Relationship of inhaled ozone concentration to acute tracheobronchial epithelial injury, site-specific ozone dose, and glutathione depletion in rhesus monkeys

Acute pulmonary epithelial injury produced by short-term exposure to ozone varies by site within the tracheobronchial tree. To test whether this variability is related to the local dose of ozone at the tissue site or to local concentrations of glutathione, we exposed adult male rhesus monkeys for 2 h to filtered air or to 0.4 or 1.0 ppm ozone generated from 18O2. Following exposure, lungs were split into lobes and specimens were selected by microdissection so that measurements could be made on airway tissue of similar branching history, including trachea, proximal (generation one or two) and distal (generation six or seven) intrapulmonary bronchi, and proximal respiratory bronchioles. One half of the lung was lavaged for analysis of extracellular components. In monkeys exposed to filtered air, the concentration of reduced glutathione (GSH) varied throughout the airway tree, with the proximal intrapulmonary bronchus having the lowest concentration and the parenchyma having the highest concentration. Exposure to 1.0 ppm ozone significantly reduced GSH only in the respiratory bronchiole, whereas exposure to 0.4 ppm increased GSH only in the proximal intrapulmonary bronchus. Local ozone dose (measured as excess 18O) varied by as much as a factor of three in different airways of monkeys exposed to 1.0 ppm, with respiratory bronchioles having the highest concentration and the parenchyma the lowest concentration. In monkeys exposed to 0.4 ppm, the ozone dose was 60% to 70% less than in the same site in monkeys exposed to 1.0 ppm. Epithelial disruption was present to some degree in all airway sites, but not in the parenchyma, in animals exposed to 1.0 ppm ozone. The mass of mucous and ciliated cells decreased in all airways, and necrotic and inflammatory cells increased. At 0.4 ppm, epithelial injury was minimal, except in the respiratory bronchiole, where cell loss and necrosis occurred, and was 50% that found in monkeys exposed to 1.0 ppm ozone. We conclude that there is a close association between site-specific O3 dose, the degree of epithelial injury, and glutathione depletion at local sites in the tracheobronchial tree.     

Distribution of SP- and CGRP-like immunoreactive nerve fibers in the lower respiratory tract of neonatal foals: evidence for loss during development

The lungs of neonatal foals contain many nerves immunoreactive for substance P and calcitonin gene-related peptide. These nerves are closely associated with the epithelium, bronchial and pulmonary vessels and the airway smooth muscle of all intrathoracic airways, including non-cartilaginous bronchioles. Activation of sensory nerves in the respiratory epithelium could thus potentially affect, via local axon reflexes, vascular and respiratory smooth muscle in neonatal equine airways. Nerves immunoreactive for these peptides are much more widely distributed within the lung than in adult horses; they may thus play a trophic role before birth, or contribute to the post-natal adaptation to breathing.     

Cellular heterogeneity of CFTR expression and function in the lung: implications for gene therapy of cystic fibrosis

Cystic fibrosis (CF) has become a paradigm disorder for the clinical testing of gene therapies in the treatment of inherited disease. In recent years, efforts directed at gene therapy of CF have concentrated on improving gene delivery systems to the airway. Surrogate endpoints for complementation of CFTR dysfunction in the lung have been primarily dependent on correction of chloride transport abnormalities. However, it is now clear that the pathophysiology of CF airways disease is far more complex than can be solely attributed to altered chloride permeability. For example, in addition to functioning as a chloride channel, CFTR also has been implicated in the regulation of other apical membrane conductance pathways through interactions with the amiloride sensitive epithelial sodium channel (ENaC) and the outwardly rectifying chloride channel (ORCC). Superimposed on this functional diversity of CFTR is a highly regulated pattern of CFTR expression in the lung. This heterogeneity occurs at both the level of CFTR protein expression within different cell types in the airway and the anatomical location of these cells in the lung. Potential targets for gene therapy of CF include ciliated, non-ciliated, and goblet cells in the surface airway epithelium as well as submucosal glands within the interstitium of the airways. Each of these distinct cellular compartments may have functionally distinct roles in processes which affect the pathogenesis of CF airways disease, such as fluid and electrolyte balance. However, it is presently unclear which of these cellular targets are most pathophysiologic relevant with regard to gene therapy. Elucidation of the underlying mechanisms of CFTR function in the airway will allow for the rational design of gene therapy approaches for CF lung diseases. This review will provide a summary of the field's current knowledge regarding CFTR functional diversity in the airway and the implications of such diversity for gene therapies of CF lung disease.     

SP-A2 gene expression in human fetal lung airways

In the present study, we characterized surfactant protein (SP)-A messenger RNA (mRNA) in mid-trimester human fetal trachea and bronchi. SP-A protein was localized by immunocytochemistry to scattered epithelial cells in the airway surface epithelium and in submucosal glands of the fetal trachea and bronchi. SP-A mRNA (2.2 kb) was detected by Northern blot analysis in human fetal trachea, as well as in primary and more distal bronchi. The levels of detectable SP-A mRNA were highest in the upper airways and were decreased in smaller bronchi in comparison. SP-A mRNA was barely detectable in the distal fetal lung tissue. In contrast, SP-A mRNA was abundant in cultured explants of distal human fetal lung tissue. SP-A1 and SP-A2 mRNA were detected by primer extension analysis in adult human lung tissue and in cultured human fetal lung explants. Only SP-A2 mRNA was detected in RNA isolated from human fetal trachea and bronchi. SP-A mRNA was localized by in situ hybridization in the fetal trachea and bronchi in scattered cells in the surface epithelium and, most prominently, in submucosal glands. Our results suggest that SP-A2, and not SP-A1, is produced in the human fetal tracheal and bronchial epithelium and in submucosal glands.     

Transgenic mice overexpressing the complement inhibitor crry as a soluble protein are protected from antibody-induced glomerular injury

We report that matrilysin, a matrix metalloproteinase, is constitutively expressed in the epithelium of peribronchial glands and conducting airways in normal lung. Matrilysin expression was increased in airway epithelial cells and was induced in alveolar type II cells in cystic fibrosis. Other metalloproteinases (collagenase-1, stromelysin-1, and 92-kD gelatinase) were not produced by normal or injured lung epithelium. These observations suggest that matrilysin functions in injury-mediated responses of the lung. Indeed, matrilysin expression was increased in migrating airway epithelial cells in wounded human and mouse trachea. In human tissue, epithelial migration was reduced by > 80% by a hydroxamate inhibitor, and in mouse tissue, reepithelialization in trachea from matrilysin-null mice was essentially blocked. In vivo observations and cell culture studies demonstrated that matrilysin was secreted lumenally by lung epithelium, but upon activation or while migrating over wounds, some matrilysin was released basally. The constitutive production of matrilysin in conducting airways, its upregulation after injury, its induction by alveolar epithelium, and its release into both lumenal and matrix compartments suggest that this metalloproteinase serves multiple functions in intact and injured lung, one of which is to facilitate reepithelialization.     

Mucous cell metaplasia in rat nasal epithelium after a 20-month exposure to ozone: a morphometric study of epithelial differentiation

The present study was designed to examine the effects of long-term ozone exposure on nasal epithelia and intraepithelial mucosubstances (IM) throughout the nasal airways of F344/N rats. Animals were exposed to 0 (controls), 0.12, 0.5, or 1.0 ppm ozone, 6 h/day, 5 days/wk, for 20 mo. Rats were killed 1 wk after the end of the exposure, and nasal tissues were processed for light and electron microscopy. Standard morphometric techniques were used to determine epithelial cell densities and the amounts of IM in the surface epithelium lining the nasal airways. No mucous cells or IM were present in the epithelia lining the nasal lateral meatus and maxillary sinus of rats exposed to 0 or 0.12 ppm ozone. In contrast, rats exposed to 0.5 or 1.0 ppm ozone had marked mucous cell metaplasia (MCM) with numerous mucous cells and conspicuous amounts of IM in the surface epithelium lining these upper airways. Ozone-induced increases in total epithelial cells (i.e., epithelial hyperplasia) were present only in rats exposed to 1.0 ppm. The results of this study indicate that rats chronically exposed to 1.0 or 0.5 ppm, but not 0.12 ppm, ozone can develop marked MCM with significant increases in IM in both proximal and distal nasal airways. The epithelial changes observed throughout the nasal passages of ozone-exposed rats may be adaptive responses in an attempt to protect the upper and lower respiratory tract from further ozone-induced injury.     

Physiologic diagnosis and function in asthma

Asthma is a condition in which there is airway hyperresponsiveness, with the propensity for widespread, reversible airways narrowing on exposure to diverse inciting factors (triggers). Inhalation of nonspecific agents such as methacholine or histamine leads to bronchoconstriction in most cases, and in some, the bronchoconstriction follows exposure to specific agents such as antigen or occupational irritants. Chest tightness and cough, which are the most common symptoms of asthma, are probably the result of inflammation mucus plugs, edema, or smooth muscle constriction in the small peripheral airways. Because major obstruction of the peripheral airways can occur without recognizable increases of airway resistance or FEV1, the physiologic alterations in acute exacerbations are generally subtle in the early stages. Poorly ventilated alveoli subtending obstructed bronchioles continue to be perfused, and as a consequence, the P(A-a)O2 increases and the PaO2 decreases. At this stage, ventilation is generally increased, with excessive elimination of carbon dioxide and respiratory alkalemia. In the more severe exacerbation, lung volume is increased and the static volume-pressure curve is shifted up (lung volume is greater) and to the left (pressure is lower) while the shape of the curve is unaltered. The airway obstruction is reversible and there is generally improvement in air flow rates following administration of beta-agonists and anti-inflammatory agents. The changes in mechanical properties are also reversible, and therapeutic intervention usually results in a shift of the PV curve downward toward the normal position, for example, a decrease in TLC and an increase in the elastic recoil pressure at any particular lung volume. Failure to take these changes into account may underestimate the impact of therapy. The PaO2 decreases (and the P(A-a)O2 increases) as the work of breathing increases, and when it becomes excessive (and/or the FEV1 falls below 20% to 25%), the PaCO2 begins to increase. Therefore, in any patient with asthma, a decreasing PaO2 and an increasing PaCO2, even into the normal range, indicates severe airway obstruction that is leading to respiratory muscle fatigue and patient exhaustion.     

Alterations of lung structure in experimental diabetes, and diabetes associated with hyperlipidaemia in hamsters

Since hyperglycaemia is known to affect normal pulmonary physiology and biochemistry and few structure-function correlations have been reported, we designed experiments on hamsters subjected to streptozotocin-induced diabetes or diabetes associated with hyperlipidaemia, and investigated the impact of these conditions on the lung structure. At time intervals ranging 2-24 weeks from the inception of disease (without correcting blood glucose with insulin), the animals were sacrificed, and plasma glucose and cholesterol assayed. The lung was processed for electron microscopy, and the structural changes of the capillary and venular endothelium, of epithelial cells, and interstitium were examined. In diabetic animals, especially after 6 weeks of disease, a gradual narrowing of approximately 35% of the capillaries and approximately 30% of the alveoli, and hyperplasia of the extracellular matrix, rich in collagen bundles, were observed. Frequently, capillaries contained adherent intravascular macrophages suggestive of an inflammatory process. The capillary endothelium was characterized by numerous plasmalemmal vesicles, often fused, well-developed synthesizing apparatus (endoplasmic reticulum and Golgi complex) and cytoskeleton, and an uneven distribution of the anionic sites on the luminal plasmalemma. The venular endothelium was particularly rich in Weibel-Palade bodies. The alveolar epithelium was often collapsed, compressing surfactant within the airspace. The lung interstitium was apparently enlarged, and the fibroblasts and contractile interstitial cells frequently contained lipid droplets. These alterations were more pronounced and occurred at a faster rate (4 weeks) in diabetes associated with hyperlipidaemia. The structural modifications reported in this study support the functional disturbances observed in association with hyperglycaemia, sustaining the conclusion that the lung is an organ affected by diabetes.     

Lung transplantation for Williams-Campbell syndrome

Williams-Campbell syndrome is a rare disorder characterized by a deficiency of cartilage in subsegmental bronchi leading to distal airway collapse and bronchiectasis. We report the first case of lung transplantation in a patient with end-stage lung disease secondary to Williams-Campbell syndrome. Although the patient did not have proximal airway collapse prior to transplantation, his posttransplant course was complicated by the development of bronchomalacia of the right and left mainstem bronchi. The patient experienced recurrent pulmonary infections and died of bacterial pneumonia 1 year after transplantation. Autopsy revealed cartilage deficiency in both right and left mainstem bronchi. A hypothesis may be made that a combination of proximal cartilage deficiency and posttransplant airway ischemia led to the development of bronchomalacia after lung transplantation. Thus, in contrast to previous reports, the cartilage deficiency in Williams-Campbell syndrome can involve both proximal and distal airways. Consequently, bilateral sequential lung transplantation may not be an effective therapeutic option in patients with this syndrome.     

Efficiency of airway macrophage recovery by bronchoalveolar lavage in hamsters: a stereological approach

Macrophages play a central role in the defence of the respiratory tract against deposited particles. In addition to the well-studied alveolar macrophages, airway macrophages have been recognized as an important clearance factor. Bronchoalveolar lavage (BAL) has been used for functional and morphological investigations of macrophages in vitro, assuming that all macrophages are removed with equal probability from the lung surface. Airway macrophages have been found in close contact with the epithelial cells. These macrophages may not be easily removed by lavage, and they might constitute a functionally different macrophage population. We have tested the hypothesis that there exists a population of macrophages in the conducting airways that resists removal by lavage. We lavaged the lungs of four hamsters and fixed the lungs, thereafter, by intravascular perfusion. The number of macrophages in the intrapulmonary conducting airways was estimated with an unbiased stereological technique, the fractionator, and compared to the number of macrophages in the airways of four hamsters whose lungs had not been lavaged prior to fixation. This in situ study revealed that, in hamster lungs, 42% of the airway macrophages were not removed by BAL and that about 5% of all macrophages in the BAL fluid were airway macrophages. Additionally, ultrastructural alterations of the airway epithelium were found. It is concluded that there exists a population of airway macrophages that resists lavage. This is an aspect which has to be considered in studies performed with macrophages obtained by BAL, since they could represent a functionally different macrophage population.     

Role of aquaporin water channels in kidney and lung

Several aquaporin-type water channels are expressed in mammalian kidney and lung: AQP1 in lung microvessels and kidney proximal tubule, thin descending limb of Henle, and vasa recta; AQP2 in apical membrane of collecting duct epithelium; AQP3 and AQP4 in basolateral membranes of airway and collecting duct epithelium; and AQP5 in alveolar epithelium. Novel quantitative fluorescence methods demonstrated very high water permeabilities of the alveolar epithelial and endothelial barriers, and moderately high water permeability across distal airways. In the kidney, water permeability is high in proximal tubule and thin descending limb of Henle, and regulated by vasopressin in collecting duct. The author's laboratory has studied the role of aquaporins in organ physiology using transgenic knockout mice lacking specific aquaporins. AQP1 null mice are mildly growth-retarded, manifest a severe urinary concentrating defect, and have reduced water permeability between airspace and capillary compartments. AQP4 null mice appear normal grossly except for a mild defect in maximum urinary concentrating ability. AQP2-deficient humans have hereditary non-X-linked nephrogenic diabetes insipidus (NDI). In transfected mammalian cells, many NDI-causing AQP2 mutants are retained in the endoplasmic reticulum. The author's laboratory has found that "chemical chaperones," that is, small compounds that promote protein folding in vitro, are able to correct defective AQP2 trafficking in cell culture models. The transgenic mouse and mammalian cell models are thus beginning to provide clues about the role of aquaporins in normal physiology and disease.     

Application of a commercial kit for detection of PCR products to quantification of human immunodeficiency virus type 1 RNA and proviral DNA

Clinical application of adenovirus-mediated or liposome-mediated gene transfer to human airway has started and the preliminary reports of cystic fibrosis conductance regulator (CFTR) gene transfer to CF patients are now available. These data showed that the duration and intensity of expression of transgene in human airway or alveoli was transient and weak. In addition, adenovirus-mediated gene transfer is suffered from immunity or nonspecific obstacles. In this sense, liposome-mediated gene transfer to airway will be hopeful but still uncertain. We have to follow all clinical reports about the gene delivery to human airways including second generation virus vectors, such as adenoassociated virus and herpes simplex virus.     

The diagnosis of ventilator-associated pneumonia: a comparison of histologic, microbiologic, and clinical criteria

STUDY OBJECTIVE: To evaluate histologic, microbiological, and clinical criteria in the recognition of ventilator-associated pneumonia (VAP) in patients who died while mechanically ventilated. METHODS: The study group consisted of 39 patients who died after a mean of 14 days of mechanical ventilation. Postmortem fiberoptic bronchoscopy (FOB) and open lung biopsy were performed with collection of specimens initiated <1 h after death. The microbiological specimens included suction catheter aspirate of tracheal secretions, FOB-guided protected specimen brush (PSB) of tracheal secretions, blindly placed PSB in a distal airway, FOB-guided PSB in a distal airway, and FOB-guided BAL fluid (BALF) in a distal airway. Qualitative bacteriologic study was performed on all specimens, and quantitative bacteriologic study was performed on all but the suction catheter aspirate of the trachea. A biopsy specimen of peripheral lung parenchyma from the same region sampled by FOB was sent for quantitative culture and histologic analysis. The BALF was analyzed for cell population and percent of neutrophils containing intracellular organisms. The clinical criteria selected for comparison with histologic and microbiological results included a temperature > or =38.5 degrees C during the 48 h prior to death, a WBC count > or =15,000/mm3 in the 48 h prior to death, presence of a bacterial or fungal pathogen on the last sputum culture, radiographic worsening in the week prior to death, and worsening gas exchange defined as a 15% decrease in the PaO2/fraction of inspired oxygen ratio in the 72 h prior to death. RESULTS: None of the quantitative cultures had a reliable positive predictive value for histologic pneumonia. None of the five clinical criteria tested showed agreement with the presence or absence of histologic pneumonia. There was a significant correlation between qualitative and quantitative microbiological results from the distal airway/FOB-guided PSB, distal airway/BALF, and quantitative culture of the lung parenchyma. Also, suction catheter aspirate of the trachea had a sensitivity of 87% in recognizing the bacterial species simultaneously present in lung parenchyma. None of the patients with histologic pneumonia had <50% neutrophils in the BALF. CONCLUSIONS: Neither the bacterial, density from the four airway quantitative cultures, nor the bacterial density from quantitative culture of lung parenchyma accurately separated the histologic pneumonia and nonpneumonia groups. No clinical criteria or combination of clinical criteria correlated with the presence or absence of histologic pneumonia. A BALF with <50% neutrophils had a 100% negative predictive value for histologic pneumonia. A BALF quantitative culture had a sensitivity of 63%, specificity of 96%, and positive predictive value of 91% in recognizing sterile lung parenchyma. Thus, BALF may have a role in excluding pneumonia/infection in the ventilated patient. Antibiotic choice for the empiric therapy of VAP can be accurately guided by the microbial population recognized through culture of a tracheal suction catheter aspirate.     

Effect of nitrogen dioxide and other combustion products on asthmatic subjects in a home-like environment

Nitrogen dioxide (NO2) is one of a number of nitrogen compounds that are by-products of combustion and occur in domestic environments following the use of gas or other fuels for heating and cooking. In this study, we examined the effect of two levels of NO2 on symptoms, lung function and airway hyperresponsiveness (AHR) in asthmatic adults and children. In addition, in the same subjects, we examined the effects of the same levels of NO2 mixed with combustion by-products from a gas space heater. The subjects were nine adults, aged 19-65 yrs, and 11 children, aged 7-15 yrs, with diagnosed asthma which was severe enough to require daily medication. All subjects had demonstrable AHR to histamine. Exposures were for 1 h on five separate occasions, 1 week apart, to: 1) ambient air, drawn from outside the building; 2) 0.3 parts per million (ppm) NO2 in ambient air; 3) 0.6 ppm NO2 in ambient air; 4) ambient air+combustion by-products+NO2 to give a total of 0.3 ppm; and 5) ambient air+combustion by-products+NO2 to give a total of 0.6 ppm. Effects were measured as changes in lung function and symptoms during and 1 h after exposure, in AHR 1 h and 1 week after exposure, and in lung function and symptoms during the week following exposure. Exposure to NO2 either in ambient air or mixed with combustion by-products from a gas heater, had no significant effect on symptoms or lung function in adults or in children. There was a small, but statistically significant, increase in AHR after exposure to 0.6 ppm NO2 in ambient air. However, there was no effect of 0.6 ppm NO2 on AHR when the combustion by-products were included in the test atmosphere nor of 0.3 ppm NO2 under either exposure condition. We conclude that a 1 h exposure to 0.3 or 0.6 ppm NO2 has no clinically important effect on the airways of asthmatic adults or children, but that 0.6 ppm may cause a slight increase in airway hyperresponsiveness.     

Mathematical modeling of the first inflation of degassed lungs

The pressure-volume (P-V) relationship of degassed lungs during the first inflation is different from that in consecutive inflations. We developed a mathematical model of the P-V curve of the first inflation by assuming that (1) central airways are open leading to many subtrees of n generations that are initially closed; (2) an airway opens when inflation pressure reaches the opening threshold pressure of that segment; and (3) the opening threshold pressures do not depend on airway generation. In this model, airway opening occurs in cascades or avalanches. To test the model which contains only two parameters, n and a pressure, P(low), at which at least one subtree completely opens, we measured the first inflation P-V curves of 15 excised and degassed rabbit lungs. By fitting these data, we found that n=17+/-5, P(low)=23+/-4 cmH2O, and that there is a wide distribution of threshold pressures for airways with diameters <2 mm. Analysis of the P-V curve in a lung which was lavaged with a liquid of constant surface tension and in which airways are presumably open demonstrated that the distribution of threshold pressures is narrow, and hence no avalanches occur during inflation. We conclude that in normal lungs the first inflation is dominated by avalanche behavior of airway opening providing information on the global distribution of threshold pressures and the average site of airway closure.     

Time-course of functional and pathological changes after a single high acute inhalation of chlorine in rats

Reactive airways dysfunction syndrome (RADS) is an asthma-like condition that follows exposure to very high concentrations of an irritant material. We assessed the time-course of pathophysiological alterations in a model of RADS. Sprague-Dawley rats were exposed to 1,500 parts per million (ppm) of chlorine for 5 min. Lung resistance (RL), responsiveness to inhaled methacholine (MCh), the airway epithelium and bronchoalveolar lavage (BAL) were assessed over a 3 month period after exposure. RL increased significantly up to 3 days after exposure, reaching a maximal change of 110+/-16% from baseline. There was a significant decrease in the concentration of MCh required to increase RL by 0.20 cmH2O x mL(-1) x s from days 1-7 after exposure. In some rats, MCh hyperresponsiveness and RL changes persisted after exposure for as long as 1 and 3 months, respectively. Histological evaluation with morphometric evaluation revealed epithelial flattening, necrosis, increase in smooth muscle mass and evidence of epithelial regeneration. BAL showed an increased number of neutrophils. The timing of maximal abnormality in the appearance of the epithelium (days 1-3) corresponded to that of the maximal functional changes. Acute high chlorine exposure results in functional and pathological abnormalities that resolve in the majority of animals after a variable period; however, these changes can persist in some animals. Functional abnormalities in the initial stages may be related to airway epithelial damage.     

Chronic obstructive pulmonary disease: role of bronchiolar mast cells and macrophages

Chronic obstructive pulmonary disease (COPD) is considered to be caused in part by smoking-induced inflammation, but it is unknown which inflammatory cells within the small airways are associated with the obstruction. We investigated the inflammatory infiltrate in the small airways of 16 current or ex-smokers with COPD (FEV1 < or = 75% predicted) and 15 without COPD (FEV1 > or = 85% predicted) in pneumectomy specimens that were removed for lung cancer. Mast cells, macrophages, neutrophils, eosinophils, T cells, and B cells were identified using immunohistochemistry on formalin-fixed, paraffin-embedded specimens. These cells were quantified in the epithelium and the remainder of the airway wall. The number of mast cells and macrophages in the epithelium, but not in the remainder of the airway wall, was significantly increased in patients with COPD. Neutrophil and T cell numbers did not differ between the groups. Only few B cells and eosinophils were present in both groups. Smoking history, perioperative steroid usage, tumor localization, or reversibility in the FEV1 to salbutamol could not account for the observed differences. We conclude that the number of epithelial mast cells and macrophages is increased in the bronchioli in smokers with airflow limitation, suggesting a role in development of COPD.