Anatomy and Physiology of the Pleura
The parietal pleura lines the chest wall and the superior surface of the diaphragm and the visceral pleura covers the lungs (). The pleural space in humans contains a small amount of fluid (0.1–0.2 ml/kg body weight) that is a filtrate from the underlying systemic circulation (Owens & Milligan, 1995
; Broaddus, 2008
). This space (10–20 μm wide) is lined by a single layer of mesothelial cells resting on a basement membrane and underlying connective tissue and blood vessels. The major routes of drainage of fluid, protein, particulates, and cells from the pleural space are through the lymphatic stomata that open between mesothelial cells on the parietal pleural lining (Hammar, 1994
; Wang, 1975
; Broaddus et al., 1988
FIGURE 1. Fluid turnover and lymphatic drainage from the pleural space. In the normal pleural space (shown here), as in other interstitial spaces of the body, liquid slowly filters from systemic capillaries and is absorbed via lymphatics (solid arrows). In the (more ...)
Effusions, an accumulation of excess liquid in the pleural space, are common features of a multitude of diseases. Transudative effusions, those not associated with inflammation or injury, usually develop due to increased hydrostatic pressure. In congestive heart failure, the most common cause of transudative effusions, increased pulmonary venous pressure leads to fluid accumulation in the interstitial spaces of the lung; the fluid then moves toward the lower pressure pleural space and leaks across the visceral pleura into the pleural space (Broaddus et al., 1990
; Owens & Milligan, 1995
). In the setting of inflammation or injury of the lung, pleura, or other organs, exudative effusions may form; these effusions contain elevated levels of protein due to the increased leakage across capillaries with increased permeability (Mutsaers et al., 2004
). Excess fluid in any part of the body may find its way to the pleural space via the interstitial tissues along pressure gradients and by moving across the permeable pleural membranes. The normal and pathological paths by which liquid, cells, and particles enter and exit the pleural space suggest pathways by which asbestos fibers may also enter and exit or fail to exit the pleural space. The study of the physiology of the pleural space is challenging; even when using laboratory animal studies, analyses of the pleural space are limited by the narrowness of the space and the difficulty in sampling without inducing inflammation or injury.
Pathways Leading to Translocation of Fibers to the Pleura
The route of translocation of fibers from the lungs to the visceral pleura, into the pleural space, and to the parietal pleura is unknown. It is postulated that asbestos fibers may migrate to the lung interstitium and visceral pleura by a paracellular route or by direct penetration across injured alveolar epithelial cells (Miserocchi et al., 2008
). Fibers may be transported to the pleural space via the lymphatics and bloodstream (Oberdörster et al., 1983
). Fibers may translocate by themselves or within macrophages. Although studies of asbestos fiber movement have not been possible due to technical limitations, it is likely that asbestos fibers translocate to the pleural space passively in the same manner as interstitial fluid. This process may be enhanced by lung inflammation induced by asbestos fibers or by mixed dust exposures that increase interstitial fluid accumulation and thus fluid movement along the interstitial spaces to the pleural space (Miserocchi et al., 2008
There are thus few studies that investigated the translocation of fibers from the lung into the pleural space. Even in the few existing studies, data from animal studies may have limited relevance for humans because of the different visceral pleural anatomy in rodents. In the rodent, the visceral pleura is “thin,” consisting mostly of a mesothelial layer and basement membrane lying directly over the alveoli. There is little submesothelial connective tissue and no pleural vasculature. In sheep and humans and other large mammals, the visceral pleura is “thick” and has a significant submesothelial connective tissue space, containing nerves and systemic blood vessels (). In contrast to the visceral pleura, the parietal pleura in different species has a constant and similar anatomy (; Light & Broaddus, 2010
). Thus, due to differences in the visceral pleura, one might postulate a difference between rodents and humans in the movement of the fibers into the pleural space. Due to similarities in the parietal pleura, one might suggest that the localization, accumulation, and actions of fibers in the parietal pleura might be similar.
These questions have been almost impossible to address using current technology but it is hoped that new tools and imaging techniques such as nuclear magnetic resonance (NMR) spectroscopy or two-photon microscopy can be developed to provide data on (1) how fibers distribute in the lungs and pleura, (2) the ultimate destination of fibers, (3) how fiber movement is enhanced, and (4) whether fibers are translocated and retained differently in animals and humans. Such techniques could be invasive, using labeled fibers that could be traced, for use in animal studies, and noninvasive for clinical studies of those exposed to asbestos. This imaging information on fiber localization would enhance diagnosis and follow-up of subjects exposed to asbestos, such as in directing where to sample tissues to assess fiber dosimetry, and how to determine preneoplastic biomarkers (Greillier et al., 2008
) that might lead to intervention and prevention of non-neoplastic and neoplastic pleural diseases.
Pleural Fiber Dosimetry in Rodents
Translocation and retention of fibrous particulates from initial sites of pulmonary deposition to extrapulmonary sites are believed to be important aspects of their potential toxicity (Dodson et al., 2003
; Suzuki & Kohyama, 1991
). Pathologic tissue responses such as edema, inflammation, or fibrosis might potentially affect translocation and retention of particulates in the body, as well as properties of particles themselves including dose, dimensions and biopersistence. Although similarities exist between animal models and humans concerning physiological processes such as interstitial fluid dynamics and lymphatic flow, there are also anatomical differences such as in visceral pleural thickness (Tyler, 1983
), as well as physiological differences such as of macrophage size and function, that need to be taken into account when comparing across animal species and when extrapolating from animals to humans (Jarabek et al., 2005
; Maxim & McConnell, 2001
). Rodents and humans also differ in particle respirability (Mossman et al., 2011
) and this limits the use of rodent models for human risk assessment based on fiber dimensions (Lippmann & Schlesinger, 1984
; Lippmann et al., 1980
Biopersistence of fibers in the lung parenchyma also influences the fiber dose that is ultimately translocated to the pleura. Biopersistence in the lung is dependent on (1) site and rate of deposition, (2) pulmonary clearance parameters, (3) solubility in lung fluids, (4) breakage rate and patterns, and (5) rates of fiber translocation and retention. Surface chemistry and diameter are important determinants of solubility. Much of the knowledge base concerning the role of biopersistence is actually derived from studies of synthetic vitreous fibers (Bernstein, 2007
; Oberdörster, 2000
Fiber characteristics also affect clearance from the lung and translocation to the pleura. Macrophage-mediated particle clearance in the lung is likely to influence translocation of particles to interstitial sites. There are important interspecies differences in particle clearance, as well as in biological effects of high pulmonary concentrations of particles, in humans and in different animal species (Bermudez et al., 2002
; Oberdörster, 2002
). In addition, the method of dose administration in experimental animals is shown to influence pleural pathology outcomes following particle exposure. In silica-exposed rats, pleural granulomas developed in animals following inhalation, but not after instillation, and the different response was likely due to differences in kinetics of particle delivery and lymphatic clearance (Henderson et al., 1995
The effects of asbestos may be altered when asbestos is mixed with other particulates, a situation common in occupational and environmental exposures. Studies by Davis and colleagues (1991)
showed that coexposure of rats to amosite asbestos and to quartz increased the incidence of amosite-induced pleural mesothelioma, presumably by elevation in fiber dosimetry and translocation through the visceral pleura. Recent studies by Bernstein and colleagues (2008)
demonstrated that coexposure of chrysotile asbestos together with nonfibrous particulates decreased fiber retention in the lungs of rats, perhaps by increasing macrophage recruitment and macrophage-mediated clearance or by inducing more inflammatory fluid movement to the pleura.
Fiber translocation in rodents appears to be rapid and may be responsible in some cases in particular for pathologic outcomes. In rats, short chrysotile asbestos fibers are found in the pleural space within a week following intratracheal instillation (Viallat et al., 1986
). Similarly, crocidolite fibers were detected in the pleural space 1 wk following inhalation (Choe et al., 1997
). In another study in rats, short fibers (<5 μm length) were found 5 d after inhalation exposure to a synthetic vitreous fiber (refractory ceramic fiber) aerosol (Gelzleichter et al., 1996
). In studies in rats and hamsters involving chronic inhalation of synthetic vitreous fibers as well as of amosite and chrysotile asbestos used as reference materials, significant inter-species differences in pleural pathology were seen (Mast et al., 1994
; McConnell, 1994
). Subsequent short-term mechanistic studies of translocation showed that the Syrian golden hamster, a species prone to development of pleural fibrosis and mesothelioma following synthetic vitreous fiber exposure, displayed greater translocation of fibers to the pleura than did similarly exposed rats (Gelzleichter et al., 1999
). The greater translocation in the Syrian golden hamster may thus have accounted for its greater susceptibility to fiber-induced toxicity.
Pleural Fiber Dosimetry in Humans
There is virtually no knowledge of the kinetics of fiber translocation and retention in the human pleura and there are few pleural fiber burden studies in occupationally exposed workers. In addition, due to loss of anatomical orientation after ashing or digestion of target tissues, it is not known where fibers reside intracellularly or extracellularly. Fibers are identified within mesothelial cells (Davis, 1974
; Fasske, 1986
; Lee et al., 1993
) but, due to technical limitations, no comprehensive studies have quantified intracellular fiber burden at the microscopic level. Although analytical transmission electron microscopy (TEM) with x-ray energy-dispersive analysis is the gold standard for quantitation and identification of asbestos fibers in tissue, in vivo studies would be enhanced greatly by nondestructive imaging approaches that could detect the presence of fibers, their chemical composition, or even the cellular response without destroying anatomical relationships.
A major data gap in understanding mechanisms of asbestos-related pleural disease is the paucity of information available to determine the dose of asbestos fibers that is deposited and retained in the pleural membranes. This overview describes the technical complexity and limitations associated with quantitation of human lung and pleural fiber burdens, as well as summarizing available data.
1. The source of tissue samples ranged from pleural biopsies obtained during diagnostic thoracoscopy (Boutin & Rey, 1993); to surgical specimens including needle biopsies, wedge biopsies, or pneumonectomy or pleural decortication samples; and to pleural and lung tissues obtained during autopsy examination (Roggli & Sharma, 2004).
2. Regardless of the source of tissue, sampling is a potential source of error since there is significant variation in anatomical distribution of fibers, especially in the parietal pleura (Roggli, 1992; Boutin et al., 1996; Mitchev, et al., 2002).
3. Tissues may be contaminated during surgical resection or at autopsy due to fibers present in fixatives, in specimen containers, on surgical gloves, or on dissecting instruments (Roggli & Sharma, 2004).
4. Light microscopy is inadequate for identification and counting of asbestos fibers. Dodson and Atkinson (2006) recommend analytical transmission electron microscopy in combination with x-ray energy-dispersive analysis and selected area diffraction techniques for specific mineralogical identification. Both coated and uncoated fibers, as well as particulates, should be analyzed and quantitated (Dodson & Atkinson, 2006).
5. A systematic approach to counting fibers of all dimensions and analysis of lung fiber burdens needs to be used, as described by the European Respiratory Society (DeVuyst et al., 1988).
6. Appropriate control populations need to be used because there is significant variability in human lung fiber burdens (Roggli, 1990). A systematic analysis of lung asbestos fiber burdens in workers with asbestos-related disease, people with asbestos exposure in households or in buildings, and control cases revealed a wide range of counts with considerable overlap between workers, other asbestos-exposed cases, and controls (Roggli & Sharma, 2004).
7. The criteria used to define and count asbestos fibers need to be stated explicitly. Some investigators only count fibers longer than 5 μm; however, the majority of asbestos fibers in human tissue samples are shorter than 5 μm (Dodson & Atkinson, 2006).
8. Tissue preparation techniques may introduce artifacts due to tissue drying or traumatic disruption of fiber bundles (Dodson & Atkinson, 2006).
Finally, although quantitation of human lung and pleural asbestos fiber burden is the only technique available to assess the dose delivered to and retained at the target tissue, there are additional considerations in interpretation of these data. Tissue fiber burden depends on the time since cessation of exposure. In addition, the fiber burden and the types of fibers in the lung may not reflect the fiber burden in the pleura. For example, shorter uncoated fibers are more readily cleared from the lungs; however, while these fibers may be decreasing in the lungs, they may be accumulating in the pleura and extrapulmonary sites (Holt, 1981
) and be associated with development of disease at these sites (Dodson & Hammer, 2006
; Dodson & Atkinson, 2006
It is important to note that the lungs of normal control cases evaluated at autopsy contain significant numbers of commercial and noncommercial asbestos fibers, as well as other particulate and fibrous minerals. This is noteworthy especially in lungs from those who resided in urban settings (). By comparing lung fiber burdens between those with pleural mesothelioma and those without, investigators showed that, although there is overlap, there is an increased risk for mesothelioma, with an elevated lung burden of certain fibers, such as crocidolite, amosite, and tremolite; due to its lower biopersistence, chrysotile may not be reliably analyzed by autopsy studies ().
Asbestos Fiber Content in Lung Tissue of an Urban Population
Lung Fiber Burdens in Malignant Mesothelioma Patients
In contrast to these and other studies of fiber burdens in the lung, only a few studies have reported asbestos fiber burdens in the pleura. In those few studies that analyzed pleural fiber burden, the results from lung and pleura differed, perhaps due to the technical problems described earlier, and appeared to indicate that pleura has a predominance of short chrysotile fibers. Sebastien et al. (1980)
concluded that lung fiber burden could not be used as an accurate reflection of pleural fiber burden. In their parietal pleural samples, most of the asbestos fibers were short chrysotile fibers. Gibbs et al. (1991)
also reported lower asbestos counts in the visceral pleura than in matched lung samples from the same patients and found mostly short chrysotile asbestos fibers.
Dodson et al. (1990)
analyzed lung tissue, lymph nodes, and pleural plaques obtained at autopsy from eight shipyard workers in Italy. Data showed both chrysotile and amphibole asbestos fibers in the lungs; however, chrysotile asbestos fibers were the most frequent type of asbestos found in pleural plaques. Most fibers in the lymph nodes and pleural plaques were shorter than 5 μm, although some fibers longer than 8 μm were present at these sites. More recently, Suzuki and his coworkers (2005)
compared asbestos fiber burdens of human mesothelioma tissues obtained following bulk tissue digestion or ashing of 25-μm tissue sections using high-resolution analytical electron microscopy. The majority of fibers were ≤5 μm long and 92.7% were ≤0.25 μm wide. Chrysotile asbestos fibers were identified most frequently in a total of 168 cases of human malignant mesotheliomas obtained from biopsy or autopsy specimens (Suzuki et al., 2005
). In an earlier study, Suzuki and Yuen (2001)
detected only short, thin chrysotile asbestos fibers in 25.7% of the lungs and in 77.4% of the mesothelial tissues of patients with malignant mesothelioma. These tissue samples were obtained from cases throughout the United States that were sent to Dr. Suzuki for pathological review and were systematically analyzed using histology, immunohistochemistry, and electron microscopy, in some cases, over a 15-yr period. As summarized succinctly by Dumortier et al. (1998)
in a letter to the editor in 1998, the size and type of asbestos fibers associated with development of diffuse malignant pleural mesothelioma remain controversial (Mossman et al., 2011
; Case et al., 2011
; Aust et al., 2011
One possible explanation for the confusion in pleural sampling came from a pioneering study carried out by Boutin et al. (1996)
. Using video-assisted fiber-optic thoracoscopy in eight asbestos-exposed patients and six unexposed cases, Dr. Boutin and colleagues (1996
) sampled specific anatomic regions of the parietal pleura identified as collecting spots for inorganic particulates and fibers that translocate to the pleural spaces. These regions are called “black spots” due to localized accumulation of carbon particles and are sites of lymphatic drainage located in the lower coastal regions of the parietal pleura and on the superior dome of the diaphragm. Using transmission electron microscopy, Boutin et al. (1996)
identified numerous amphibole as well as chrysotile fibers at black spots, and 22.5% were ≥5 μm long. The mean asbestos fiber concentration in the 8 exposed cases was 12.4 ± 9.8 × 106
fibers/g dry lung tissue, 4.1 ± 1.9 × 106
fibers/g black spots on the parietal pleura, and 0.5 ± 0.2 × 106
fibers/g normal parietal pleura, using bleach digestion of lung tissue and low-temperature ashing of pleural tissue samples. Evidence indicated that asbestos fibers accumulate in focal areas of the parietal pleura and that these “black spots” are the most likely anatomic origin of diffuse malignant mesothelioma. In a subsequent study of black spots analyzed from 150 consecutive autopsies of urban residents in Brussels, Belgium, the histopathological appearance of black spots showed chronic inflammation with lymphocytes, plasma cells, and macrophages with a variety of particulates and fibers both intracellularly and extracellularly (Mitchev et al., 2002
). Of note, there was no anatomic relationship between black spots and parietal pleural plaques. Black spots were present in 92.7% of these cases; these lesions were more numerous in older cases and in males. Evidence indicated that these cases may have had greater exposure to coal dust used for home heating and in industry. In this case series, asbestos bodies >1000/g dry lung were found in 15 of 97 cases studied; unfortunately, pleural samples were not analyzed for the presence of asbestos fibers (Mitchev et al., 2002
). The discrepancy between studies of pleural fiber burden and distribution may thus be explained by the inhomogeneity of fiber deposition in the parietal pleura. Since this important observation of the localization of pleural fibers in black spots, almost no studies addressed pleural fiber burden to clarify which fibers are present and which fibers are associated most closely with asbestos-induced pleural disease, whether neoplastic or non-neoplastic.