ToxSci Advance Access originally published online on May 12, 2006
Toxicological Sciences 2006 92(2):545-559; doi:10.1093/toxsci/kfl016
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Modulators of Cigarette Smoke–Induced Pulmonary Emphysema in A/J Mice
Lovelace Respiratory Research Institute, Albuquerque, New Mexico 87108
1 To whom correspondence should be addressed at Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive, SE Albuquerque, NM 87108. Fax: (505) 348-8567. E-mail: tmarch{at}lrri.org.
Received March 15, 2006; accepted May 10, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice develop pulmonary emphysema after chronic exposure to cigarette smoke (CS). In this study, the influence of gender, exposure duration, and concentration of CS on emphysema, pulmonary function, inflammation, markers of toxicity, and matrix metalloproteinase (MMP) activity was examined in A/J mice. Mice were exposed to CS at either 100 or 250 mg total particulate material/m3 (CS-100 or CS-250, respectively) for 10, 16, or 22 weeks. Evidence of emphysema was first seen in female mice after 10 weeks of exposure to CS-250, while male mice did not develop emphysema until 16 weeks. Female mice exposed to CS-100 did not have emphysema until 16 weeks, suggesting that disease development depends on the concentration and duration of exposure. Airflow obstruction and increased pulmonary compliance were observed in mice exposed to CS-250 for 22 weeks. Decreased elasticity was likely the major contributor to airflow obstruction because substantial remodeling of the conducting airways, beyond mild mucous cell hyperplasia, was lacking. Exposure to CS increased the number of macrophages, neutrophils, lymphocytes (B cells and activated CD4- and CD8-positive T cells), and activity of MMP-2 and -9 in the bronchoalveolar lavage fluid (BALF). Treatment with antioxidants N-acetylcysteine or epigallocatechin gallate (EGCG) did not decrease emphysema severity, but EGCG slightly decreased BALF inflammatory cell numbers and lactate dehydrogenase activity. Inflammation and emphysema persisted after a 17-week recovery period following exposure to CS-250 for 22 weeks. The similarities of this model to the human disease make it promising for studying disease pathogenesis and assessing new therapeutic interventions.
Key Words: A/J mice; animal models; cigarette smoke; pulmonary emphysema; morphometry; pulmonary function.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chronic obstructive pulmonary disease (COPD) is currently the fourth leading killer of adults in the United States and will likely be the third leading cause of death worldwide by 2020 (Viegi et al., 2001
). The disease is insidious with most patients presenting with symptoms only after irreversible lung damage has occurred, and death often occurs following acute exacerbations (Rodriguez-Roisin, 2000). Cigarette smoking is by far the biggest cause of COPD; however, not all smokers go on to develop clinically significant reduction in lung function. Some studies have suggested that part of this differential susceptibility is due to "dose" of cigarette smoke (CS), as the disease usually manifests with greater severity after many decades of smoking (Cosio and Guerassimov, 1999; Viegi et al., 2001). Other predispositions for the disease, such as genetic differences in extracellular matrix repair and turnover, detoxification, and inflammatory responses, are also being studied.
The incidence of pulmonary emphysema as part of the COPD complex (including chronic bronchitis) is difficult to assess because most COPD patients have both emphysema and bronchitis/bronchiolitis to some degree. Emphysema is primarily a pathologically described inflammatory disease characterized by the destruction of parenchyma distal to the terminal bronchioles without significant fibrosis (Snider, 1986). Palliative treatments include bronchodilators for improving airflow along with corticosteroids and antibiotics for preventing or treating exacerbations (Groneberg and Chung, 2004). Reversal of emphysema has been shown in animal models of the disease by treatment with retinoic acid (Massaro and Massaro, 1997); however, this has not been recapitulated in the clinical environment (Mao et al., 2002). Prevention rather than treatment of emphysema in animals has been attempted more often, and animals have been treated with a variety of anti-inflammatory and antiproteolytic drugs either prior to or concurrently with induction of the disease (Churg et al., 2003; Martorana et al., 2005; Selman et al., 2003). Whether or not such drugs might help patients with moderate to severe airflow obstruction remains to be determined.
Modeling of emphysema in the past used intratracheal instillation of proteolytic enzymes, such as pancreatic and neutrophil-derived elastases, to induce the lesion in a variety of animal species. This proteolytic process leads to destruction of the extracellular matrix and is purportedly similar to that induced by inflammatory responses to CS. This type of model has helped to bolster the hypothesis that emphysema develops from an imbalance in proteinase and antiproteinase activity (Snider, 1986). More recently, models have been produced by chronically exposing animals to CS, and strain differences among mice as well as transgenic animals have provided some insights into the disease pathogenesis (Groneberg and Chung, 2004). The A/J mouse develops emphysema more readily following CS exposure than other strains of mice (March et al., 2005).
The primary purpose of this study was to characterize the time-course for development of emphysema in A/J mice exposed to CS. We postulated that longer duration of CS exposure would be associated with development of more severe disease. A tangential study was also done to determine the disease response after lowering the concentration of CS over the same time-course. Further, because there is some indication that women may be more susceptible to CS-induced COPD (Langhammer et al., 2003; Viegi et al., 2001), the influence of gender on the disease process was evaluated. Other assessed parameters included pulmonary function, inflammation, matrix metalloproteinase (MMP) activity, and development of mucous cell hyperplasia in the conducting airways. The ability of CS from two different types of research cigarettes (filtered vs. nonfiltered) to produce emphysema was also tested. Additionally, the preventive effect of orally administered antioxidants on development of the disease was assessed. Finally, mice were chronically exposed to CS and then held for a "recovery" period in order to assess pulmonary carcinogenesis (Witschi, 2005) and persistence of the emphysema lesion.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals, exposures, and treatments.
All procedures were conducted under protocols approved by an Institutional Animal Care and Use Committee in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. The study design is depicted in Table 1 and represents CS exposures conducted over a 2-year period. Male and female A/J mice (Jackson Laboratories, Bar Harbor, ME) at 8–10 weeks of age were exposed whole body to diluted and slightly aged mainstream CS generated by an AMESA Type 1300 smoking machine from 1R3 nonfiltered research cigarettes (27.1 mg total particulate material [TPM] yield per cigarette; University of Kentucky Tobacco Research and Development Center [UKTRDC], Lexington, KY) or to filtered air (FA, control) as described (March et al., 2005
). Mice were acclimated to exposure during the first week by delivering CS targeted at 100 mg TPM/m3 (CS-100) and exposed following acclimation at a concentration of 250 mg TPM/m3 (CS-250) for up to 22 weeks. A group of male mice was exposed to FA or CS-100 during acclimation and to CS-250 for a total of 22 weeks of exposure and then held unexposed for an additional 17 weeks during a recovery period (housed in shoe box cages) in a protocol designed primarily to assess carcinogenicity (Witschi, 2005). Mice in this study were used for assessment of both emphysema and the carcinogenicity of CS and, thus, to allow a direct comparison to previous studies (Witschi, 2005), only males were used.
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Only female mice were used in additional experimental groups because preliminary data has suggested that female mice of different strains might be more susceptible to CS-induced emphysema (March et al., 2005
). One group of female mice was acclimated to CS exposure at a low concentration of 50 mg TPM/m3 during the first week and then further exposed to CS-100 for 10, 16, or 22 weeks. Two additional groups of female mice, one exposed to CS-250 and the other exposed to FA for 16 weeks, were treated with the green tea–derived polyphenol antioxidant epigallocatechin gallate (EGCG; Sigma, St Louis, MO) by administration of the compound in the drinking water at 50 µg/ml (estimated dose equivalent to that received by a heavy drinker of green tea; Muto et al., 1999). Lastly, a group of 8- to 10-week-old female A/J mice was exposed for the first week to CS-100 and further for a total of 10 weeks to CS-250 from filtered 2R4F research cigarettes with a nominal yield of 11.7 mg TPM per cigarette (UKTRDC, Lexington, KY). Emphysema in these mice was compared to that in mice exposed to CS-250 from the nonfiltered 1R3 research cigarettes. In addition to monitoring CS TPM, particle sizes were characterized, and the vapor phase of CS atmospheres from each of the two types of cigarettes was compared for concentrations of carbon monoxide, nitrogen oxides, and total hydrocarbons as described (McDonald et al., 2004). A subset of animals exposed to CS-250 from the filtered research cigarettes was treated with the antioxidant thiol N-acetylcysteine (NAC) in the drinking water at 1 mg/ml (estimated 20-fold greater than effective human dose; Pela et al., 1999; van Overveld et al., 2005) concurrent to the exposure (Table 1).
Necropsy, lavage, and tissue analysis.
Lungs from mice designated for morphometry of emphysema were inflated with 4% buffered paraformaldehyde or 10% neutral buffered formalin at a constant hydrostatic pressure of 25 cm for 6 h as described (March et al., 2005). Lungs were fixed further by immersion in fixative for 48–72 h. The volume of fixed whole lungs (VL) was determined, and left lung lobes were trimmed, histoprocessed, sectioned, and stained with hematoxylin and eosin (H&E) as described (March et al., 2005). The amount of tissue shrinkage due to histoprocessing was determined in five randomly selected animals per group as described (March et al., 2005).
Sections of lungs were qualitatively assessed by light microscopy for the severity of emphysema. Emphysema was described as irregular, multifocal expansion of alveolar airspaces and alveolar ducts due to destruction of parenchymal tissue (Snider, 1986). The irregular nature of airspace enlargement was given emphasis in that alveolar septal destruction occurred nonuniformly, and alveoli of normal size were often juxtaposed to greatly expanded airspaces. Morphometry was used to quantify emphysema as described previously (March et al., 2005). Briefly, microscopic fields were chosen by a systematic random method, and emphysema was assessed in these fields with computer-aided measurements. The most commonly used indicator of alveolar airspace size, the mean linear intercept (Lm, in µm), was calculated. The Lm was corrected for histoprocessing shrinkage by dividing it by the group-average linear shrinkage factor. Using the VL and Lm, the total alveolar surface area (Sa, in cm2) was calculated. From point counting, the fractional volume densities of alveolar septa (VVspt) and alveolar airspace (VVair) were also measured and multiplied by VL to yield total volume of a particular component for the lung. The ratio of Sa to VL was also evaluated. Additionally, in a randomly selected subset of female mice from the 16-week time point exposed to FA or CS-250 (Table 1), sections of 5 µm thickness from the paraffin blocks described above were stained with alcian blue (at pH = 2.5 for acid mucosubstances) in combination with periodic acid-Schiff (AB-PAS). The volume density of epithelial mucosubstances was then determined (March et al., 1999) in cross-sections of the axial airway within the two largest lung sections (comprising the airway near the hilus).
In separate groups of animals (Table 1), mice were killed, tracheas cannulated, left main stem bronchi temporarily clamped, and right lungs lavaged three times with 0.5 ml phosphate-buffered saline (PBS). The bronchoalveolar lavage fluid (BALF) was assessed for the number of inflammatory cells and the activity of MMPs assessed in the noncellular supernatant fraction as described (March et al., 2002; Seagrave et al., 2004). Lavage fluid was also analyzed as described (March et al., 2002) for amount of total protein as well as the activities of lactate dehydrogenase (LDH) and alkaline phosphatase (AP) as indicators of injury. In another group of mice, whole lungs were lavaged three times with 0.7 ml of PBS, and whole-lung tissue was enzyme digested as described (Wilder et al., 2001) with the modification that tissue was treated with a proteinase mixture (Liberase Blendzyme 3, Roche Applied Science, Indianapolis, IN) at 0.28 mg/ml in 5% FBS/RPMI media and with DNAse (Sigma, 30 µg/ml) for 60 min. Total cells from BALF (0.2 x 106 to 0.6 x 106 per sample) and digested tissue (1 x 106 per sample) were counted with a hemacytometer and then incubated for 10 min on ice with FcBlock (rat anti-mouse CD16/CD32) and subsequently immunostained for subsets of activated T lymphocytes using CD4-FITC (GK1.5) and CD8-PerCP (Ly2) in combination with either CD62L-PE (phycoerythrin) (Mel-14) and CD25-APC (allophycocyanin) (PC61) or CD69-PE (H1.253) and CD44-APC (PGP-1, Ly24). Antibody CD45R/B220-PerCP (RA3-6B2) was used to stain B lymphocytes in a separate tube. All antibodies were purchased from BD Biosciences (San Diego, CA), and their appropriately labeled rat or hamster (CD69-PE only) immunoglobulin isotype controls were used to stain pools of BALF and lung cells. Data were collected on a BD Biosciences FACSCalibur and analyzed using CellQuest Pro software. Gates were set to enumerate small mononuclear leukocytes and to distinguish those cells expressing fluorescence above that observed from isotype control–stained cells. Data were reported as the absolute number of specifically stained cells in a given sample.
Pulmonary function testing.
Pulmonary function testing was performed on mice after 22 weeks of exposure to CS-250 essentially as described (Harkema et al., 1996). The number of surviving female mice originally designated for pulmonary function tests was few, such that statistically powerful results from females were precluded. Thus, male mice were anesthetized with an ip injection of xylazine and ketamine (0.01 and 0.08 mg/kg, respectively), orotracheally intubated with a 22-gauge catheter, and maintained under inhalation anesthesia at a baseline respiration frequency of 110–120 breaths/min with 1.5–2.5% halothane in a 50:50 oxygen:nitrous oxide mix. Mice were placed in a heated plethysmograph box (approximately 500 ml volume). Transpulmonary pressure (Ptp) was measured with a fluid-filled 22-gauge esophageal cannula connected to a differential pressure transducer (MPX11DP, Motorola, Phoenix, AZ). Respiratory flows were measured with a pneumotachograph composed of a differential pressure transducer (MP45, Validyne, Northridge, CA) connected across nine layers of stainless steel, 316-mesh wire cloth covering a 0.7-cm-diameter hole in the plethysmograph wall.
The Ptp and flow signals were conditioned by preamplifiers (Buxco Electronics, Sharon, CT) and routed to a PC-based monitor and data collection system (AcqKnowledge 3.7.2, BioPac Systems, Inc, Goleta, CA). Once stable, the values for flow, volume, and frequency during spontaneous breathing were measured, and tidal volume (VT), minute volume (VE), total pulmonary resistance (RL), and dynamic lung compliance (Cdyn) were calculated. Static lung compliance (Cstat) was measured by observing the volume needed to inflate lungs to Ptp = 20 cm H2O. The diffusing capacity for carbon monoxide (DLCO) was then measured by a modified single-breath method as described (Harkema et al., 1996). Following this, mice were injected with 70 µg/kg vecuronium bromide (Ben Venue Labs, Inc, Bedford, OH) ip to arrest spontaneous breathing. A quasistatic deflation from total lung capacity to residual volume was performed by inflating the lung to a pressure of + 30 cm H2O and deflating slowly until cessation of flow occurred. Inspiratory capacity, vital capacity, and the expiratory reserve volume were determined. Quasistatic chord compliance (Cqs) was calculated from slopes of the steepest portion of the volume versus Ptp curves, covering a pressure range of 3.4–8.0 cm H2O (see the "Results" section). A forced expiration maneuver was done by inflating the lung to + 30 cm H2O and deflating to approximately – 45 cm H2O. Forced vital capacity (FVC) and peak expiratory flow rate (PEFR) were calculated, and volumes and rates at various time points were related to the FVC (i.e., forced expiratory volume at 0.05 s [FEV0.05] as a percentage of FVC). Two or three forced maneuvers were made per mouse, and the average values were reported. Mice were euthanized following the procedure.
Statistical analysis.
Several comparisons were made by the general linear models ANOVA procedure (SAS Institute, Inc, Cary, NC) often using three independent variables (e.g., exposure, gender, and duration). Data were tested for normal distribution (Kolmogorov-Smirnov test) and equal variance (Levene's median test or two-sample F-test). Where the tests failed (p < 0.05), data were transformed by ranking prior to ANOVA. Interactions of the independent variables were of particular interest. For example, the effect of an interaction between exposure and duration might have suggested progressive worsening of the disease process over time. Pairwise comparisons between individual groups, post hoc to ANOVA, were performed using a least square means procedure with the Tukey-Kramer adjustment for multiple comparisons, and the significance level was set at p < 0.05. In other instances, end points from two groups (FA control and CS exposed, generally) were compared by Student's two-tailed unpaired t-test or the nonparametric Mann-Whitney rank sum test with statistical significance set at p < 0.05.
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RESULTS |
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Histopathology and Morphometry
Effect of gender, exposure duration, and CS concentration.
Comparisons were made between male and female mice exposed either to FA or to CS-250 for 10, 16, or 22 weeks. Similar to previous results (March et al., 2002
, 2005), CS exposure caused mild to moderate, irregular, multifocal enlargement of airspaces throughout the parenchyma, such that alveoli of normal size were often juxtaposed to large, expanded airspaces (Fig. 1). The organization of parenchymal acini was often disrupted or unrecognizable. Female mice exposed to CS-250 had features of irregular alveolar airspace enlargement at 10 weeks while effects in male mice were not readily apparent until 16 weeks (Fig. 1). Female mice exposed to CS-100 did not have substantial airspace enlargement at the 10-week time point, but at 16 and 22 weeks, emphysema in these mice was morphologically similar to that of female mice exposed to CS-250 (Fig. 1).
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An increase in the Lm was regarded as the main morphometric indicator of emphysema because it is the best estimate of alveolar size in this type of morphometry. For the CS-250 exposure, ANOVA demonstrated that the Lm was affected by an interaction between the independent variables of exposure (FA vs. CS-250), gender, and duration of exposure (10–22 weeks; Table 2). Pairwise tests demonstrated that the CS-induced increase in Lm was significant only in females and was different from FA control values at each of the time points. In contrast, CS-250 caused an increase in Lm in the lungs from male mice only at the 16- and 22-week time points, but these increases were not significant by pairwise tests. This suggested that the response was more robust in females, in parallel to the histopathologic findings (Fig. 1). Age-related enlargement of alveoli was seen in FA- and CS-250–exposed mice of both genders. The significant effect of an interaction between duration and exposure in females suggested that alveolar enlargement was different than expected from either aging or CS exposure alone. However, the severity of the emphysema in the females did not clearly worsen during the study period because the CS-induced increase in Lm over mean FA control values was similar at 10, 16, and 22 weeks (24, 25, and 22% enlargement, respectively).
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Other morphometric measures of the parenchyma included the total volume of alveolar septa (VVspt x VL), alveolar airspace (VVair x VL), and alveolar surface area (Sa). As seen in previous experiments (March et al., 2002
, 2005), CS-250 caused a significant increase in the VVair x VL of approximately 1.2-fold in males and 1.3-fold in females when averaged over the entire exposure period (Table 2). The magnitude of increase in VVair x VL was similar to the CS-induced increase in VL. There were also increases in the volume of alveolar septa (VVspt x VL) associated with CS exposure, and in males this increase was similar to the increase in VL (1.2-fold, averaged over the whole exposure period). The VVspt x VL in females increased only slightly (1.1-fold) consistent with the enlargement of VL being primarily due to increased parenchymal airspace rather than an increase in septal tissue. Similarly, there was a significant effect of exposure on the total alveolar surface area (Sa), attributable primarily to the increased Sa in the CS-exposed males at the 10-week time point (Table 2). Differences in Sa were not substantial at the other time points in either gender and in fact were not apparent in the females at the 16- and 22-week time points. The ratio of alveolar surface area to lung volume (Sa:VL) was assessed as an indicator of tissue loss per unit volume of lung. The ratio was decreased at all time points for the CS-exposed females, but only significantly at the 10-week time point (Table 2). The ratio for the males was decreased to a lesser degree (16 and 22 weeks, only), but the decreases were not significant. Other morphometric parameters in the gender-duration experiment, including lung weight (LW) and body weight (BW), were also affected by CS-250 exposure, gender, and duration but without significant interaction among the variables (Table 2). The CS-250 exposure increased LW and decreased BW, while gender and duration influences were as expected for male and female mice aging over a 3-month period.
Morphometry of lungs from mice that were exposed to CS-100 for 10 to 22 weeks revealed responses intermediate to those elicited by exposure at the high concentration of CS-250. Significant enlargement of Lm in the CS-100 group was demonstrated, and similar to the histopathologic findings (Fig. 1), this was apparent after 16 or 22 weeks of exposure, but not after 10 weeks (Fig. 2A). There was no interaction of the CS exposure with duration. Instead, Lm averaged over the entire period in the CS-100–exposed mice was significantly greater than that of the FA control group and significantly less than that induced by exposure to CS-250 (p < 0.05, least square means test). This was reiterated by VL enlargement (Fig. 2B). The ratio of Sa:VL was also significantly decreased by CS exposure at both concentrations, but there was no difference for this parameter between the two CS exposure levels (Fig. 2C). The VVair x VL was not increased significantly by CS-100 in contrast to the increase apparent in the CS-250 group (Fig. 2D). The VVspt x VL was not affected by CS exposure but was affected by duration with the average value at 16 weeks lower than at the 22-week time point (Fig. 2E). Significant CS-induced decreases in BW followed a pattern seen with the Lm; mice exposed to CS-100 had mean weights that were between those of the FA- and CS-250–exposed groups (Fig. 2F). In summary, exposure to CS-100 was associated with intermediate enlargement of Lm and VL (11% greater than FA for both parameters over the entire period) compared to that induced by CS-250 (23 and 26% greater than FA, respectively), suggesting induction of less severe emphysema.
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Histopathologic inflammation and mucus production.
Mild to moderate pulmonary inflammation was present in all CS-exposed mice and was characterized by exudates of macrophages and neutrophils among cell debris in scattered alveolar lumina (Fig. 3). Inflammatory cells were numerous in airspaces near broncholoalveolar duct junctions. Alveolar septa were sometimes thickened by infiltrates of similar cells along with other mononuclear leukocytes. Infiltrates of macrophages, lymphocytes, and fewer neutrophils were common in the perivascular interstitium; thin-walled venules deep in the parenchyma were often affected (Fig. 3). Smaller numbers of mononuclear leukocytes and rare neutrophils were often present in the interstitium around terminal bronchioles and associated arterioles and lymphatic vessels. Larger airways were unaffected or only slightly affected by inflammation; however, the number of cells containing mucus was increased in the lining epithelium of sections stained with AB-PAS (Fig. 4). These cells also had more abundant amounts of mucus per cell. Morphometry revealed a fivefold increase in the volume density of AB-PAS–positive mucosubstances in CS-exposed mice over FA controls (Fig. 4).
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Effect of a recovery period.
The effect of a recovery period on emphysema was assessed in male mice exposed to CS-250 for 22 weeks and then held without exposure for an additional 17 weeks. One purpose of this experiment was to determine the carcinogenicity of mainstream CS in the A/J mouse in a protocol similar to that established previously (Witschi, 2005
). Mice exposed to FA for 22 weeks then held for 17 weeks had a 17% incidence (five mice of n = 29 survivors) of lung tumors characterized as adenomas (photomicrographs not shown), while those exposed to CS-250 for 22 weeks and held for a 17-week recovery had a tumor incidence of 20% (six of n = 30; p = 1.0, Fisher's exact test, comparison to FA). The lungs of one mouse in the FA group had two tumors while the remaining four mice with lung tumors in the group each had one. Mice with lung tumors in the CS-250 group each had only one tumor. Tumor incidence in this experiment was low for control animals, but multiplicity (number of tumors per lung) was similar in comparison to previous work (Witschi, 2005). Histopathologically, irregular airspace enlargement, alveolar septal destruction, and inflammation were present in lungs from the CS-250 plus recovery group, and the severity of the emphysema was similar to that of mice exposed to CS-250 for 22 weeks alone (Fig. 5). In corroboration of the histopathology (Fig. 5), morphometry demonstrated a significant increase in the Lm in mice held for recovery (Table 3). The increase was paralleled by significant increases in the VL and the VVair x VL of approximately 1.2-fold compared to the FA-exposed control group. These fold increases were similar to those in CS-250–exposed males at the 22-week time point (Table 2). Unlike the measures in males at the 22-week time point, the VVspt x VL and Sa were not increased, and the Sa:VL was significantly decreased. Another notable exception was that the mean BW of mice formerly exposed to CS-250 was no different from that of the FA group (Table 3). Thus, the recovery period allowed regains in BW, while restoration of the damaged pulmonary parenchyma did not occur.
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Effect of antioxidant treatment and CS from filtered cigarettes.
Female A/J mice were treated with the polyphenol EGCG concurrently with exposure to CS-250 for 16 weeks. Emphysema was similar in all CS-exposed mice regardless of the EGCG treatment (photomicrographs not shown). The lack of a treatment effect was corroborated by morphometry (below, Table 4). Female A/J mice were also exposed to CS-250 from filtered, 2R4F research cigarettes for 10 weeks. Emphysema in these animals was both histopathologically (photomicrographs not shown) and morphometrically similar (below, Table 4) to that induced by exposure to CS-250 from nonfiltered 1R3 research cigarettes. This suggested that equivalent development of the disease did not depend on the type of cigarette used to generate the exposure atmospheres, as long as concentrations of particulate material were similar. A subset of animals exposed to CS from the 2R4F cigarettes was also treated with NAC throughout the 10-week duration. The antioxidant had no ameliorating effect on the histopathologic evidence of emphysema (photomicrographs not shown) or the morphometry for alveolar airspace expansion (below, Table 4).
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Particulate material concentrations (target of 250 mg TPM/m3), particle sizes, and yields of vapor-phase components from the filtered and nonfiltered cigarettes were compared. Particulate concentrations between the two types of cigarettes were not different (1R3s: 250 ± 23 TPM/m3, 2R4Fs: 237 ± 15 TPM/m3, n = 12 samplings each; p > 0.05, t-test). By impactor analysis, 66 and 83% of the particulate mass in CS-250 from 1R3 and 2R4F cigarettes, respectively, was from particles less than 500 nm in diameter. By differential mobility analysis in the 15- to 600-nm range, particles in the two types of CS were of similar size (count median diameter of 240 nm and geometric standard deviation [GSD] of 1.5 in each; calculated mass median aerodynamic diameter = 400 nm and GSD = 1.4, assuming unit density). Vapor-phase samples from each of the cigarettes contained similar concentrations of carbon monoxide (1R3s: 326 ± 6 ppm, n = 5; 2R4Fs: 345 ± 25 ppm, n = 6; p > 0.05) and nitrogen oxides (1R3s: 4.7 ± 0.5 ppm, 2R4Fs: 5.2 ± 0.4 ppm, n = 6 each; p > 0.05). Significantly greater (32%) vapor-phase concentrations of total hydrocarbons were generated from the filtered cigarettes (1R3s: 44 ± 4 ppm, 2R4Fs: 58 ± 4 ppm, n = 6 each; p < 0.0001, t-test) at the target concentration of 250 mg TPM/m3.
Emphysema was similar after exposure to CS-250 from filtered and nonfiltered cigarettes for 10 weeks based on enlargement of the Lm (Table 4). Additionally, neither NAC nor EGCG prevented the increase in Lm associated with CS-250 exposure (Table 4). Increases in Sa (NAC) and VVspt x VL (EGCG) in CS-exposed mice were paralleled by increases in VL. However, the alveolar airspace (VVair x VL) was also significantly greater in the NAC-treated, CS-exposed mice at 1.1-fold over the mean value for mice exposed to CS-250 alone and 1.5-fold over that of FA-exposed mice (Table 4), suggesting that much of the VL enlargement could be attributed to an increase in alveolar airspace.
Pulmonary Function Tests
Male mice were assessed for changes in pulmonary function after 22 weeks of exposure to CS-250. This exposure caused a mild decrease in forced expiratory flow rate and an increase in pulmonary compliance (Table 5, Fig. 6). Notably, PEFR was reduced, the FVC was increased, and both the FEV0.05 and the FEV0.05/FVC ratio were significantly decreased in the CS-250 group. Because histopathology demonstrated no obstruction of the conducting airways, the decreased airflow might have been primarily due to decreased elastic recoil (increased compliance; Table 5, Fig. 6), which has been suggested as typical for rodent models of "pure" emphysema (Costa et al., 1992). The mean DLCO in animals exposed to CS-250 was not different from that of FA-exposed controls. This suggested that the exposure caused no net loss in gas exchange tissue; this was consistent with the morphometry results on Sa (above). However, DLCO normalized to the volume necessary for lung inflation at 20 cm H2O pressure was 15% less in the CS-exposed mice (data not shown, p = 0.03, t-test).
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volume/BALF Analysis
Inflammatory cells.
The number of BALF inflammatory cells was increased at all time points by CS exposure when comparing gender and duration effects of exposure to CS-250 for 10 to 22 weeks (Table 6). There were no differences between genders in the inflammatory response at any time point. Differential cell counts showed that most of the inflammatory cells were macrophages and neutrophils in approximately equal numbers. Few lymphocytes were observed. The CS-250 exposure was associated with 90- to 1800-fold elevations in BALF neutrophils, and lymphocytes were elevated 14- to 80-fold compared to FA-exposed mice. Macrophages were increased by CS-250 exposure three- to eightfold over mean control values. The effect of exposure interacted with that of duration on the neutrophil and lymphocyte counts, generally because of a decrease in cell numbers (in females) from 16 to 22 weeks, but neutrophil numbers were similar at each of the time points.
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Treatment with the antioxidant EGCG was associated with a significant reduction in BALF total inflammatory cells and macrophages without an interaction with exposure, i.e., cell numbers averaged from both the CS- and FA-exposed groups were lessened by EGCG treatment in comparison to groups that received no treatment (Fig. 7A). The slight reduction of neutrophils by EGCG treatment in BALF from CS-exposed mice was not significant in comparison to untreated CS-exposed mice (Fig. 7A). The CS-induced elevation of lymphocytes was not affected by EGCG treatment. Cells in BALF from male A/J mice exposed to CS-250 for 22 weeks and then held for a 17-week recovery period were also counted. The CS-induced elevations in neutrophils and lymphocytes were persistent after the recovery period (Fig. 7B), albeit at much lower levels when compared to the male mice exposed to CS-250 for 22 weeks (Table 6).
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BALF lymphocyte subsets.
Exposure to CS-250 for 16 weeks increased the number of BALF mononuclear inflammatory cells as measured by flow cytometry, but the corollary was not evident in the lung tissue (Table 7). Total cells in the lung tissue digests (inflammatory cells, including neutrophils, and other cell types) were significantly increased by CS-250 exposure. The insignificant reduction in lung tissue mononuclear leukocytes may have been in part due to the infiltration of neutrophils as observed in histologic sections. The CS-induced increase in BALF mononuclear cells was paralleled by increases in all the CD4- and CD8-positive T lymphocytes including activated cells (CD69 and CD25 positive, low expression of CD62L) and memory cells (CD44 positive, high expression). In lung tissue, total CD4- and CD8-positive cells were slightly but insignificantly decreased, and only some of the subsets of activated lymphocytes were significantly increased. Interestingly, the CD4:CD8 ratio in cells from lung tissue was decreased by 17% (p = 0.0495); in the BALF an 11-fold increase in the same ratio was induced by CS exposure (p = 0.0460, rank sum tests). The B220-positive B lymphocytes were significantly elevated only in the BALF after exposure to CS-250 (Table 7).
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BALF protein and MMPs.
Exposure of both genders of mice to CS-250 for 10 to 22 weeks elevated total protein and increased activities of LDH and AP in the cell-free BALF supernatant (data not shown). The BALF protein was affected by a significant interaction between the independent variables of exposure, duration, and gender (p = 0.02, ANOVA on ranked data). However, protein concentrations among the CS-exposed mice were approximately threefold greater than those for FA controls in both genders and at all time points. Similarly, CS-250 caused approximately twofold elevations over FA control mean values in the activity of LDH without any striking differences between the genders and time points despite a significant effect of a three-way interaction (p = 0.003, ranked data). The AP activity was approximately three- to fourfold greater in most CS-exposed groups and was affected by interactions between exposure and duration (p = 0.001, ranked data) and between exposure and gender (p = 0.006). Similar to total protein and LDH, there were no large differences in AP activity among the groups that might indicate strong gender or duration influences despite some statistically significant pairwise differences among the groups.
Part of the CS-induced elevation of proteins in the BALF supernatant was due to increases in MMPs. The activities of MMP-2, a proenzyme form of MMP-2, and MMP-9 were measured by zymography. Due to variation between the assays, duration effects could not be analyzed, but exposure to CS-250 elevated MMP-2 activity four- to eightfold in both genders at all time points (data not shown, p < 0.0001, two-way ANOVA on ranked data for exposure and gender effects). The proenzyme form of MMP-2 was also elevated four- to sevenfold over FA control values by exposure to CS-250 at the three time points (data not shown, p < 0.0001, two-way ANOVA on ranked data). The greatest increase in CS-induced activity occurred with MMP-9, where mean CS-250 values were up to 35-fold greater than FA control values (data not shown, p < 0.0001, ANOVA, ranked data). Significant effects of gender were evident for MMP-9 activity at 16 weeks (females greater than males) and for all the analyzed MMP activities at 22 weeks (males greater than females), although none of these gender-related differences were large.
Protein and enzyme activities were also measured in BALF supernatant from female mice exposed to CS-250 for 16 weeks and treated concurrently with EGCG and results compared to those from the mice described above. The CS exposure increased protein concentration and activities of LDH, AP, and the MMPs without regard to the EGCG treatment (data not shown; p < 0.0001 for the exposure effect, two-way ANOVA on ranked data). The EGCG treatment reduced LDH activity slightly in BALF from both FA- and CS-exposed mice (7 and 21% reductions, respectively; p = 0.001 for the treatment effect, ANOVA on ranked data) but did not affect total protein concentration or AP activity (data not shown). These results suggested that EGCG might protect cells from some CS-induced toxicity. In contrast, EGCG treatment increased the activities of MMP-2 (p < 0.0001) and the proenzyme form of MMP-2 (p < 0.05 for the treatment effect, ANOVA on ranked data) in both FA- and CS-exposed mice by four- to sevenfold (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The major conclusions from these studies are that CS-induced emphysema occurs more readily in female mice than in males, the emphysema severity is dependent on the concentration of CS TPM, and the morphology of emphysema in mice is corroborated by functional changes that are characteristic of COPD. Mucous cell hyperplasia and hypertrophy are present in the epithelium of large, conducting airways of CS-exposed mouse lungs, and inflammatory cell infiltrates and elaboration of MMPs in lungs from CS-exposed mice are similar to those observed in human smokers with COPD (Finkelstein et al., 1995
; Hunninghake and Crystal, 1983; Jeffery, 1999; Ohnishi et al., 1998; Segura-Valdez et al., 2000). Further, allowing mice to recover from CS exposure is not associated with reversal of emphysema, and CS-induced pulmonary inflammation persists in recovery. The induction of this lesion by CS makes the mouse a relevant model to study human emphysema (Groneberg and Chung, 2004).
Unpublished studies in our institution have suggested that female mice of different strains are more susceptible than male mice to CS-induced emphysema, and enhanced susceptibility of women to CS-induced COPD has also been suggested (Langhammer et al., 2003; Viegi et al., 2001). Our previously published work, however, demonstrated no effect of gender on the induction of emphysema in CS-exposed A/J mice (March et al., 2005). Results presented here (Table 2) are seemingly contradictory, but in the previous work, the effect of gender was assessed at only one time point (March et al., 2005), and males in the present study required longer exposure than females in order to develop significant (and slightly less severe) emphysema. Because of the duration effect, the morphometric analysis was better able to discern the influence of gender on CS-induced emphysema.
Structural changes in the pulmonary parenchyma of the CS-exposed mice of this study were supported by functional changes typical of COPD, including increased compliance and mild decrements in expiratory flow. Other investigators described decreased pulmonary "elastance" (increased compliance) in the AKR mouse following exposure to CS for 6 months, but no elastance change was observed in similarly exposed A/J mice or mice of several other strains (Guerassimov et al., 2004). Other investigations also showed no correlation between compliance and pathologic features of emphysema in CS-exposed A/J mice (Foronjy et al., 2005). A reason for the contrary findings depicted here is not apparent, but the exposure procedures here may have induced emphysema at a severity detectable by function tests.
Indicators of pulmonary inflammation in CS-exposed mice, particularly the CS-induced elevation in CD8-positive T lymphocytes, were similar to findings in people. Numerous CD8-positive T lymphocytes have been found in the interstitium and mucosa of large and small conducting airways as well as in the parenchyma of human smokers with COPD, and the number of these cells has been correlated with the severity of decrements in FEV over time (Cosio et al., 2002; O'Shaughnessy et al., 1997; Saetta et al., 1998, 1999). The CD8-positive cells have been speculated to be either directly responsible for tissue destruction or the orchestrators of other types of destructive inflammatory cells in a process analogous to an autoimmune disease (Cosio et al., 2002).
Neutrophil and macrophage numbers in lung tissue, sputum, or BALF from humans with COPD vary depending on methodology, and such discrepancies have led to confusion about roles of specific cell types in the pathogenesis of the disease (Cosio et al., 2002). Some studies have demonstrated increased neutrophils in the BALF of COPD patients (Hunninghake and Crystal, 1983; Martin et al., 1985; Thompson et al., 1989), while neutrophils in histopathologically examined parenchymal tissue of human COPD patients have not always correlated well with severity of emphysema (Finkelstein et al., 1995; Saetta et al., 1999). In the current experiments, there were no large differences among the CS-exposed groups in the number of BALF inflammatory cells over the 10- to 22-week duration (Table 6). Subsets of T cells, however, were measured only at 16 weeks. If infiltrates of CD8-positive cells confer the progressive worsening of the disease, as implicated in humans, then infiltrates of such cells early in the exposure of animals could serve as a biomarker for identifying when the balance is tipped toward production of the disease.
The lack of an increase in macrophages and neutrophils from 10 to 22 weeks might be similar to a response demonstrated previously in CS-exposed mice (Gairola, 1986), where BALF total cells and neutrophils gradually increased from 1 to 9 weeks of exposure to reach a plateau in cell numbers from 10 to 16 weeks. This inflammation plateau may be related to the pathogenesis of the emphysema lesion because there was no progressive enlargement of parenchymal airspace from the 10- to the 22-week time point. Perhaps this apparent inflammation plateau allowed reparative processes to occur or that the inflammation severity was not sustained above a threshold that would allow progression of emphysema, and additional airspace enlargement from 10 to 22 weeks was mostly related to normal aging processes as indicated in FA-exposed mice. In similarity, progressive worsening of the disease does not always occur in human smokers with COPD (Groneberg and Chung, 2004).
Interestingly, emphysema and low levels of inflammation persisted in male mice held for a 17-week recovery period following 22 weeks of CS exposure. This is similar to findings in people who have quit smoking (Hogg et al., 2004). Persistence of emphysema following recovery has also been demonstrated in a CS-induced guinea pig model (Wright and Sun, 1994). Inflammation in the "recovered" lung suggested that ongoing tissue destruction might be responsible for persistence of emphysema. Obviously, proinflammatory stimuli remained in the lungs after the recovery period. Persistent inflammatory cell infiltration and the subsequent release of proteinases might have been induced by chemotactic peptides from the damaged parenchymal extracellular matrix (Houghton et al., 2006). Whether or not the persistent inflammation counteracted any reparative process requires further study.
Other biomarkers that might be predictive of emphysema pathogenesis could include quantitative measures of MMP activity. Proteolysis of the extracellular matrix in the parenchyma might change during development of the lesion. There was no apparent progression of the disease over the limited observation period. However, MMP activity might have reached a critical level during the initial development of parenchymal damage. Past results indicate that activity of MMPs in BALF is increased to a similar extent as observed here after 6 weeks of CS exposure (Seagrave et al., 2004), and MMPs at earlier time points need evaluation.
The effect of the CS concentration on emphysema development was a major interest in this study. Deposition in the respiratory tract of a mouse exposed to CS-250 was estimated to be 450 mg TPM/kg BW/week (March et al., 2005). This is equivalent to a person smoking approximately 15 packs of high tar-yielding cigarettes/day (e.g., 1R3 research cigarettes) assuming that the entire particulate yield of a cigarette is inhaled by the smoker. With this massive concentration, mice develop the lesion after a much shortened exposure period relative to humans. Reducing the concentration of CS was done in an attempt to generate a concentration-response effect in mice. Histopathologically, mice exposed to CS-100 had little or no emphysema after 10 weeks of exposure, while their counterparts exposed to CS-250 readily developed the lesion. With longer duration, the lower concentration did cause emphysema (Figs. 1 and 2). There was a significant trend by regression analysis for increasing Lm with increasing CS concentration and duration (data not shown). However, the exposure caused a plateau of the emphysema response in mice exposed to CS-250 for 10–22 weeks, i.e., mice exposed to CS-250 had a similar increase in the Lm above FA controls at all time points. Thus, exposing female A/J mice for more than 10 weeks (6 h/day, 5 days/week) at 250 mg TPM/m3 is not necessary, while exposing them at 100 mg TPM/m3 requires 16 weeks or more to attain significant emphysema.
The shortcoming of the A/J mouse model was that it did not mimic all features of COPD or emphysema in humans. The histopathologic findings of emphysema were generally mild to moderate in severity. There was no apparent loss of gas exchange tissue as measured morphometrically by Sa and VVspt x VL and physiologically by DLCO. However, in this and past experiments (March et al., 2002, 2005), CS did not, in most instances, cause any increase in Sa or VVspt x VL despite significant increases in VL. This suggested that overt hyperplasia and hypertrophy of parenchymal tissue did not occur as might be expected in rodents following inhalation exposure to toxicants (Costa et al., 1992). Alveolar septa within enlarged lungs may have been stretched because of decreased elastic recoil, and this might have given the false impression of increased septal tissue. The morphometry was inadequate to discern tissue stretching because the intercept counting method did not account for alveolar septal thickness.
Another shortcoming of the model was the lack of substantial histopathologic change in the conducting airways. The lungs from CS-exposed mice lacked one of the major components of COPD, namely inflammation of the large conducting airways (chronic bronchitis). There were minimal inflammatory cell infiltrates around terminal bronchioles and at broncholoalveolar duct junctions, but extensive inflammation and remodeling of the small airways were also lacking. Such small airway disease is regarded as the major contributor to airflow obstruction in people with COPD (Hogg et al., 2004). Anatomic differences might explain some of this lack of correlation because mice lack cartilaginous support structures and submucosal mucous glands in the largest intrapulmonary conducting airways and have fewer generations of airway branches preceding the parenchyma. Despite the lack of inflammation, there was a significant increase in mucosubstances in mice exposed to CS for 16 weeks (Fig. 4). The increase was not substantial given that it did not result in any demonstrable airway obstruction; however, the mucous cell hyperplasia and hypertrophy paralleled those seen in humans with COPD.
Treatment of CS-exposed mice with the antioxidants EGCG or NAC did not prevent the development of emphysema in CS-exposed mice. The green tea–derived polyphenol EGCG decreased inflammatory cells in the BALF to a modest extent. In COPD patients, treatment with the mucolytic sulfhydryl antioxidant NAC has decreased pulmonary inflammation (van Overveld et al., 2005), but in the current studies inflammation in CS-exposed mice treated with NAC was not quantified. In corroboration of the present work, NAC was ineffective in preventing exacerbations and lung function decline in COPD patients (Decramer et al., 2005), while the opposite effect was demonstrated in other work (Pela et al., 1999). Neither doses for the antioxidants in the present studies were optimized nor were any pharmacokinetic analyses performed to determine effects of CS exposure on these drugs, and the high concentrations of CS used may have overwhelmed any protective effect of these compounds. Thus, the effect of antioxidants on COPD in general and emphysema in particular requires further clarification.
The pathologic and physiologic changes in CS-exposed mice of the current set of studies were similar to findings in people with COPD. Emphysema is a pathologically defined disease, while its functional consequence of expiratory airflow obstruction is manifested in the clinical syndrome of COPD. The progression of some aspects of the disease in people can be monitored with pulmonary function, but the pathogenesis of parenchymal tissue destruction is not as easily evaluated. Several important candidate biomarkers of cellular and biochemical changes reflecting the pathogenesis of the disease were characterized in this study. The biomarkers of inflammation, particularly CD8-positive T-cell infiltrates, and of tissue destruction, such as expression of MMPs, may prove to be useful surrogates for the histopathology of emphysema. Through better characterization in animals, these and other markers may predict the course of the disease and may be useful in future studies of COPD in animals and humans exposed to CS.
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ACKNOWLEDGMENTS |
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The authors wish to thank the excellent technical staff for production of this article including those in the Animal Care, Aerosol Science, and Technical Communications Units. This work was funded by the Tobacco Master Settlement through a cooperative research agreement with the University of New Mexico.
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