four groups (NRS-treated FA-exposed, NRS-treated O₃-exposed, ANS-treated FA-exposed, and ANS-treated O₃-exposed) at each time point were analyzed using ANOVA and the Student-Newman-Keuls test (SAS, Version 6.01; SAS Institute Inc., Cary, NC). We used the nonparametric Kruskal-Wallis and Mann-Whitney U statistical tests to evaluate the semiquantitative scoring system. We analyzed all other data by ANOVA and Fisher's least significant difference test (SYSTAT 5 for the Macintosh, Version 5.2; SYSTAT, Inc., Evanston, IL). We present all data as means ± SEM unless otherwise stated. Statistical significance is accepted for P < 0.05.

	Results

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Peripheral Blood Smears

The percentages of circulating monocytes, lymphocytes, and neutrophils are shown in Figure 2. The ANS significantly decreased the mean percentage of neutrophils in the peripheral blood to less than 2.5% of the total white blood cells (WBCs) in both ANS-treated groups (FA exposed and O₃ exposed). Both the NRS-treated rats exposed to FA and the NRS-treated rats exposed to O₃ had neutrophil values that were higher than expected for Wistar rats. The NRS-treated rats exposed to FA had a mean neutrophil percentage of 58 ± 3.7, whereas NRS-treated rats exposed to O₃ had a percentage of 64 ± 2.1. The mean neutrophil percentage for normal male rats is reported to be 12 with a range of 2 to 22 ± 2 SD (19). Eosinophils and basophils accounted for < 1% of the total WBC population and do not show up in Figure 2. Neutrophil depletion with ANS in an earlier pilot study showed no difference in total lymphocyte or monocyte numbers in NRS and ANS groups. Neutrophils were reduced to less than 2% of the WBCs by 12 h (beginning of O₃ exposure) and remained depressed through 48 h, which covered all of the experimental time (16 h) reported here.

View larger version (21K): [in this window] [in a new window]   Figure 2.   Percentage of total WBCs for circulating neutrophils, lymphocytes, and monocytes in peripheral blood from neutrophil-sufficient control rats treated with NRS and neutrophil-depleted rats treated with ANS. FA-exposed NRS controls and ANS-treated animals were exposed to FA for 16 h. O3-exposed NRS controls and ANS-treated animals were exposed to 1.0 ppm O3 for 8 h followed by an 8-h FA postexposure period. Each bar represents the mean ± SEM of 9 to 10 animals. *Significantly different (P < 0.05) from corresponding control rats.

Effect of Neutrophil Depletion on Breathing Pattern

There were no significant differences in baseline VT, f, and E values between any of the groups (Table 2). Results of the time-course data for VT, f, and E were reported as percent change to minimize the small effect of weight gain over the 3 wk of experimentation. There were no significant changes in VT, f, or E in NRS- or ANS-treated rats exposed to only FA (Figure 3). O₃ exposure in the NRS-treated and ANS-treated groups resulted in a similar progressive decrease in VT and increase in f. VT was significantly different for both groups compared to the corresponding FA-exposed group beginning at 3 h of experimental time (3 h of O₃ exposure) with a maximal change at 8 h of experimental time (8 h of exposure). The NRS-treated O₃-exposed rats had a maximal change of 22.2 ± 2.51% in VT, whereas the ANS-treated O₃-exposed rats showed a 26.6 ± 2.6% change in VT at 8 h of experimental time (8 h of exposure). Breathing frequency was significantly different for both O₃-exposed groups compared with the corresponding FA-exposed group beginning at 5 h of experimental time (5 h of exposure) with maximal change occurring at 8 h of experimental time (8 h of exposure). The NRS-treated rats exposed to O₃ showed a 29.2 ± 3.13% change in f, whereas the ANS-treated O₃- exposed rats showed a 31.7 ± 2.85% change in f at 8 h of experimental time (8 h of exposure). After discontinuation of the O₃ (at 8 h of experimental time), VT in the O₃-exposed NRS-treated group and ANS-treated group tended to increase but remained significantly different from the corresponding FA-exposed groups at all postexposure time points except one (NRS-treated O₃-exposed rats at 15 h experimental time [7 h after exposure]). Breathing frequency in the NRS-treated O₃-exposed rats showed a return toward baseline that was no longer significantly different from the NRS-treated FA-exposed rats after 11 h of experimental time (3 h after exposure). The ANS-treated O₃-exposed group showed a similar but less abrupt decrease in breathing frequency that remained significantly different from the ANS-treated FA-exposed group during the entire postexposure period. When compared with NRS-treated rats exposed to O₃, ANS-treated O₃-exposed rats showed a greater percent change in VT and f that began early in the exposure period and lasted until the end of the postexposure period. The differences in responses between the two groups were greatest in the postexposure period, especially with regard to f. There were significant differences in f between the NRS-treated rats exposed to O₃ and the ANS-treated rats exposed to O₃ at 5, 11, 13, and 15 h of experimental time (5 h of exposure and 3, 5, and 7 h postexposure, respectively). The only significant difference in VT between the same two groups was at 16 h of experimental time (8 h after exposure). There was a significant difference in E between the ANS-treated FA-exposed group and the ANS-treated O₃-exposed group at 4 h of experimental time (4 h of exposure).

View larger version (36K): [in this window] [in a new window]   Figure 3.   Time course of effect of FA and O3 on VT, f, and E in neutrophil-sufficient controls treated with NRS (a, c, e) and neutrophil-depleted rats treated with ANS (b, d, f ). Values are means ± SEM for 9 to 10 animals. NRS-treated (open triangles) and ANS-treated (open squares) rats were exposed to FA for 16 h. NRS-treated ( filled triangles) and ANS-treated ( filled squares) rats were exposed to 1 ppm O3 for 8 h followed by an 8-h FA postexposure period. *Significantly different (P < 0.05) from corresponding FA-exposed group. §Significantly different (P < 0.05) from corresponding NRS-treated group exposed to O3 at same time point. For clarity, we have omitted the opposite SEM bars.

Morphology of Nasal Cavity and Lungs

The scoring system allowed accurate assessment of inflammatory/exudative changes within the nasal cavity, bronchi, terminal bronchioles, and proximal alveolar ducts, and epithelial changes within the nasal cavity, bronchi, and terminal bronchioles. The extent and severity of 17 features were morphologically evaluated by the grading scheme (Table 1). Overall, both NRS- and ANS-treated groups exposed to O₃ showed more severe epithelial degeneration/necrosis and inflammation in the nasal cavity, bronchi, and distal airways than did the corresponding FA- exposed groups. Moreover, the ANS-treated rats exposed to O₃ showed more epithelial necrosis in the nasal cavity, bronchi, and distal airways than did the NRS-treated rats exposed to O₃. In contrast, compared with ANS-treated rats exposed to O₃, NRS-treated rats exposed to O₃ showed more severe and extensive inflammation throughout the respiratory tract.

We graded only the nasal transitional epithelium, because it appears to be the most responsive epithelial cell type in the nasal cavity to the effects of O₃ exposure (1). The nasal transitional epithelium is a nonciliated cuboidal epithelium that lines the lateral meatus in both nasal passages. This simple surface epithelium has few or no mucous goblet cells in the proximal half of the rat nose. Both the NRS-treated group exposed to FA and the ANS-treated group exposed to FA showed occasional mild transitional epithelial necrosis and sloughing with minimal intraepithelial infiltration of neutrophils. We attributed these unremarkable epithelial lesions to the design of our head-only inhalation system. Because we were unable to humidify the air prior to its entry into the head-only apparatus, we relied solely on the rat's nasal cavity to humidify the air for the entire 16-h experimental period. NRS- and ANS-treated rats exposed to O₃ showed significantly more necrosis of transitional epithelium than did the corresponding FA-exposed groups. The extent of transitional epithelial necrosis and sloughing tended to be greater in the ANS-treated rats than in the NRS-treated rats following O₃ exposure. In contrast, the severity of the necrosis was greater in the NRS-treated rats than in the ANS-treated rats. Histologic changes in the transitional epithelium ranged from epithelial attenuation to sloughing of epithelial cells into the nasal lumen with associated coagulative necrosis primarily along the nasoturbinates and maxilloturbinates. The inflammatory changes in the underlying lamina propria/submucosa were characterized by varying numbers of neutrophils. The acute inflammatory response tended to be more severe in the NRS-treated rats exposed to O₃ than in ANS-treated rats exposed to O₃. Extension of the neutrophilic infiltrate into the transitional epithelium was significantly greater in the NRS-treated O₃-exposed group than in the NRS-treated group exposed to FA. In addition, the intraepithelial inflammatory lesion and luminal exudate tended to be more severe in the NRS-treated rats exposed to O₃ than in the ANS-treated rats exposed to O₃.

Changes to the bronchial epithelium were statistically more extensive and severe in both NRS- and ANS-treated rats exposed to O₃ when compared with the corresponding FA-exposed groups. The ANS-treated O₃-exposed group showed more severe and extensive bronchial epithelial necrosis than did NRS-treated rats exposed to O₃, although the difference was not statistically significant. Histologically, the bronchial epithelium showed swelling and vacuolation with occasional sloughing of necrotic cells into the lumen. An accumulation of neutrophils either diffusely distributed or in variably sized aggregates around bronchi and in perivascular spaces was the main bronchial inflammatory change. The inflammatory response was more extensive and severe in the NRS-treated rats exposed to O₃ than in the ANS-treated group exposed to O₃ but was not statistically different.

Both the NRS-treated and ANS-treated rats exposed to O₃ showed significantly more extensive and severe epithelial changes in terminal bronchioles than did the corresponding FA-exposed groups. The extent of epithelial vacuolation, necrosis, and sloughing within the terminal bronchioles was statistically greater in the ANS-treated O₃-exposed rats than in NRS-treated rats exposed to O₃. The severity of the epithelial lesions tended to be greater in the ANS-treated O₃-exposed rats (P = 0.055). Histologic changes in terminal bronchioles were similar to those observed in the bronchi, including epithelial vacuolation, necrosis, and occasional sloughing into the lumen. Inflammatory changes in and around terminal bronchioles and adjacent alveolar septa were most severe in the NRS-treated O₃-exposed rats and were statistically greater than those in the NRS-treated rats exposed to FA. The predominant inflammatory change was an influx of neutrophils.

Morphometry of Lungs

We assessed airway epithelial repair following acute O₃ inhalation by applying unbiased morphometric techniques described in detail elsewhere (18). We used incorporation of BrdU into replicating cells as an index of epithelial repair. Figure 4 shows the number of BrdU-labeled cells per square millimeter of terminal bronchiolar epithelial basement membrane for all four groups. The number of BrdU-labeled epithelial cells for both ANS- and NRS-treated rats exposed to O₃ was significantly greater than it was for the corresponding FA-exposed groups. The total number of BrdU-positive cells per square millimeter of epithelial basement membrane for the ANS-treated rats exposed to O₃ was statistically less than the NRS-treated rats exposed to O₃ (a decrease of 43% in ANS-treated rats compared with NRS-treated rats).

View larger version (16K): [in this window] [in a new window]   Figure 4.   Total number of BrdU cumulative label-positive cells per square millimeter epithelial basement membrane (Ns, #/mm2) in neutrophil-sufficient control rats treated with NRS and neutrophil-depleted rats treated with rabbit ANS. FA-exposed NRS controls and ANS-treated animals were exposed to FA for 16 h. O3-exposed NRS controls and ANS-treated animals were exposed to 1 ppm O3 for 8 h followed by an 8-h FA postexposure period. Each bar represents the mean ± SEM of 9 to 10 animals. The differences between O3-exposed NRS controls and ANS-treated rats is shown as percent change. *Significantly different (P < 0.05) from corresponding FA control. §Significantly different (P < 0.05) from corresponding O3 control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The results of this study supported our overall hypothesis that neutrophils contribute to the repair process by enhancing the removal of O₃-injured airway epithelial cells. The design of our experiments enabled us to compare the physiologic response to O₃ inhalation in neutrophil- depleted rats treated with ANS and neutrophil-sufficient rats treated with NRS. Both groups showed a similar decrease in VT and increase in f with maximal percent change occurring at 8 h of O₃ exposure. In addition, neutrophil-depleted rats exposed to O₃ showed a statistically greater percent change in f in most of the postexposure period compared with the neutrophil-sufficient rats exposed to O₃. Both ANS- and NRS-treated rats exposed to O₃ showed more severe epithelial degeneration/necrosis and inflammation in the nasal cavity, bronchi, and distal airways than did the corresponding FA-exposed groups. In contrast, with O₃ exposure, the ANS-treated rats showed a greater extent of epithelial necrosis in the nasal cavity, bronchi, and distal airways than did the NRS-treated rats. Finally, within the terminal bronchioles, the total number of BrdU-labeled cells per square millimeter of epithelial basement membrane for the ANS-treated rats exposed to O₃ was statistically less than that in the NRS-treated rats exposed to O₃. Thus, our data implies that neutrophils enhance the proliferation of terminal bronchiolar epithelial cells following O₃ inhalation, possibly through the removal of O₃-injured epithelial cells.

Short-term O₃ inhalation is characterized by epithelial injury, especially type I alveolar and ciliated epithelial cells in the centriacinar regions, and inflammation in both humans (20, 21) and animals (2, 3, 20). Epithelial necrosis and sloughing are seen along terminal bronchioles and adjacent alveolar ducts with an associated influx of inflammatory cells, mural edema, and fibrin deposition (3, 22). Injured epithelial cells have also been proposed as a source of leukocyte chemotaxins (23). Although several chemoattractants produced by epithelial cells have been characterized, interleukin (IL)-8 appears to be the most potent neutrophil chemokine released by epithelial cells (24). IL-8 is known to induce neutrophil migration through both vascular endothelium and pulmonary epithelium (25). Although IL-8 is not found in rats, chemokine-induced neutrophil chemoattractant (CINC) is produced by type II epithelial cells in response to lipopolysaccharides, IL-1, and tumor necrosis factor  (26). Thus, following the release of IL-8 or CINC from injured epithelial cells, the recruitment of neutrophils into the area of injury may serve a beneficial role by assisting in the destruction and/or removal of O₃-injured epithelial cells. We believe that a consequence of this proinflammatory effect, especially the influx of neutrophils, is an enhancement of the onset of tissue repair. Supporting data for this supposition came from a previous study in this laboratory using morphometry and electron microscopic identification of degenerating and necrotic epithelial cells in neutrophil- depleted (ANS) rats exposed to 8 h of 1 ppm O₃ (5). ANS rats showed a substantial number of degenerating and necrotic cells 16 h following exposure, whereas NRS rats showed none. Furthermore, the significant decrease in ciliated cells compared with control cells was observed only immediately after ozone exposure (0 h postexposure) in NRS rats, continued to be significantly decreased at all times to 16 h postexposure in ANS rats. Because ciliated cells differentiate from nonciliated bronchiolar cells following ozone exposure, we speculate that delayed removal of necrotic and degenerating ciliated cells would be reflected by a decreased proliferation of nonciliated bronchiolar cells following ozone exposure.

A single intraperitoneal injection of ANS successfully reduced circulating neutrophils in this study. Both the NRS-treated rats exposed to FA and those exposed to O₃ had neutrophil values elevated above the normal mean for Wistar rats (19). Because all exposed rats showed no evidence of infectious respiratory disease and appeared to be in good health, we attributed the higher levels of circulating neutrophils to a stress neutrophilia induced by the long-term anesthesia (27). Other studies using polyclonal rabbit antirat neutrophil antiserum showed similar neutrophil depletion to this study (28, 29). Both of these studies showed a decrease in lymphocytes, although the decrease in lymphocytes was not statistically significant. Our preliminary data in three rats showed no decreases in monocytes or lymphocytes with the antibody used in this study. The efficacy of neutrophil depletion by antirat neutrophil antiserum is supported by similar microvascular responses to intestinal ischemia/reperfusion in rats by neutrophil depletion and monoclonal antibodies to either intercellular adhesion molecule-1 or CD11/CD18 (30). Although we are not aware of a study that examined the removal of antibody-treated neutrophils in vivo, it seems likely that they would be removed by the mononuclear phagoctye (reticuloendothelial) system of the liver and spleen similar to the usual disposal mechanism for circulating apoptotic neutrophils (31). There is no data that show the rat lung is involved in this mechanism of neutrophil clearance, and we did not observe an increase in neutrophil sequestration in the pulmonary vasculature of any of our ANS-treated rats. One final concern is whether the mechanism of neutrophil depletion influences the endothelium or adjacent structures of the lung. The role of neutrophil depletion on the mechanisms of pulmonary vascular smooth muscle relaxation was recently investigated (32). Antibody-mediated depletion did not impair endothelial-dependent or -independent cyclic guanosine monophosphate-mediated pulmonary vasorelaxation.

To avoid redistribution of intraluminal inflammatory cells and sloughed epithelial cells (14), we did not collect bronchoalveolar lavage fluid in this study. This was also the primary reason we elected to use intravascular perfusion fixation for the nasal cavity and lungs. This proved extremely beneficial in interpreting lesions using the grading scheme, because the rats in this study showed mild centriacinar lesions compared with the more severe lesions in the chamber-exposed, conscious rats in previous studies in our laboratory, although the dose and exposure period were the same (5, 17). We attributed the mild centriacinar lesions in this study to the anesthesia-induced decrease in f and E compared with that observed in conscious restrained rats (33). The decrease in f and E may have resulted in a decreased dose delivery of O₃ to the airways, thereby causing less epithelial damage and inflammation.

Studies investigating cell kinetics of the lung have shown the actual number of airway and alveolar epithelial cells in the process of division in the normal lung to be very small (34). However, animals surviving the initial damage from an inhaled irritant such as O₃ enter a phase of cellular proliferation in order to repair the injured airway epithelium. Evans and colleagues (35) were the first to show that the proliferative response of type II epithelial cells could be used as an indirect means to quantify acute damage to the alveolar epithelium. In previous studies involving lung cell kinetics, pulse-labeling techniques were employed to measure the incorporation of tritiated thymidine into the DNA of replicating epithelial cells (36). Measuring BrdU incorporation into DNA during epithelial cell proliferation has been shown to be a sensitive and quantitative indicator of O₃ exposure throughout the respiratory tract of rats, including the nasal cavity, large airways, and terminal bronchioles (11, 37). In the present study, we used the incorporation of BrdU into proliferating terminal bronchiolar epithelial cells as an index of repair.

Data from a pilot study in our laboratory using pulse labeling in chamber-exposed rats showed peak BrdU labeling in terminal bronchioles occurred at 16 h postexposure following an 8-h O₃ exposure period. After careful assessment of these data and consideration of pulse label data in monkey airways that showed a maximal epithelial label at 12 h following an 8-h exposure to 0.96 ppm O₃ (2), we selected an exposure period of 1 ppm O₃ for 8 h followed by an 8-h postexposure of FA for this study. Previous studies in our laboratory had shown that 1 ppm O₃ for an 8-h exposure period was higher than ambient levels but necessary to give us sufficient injury to attain measurable values for some of the endpoints (3). In addition, results of a second pilot study had shown that a 16-h period of anesthesia did not adversely affect the physiologic parameters we were interested in collecting, nor did it appear to alter lung tissue when examined histologically. Thus, osmotic minipumps were used in this study to maximize the sensitivity for measuring any potential proliferative response in terminal bronchiolar epithelium through continuous labeling. Haschek and coworkers (38) introduced the technique of continuous in vivo labeling of dividing cells in the lung using osmotic minipumps. Continuous or cumulative labeling indices accurately record acute bursts of cell proliferation and represent an integrated measurement of cell proliferation over a defined period of time (38).

To the best of our knowledge, this is the first study to assess quantitatively BrdU labeling of terminal bronchiolar epithelium using the optical disector. The disector and its counting rules were originally described in terms of the presence of sectional profiles of objects on two appropriately spaced, physically separate sections (physical disector) (39). The physical disector technique involves the time-consuming and difficult task of identifying and properly positioning corresponding parts of two separate sections (i.e., determining if a cell can be seen on one section but not the other). In contrast, the optical disector method counts cells directly in a measurable volume (18). The major advantage of the optical disector over the physical disector is that the optical sections are always positioned in register for comparison (40).

To our knowledge, this is also the first study to examine the role that neutrophils play in breathing pattern changes induced by O₃ inhalation. A previous chamber exposure study in our laboratory (5), while demonstrating a significantly prolonged presence of necrotic airway epithelial cells, did not allow us to monitor the ventilatory responses in neutrophil-depleted and neutrophil-sufficient rats. Our rationale for examining breathing pattern changes in the present study were twofold. First, given the delayed onset of O₃-induced rapid shallow breathing (Figure 3) we thought it possible that neutrophils might play a role and/ or modulate the cascade of events that leads to its development. We have proposed that airway epithelial injury induced by O₃ inhalation initiates the release of eicosanoic inflammatory mediators. One or more of these mediators excite lung sensory C fibers whose afferent endings are located within the walls of the conducting airways and are known to produce the protective reflex of rapid, shallow breathing (7, 8, 33). The cell type that releases these eicosanoic inflammatory mediators and the role that neutrophils play in this process is unknown. Second, if neutrophils have an effect on the development of O₃-induced rapid, shallow breathing, then this potential difference in the breathing response to O₃ inhalation in NRS- and ANS-treated rats would be expected to result in a different distribution pattern of O₃ dose within the airway (10) and thus require us to reevaluate the morphologic and morphometric data collected in this study, as well as the data collected in our previous chamber study (5).

The neutrophil-depleted rats and neutrophil-sufficient rats showed similar decreases in VT and increases in f with maximal percent change occurring at 8 h of exposure. At 8 h of exposure, f in the ANS-treated group had increased from the preexposure value of 87 breaths/min to 114 breaths/min (an increase of 24%), whereas f in the NRS-treated group went from 82 breaths/min preexposure to 106 breaths/min (an increase of 23%). On the basis of these observations, it appears that neutrophils alone do not influence the release of mediators from O₃-injured airway epithelium that leads to the excitation of airway C fibers and the development of rapid, shallow breathing. In addition, because the breathing pattern in response to O₃ inhalation was similar in ANS- and NRS-treated rats, we speculate that the distribution and deposition pattern of O₃ was not different between groups and provides for a valid comparison.

In contrast to the breathing pattern during O₃ inhalation, there was a significant difference in breathing pattern in the ANS- and NRS-treated rats during the 8-h recovery period. Neutrophil-depleted rats exposed to O₃ showed a greater percent change in f that began at 2 h after the end of O₃ exposure and lasted until the end of the postexposure period when compared with the neutrophil-sufficient rats exposed to O₃ (Figure 3). A similar but weaker delayed recovery was seen in VT with only the 8-h postexposure value being significantly different. We propose that the greater and prolonged f and VT responses in the neutrophil-depleted rats exposed to O₃ may be explained by the following pathophysiologic mechanism. With no neutrophils in the distal airways to assist with the destruction and/or removal of O₃-injured cells, epithelial repair is delayed and inflammation is sustained. This leads to a prolonged release of inflammatory mediators that continue to excite sensory lung C fibers in close proximity to the injured airway epithelium (41). This prolonged stimulation of C fibers would maintain the centrally mediated rapid, shallow breathing reflex, thereby causing the more prolonged f and VT responses observed in the neutrophil- depleted rats.

In conclusion, our findings support the hypothesis that neutrophils facilitate the repair process by assisting with the destruction and/or removal of O₃-injured airway epithelial cells. ANS-treated rats exposed to O₃ showed more epithelial necrosis in the nasal cavity, bronchi, and distal airways than did the NRS-treated rats. In addition, our results showed significantly more BrdU-labeled cells for the neutrophil-sufficient rats compared with the neutrophil-depleted rats following O₃ exposure. We believe these data suggest that neutrophils accelerate the onset of tissue repair by enhancing the removal of O₃-injured airway epithelial cells, and in so doing aid the reduction of inflammatory mediators that excite airway C fibers.