Extrapulmonary Airways Research Articles

Ozone exposure upregulates the expression of host susceptibility protein TMPRSS2 to SARS-CoV-2

SARS-CoV-2, a novel coronavirus and an etiologic agent for the current global health emergency, causes acute infection of the respiratory tract leading to severe disease and significant mortality. Ever since the start of SARS-CoV-2, also known as the COVID-19 pandemic, countless uncertainties have been revolving around the pathogenesis and epidemiology of the SARS-CoV-2 infection. While air pollution has been shown to be strongly correlated to increased SARS-CoV-2 morbidity and mortality, whether environmental pollutants such as ground-level ozone affects the susceptibility of individuals to SARS-CoV-2 is not yet established. To investigate the impact of ozone inhalation on the expression levels of signatures associated with host susceptibility to SARS-CoV-2, we analyzed lung tissues collected from mice that were sub-chronically exposed to air or 0.8 ppm ozone for three weeks (4 h/night, 5 nights/week), and analyzed the expression of signatures associated with host susceptibility to SARS-CoV-2. SARS-CoV-2 entry into the host cells is dependent on the binding of the virus to the host cellular receptor, angiotensin-converting enzyme (ACE2), and its subsequent proteolytic priming by the host-derived protease, transmembrane protease serine 2 (TMPRSS2). The Ace2 transcripts were significantly elevated in the parenchyma, but not in the extrapulmonary airways and alveolar macrophages, from ozone-exposed mice. The TMPRSS2 protein and Tmprss2 transcripts were significantly elevated in the extrapulmonary airways, parenchyma, and alveolar macrophages from ozone-exposed mice. A significant proportion of additional known SARS-CoV-2 host susceptibility genes were upregulated in alveolar macrophages and parenchyma from ozone-exposed mice. Our data indicate that the unhealthy levels of ozone in the environment may predispose individuals to severe SARS-CoV-2 infection. Given the severity of this pandemic and the challenges associated with direct testing of host-environment interactions in clinical settings, we believe that this ozone exposure-based study informs the scientific community of the potentially detrimental effects of the ambient ozone levels in determining the host susceptibility to SARS-CoV-2.

SOX21 modulates SOX2-initiated differentiation of epithelial cells in the extrapulmonary airways.

SOX2 expression levels are crucial for the balance between maintenance and differentiation of airway progenitor cells during development and regeneration. Here, we describe patterning of the mouse proximal airway epithelium by SOX21, which coincides with high levels of SOX2 during development. Airway progenitor cells in this SOX2+/SOX21+ zone show differentiation to basal cells, specifying cells for the extrapulmonary airways. Loss of SOX21 showed an increased differentiation of SOX2+ progenitor cells to basal and ciliated cells during mouse lung development. We propose a mechanism where SOX21 inhibits differentiation of airway progenitors by antagonizing SOX2-induced expression of specific genes involved in airway differentiation. Additionally, in the adult tracheal epithelium, SOX21 inhibits basal to ciliated cell differentiation. This suppressing function of SOX21 on differentiation contrasts SOX2, which mainly drives differentiation of epithelial cells during development and regeneration after injury. Furthermore, using human fetal lung organoids and adult bronchial epithelial cells, we show that SOX2+/SOX21+ regionalization is conserved. Lastly, we show that the interplay between SOX2 and SOX21 is context and concentration dependent leading to regulation of differentiation of the airway epithelium.

Airway basal stem cells generate distinct subpopulations of PNECs.

SUMMARYPulmonary neuroendocrine cells (PNECs) have crucial roles in airway physiology and immunity by producing bioactive amines and neuropeptides (NPs). A variety of human diseases exhibit PNEC hyperplasia. Given accumulated evidence that PNECs represent a heterogenous population of cells, we investigate how PNECs differ, whether the heterogeneity is similarly present in mouse and human cells, and whether specific disease involves discrete PNECs. Herein, we identify three distinct types of PNECs in human and mouse airways based on single and double positivity for TUBB3 and the established NP markers. We show that the three PNEC types exhibit significant differences in NP expression, homeostatic turnover, and response to injury and disease. We provide evidence that these differences parallel their distinct cell of origin from basal stem cells (BSCs) or other airway epithelial progenitors.

Caveolin-3 differentially orchestrates cholinergic and serotonergic constriction of murine airways

The mechanisms of controlling airway smooth muscle (ASM) tone are of utmost clinical importance as inappropriate constriction is a hallmark in asthma and chronic obstructive pulmonary disease. Receptors for acetylcholine and serotonin, two relevant mediators in this context, appear to be incorporated in specialized, cholesterol-rich domains of the plasma membrane, termed caveolae due to their invaginated shape. The structural protein caveolin-1 partly accounts for anchoring of these receptors. We here determined the role of the other major caveolar protein, caveolin-3 (cav-3), in orchestrating cholinergic and serotonergic ASM responses, utilizing newly generated cav-3 deficient mice. Cav-3 deficiency fully abrogated serotonin-induced constriction of extrapulmonary airways in organ baths while leaving intrapulmonary airways unaffected, as assessed in precision cut lung slices. The selective expression of cav-3 in tracheal, but not intrapulmonary bronchial epithelial cells, revealed by immunohistochemistry, might explain the differential effects of cav-3 deficiency on serotonergic ASM constriction. The cholinergic response of extrapulmonary airways was not altered, whereas a considerable increase was observed in cav-3−/− intrapulmonary bronchi. Thus, cav-3 differentially organizes serotonergic and cholinergic signaling in ASM through mechanisms that are specific for airways of certain caliber and anatomical position. This may allow for selective and site-specific intervention in hyperreactive states.

Caveolin-1: Functional Insights into Its Role in Muscarine- and Serotonin-Induced Smooth Muscle Constriction in Murine Airways.

An increased bronchoconstrictor response is a hallmark in the progression of obstructive airway diseases. Acetylcholine and 5-hydroxytryptamine (5-HT, serotonin) are the major bronchoconstrictors. There is evidence that both cholinergic and serotonergic signaling in airway smooth muscle (ASM) involve caveolae. We hypothesized that caveolin-1 (cav-1), a structural protein of caveolae, plays an important regulatory role in ASM contraction. We analyzed airway contraction in different tracheal segments and extra- and intrapulmonary bronchi in cav-1 deficient (cav-1−/−) and wild-type mice using organ bath recordings and videomorphometry of methyl-beta-cyclodextrin (MCD) treated and non-treated precision-cut lung slices (PCLS). The presence of caveolae was investigated by electron microscopy. Receptor subtypes driving 5-HT-responses were studied by RT-PCR and videomorphometry after pharmacological inhibition with ketanserin. Cav-1 was present in tracheal epithelium and ASM. Muscarine induced a dose dependent contraction in all airway segments. A significantly higher Emax was observed in the caudal trachea. Although, caveolae abundancy was largely reduced in cav-1−/− mice, muscarine-induced airway contraction was maintained, albeit at diminished potency in the middle trachea, in the caudal trachea and in the bronchus without changes in the maximum efficacy. MCD-treatment of PLCS from cav-1−/− mice reduced cholinergic constriction by about 50%, indicating that cholesterol-rich plasma domains account for a substantial portion of the muscarine-induced bronchoconstriction. Notably, cav-1-deficiency fully abrogated 5-HT-induced contraction of extrapulmonary airways. In contrast, 5-HT-induced bronchoconstriction was fully maintained in cav-1-deficient intrapulmonary bronchi, but desensitization upon repetitive stimulation was enhanced. RT-PCR analysis revealed 5-HT1B, 5-HT2A, 5-HT6, and 5-HT7 receptors as the most prevalent subtypes in the airways. The 5-HT-induced-constriction in PCLS could be antagonized by ketanserin, a 5-HT2A receptor inhibitor. In conclusion, the role of cav-1, caveolae, and cholesterol-rich plasma domains in regulation of airway tone are highly agonist-specific and dependent on airway level. Cav-1 is indispensable for serotonergic contraction of extrapulmonary airways and modulates cholinergic constriction of the trachea and main bronchus. Thus, cav-1/caveolae shall be considered in settings such as bronchial hyperreactivity in common airway diseases and might provide an opportunity for modulation of the constrictor response.

The Search of Vagal Pulmonary Pleural Innervation

The visceral pleura envelops the lung lobes. The visceral and parietal pleurae form the pleural cavity with negative pressure to keep the normal respiration possible. The lungs are complicatedly innervated by bilateral vagal and spinal nerves that have the sensory and motor components. Pulmonary vagal sensory receptors have been identified within the tissues in the extrapulmonary and intrapulmonary airways. However, vagal vs. spinal innervation in the pulmonary visceral pleura has been undecided. Möllgaard (1912) determined the neurons that innervate the lungs by whole or partial extirpation of the lung lobe. Larsell (1922) examined the vagal degeneration in the lungs and supported the vagal origin. However, Larsell and Coffey (1928) turned to the spinal origin when they inserted the balloon between the lung lobe and the chest wall or diaphragm and found no change in the rate and depth of inspiration. With vagotomy and excision of the second and third thoracic spinal ganglia, Honjin (1956) determined the vagal and rejected the spinal origin. With vagotomy, Pintelon and colleague (2007) identified non-vagal ＂visceral pleura receptors＂. Recently, we identified the classical hilar and novel non-hilar vagal pleural innervation pathways and two kinds of vagal nerve endings in the visceral pleura and triangular ligaments. Most of the areas that face the dorsal thoracic cavity have no vagal innervation, whereas the interlobar areas and those areas that face the heart receive bilateral or unilateral vagal innervation with a left-vagus-rostral-lung vs. right-vagus-caudal-lung lateralized innervation pattern. The experimental considerations and meanings for the vagal innervation pattern are discussed.

Naphthalene cytotoxicity in microsomal epoxide hydrolase deficient mice.

Naphthalene (NA) is a ubiquitous pollutant to which humans are widely exposed. 1,2-Dihydro-1,2-dihydroxynaphthalene (NA-dihydrodiol) is a major metabolite of NA generated by microsomal epoxide hydrolase (mEH). To investigate the role of the NA-dihydrodiol and subsequent metabolites (i.e. 1,2-naphthoquinone) in cytotoxicity, we exposed both male and female wild type (WT) and mEH null mice (KO) to NA by inhalation (5, 10, 20 ppm for 4h). NA-dihydrodiol was ablated in the KO mice. High-resolution histopathology was used to study site-specific cytotoxicity, and formation of naphthalene metabolites was measured by HPLC in microdissected airways. Swollen and vacuolated airway epithelial cells were observed in the intra- and extrapulmonary airways of all mice at and below the current OSHA standard (10 ppm). Female mice may be more susceptible to this acute cytotoxicity. In the extrapulmonary airways, WT mice were more susceptible to damage than KO mice, indicating that the metabolites associated with mEH-mediated metabolism could be partially responsible for cytotoxicity at this site. The level of cytotoxicity in the mEH KO mice at all airway levels suggests that non-mEH metabolites are contributing to NA cellular damage in the lung. Our results indicate that the apparent contribution of mEH-dependent metabolites to toxicity differs by location in the lung. These studies suggest that metabolites generated through the mEH pathway may be of minor importance in distal airway toxicity and subsequent carcinogenesis from NA exposure.

Novel multi-functional europium-doped gadolinium oxide nanoparticle aerosols facilitate the study of deposition in the developing rat lung.

Ambient ultrafine particulate matter (UPM), less than 100 nm in size, has been linked to the development and exacerbation of pulmonary diseases. Age differences in susceptibility to UPM may be due to a difference in delivered dose as well as age-dependent differences in lung biology and clearance. In this study, we developed and characterized aerosol exposures to novel metal oxide nanoparticles containing lanthanides to study particle deposition in the developing postnatal rat lung. Neonatal, juvenile and adult rats (1, 3 and 12 weeks old) were nose only exposed to 380 μg m(-3) of ∼30 nm europium doped gadolinium oxide nanoparticles (Gd2O3:Eu(3+)) for 1 h. The deposited dose in the nose, extrapulmonary airways and lungs was determined using inductively-coupled plasma mass spectroscopy. The dose of deposited particles was significantly greater in the juvenile rats at 2.22 ng per g body weight compared to 1.47 ng per g and 0.097 ng per g for the adult and neonate rats, respectively. Toxicity was investigated in bronchoalveolar lavage fluid (BALF) by quantifying recovered cell types, and measuring lactate dehydrogenase activity and total protein. The toxicity data suggests that the lanthanide particles were not acutely toxic or inflammatory with no increase in neutrophils or lactate dehydrogenase activity at any age. Juvenile and adult rats had the same mass of deposited NPs per gram of lung tissue, while neonatal rats had significantly less NPs deposited per gram of lung tissue. The current study demonstrates the utility of novel lanthanide-based nanoparticles to study inhaled particle deposition in vivo and has important implications for nanoparticles delivery to the developing lung either as therapies or as a portion of particulate matter air pollution.

Pulmonary surfactant in the airway physiology: A direct relaxing effect on the smooth muscle

Beside alveoli, surface active material plays an important role in the airway physiology. In the upper airways it primarily serves in local defense. Lower airway surfactant stabilizes peripheral airways, provides the transport and defense, has barrier and anti-edematous functions, and possesses direct relaxant effect on the smooth muscle. We tested in vitro the effect of two surfactant preparations Curosurf® and Alveofact® on the precontracted smooth muscle of intra- and extra-pulmonary airways. Relaxation was more pronounced for lung tissue strip containing bronchial smooth muscle as the primary site of surfactant effect. The study does not confirm the participation of ATP-dependent potassium channels and cAMP-regulated epithelial chloride channels known as CFTR chloride channels, or nitric oxide involvement in contractile response of smooth muscle to surfactant.By controlling wall thickness and airway diameter, pulmonary surfactant is an important component of airway physiology. Thus, surfactant dysfunction may be included in pathophysiology of asthma, COPD, or other diseases with bronchial obstruction.

Tissue sensitivity of the rat upper and lower extrapulmonary airways to the inhaled electrophilic air pollutants diacetyl and acrolein.

The target site for inhaled vapor-induced injury often differs in mouth-breathing humans compared with nose-breathing rats, thus complicating the use of rat inhalation toxicity data for assessment of human risk. We sought to examine sensitivity of respiratory/transitional nasal (RTM) and tracheobronchial (TBM) mucosa to two electrophilic irritant vapors: diacetyl and acrolein. Computational fluid dynamic physiologically based pharmacokinetic modeling was coupled with biomarker assessment to establish delivered dose-response relationships in RTM and TBM in male F344 rats following 6 h exposure to diacetyl or acrolein. Biomarkers included glutathione status, proinflammatory and antioxidant gene mRNA levels, and nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Modeling revealed that 0.0094-0.1653 μg acrolein/min-cm(2) and 3.9-21.6 μg diacetyl/min-cm(2) were deposited into RTM/TBM. Results indicate RTM and TBM were generally of similar sensitivity to diacetyl and acrolein. For instance, both tissues displayed induction of antioxidant and proinflammatory genes, and nuclear accumulation of Nrf2 after electrophile exposure. Hierarchical cellular response patterns were similar in RTM and TBM but differed between vapors. Specifically, diacetyl exposure induced proinflammatory and antioxidant genes concomitantly at low exposure levels, whereas acrolein induced antioxidant genes at much lower exposure levels than that required to induce proinflammatory genes. Generally, diacetyl was less potent than acrolein, as measured by maximal induction of transcripts. In conclusion, the upper and lower extrapulmonary airways are of similar sensitivity to inhaled electrophilic vapors. Dosimetrically based extrapolation of nasal responses in nose-breathing rodents may provide an approach to predict risk to the lower airways of humans during mouth-breathing.

P155 Visualisation of Airway Nerves in Chronic Cough: Towards the Identification of the Human ‘Cough Receptor’

Introduction The spinal and vagal innervation of the respiratory tract is well defined, particularly in animals. These discoveries include the identification of pathways involved in provoking cough and descriptions of the guinea pig ‘cough receptor’. However, many of the immunochemical features of the airway afferents described in animals have yet to be defined in humans, yet plasticity of these airway afferents may be important in the pathophysiology of chronic cough. Objectives To define and characterise the innervation present in bronchoscopic biopsies from patients with chronic cough. We aimed to carry out the first ever whole mount immunohistochemical studies of airway nerves in cough patients, a technique that should improve visualisation of these neuronal structures and their sites of termination. Methods Biopsy tissue was gifted from patients undergoing clinical investigation for chronic cough. Tissue, sampled from throughout the extrapulmonary airways, was immediately fixed in 4% paraformaldehyde. Non-specific antibody binding was blocked using 10% normal serum and 1% skimmed milk powder, diluted in PBS, before application of primary antibodies. Polyclonal rabbit anti-PGP9.5 (Ultraclone, UK) and monoclonal mouse anti-neurofilament (NF200, Leica Biosystems, UK) were applied at a dilution of 1:1000 and 1:200 respectively. Primary antibody binding was detected using appropriate Alexa-fluor conjugated secondary antibodies and whole mount preparations were visualised using epifluorescence and confocal microscopy. Images of biopsy staining were subject to morphometric analysis. Results Many epithelial, subepithelial and intramuscular fibres were detected using both antibodies. Co-staining revealed that only PGP9.5 defined pulmonary neuroendocrine cells and better elucidated varicose epithelial fibres. A proportion of all fibre types were only immunopositive for one marker. Notably NF200 exclusively detected some epithelial fibres and a group of larger diameter neurons (O3–6 µm) some of which are consistent with observations of Aδ-fibre airway mechanoreceptors. Conclusions We have described neuronal structures in whole-mount preparations of tissue from patients suffering from chronic cough. Notably, we described a population of PGP9.5 negative fibres within the airways. Some of these fibres are morphologically consistent with thinly myelinated Aδ-fibres described in animal models as ‘cough receptors’. These ongoing studies could help define the plasticity of airway nerves in patients with chronic cough.

486 Prenatal Allergen Exposure Facilitated Airway Remodeling by Airborne Allergen Stimuli in Postnatal Life

BackgroundMurine asthma models are mainly created through adulthood sensitization, but lack airway remodeling hallmarks.AimsThis study aimed to examine airway remodeling in the prenatally-sensitized murine asthma model.MethodsFVB/N fetuses were exposed to...

Vagal afferent nerves regulating the cough reflex

Coughing is initiated by activation of mechanically and chemically sensitive vagal afferent nerves innervating the airways. All afferent nerve subtypes innervating the airways can modulate the cough reflex. Rapidly adapting and slowly adapting stretch receptors (RARs and SARs, respectively) innervating the intrapulmonary airways and lung may enhance and facilitate coughing. Activation of intrapulmonary C-fibers has been shown to inhibit coughing in anesthetized animals. Extrapulmonary C-fibers and RARs can initiate coughing upon activation. C-fiber-dependent coughing is uniquely sensitive to anesthesia. Tracheal and bronchial C-fibers may also interact with other afferents to enhance coughing. Recent studies in anesthetized guinea pigs have identified a myelinated afferent nerve subtype that can be differentiated from intrapulmonary RARs and SARs and play an essential role in initiating cough. Whether these "cough receptors" are the guinea pig equivalent of the irritant receptors described in the extrapulmonary airways of other species is unclear.

Biphasic effect of extracellular ATP on human and rat airways is due to multiple P2 purinoceptor activation

BackgroundExtracellular ATP may modulate airway responsiveness. Studies on ATP-induced contraction and [Ca2+]i signalling in airway smooth muscle are rather controversial and discrepancies exist regarding both ATP effects and signalling pathways. We compared the effect of extracellular ATP on rat trachea and extrapulmonary bronchi (EPB) and both human and rat intrapulmonary bronchi (IPB), and investigated the implicated signalling pathways.MethodsIsometric contraction was measured on rat trachea, EPB and IPB isolated rings and human IPB isolated rings. [Ca2+]i was monitored fluorimetrically using indo 1 in freshly isolated and cultured tracheal myocytes. Statistical comparisons were done with ANOVA or Student's t tests for quantitative variables and χ2 tests for qualitative variables. Results were considered significant at P < 0.05.ResultsIn rat airways, extracellular ATP (10-6–10-3 M) induced an epithelium-independent and concentration-dependent contraction, which amplitude increased from trachea to IPB. The response was transient and returned to baseline within minutes. Similar responses were obtained with the non-hydrolysable ATP analogous ATP-γ-S. Successive stimulations at 15 min-intervals decreased the contractile response. In human IPB, the contraction was similar to that of rat IPB but the time needed for the return to baseline was longer. In isolated myocytes, ATP induced a concentration-dependent [Ca2+]i response. The contractile response was not reduced by thapsigargin and RB2, a P2Y receptor inhibitor, except in rat and human IPB. By contrast, removal of external Ca2+, external Na+ and treatment with D600 decreased the ATP-induced response. The contraction induced by α-β-methylene ATP, a P2X agonist, was similar to that induced by ATP, except in IPB where it was lower. Indomethacin and H-89, a PKA inhibitor, delayed the return to baseline in extrapulmonary airways.ConclusionExtracellular ATP induces a transient contractile response in human and rat airways, mainly due to P2X receptors and extracellular Ca2+ influx in addition with, in IPB, P2Y receptors stimulation and Ca2+ release from intracellular Ca2+ stores. Extracellular Ca2+ influx occurs through L-type voltage-dependent channels activated by external Na+ entrance through P2X receptors. The transience of the response cannot be attributed to ATP degradation but to purinoceptor desensitization and, in extrapulmonary airways, prostaglandin-dependent PKA activation.

ESTROUS CYCLE ALTERS NAPHTHALENE METABOLISM IN FEMALE MOUSE AIRWAYS

Previous studies have shown variability in naphthalene cytotoxicity, expression of CYP2F2 gene and protein, and naphthalene metabolism in random cycling female mice (NIH:Swiss). CYP2F2 metabolizes naphthalene to cytotoxic metabolites in lungs of mice. This study was designed to address the question: do hormonal changes associated with the estrous cycle alter metabolism of naphthalene in the lung? Adult virgin female mice were manipulated into defined stages of the reproductive cycle: estrus, proestrus, and noncycling. Cycling was confirmed by cytology on vaginal swabs. At specific cycle times, extrapulmonary (tracheal and bronchial) and intrapulmonary (bronchiolar) conducting airways were microdissected from the lung parenchyma and incubated with naphthalene, and the products of naphthalene metabolism were trapped and measured using high-performance liquid chromatography. Circulating estradiol levels were measured at necropsy using an enzyme-linked immunosorbent assay. CYP2F2 gene expression was determined by airway level using real-time reverse transcription-polymerase chain reaction and did not vary by estrous cycle stage in intrapulmonary airways but did in extrapulmonary airways. Metabolism of naphthalene varied significantly by estrous cycle stage with the highest level of total metabolism occurring in proestrus (when estrogen is lowest) in intrapulmonary airways. Total activity and metabolite profiles in both extrapulmonary and intrapulmonary airways were affected by cycle stage. We conclude that the hormonal patterns associated with different stages of the estrous cycle 1) alter metabolism of naphthalene in the lungs of mice and 2) alter naphthalene metabolism differentially in extrapulmonary versus intrapulmonary airways.

Subtypes of vagal afferent C-fibres in guinea-pig lungs.

An ex vivo, vagally innervated, lung preparation was used to address the hypothesis that vagal C-fibres comprise at least two distinct phenotypes. Histological and extracellular electrophysiological experiments revealed that vagal C-fibres innervating the pulmonary system are derived from cell bodies situated in two distinct vagal sensory ganglia. The jugular (superior) ganglion neurones project C-fibres to both the extrapulmonary airways (larynx, trachea and bronchus) and the lung parenchymal tissue. By contrast, C-fibres from nodose (inferior) neurones innervate primarily structures within the lungs. Histologically, nodose neurones projecting lung C-fibres were different from the jugular neurones in that they were significantly less likely to express neurokinins. The nerve terminals within the lungs of both nodose and jugular C-fibres responded with action potential discharge to capsaicin and bradykinin application, but only the nodose C-fibre population responded with action potential discharge to the P2X selective receptor agonist alpha,beta-methylene-ATP. Whole cell patch clamp recording of capsaicin-sensitive nodose and jugular ganglion neurones retrogradely labelled from the lung tissue revealed that, like the nerve terminals, lung specific nodose C-fibre neurones express functional P2X receptors, whereas lung specific jugular C-fibres do not. The data support the hypothesis that both neural crest-derived neurones (jugular ganglia) and placode-derived neurones (nodose ganglia) project C-fibres in the vagus, and that these two C-fibre populations represent distinct phenotypes.

Leukotrienes mediate neurogenic inflammation in lungs of young rats infected with respiratory syncytial virus.

Respiratory syncytial virus (RSV) infection potentiates neurogenic inflammation in rat airways. Because some vascular effects of sensory nerves are mediated by cysteinyl leukotrienes (cysLTs), we studied whether the receptor antagonist montelukast inhibits neurogenic plasma extravasation in RSV-infected rats. Pathogen-free rats were inoculated at 2 wk (weanlings) or 12 wk (adults) of age with RSV or virus-free medium and treated with montelukast or its vehicle starting 1 day before inoculation. Five days postinoculation, we measured the extravasation of Evans blue-labeled albumin in the respiratory tract after stimulation of sensory nerves with capsaicin. Montelukast had no effect in the extrapulmonary airways but abolished albumin extravasation in the intrapulmonary airways of RSV-infected rats, with a larger effect in weanlings than in adults. Increased concentrations of 5-lipoxygenase-encoding mRNA and cysLTs, as well as numerous mast cells, were detected in the lung tissues of RSV-infected weanling rats. These observations suggest that the release of neuropeptides from capsaicin-sensitive sensory nerves and nonneuronal cells in the lungs of RSV-infected young rats increases vascular permeability by promoting the release of leukotrienes from mast cells.

Endogenous pulmonary nitric oxide in the regulation of airway microvascular leak.

Endogenous nitric oxide (NO) is an important modulator of airway function, but its role in the regulation of airway microvascular leak (AMVL) remains unclear. Thus we assessed the effects of NO synthase (NOS) inhibition on expired NO (ENO) levels and on AMVL measured by the Evans blue dye technique in guinea pigs. In control unsensitized animals, systemic NG-nitro-L-arginine methyl ester (L-NAME) reduced ENO by 70 +/- 8% (P < 0.01) and reduced AMVL by 92 +/- 1 and 44 +/- 17% (P < 0.05 for both) in the extrapulmonary and intrapulmonary airways, respectively. In animals sensitized and challenged with intratracheal antigen, markedly increased levels of AMVL and ENO were similarly attenuated by L-NAME. In contrast, aminoguanidine, a relatively selective type II NOS inhibitor, reduced ENO in both antigen-sensitized and control unsensitized animals by 39 +/- 3% (P < 0.01) but had no effect on AMVL. These data indicate that endogenous pulmonary NO contributes to both basal and antigen-stimulated levels of AMVL in guinea pigs and that this NO-dependent activity does not appear to be derived from type II NOS.

Ultrastructure of the aqueous lining layer in hamster airways: Is there a two-phase system?

For particle retention and clearance, the structure and surface properties of the airway lining layer are important. Due to difficulties of its preservation, structural analysis has been hampered, and, hence, the existence of two distinct and continuous phases and how much osmiophilic material is available are unclear. It was the objective of this study to investigate the ultrastructure of the aqueous lining layer in the intrathoracic conducting airways of hamsters. By means of transmission electron microscopy, we investigated the ultrastructure of the airway lining layer in hamsters whose lungs have been fixed by the application of fixative dissolved in nonpolar fluorocarbon, either by instillation via the trachea or injection into the gas exchange parenchyma, together with intravascular perfusion of aqueous fixatives. The results were compared to lungs fixed by intravascular perfusion only. In twelve hamsters, the airway lining layer was found to consist of an aqueous phase and was coated by an osmiophilic film that follows fairly closely the upper-extending contours of cilia protruding from epithelial cells. Substantially less osmiophilic material was preserved in extrapulmonary airways and when nonaqueous fixative was injected. We found that the aqueous lining layer of the intrathoracic airways in hamsters essentially surrounds and covers the cilia, the microvilli, and any other structures like macrophages or deposited particles contained in it and is coated by an osmiophilic film of variable thickness. In healthy animals, a gel phase is expected to be very thin, not clearly separated from the periciliary fluid, and located just beneath the osmiophilic film.

Heterogeneity of airway smooth muscle at tissue and cellular levels

Heterogeneity of smooth muscle behavior is rapidly emerging as an important phenomenon. However, it is only recently that investigators have begun to explore its physiological and pathogenic importance. Stenmark's group recently reported the existence of four subpopulations of smooth muscle cells in pulmonary artery, and noted that only a specific group of cells was likely responsible for the hyperplasia of smooth muscle in the hypertensive artery. Our studies reported here were focused on determining whether heterogeneity of contractile properties existed in airway smooth muscle cells. Firstly, mechanical properties of muscle strips, free of cartilage and epithelium, were compared among the different generations of canine airway from trachea down to bronchial generation 6. Two distinct groups of smooth muscle were detected from these airways: (i) an extrapulmonary group, consisting of muscle strips from the trachea, and bronchial generation 1 and 2, characterized by higher maximum shortening capacity (delta Lmax) and zero-load velocity (V0) of shortening; (ii) an intrapulmonary group, containing bronchi from generation 3 to 6, with lower delta Lmax and V0. This led to the implication that smooth muscles from these two groups consisted of different types of cells. Secondly, single smooth muscle cells were isolated from trachea and bronchus (from generation 3 to 6) enzymatically, and the unloaded shortening capacity of fresh isolated cells was measured employing electrical field stimulation at room temperature. Two types of cells were identified from these preparations based on the length of muscle cells: type I, with a mean length of 110 +/- 3 microns (SE), predominated in trachea (up to 84%); type II, with a mean length of 200 +/- 9 microns (SE), accounted for 58% in bronchus. The mechanical properties of these two types of cells were also different. Type I cells shortened by 28% of their original lengths in response to maximal stimulation, while most type II cells (90% of total type II cells) shortened 16% (type IIA), and the remainder shortened 58% (type IIB). Because of the paucity of information about the orientation and arrangement of these different types of smooth muscle cells in the airway tree, it is difficult to apply these data directly for interpretation of differences in mechanical properties of smooth muscle cells obtained from strips from extrapulmonary and intrapulmonary airways. Existence of great numbers of cells with lower shortening capacity in intrapulmonary airways may be the major factor responsible for lower mechanical performance of these airway smooth muscle strips. The relatively lower mechanical performance of intrapulmonary airway smooth muscle may be of importance in preventing excessive narrowing of these airways during muscle contraction and optimizing airflow.