Airway Branch Points Research Articles

Bronchus-associated macrophages efficiently capture and present soluble inhaled antigens and are capable of local Th2 cell activation.

In allergic asthma, allergen inhalation leads to local Th2 cell activation and peribronchial inflammation. However, the mechanisms for local antigen capture and presentation remain unclear. By two-photon microscopy of the mouse lung, we established that soluble antigens in the bronchial airway lumen were efficiently captured and presented by a population of CD11c+ interstitial macrophages with high CX3CR1-GFP and MHC class II expression. We refer to these cells as Bronchus-Associated Macrophages (BAMs) based on their localization underneath the bronchial epithelium. BAMs were enriched in collagen-rich regions near some airway branchpoints, where inhaled antigens are likely to deposit. BAMs engaged in extended interactions with effector Th2 cells and promoted Th2 cytokine production. BAMs were also often in contact with dendritic cells (DCs). After exposure to inflammatory stimuli, DCs migrated to draining lymph nodes, whereas BAMs remained lung resident. We propose that BAMs act as local antigen presenting cells in the lung and also transfer antigen to DCs.

Consider the lung as a sensory organ: A tip from pulmonary neuroendocrine cells.

While the lung is commonly known for its gas exchange function, it is exposed to signals in the inhaled air and responds to them by collaborating with other systems including immune cells and the neural circuit. This important aspect of lung physiology led us to consider the lung as a sensory organ. Among different cell types within the lung that mediate this role, several recent studies have renewed attention on pulmonary neuroendocrine cells (PNECs). PNECs are a rare, innervated airway epithelial cell type that accounts for <1% of the lung epithelium population. They are enriched at airway branch points. Classical in vitro studies have shown that PNECs can respond to an array of aerosol stimuli such as hypoxia, hypercapnia and nicotine. Recent in vivo evidence suggests an essential role of PNECs at neuroimmunomodulatory sites of action, releasing neuropeptides, neurotransmitters and facilitating asthmatic responses to allergen. In addition, evidence supports that PNECs can function both as progenitor cells and progenitor niches following airway epithelial injury. Increases in PNECs have been documented in a large array of chronic lung diseases. They are also the cells-of-origin for small cell lung cancer. A better understanding of the specificity of their responses to distinct insults, their impact on normal lung function and their roles in the pathogenesis of pulmonary ailments will be the next challenge toward designing therapeutics targeting the neuroendocrine system in lung.

Lobe-Specific Gene Vector Delivery to Rat Lungs Using a Miniature Bronchoscope.

For respiratory research utilizing gene vector delivery to the lung, the size of rodent models has typically necessitated relatively "blind" dosing via the nose, via an endotracheal tube, or through a surgical incision into the trachea. This commonly results in a limited ability to dose specific small regions of the lung reliably, and contributes to high levels of transduction variability between animals. The resultant poor reliability, reproducibility, and high variability compromises statistical capability, and so demands greater animal sample sizes than should be feasible. The first reliable targeted gene vector dosing of small regions in rat lungs has been designed and successfully implemented using a miniature rigid bronchoscope containing a working channel. Using this setup, this technique can currently access airway branches down to at least the fourth generation in the lungs of rats >200 g in body weight, allowing dosing and re-dosing of specific lobes via airway branch points in the lung tree. Here, the protocol for performing this minimally invasive technique is reported, along with the effect of delivering vesicular stomatitis virus G pseudotyped lentivirus to selected lung lobes. Examples of other applications, such as delivery of agar beads, are also shown. It is expected that the availability of this technique will substantially enhance gene vector studies in rat models for a range of lung diseases.

Finding a role for PNECs in asthma

Immunology Pulmonary neuroendocrine cells (PNECs) are a rare cell type located in airway and alveolar epithelia and are often in contact with sensory nerve fibers. They have a wide phylogenic distribution and are found even in the relatively primitive lungs of amphibia and reptiles, suggesting a critical function. Sui et al. found that mice lacking PNECs have suppressed type 2 (allergic) immune responses. PNECs were observed in close proximity to group 2 innate lymphoid cells (ILC2s) around airway branch points. The PNECs enhanced ILC2 activity by secreting CGRP (calcitonin gene-related peptide). They also induced goblet-cell hyperplasia via the neurotransmitter GABA (γ-aminobutyric acid). Interestingly, human asthma patients were found to have increased PNEC numbers, suggesting a potential therapeutic target for the treatment of asthma. Science , this issue p. [eaan8546][1] [1]: /lookup/doi/10.1126/science.aan8546

Pulmonary neuroendocrine cells amplify allergic asthma responses.

Pulmonary neuroendocrine cells (PNECs) are rare airway epithelial cells whose function is poorly understood. Here we show that Ascl1-mutant mice that have no PNECs exhibit severely blunted mucosal type 2 response in models of allergic asthma. PNECs reside in close proximity to group 2 innate lymphoid cells (ILC2s) near airway branch points. PNECs act through calcitonin gene-related peptide (CGRP) to stimulate ILC2s and elicit downstream immune responses. In addition, PNECs act through the neurotransmitter γ-aminobutyric acid (GABA) to induce goblet cell hyperplasia. The instillation of a mixture of CGRP and GABA in Ascl1-mutant airways restores both immune and goblet cell responses. In accordance, lungs from human asthmatics show increased PNECs. These findings demonstrate that the PNEC-ILC2 neuroimmunological modules function at airway branch points to amplify allergic asthma responses.

Establishing a Normative Atlas of the Human Lung: Computing the Average Transformation and Atlas Construction

To establish the range of normal for quantitative computed tomography (CT)-based measures of lung structure and function, we seek to develop methods for matching pulmonary structures across individuals and establishing a normative human lung atlas. In our previous work, we have presented a three-dimensional (3D) image registration method suitable for pulmonary atlas construction based on CT datasets. The method has been applied to a population of normative lungs in multiple experiments and, in each instance, has resulted in significant reductions in registration errors. This study is a continuation to our previous work by presenting a method for synthesizing a computerized human lung atlas from previously registered and matched 3D pulmonary CT datasets from a population of normative subjects. Our method consists of defining the origin of the atlas coordinate system; defining the nomenclature and labels for anatomical structures within the atlas system; computing the average transformation based on the displacement fields to register individual subject to the common template subject; constructing the atlas by deforming the template with the average transformation; and calculating shape variations within the population. The feasibility of pulmonary atlas construction was evaluated using CT datasets from 20 normal volunteers. Substantial reductions in shape variability were demonstrated. In addition, the constructed atlas depends only slightly on a specific subject being selected as the template. These results indicate the framework is a robust and valid method for pulmonary atlas construction based on CT scans. The atlas consists of a grayscale CT dataset of the template, a labeled mask dataset of the template (ie, lungs, lobes, and lobar fissures are labeled with different gray levels), a data set representing the population's average shape, datasets representing the population's shape variations (ie, the magnitude of standard deviation), a data structure to contain the labels and coordinates of major airway branchpoints, and the labels of the mask dataset, and a reference coordinate system for each lung. A computerized human lung atlas representing by the average shape of a population of twenty normal subjects was constructed and visualized. The atlas provides a basis for establishing regional ranges of normative values for structural and functional measures of the human lung. In the future, we plan to use the computerized human lung atlas to help detect and quantify early signs of lung pathology.

Pulmonary CT image registration and warping for tracking tissue deformation during the respiratory cycle through 3D consistent image registration

Tracking lung tissues during the respiratory cycle has been a challenging task for diagnostic CT and CT-guided radiotherapy. We propose an intensity- and landmark-based image registration algorithm to perform image registration and warping of 3D pulmonary CT image data sets, based on consistency constraints and matching corresponding airway branchpoints. In this paper, we demonstrate the effectivenss and accuracy of this algorithm in tracking lung tissues by both animal and human data sets. In the animal study, the result showed a tracking accuracy of 1.9 mm between 50% functional residual capacity (FRC) and 85% total lung capacity (TLC) for 12 metal seeds implanted in the lungs of a breathing sheep under precise volume control using a pulmonary ventilator. Visual inspection of the human subject results revealed the algorithm's potential not only in matching the global shapes, but also in registering the internal structures (e.g., oblique lobe fissures, pulmonary artery branches, etc.). These results suggest that our algorithm has significant potential for warping and tracking lung tissue deformation with applications in diagnostic CT, CT-guided radiotherapy treatment planning, and therapeutic effect evaluation.

Expression of Hoxb‐5 during human lung development and in congenital lung malformations

We have previously shown that the Hox gene Hoxb-5 is necessary for normal mouse lung branching morphogenesis. Abnormal Hoxb-5 regulation causes specific alterations in airway branching. We hypothesized that Hoxb-5 is similarly involved in human lung branching morphogenesis, and is abnormally expressed in bronchopulmonary sequestration (BPS) and congenital cystic adenomatoid malformation (CCAM), both of which are congenital lung malformations with abnormal airway development. The temporal, spatial, and cellular expression of the Hoxb-5 protein was evaluated in normal human lung and BPS and CCAM tissue using Western blot analysis and immunocytochemistry. The expression of Hoxb-5 during human lung development showed strong similarities to that during mouse lung development. Western blots showed high Hoxb-5 protein levels in the pseudoglandular period (PSG), decreased but sustained levels in the canalicular period (CAN), and negligible levels during the alveolar period (ALV). Immunocytochemistry showed Hoxb-5 protein expression in mesenchymal cells around branching airways in the pseuodglandular period, subepithelial fibroblast localization (especially at airway branch points) in the CAN and minimal expression in the ALV. In BPS and CCAM tissue, Hoxb-5 protein levels were increased compared to age- and developmentally-matched lung tissue, and were more similar to the PSG and CAN with Hoxb-5-positive cells in mesenchyme surrounding abnormally branched airways. Hoxb-5 expression during human lung branching morphogenesis, which is similar to that observed in mouse lung development, indicates that it plays a role in controlling airway patterning. This notion is supported by results from BPS and CCAM tissue, in which Hoxb-5 is maintained in a manner typical of an earlier developmental stage and is associated with development of abnormal lung tissue.

Dihydrotestosterone stimulates branching morphogenesis, cell proliferation, and programmed cell death in mouse embryonic lung explants.

Early gestation lung development is characterized by branching morphogenesis of the airways and basic lung structure formation. Androgens delay late-gestation lung development if the androgen exposure begins in early gestation. We hypothesized that there would be effects of early gestation androgens on lung development. Embryonic mouse lungs (d 11.5) were cultured with dihydrotestosterone (DHT), DHT plus flutamide, or with nothing as controls. Branching morphogenesis was significantly increased after 24, 48, and 72 h of culture. This effect was blocked by simultaneous flutamide treatment. Fetal sex did not influence the DHT response. DHT increased cell proliferation as measured by [3H]thymidine incorporation into DNA. Autoradiography showed prominent [3H]thymidine labeling of epithelia and mesenchyme in regions of new bud formation. DHT treatment significantly increased the thymidine-labeling index of fibroblasts and airway epithelial cells. Programmed cell death, which is found in developing organs in association with cell proliferation during structure formation and tissue remodeling, was studied using terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling assay. In control lungs, programmed cell death occurred in the peripheral mesenchyme surrounding newly forming buds and underlying airway branch points. DHT treatment increased programmed cell death in association with increased branching morphogenesis. Evaluation of near-adjacent sections (control and DHT-treated lungs) showed that apoptotic mesenchymal cells were flanked by [3H]thymidine-labeled fibroblasts and epithelial cells, suggesting a coordination of these processes in the progression of branching morphogenesis. We conclude that androgen enhances the process of early lung morphogenesis by increasing cell proliferation and programmed cell death and by promoting the structural progression of branching morphogenesis.

Neuroepithelial Bodies of Pulmonary Airways Serve as a Reservoir of Progenitor Cells Capable of Epithelial Regeneration

Remodeling of the conducting airway epithelium is a common finding in the chronically injured lung and has been associated with increased risk for developing lung cancer. Pulmonary neuroendocrine cells and clusters of these cells termed neuroepithelial bodies (NEBs) play a central role in each of these processes. We previously developed an adult mouse model of airway injury and repair in which epithelial regeneration after naphthalene-induced Clara cell ablation occurred preferentially at airway branch points and gave rise to nascent Clara cells. Continued repair was accompanied by NEB hyperplasia. We now provide the following evidence that the NEB microenvironment serves as a source of airway progenitor cells that contribute to focal regeneration of the airway epithelium: 1) nascent Clara cells and NEBs localize to the same spatial domain; 2) within NEB, both Clara cell secretory protein- and calcitonin gene-related peptide-immunopositive cells are proliferative; 3) the NEB microenvironment of both the steady-state and repairing lung includes cells that are dually immunopositive for Clara cell secretory protein and calcitonin gene-related peptide, which were previously identified only within the embryonic lung; and 4) NEBs harbor variant Clara cells deficient in cytochrome P450 2F2-immunoreactive protein. These data suggest that the NEB microenvironment is a reservoir of pollutant-resistant progenitor cells responsive to depletion of an abundant airway progenitor such as the Clara cell.

Ontogeny of Clara cell-specific protein and its mRNA: their association with neuroepithelial bodies in human fetal lung and in bronchopulmonary dysplasia.

Clara cell-specific 10-KD protein (CCSP) is an abundant product of nonciliated bronchiolar epithelial (Clara) cells in the lung. We have determined the temporal-spatial distribution of CCSP and its mRNA in developing human lung and in neonatal lung disease, using immunohistochemistry and in situ hybridization. CCSP immunoreactivity was found in nonciliated bronchiolar epithelial cells from 12 weeks of gestation onward. Tracheal and bronchial epithelia showed positive immunoreactivity at each gestational week after 15 weeks and 14 weeks, respectively. CCSP mRNA was seen in the bronchial and bronchiolar epithelia from 16 weeks onward and was detected in the trachea from 19 through 23 weeks of gestation. CCSP immunoreactivity and mRNA were present in nonciliated single cells of bronchial and bronchiolar epithelia in fetuses and in infants with and without lung disease. CCSP- and CCSP mRNA-containing epithelial cells also formed dusters around neuroepithelial bodies (NEBs), especially at airway branch points, suggesting that NEBs and Clara cells might interact during development and during pulmonary regeneration. Because of evidence of overlapping of some but not all cells expressing CCSP, SP-A, and pro-SP-B during lung development, a common cell lineage is proposed, with subsequent divergence of phenotypes.

Bombesin and [Leu8]phyllolitorin promote fetal mouse lung branching morphogenesis via a receptor-mediated mechanism.

Pulmonary neuroendocrine cells are localized predominantly at airway branchpoints. Previous work showed that gastrin-releasing peptide (GRP), a major pulmonary bombesin-like peptide, occurred in neuroendocrine cells exclusively in branching human fetal airways. We now demonstrate that GRP and GRP receptor genes are expressed in fetal mouse lung as early as embryonic day 12 (E12), when lung buds are beginning to branch. By in situ hybridization, GRP receptor transcripts were at highest levels in mesenchymal cells at cleft regions of branching airways and blood vessels. To explore the possibility that bombesin-like peptides might play a role in branching morphogenesis, E12 lung buds were cultured for 48 hr in serum-free medium. In the presence of 0.10-10 microM bombesin, branching was significantly augmented as compared with control cultures, with a peak of 94% above control values at 1 microM (P < 0.005). The bombesin receptor antagonist [Leu13- psi(CH2NH)Leu14]bombesin alone (100 nM) had no effect on baseline branching but completely abolished bombesin-induced branching. A bombesin-related peptide, [Leu8]phyllolitorin also increased branching (65% above control values at 10 nM, P < 0.005). [Leu8]Phyllolitorin also significantly augmented thymidine incorporation in cultured lung buds. Fibronectin, which is abundant at branchpoints, induces GRP gene expression in undifferentiated cell lines. These observations suggest that BLPs secreted by pulmonary neuroendocrine cells may contribute to lung branching morphogenesis. Furthermore, components of branchpoints may induce pulmonary neuroendocrine cell differentiation as part of a positive feedback loop, which could account in part for the high prevalence of these cells at branchpoints.