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DOI: 10.1080/01926230601132055
Comparative Pathology of Environmental Lung Disease: An Overview
1 Respiratory Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada Correspondence: Address correspondence to: Francis H. Y. Green, Professor, Pathology and Laboratory Medicine, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada; e-mail:fgreen{at}ucalgary.ca
Environmental factors play a major role in a majority of lung diseases. Asthma, chronic obstructive pulmonary disease (COPD), lung cancer, and many interstitial lung diseases are influenced or caused by environmental factors. Animals and humans may respond differently to the same agent, and a study of the comparative pathology between the two is useful for optimizing animal models of environmental lung disease and for evaluating their predictive value in carcinogenicity studies. This overview describes the most common nonneoplastic pathologic pulmonary responses to inhaled environmental agents in the human and contrasts them with the responses observed in rats exposed to the same agents. We show both similarities and difference in response to the same agents; furthermore, both species have unique responses to some agents (for example, progressive massive fibrosis in the human and proliferative squamous lesions in the rat). Quantitative analysis of the grades of response to three environmental particulate dusts revealed differences between the 2 species at the cellular level. Specifically, acute intra-alveolar inflammation, alveolar epithelial hyperplasia, and alveolar lipoproteinosis were all greater in rats than in humans exposed to the same agents. These differences may account for differences between the 2 species in carcinogenic response to nonfibrous particulates.
Key Words: Pneumoconiosis • occupational • environmental lung disease • comparative pathology • rat • human
The mammalian lung can respond in a limited number of ways to injury. In the human lung, these responses can be classified into those affecting the pulmonary parenchyma and those affecting the airways, corresponding to the broad clinical classifications of restrictive and obstructive lung disease, respectively. The histologic criteria necessary for the diagnosis and classification of human occupational/environmental lung disease have been established over many decades of careful observation and research. For some diseases (coal workers pneumoconiosis, asbestosis, silicosis, and silicate pneumoconiosis) diagnostic criteria have been established by panels of experts (Kleinerman et al., 1979; Craighead et al., 1982, 1988). In animals the lesions induced by the same agents are largely classified descriptively (Lee et al., 1989; Renne et al., 2003). Recently, however, diagnostic criteria for the general pulmonary reactions to toxic compounds have been developed (Renne et al., 2003). Many lesions in humans are agent specific (e.g., the silicotic nodule) whereas others are more generic and result from exposure to a variety of agents, e.g., small airways disease, which is seen in association with exposures ranging from gases such as ozone to cigarette smoke and mineral dusts (Churg and Wright, 1983; Churg et al., 2003). Moreover, many agents produce site-specific lesions, e.g., ozone or coal mine dust, both of which affect the central portions of the acinus. This reflects, in large part, the interactions between lung geometry, ventilation patterns, and the physicochemical properties of the inhaled substance. More than one type of lesion may be produced by a given agent, for example, coal mine dust exposure can cause lesions as diverse as the dust macule, progressive massive fibrosis (PMF) and emphysema. Furthermore, pre-existing diseases may be aggravated by inhaled dusts and fumes, examples being the relationship between silica exposure and the tubercle bacillus giving rise to the distinct disease silicotuberculosis (Green and Vallyathan, 1998) and exacerbation of asthma by wood smoke (Tesfaigzi et al., 2005). Although some lesions may be sufficiently specific by themselves to justify a disease diagnosis, for example the silicotic nodule and emphysema; other diseases require the presence of a constellation of histologic features in order to make a diagnosis, for example small airway disease (SAD) and asthma. In general, there are good animal models for the former diseases, whereas animal models for the latter type of disease may lack one or more of the diagnostic features. There appear to be differences in the ways in which species react to inhaled toxic substances (Mauderly, 1996), though these are only recently being systematically characterized (Nikula et al., 1997; Hahn et al., 1998; March et al., 2000). This may be important as animal models are relied upon for assessing carcinogenicity and for understanding mechanisms of disease and progression. The purpose of this review is to define and illustrate the more common environmental nonneoplastic lesions seen in human lungs and contrast these with lesions seen in the rat following inhalation exposure to the same or similar agents. Because the majority of human environmental and occupational lung diseases have long latencies, we will focus on chronic inhalation studies. The rat will be the primary species of comparison as a majority of chronic inhalation studies have been done using this species. Studies using canines (Calderon-Garciduenas et al., 2001) and primates are discussed elsewhere. An assessment of in vitro systems for assessing toxicity and their relevance to human disease is also beyond the scope of this work. The reader is referred to excellent symposia and other sources focusing on various aspects of these subjects (Lippmann and Schlesinger, 1984; EHP, 1992; Ghanem et al., 2004; Seagrave et al., 2006) for further information. Finally, there are several lesions observed in the rat and human that appear unique to each species; for example, focal histiocytic lesions and squamous cystic lesions are relatively common in rats (Boorman et al., 1996; Nikula et al., 2000) but seen rarely, if at all, in humans (Schultz, 1996). Conversely, the most severe form of pneumoconiosis in humans, progressive massive fibrosis (PMF), has yet to be described in an animal model. This is followed by a comparative study of tissue responses in humans and rats exposed to coal dust, talc, and silica. We show that humans and rats differ fundamentally in the ways they react to these particulates.
The spectrum of nonneoplastic responses in the human lung associated with inhalation exposure to environmental agents are listed in the left-hand column of Table 1. These responses are described next and contrasted with the equivalent response in the rat.
Dust Accumulation Without Fibrosis Exposure to ambient particulates at low concentration results in the gradual accumulation of carbonaceous and mineral dust particles in the lungs of all humans. The dust is found in alveolar macrophages within airways and alveoli; later, it is seen in macrophages in the peribronchial, perivascular, interstitial, and subpleural connective tissues. Transport of dust by lymphatic vessels leads to accumulation of dust in the draining lymph nodes. A non-occupationally-exposed North American urban dweller will accumulate on average 0.5 x 109 mineral dust particles per gram of dry lung during an average lifetime, with a range from 71 to 1860 x 106 particles per gram of dry lung (Stettler et al., 1991). Higher concentrations are found in males compared to females and dust content is positively correlated with age (Stettler et al., 1991), smoking (Churg et al., 1985), and urban pollution (Brauer et al., 2001). Similar values have been obtained by Abraham et al. (1991), Churg and Wiggs (1987), and Dumortier et al. (1994) using different techniques. These levels of dust accumulation do not reflect the total dust burden as the data do not include the nonmineral or carbonaceous dust particles. The latter are likely to be present in greater numbers than mineral dust particles, particularly in the smoking population. The major mineral species present in the normal human lung are a variety of silicates, silica (quartz), and metal oxides (Churg et al., 1987; Abraham et al., 1991; Stettler et al., 1991; Pairon et al., 1994). Mineral fibers including asbestos are present in almost all lungs but constitute less than 10% of the total mineral dust burden (Churg and Wiggs, 1985). The size distribution of nonfibrous minerals varies slightly from lobe to lobe in the human lung (Churg et al., 1987) but the reported geometric mean diameters of particles extracted from human lung of 0.6 µm ± 2.1 µm (Churg and Wiggs, 1987), and 0.6 µm ± 2.35 µm (Stettler et al., 1991) show remarkable concordance. These data do not reflect nanoscale particles that are not captured by the dust extraction techniques used. When appropriate methods are used, large numbers of nanoparticles are found in the human lung (Brauer et al., 2001). Histological assessment of lungs from the above-referenced studies show no or minimal evidence of lung fibrosis, suggesting that the human lung can accumulate a considerable burden of dust without adverse effect. It is not known whether the human lung achieves a "steady state" with regard to dust burden where newly deposited dust equals dust removed from the lungs by clearance and dissolution. The positive association of lung dust with age noted in Stettler’s et al. study (1991) would argue for a more complex model. A 2-compartment model would combine steady state conditions in the intra-alveolar and intra-airway compartments with an interstitial compartment where dust is effectively sequestered from clearance mechanisms. The many studies comparing lung dust burden in occupationally-exposed populations with non-occupationally-exposed control groups show that clinical pneumoconiosis only develops when the dust burden is several orders of magnitude above background levels (Abraham et al., 1991; Stettler et al., 1991). There may not be an abrupt threshold, above which a person is likely to develop the disease, as studies of silica-exposed men working in quarries (Vallyathan and Craighead, 1981) who were asymptomatic during life and had normal chest X-rays and young farm workers dying from nonrespiratory disease (Pinkerton et al., 2000) had subtle pathologic abnormalities in their lungs at autopsy. Thus, tissue response and collagen accumulation may be gradual and incremental in response to increasing lung dust burden. In truth, the nature of the dust burden-response relationship is poorly understood for humans at the low end of the dose-response relationship. This is an important question to address as the available evidence in rats indicates that disease results after a certain dust-burden threshold is achieved, the so-called "dust overload" hypotheses (Morrow, 1988; Muhle et al., 1990). This observation may not be true for all cases. Recent data in rats exposed by inhalation to silica showed no evidence for a dust overload effect (Porter et al., 2004). Accumulation of dust containing macrophages in and adjacent to respiratory bronchioles/alveolar ducts and in the perivascular and subpleural interstitium is also a universal response to inhaled dust in animals. As in the human, fibrosis may be absent following exposure to nuisance and carbonaceous dusts. These changes closely parallel those seen in humans.
Lymph Node Fibrosis
Macules
The lesions develop in persons exposed to relatively ‘inert’ dusts, and at exposure concentrations high enough to overload alveolar clearance mechanisms. For coal miners, this level is considered to be approximately 1 mg/m3 based on epidemiologic studies (US DHHS, 1995). Morphologic examination of the lungs of young miners killed in accidents indicate that the macules develop in association with collections of tightly packed and pigmented macrophages in alveoli adjacent to the respiratory bronchioles (Green and Vallyathan, 1998). Collapse of these alveoli and incorporation of the macrophages into the walls of the respiratory bronchioles may be one mechanism for growth of the lesion. Direct penetration of particles impacted on the walls of the respiratory bronchioles into the interstitium also contributes to their growth (Brody et al., 1982; Churg et al., 1996). A similar preferential accumulation of dust in the centriacinar portions of the lung lobules is seen in several animal species (mice, rats, hamsters) exposed to nuisance and relatively nonfibrogenic dusts (Busch et al., 1981; Heinrich et al., 1986; Lewis et al., 1989; Muhle et al., 1990; Schultz, 1996). Compaction of dust particles within macrophages in alveoli adjacent to alveolar ducts, together with interstitial localization of dust is also seen (Figure 2A). Fibrosis is usually mild and the lesion may be associated with centriacinar emphysema (Heinrich et al., 1986). The latter is less prominent than is seen in the human lesion. Epithelial hyperplasia, acute inflammation and lipoproteinosis, common features of the rodent lesions (Figures 2B and 2C) are rare or muted in the human.
Small Airways Disease These lesions are more diffuse than the dust macules described here but occupy a similar region of the lung. They differ from the dust macule in that they cause greater fibrosis and are not associated with dilatation or destruction of the bronchiolar wall (focal emphysema). They are perhaps the most common lung lesion produced by exposure to environmental dusts and fumes in the human. The affected small airways include the membranous bronchioles, 3 orders of respiratory bronchioles and occasionally the alveolar ducts. Cigarette smoking and mineral dust exposures are the most common causes of small airway disease (Churg et al., 2003). It is the earliest lesion to characterize asbestosis (Craighead et al., 1982). The first-order respiratory bronchiole is the most severely affected (Pinkerton et al., 2000). Small airway disease is associated with chronic airflow obstruction when the process is severe (Wright et al., 1992). In cigarette smokers, the lesion is characterized by fibrosis and mild muscular hyperplasia of the small airways with chronic inflammatory cell infiltration, most commonly lymphocytes, and mucous metaplasia of the lining epithelium. Collections of macrophages are seen within the small airways and adjacent alveoli and, in cigarette smokers, these macrophages have a characteristic tan-colored cytoplasm and contain small carbonaceous particles. They are referred to as "smokers’ macrophages." Small airway disease produced by mineral dusts including both fibrous minerals (such as asbestos) and nonfibrous minerals is similar to that seen in cigarette smokers except the degree of fibrosis is greater and in nonsmokers the characteristic smokers’ macrophages are not found. This region of the lung is a primary site of deposition for small particles, which become translocated through the wall where they initiate inflammatory and fibrotic responses (Brody et al., 1982; Churg et al., 1996). The small airways in rodents have a different architecture and lack well-defined respiratory bronchioles. Nonetheless, the small airways leading to the acini are similarly affected by exposures to dusts and fumes and the pathologic features are similar to those seen in humans.
Nodules The silicotic nodule is the classic nodule (Figure 3A). This nodule is sharply circumscribed from the surrounding tissues and the collagen has a whorled "onion skin" appearance in which varying amounts of dust are embedded. Polarizing microsocopy usually shows crystalline particles consistent with silica in the centers and periphery of these lesions.
Several variants of nodules are recognized (Green and Churg, 1998). The mixed dust nodule is more stellate in shape and has irregularly arranged collagen fibers in which dust particles are enmeshed (Honma et al., 2004). These nodules develop in association with less fibrotic silicate dusts but usually a significant quantity of silica is also found within them. A third type of nodule has a granulomatous component and is seen in association with mineral dusts that form plates or sheets, such as talc, which appear to stimulate a giant cell response. Immature silicotic nodules also may have a granulomatous appearance prior to their becoming mature and fibrotic (Figure 3B). Similar lesions may be observed in animals, however as animal lesions are generally less old (immature), they rarely become as densely fibrotic as those seen in humans. Granulomatous nodules predominate in rats (Saffiotti and Stinson, 1988) (Figure 3C) and the surrounding lung frequently shows alveolar lipoproteinosis (Renne et al., 2003) (Figure 3D).
Progressive Massive Fibrosis They have been described in association with exposure to silica, coal mine dust, nonfibrous silicates and carbon black (Green and Churg, 1998). The lesions can become very large, occupying one or more lobes, and are usually bilateral. On the cut surface, the lesions are rubbery black in appearance, they may contain remnants of old nodular lesions including silicotic lesions, and they tend to cavitate due to central necrosis (Figure 4A). They also can become secondarily infected with mycobacterial or fungal organisms (Green and Vallyathan, 1998). These lesions have not been described in wild or laboratory animals.
Diffuse Interstitial Fibrosis In humans, this pathology is seen in response to a number of fibrogenic dusts. The most well known of these being asbestos but other dusts, including silica, silicates, and coal dust (Craighead et al., 1982, 1988; McConnochie et al., 1988) can produce this type of response. Diffuse fibrosis may be seen in association with focal lesions, such as macules or nodules. There is a variable degree of chronic inflammation, but this is not a prominent feature. The fibrosis includes collagens, elastic fibers, and smooth muscle cells and extends into and between the alveolar walls. In the late stages, the lung architecture becomes remodeled into thick-walled cystic spaces, which may or may not communicate with the conducting airways. These spaces commonly are lined by metaplastic epithelium of cuboidal, ciliated, columnar, mucous, or squamous types. In humans, interstitial fibrosis, from whatever cause, is associated with an increased risk of lung cancer, particularly adenocarcinomas (Park et al., 2001). Interstitial fibrosis similar to that seen in humans is seen in rats and metaplastic and hyperplastic changes of the lining epithelium are also common. The profound remodeling seen in humans, known as "honeycomb lung," is, however, not reported in rats. This difference may reflect the tendency for inhaled particles to be retained more in the interstitium of humans and more in the alveoli of rats (Nikula et al., 2001). The shorter life span of rats compared to the human may also be a factor.
Lipoproteinosis
Diffuse Alveolar Injury and Bronchiolitis Obliterans
Alveolar Epithelial Hyperplasia
Cholesterol Granulomata
Proliferative Squamous Lesions
Emphysema
We sought to directly compare the tissue responses in the human and the rat to a range of nonfibrous mineral dusts of varying fibrogenicity in the human. The dusts were coal dust (low and high dose), talc, and silica.
Selection of Pathology Materials The source of the materials is shown in Table 2. Criteria for selection of human pathology materials was based on a known exposure to dust aerosols, a history of not smoking cigarettes, and a lack of major confounding diseases, such as primary or metastatic cancer of the lung or pneumonia. Cases of coal worker’s pneumoconiosis were obtained from the National Coalworker’s Autopsy Study operated by the National Institute for Occupational Safety and Health (NIOSH), Morgantown, WV. Human pathology materials from individuals exposed to talc and silica were obtained from pathology archives at NIOSH, Morgantown and the University of Calgary Medical School, Calgary, Alberta.
The Internal Review Board of The Lovelace Institutes (currently known as the Lovelace Respiratory Research Institute) certified that the project qualified as exempt from the requirements of Title 45, CFR Part 46, Protection of Human Subjects. Medical histories and questionnaire data were used to eliminate smokers from the selected populations. In addition, if the histologic appearance of lung showed features of cigarette smoke exposure the case was exluded.
Selection of Rodent Materials By the very nature of the exposure situations, documentation of exposure to dust aerosols was excellent in studies with rats and poor in human populations. For rats, the exposure concentrations, exposure durations and aerosol characteristics were known. For humans, the exposures were only qualitatively known, based on work history and occasional air sampling. This documentation was best for workers in the NCWAS. The characteristics of the exposures in each group selected are shown in Table 3.
Pathology Documentation The cellular responses in the lungs were graded for severity using a standardized grading system developed to document the similarities and differences in response between humans and rats. Key morphologic changes documented were visible dust, inflammation, fibrosis, granulomas, alveolar macrophages numbers, proteinosis, alveolar epithelial hyperplasia, bronchiolar metaplasia, squamous metaplasia, squamous cysts, emphysema, vascular changes, and neoplasia. The severity of the reactions was also graded from 1 to 4 based on a subjective evaluation. Both a medical (FHYG) and a veterinary pathologist (FFH) graded the slides using a standard light microscope.
Exposure to coal mine dust produced a graded response in fibrosis for both humans and rats (Figure 5). In both species, high-dose exposure caused more fibrosis than low dose exposure with the greatest difference being seen in the rats. In humans, the most fibrogenic dust was silica, whereas talc produced an intermediate grade of fibrosis, similar to that seen for high dose coal dust exposure. In rats, talc was associated with a higher grade of fibrosis than either coal dust or silica. The fibrogenic response to silica was relatively low in the rat, lying between the fibrogenic response to low and high dose coal dust.
Granulomas were not seen following exposure to coal mine dust in the human (Figure 6). Low grades of granulomas were associated with exposure to both talc and silica in the human with the greatest response to talc. A very low granulomatous response to low and high dose coal dust was seen in the rat with an intermediate response to talc, both very similar in degree to that seen in the human. The major difference was seen for silica, which caused a florid high-grade granulomatous response in the rat.
Major differences were recorded between humans and rats in the acute intra-alveolar inflammatory response to the dusts (Figure 7). For humans, none of the dusts provoked a significant inflammatory response. Rats, by contrast, showed an acute inflammatory response to all of the dusts. Coal dust was the least inflammatory, talc produced an intermediate response, and silica was associated with a high grade of inflammation. A dose-response relationship for coal dust was seen.
Degenerative changes were also markedly different between the species in response to the dusts. In the human, alveolar lipoproteinosis was seen occasionally in subjects exposed to talc and silica but not coal mine dust (Figure 8). Lipoproteinosis was seen in the lungs of occasional sections from rats exposed to the higher level of coal dust. The lipoproteinosis response was dramatically increased in rats exposed to talc and silica, being seen in all animals examined.
Alveolar epithelial hyperplasia was seen at low levels in humans exposed to all 4 dusts (Figure 9) with the greatest response seen in talc-exposed individuals. By contrast, apart from low coal dust exposure, which produced a mild epithelial hyperplastic response in the rat, high-dose coal, talc, and silica exposure were all associated with a moderate (high-dose coal, talc) or high-grade epithelial response (silica).
This review illustrates some of the characteristic pathologic responses to inhaled pollutants in the human and compares them to the lesions seen in rodents exposed to the same agents. It reveals both similarities and differences in response. A major difference was that there were unique species-specific responses to the same dusts. Responses such as progressive massive fibrosis (PMF) or rheumatoid pneumoconiosis (Green and Churg, 1998), which are of great importance to the exposed human, are not described in animals. Conversely, keratin cysts, which appear common in rats, are not seen in humans exposed to environmental pollutants (Boorman et al., 1996). In addition, there were differences in prevalence between rats and humans for alveolar lipoproteinosis, cholesterol granulomata, and giant cell granulomas, all of which were more common in the rat than the human. Although both species accumulated dusts within similar compartments (intra-alveolar, interstitial, subpleural, and around the broncho-vascular bundles), morphometric analysis has shown that the relative amounts of interstitial and intraluminal dust differ markedly between humans and rats with a predominance of dust being found in the interstitium in man and intraluminarly in the rat (Nikula et al., 2001). The dose of particles did not alter the predominant site of particle retention and in both species macrophages were the principal phagocytic cell. The centriacinar region was the primary site of dust deposition and the primary target of injury in both species studied. However, because the rat lacks respiratory bronchioles, the distinction between macules and small airway disease in the human was attenuated in the rat. The most profound differences, however, lay at the cellular level. The response of rats to centriacinar deposition of inert and fibrogenic dusts was different from that seen in the human, with the possible exception of fibrosis. In both species, fibrosis was proportional to exposure and the relative ranking of the fibrosis followed a similar order in rats and man. The apparent exception was for the grading of fibrosis in rats exposed to silica. This may have resulted from the observation that the silica caused a primarily granulomatous response in the rats used in this study (Figure 6). In the early stages of silicosis in the human, the response may also be granulomatous, which matures into the classic fibrotic nodules over several decades (Craighead et al., 1988). Studies of rats following inhalation exposure have shown that the granulomatous response matures into a fibrotic one (Langley et al., 2004; Porter et al., 2004). Acute intraluminal inflammatory and degenerative changes were much more severe in rats than humans and these inflammatory changes were associated with lipoproteinosis and cholesterol granulomas. In addition, the rats showed much greater alveolar epithelial hyperplasia than humans and, in addition, showed dysplastic and early neoplastic change in response to both fibrogenic and relatively nonfibrogenic dusts. This pattern of epithelial changes is not seen in humans. Asbestos (Craighead et al., 1988) and, to a lesser extent, silica (Kurihara and Wada, 2004) are associated with epithelial hyperplasia in the human (Craighead et al., 1982, 1988) and asbestos is strongly linked to increased risk for lung cancer and mesothelioma (Hodgson and Darnton, 2000). By contrast, humans exposed to relatively nonfibrogenic dusts, such as coal mine dust, do not develop proliferative epithelial lesions and coal miners have lower than expected rates for lung cancer when adjusted for their smoking histories (Kuempel et al., 1995; Ghariem et al., 2004). These findings in humans indicate that only dusts that initiate epithelial hyperplastic changes are associated with increased risk for lung cancer. Rats, on the other hand, appear to develop proliferative epithelial lesions in response to particulate dusts of all types. In conclusion, rats and humans reveal many important differences in how they respond to nonfibrous inhaled particles. These differences must be considered when developing rat models of human pulmonary diseases. In addition some of these may account for the differences in carcinogenicity between humans and rats in response to inhaled particulates (Dungworth et al., 1994; Mauderly, 1996; Hahn et al., 1998).
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Abstract
Introduction
