Nonciliated Bronchiolar Cells Research Articles

The multiple facets of the club cell in the pulmonary epithelium.

The non-ciliated bronchiolar cell, also referred to as "club cell", serves as a significant multifunctional component of the airway epithelium. While the club cell is a prominent epithelial type found in rodents, it is restricted to the bronchioles in humans. Despite these differences, the club cell's importance remains undisputed in both species due to its multifunctionality as a regulatory cell in lung inflammation and a stem cell in lung epithelial regeneration. The objective of this review is to examine different aspects of club cell morphology and physiology in the lung epithelium, under both normal and pathological conditions, to provide a comprehensive understanding of its importance in the respiratory system.

Sex differences in vanadium inhalation effects in non-ciliated bronchiolar cells.

The non-ciliated bronchiolar cell (NCBC) is responsible for the defense of the lung and responds to negative stimuli such as exposure to toxic pro-oxidant substances, which triggers the hyperproduction and hypersecretion of mucins and CC16 protein. The literature demonstrates that physiological and pathological responses in the lung can be influenced by the organism's sex. The objective of this report was to evaluate response differences to vanadium (V) inhalation in male and female CD-1 mice. Mice were exposed to V for four weeks. Hyperplasia of bronchiolar epithelium, small inflammatory foci and sloughing of the NCBC were observed, without changes between sexes and throughout the exposure time. Mucosecretory metaplasia was found in both males and females, however it was more drastic in males. The expression of CC16 increased in both sexes. This study demonstrated a different susceptibility between male and female mice exposed to V inhalation regarding mucosecretory metaplasia.

The role of the non-ciliated bronchiolar cell in tolerance to inhaled vanadium of the bronchiolar epithelium.

The Non-Ciliated Bronchiolar Cell (NCBC) is responsible for the defense and maintenance of the bronchiolar epithelium. Several cellular defense mechanisms have been associated with an increase in the secretion of CC16 and changes in the phenotype of the cell; these mechanisms could be linked to tolerance to the damage due to exposure to inhaled Particulate Matter (PM) of the epithelium. These defense mechanisms have not been sufficiently explored. In this article, we studied the response of the NCBC to inhaled vanadium, an element which adheres to PM. This response was measured by the changes in the phenotype of the NCBC and the secretion of CC16 in a mouse model. Mice were exposed in two phases to different vanadium concentrations; 1.27 mg/m³ in the first phase and 2.56 mg/m³ in the second phase. Mice were sacrificed on the 2nd, 4th, 5th, 6th and 8th weeks. In the second phase, we observed the following: sloughing of the NCBC, hyperplasia and small inflammatory foci remained without changes and that the expression of CC16 was higher in this phase than in phase I. We also observed a change in the phenotype with a slow decrease in both phases. The increase in the secretion of CC16 and the phenotype reversion could be due to the anti-inflammatory activity of CC16. The changes observed in the second phase could be attributed to the tolerance to inhaled vanadium.

A Novel Dynamic Model Describing the Spread of the MERS-CoV and the Expression of Dipeptidyl Peptidase 4.

The Middle East respiratory syndrome (MERS) coronavirus, a newly identified pathogen, causes severe pneumonia in humans. MERS is caused by a coronavirus known as MERS-CoV, which attacks the respiratory system. The recently defined receptor for MERS-CoV, dipeptidyl peptidase 4 (DPP4), is generally expressed in endothelial and epithelial cells and has been shown to be present on cultured human nonciliated bronchiolar epithelium cells. In this paper, a class of novel four-dimensional dynamic model describing the infection of MERS-CoV is given, and then global stability of the equilibria of the model is discussed. Our results show that the spread of MERS-CoV can also be controlled by decreasing the expression rate of DPP4.

Ultrastructure of Developing Feline Nonciliated Bronchiolar Epithelial Cells

The ultrastructure of the developing bronchiolar cell was studied in six age groups: prenatal (60 d post-conception); postnatal (1-, 7-, 14- and 21-day-old); and adult. Following intratracheal fixation, the lung tissue was processed for scanning and transmission electron microscopy. The lining of terminal bronchioles consists of cuboidal to columnar nonciliated bronchiolar cells (NBCs) and ciliated with or without microvilli. NBCs were recognized by indented centrally located nucleus. The apical surface extended beyond the surface of neighboring cells and was covered by minute microvilli, except in prenatal kittens. The NBCs of the adult were characterized by abundant mitochondria and glycogen inclusions. In prenatal kittens, the cytoplasm was filled with patches of alpha and beta form of glycogen. Postnatally, glycogen was reduced in quantity, became scattered throughout the cytoplasm and was predominantly of the beta form. Islands of cytoplasm, separated from the apical cytoplasm were observed in the lumen of adult bronchioles. This suggests an apocrine mode of secretion. The NBCs attain maturity by three weeks of age.

IL-1β expression in the distal lung epithelium disrupts lung morphogenesis and epithelial cell differentiation in fetal mice

Perinatal inflammation and the inflammatory cytokine IL-1 can modify lung morphogenesis. To examine the effects of antenatal expression of IL-1β in the distal airway epithelium on fetal lung morphogenesis, we studied lung development and surfactant expression in fetal mice expressing human IL-1β under the control of the surfactant protein (SP)-C promoter. IL-1β-expressing pups suffered respiratory failure and died shortly after birth. IL-1β caused fetal lung inflammation and enhanced the expression of keratinocyte-derived chemokine (KC/CXCL1) and monocyte chemoattractant protein 3 (MCP-3/CCL7), the calgranulins S100A8 and S100A9, the acute-phase protein serum amyloid A3, the chitinase-like proteins Ym1 and Ym2, and pendrin. IL-1β decreased the percentage of the total distal lung area made up of air saccules and the number of air saccules in the lungs of fetal mice. IL-1β inhibited the expression of VEGF-A and its receptors VEGFR-1 and VEGFR-2. The percentage of the cellular area of the distal lung made up of capillaries was decreased in IL-1β-expressing fetal mice. IL-1β suppressed the production of SP-B and pro-SP-C and decreased the amount of phosphatidylcholine and the percentage of palmitic acid in the phosphatidylcholine fraction of lung phospholipids, indicating that IL-1β prevented the differentiation of type II epithelial cells. The production of Clara cell secretory protein in the nonciliated bronchiolar (Clara) cells was likewise suppressed by IL-1β. In conclusion, expression of IL-1β in the epithelium of the distal airways disrupted the development of the airspaces and capillaries in the fetal lung and caused fatal respiratory failure at birth.

Serum concentration of surfactant protein D in horses with lower airway inflammation

Surfactant protein D (SP-D), mainly synthesised by alveolar type II cells and nonciliated bronchiolar cells, is one important component of innate pulmonary immunity. In man, circulating concentrations of SP-D are routinely used as biomarkers for pulmonary injury. To date, serum SP-D levels have only been investigated in horses in an experimental model of bacterial airway infection. To compare serum SP-D concentrations at rest and after exercise in horses with and without inflammatory airway disease (IAD). Venous blood samples were collected from 42 Standardbred racehorses at rest and 60 min after performing a standardised treadmill exercise test. Tracheal wash and bronchoalveolar lavage fluid (BALF) samples were collected after exercise. Based on BALF cytology, 22 horses were defined as IAD-affected and 20 classified as controls. Serum SP-D concentrations were assessed using a commercially available ELISA kit and statistically compared between groups of horses and sampling times. Serum concentrations of SP-D in IAD-affected horses were significantly higher than those of control horses, both at rest and after exercise. Within the IAD-affected group, no significant correlation was found between serum SP-D concentrations and BALF cytology. Within each group of horses (IAD and control), no significant influence of exercise was found on serum SP-D levels. This is the first study determining serum SP-D concentrations in a noninfectious, naturally occurring form of lower airway inflammation in horses. The results highlight that IAD is associated with a detectable, though moderate, increase of circulating SP-D levels. Serum concentration of surfactant protein D could represent a potentially valuable and readily accessible blood biomarker of equine lower airway inflammation.

S138 Clara cells inhibit alveolar epithelial wound repair through a TRAIL-dependent apoptosis mechanism

IntroductionAlveolar bronchiolization, a hyperproliferation of ciliated and non-ciliated (Clara) bronchiolar cells and their extension into the alveolar region, is a common feature of idiopathic pulmonary fibrosis. The role of this...

Highly Pathogenic Avian Influenza Virus H7N7 Isolated From a Fatal Human Case Causes Respiratory Disease in Cats but Does Not Spread Systemically

Highly pathogenic avian influenza viruses (HPAIV) of the H5 and H7 subtypes primarily infect poultry but are occasionally transmitted to humans and other mammalian species, often causing severe disease. Previously we have shown that HPAIV H5N1 causes severe systemic disease in cats. In this study, we investigated whether HPAIV H7N7 isolated from a fatal human case is also able to cause disease in cats. Additionally, we compared the cell tropism of both viruses by immunohistochemistry and virus histochemistry. Three domestic cats were inoculated intratracheally with HPAIV H7N7. Virus excretion was restricted to the pharynx. At necropsy, 7 days post inoculation, lesions were restricted to the respiratory tract in all cats. Lesions consisted of diffuse alveolar damage and colocalized with virus antigen expression in type II pneumocytes and nonciliated bronchiolar cells. The attachment patterns of HPAIV H7N7 and H5N1 were similar: both viruses attached to nonciliated bronchiolar epithelial cells, type II pneumocytes, as well as alveolar macrophages. These data show for the first time that a non-H5 HPAIV is able to infect and cause respiratory disease in cats. The failure of HPAIV H7N7 to spread beyond the respiratory tract was not explained by differences in cell tropism compared to HPAIV H5N1. These findings suggest that HPAIV H5N1 possesses other characteristics that allow it to cause systemic disease in both humans and cats.

Surfactant Protein B Propeptide Contains a Saposin-Like Protein Domain with Antimicrobial Activity at Low pH

Surfactant protein B (SP-B) proprotein contains three saposin-like protein (SAPLIP) domains: a SAPLIP domain corresponding to the mature SP-B peptide is essential for lung function and postnatal survival; the function of SAPLIP domains in the N-terminal (SP-BN) and C-terminal regions of the proprotein is not known. In the current study, SP-BN was detected in the supernatant of mouse bronchoalveolar lavage fluid (BALF) and in nonciliated bronchiolar cells, alveolar type II epithelial cells, and alveolar macrophages. rSP-BN indirectly promoted the uptake of bacteria by macrophage cell lines and directly killed bacteria at acidic pH, consistent with a lysosomal, antimicrobial function. Native SP-BN isolated from BALF also killed bacteria but only at acidic pH; the bactericidal activity of BALF at acidic pH was completely blocked by SP-BN Ab. Transgenic mice overexpressing SP-BN and mature SP-B peptide had significantly decreased bacterial burden and increased survival following intranasal inoculation with bacteria. These findings support the hypothesis that SP-BN contributes to innate host defense of the lung by supplementing the nonoxidant antimicrobial defenses of alveolar macrophages.

GP130-STAT3 Regulates Epithelial Cell Migration and Is Required for Repair of the Bronchiolar Epithelium

Following injury, bronchiolar cells undergo rapid squamous metaplasia, followed by proliferation and re-establishment of the complex columnar epithelium that is characteristic of the normal airway. Mechanisms that regulate the repair of bronchiolar epithelium are of considerable relevance for understanding the pathogenesis of both acute and chronic lung diseases associated with airway remodeling. This study was designed to identify the role of the GP130-STAT3 signaling pathway during repair of the bronchiolar epithelium. STAT3 (signal transducer and activator of transcription 3) and GP130 (glycoprotein 130) were each selectively deleted from the pulmonary epithelial cells of transgenic mice in vivo, producing Stat3(Delta/Delta) and Gp130(Delta/Delta) mice, respectively. Airway injury was induced in adult mice by administration of naphthalene, a toxicant of nonciliated respiratory epithelial cells (Clara cells). Nuclear STAT3 staining was induced in bronchiolar epithelial cells following naphthalene-mediated injury in control (Stat3(flox/flox)) mice. Whereas nearly complete repair of the bronchiolar epithelium was observed in control mice within 13 days, restoration of cell shape, cell density, and the pattern of ciliated and nonciliated cells did not occur in the peripheral bronchioles of either Stat3(Delta/Delta) or Gp130(Delta/Delta) mice. Expression of dominant-negative STAT3 inhibited airway epithelial cell migration during repair in vitro; wild-type STAT3 expression activated such migration. In the present study, we show that GP130-STAT3 signaling functions in a cell-autonomous manner to restore cell shape and numbers required for repair of the bronchiolar epithelium following injury.

H5N1 Virus Attachment to Lower Respiratory Tract

Highly pathogenic avian influenza virus (H5N1) may cause severe lower respiratory tract (LRT) disease in humans. However, the LRT cells to which the virus attaches are unknown for both humans and other mammals. We show here that H5N1 virus attached predominantly to type II pneumocytes, alveolar macrophages, and nonciliated bronchiolar cells in the human LRT, and this pattern was most closely mirrored in cat and ferret tissues. These findings may explain, at least in part, the localization and severity of H5N1 viral pneumonia in humans. They also identify the cat and the ferret as suitable experimental animals based on this criterion.

Sex differences in bronchiolar epithelium response after the inhalation of lead acetate (Pb)

In order to identify if there were sex differences in lead (Pb) lung concentrations and in bronchiolar response after its inhalation, a mice inhalation model was conducted. Sixty CD-1 adult mice from each sex inhaled separately, lead acetate 0.1 M for 1 h, thrice weekly during 15 days. Animals were evaluated for Pb-lung concentrations by atomic absorption spectrometry and for morphological evaluation by scanning electron microscopy (SEM). Higher Pb-lung concentrations were determined in females, however, more cell damage was found in males, finding that correlated with an increased loss of the nonciliated bronchiolar cells (NCBC) more sloughing and necrosis. Differences in particle clearance, oxidative stress handling, cytokines pathway activation and cytochrome P450 enzymes activity, all influenced by sex hormones, might be a possible explanation for our findings. The relevance of further studies in this field is stressed, as well as its relation to the different development expected for each sex in disease evolution, possible complications and treatment response.

Inhalation of cadmium, lead or its mixture: Effects on the bronchiolar structure and its relation with metal tissue concentrations

The human population in the industrialized world is constantly exposed to chemical mixtures of pollutants such as metals; information about the consequences of the interactions of these compounds on health is scarce. The current study examines the effects of the inhalation of lead (Pb), cadmium (Cd) and Pb–Cd mixture in mice models analyzing the metal concentrations in lung, and the morphological modifications in the bronchiolar epithelium identified by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) after 4 weeks of inhalation. Our results showed that metal concentrations in lung were higher compared to controls; however, Pb concentrations drastically decrease with the mixture. This reduction was also observed in the inhalation chamber. These data correlate with the morphological alterations observed, which consisted of flattened and decreased number of nonciliated bronchiolar cells (NCBC), bald ciliated cells and bundles of NCBC. These modifications were mainly given by Cd, alone or in combination with Pb. The clusters formed by NCBC cells suggest cell proliferation which probably means that after metal inhalation, the cells enhance their proliferative capacity in order to repopulate the bronchiolar wall.

Impaired recovery from naphthalene-induced bronchiolar epithelial injury in mice exposed to aged and diluted sidestream cigarette smoke

The effect of sidestream tobacco smoke combined with other pollutants is largely unknown. Previously, we found that distal airway epithelial repair was inhibited in mice exposed to sidestream tobacco smoke (TS) for 5 days followed by single exposure to naphthalene (NA), a common polycyclic aromatic hydrocarbon found in cigarette smoke, diesel exhaust, and pesticide formulations. The main injury target of NA is the nonciliated (Clara) bronchiolar cell. NA injury normally resolves in two weeks. Repair in mice exposed to TS and NA was unresolved in the distal bronchioles 14 days post-NA injury. We hypothesized that repair inhibition persisted as a first step towards long-term airway remodeling and expanded the previous study by evaluating repair 21 days after acute NA injury. Repair was evaluated using high resolution histopathology, TEM, and quantitative morphometry. In animals exposed to TS and NA, repair was still impaired; re-differentiation of Clara cells at the bronchoalveolar duct junction was incomplete, indicating repair was continuing. Compared to 14 days post-NA-injury, repair at 21 days post-NA treatment was more extensive. Animals exposed only to TS had epithelium similar to controls. While TS exposure impairs bronchiolar epithelial repair after NA exposure, this effect appears to be slowly resolving over time.

Metal mixture inhalation (Cd-Pb) and its effects on the bronchiolar epithelium. An ultrastructural approach

The current study explores the effects of the inhalation of lead (Pb), Cd and its mixture (Pb-Cd) in a mice model, analysing metal concentrations in the lung, and the morphological modifications in the bronchiolar epithelium identified by scanning electron microscopy after eight weeks of inhalation. Our results indicate that metal concentrations in lung were higher compared to controls; however, Pb concentrations drastically decrease in the mixture. This reduction was also observed in the inhalation chamber. The main changes observed in the bronchiole were mostly in the mixture. The modifications were mainly given by Cd alone and in the mixture, with a decreased number of nonciliated bronchiolar cells and an increased number of bundles of dividing cells. The additive effect of Pb-Cd is suggested, as the extensive damage observed was more evident when mice were exposed to the mixture, and the results endured more research in the area of inhaled mixtures.

Reversibility of Pulmonary Abnormalities by Conditional Replacement of Surfactant Protein D (SP-D) in Vivo

Surfactant protein D (SP-D) gene-targeted mice develop severe pulmonary disease associated with emphysema, pulmonary lipidosis, and foamy macrophage infiltrations. To determine the potential reversibility of these abnormalities, transgenic mice were developed in which SP-D was conditionally replaced in the respiratory epithelium of SP-D(-/-) mice. SP-D was not detected in the absence of doxycycline. Treatment with doxycycline after birth restored pulmonary SP-D concentrations and corrected pulmonary pathology at adulthood. When SP-D was replaced in adult SP-D(-/-) mice, alveolar SP-D was restored within 3 days, pulmonary lipid abnormalities were corrected, but emphysema persisted. In corrected adult SP-D(-/-) mice, loss of SP-D caused focal emphysema and pulmonary inflammation but did not cause phospholipid abnormalities characteristic of SP-D(-/-) mice. Thus, abnormalities in surfactant phospholipid homeostasis and alveolar macrophage abnormalities were readily corrected by restoration of SP-D. However, once established, emphysema was not reversed by SP-D. SP-D-dependent processes regulating surfactant lipid homeostasis were disassociated from those mediating emphysema.

Immunolocalization of sonic hedgehog (Shh) in developing mouse lung.

Expression of sonic hedgehog (Shh) is required for normal development of the lung during embryogenesis. Loss of Shh expression in mice results in tracheoesophageal fistula, lung hypoplasia, and abnormal lung lobulation. To determine whether Shh may play a role later in lung morphogenesis, immunostaining for Shh was performed in mouse lung from embryonic day (E) 10.5 to postnatal day (PD) 24. Shh was detected in the distal epithelium of the developing mouse lung from E10.5 to E16.5. From E16.5 until PD15, Shh was present in epithelial cells in both the peripheral and conducting airways. Although all cells of the developing epithelium uniformly expressed Shh at E10.5, Shh expression was restricted to subsets of epithelial cells by E16.5. Between E16.5 and PD15, non-uniform Shh staining of epithelial cells was observed in the conducting airways in a pattern consistent with the distribution of non-ciliated bronchiolar cells (i.e., Clara cells) and the Clara cell marker CCSP. Shh did not co-localize with hepatocyte nuclear factor/forkhead homologue-4 (HFH-4), beta-tubulin, or with the presence of cilia. These results support the concept that Shh plays a distinct regulatory role in the lung later in morphogenesis, when it may influence formation or cytodifferentiation of the conducting airways.

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.

Catalase immunocytochemistry allows automatic detection of lung type II alveolar cells.

In mammalian lung, type II pneumocytes are especially critical in normal alveolar functioning, as they are the major source of surfactant and the progenitors of type I alveolar cells. Moreover, they undergo proliferation and transformation into type I cells in most types of cellular injury, where flattened type I pneumocytes are selectively destroyed. Hyperplasia of alveolar type II cells has also been described in some human chronic lung diseases. In lung, type II pneumocytes and non-ciliated bronchiolar cells are the unique cell types that contain a considerable amount of peroxisomes. Due to the presence of dihydroxyacetone phosphate acyltransferase and non-specific lipid-transfer protein, these organelles have been suggested to be involved in the synthesis and/or transport of the lipid moiety of surfactant. In the present research, the peroxisomal marker enzyme catalase was immunolocalised at the light microscopic level, utilising the avidin-biotin complex method, in lung specimens excised from newborn, adult and aged rats. In all the examined stages the immunoreactivity was so selective for type II pneumocytes it allowed quantitation of these cells by an automated detection system. This was accomplished on specimens from newborn rat lung, in which labelled alveolar cells were counted by a grey level-based procedure and their main morphometric parameters were determined.