Isolation of alveolar epithelial cells from lung tissue obtained at autopsy

Summary Lung alveolar epithelial cells have been studied in a variety of laboratory animal models, and studies of human alveolar epithelial cells are important for comparison to information obtained from animal studies. Autopsy material is a source of human cells for study. Studies of human autopsy material revealed variables that negatively affected the yield of viable cells. For specimens from adults, these included death greater than 12 h before cell isolation, obvious severe lung fibrosis, longstanding metabolic disorders, and lung congestion indicated by weight of the right middle lobe greater than 150 g. Samples from children yielded significant numbers of viable cells up to 18 h after death. For 17 specimens that conformed to the above criteria, approximately 8.5×10⁶ alveolar cells were obtained per gram of tissue (tissue weights ranged from 30 to 108 g) using a procedure involving instillation of proteases into the airways. The cells could be further fractionated, and 10 to 15% of the mixed cells obtained were type II pneumocytes. Analysis of NADPH cytochrome-c-reductase distribution in subcellar fractions provided evidence that the cells obtained were intact. Phospholipid enzyme activities and synthetic activity were within the ranges previously found in laboratory studies of freshly obtained animal lungs. These results suggest that significant numbers of viable and functional human lung cells, including type II pneumocytes, can be obtained from autopsy material. This research was supported by a grant (HL 33083) from the National Heart, Lung and Blood Institute, Bethesda, MD.     

Ozone exposure alters tracheobronchial mucociliary function in humans

Mucociliary function is a primary defense mechanism of the tracheobronchial airways, and yet the response of this system to an inhalational hazard, such as ozone, is undefined in humans. Utilizing noninvasive techniques to measure deposition and retention of insoluble radiolabeled particles on airway mucous membranes, we studied the effect on mucus transport of 0.2 and 0.4 ppm ozone compared with filtered air (FA) in seven healthy males. During 2-h chamber exposures, subjects alternated between periods of rest and light exercise with hourly spirometric measurement of lung function. Mechanical and mucociliary function responses to ozone by lung airways appeared concentration dependent. Reduction in particle retention was significant (P less than 0.005) (i.e., transport of lung mucus was increased during exposure to 0.4 ppm ozone and was coincident with impaired lung function; e.g., forced vital capacity and midmaximal flow rate fell by 12 and 16%, respectively, and forced expiratory volume at 1 s by 5%, of preexposure values). Regional analysis indicated that mucus flow from distal airways into central bronchi was significantly increased (P less than 0.025) by 0.2 ppm ozone. This peripheral effect, however, was buffered by only a marginal influence of 0.2 ppm ozone on larger bronchi, such that the resultant mucus transport for all airways of the lung in aggregate differed only slightly from FA exposures. These data may reflect differences in regional diffusion of ozone along the respiratory tract, rather than tissue sensitivity. In conclusion, mucociliary function of humans is acutely stimulated by ozone and may result from fluid additions to the mucus layer from mucosal and submucosal secretory cells and/or alteration of epithelial permeability.     

Immunohistological characterization of leukocytes in the lungs of healthy mice and after bacterial intratracheal infection

Leukocytes in the peripheral lung parenchyma of mice have not been characterized histologically during bacterial infection. The aim of this study was to investigate (a) the immunohistological characteristics of healthy murine lungs and (b) the cell kinetics during acute inflammation. BALB/c and MF1 mice were examined; as well as transgenic mice with the gene defect of cystic fibrosis (CF) in the airways as an animal model for this disease. MF1 mice served as controls for the transgenic animals. Lavaged and perfused lungs were snap frozen. B and T lymphocytes, CD4+ and CD8+ cells, dendritic cells, neutrophils and a subset of macrophages were enumerated on cryostat lung sections. The lung tissue and bronchoalveolar lavage (BAL) of BALB/c mice, infected intratracheally with Haemophilus influenzae type b (Hib), were studied at different time points after infection. In the lungs of healthy mice, including CF mice, the largest population was that of T cells, CD4+ cells being always more frequent than CD8+ cells. During acute inflammation the number of neutrophils in the lung parenchyma and BAL increased strongly within the first hours after bacterial instillation and reached baseline levels within one week. This study provides a semi-quantitative analysis of immunocompetent cells in normal and infected murine lung tissue. Differences in cell numbers are found between different strains. Moreover, the cellular reaction during Hib infection in mouse lungs is dominated by neutrophils, as expected in a primary immune response. In uninfected CF mice the numbers and distribution of immune cells in the lung tissue are normal, indicating that the cellular defense is adequate.     

How far does ozone penetrate into the pulmonary air/tissue boundary before it reacts?

A simple method is suggested for calculating the time it takes ozone to traverse a biological region, such as a bilayer or a cell, and comparing this time to the halflife of ozone within that region. For a bilayer the calculations suggest that most of the ozone reacts within a bilayer, but a fraction may exit unreacted. For the lung lining fluid layer (LLFL), the calculations show that ozone cannot cross this layer without reacting where the LLFL is thicker than about 0.1 microns. However, since the LLFL varies from 20 to 0.1 microns in thickness with patchy areas in the lower airways that are virtually uncovered, some ozone could reach underlying cells, particularly in the lower airways. For cells (such as alveolar type I epithelial cells), the calculations show that ozone reacts within the cell too rapidly to pass through and exit unreacted from the other side. These calculations have implications for ozone toxicity. In vivo, the toxicity of ozone is suggested to result from the effects of a cascade of products that are produced in the reactions of ozone with primary target molecules that lie close to the air/tissue boundary. These products, which have a lower reactivity and longer lifetime than ozone itself, can transmit the effects of ozone beyond the air/tissue interface. The variation in thickness of the LLFL may modulate the species causing damage to the cells below it. In the lower airways, where the LLFL is thin and patchy, more cellular damage may be caused by ozone itself; in the upper airways where the LLFL is thicker, secondary products (such as aldehydes and hydrogen peroxide) may cause most of the damage. In vitro studies must be designed in an attempt to model the lung physiology. For example, if cells in culture are studied, and if the cells are exposed to ozone while under a supporting medium solution that contains ozone-reactive substances, then the cells may be damaged by products that are formed in the reactions of ozone with the cell medium rather than by ozone itself.     

Ambient ozone primes pulmonary innate immunity in mice

Exposure to ozone in air pollution in urban environments is associated with increases in pulmonary-related hospitalizations and mortality. Because ozone also alters clearance of pulmonary bacterial pathogens, we hypothesized that inhalation of ozone modifies innate immunity in the lung. To address our hypothesis, we exposed C57BL/6J mice to either free air or ozone, and then subsequently challenged with an aerosol of Escherichia coli LPS. Pre-exposure to ozone resulted in enhanced airway hyperreactivity, higher concentrations of both total protein and proinflammatory cytokines in lung lavage fluid, enhanced LPS-mediated signaling in lung tissue, and higher concentrations of serum IL-6 following inhalation of LPS. However, pre-exposure to ozone dramatically reduced inflammatory cell accumulation to the lower airways in response to inhaled LPS. The reduced concentration of cells in the lower airways was associated with enhanced apoptosis of both lung macrophages and systemic circulating monocytes. Moreover, both flow cytometry and confocal microscopy indicate that inhaled ozone causes altered distribution of TLR4 on alveolar macrophages and enhanced functional response to endotoxin by macrophages. These observations indicate that ozone exposure increases both the pulmonary and the systemic biologic response to inhaled LPS by priming the innate immune system.     

Compliance of peripheral airways deduced from morphometry.

Insights into airway mechanics were sought by applying morphometric techniques to rabbit lungs fixed at several lung recoil pressures. Rabbits were treated with either nebulized carbachol followed by iv administration of carbachol or with saline solution (sham). The lungs were held at one of six values of positive end-expiratory pressure (PEEP; 10, 7, 4, 2, 0, and -4 cmH(2)O) while the animal was killed and formalin was circulated through the lungs. The lungs were removed and left in a bath of formalin for 24 h. Standard airway morphometric measurements were made on membranous bronchiole slices taken from representative blocks of tissue. Reductions in PEEP produced the expected reductions in lumen area in the carbachol-treated airways but not in the sham-treated airways for PEEP > 2 cmH(2)O. Sham-treated airways remained more open than expected until they collapsed into an oval shape at PEEPs between 4 and 2 cmH(2)O. The carbachol-treated airways exhibited this behavior at PEEP = -4 cmH(2)O. The smallest airways, which had relatively thicker walls, collapsed less than larger airways. We postulate that this behavior implies that peribronchial stress is greater than lumen pressure on collapse into the oval shape. Resistance to buckling increases with the thickness-to-radius ratio of the airway wall, which explains why the smallest airways are the most open. The development of epithelial folds appeared to follow the theoretical prediction of a previous study (Lambert RK, Codd SL, Alley MR, and Pack RJ. J Appl Physiol 77: 1206-1216, 1994).     

Alternatives for lung research: stuck between a rat and a hard place

The respiratory system acts as a portal into the human body for airborne materials, which may gain access via the administration of medicines or inadvertently during inhalation of ambient air (e.g. air pollution). The burden of lung disease has been continuously increasing, to the point where it now represents a major cause of human morbidity and mortality worldwide. In the UK, more people die from respiratory disease than from coronary heart disease or non-respiratory cancer. For this reason alone, gaining an understanding of mechanisms of human lung biology, especially in injury and repair events, is now a principal focus within the field of respiratory medicine. Animal models are routinely used to investigate such events in the lung, but they do not truly reproduce the responses that occur in humans. Scientists committed to the more robust Three Rs principles of animal experimentation (Reduction, Refinement and Replacement) have been developing viable alternatives, derived from human medical waste tissues from patient donors, to generate in vitro models that resemble the in vivo human lung environment. In the specific case of inhalation toxicology, human-oriented models are especially warranted, given the new REACH regulations for the handling of chemicals, the rising air pollution problems and the availability of pharmaceutically valuable drugs. Advances in tissue-engineering have made it feasible and cost-effective to construct human tissue equivalents of the respiratory epithelia. The conducting airways of the lower respiratory system are a critical zone to recapitulate for use in inhalation toxicology. Three-dimensional (3-D) tissue designs which make use of primary cells, provide more in vivo-like responses, based on the targeted interactions of multiple cell types supported on artificial scaffolds. These scaffolds emulate the native extracellular matrix, in which cells differentiate into a functional pulmonary tissue. When 3-D cell cultures are employed for testing aerosolised chemicals, drugs and xenobiotics, responses are captured that mirror the events in the in situ human lung and provide human endpoint data.     

Convective and diffusive gas transport in canine intrapulmonary airways.

The significance of convective and diffusive gas transport in the respiratory system was assessed from the response of combined inert gas and particle boluses inhaled into the conducting airways. Particles, considered as "nondiffusing gas," served as tracers for convection and two inert gases with widely different diffusive characteristics (He and SF6) as tracers for convection and diffusion. Six-milliliter boluses labeled with monodisperse di-2-ethylhexyl sebacate droplets of 0.86-microns aerodynamic diameter, 2% He, and 2% SF6 were inspired by three anesthetized mechanically ventilated beagle dogs to volumetric lung depths up to 170 ml. Mixing between inspired and residual air caused dispersion of the inspired bolus, which was quantified in terms of the bolus half-width. Dispersion of particles increased with increasing lung depth to which the boluses were inhaled. The increase followed a power law with exponents less than 0.5 (mean 0.39), indicating that the effect of convective mixing per unit volume was reduced with depth. Within the pulmonary dead space, the behavior of the inert gases He and SF6 was similar to that of the particles, suggesting that gas transport was almost solely due to convection. Beyond the dead space, dispersion of He and SF6 increased more rapidly than dispersion of particles, indicating that diffusion became significant. The gas and particle bolus technique offers a suitable approach to differential analysis of gas transport in intrapulmonary airways of lungs.     

The design and fabrication of three-chamber microscale cell culture analog devices with integrated dissolved oxygen sensors

Whole animal testing is an essential part in evaluating the toxicological and pharmacological profiles of chemicals and pharmaceuticals, but these experiments are expensive and cumbersome. A cell culture analog (CCA) system, when used in conjunction with a physiologically based pharmacokinetic (PBPK) model, provides an in vitro supplement to animal studies and the possibility of a human surrogate for predicting human response in clinical trials. A PBPK model mathematically simulates animal metabolism by modeling the absorption, distribution, metabolism, and elimination kinetics of a chemical in interconnected tissue compartments. A CCA uses mammalian cells cultured in interconnected chambers to physically represent the corresponding PBPK. These compartments are connected by recirculating tissue culture medium that acts as a blood surrogate. The purpose of this article is to describe the design and basic operation of the microscale manifestation of such a system. Microscale CCAs offer the potential for inexpensive, relatively high throughput evaluation of chemicals while minimizing demand for reagents and cells. Using microfabrication technology, a three-chamber ("lung"-"liver"-"other") microscale cell culture analog (microCCA) device was fabricated on a 1 in. (2.54 cm) square silicon chip. With a design flow rate of 1.76 microL/min, this microCCA device achieves approximate physiological liquid-to-cell ratio and hydrodynamic shear stress while replicating the liquid residence time parameters in the PBPK model. A dissolved oxygen sensor based on collision quenching of a fluorescent ruthenium complex by oxygen molecules was integrated into the system, demonstrating the potential to integrate real-time sensors into such devices.     

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.     

Development of a microscale cell culture analog to probe naphthalene toxicity

Prediction of human response to drugs or chemicals is difficult as a result of the complexity of living organisms. We describe an in vitro model that can realistically and inexpensively study the adsorption, distribution, metabolism, elimination, and potential toxicity (ADMET) of chemicals. A microscale cell culture analog (microCCA) is a physical replica of the physiologically based pharmacokinetics (PBPK) model. Such a microfabricated device consists of a fluidic network of channels to mimic the circulatory system and chambers containing cultured mammalian cells representing key functions of animal "organ" systems. This paper describes the application of a two-cell system, four-chamber microCCA ("lung"-"liver"-"other tissue"-"fat") device for proof-of-concept study using naphthalene as a model toxicant. Naphthalene is converted into reactive metabolites (i.e., 1,2-naphthalenediol and 1,2-naphthoquinone) in the "liver" compartment, which then circulate to the "lung" depleting glutathione (GSH) in lung cells. Such microfabricated in vitro devices are potential human surrogates for testing chemicals and pharmaceutics for toxicity and efficacy.     

Uncertainties on lung doses from inhaled plutonium

In a recent epidemiological study, Bayesian uncertainties on lung doses have been calculated to determine lung cancer risk from occupational exposures to plutonium. These calculations used a revised version of the Human Respiratory Tract Model (HRTM) published by the ICRP. In addition to the Bayesian analyses, which give probability distributions of doses, point estimates of doses (single estimates without uncertainty) were also provided for that study using the existing HRTM as it is described in ICRP Publication 66; these are to be used in a preliminary analysis of risk. To infer the differences between the point estimates and Bayesian uncertainty analyses, this paper applies the methodology to former workers of the United Kingdom Atomic Energy Authority (UKAEA), who constituted a subset of the study cohort. The resulting probability distributions of lung doses are compared with the point estimates obtained for each worker. It is shown that mean posterior lung doses are around two- to fourfold higher than point estimates and that uncertainties on doses vary over a wide range, greater than two orders of magnitude for some lung tissues. In addition, we demonstrate that uncertainties on the parameter values, rather than the model structure, are largely responsible for these effects. Of these it appears to be the parameters describing absorption from the lungs to blood that have the greatest impact on estimates of lung doses from urine bioassay. Therefore, accurate determination of the chemical form of inhaled plutonium and the absorption parameter values for these materials is important for obtaining reliable estimates of lung doses and hence risk from occupational exposures to plutonium.     

Respiratory mechanics in the normal dog determined by expiratory flow interruption

We recently proposed an eight-parameter model of the respiratory system to account for its mechanical behavior when flow is interrupted during passive expiration. The model consists of two four-parameter submodels representing the lungs and the chest wall, respectively. The lung submodel consists of an airways resistance together with elements embodying the viscoelastic properties of the lung tissues. The chest wall submodel has similar structure. We estimated the parameters of the model from data obtained in four normal, anesthetized, paralyzed, tracheostomized mongrel dogs. This model explains why lung tissue and chest wall resistances should be markedly frequency dependent at low frequencies and also permits a physiological interpretation of resistance measurements provided by the flow interruption method.     

A new porcine model of reperfusion injury after lung transplantation

Rodent models have been described to investigate lung preservation and reperfusion injury but have significant disadvantages. In large animals single lung transplant studies are probably optimal but problems remain over the ability to rigorously separate the lungs for assessment while promoting medium to long-term animal survival for meaningful investigation. Our aim was to develop a novel and refined large animal model to assess reperfusion injury in the transplanted lung, overcoming the difficulties associated with existing models. Specifically, small animal models of lung transplantation usually have short perfusion times (often one hour) and include extracorporeal circuits while larger animal models often require the contralateral lung to be excluded after transplantation-an unphysiological situation under which to evaluate the graft. A porcine model of left lung allotransplantation was developed in which native and donor lungs are individually ventilated. Sampling catheters placed within the graft lung allowed specimen withdrawal without mixing of blood from the contralateral lung after reimplantation. The model permits a variety of clinical scenarios to be simulated with the native lung supporting the animal irrespective of function in the graft. This model has been used in over 60 transplant procedures with a postoperative survival time of 12 h being readily achieved. The mean operating time was 2.6 h. The mortality rate is 4% in our series. We have found the model to be reliable, reproducible and flexible. We propose this model as an adaptable investigation for evaluating lung reperfusion injury and preservation.     

Estimation of Incremental Unit Risks for Inhaled Nickel Subsulfide and Nickel Oxide,Based on a Combination of Human and Animal Data

Both animal and human exposure–response data are used to estimate the incremental unit risks (IURs) of lung cancer for Ni₃S₂ and NiO, which are constituent compounds of nickel refinery dust. The animal experiments are used to determine relative lung cancer potencies for Ni₃S₂ and NiO, and the human epidemiological data are used as the best estimate of overall risk for refinery dust exposure. The animal data for Ni₃S₂ are fit with a linear model, while the nonlinear animal data for NiO are fit with a Weibull model. The lower 95% confidence limit at a 5% point of departure is used to calculate a tumorigenic potency ratio of Ni₃S₂ to NiO of 5.6. Analyses of actual nickel refinery dust indicate the weight% of Ni₃S₂ and NiO to be 82% and 9%, respectively. This information is used with the previously determined IUR for nickel refinery dust to calculate IUR_NiO = 5.1 × 10^?5 (μg Ni/m³)^?1 and IUR_Ni3S2 = 2.9 × 10^?4 (μg Ni/m³)^?1.     

Pulmonary gas mixing during spontaneous breathing and at high-frequency ventilation

This work is intended to estimate the contribution of either laminar or turbulent dispersion during spontaneous breathing on one hand, and at high-frequency pulmonary ventilation on the other. For that purpose, we performed a computer simulation of a mathematical model of gas transport in the human airways governed by a combination of axial convection and longitudinal dispersion. Calculations were carried out by incorporating two dispersion coefficients, proposed by Taylor and Scherer respectively, into the mathematical model. Moreover, computations were performed with five constant flow rates and two inert heavy (SF₆) and light (He) gases to enhance the effect of mixing. It is concluded that Taylor laminar dispersion cannot play a significant role in the human airways; however, it seems that convective gas mixing with disturbed dispersion - corresponding to a regime of quasi-steady state-can account for most gas transport during spontaneous respiration and high-frequency ventilation.     

Bridging epidemiology and model organisms to increase understanding of endocrine disrupting chemicals and human health effects

Concerning temporal trends in human reproductive health has prompted concern about the role of environmentally mediated risk factors. The population is exposed to chemicals present in air, water, food and in a variety of consumer and personal care products, subsequently multiple chemicals are found human populations around the globe. Recent reviews find that endocrine disrupting chemicals (EDCs) can adversely affect reproductive and developmental health. However, there are still many knowledge gaps. This paper reviews some of the key scientific concepts relevant to integrating information from human epidemiologic and model organisms to understand the relationship between EDC exposure and adverse human health effects. Additionally, areas of new insights which influence the interpretation of the science are briefly reviewed, including: enhanced understanding of toxicity pathways; importance of timing of exposure; contribution of multiple chemical exposures; and low dose effects. Two cases are presented, thyroid disrupting chemicals and anti-androgens chemicals, which illustrate how our knowledge of the relationship between EDCs and adverse human health effects is strengthened and data gaps reduced when we integrate findings from animal and human studies.     

Animal carcinogenicity studies: implications for the REACH system

The 2001 European Commission proposal for the Registration, Evaluation and Authorisation of Chemicals (REACH) aims to improve public and environmental health by assessing the toxicity of, and restricting exposure to, potentially toxic chemicals. The greatest benefits are expected to accrue from decreased cancer incidences. Hence the accurate identification of chemical carcinogens must be a top priority for the REACH system. Due to a paucity of human clinical data, the identification of potential human carcinogens has conventionally relied on animal tests. However, our survey of the US Environmental Protection Agency's (EPAs) toxic chemicals database revealed that, for a majority of the chemicals of greatest public health concern (93/160, i.e. 58.1%), the EPA found animal carcinogenicity data to be inadequate to support classifications of probable human carcinogen or non-carcinogen. A wide variety of species were used, with rodents predominating; a wide variety of routes of administration were used; and a particularly wide variety of organ systems were affected. These factors raise serious biological obstacles that render accurate extrapolation to humans profoundly difficult. Furthermore, significantly different International Agency for Research on Cancer assessments of identical chemicals, indicate that the true human predictivity of animal carcinogenicity data is even poorer than is indicated by the EPA figures alone. Consequently, we propose the replacement of animal carcinogenicity bioassays with a tiered combination of non-animal assays, which can be expected to yield a weight-of-evidence characterisation of carcinogenic risk with superior human predictivity. Additional advantages include substantial savings of financial, human and animal resources, and potentially greater insights into mechanisms of carcinogenicity.