Asthma/Allergic Airways Disease: Does Postnatal Exposure to Environmental Toxicants Promote Airway Pathobiology?

Abstract

The recent, dramatic increase in the incidence of childhood asthma suggests a role for environmental contaminants in the promotion of interactions between allergens and the respiratory system of young children. To establish whether exposure to an environmental stressor, ozone (O₃), and an allergen, house dust mite (HDMA), during early childhood promotes remodeling of the epithelial-mesenchymal trophic unit (EMTU) of the tracheobronchial airway wall by altering postnatal development, infant rhesus monkeys were exposed to cyclic episodes of filtered air (FA), HDMA, O₃, or HDMA plus O₃. The following alterations in the EMTU were found after exposure to HDMA, O₃, or HDMA plus O₃: (1) reduced airway number; (2) hyperplasia of bronchial epithelium; (3) increased mucous cells; (4) shifts in distal airway smooth muscle bundle orientation and abundance to favor hyperreactivity; (5) interrupted postnatal basement membrane zone differentiation; (6) modified epithelial nerve fiber distribution; and (7) reorganization of the airway vascular and immune system. Conclusions: cyclic challenge of infants to toxic stress during postnatal lung development modifies the EMTU. This exacerbates the allergen response to favor development of intermittent airway obstruction associated with wheeze. And, exposure of infants during early postnatal lung development initiates compromises in airway growth and development that persist or worsen as growth continues, even with cessation of exposure.

Introduction

Asthma is a health problem affecting both developed and developing countries. As of 2000, it was estimated that 100 to 150 million people have the disease worldwide (World Health Organization, 2000). The economic impact of asthma is quite high. In the United States alone, direct and indirect health care costs have been estimated to be 16.1 billion dollars (American Lung Association, 2005). The disease is characterized by chronic inflammation, impaired airflow, and remodeling of the airways, coupled with breathlessness and wheezing. Asthma often develops in childhood when the lung is still maturing. In the United States, asthma is the most prevalent chronic disorder of children (American Lung Association, 2005). Unfortunately, asthma early in life leads to a deficiency in pulmonary function that may not return to normal in adulthood, even if the asthmatic becomes asymptomatic (Gold et al., 1994). Because of the detrimental effect of asthma, there is a great deal of work being done to understand how it develops. The purpose of this review is to discuss which early life influences may affect airway structure and function and how postnatal exposure to an air pollutant, ozone, may alter airway development leading to the development of asthma.

One of the factors contributing to asthma susceptibility may be the long maturation period of the lung. Although the human lung needs to be sufficiently formed at birth to perform its primary function of gas exchange, lung development continues for an extensive period (8–12 years) after birth (Burri, 1997), and lung function continues to change in adolescence (Gauderman et al., 2004). Driven by increases in body size and associated metabolic demands, multifold increases in overall size, active cellular differentiation, cell division, and alveolar formation all occur during the 8–12 year period of postnatal lung development (Fanucchi and Plopper, 2004).

The growth of the parenchymal gas exchange area has been well characterized during this time period (Massaro and Massaro, 1996; McGowan and Synder, 2004), yet the same is not the case for the conducting airways where asthma manifests itself. Cudmore et al. (1962) and Phalen et al. (1978, 1985) conducted detailed, quantitative studies of lung casts from a limited number of humans at various postnatal ages. Both groups produced linear regressions that suggest that as body size increases there is a corresponding increase in airway diameter and length. The airways consist of epithelium, mucus-producing glands, cartilage, fibroblasts, nerves, interstitial vasculature, extracellular matrix, immune cells, and smooth muscle cells.

As the airways change in size and increase in surface area, there must be active transformation of all of these components within the airway wall. The process by which airways grow is not well delineated, especially in regard to the organization of the 3-dimensional architecture and the mesenchymal components of the airway wall. Because asthma involves remodeling of the airway wall, such as thickening of the reticular basement membrane and increases in smooth muscle, it would be helpful if more information was known about how the individual components of the airways develop during childhood and adolescence. This type of information would assist us in understanding how normal developmental events are changed to cause asthma.

For example, while atopic infants with reversible airflow obstruction do not have reticular basement membrane thickening similar to adults (Payne et al., 2003; Saglani et al., 2005), older asthmatic children (6–16 years) do have reticular basement membrane thickening. At what point along the continuum of airway development from infancy to childhood does the basement membrane change and when is the change irreversible and related to compromised airway function?

Asthma is thought to be caused by a combination of ongoing lung development, genes (not discussed here) and environmental factors resulting in the alteration of the normal development and function of the lung (Holt et al., 2004). The longer period of human lung maturation (pre- and postnatally) provides ample time for normal lung development to be challenged and perturbed. Environmental factors associated with disturbing normal postnatal lung growth and development include pathogens, such as the respiratory syncytial virus (Lemanske, 2004); allergens, such as from house dust mites (Sporik et al., 1992; Richardson et al., 2005; Thorne et al., 2005); endotoxin (Thorne et al., 2005); and environmental air pollutants, such as oxidant gases, particles, acid vapor, elemental carbon, and cigarette smoke (Tager et al., 1983; Frischer et al., 1999; Peters et al., 1999a, 1999b). Children, especially those living in urban areas, are often exposed to many, if not all of these factors. Numerous epidemiologic studies have demonstrated an association between children living in major industrialized urban areas (e.g., Los Angeles or Mexico City) and the development of childhood respiratory diseases and decreased lung function (Romieu et al., 1996; Peters et al., 1999a, 1999b; Calderon-Garciduenas et al., 2003; Gauderman et al., 2004). These studies demonstrate that the lung is especially sensitive to noxious stimuli during postnatal lung development.

This review summarizes how we have addressed the following two key questions regarding the potential for the development of allergic airways disease in infants and how polluted air environments may influence susceptibility. First, does repeated exposure to environmental contaminants alter postnatal growth and development? Second, does removal from contaminated air reverse the response?

Experimental Model for Environmental Postnatal Airways Disease

To define how environmental contaminants may influence the ability of developing lungs to resist the impact of allergens and other contaminants, we developed a model of allergic airways disease in the adult rhesus monkey (Schelegle et al., 2001) that reflects all the criteria used by the National Heart, Lung, and Blood Institute’s (NHLBI) National Asthma Education and Prevention Program Clinical Practice Guidelines. Based on the NHLBI definition, we found that asthma can be produced in rhesus monkeys following exposure to house dust mite allergen (HDMA) (Schelegle et al., 2001). The monkeys develop a positive skin test for HDMA, with elevated levels of IgE in the serum and IgE-positive cells within the tracheobronchial airway walls.

The animals exhibit impaired airflow after the inhalation of aerosolized allergen that is associated with cough, rapid shallow breathing, and decreased arterial oxygen saturation, all of which are reversible by treatment with aerosolized albuterol. Further, serum histamine concentrations are elevated in sensitized monkeys after allergen exposure. In sensitized monkeys, shed epithelial cells are detectable in bronchoalveolar lavage immediately after allergen aerosol challenge. As with human asthmatics, the majority of the shed cells are ciliated cells. Immune cells, especially eosinophils, increase markedly in abundance in airway exudates and bronchoalveolar lavage. There are also elevations in CD 25 expression on CD 4+ lymphocytes in both lavage and serum.

The animals develop nonspecific airways responsiveness, which is reflected as a fourfold reduction in the dose of histamine aerosol required to produce a 150% increase in airway resistance. There is marked mucous cell hyperplasia accompanied by general epithelial hypertrophy in both intra- and extrapulmonary conducting airways. The basement membrane zone is markedly thickened in most of the intrapulmonary bronchi. This thickened basement membrane zone appears to be characteristic of conducting airways only in primate asthma models and plays a key role in modulating signaling within the airway wall. There are marked accumulations of eosinophils in both the epithelial and subepithelial matrix compartments as well. All of these histological features are focal and distributed throughout the conducting airway trees.

One of the reasons for choosing the rhesus monkey as a model for these studies is the organization of the tracheobronchial airway tree (Figure 1; Plopper and Harkema, 2005). The branching pattern and distribution of airways in the rhesus monkey are more similar to humans than rodents are to humans. The transition zone between the gas exchange area and the conducting airways is very different in primates, both human and nonhuman, than it is in laboratory rodents. Specifically, the extensive transition zone in primates includes a substantial number of branches with alveolarized tissue on one side for gas exchange and mucociliary epithelium on the other side. In laboratory rodents, this transition occupies only one branch of airway at the most.

The experimental protocol that we have used to evaluate the susceptibility of developing lungs in postnatal rhesus monkeys includes beginning exposure early during the postnatal period (30 days after birth) and ending at approximately 6 months of age (Schelegle et al., 2003b). The standard five month exposure protocol involves repeated cycles of exposure to ozone (O₃), 5 days in succession followed by 9 days in filtered air (FA), at a concentration resembling that of Mexico City (0.5 ppm, 8 hours/day). The allergen exposure, using HDMA, is for approximately 3 days (2 hours per day) followed by 11 days of FA. The 3 days of allergen exposure are conducted on the last 3 days of O₃ exposure. Animals are housed in a FA environment and then exposed to O₃, HDMA, or a combination for up to 11 cycles.

To evaluate the potential for recovery, we have followed the 5 months of exposure with another 6 months in FA until the monkeys are 12 months of age. Each study has a group of animals that are housed in a FA environment for the entire period of this study.

Physiologic and Immunologic Responses

Infant animals exposed to HDMA alone have increases in baseline airway resistance (Figure 2; Schelegle et al., 2003b). O₃ plus HDMA more than doubles baseline resistance. A similar pattern occurs with nonspecific airways hyperresponsiveness to histamine (Figure 3); the combination of O₃ and HDMA greatly elevates the responsiveness. We have observed increases in eosinophils in bronchoalveolar lavage in response to O₃ but not to HDMA in the infant rhesus monkeys (Schelegle et al., 2003b). However, the combination exposure greatly increases the abundance of eosinophils in bronchoalveolar lavage (Figure 4).

Airway Growth

To evaluate the extent of remodeling, or alterations in airway development and growth, we use a sampling method in which we microdissect open the conducting airway tree in fixed lungs, count each airway branch, and identify each sample taken from the lung based on its branching history (Plopper, 1990). One of the most striking features that we have observed with remodeling of the airways in infant rhesus monkeys is changes in normal growth. As illustrated in Figure 5, the airways increase by a third in diameter and by twice in length between 1 month of age and 6 months of age (Tran et al., 2004a). The growth pattern of distal airways is exacerbated to a mild extent by exposure to HDMA alone, but is very markedly changed by exposure to O₃ or a combination of HDMA and O₃. The combination exposure inhibits growth in diameter, yet promotes lengthwise growth.

This combination results in longer, narrower airways with higher intrinsic theoretically calculated resistance. Exposure to O₃ causes infant monkeys to have a decrease in the number of conducting airway generations between the trachea and the gas exchange area, as represented by the location of the most proximal respiratory bronchiole (Fanucchi et al., 2006). We counted the number of generations to the first respiratory bronchiole in 4 different lobes of fixed lungs and found a reduction of as many as 6 generations of conducting airways following exposure to O₃ with or without HDMA (Figure 6). A recovery period of an additional 6 months of FA did not have a substantial impact on recouping the number of airway generations that were lost (Figure 6).

The Epithelial Mesenchymal Trophic Unit (Emtu)

The concept of the epithelial-mesenchymal trophic unit (EMTU) was developed as a framework for defining the cellular and metabolic mechanisms regulating the response to injury in a complex biological structure, such as the tracheobronchial airway tree, and for identifying the mechanisms that regulate airway remodeling in allergic airways disease (Figure 7; Evans et al., 1999; Holgate et al., 2000). The EMTU is made up of several tissue compartments.

The epithelial compartment of the airway wall is comprised of surface epithelium and submucosal glands. The interstitial compartment includes the basement membrane zone, smooth muscle, cartilage and vasculature. The nervous compartment includes the afferent and efferent nerve processes that interdigitate between the smooth muscle, the subepithelial matrix and the epithelium, and the regulating neurons of the ganglia and brain stem. The vascular compartment includes capillaries; arterioles and venules, primarily from the bronchial circulation; and lymphatic vessels. The immune compartment includes both inflammatory cells and migratory cells involved in the regulation of immune responses.

Functionally, the EMTU is based on the assumption that the various compartments of the airway actively interact with each other, i.e., the biological function of cells in one compartment is regulated by the functions of the cell populations in the other compartments, and when one compartment is injured the others also respond. In the steady state, these compartments establish a baseline trophic interaction that is disrupted during acute injury and repair and is reset by successive cycles of injury, inflammation and repair, which is characteristic of chronic airways diseases, such as asthma. In addition, we believe that each airway segment, or generation, within the branching architecture of the tracheobronchial airways is a unique biological entity whose properties may differ from those of neighboring branches and that all the components of the airway wall, both cellular and acellular, play a role in both injury and repair responses.

Airway Epithelium

The epithelium of tracheobronchial airways in rhesus monkeys develops both prenatally and postnatally (Plopper and Harkema, 2005). Epithelial development includes increases in the phenotypes of cells that are present, transitioning from a simple columnar epithelium filled with glycogen to a ciliated and nonciliated epithelium, and finally to an epithelium that includes a basal cell population. While most of this differentiation occurs prenatally, a substantial part of the mucous cell differentiation, particularly in distal airways, occurs postnatally (Plopper and Harkema, 2005).

Exposure of infant monkeys to O₃ and HDMA from 1 to 6 months of age modifies the conducting airway epithelium by increasing the number and size of mucous cells and by incorporation of a large number of eosinophils into the luminal epithelium (Figure 8). If these same animals are subsequently exposed to FA for 6 months, the now 12-month-old animals reestablish an almost steady-state mucous cell composition in proximal airways, but the organization of the airway epithelium is significantly disrupted (Figure 9). In more distal airways (Figure 10), there is a dramatic increase in mucous cell abundance in response to exposure to O₃, HDMA, or O₃ and HDMA, (Schelegle et al., 2003b). There is also mucous and nonmucous cell hyperplasia in respiratory bronchioles and a disruptive change in the relationship between alveolar tissue and nonalveolarized aspects of the airway wall (Fanucchi et al., 2006).

Basement Membrane Zone

The basement membrane zone (BMZ) of the tracheobronchial airway wall organizes and develops postnatally (Evans et al., 2002). At the time of birth of the rhesus monkey, the BMZ beneath the tracheobronchial epithelium is a thin sheet representing the basal lamina. During the first 6 months to 1 year of life, this zone grows and changes with the addition of new matrix components and an increase in the overall thickness. As illustrated in Figure 11, exposure to HDMA for 5 months beginning when the infant monkeys are 1 month old accelerates the rate at which BMZ material, such as collagen I, is deposited. Following HDMA exposure, the epithelial contact surface is a smooth uniform boundary for attachment of basal and columnar cells, but the side facing the attenuated fibroblast layer becomes highly irregular.

There is also a marked change in the chemical composition of the BMZ and in the types of signaling molecules that are stored there. Exposure to O₃ and HDMA completely disrupts the differentiation of the BMZ, with many areas being highly thickened and irregular, such as occurs with exposure to HDMA alone, and other areas being extremely thin as observed after exposure to O₃ alone (Figure 11). Between 6 and 12 months of age, the BMZ of the rhesus monkey doubles in size and increases in complexity. Six months of exposure to FA subsequent to a 5-month exposure to O₃, HDMA or both does not provide sufficient time for the normal developmental and growth processes of the BMZ to compensate for the previous disruption (Evans et al., 2004).

Epithelial Innervation

In midlevel airways in the rhesus monkey, innervation of the epithelial compartment is a postnatal event. Soon after birth, components of the wall that express markers for nervous tissue consist primarily of neuroendocrine-like cells. By 6 months of age, this changes, and a fine arborization of nerve processes can be detected (Figure 12) (Larson et al., 2004). Five months of exposure to O₃, HDMA or both results in a marked reduction in the density and distribution of nerve fibers and an elevation of neuroendocrine-like cells (Figure 12). The normal process of growth and development after 6 months of age is for the density of the nerve fibers to be reduced as the airways increase in size (Kajekar et al., 2006). Six months of exposure to FA subsequent to a 5-month exposure to O₃, HDMA or both results in a more than doubling of the density of the nerve fibers within the epithelium and an increase in the neuroendocrine-like cells as compared with unexposed animals (Kajekar et al., 2006).

Airway Smooth Muscle

The growth, differentiation, and organization of smooth muscle fibers within the tracheobronchial airway wall involves a significant number of changes during the postnatal period (Tran et al., 2004a). As the airways grow, the number of smooth muscle fibers and the bundles into which they are organized increase dramatically. This results in a uniform density of bundles within the airway wall regardless of airway size. Another major feature of airway growth is changes in the 3-dimensional orientation of the smooth muscle bundles. Early in postnatal development, the bundles are arranged primarily perpendicular to the long axis of the airway.

By 90 days of age, a large percentage of the fibers are oriented greater than 30° from perpendicular. Exposure to O₃, HDMA or both disrupts this developmental process (Tran et al., 2004b). In terminal bronchioles of FA control 6-month-old monkeys, the majority of the smooth muscle bundles (~74%) around the airway are oriented at an angle less than 15° perpendicular to the long axis of the airway and only a very small percentage (~3%) of bundles were found at an angle greater than 30° (Figure 13). In O₃ exposed infant monkeys, only 43.0% of the terminal bronchiole smooth muscle bundles were oriented at an angle less than 15° perpendicular to the long axis of the airway, but there were 12% of bundles oriented at an angle of greater than 30°.

There was no significant difference in terminal bronchiole smooth muscle bundle thickness or abundance between FA and O₃ exposed monkeys, respectively. An opposite pattern of smooth muscle bundle orientation was present in the most proximal respiratory bronchiole. Only half of the smooth muscle bundles in the proximal respiratory bronchioles of FA control monkeys were oriented around the airway at an angle less than 15° perpendicular to the long axis of the airway, and ~16% of bundles were oriented at an angle of greater than 30°. In the first respiratory bronchiole of ozone-exposed monkeys, ~65% of the smooth muscle bundles were oriented at an angle less than 15° perpendicular to the long axis of the airway and only 5% of the bundles oriented at an angle of greater than 30°.

As in the terminal bronchioles, however, there were no significant differences in smooth muscle bundle thickness or abundance in the first respiratory bronchioles between FA control monkey and O₃-exposed monkeys, respectively (Fanucchi et al., 2006). Six months of exposure to FA subsequent to a 5-month exposure to O₃, HDMA or both does not provide sufficient time for the normal developmental and growth processes to adjust bundle size and orientation to match the configuration in unexposed, age-matched controls.

Airway Vasculature

Schematically, bronchial vasculature is part of the EMTU, and the role of vascular remodeling in the pathogenesis asthma is an evolving concept. Recent studies have described an increase in bronchovascular density in patients with asthma and asthma-like disease (Hoshino et al., 2001). To define the temporal and spatial aspects of vascular remodeling in the airway, we used a design-based stereological method to estimate bronchial vessels in juvenile monkeys exposed to O₃ and HDMA. Bronchial vascular surface area and density were significantly increased at mid-level airways in monkeys exposed to HDMA (Figure 14; Avdalovic et al., 2006). Gene expression of vascular endothelial growth factor (VEGF) was also significantly increased in distal airway levels in HDMA-exposed monkeys. Changes in vascular surface area and density were not as significant in O₃-exposed monkeys, and the combination of O₃ and HDMA led to similar changes as those seen in HDMA-exposed only monkeys (Avdalovic et al., 2003). These results imply that HDMA may stimulate an increase in bronchovascular density and that O₃ exposure is not additive.

Airway Immune System

The organization and distribution of immune and inflammatory cells within the infant tracheobronchial airway wall is very specific for the airway branch in which it is evaluated (Miller et al., 2005; Miller, 2006). Figure 4 shows that eosinophil frequency within airway lavage is significantly elevated in response to combined exposure to O₃ and HDMA, with no significant differences between animal groups exposed to FA (sensitized or nonsensitized), HDMA alone, and O₃ alone (Schelegle et al., 2003a). Comparatively, the density of eosinophil populations within epithelial and interstitial compartments does not necessarily reflect abundance within the airway lumen relative to exposure history (Figure 15A, 15B, top figures).

Within the epithelial and interstitial compartments, the volume of eosinophils from HDMA exposed animals is significantly elevated as compared with FA exposed animals, although exposure to both resulted in less abundance than exposure to HDMA alone. To add another level of complexity, the distribution of eosinophil populations within epithelial and interstitial compartments is variable, depending on where within the airway tree they are located (Figure 15A, 15B, bottom figures). Density of eosinophils within epithelium is highly dependant upon prior exposure to HDMA and appears to occur preferentially in distal airways. Density of eosinophils within interstitium is significantly dependent upon airway generation; eosinophils preferentially accumulate within the trachea and the most proximal intrapulmonary airways.

Summary and Conclusions

In summary, evaluation of the pathobiology of airway remodeling in growing lungs of neonates, using an animal model where exposure to allergen generates reactive airways disease with all the hallmarks of asthma in humans, illustrates that exposure to environmental pollutants and allergens early in life produces a large number of disruptions of fundamental growth and differentiation processes. All the compartments of the epithelial mesenchymal trophic unit are changed, including acceleration of mucous cell development, disruption of basement membrane growth and reorganization, alterations in the organization and orientation of airway smooth muscle, down-regulation of innervation of the epithelial compartment, and disruption of the sites of residence for migratory inflammatory and immune cells.

In addition, airway remodeling in neonatal lungs also involves restriction in the growth of tracheobronchial airways as well as fundamental alterations in branching number. Most of these disruptions do not appear to be easily correctable by subsequent extended periods in an environment free of either oxidant stressors or allergens.

While epidemiological studies have provided evidence of which environmental factors influence the development of asthma, there is a dearth of information on how these factors change normal airway development to result in the asthmatic condition. A major challenge is to determine when the airways change irreversibly along the disease continuum. Currently, we are in the process of evaluating the impact of early life exposure to oxidants and allergens on respiratory health over the long term and the potential implications for chronic lung disease in the adult, including increased susceptibility to infectious diseases, COPD, and chronic bronchitis. Meaningful studies of the mechanisms regulating growth and differentiation of the airways during lung development are needed.

This knowledge will help establish whether there are windows of susceptibility to asthma when infants and children should avoid exposure to harmful environmental factors. Such information is significant for public health as the basis for developing intervention strategies that can minimize childhood susceptibility. Major epidemiological studies, such as the National Children’s Study (2005) that will follow children from birth to age 21, will help answer some of these questions. In conclusion, airways are complex structures that change by growing and differentiating for a significant time during postnatal life. The key to understanding how early life exposures cause asthma is to understand normal airway growth processes so that mechanisms behind the airway changes that occur in asthma can be determined.