Terminal Bronchiolar Cells Research Articles

An optimized method to visualize the goblet cell-associated antigen passages and identify goblet cells in the intestine, conjunctiva, and airway

The goblet cell-associated antigen passages (GAPs), formed in the intestine and conjunctiva, contribute to immune responses by delivering antigens to immune cells. It has been proved that dysfunctions of GAPs may contribute to intestinal inflammation and dry eye disease. GAPs were first discovered in the intestine with the help of intravital two-photon microscopy. The imaging of GAPs was subsequently captured in a frozen section and whole-mount immunofluorescent staining. Both protocols have their features. This study provides a new protocol for visualizing GAPs with better mucus preservation by combining 4% PFA and methanol-Carnoy’s solution (PFA-MC). In addition, embedding samples in paraffin provides an opportunity for antigen retrieval for immunohistochemistry analysis. Using this protocol, we have successfully visualized GAPs in the intestine, intestinal organoids, conjunctiva, and trachea. It is worth mentioning that the visualization of GAPs in intestinal organoids and airways has rarely been discussed before, which may assist researchers in their studies. Moreover, we also discovered the active uptake of dextran of club cells in terminal bronchioles, which may provide a new direction for studying the function of club cells. Overall, we developed a new method to visualize GAP formation and identify GCs simultaneously in the intestine, organoid, conjunctiva, and airway.

Higher expression levels of aquaporin (AQP)1 and AQP5 in the lungs of arid-desert living Lepus yarkandensis.

Water transport across epithelial cells that line the airways and alveoli is a crucial component of lung physiology. Aquaporins (AQPs) facilitate water transport across the air space-capillary barrier in the distal lung. However, the roles of lung AQPs in desert animal adaptation to dry airstream environments are still unclear. A hare (Lepus yarkandensis) only lives in the Tarim Basin, and its living environment is an arid climate with rare precipitation. We studied cellular localization and expression levels of AQP1, AQP3, AQP4 and AQP5 in L.yarkandensis lungs by immunohistochemistry, quantitative real-time polymerase chain reaction and Western blot. The lung of rabbits (Oryctolagus cuniculus) that inhabit in mesic environment was similarly studied. Obtained results in two species of animals were compared to investigate whether AQPs in the lung altered expression in the animal living in arid region. AQP1 was localized to the endothelial cells in capillaries and venules surrounding terminal bronchioles and alveoli. AQP5 was localized to the ciliated columnar cells in terminal bronchioles and the alveolar type I cells in the alveolus. Quantitative real-time PCR analysis showed higher AQP1 and AQP5 mRNA levels in L.yarkandensis compared to O.cuniculus. Similar results were obtained by Western blot. These results revealed that the higher expression levels of AQP1 and AQP5 played a significant role in water transport in the lungs of arid-desert living L.yarkandensis and might accelerate water transport from capillary compartments to the airspace.

Distribution of Basal/Myoepithelial Markers in Benign and Malignant Bronchioloalveolar Proliferations of the Lung

We investigated the staining pattern of commonly used basal cell/myoepithelial markers, such as p63 (a p53-homologous nuclear protein), basal cell-specific cytokeratin antibody (34betaE12, K903), and smooth muscle myosin heavy chain (SMMHC) in benign and malignant bronchioloalveolar proliferations of the lung. We studied 85 lung lesions consisting of 35 bronchioloalveolar carcinoma, 30 well-differentiated adenocarcinoma, and 20 cases of benign lung lesions. In normal lung, p63, K903, and SMMHC decorated the basal cells of large and small airways and occasional cells of terminal bronchioles. In reactive processes, a distinctive staining pattern was present in 19/20 (95%) of the cases characterized by staining of basal cells of the airways and bronchiolar epithelium and squamous metaplastic epithelium for p63 and K903, whereas 12/20 (60%) stained with SMMHC. Respiratory ciliated cells, alveolar epithelial cells, and nonepithelial cells were negative. In bronchioloalveolar carcinoma, a discontinuous peripheral rim of p63-immunoreactive cells was retained surrounding and intermingled with the malignant bronchioloalveolar proliferation in 31/35 (88.5%) cases, SMMHC in 28/35 (80%) cases, and K903 in 20/35 (57%) cases. For adenocarcinoma, a majority of the cases (28/30, 93%) were negative for p63 and K903; however, SMMHC showed artifactual staining in the desmoplastic stroma in 6/30 (20%) cases. Our results highlighted the differential expression of basal cell markers across various bronchioloalveolar lesions. The staining pattern of basal cells in bronchioloalveolar carcinoma supports that these neoplasms may actually be carcinoma in-situ.

Distal Airways in Mice Exposed to Cigarette Smoke

Cigarette smoke (CS) is the main risk factor for chronic obstructive pulmonary disease (COPD). Terminal bronchioles are critical zones in the pathophysiology of COPD, but little is known about the cellular and molecular changes that occur in cells lining terminal bronchioles in response to CS. We subjected C57BL/6 mice to CS (6 d/wk, up to 6 mo), looked for morphologic changes lining the terminal bronchioles, and used laser capture microdissection to selectively isolate cells in terminal bronchioles to study gene expression. Morphologic and immunohistochemical analyses showed that Clara cell predominance remained despite 6 months of CS exposure. Since Clara cells have a role in protection against oxidative stress, we focused on the expression of antioxidant/detoxification genes using microarray analysis. Of the 35 antioxidant/detoxification genes with at least 2.5-fold increased expression in response to 6 months of CS exposure, 21 were NF-E2-related factor 2 (Nrf2)-regulated genes. Among these were cytochrome P450 1b1, glutathione reductase, thioredoxin reductase, and members of the glutathione S-transferase family, as well as Nrf2 itself. In vitro studies using immortalized murine Clara cells (C22) showed that CS induced the stabilization and nuclear translocation of Nrf2, which correlated with the induction of antioxidant and detoxification genes. Furthermore, decreasing Nrf2 expression by siRNA resulted in a corresponding decrease in CS-induced expression of several antioxidant and detoxification genes by C22 cells. These data suggest that the protective response by Clara cells to CS exposure is predominantly regulated by the transcription factor Nrf2.

CLARA CELL PROTEIN CC16 AS A BIOLOGICAL MARKER OF LUNG INJURY AMONG QUARRY WORKERS EXPOSED TO PM2.5

Objective: evaluation of the use of Clara cell protein (CC16) level in serum as an early indicator of the adverse respiratory health effects of particulate matter (PM2.5) exposure.Methods: Detailed assessment questionnaire was used for interviewing 54 individuals divided into workers exposed to particulates in the atmosphere of stone quarriesand a control group. The studied population was subjected to clinical examination and pulmonary function testing in order to diagnose airway diseases and lung irritation. Laboratory investigations were done in the form of Clara cell protein and immunoglobul in A (IgA) estimation in serum using the immunoassay technique. Results: According to the thorough occupational history and clinical examination,respiratory system affection was detected among the exposed population in the form of frequent asthmatic attacks, chronic bronchitis and chronic obstructive pulmonary disease. Pulmonary function tests demonstrated marked decrease in the expiratory flow rate 25-75% (FEF 25-75%) suggesting irritation and affection of the small airways correlating to duration of exposure more than smoking index. Estimation of the pneumoprotein CC16 which is produced by the epithelial cells in terminal bronchioles revealed marked elevation in serum of exposed group (149.61 ± 22.64 pg/mi) than among the control group (107.08 ± 12.18 pg/ml). Similarly, the humoral immune defense involving the secretory immunoglobulin A (slgA) predominant in mucosal secretions was markedly elevated among the exposed than the control groups with mean values of 372.41 ± 232.87 IU/ml and 143.76 ± 83.531U/ml, respectively. The serurn CC 16 and IgA were both significantly negatively correlated with flow rates.Worth-mentioning, is the increased CC 16 serum levels with ageing which influences the functionality of the lungs and immune systern as evidenced by the decline in lung volumes, lung capacity and flow rates and by decreased IgA in serum. Conclusion: The results suggest that damage of Clara cells in the small airways and the increased epithelial barrier permeability are ultimate consequences of exposure to particulates in air at the workplace. Accordingly, the elevated serum CC16 asa result of increased diffusion of this type of pneumoprotein into the circulation might be used as an early non-invasive biological indicator for lung affection. Aim of the Work: This study was designed to investigate the cellular and humoraldefense mechanisms of the respiratory tract in workers exposed to particulate air pollution in quarries in an attempt to evaluate the importance of using CC16 as a biologic marker for respiratory injury in comparison to pulmonary function tests.

Gelatinase B Is Required for Alveolar Bronchiolization After Intratracheal Bleomycin Instillation

Increased expression of matrix metalloproteinases, particularly gelatinase B (matrix metalloproteinase-9), has been described in the lungs in pulmonary fibrosis. Intratracheal bleomycin is often used experimentally to produce lesions resembling human fibrosing alveolitis. To assess the role of gelatinase B in bleomycin-induced fibrosing alveolitis, we instilled bleomycin intratracheally into gelatinase B-deficient mice and gelatinase B +/+ littermates. Twenty-one days after bleomycin instillation, the two groups of mice were indistinguishable in terms of pulmonary fibrosis histologically and total lung collagen and elastin. However, the lungs of gelatinase B-deficient mice showed minimal alveolar bronchiolization, while bronchiolization was prominent in the lungs of gelatinase B +/+ mice. Gelatinase B was identified immunohistochemically in terminal bronchiolar cells and bronchiolized cells 7 days and 14 days after bleomycin instillation in gelatinase B +/+ mice, and whole-lung gelatinase B messenger RNA was increased at the same time. Many bronchiolized cells displayed Clara cell features by electron microscopy and by immunostaining with antibody to Clara cell-specific protein. Some bronchiolized cells stained with antibody to forkhead/winged helix transcription factor 4, a factor associated with the ciliated cell phenotype, but none stained for an alveolar type-II cell marker, surfactant protein C. Thus, fibrosing alveolitis develops after intratracheal bleomycin instillation irrespective of gelatinase B. However, gelatinase B is required for alveolar bronchiolization, perhaps by facilitating migration of Clara cells and other bronchiolar cells into regions of alveolar injury.

Prior exposure to aged and diluted sidestream cigarette smoke impairs bronchiolar injury and repair.

The bronchiolar injury/repair response to naphthalene (NA) in mice includes acute distal airway epithelial injury that is followed by epithelial proliferation and redifferentiation, which result in repair of the epithelium within 14 days. To test whether prior exposure to aged and diluted sidestream cigarette smoke (TS) would alter the injury/repair response of the airway epithelium, adult mice were exposed to either filtered air (FA) or smoke for 5 days before injection with either corn oil carrier (CO) or naphthalene. Mice were killed 1 and 14 days after naphthalene injury. Lung and lobar bronchus were examined and measured using high-resolution epoxyresin sections. The control group (FACOFA) that was exposed to filtered air/corn oil/filtered air contained airway epithelium similar to untreated controls at all airway levels. The group exposed to tobacco smoke/corn oil/filtered air (TSCOFA) contained some rounded cells in the small airways and some expansion of the lateral intercellular space in the larger airways. Necrotic or vacuolated cells were not observed. As expected, the epithelium in the group exposed to filtered air/naphthalene/filtered air (FANAFA) contained many light-staining vacuolated Clara cells and squamated ciliated cells within distal bronchioles during the acute injury phase. Repair (including redifferentiation of epithelial cells and restoration of epithelial thickness) was nearly complete 14 days after injury. The extent of Clara cell injury, as assessed in lobar bronchi, was not different between the four groups. Although the FANAFA group contained greater initial injury in the distal airways at 1 day, the group exposed to tobacco smoke/naphthalene/filtered air (TSNAFA) had the least amount of epithelial repair at 14 days after naphthalene treatment; many terminal bronchioles contained abundant squamated undifferentiated epithelium. We conclude that tobacco smoke exposure prior to injury (1) does not change the target site or target cell type of naphthalene injury, since Clara cells in terminal bronchioles are still selectively injured; (2) results in slightly diminished acute injury from naphthalene in distal bronchioles; and (3) delays bronchiolar epithelial repair.

Gelatinase B Is Required for Alveolar Bronchiolization after Intratracheal Bleomycin

Increased expression of matrix metalloproteinases, particularly gelatinase B (MMP-9), has been described in the lungs in pulmonary fibrosis. Intratracheal bleomycin is often used experimentally to produce lesions resembling human fibrosing alveolitis. To assess the role of gelatinase B in bleomycin-induced fibrosing alveolitis, we instilled bleomycin intratracheally into gelatinase B-deficient mice and gelatinase B+/+ littermates. Twenty-one days after bleomycin the two groups of mice were indistinguishable in terms of pulmonary histology and total lung collagen and elastin. However, the lungs of gelatinase B-deficient mice showed minimal alveolar bronchiolization, whereas bronchiolization was prominent in the lungs of gelatinase B+/+ mice. Gelatinase B was identified immunohistochemically in terminal bronchiolar cells and bronchiolized cells 7 and 14 days after bleomycin in gelatinase B+/+ mice, and whole lung gelatinase B mRNA was increased at the same times. Many bronchiolized cells displayed Clara cell features by electron microscopy. Some bronchiolized cells stained with antibody to helix transcription factor 4, a factor associated with the ciliated cell phenotype. Thus, fibrosing alveolitis develops after intratracheal bleomycin irrespective of gelatinase B. However, gelatinase B is required for alveolar bronchiolization, perhaps by facilitating migration of Clara cells and other bronchiolar cells into the regions of alveolar injury.

Comparative ultrastructure of Clara cells in neonatal and older cattle

ABSTRACT Calf lungs were fixed with glutaraldehyde and examined by scanning (SEM) and transmission (TEM) electron microscopy to compare the ultrastructure of Clara cells in terminal bronchioles of neonatal calves and older cattle. In the neonatal calf, SEM revealed numerous smooth-surfaced Clara cells protruding above a similar number of ciliated cells, whereas in older animals the surface of Clara cells was lobulated. Thin sections examined by TEM revealed numerous cuboidal to columnar Clara cells with indented nuclei and a pale cytoplasm filled with faintly granular glycogen in the neonatal calf. Some cells were characterized by apical dense and/or pale membrane-bound granules or secretory droplets. Many cells had an apical tubular network of cisternae that were partly smooth and partly decorated with ribosomes. Ultrastructural comparison of Clara cells in a 2-day-old calf with those of 14- and 19-day-old, 4- and 5.5-month-old, and 3.5-year-old cattle revealed a striking reduction in the amount of glycogen per cell after 14 days. The number of cells with apical granules was small at all ages, and the density of the secretory granules varied greatly in different cells. A variable amount of smooth endoplasmic reticulum (SER) was present but was less prominent than cisternae of ribosomal endoplasmic reticulum (RER). In older cattle, the limited amount of SER compared to the RER and secretory granules suggests that bovine Clara cells are more likely to be secretory than detoxifying. J. Morphol. 244:143–151, 2000. © 2000 Wiley-Liss, Inc.

Dose-Related Airway-Selective Epithelial Toxicity of 1-Nitronaphthalene in Rats

Nonciliated (“Clara”) cells of terminal bronchioles are one of the principal targets of bioactivated pulmonary toxicants. However, we have recently observed cytotoxicity in proximal tracheobronchial airways with high doses of naphthalene. To test if this was true for other xenobiotics, 1-nitronaphthalene (25, 50, 100, or 150 mg/kg) was injected ip into male Sprague–Dawley rats and cytotoxicity characterized 24 hr later by high-resolution histopathology along a defined airway path extending from the trachea to the terminal bronchioles. At 25 mg/kg, only nonciliated cells in minor daughter airways were swollen and necrotic. At 50 mg/kg there was near complete loss of nonciliated cells in most bronchioles, minor daughter airways, and tracheas. Marked damage to ciliated cells was observed at doses of 100 and 150 mg/kg. Denudation of the basement membrane was common in the trachea. We conclude for 1-nitronaphthalene cytotoxicity in the lung that: (1) nonciliated cells of the distal bronchioles are not the only target, (2) the threshold for injury in nonciliated cells is lower than that for ciliated cells, (3) the response is airway selective, (4) the response is dose-related.

Dose-Dependent Tolerance to Ozone: III. Elevation of Intracellular Clara Cell 10-kDa Protein in Central Acini of Rats Exposed for 20 Months

Understanding factors that promote pulmonary tolerance to long-term oxidant injury is essential to evaluating health risks in humans. One such factor may be Clara cell 10-kDa protein (CC10), a protein secreted by nonciliated cells in distal conducting airways thought to have an anti-inflammatory action against inhaled xenobiotics. Using standard immunohistochemical techniques and laser scanning confocal microscopy, we assessed changes in CC10 abundance in the centriacinar region of the rat following 20 months′ exposure to 0.12 and 1.00 ppm ozone. Three zones of reflectance intensity (high, medium, and low), directly related to CC10 density, were used to distinguish between the two major subcellular compartments where CC10 is distributed: granules and endoplasmic reticulum. Low levels of ozone (0.12 ppm) had no significant effect on the cellular distribution or abundance of CC10 in nonciliated epithelium in the centriacinar region. In contrast, 1.00 ppm ozone not only elevated cellular volume of granule-based CC10, but also elevated the protein′s concentration within the granules and increased the number of granules per cell profile. The proportion of nonciliated cells in terminal bronchioles increased significantly at the expense of ciliated cells. This combination of factors led to a threefold increase in CC10 stored per unit surface area of epithelium in terminal bronchioles. The nonciliated cells in ozone-induced respiratory bronchioles contained a distribution of CC10 similar to that of bronchiolar nonciliated cells in control animals. We conclude that ozone-induced tolerance may be related to the increased abundance and wider distribution of CC10 in central acini of rats following long-term ozone exposure.

Pulmonary cellular effects in rats following aerosol exposures to ultrafine Kevlar aramid fibrils: Evidence for biodegradability of inhaled fibrils

Previous chronic inhalation studies have shown that high concentrations of Kevlar fibrils produced fibrosis and cystic keratinizing tumors in rats following 2-year inhalation exposures. The current studies were undertaken to evaluate mechanisms and to assess the toxicity of inhaled Kevlar fibrils relative to other reference materials. Rats were exposed to ultrafine Kevlar fibers (fibrils) for 3 or 5 days at concentrations ranging from 600–1300 fibers/cc (gravimetric concentrations ranging from 2–13 mg/m 3). A complete characterization of the fiber aerosol and dose was carried out. These measurements included gravimetric concentrations, mass median aerodynamic diameter, fiber numbers, and count median lengths and diameters of the aerosol. Following exposures, cells and fluids from groups of sham- and fiber-exposed animals were recovered by bronchoalveolar lavage (BAL). Alkaline phosphatase, lactate dehydrogenase (LDH), protein, and N-acetyl glucosaminidase (NAG) values were measured in BAL fluids at several time points postexposure. Alveolar macrophages were cultured and studied for morphology, chemotaxis, and phagocytosis by scanning electron microscopy. The lungs of additional exposed animals were processed for deposition, cell labeling, retained dose, and lung clearance studies, as well as fiber dimensions (from digested lung tissue), histopathology, and transmission electron microscopy. Five-day exposures to Kevlar fibrils elicited a transient granulocytic inflammatory response with concomitant increases in BAL fluid levels of alkaline phosphatase, NAG, LDH, and protein. Unlike the data from silica and asbestos exposures where inflammation persisted, biochemical parameters returned to control levels at time intervals between 1 week and 1 month postexposure. Macrophage function in Kevlar-exposed alveolar macrophages was not significantly different from sham controls at any time period. Cell labeling studies were carried out immediately after exposure, as well as 1 week and 1 month postexposure. Increased pulmonary cell labeling was measured in terminal bronchiolar cells immediately after exposure but returned to control values 1 week later. Fiber clearance studies demonstrated a transient increase in the numbers of retained fibers at 1 week postexposure, with rapid clearance of fibers thereafter. The transient increase in the number of fibers could be due to transverse cleaving of the fibers, since the average lengths of retained fibers continued to decrease over time. In this regard, a progressive decrease in the mean lengths and diameters of inhaled fibers was measured over a 6-month postexposure period. Mean fiber lengths decreased from 12.5 to 7.5 μm during this time period; mean fiber diameters decreased from 0.33 to 0.24 μm over the same period. This pattern suggests fiber degradability and is similar to results reported in fiberglass-exposed rats. Histopathologic analysis revealed no pathologic effects in the lungs of Kevlar-exposed rats. The results indicate that the pulmonary toxicity of ultrafine Kevlar fibrils is similar to effects measured in wollastonite and carbon fiber-exposed rats and differs significantly from results observed in crystalline silica or asbestos-exposed animals.

Pulmonary inflammation and epithelial injury in response to acute ozone exposure in the rat

To document the time course of the inflammatory response and epithelial injury in the lung following and 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 ( V s) 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).

Attenuation of perfluoropolymer fume pulmonary toxicity: Effect of filters, combustion method, and aerosol age

Thermal decomposition products of some perfluorinated polymers are toxic to experimental animals in small-scale combustion toxicity tests; the toxicity is dependent upon the heating procedure, combustion temperature, and other experimental conditions. In the current studies we investigated the time course of fume generation and exposure on pulmonary effects in rats following a 30-min exposure to perfluoropolymer decomposition products (i.e., fume concentration = 0.2 mg/m 3 of tetrafluoroethylene/hexafluoropropylene copolymer (FEP)) pyrolyzed with either static or dynamic airflows. In the first set of experiments, five different groups of rats were exposed to FEP fumes in a static combustion toxicity test system. Three groups were exposed to unfiltered FEP fumes during 0- to 15-, 15- to 30-, and 0- to 30-min intervals, respectively, and one to a filtered (particle-free) atmosphere of combusted FEP for 30 min. Sham-exposed rats constituted the control group. Immediately after exposure, the rats were sacrificed and their lungs weighed and lavaged or perfused to assess indices of cytotoxicity. Our results showed that lung weights, markers of inflammation, and pulmonary hemorrhage and alkaline phosphatase, β-glucuronidase, lactate dehydrogenase, and protein levels in bronchoalveolar lavage fluids were significantly elevated in all unfiltered FEP-exposed groups compared to those in either the rats exposed through filters or controls ( P < 0.01). In a second set of experiments using a dynamic pyrolysis toxicity test system, rats were exposed for 30 min to FEP-pyrolyzed fumes which were either freshly generated or aged for 1 or 5 min prior to delivery to the animal's breathing zone. Subsequently, lung cytotoxicity parameters were measured. Rats exposed directly to the fresh fumes demonstrated toxic effects consistent with those described above ( P < 0.01), but the pulmonary toxicity of aged (i.e., 1 or 5 min delay) FEP fumes was diminished in a time-dependent manner, suggesting that the toxicant was unstable. Histopathological studies correlated with biochemical results and revealed that inhalation of unfiltered or freshly generated FEP fumes produced a severe lung injury characterized by the development of alveolar and intersititial edema, intraalveolar hemorrhage, congestion, and fibrin deposition. Electron microscopy studies demonstrated severe damage to terminal bronchiolar cells and detachment of Type I epithelial and endothelial cells in pulmonary regions. The severity of pathology observed in lungs of rats exposed to 1-min aged fumes was intermediate between unfiltered/unaltered fume-exposed animals and sham controls. The results of these studies demonstrate that the lung toxicity of perfluoropolymer fumes is associated with the aerosol phase generated in perfluoropolymer pyrolysis.

Effects of low levels of NO 2 on terminal bronchiolar cells and its relative toxicity compared to O 3

This report describes structural changes occurring in the terminal bronchioles of rats exposed to low levels of NO 2 continuously for 6 weeks. In addition, the relative susceptibility of epithelial cells to oxidants and the comparative toxicity of NO 2 and O 3 are discussed. Terminal bronchioles isolated from rats exposed 5 days/week to 2.0 ppm NO 2 (plus two 1-hr daily spikes to 6.0 ppm) were found to have 19% less ciliated cells per unit area of epithelial basement membrane. The remaining ciliated cells had a reduced mean surface area (−29%). The shape of the Clara cell changed with reduced size of the dome protrusions but increased cell contact with the basement membrane. These data indicate that exposure to 2.0 ppm NO 2 (+ spikes) for 6 weeks caused injuries to cilia and ciliated cells and possible Clara cell differentiation in the terminal bronchioles of adult rats. Exposures of adult or juvenile rats to 0.5 ppm NO 2 (+ two 1-hr daily spikes 5 days/week to 1.5 ppm) did not cause morphologically measurable injuries in the terminal bronchioles. The severity of the concentration-dependent epithelial cell reactions to NO 2 and O 3 in adult rat terminal bronchioles were compared to those occurring in the proximal alveolar regions (PAR). Epithelial cells in the PAR appeared to be more susceptible to oxidant insult since both 0.5 ppm NO 2 and 0.25 ppm O 3 were found to cause epithelial injury only in the PAR. Comparison of epithelial reactions to 6-week exposures to either NO 2 or O 3 indicated that 0.25 ppm O 3 caused four times as much increase in the number of type I epithelial cells as did 2 ppm (+ spikes) NO 2. Therefore, O 3 could be 40 times more toxic than NO 2 in the PAR on the basis of the inspired concentration and the focal response. On the other hand, there was no loss of ciliated cells following the 0.25 ppm O 3 exposure. This suggests that the ratio of O 3 to NO 2 toxicity in the terminal bronchioles is considerably less than 10. The relative toxicity of the two oxidant gases appears to be site specific.

Early cytokinetic and morphological response of rat lungs to inhaled benzo(a)pyrene, gallium oxide, and SO2

The purpose of this study was to determine if the early cytokinetic and morphological responses of the lung to inhaled particles and an irritant gas are altered by the presence of a carcinogen. Fischer-344 rats were exposed by nose-only inhalation to a combination of benzo(a)pyrene (B(a)P) (7 mg/m3) adsorbed onto gallium oxide particles (22 mg/m3) and SO2 (5 ppm) or to gallium oxide particles and SO2 alone for 2 h per day for 9 days. Serial sacrifices were performed up to 28 days after the beginning of the exposure. Cell proliferation in the lungs was studied in 1-μm-thick glycol methacrylate autoradiographs of tritiated thymidine-labeled tissues. Cytokinetic and morphological changes in the two groups were similar. However, there were several changes associated only with B(a)P treatment such as increased proliferation in most cell types at 8 or 11 days and foci of large proliferating cuboidal cells in the alveolar region. Cell proliferation in airway and alveolar regions of lungs in both groups increased during exposure and decreased subsequently. Small foci of hyperplastic alveolar cells and hypertrophic terminal bronchiolar cells were present in both groups although they occurred more frequently in the group that received B(a)P. These cytokinetic and morphological changes were interpreted as a repair response of the lungs to a toxic insult. Whether the differences between the two treatments were related to the carcinogenic nature of B(a)P or to the toxic nature of B(a)P will be determined in a long-term study.

Neoplastic and nonneoplastic effects of vinyl chloride in mouse lung.

Neoplastic effects of vinyl chloride were studied in lungs of 27 mice exposed to vinyl chloride monomer at 2500 and 6000 ppm for 5 and 6 months (large doses and long-term exposure). Pulmonary tumors were observed in 26 of 27 experimental animals. Light microscopy showed the tumors to be multiple and arranged in either tubulo-papillary or adenomatous formations. Although occasional mitotic divisions and invaginations into the bronchiolar lumen were observed, no metastases were found. By electron microscopy, short microvilli, tight junctions between two adjacent cells, appearance of osmiophilic lamellar bodies, large mitochondria of irregular shape, well developed Golgi complexes, continuous or discontinuous basement membranes, occasional appearance of "sequestration" and of crystalloids and lack of both cilia and mucous secretory granules were observed as characteristic features of the neoplastic cells. Some of the cells were poorly differentiated and were equipped with poorly developed organoids, without formation of osmiophilic lamellar bodies. The pulmonary tumors corresponded to "alveologenic" tumors. It is suggested that the neoplastic cells were transformed from type II alveolar epithelium via its hyperplastic form. Nonneoplastic effects of the chemical were also studied in the 27 mice. Major light microscopic alterations observed were proliferation and hypertrophy of the terminal bronchiolar cells, consisting of ciliated and Clara cells, hypersecretion of the epithelial mucin in the goblet cells of both the bronchial and the proximal bronchiolar epithelium, hyperplasia of alveolar epithelium, mobilization of alveolar macrophages and occasional presence of peribronchial or bronchiolar chronic inflammation. Electron microscopically, Clara cells of the terminal bronchiolar epithelium showed proliferation of the rough and smooth surfaced endoplasmic reticulum and appearance of large and abnormally shaped mitochondria. Similar alterations were found in the ciliated cells. Submicroscopic changes of pulmonary alveoli were represented by focal thickening of the basement membrane, multiple foci of hyperplastic type II cell (the precondition of the alveologenic tumor), active discharge of osmiophilic lamellar bodies from the type II cell and phagocytosis of the bodies by macrophages, appearance of cholesterol crystalloids in the macrophages, degeneration of alveolar septal cells and occasional appearance of a large nucleus with swelling of the capillary endothelium. The neoplastic effect of vinyl chloride of smaller doses (100, 10, 1 and 0 [control] ppm) and shorter exposure (four weeks) was studied in lungs of 120 mice. Our preliminary observation indicated that sacrificed animals at 40 weeks after the exposure showed productions of the alveologenic tumor in 5 of 9 (100 ppm), 2 of 9 (10 ppm), 1 of 9 (1 ppm) and 0 of 10 (control = 0 ppm). A dose-response relation was considered in the incidence of the alveologenic tumor production of vinyl chloride. It is concluded that mouse lung is an extremely sensitive indicator of the oncogenicity of vinyl chloride.

Neoplastic and Nonneoplastic Effects of Vinyl Chloride in Mouse Lung

Neoplastic effects of vinyl chloride were studied in lungs of 27 mice exposed to vinyl chloride monomer at 2500 and 6000 ppm for 5 and 6 months (large doses and long-term exposure). Pulmonary tumors were observed in 26 of 27 experimental animals. Light microscopy showed the tumors to be multiple and arranged in either tubulo-papillary or adenomatous formations. Although occasional mitotic divisions and invaginations into the bronchiolar lumen were observed, no metastases were found. By electron microscopy, short microvilli, tight junctions between two adjacent cells, appearance of osmiophilic lamellar bodies, large mitochondria of irregular shape, well developed Golgi complexes, continuous or discontinuous basement membranes, occasional appearance of “sequestration” and of crystalloids and lack of both cilia and mucous secretory granules were observed as characteristic features of the neoplastic cells. Some of the cells were poorly differentiated and were equipped with poorly developed organoids, without formation of osmiophilic lamellar bodies. The pulmonary tumors corresponded to “alveologenic” tumors. It is suggested that the neoplastic cells were transformed from type II alveolar epithelium via its hyperplastic form. Nonneoplastic effects of the chemical were also studied in the 27 mice. Major light microscopic alterations observed were proliferation and hypertrophy of the terminal bronchiolar cells, consisting of ciliated and Clara cells, hypersecretion of the epithelial mucin in the goblet cells of both the bronchial and the proximal bronchiolar epithelium, hyperplasia of alveolar epithelium, mobilization of alveolar macrophages and occasional presence of peribronchial or bronchiolar chronic inflammation. Electron microscopically, Clara cells of the terminal bronchiolar epithelium showed proliferation of the rough and smooth surfaced endoplasmic reticulum and appearance of large and abnormally shaped mitochondria. Similar alterations were found in the ciliated cells. Submicroscopic changes of pulmonary alveoli were represented by focal thickening of the basement membrane, multiple foci of hyperplastic type II cell (the precondition of the alveologenic tumor), active discharge of osmiophilic lamellar bodies from the type II cell and phagocytosis of the bodies by macrophages, appearance of cholesterol crystalloids in the macrophages, degeneration of alveolar septal cells and occasional appearance of a large nucleus with swelling of the capillary endothelium. The neoplastic effect of vinyl chloride of smaller doses (100, 10, 1 and 0 [control] ppm) and shorter exposure (four weeks) was studied in lungs of 120 mice. Our preliminary observation indicated that sacrificed animals at 40 weeks after the exposure showed productions of the alveologenic tumor in 5 of 9 (100 ppm), 2 of 9 (10 ppm), 1 of 9 (1 ppm) and 0 of 10 (control = 0 ppm). A dose-response relation was considered in the incidence of the alveologenic tumor production of vinyl chloride. It is concluded that mouse lung is an extremely sensitive indicator of the oncogenicity of vinyl chloride.

Nonneoplastic effects of vinyl chloride in mouse lung

In a previous study, a high incidence of pulmonary tumors (alveologenic neoplasia in mouse lung exposed to vinyl chloride at heavy dose (2500 and 6000 ppm) for long durations (5 and 6 months) was reported ( Y. Suzuki, 1978, Environ. Res. 16, 285–301 ). In the present study, nonneoplastic effects in mouse lung were investigated by light and electron microscopy. As major light microscopic alterations, proliferation and hypertrophy of the terminal bronchiolar cells, consisting of ciliated and Clara cells, hypersecretion of the epithelial mucin in the goblet cells of both the bronchial and the proximal bronchiolar epithelium, hyperplasia of alveolar epithelium, mobilization of alveolar macrophages, and occasional presence of peribronchial or bronchiolar chronic inflammation, were observed. Electron microscopically, Clara cells of the terminal bronchiolar epithelium showed proliferation of the rough and smooth surfaced endoplasmic reticulum and appearance of large and abnormally shaped mitochondria. Similar alterations were found in the ciliated cells. Submicroscopic changes of pulmonary alveoli were represented by focal thickening of the basement membrane, multiple foci of hyperplastic type II cell (the precondition of the alveologenic tumor), active discharge of osmiophilic lamellar bodies from the type II cell and phagocytosis of the bodies by macrophages, appearance of cholesterol crystalloids in the macrophages, degeneration of alveolar septal cells and occasional appearance of a large nucleus with swelling of the capillary endothelium.

Chapter 4 Explant Culture of Human Peripheral Lung

This chapter describes the explant culture of human peripheral lung. Fragments of fetal or adult lung have been cultured from human or animal species and on various substrates such as membrane filters on stainless steel grids, lens paper. Prior to culture, the lung specimens consist of typical alveolar structures lined with epithelial cells. The alveoli in specimens from some patients are nearly filled with macrophages, whereas in other cases they contain only a small number of macrophages. Connective tissue composed of fibroblast cells, collagen, and elastin often surrounds the alveolar structures. Terminal bronchiolar cells and endothelial cells are also observed. The alveoli of fresh tissue contained type-1 epithelial cells with extended cytoplasm and type-2 epithelial cells. Gaseous exchange occurs across the cytoplasm of the type-1 cells. Type-2 cells contained intracytoplasmic lamellar inclusion bodies that corresponded to the darkly stained inclusions observed in the cytoplasm of these cells with light microscopy. The alveolar walls remained intact in the peripheral portions of the explants for relatively long periods of time.