Tracheobronchial epithelium in the adult rhesus monkey: a quantitative histochemical and ultrastructural study

Previous studies of the intrapulmonary conducting airways of sheep and rabbit have demonstrated marked diversity in the epithelial populations lining them. Because studies of trachea and centriacinar regions of macaque monkeys suggested that primates may be even more diverse, the present study was designed to characterize the epithelial population throughout the airway tree of one primate species, the rhesus monkey. Trachea and intrapulmonary airways of the right cranial and middle lobes of glutaraldehyde/paraformaldehyde-infused lungs of five adult rhesus monkeys were microdissected following the axial pathway. Each branch was assigned a binary number indicating its specific location within the tree. The trachea and six generations of intrapulmonary airway from the right cranial lobe were evaluated for ultrastructure and quantitative histology as were those of the right middle lobe for quantitative carbohydrate histochemistry. Four cell types were identified throughout the tree: ciliated, mucous goblet, small mucous granule, and basal. The tallest epithelium lined the trachea; the shortest, the respiratory bronchiole. The most cells per unit length of basement membrane were in proximal intrapulmonary bronchi; the least, in the respiratory bronchiole. The nonciliated bronchiolar epithelial or Clara cell was restricted to respiratory bronchioles. Sulfomucins were present in the vast majority of surface goblet cells in the trachea and proximal bronchi. In proximal bronchi, neutral glycoconjugates predominated in glands and acidic glycoconjugates in surface epithelium. In terminal and respiratory bronchioles the ratio of acidic glycoconjugate to neutral glycoconjugate equaled that in proximal bronchi, although glands were not present. Sulfomucins were minimal in terminal airways. We conclude that the characteristics of the epithelial lining of the mammalian tracheobronchial airway tree are very species-specific. The lining of the rhesus monkey does not have the diversity in cell types in different airway generations observed in sheep and rabbit. Also, the populations lining these airways in the rhesus are very different from either the sheep or rabbit in number, proportions of different cell types, glycoconjugate content, and distribution of specific cell types.     

Methacholine responsiveness of proximal and distal airways of monkeys and rats using videomicrometry.

Rat and monkey are species that are used in models of human airway hyperresponsiveness. However, the wall structures of rat and monkey airways are different from each other, with that of the monkey more closely resembling that of humans. We hypothesized that differences in wall structure would explain differences in airway responsiveness. Using videomicrometry, we measured airway luminal area in lung slices to compare proximal and distal airway responsiveness to methacholine in the rat and monkey. The airway type was then histologically identified. Proximal airways of the young rat and monkey were equally responsive to methacholine. In contrast, respiratory bronchioles of monkeys were less responsive than were their proximal bronchi, whereas the distal bronchioles of rats were more responsive than their proximal bronchioles. Both proximal and distal airways of younger monkeys were more responsive than those of older monkeys. Airway heterogeneity in young monkeys was greatest with regard to degree of airway closure of respiratory bronchioles. We conclude that responsiveness to methacholine varies with airway wall structure and location.     

Morphogenesis of the respiratory bronchiole in rhesus monkey lungs

The epithelium of the respiratory bronchiole in the adult rhesus monkey consists of two populations: a pseudostratified epithelium with basal, mucous goblet, and ciliated cells located near the pulmonary artery (PA); and a simple cuboidal epithelium composed only of nonciliated bronchiolar epithelial (or Clara) cells in areas away from the PA. This study describes the pattern of differentiation of these two epithelial populations, and their relationship to the PA and to the time of appearance of alveoli in the respiratory bronchiole of the rhesus monkey during the period of 90-125 days gestational age (DGA). These events were related to changes in the adjacent parenchyma. Dissected airways of infusion-fixed, critical-point-dried lungs were evaluated by scanning microscopy followed by light microscopy of the same airways. At 54% of gestation (90 DGA), the distal airway was lined by a mixture of ciliated and nonciliated cells. By 67% of gestation (110 DGA), the ciliated cells were confined to the epithelium over the PA. The underlying connective tissue initially was cellular containing few fibers but was fibrous by 76% of gestation (125 DGA). Alveolarization began near the most distal cartilage at 57% of gestation (95 DGA), the same period at which secondary septation occurred in the distal acinus. Thus, alveolarization occurred simultaneously in two centers: 1) the proximal centriacinar region in the vicinity of the most distal cartilage and 2) the distal lung parenchyma. The duration of centriacinar alveolarization was short, approximately 5 days.     

Novel method for isolation of murine clara cell secretory protein-expressing cells with traces of stemness

Clara cells are non-ciliated, secretory bronchiolar epithelial cells that serve to detoxify harmful inhaled substances. Clara cells also function as stem/progenitor cells for repair in the bronchioles. Clara cell secretory protein (CCSP) is specifically expressed in pulmonary Clara cells and is widely used as a Clara cell marker. In addition CCSP promoter is commonly used to direct gene expression into the lung in transgenic models. The discovery of CCSP immunoreactivity in plasma membranes of airway lining cells prompted us to explore the possibility of enriching Clara cells by flow cytometry. We established a novel and simple method for the isolation of CCSP-expressing cell Clara cells using a combination of mechanical and enzymatic dissociation followed by flow cytometry sorting technology. We showed that ～25% of dissociated cells from whole lung expressed CCSP. In the resulting preparation, up to 98% of cells expressed CCSP. Notably, we found that several common stem cell markers including CD44, CD133, Sca-1 and Sox2 were expressed in CCSP(+) cells. Moreover, CCSP(+) cells were able to form spheroid colonies in vitro with 0.97‰ efficiency. Parallel studies in vivo confirmed that a small population of CCSP(-)expressing cells in mouse airways also demonstrates stem cell-like properties such as label retention and harboring rare bronchioalveolar stem cells (BASCs) in terminal bronchioles (TBs). We conclude that CCSP(+) cells exhibit a number of stem cell-like features including stem cell marker expression, bronchosphere colony formation and self-renewal ability. Clara cell isolation by flow cytometry sorting is a useful method for investigating the function of primary Clara cells in stem cell research and mouse models.     

CCSP modulates airway dysfunction and host responses in an Ova-challenged mouse model

Clara cell secretory protein (CCSP) is synthesized by nonciliated bronchiolar cells in the lung and modulates lung inflammation to infection. To determine the role of CCSP in the host response to allergic airway disease, CCSP-deficient [(-/-)] mice were immunized twice with ovalbumin (Ova) and challenged by Ova (2 or 5 mg/m(3)) aerosol. After 2, 3, and 5 days of Ova aerosol challenge (6 h/day), airway reactivity was increased in CCSP(-/-) mice compared with wild-type [CCSP(+/+)] mice. Neutrophils were markedly increased in the bronchoalveolar lavage fluid of CCSP(-/-) Ova mice, coinciding with increased myeloperoxidase activity and macrophage inflammatory protein-2 levels. Lung histopathology and inflammation were increased in CCSP(-/-) compared with wild-type mice after Ova challenge. Mucus production, as assessed by histological staining, was increased in the airway epithelium of CCSP(-/-) Ova mice compared with that in CCSP(+/+) Ova mice. These data suggest a role for CCSP in airway reactivity and the host response to allergic airway inflammation and provide further evidence for the role of the airway epithelium in regulating airway responses in allergic disease.     

Clara cell secretory protein modulates lung inflammatory and immune responses to respiratory syncytial virus infection

Clara cell secretory protein (CCSP) has been shown to have anti-inflammatory and immunomodulatory functions in the lung. Respiratory syncytial virus (RSV) is the most common cause of respiratory infection in infants and young children. RSV usually infects small airways and likely interacts with the Clara cells of bronchioles. To determine a possible role for CCSP during acute RSV infection, CCSP-deficient (CCSP(-/-)) and wild-type (WT) mice were intratracheally infected with RSV and the lung inflammatory and immune responses to RSV infection were assessed. RSV-F gene expression was increased in the lungs of CCSP(-/-) mice as compared with WT mice following RSV infection, consistent with increased viral persistence. Lung inflammation was significantly increased in CCSP(-/-) mice as compared with WT mice after infection. Moreover, although the levels of Th1 cytokines were similar, the levels of Th2 cytokines and neutrophil chemokines were increased in the lungs of CCSP(-/-) mice following infection. Physiologic endpoints of exacerbated lung disease, specifically airway reactivity and mucus production, were increased in CCSP(-/-) mice after RSV infection. Importantly, restoration of CCSP in the airways of CCSP(-/-) mice abrogated the increased viral persistence, lung inflammation, and airway reactivity. These findings suggest a role for CCSP and Clara cells in regulating lung inflammatory and immune responses to RSV infection.     

实验动物呼吸系统主要器官比较组织学研究

目的比较实验动物呼吸系统主要器官的组织学特征,为制定实验动物病理检测标准、以及毒理学、新药安全性评价提供依据。方法选取实验动物质量国家检测标准检测合格的恒河猴30只、昆明小鼠20只、SD大鼠20只、日本大耳白兔18只、比格犬16只、树鼩20只。除昆明小鼠采用颈椎脱臼致死外,其余动物麻醉后放血处死和病理解剖,对气管、肺脏进行病理大体检查和取材,常规病理制片,进行HE染色、特殊染色和免疫组化染色,显微镜下观察气管、肺脏的组织结构和细胞结构异同。结果 （1）实验动物气管上皮杯状细胞有差异：恒河猴、比格犬、日本大耳白兔杯状细胞较多,大鼠、小鼠、树鼩则较少或无。上皮分泌的黏液类型以中性黏液为主,比格犬杯状细胞分泌的黏液类型有中性黏液和酸性黏液。（2）实验动物黏膜下腺泡分布有差异：比格犬黏膜下层的腺泡最多,恒河猴、大鼠、小鼠、树鼩腺泡数量偏少,日本大耳白兔黏膜下层的混合腺泡最少。（3）实验动物的肺内支气管分支有差异：比格犬、恒河猴、日本大耳白兔由叶支气管、段支气管、小支气管、细支气管、终末细支气管和呼吸性细支气管组成,树鼩、大鼠、小鼠只由细支气管、终末细支气管和呼吸性细支气管组成。（4）实验动物细支气管组织结构有差异：恒河猴、比格犬的细支气管平滑肌为完整环形平滑肌层,没有缺失,而大鼠、小鼠、树鼩及日本大耳白兔的细支气管平滑肌薄或缺失。恒河猴、树鼩、大鼠细支气管有少量杯状细胞,其余实验动物均无杯状细胞。（5）实验动物Clara细胞形态有差异：比格犬Clara细胞呈立方形,其余动物呈柱状。结论实验动物呼吸系统组织结构的质是相同的,差异在于量的不同。研究人员在制定病理学检测标准、实验研究、药物安全性评价时应予充分考虑。     

Overexpression of lunatic fringe does not affect epithelial cell differentiation in the developing mouse lung

The Notch/Notch-ligand pathway regulates cell fate decisions and patterning in various tissues. Several of its components are expressed in the developing lung, suggesting that this pathway is important for airway cellular patterning. Fringe proteins, which modulate Notch signaling, are crucial for defining morphogenic borders in several organs. Their role in controlling cellular differentiation along anterior-posterior axis of the airways is unknown. Herein, we report the temporal-spatial expression patterns of Lunatic fringe (Lfng) and Notch-regulated basic helix-loop-helix factors, Hes1 and Mash-1, during murine lung development. Lfng was only expressed during early development in epithelial cells lining the larger airways. Those epithelial cells also expressed Hes1, but at later gestation Hes1 expression was confined to epithelium lining the terminal bronchioles. Mash-1 displayed a very characteristic expression pattern. It followed neural crest migration in the early lung, whereas at later stages Mash-1 was expressed in lung neuroendocrine cells. To clarify whether Lfng influences airway cell differentiation, Lfng was overexpressed in distal epithelial cells of the developing mouse lung. Overexpression of Lfng did not affect spatial or temporal expression of Hes1 and Mash-1. Neuroendocrine CGRP and protein gene product 9.5 expression was not altered by Lfng overexpression. Expression of proximal ciliated (beta-tubulin IV), nonciliated (CCSP), and distal epithelial cell (SP-C, T1alpha) markers also was not influenced by Lfng excess. Overexpression of Lfng had no effect on mesenchymal cell marker (alpha-sma, vWF, PECAM-1) expression. Collectively, the data suggest that Lunatic fringe does not play a significant role in determining cell fate in fetal airway epithelium.     

Temporal and spatial distribution of ciliogenesis in the tracheobronchial airways of mice

Little is known about ciliogenesis as it proceeds through the entire airway tree, from the trachea to the terminal bronchioles, especially during the postnatal period. The purpose of this study was to define the spatial and temporal (prenatal and postnatal) pattern of normal cilia development in the mouse. Three airway generations representing the entire airway tree were examined: trachea, lobar bronchi, and terminal bronchiole. Ciliated cells in lung lobe whole mounts were labeled with a fluorescent dye for confocal microscopy, and ciliated cell surface density was measured for each airway generation and age. The same samples were examined by scanning electron microscopy to verify the appearance of ciliated cells among the differentiating epithelium of the airways. Ciliated cells were first detected in the trachea and lobar bronchi at 16 days gestational age (DGA) and in the terminal bronchioles at 18 DGA. Ciliated cell surface density increased with prenatal and postnatal age at all airway levels. However, the ciliated cell surface density of the trachea and lobar bronchi was always greater compared with the terminal bronchiole. In conclusion, the study revealed that in developing tracheobronchial airways of the mouse: 1) Ciliogenesis differs temporally and spatially by airway generation; 2) Ciliated cell surface density increases with age in all airway generations, but density decreases in a proximal to distal direction; and 3) A significant portion of ciliogenesis continues after birth. This study provides a healthy basis for investigations of neonatal pulmonary disease or pollutant toxicity affecting cilia and its functions.     

Immunohistochemical mapping of endothelin in the developing and adult mouse lung.

Endothelins (ET) are a family of regulatory peptides displaying, among other abilities, potent constrictor actions. We studied the perinatal time course expression and distribution of ET in the mouse airway epithelium. In fetal mouse, ET-immunoreactivity (IR) appeared earlier (gestational Day 18) in the epithelium of upper (bronchi and large bronchioles) than in lower airways, being scarce and mainly located in the apical cytoplasm. As the lung developed, ET-IR became gradually stronger and extended throughout the cell in both bronchi and bronchioles. ET-IR was found in most airway epithelial cells. Clara cells were positive for ET, whereas ciliated and endocrine cells were not. In adult lungs, part of the myocytes and parenchymal cells also showed ET-IR. In both developing and adult mouse lungs, the cell distribution of ET-IR in the epithelium is compatible with apical and/or basal secretion. The presence of ET in mouse airway epithelium during the perinatal period may indicate a role for ET as a growth factor in lung development and its involvement in control of lung ventilation at birth.     

Cre-mediated recombination in mouse Clara cells

Clara cells are nonciliated secretory cells lining the respiratory epithelium and are easily identified by the expression of Clara cell secretory protein (CCSP). To investigate molecular mechanism(s) regulating Clara cell function in the lungs, Cre recombinase was inserted into exon 1 of the CCSP, generating two novel mouse models, CCSP(Cre-Neo) and CCSP(Cre). These two models differ only by the inclusion of the neomycin resistance gene. These mice were bred to the R26R reporter mouse to investigate the tissue and cell specificity of Cre-mediated recombination. The efficiency of Cre recombination in the CCSP(Cre) mouse model was higher than in the CCSP(Cre-Neo) mouse model. Recombination was detected at D 4.5 in CCSP(Cre-Neo)/R26R mice and at D 0.5 in CCSP(Cre)/R26R mice. The CCSP(Cre-Neo) and CCSP(Cre) mouse models provide valuable tools for the ablation of genes in the postnatal mouse Clara cells.     

Smooth muscle development during postnatal growth of distal bronchioles in infant rhesus monkeys.

Development of smooth muscle in conducting airways begins early in fetal life. Whereas the pattern and regulation of smooth muscle differentiation are well-defined, the impact of airway growth on the process is not. To evaluate the transformations in organization during postnatal growth, smooth muscle bundle organization (size, abundance, and orientation) was mapped in five generations of distal airways of infant rhesus monkeys (5 days and 1, 2, 3, and 6 mo old). On the basis of direct measurement of the bronchiole proximal to the terminal bronchiole, length increased by 2-fold, diameter by 1.35-fold, and surface area by 2.8-fold between 5 days and 6 mo of age. Smooth muscle bundle size was greater in proximal bronchioles than in respiratory bronchioles and did not change with age. However, relative bundle size decreased in proportion to airway size as the airways grew. Relative bundle abundance was constant regardless of airway generation or age. The distribution of smooth muscle bundle orientation changed with age in each airway generation, and there were significant changes in the terminal and respiratory bronchioles. We conclude that smooth muscle undergoes marked organizational changes as airways grow during postnatal development.     

The regulated expression of mRNA for Clara cell protein in the developing airways of the rat, as revealed by tissue in situ hybridization.

Tissue in situ hybridization has been used on sections of developing rat lung to follow the cellular sites of mRNA expression for a protein identified only in bronchiolar Clara cells. The mRNA for this Clara cell protein (CCP) was first detected on gestational day 16 in only one of the two types of tubules existing in the lung at this developmental stage. During the next 2 days CCP mRNA expression increased uniformly only in the epithelium lining the respiratory tubules. By gestational day 19, CCP mRNA expression became limited to secretory epithelial cells lining the bronchi, and terminal bronchioles. By neonatal day 1, an intense hybridization signal was observed along all of the conducting airways, but it was irregular due to the fact that expression of the CCP gene was limited to the secretory epithelial cells. In adult rats, CCP mRNA was expressed not only in secretory cells of the intrapulmonary airways at all anatomical levels, but also in secretory epithelial cells lining the trachea and its glands, as well as in specific alveolar cells thought to be type II pneumocytes. These findings demonstrate that the regulation of the CCP gene during lung development is a complicated process and that the expression of CCP mRNA does not parallel exactly the sequential development of the airways.     

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

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

Microscopic and ultrastructural anatomy of the trachea and bronchi of Melopsittacus undulatus (Aves,Psittaciformes)

Summary The normal microscopic pattern and ultrastructure of the lower trachea and the primary and secondary bronchi of the budgerigar (Melopsittacus undulatus) are described. The trachea is lined by mucociliary pseudostratified columnar epithelium with simple acinar mucous glands; epithelium in primary and secondary bronchi becomes progressively lower and less pseudostratified, and mucous cells less aggregated. The wall structure shows a parallel simplification; tracheal elements are comprised of osseous metaplastic cartilage with bone marrow between cancellous trabeculae, whereas distal secondary bronchial walls are principally comprised of smooth muscle. Mucous cells are similar to those described in mammalian, and other avian respiratory mucosae. Ciliated cells are similar to those known in other avian airways. No brush cells or Clara cells are observed.     

Airway level at which edema liquid enters the air space of isolated dog lungs

To identify lung units associated with liquid leakage into the air space in high-pressure pulmonary edema, we perfused air-inflated dog lung lobes with albumin solution to fill the loose peribronchovascular interstitium. Next, we perfused the lobes for 90 s with fluorescent albumin solution then froze the lobes in liquid nitrogen. This procedure confined the fluorescent perfusate to the liquid flux pathway between the circulation and the air space and eliminated the previously filled peribronchovascular cuffs as a source of the fluorescence that entered the air space. We divided each frozen lobe into three horizontal layers and prepared fluorescence-microscopic sections of each layer. In the most apical layers where alveolar flooding was minimal, 10.6 +/- 21.0% (SD) of alveolar ducts were either fluorescence filled or air filled and continuous with fluorescence-filled alveoli. In the same layers, 11.0 +/- 19.0% of respiratory bronchioles were similarly labeled. No terminal bronchioles in these layers were fluorescence labeled. This suggested that the fluorescent albumin entered the air space across the epithelium of respiratory bronchioles, alveolar ducts, or their associated alveoli. To simulate an alternative explanation, i.e., that fluorescence first entered central airways then flowed into peripheral air spaces, we prepared two additional lobes that we first partially inflated with fluorescent albumin then filled to capacity with air. This pushed the fluorescent solution along the airways into the lung periphery. In these lobes the ciliary lining of bronchi and terminal bronchioles was fluorescence coated. By comparison, cilia in fluorescence-perfused lobes were not coated. We conclude that alveolar flooding in hydrostatic pulmonary edema occurs across the epithelium of alveolar ducts, respiratory bronchioles, or their associated alveoli.(ABSTRACT TRUNCATED AT 250 WORDS)     

CCSP regulates cross talk between secretory cells and both ciliated cells and macrophages of the conducting airway

Pulmonary host defense employs a combination of biochemical and biophysical activities to recognize, inactivate, and mediate clearance of environmental agents as well as modulate the overall response to such challenge. Dysregulation of the inflammatory arm of this response is associated with chronic lung diseases (CLD) including cystic fibrosis and chronic obstructive lung disease. Although mechanisms mediating immunoregulation are incompletely characterized, decrements in levels of the nonciliated secretory cell product Clara cell secretory protein (CCSP) in numerous CLD and identification of proinflammatory state in mice homozygous for a null allele of the CCSP gene (CCSP-/-) suggest a central role for the nonciliated secretory cell in this process. In an effort to determine the molecular basis for immunoregulatory defects associated with CCSP deficiency, we utilized difference gel electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight to compare the proteomes of wild-type and CCSP-/- mice. We demonstrate a shift in the isoelectric point of the immunomodulatory protein annexin A1 (ANXA1) to more acidic isoforms in CCSP-/- mice. Similar ANXA1 mRNA and protein abundance in wild-type and CCSP-/- tissue and identical localization of ANXA1 protein to alveolar macrophages and the ciliary bed of ciliated cells demonstrated that CCSP deficiency was associated exclusively with altered posttranslational modification of ANXA1. These results suggest that both long- and short-range paracrine signaling between nonciliated secretory cells and cells of the immune system and epithelium impact modification of cell type-specific proteins and implicate nonciliated secretory cells in a regulatory axis that might integrate critical aspects of host defense.