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.

Acute injury to differentiating Clara cells in neonatal rabbits results in age-related failure of bronchiolar epithelial repair.

Nonciliated bronchiolar (Clara) cells are progenitor cells during lung development. During differentiation, they have a heightened injury susceptibility to environmental toxicants bioactivated by cytochrome P450 monooxygenase. When neonatal rabbits are treated with the P450-mediated cytotoxicant 4-ipomeanol (IPO), abnormal bronchiolar epithelium results. This study establishes the impact of IPO cytotoxicity on 3 stages of rabbit Clara cell differentiation, early (2.5 and 5 days postnatal [DPN]), intermediate (7 and 9 DPN), and late (15 and 21 DPN), and relates the cytotoxicity to the extent of bronchiolar repair. Neonates received a single dose of IPO (5 mg/kg) and were assessed by qualitative pathology 48 hours later for injury or at 4 weeks for repair. IPO injured the 3 stages of Clara cell differentiation to the same degree; epithelium was swollen, exfoliated, and squamated. Epithelial repair differed among the 3 stages. Bronchioles of animals treated during early and intermediate stages had simple squamous and irregularly shaped cuboidal cells. Animals treated during late stages were similar to controls. Thus, differentiating Clara cells are susceptible to injury by the P450-mediated cytotoxicant IPO, but the extent of repair varies based on when the initial injury occurs.

Relationship of inhaled ozone concentration to acute tracheobronchial epithelial injury, site-specific ozone dose, and glutathione depletion in rhesus monkeys.

Acute pulmonary epithelial injury produced by short-term exposure to ozone varies by site within the tracheobronchial tree. To test whether this variability is related to the local dose of ozone at the tissue site or to local concentrations of glutathione, we exposed adult male rhesus monkeys for 2 h to filtered air or to 0.4 or 1.0 ppm ozone generated from 18O2. Following exposure, lungs were split into lobes and specimens were selected by microdissection so that measurements could be made on airway tissue of similar branching history, including trachea, proximal (generation one or two) and distal (generation six or seven) intrapulmonary bronchi, and proximal respiratory bronchioles. One half of the lung was lavaged for analysis of extracellular components. In monkeys exposed to filtered air, the concentration of reduced glutathione (GSH) varied throughout the airway tree, with the proximal intrapulmonary bronchus having the lowest concentration and the parenchyma having the highest concentration. Exposure to 1.0 ppm ozone significantly reduced GSH only in the respiratory bronchiole, whereas exposure to 0.4 ppm increased GSH only in the proximal intrapulmonary bronchus. Local ozone dose (measured as excess 18O) varied by as much as a factor of three in different airways of monkeys exposed to 1.0 ppm, with respiratory bronchioles having the highest concentration and the parenchyma the lowest concentration. In monkeys exposed to 0.4 ppm, the ozone dose was 60% to 70% less than in the same site in monkeys exposed to 1.0 ppm. Epithelial disruption was present to some degree in all airway sites, but not in the parenchyma, in animals exposed to 1.0 ppm ozone. The mass of mucous and ciliated cells decreased in all airways, and necrotic and inflammatory cells increased. At 0.4 ppm, epithelial injury was minimal, except in the respiratory bronchiole, where cell loss and necrosis occurred, and was 50% that found in monkeys exposed to 1.0 ppm ozone. We conclude that there is a close association between site-specific O3 dose, the degree of epithelial injury, and glutathione depletion at local sites in the tracheobronchial tree.

Comparison of the distinct effects of epidermal growth factor and betamethasone on the morphogenesis of the gas exchange region and differentiation of alveolar type II cells in lungs of fetal rhesus monkeys.

To compare the effects of epidermal growth factor (EGF) and betamethasone on the morphogenesis of the gas exchange region and the differentiation of the alveolar type II cell during fetal lung development, fetal rhesus monkeys (78% gestation) were treated in utero with EGF (5.33 mg/kg total dose), beta-methasone (2.6 mg/kg total dose) or the carrier, saline (control), every other day for 7 days. EGF-treated monkeys had significantly increased body and adrenal weights. Betamethasone-treated monkeys had significantly decreased body and adrenal weights. Exogenous EGF reduced cytoplasmic glycogen and increased the cytoplasmic organelle and SP-A content within alveolar type II cells. In contrast, exogenous betamethasone did not alter alveolar type II cell cytodifferentiation. Neither EGF nor betamethasone treatment significantly altered the structure of the gas exchange region as shown by a lack of change from controls in alveolar airspace size or in the fraction of the gas exchange region that was potential airspace. We conclude that at clinically relevant doses, EGF greatly accelerates the maturation of alveolar type II cells, whereas betamethasone does not. Exogenous EGF may act directly on alveolar type II cells because these cells contain EGF receptor. Neither EGF nor betamethasone had dramatic effects on the morphogenesis of the gas exchange region.

Characterization of the cytochrome P-450 monooxygenase system in nonciliated bronchiolar epithelial (Clara) cells isolated from mouse lung.

The nonciliated bronchiolar epithelial (Clara) cell of the mouse is highly susceptible to toxicants that undergo metabolic activation, presumably because this cell type has high levels of cytochrome P-450 monooxygenases. As a first step in further defining the role of Clara cells in pulmonary xenobiotic activation and detoxication, we have isolated Clara cells (75 to 80% purity) and characterized them morphologically and biochemically. The identity of Clara cells, confirmed by transmission electron microscopy, was based on several features, including abundant agranular endoplasmic reticulum, large mitochondria, and dense secretory granules. Immunocytochemistry of isolated mouse cells showed that the majority were positive with antibodies against three major components of the pulmonary cytochrome P-450 monooxygenase system, cytochrome P-450 isozymes 2 (IIB), 5 (IVB), and NADPH cytochrome P-450 reductase, purified from rabbit lung. The isolated cells also showed a positive reaction with an antibody against the cytochrome P-450 isozyme that is active in the stereoselective metabolism of naphthalene, cytochrome P-450 mN (mN). Immunocytochemistry using the antibody against cytochrome P-450 isozyme 6 (IA1), purified from rabbit lung, showed no reaction in the isolated cells. The presence of intact cytochrome P-450 protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blots of homogenates of isolated cell preparations. The N-demethylation of benzphetamine and epoxidation of naphthalene occurred at easily measurable rates in incubations of isolated Clara cells. In contrast, diols, quinones, and monohydroxylated benzo(a)pyrene metabolites, analyzed by high performance liquid chromatography, were undetectable in extracts of Clara cells incubated with 3H-labeled substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

A morphometric study of lung maturation in the adrenocorticotrophin-infused fetal lamb.

Adrenocorticotrophin (ACTH) effects on lung development were studied in sheep. ACTH was infused into 10 fetal lambs at 129 days of gestation at a rate of 29, 110 or 250 micrograms/day for 72 h, a time course which was independent of the mechanical and hormonal forces associated with labor. Lungs were compared with those of normal fetuses at term (day 147). ACTH accelerates maturation of components of lung structure toward term values. Different components of cytodifferentiation of the type II alveolar cell showed different levels of sensitivity. The degree of acceleration, not the nature of the changes, was influenced by dose.

Differentiation of tracheal epithelium during fetal lung maturation in the rhesus monkey Macaca mulatta.

Of the eight categories of epithelial cells identified in pulmonary conducting airways, four are found in the trachea of adult primates: basal, mucous goblet, intermediate, and ciliated cells. While their ultrastructure is well characterized, little is understood about their origin or differentiation. This study describes the pattern of differentiation of the tracheal luminal epithelium in a species of nonhuman primate, the rhesus monkey, Macaca mulatta. Tracheas of 57 fetal and postnatal rhesus were fixed with glutaraldehyde/paraformaldehyde: ten at 29-54 days gestational age (GA), ten at 59-80 days GA (pseudoglandular stage), sixteen at 82-130 days GA (canalicular stage), ten at 141-168 days GA (saccular stage), eight at 1-134 days postnatal, and three adults (2 yr 11 months to 11 yr 11 months). Slices taken proximal to the carina were processed for electron microscopy by a selective embedding procedure. In the youngest fetuses, essentially one population of cells lined the tracheal epithelial surface. These cells were columnar in shape with a central nucleus, few organelles, and large amounts of cytoplasmic glycogen. At 46 days GA, ciliated cells were observed on the membranous side of the trachea. Some nonciliated cells had concentrations of organelles in the most apical portion of their cytoplasm. At 59 days GA, membrane-bound cored granules were intermixed with organelles in the apices of some glycogen-filled cells. They were observed first on the cartilaginous side. Between 59 and 100 days GA, a large number of cell forms which appeared to be transitional between ciliated, secretory, basal, and undifferentiated cells were present. These included ciliated cells with electron-lucent inclusions resembling mucous granules. Mucous secretory cells were more numerous and had more granules and less glycogen in older fetuses. By 105 days GA, few of the secretory cells had significant amounts of glycogen and the cytoplasm was condensed. Secretory granules were very abundant in some cells and minimal in others. The Golgi apparatus was prominent. In animals 120 days GA and older, small mucous granule cells and basal cells resembling these cells in adults were present. By 134 days postnatal age, the epithelium resembled that in adults. We conclude that most of the differentiation of tracheal epithelium in the rhesus monkey occurs prior to birth; the cells differentiate in the following sequences: ciliated, mucous goblet, small mucous granule, basal; and basal and small mucous granule cells do not play a role in ciliated and mucous cell formation in the fetus.

Peribronchiolar fibrosis in lungs of cats chronically exposed to diesel exhaust.

This study reports the quantitative changes in the pulmonary proximal acinar region following chronic exposure to diesel exhaust and following an additional 6 months in clean air. Cats (13 months of age) from a minimum disease colony were exposed to clean air (eight cats for 27 months and nine cats for 33 months), diesel exhaust for 8 hours/day, 7 days/week (nine cats for 27 months), or diesel exhaust for 27 months followed by 6 months in clean air (10 cats). Morphologic and morphometric evaluation using light microscopy and scanning and transmission electron microscopy revealed two major exposure-related lesions in proximal acinar regions of lungs of cats: peribronchiolar fibrosis associated with significant increases in lymphocytes, fibroblasts, and interstitial macrophages containing diesel particulate-like inclusions and bronchiolar epithelial metaplasia associated with the presence of ciliated and basal cells and alveolar macrophages containing diesel particulate-like inclusions. Peribronchiolar fibrosis was greater at the end of the 6 months in clean air following exposure, whereas the bronchiolar epithelial metaplasia was most severe at the end of exposure. Following an additional 6 months in clean air the epithelium more closely resembled the control epithelial cell population. The labeling index of terminal bronchiolar epithelium was significantly increased at the end of exposure but was not significantly different from controls or exposed cats following an additional 6 months in clean air. The ultrastructural appearance of epithelial cells remained relatively unchanged following diesel exhaust exposure with the exception of diesel particulate-like inclusions. Total lung collagen, expressed as hydroxyproline per left caudal lobe, was apparently increased (although the difference was not significant) in lungs of cats allowed to recover 6 months in clean air. Newly synthesized collagen (evaluated as the amount of cross-link-derived aldehydes in collagen) was significantly increased to more than twice the control values. The ratio of collagen aldehydes to hydroxyproline was also significantly increased. These observations imply that chronic exposure to diesel exhaust has a persistent fibrogenic effect on the proximal acinar region of the lung.

Centriacinar alterations in lungs of cats chronically exposed to diesel exhaust.

This study describes the morphologic changes in the centriacinar regions of lungs following long-term exposure of cats to diesel exhaust. Nine male cats (13 months of age) from a minimal disease colony were exposed to diesel exhaust for 8 hours/day, 7 days/week for 27 months. Eight cats were exposed to filtered air. Following exposure, the animals were killed by exsanguination and the lungs and trachea removed from the chest by thoracotomy, weighed, and fixed via tracheal cannula with glutaraldehyde/paraformaldehyde (550 mOsmoles, pH 7.4) at 30 cm of pressure. Centriacinar regions were selected from fixed tissue, the airways bisected, and complementary tissue halves processed by a large block method for high resolution light microscopy and for scanning electron microscopy. Compared with controls, diesel-exposed cats had lower fresh lung and kidney weights and lower fixed volumes of the right cranial lobe. The volume fractions of pulmonary parenchyma and nonparenchyma were unchanged. Epithelium of terminal and respiratory bronchioles in exposed cats consisted of three types of cells (ciliated, basal, and Clara cells), compared with only one type (Clara cells) in controls. Carbon-laden macrophages were found filling alveolar and interstitial spaces in exposed animals. Type 2 pneumocyte hyperplasia was present in proximal interalveolar septa. More distal alveolar ducts and the majority of the rest of the parenchyma were unchanged from controls. We concluded that exposure to diesel exhaust produces changes in both epithelial and interstitial tissue compartments and that the focus of these lesions in peripheral lung is the centriacinar region where alveolar ducts join terminal conducting airways.