* Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina;
Instituto Nacional de Pediatría, Mexico City 14410;
Department of Radiology and
Center for Environmental Medicine and Lung Biology, University of North Carolina, Chapel Hill, North Carolina 275997310; and
¶ National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Received July 21, 2000; accepted December 14, 2000
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ABSTRACT |
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Key Words: dogs; air pollution; lungs; particulate matter; ultrafine particulate matter; ozone; endothelial and epithelial lung dysfunction; chronic lung inflammation; lung remodeling.
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INTRODUCTION |
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MATERIALS AND METHODS |
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Northeast MC, on the other hand, is characterized by pollutant emissions from light and medium industries and high vehicular traffic, both on paved and unpaved roads (Edgerton et al., 1999), with high PM values. PM10 averages on the order of 235 µg/m3 (Cicero-Fernandez et al., 1993
); the PM2.5 annual average is 44 µg/m3 (Edgerton et al., 1999
). Concentrations are above the standards for O3 (71.9% of days), NO2 (15.8% of days), and CO; VOC averages 3130 ppbC (Edgertonet al., 1999
). The high persistent PM concentrations in the NE are due to both entrainment and near-surface convergence (Fast and Zhong, 1998
); these authors make a point that is interesting to emphasize regarding comparisons of health effects between MC and other midlatitude polluted cities: ozone exceedences in MC occur anytime during the entire year regardless of the season because of its subtropical latitude and high altitude. In MC, chlorotic banding and mottling of 2 pine species native to the area have been well documented in the past, and have persisted up to the present time. The chlorotic banding likely represents a response to periodic random acute or episodic exposure to oxidants, while the mottling is considered a response to chronic oxidative stress (de Bauer and Krupa, 1990
).
Cuernavaca is the capital city in the state of Morelos. It extends over 31 km2 and is located 60 km south, downwind from MC, at 1154 m above sea level. It has a temperate climate, an average year-round temperature of 1020°C, and receives 1200 mm of rain annually. Its 316,782 inhabitants are mostly dedicated to service, business, industry, and commerce sectors. Twenty-four percent of the 4086 industries in the state of Morelos are located in Cuernavaca; air pollution is related to vehicular traffic and the generation of PM by the cement and brick industries in town, as well as the open field burning of garbage. A clear oxidant-induced vegetation injury gradient has been established between MC and Cuernavaca (de Bauer and Krupa, 1990). Tlaxcala is the capital city in the state of Tlaxcala, located 114 km east of MC at 2252 m above sea level. It has a temperate climate, an average year-round temperature of 16°C and receives 700 mm of rain annually. It has 63,423 inhabitants and various industries including textile, plastics, foods, and drinks. Tuxpam is a small port on the Gulf of Mexico, 390 km northeast of MC, 14 m above sea level. It has a tropical climate with average temperatures in the range of 2436°C, and a relative humidity above 70%. Its 127,622 inhabitants are dedicated to commerce, service, fishing, and business sectors.
Atmospheric pollutant data.
For the purpose of this work, MC's atmospheric pollutant data was obtained from 2 representative pollutant monitoring stations located in the SW and NE areas, and from the available literature. In the case of the less polluted cities, data were obtained from the Subsecretaria de Ecologia. The data are representative of the air pollution patterns corresponding to the collection period of the dogs (19972000).
Necropsy and tissue preparation.
Animals were examined by a veterinarian before they were euthanized. A brief clinical exam included cardiac and lung fields auscultation, and abdominal and peripheral lymph node palpation. A gross external description was done after their demise, with special focus on age and nutritional status. The animals were weighed and a restricted autopsy was done. Immediately after death, the trachea, extrapulmonary bronchi and lungs, along with the heart, were excised intact from the thoracic cavity. The abdominal cavity was opened and examined; samples of liver, spleen, and kidneys were obtained, and immediately immersed in 10% neutral formaldehyde. The cardiorespiratory block was transported on ice to the laboratory where it was inspected for gross lesions and photographed when necessary. In a group of 50 animals (M, n = 43; Tuxpam, n = 7), a bronchoalveolar lavage (BAL) was performed immediately upon arrival at the laboratory (average 2 h after death). The lungs were filled by intratracheal instillation of warm (37°C) calcium and magnesium-free Dulbecco's phosphate buffered saline (Sigma Chemical Co., St. Louis, MO), at a ratio of 35 ml/kg body weight. The saline was aspirated and re-injected twice more, and the BAL fluid was recovered in cold centrifuge tubes. BAL fluid was centrifuged at 350 g for 15 min at 4°C. The BAL procedure in these animals was followed by formaldehyde fixation. The lungs were inflated via the trachea with 10% neutral formaldehyde at a pressure of 25 cm H2O of a column of fixative solution. The pressure was maintained for a minimum of 4 h following which the trachea was ligated and the lungs were immersed in 10% neutral formaldehyde solution an average of 1 week prior to taking sections. In a group of 60 dogs from the 4 selected cities, electron microscopic samples were taken from unlavaged right lungs in the fresh state. A fragment of the caudal right lobe was cut in small pieces, immersed in glutaraldehyde (2.5%), embedded in plastic, and conventionally processed for electron microscopy. A 0.1 M cacodylate buffer was used in the tissue processing. For light microscopy (LM), blocks of tissue 2 x 2 x 1 cm were taken from trachea, carina, right and left main bronchi, and right caudal and cranial lobes. Peribronchial and peritracheal lymph nodes were macroscopically described and sections for LM taken. Paraffin sections 5 µm thick were cut and routinely stained with hematoxylin and eosin. Special stains included: Masson's trichrome for collagen, Prussian blue for the detection of Fe3+, periodic acid-Schiff and luxol fast blue (PAS/LFB) for the detection of polysaccharides and mucosubstances containing hexoses or deoxyhexoses, and elastic Verhoeff stain. The histological parameters evaluated in each section included: tracheal and bronchial epithelial and smooth muscle hyperplasia, terminal and respiratory bronchiolar epithelial hyperplasia, bronchiolar smooth muscle hyperplasia, dilatation of terminal and respiratory bronchioles, alveolar macrophage hyperplasia, peribronchiolar fibrosis, septal thickening, microthrombi, PMN margination in capillaries and venules, and peribronchiolar and perivascular chronic inflammatory infiltrates. Sections were scored from 03; absent corresponded to 0, and the most severe findings to 3. For the microthrombi we evaluated 0 negative and 1 positive finding. All sections were read blindly by experienced observers.
BAL cell pellet studies.
Cell viability was assessed by the trypan blue exclusion method and the propidium iodide (PI) exclusion assay. Fifty microliters of the cell pellet suspension were taken to perform a cell count with differential and trypan blue dye exclusion. An aliquot of 100 µl was used to test viability with the PI assay. 200 µl was used for DNA cell cycle analysis in an EPICS Profile II Coulter flow cytometer and a 200 µl sample was used for electron microscopic studies.
BAL DNA cell cycle analysis.
The PI method was used for DNA cell cycle analysis (Darzynkiewicz et al., 1994). Processed samples were analyzed using the Multicycle analysis software, a cell cycle analysis program that is based on a procedure of curve fitting that separates the Go/G1, S, and G2/M phases of a cell cycle in one DNA histogram. The DNA synthesis phase (%S or the portion of the cell cycle in which DNA is replicated) was determined for each BAL sample. The cell cycle analysis model used was a zero-order S phase with sliced nuclei debris correction, which results in increased reproducibility (Bergers et al., 1997
).
BAL differential cytology.
Cells obtained by lavage (1 x 104 ) were sedimented on glass slides using a cytocentrifuge (Cytospin-3, Shandon, Pittsburg, PA) at 350 g for 8 min. The slides were stained with Papanicolau stain (Sigma Chemical Co., St. Louis, MO). Differential counts for M, white blood cells (WBC), and epithelial cells were determined by microscopic examination (Mayer et al., 1990
).
Computarized axial tomography of lungs.
Computed tomography (CT) was done in isolated inflation-fixed, air dried, postmortem lungs of MMC dogs (n: 11), including n: 5 from SW and n: 6 from NE. The lungs were visualized in toto and then sectioned along the plane of the CT image. The CT findings were correlated with histological changes in these sets of lungs.
Statistics.
Statistical analyses were performed using the Instat program (Graph Pad, San Diego, CA). The following statistical procedures were used: (1) one-way ANOVA. The Tukey-Kramer multiple comparisons test was used to establish the differences in values of synthesis phase (%S), and the % viabilities for BAL cells for control vs. MC dogs. (2) Pearson correlation was used to assess the strength of association between the lung perivascular mononuclear cell infiltrates, and the bronchiolar epithelial and smooth muscle cell hyperplasia. (3) Unpaired t-test was used to compare the differences in the numbers of cells per ml in BAL, and the significance of the histological endpoints among the different geographical locations. Data are expressed as mean values ± SD. Significance was assumed at p < 0.05.
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RESULTS |
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BAL viability and cell numbers.
There were no differences in cell viability between SW and NE dogs (92.8 ± 8 vs. 92.5 ± 6.6) and the control dogs (93.5 ± 4.1). MC dogs had 27 ± 3.4 x 106 cells per ml of lavage fluid, while control dogs had 13.4 ± 5.2 x 10 6 cells (p < 0.0001).
DNA cell cycle analysis.
We analyzed all BAL samples available with a CV Go/G1 peak < 5. The average of % S phase cells was defined as the average S-phase value of a DNA-diploid cell cycle. A significant difference was seen in the % S of alveolar macrophages between lower and higher polluted cities (p < 0.0001). Dogs from the lower polluted city (Tuxpam) had an S% value of 4.9 ± 4.4, while SW and NEMMC dogs had similar values of 22.5 ± 4.5 and 22.06 ± 7.3 respectively.
Clinical and gross pathological observations.
All the animals in this study were apparently healthy without evidence of overt respiratory or cardiovascular disease. Approximately 16% of animals included in each geographical location had a low weight for size, without evidence of severe malnutrition or clinical sickness. There were no overweight dogs in this study.
Gross morphology of the lungs.
No gross abnormalities were seen in the lungs of animals from Tuxpam. Lungs from Tlaxcala and Cuernavaca displayed discrete areas of pleural thickening and anthracotic macules. Lungs from MC dogs displayed patchy whitish pleural areas with prominent anthracotic macules. These pleural changes were seen in every lobe and had no apparent relationship with the ribs or the lung apices. The changes were particularly prominent in older dogs. Enlarged, congested, anthracotic pulmonary hiliar lymph nodes were observed in all MC dogs and in NE dogs, lymph nodes were larger and completely replaced by black pigmentation. No evidence of intercurrent lung disease was seen in any of the animals.
Lung histopathology.
The normal histologic and ultrastructural morphology of the dog lung has been described by several investigators (Pinkerton et al., 2001; Plopper and Hyde, 1992
; Takenaka et al., 1998
). A summary of relevant lung findings is seen in Tables 2 and 3
. Relevant findings were confined to pleura, terminal and respiratory bronchioles, centriacinar regions, and lung parenchymal blood vessels.
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Conducting airways.
In MC and Cuernavaca dogs, trachea and bronchi exhibited focal ciliated cell loss, patchy areas with basal and goblet cell hyperplasia, and scattered epithelial and submucosal mononuclear cell infiltration. Submucosal fibrosis was present in MC dogs, while a moderate increase in the smooth muscle layer around large bronchi was prominent in NE dogs and in a small number of older SW dogs.
Terminal and respiratory bronchioles.
A predominant finding in MC and Cuernavaca dogs was respiratory bronchiolar epithelial hyperplasia. The hyperplastic bronchiolar lesions consisted of either diffuse epithelial cell proliferation or focal micronodular epithelial projections (Figs. 4A and 4B). The hyperplasia was predominantly composed of nonciliated Clara cells (Fig. 4C
). In the samples with focal hyperplasia there was a close relationship between the epithelial changes and the presence of macrophages laden with PM (Fig. 4B
). Neovascularization was prominent in terminal bronchioles from MC dogs and to lesser extent Cuernavaca dogs (Fig. 3B
). Smooth muscle hyperplasia was salient in terminal and respiratory bronchioles of SWMMC, NEMMC, and Cuernavaca dogs (Figs. 4A4C
). Chronic mononuclear cell infiltrates along with macrophages loaded with PM were commonly surrounding the bronchiolar walls and extending into adjacent vascular structures (Fig. 4B
). Patchy proximal acinar air space enlargement was centered on respiratory bronchioles and alveolar ducts, and was accompanied by an apparent loss of interalveolar septae and emphysematous-like irregular areas. It was more frequent in NE dogs regardless of age (p < 0.01).
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Alveolar type I cells.
An increased luminal and abluminal pinocytotic activity in type I alveolar cells was noticed in MC and Cuernavaca dogs. Ultrafine electron-dense particles were present in association with the pinocytotic vesicles (both on the luminal and abluminal sides), in filopodia engulfing the particles, and free in the cytosol (Figs. 5D and 5E).
Septal fibrotic reaction.
Septal focal fibrosis was present mostly in MC dogs, but to a lesser extent also in Cuernavaca dogs. It was mostly associated with alveolar type II and macrophage hyperplasia, and was more prominent in older dogs. Trichrome staining revealed fibroconnective tissue in alveolar septae; these findings were confirmed by electron microscopy (Figs. 5B and 5C). An increase in the numbers of cells displaying metachromatic granules was seen in association with the fibrotic areas stained with toluidine blue. EM showed that these cells corresponded to partially degranulated mast interstitial cells. MC dogs displayed numerous interstitial macrophages with cytoplasmic processes surrounding clusters of PM (Fig. 5F
). PM in interstitial M
was a striking finding in NEMMC dogs, the internalized condensed ultrafine particles were mostly free in the cytoplasm (Fig. 5F
). Foci of osseous metaplasia were seen in 2 NE and 1 Cuernavaca dogs.
Inflammation.
A striking finding in MC and to lesser degree in Cuernavaca dogs, was the presence of moderate to marked infiltration by mononuclear cells of vascular walls and perivascular spaces of pulmonary veins and arterioles. Mononuclear cells were seen along M laden with coarse PM. Chronic inflammatory cells were present primarily around terminal and respiratory bronchioles and in adjacent vascular structures (Figs. 4B and 4D
). Low-grade focal pneumonitis was confined to end-airway structures. Aggregates of AM, scattered PMN, and fibrinocellular debris were seen within a few alveoli.
Vascular changes.
Alongside the scattered infiltration of small pulmonary veins and arterioles by mononuclear cells and macrophages (SW, 83%; NE, 78 %; Cuernavaca, 36%), MC dogs displayed platelet micro-thrombi, and PMN endothelial margination, particularly prominent in septal capillaries and venules (Figs. 4D4F). The changes were particularly striking in SW dogs (Fig. 4F
). EM showed the presence in septal capillary lumen of PMN with a decreased amount of intracytoplasmic granules (Fig. 6D
). Endothelial cells displayed numerous pinocytic vesicles both in the luminal and abluminal surfaces. Ultrafine PM was present both in relation with the pinocytic vesicles as well as free in the cytosol. The endothelial cells send multiple thin projections of cytoplasm into the lumen (anemone-like), as well as larger amounts of cytoplasm with striking pinocytic activity (Figs. 5E and 6C
). Interestingly, the anemone-like cytoplasmic projections mostly originated at the zonula occludentes (Fig. 6A
). Large macrophage-like cells with irregular shape, abundant cytoplasm containing lysosomal bodies, and free ultrafine PM in the cytosol displayed extensive areas of interaction with the capillary endothelium (Figs. 5E, 6B, and 6C
). No fused membranes were seen between the macrophage-like cells and the endothelial surface, however membrane adhesive complexes were present, with intercellular separations between 1214 nm (Fig. 5E
). The macrophage-like cells were seen on the thick side of the alveolar septum. Endothelial cell junctions were intact.
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Postmortem CT.
CT scanning was performed on formaldehyde-fixed lungs. Formaldehyde's density is nearly the same density as the lung parenchyma by CT, limiting the evaluation to the presence or absence of hyperdense nodules. Small hyperdense areas were seen in the lung parenchyma, and subpleural regions. Histologically, the hyperdense areas corresponded to conglomerates of densely packed macrophages loaded with gold-brown and coal-like particles.
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DISCUSSION |
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Pulmonary vascular endothelial and epithelial cells are crucial barriers that keep lung tissue functional and play a dynamic role in the lung inflammatory responses (Castranova et al., 1988; Driscoll et al.,1997
; Majno and Joris, 1996
; Ryan, 1986
; Saffiotti, 1996
; Simon and Paine, 1995
; Ward and Hunninghake, 1998
). Damage of the capillary endothelial cells and type I alveolar cells are the earliest changes of lung toxicity by diesel exhaust particulates; these cell injuries lead to alveolar edema and a subsequent inflammatory response (Ichinose et al., 1995
). Endothelial damage is a main feature of acute lung injury and in microangiopathies where the endothelial injury is accompanied by the production of cytokines such as IL-10, IL-12, tumor necrosis factor (TNF-
) and interferon-
, in turn affecting the expression of adhesion molecules and chemokines (IL-8) (Kawanami et al., 1995
; McGuirre et al., 1981; Vaillant et al., 1996
). Impairment of pulmonary vessel's structure and function has been described in patients with mild chronic obstructive pulmonary disease (COPD), suggesting the potential involvement of an inflammatory process in the pathogenesis of pulmonary vascular abnormalities in the early stage of COPD (Peinado et al., 1999
). Our findings of vascular pulmonary and myocardial prominent PMN margination and microthrombi formation, as well as fibrin vascular deposition and breakdown of RBC could be related to endothelial dysfunction in these highly exposed dogs (Bevilacqua and Gimbrone, 1987
; Chen and Manning, 1995
; Sibille and Reynolds, 1990
; Zomas et al., 1998
). Activated endothelium induces the presence of pro- and anticoagulant factors, and expression of a variety of adhesion molecules (Bombeli et al., 1997
; Krishnaswamy et al., 1999
; Ward and Hunninghake, 1998
). Circulating PMN adherent to endothelial cells can damage these endothelial cells and produce the capillary leak that is central to the evolution of a systemic inflammatory response to multiple organ failure (Chen and Christou, 1998
). Evidence of lung capillary leakage is present in MC dogs; RBC are seen in alveolar spaces and in BAL macrophages.
A striking finding in the lung capillaries of MC and Cuernavaca dogs was the presence of partially degranulated PMN attached to the endothelial walls. Leukocytes within the circulation are in dynamic equilibrium with a marginated pool, thought to reside mainly within the pulmonary capillaries (Downey et al., 1990). Environmental factors such as inhalation of cigarette smoke delay and activate PMN traveling through the lung capillaries (Brown et al., 1995
; Hogg, 1994
). As in the rat model of cigarette smoke, where electron microscopy shows lung capillary damage with adherent PMN, in canines PMN could also contribute to the alveolar wall damage (Terashima et al., 1999
). The result of capillary leukocyte sequestration appears to be impaired alveolar capillary perfusion (Kuebler et al., 2000
).
In the model of alveolar fibrosis and capillary alteration in rat silicosis (Kawanami et al., 1995), peribronchiolar vessels were suggested as likely sources for renewal of damaged alveolar capillary endothelium, an observation that coincides with the neovascularization in terminal bronchioles in MC canines. In sharp contrast with the histopathological picture of acute lung injury, where early in its course large numbers of PMN are sequestered in and then migrate from the pulmonary capillaries towards the alveolar spaces, in our material we were unable to visualize the migration of these adherent PMN into the alveolar space, and further the numbers of PMN in BAL were low. Thus, migration from the lung capillaries to the alveolar spaces is not taking place in these chronically exposed dogs, an observation that coincides with the increased numbers of macrophages, but not PMN, in the sputum-induced samples obtained from healthy children and adults living in MC (L. Calderón-Garcidueñas, personal observation).
The EM findings in the surfactant profiles of type II alveolar cells are also important to point out, since injury to type II cells can alter their ability to synthesize, secrete, and recycle surfactant (Griese, 1999; Simon and Paine, 1995
). Differentiation of alveolar type II to I cells implies that type II cells lose their apical microvilli and surfactant-containing lamellar bodies (Smith, 1983
). Further, during cell division many of the surfactant-containing lamellar bodies are also lost (Smith, 1983
). In relation to surfactant we observed alveolar macrophages with numerous phagocytized lamellar-like bodies, suggesting that ingestion of potentially recyclable surfactant by macrophages could contribute to the slow loss of surfactant lipids from the recycling pool, as pointed out by Miles et al. (1985).
The accumulation of PM in lymph nodes draining conducting airways is also worth comment (Adamson and Prieditis, 1998). PM is in contact with highly proliferating lymphoid cells and potential mutagenic materials are likely present. There has been a rise in the incidence of non-Hodgkins lymphomas in Europe, Canada, the U. S., and Mexico; workers in wood, petroleum, solvent, plastic, and ceramic industries have an excess risk (Cartwright et al., 1999
; Mohar et al., 1997
; Scherr et al., 1992
). Relevant to this work in canines, the highest incidence of malignant neoplasms in MC corresponded to boys living in the southern areas of metropolitan MC (Fajardo-Gutierrez et al., 1997
). Lastly, cell proliferation in BAL cells, as determined by the percentage of cells in S phase was
22% in MC dogs vs.
4.9% in dogs from less polluted locations, suggesting that the marked increase in the numbers of alveolar macrophages is due to in situ cell division (Spurzem et al., 1987
). These marked increments in macrophage cell proliferation are further evidence of an ongoing pulmonary injury (Evans and Shami, 1989
).
In summary, dogs naturally exposed to PM and ozone, as well as other ambient air pollutants, in concentrations at or above the current standards are displaying evidence of lung epithelial and endothelial pathology along with lung remodeling and altered repair with focal fibrosis. Therefore, the studies presented here may be useful to build future hypothesis-driven mechanistic studies to explain the biological plausibility of the epidemiological data suggesting increased cardiorespiratory morbidity and mortality in susceptible populations, and to gain a better understanding of the effects of air pollution on the respiratory system in children in both developing and developed countries.
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ACKNOWLEDGMENTS |
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NOTES |
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The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U. S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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