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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.

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To test the hypothesis that neutrophil influx is important for the removal of necrotic airway epithelial cells, rhesus monkeys were treated with a function-blocking monoclonal antibody (MAb) against CD18 followed by exposure to ozone or filtered air. CD18 MAb-treated, ozone-exposed monkeys showed a significant inhibition of neutrophil emigration and an accumulation of necrotic airway epithelial cells. In a subsequent experiment, monkeys were given CD18 MAb or an isotype control immunoglobulin before ozone or filtered-air exposure. Complement 5a was instilled into lobes of the right lung at the end of the exposure. Lavage neutrophils were significantly elevated in the right lobes compared with those in the contralateral left lobes; consequently, there were significantly fewer necrotic cells in the airways of the right lung, whereas large aggregations of necrotic cells were observed in the contralateral airways of the left lung. These data indicate that neutrophil influx in ozone-induced injury in primates is CD18 dependent and that neutrophils contribute to the repair of airway epithelium by removal of injured epithelial cells.

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Gamma delta intraepithelial lymphocytes are thought to coordinate responses to pathogens that penetrate the epithelial barrier. To directly test this, mice were inoculated with Nocardia asteroides. At doses that were nonlethal for control mice, gamma delta-deficient mice became severely ill and died within 14 days. Histologic examination of these lungs demonstrated the presence of severe tissue damage and unimpeded bacterial growth in the gamma delta-deficient mice compared with neutrophilic lesions and clearance of the organism in control mice. Interestingly, ozone exposure that targets a comparable lung region also resulted in diffuse epithelial necrosis associated with a similar lack of neutrophil recruitment in gamma delta-deficient mice. These data demonstrate that gamma delta intraepithelial lymphocytes can protect the host from pathogenic and nonpathogenic insults by targeting the inflammatory response to epithelial necrosis.

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To test the hypothesis that neutrophils enhance the repair of ozone (O3)-injured airway epithelium, we investigated breathing pattern responses and airway epithelial injury and repair in rats depleted of neutrophils using rabbit antirat neutrophil serum (ANS) and control rats treated with normal rabbit serum (NRS). Thirty-seven Wistar rats were exposed to O3 (1 ppm) or filtered air (FA) for 8 h followed by 8 h in FA. O3-exposed NRS- and ANS-treated rats showed similar progressive decreases in tidal volume and increase in breathing frequency, with maximal changes occurring at 8 h of exposure, whereas FA-exposed rats showed no significant changes. O3-exposed ANS-treated rats showed more epithelial necrosis in the nasal cavity, bronchi, and distal airways than did O3-exposed NRS-treated rats. Incorporation of 5-bromo-2-deoxyuridine (BrdU), a measure of cellular proliferation, was assessed using an optical disector to count BrdU- labeled terminal bronchiolar epithelial cells. O3-exposed ANS-treated rats had significantly less BrdU- labeled epithelial cells than did O3-exposed NRS-treated rats. We conclude that neutrophils contribute to the repair process by enhancing the proliferation of O3-injured airway epithelial cells.

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To assess the role of lung sensory C fibers during and after inhalation of 1 part/million ozone for 8 h, we compared breathing pattern responses and epithelial injury-inflammation-repair in rats depleted of C fibers by systemic administration of capsaicin as neonates and in vehicle-treated control animals. Capsaicin-treated rats did not develop ozone-induced rapid, shallow breathing. Capsaicin-treated rats showed more severe necrosis in the nasal cavity and greater inflammation throughout the respiratory tract than did control rats exposed to ozone. Incorporation of 5-bromo-2'-deoxyuridine (a marker of DNA synthesis associated with proliferation) into terminal bronchiolar epithelial cells was not significantly affected by capsaicin treatment in rats exposed to ozone. However, when normalized to the degree of epithelial necrosis present in each rat studied, there was less 5-bromo-2'-deoxyuridine labeling in the terminal bronchioles of capsaicin-treated rats. These observations suggest that the ozone-induced release of neuropeptides does not measurably contribute to airway inflammation but may play a role in modulating basal and reparative airway epithelial cell proliferation.

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To test the hypothesis that lung sensory C fibers protect the small distal airways and alveoli from oxidant injury, we compared the effects of inhalation of ozone (1 ppm) or filtered air for 8 h on lung injury and lung inflammation in four groups of rats: (1) normal rats exposed to filtered air; (2) normal rats exposed to ozone; (3) rats treated as neonates with capsaicin (50 mg/kg, intraperitoneally) and subsequently exposed to filtered air; and (4) rats treated as neonates with capsaicin and subsequently exposed to ozone. All rats were allowed to recover in filtered air for 0, 4, 16, and 40 h before necropsy. Rats exposed to filtered air (Groups 1 and 3) showed normal airway and parenchyma structure. Normal untreated rats exposed to ozone showed a random distribution of mild, interstitial inflammatory changes and epithelial necrosis of bronchi and bronchiolar epithelium. However, rats treated with capsaicin and subsequently exposed to ozone demonstrated severe acute interstitial inflammation and epithelial coagulate necrosis in all airways, especially in small, peripheral airways and parenchyma; all of these changes were statistically significant. These findings support our hypothesis that lung sensory C fibers protect the distal airways from oxidant injury during acute ozone inhalation.

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To test the hypothesis that neutrophils contribute to acute, ozone-induced epithelial damage in the lung, rats were depleted of their circulating neutrophils by intraperitoneal injection of a rabbit anti-rat neutrophil serum (ANS) 12 hr prior to an 8-hr exposure to 1.0 ppm ozone. Additional rats were given an injection of normal rabbit serum (NRS) prior to ozone exposure. Exposures were followed by postexposure periods in filtered air for 0, 4, or 16 hr. Control rats were given either ANS or NRS and then exposed only to filtered air. Analysis of bronchoalveolar lavage fluid (BALF) from NRS-treated rats revealed a significant increase in total neutrophils above that of controls at the 4- and 16-hr postexposure times, with a peak increase at 4 hr postexposure. In contrast, there was almost total ablation of the BALF neutrophil response in the ANS-treated rats at all times. Ozone caused an increase in BALF protein, fibronectin, and interleukin-6 above those in controls in both the NRS- and ANS-treated rats, but the only significant difference between the two groups was a level of fibronectin in the neutrophil-depleted animals higher than that in the neutrophil-sufficient animals at the 0-hr postexposure time. Electron microscopic morphometry on lungs fixed by intravascular perfusion demonstrated no significant differences in the volume per surface area epithelial basal lamina (Vs) of necrotic and degenerating epithelial cells in central acini between the neutrophil-depleted and neutrophil-sufficient animals. From these results, we concluded that neutrophils do not play a detectable role in contributing to the early epithelial damage in the lung caused by an acute exposure to ozone.

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To document the time course of the inflammatory response and epithelial injury in the lung following an acute ozone exposure, rats were exposed to 1.0 ppm ozone for periods between 4 and 24 hr. Some of the exposures were followed by postexposure periods in filtered air for up to 20 hr. Bronchoalveolar lavage fluid (BALF) analysis and electron microscopic morphometry on centriacinar regions of lungs fixed by intravascular perfusion were used to assess the degree of pulmonary inflammation and epithelial cell necrosis. Total protein and numbers of neutrophils and epithelial cells in BALF increased as the duration of ozone exposure increased, while BALF macrophages decreased. Quantitation of the neutrophil response in centriacinar lung regions (capillary, interstitial, and epithelial/luminal compartments of the terminal bronchiole and proximal alveolar duct) by morphometry generally correlated with the BALF analysis, and revealed a greater volume per surface area epithelial basal lamina (Vs) of neutrophils in the terminal bronchiole compartments compared to proximal alveoli. Necrosis of epithelial cells in terminal bronchioles, primarily ciliated cells, occurred as early as 4 hr after initiation of ozone exposure, before marked neutrophil migration, and continued during periods of maximal neutrophil influx. We concluded that the early epithelial necrosis in terminal bronchioles during the first few hours of ozone exposure was primarily due to direct ozone toxicity, but could not rule out the possibility of neutrophils contributing to the injury at later time points, especially between 8 and 12 hr of exposure (during periods of maximal neutrophil migration).

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Multiple reactive oxygen species-induced epithelial injury by glucose, glucose oxidase, and lactoperoxidase instillation in the lung results in a progressive interstitial fibrosis. To test the hypothesis that multiple pulmonary inflammatory responses alone would not result in fibrosis, three sequential inflammatory reactions were produced at weekly intervals in hamster lungs via intratracheal instillation of human recombinant C5a. Numbers of neutrophils and total inflammatory cells in bronchoalveolar lavage (BALF) increased significantly at 24 h after each C5a treatment compared with saline controls. Neutrophils increased by 3-, 33-, and 34-fold compared with the corresponding controls at 24 h after the first, second, and third doses, respectively, but returned to control levels by six days postinstillation. LTB4 levels increased by 24% and 20% compared with the corresponding controls at 24 h after the first and second doses but were not different from controls at other times. Hydroxyproline levels in treated animals did not differ significantly from control levels throughout the study. Protein levels were significantly increased at 24 h after the second and third doses and six days after the third dose compared with the corresponding controls. Occasional foci of neutrophils in alveolar spaces were observed at 24 h after each dose, but they decreased in frequency after six days. No foci of neutrophils were observed six days after the final dose, although some epithelial degeneration was observed by transmission electron microscopy. Our results indicate that pulmonary inflammation resulting from repeated influx of neutrophils in response to multiple instillations of C5a in the lung does not cause sufficient injury to result in pulmonary fibrosis.

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Because in vitro studies have shown inhibition of fibroblast proliferation and collagen synthesis by interferon, we tested the hypothesis that murine gamma interferon inhibits bleomycin-induced pulmonary fibrosis in mice. Mice were divided into the following groups: saline plus vehicle (27), saline plus interferon (29), bleomycin plus vehicle (26), and bleomycin plus interferon (26). Bleomycin or saline were given intratracheally once at the beginning of the experiment and vehicle (phosphate-buffered saline) or interferon was given intramuscularly daily. Mice were killed at 14 or 21 days of the experiment. About half of the mice from each group were used for collagen biochemistry and half for bronchoalveolar lung lavage, transmission electron microscopy (TEM), and morphometry. Hydroxyproline content showed a significant reduction in bleomycin plus interferon compared to bleomycin plus vehicle mice at 21 days. The saline plus vehicle and saline plus interferon mice showed no difference in hydroxyproline content. Similarly, bronchoalveolar lavage showed no differences between saline plus vehicle and saline plus interferon mice; however, all mice treated with bleomycin showed significant increases in total cells as compared to saline treated mice. At 14 and 21 days in bronchoalveolar lavage there were significantly more lymphocytes in bleomycin plus interferon compared to bleomycin plus vehicle mice. In bronchoalveolar lavage, there were usually fewer neutrophils, monocytes and macrophages in bleomycin plus interferon compared to bleomycin plus vehicle mice. Morphometric estimates of the volume of lesion within lung showed no significant differences among the bleomycin treated groups. Stainable collagen fibers were less, but not significantly, in the bleomycin plus interferon compared to bleomycin plus vehicle mice. The number of fibroblasts per volume of lesion was significantly decreased at 14 and 21 days in bleomycin plus interferon compared to bleomycin plus vehicle mice. The total volume of lymphocytes in interstitial lesions was significantly greater at 14 and 21 days in bleomycin plus interferon mice compared to bleomycin plus vehicle mice. These results suggest an inhibitory action of gamma interferon on collagen accumulation and fibroblast proliferation associated with lymphocyte accumulation in the lungs of mice following bleomycin administration.

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