Abstract
The guinea pig model of cigarette smoke (CS)-induced lung injury is known to exhibit many pathophysiological similarities to chronic obstructive pulmonary disease (COPD), but the expression profiles of inflammatory mediators in the lung are poorly understood.
Quantitative real-time RT-PCR was used in this study to investigate the pulmonary expression profiles of cytokine and chemokine mRNA in response to single or repeated CS exposure in guinea pigs.
一个CS暴露不引起明显的inflammatory cell infiltration into the lungs, but it led to significant increases in the mRNA expression of tumour necrosis factor-α, interleukin (IL)-1β, IL-8, and monocyte chemoattractant protein (MCP)-1, and decreases in IL-5 and granulocyte-macrophage colony-stimulating factor. Repeated CS exposure induced many features of COPD, such as marked accumulation of macrophages and neutrophils, augmented protease activities, lung structural alterations and increased airway resistance, accompanied by significant increases in the mRNA expression of IL-1β and MCP-1 and decreases in IL-2, IL-5, transforming growth factor-β, and eotaxin.
In conclusion, in guinea pigs, inflammatory mediator changes in the lungs following cigarette smoke exposure are largely similar to those reported for smokers and/or chronic obstructive pulmonary disease patients. This model will therefore be useful to further understand the pathogenesis of chronic obstructive pulmonary disease.
- Animal model
- chemokines
- chronic obstructive pulmonary disease
- cigarette smoking
- cytokines
- gene expression
Chronic obstructive pulmonary disease (COPD) is characterised by slowly progressive and largely irreversible airflow limitation due to chronic bronchitis, emphysema, or both. It is currently one of the most common causes of death in Western countries. With the exception of genetic aetiology, including α1-antitrypsin deficiency, chronic pulmonary inflammation caused by long-term cigarette smoking is the most accepted pathogenesis. Inflammatory cells that play central roles in COPD include macrophages, neutrophils, and T-lymphocytes. Various inflammatory mediators derived from these cells, such as cytokines/chemokines (tumour necrosis factor (TNF)-α, interleukin (IL)-8, and monocyte chemoattractant protein (MCP)-1,etc.), lipids (leukotriene B4,etc.), and proteases (matrix metalloproteinase (MMP)-2, 8, and 9,etc., which are counteracted by tissue inhibitors of matrix metalloproteinases (TIMPs)), are suggested to contribute to disease development1,2.However, compared with other diseases of the lung, such as asthma, the detailed pathology of COPD is still relatively unknown.
Previous studies have demonstrated the possible induction of COPD-like lung injuries, by exposure to lipopolysaccharides, ozone, nitrogen dioxide, sulphur dioxide, cigarette smoke (CS), or other environmental pollutants, in several animal species3–7.Among them, guinea pig CS models are considered to be adequate for further investigations of COPD because of the anatomical and pathophysiological similarities to human COPD6.人们普遍认为短期CS世博会sure causes increased pulmonary permeability and accumulation of inflammatory cells6,8–12, and long-term exposure causes emphysematous airspace enlargement in the guinea pig models12–15.The guinea pig CS models have contributed to the current understanding of histological and physiological aspects of smoke-associated lung disease, but the underlying molecular mechanisms, including local production of cytokines and chemokines, are poorly understood because of the limited availability of research tools, such as specific antibodies for guinea pig proteins.
在这项研究中,定量实时rt - pcr中欧ology was used to investigate the gene expression patterns of cytokines (TNF-α, IL-1β, IL-2, IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF)-β) and chemokines (IL-8, MCP-1 and eotaxin) in both the acute and chronic phases of a guinea pig CS model. Pathological and physiological analyses were also performed for this model to investigate the relationship between those cytokine and chemokine expression patterns and the diseased states.
MATERIALS AND METHODS
Animals
Male Hartley strain guinea pigs were purchased from SLC (Shizuoka, Japan). The animals were maintained in ordinary animal cages, with food and water availablead libitum.Animals that were 7 weeks old and weighed 400−600 g at the start of the experiments were used. Experiments were performed in accordance with the regulations of the Animal Ethical Committee of Yamanouchi Pharmaceutical.
Study design
Acute phase model
Animals were singly exposed to CS. A bronchoalveolar lavage (BAL) was performed 3, 24, or 48 h after the exposure. Lung extraction for mRNA expression analysis was performed 2–3 h after exposure, in separate animals.
Chronic phase model
Animals were repeatedly exposed to CS for 4 weeks. Body weight or airway resistance was measured twice or once a week, respectively, for the duration of the experiment. BAL and lung extraction for mRNA expression and histopathological analyses were performed 2–3 h after the last exposure. The lung extraction was performed in separate animals. Detailed methods are described below. For all studies, the numbers of animals are indicated in the figure legends.
Cigarette smoke exposure
CS exposure was conducted using a set of CS generator SG-200 and inhalation apparatus made up of 20 chambers (Sibata Scientific Technology Ltd, Tokyo, Japan). A commercially available nonfilter cigarette was used (Peace brand cigarettes; Japan Tobacco Inc., Tokyo, Japan) and, according to the manufacturer's specifications, each cigarette contained 2.4 mg of nicotine and 24 mg of tar. Referring to previous studies8–14, the smoking conditions used in this study were determined based on the levels of BAL fluid (BALF) protein and total cell count. Each animal was placed into an individual chamber and exposed to diluted CS in a conscious and restrained state. In the acute phase experiment, animals were exposed to the smoke of a total of 10 cigarettes during a 40-min period. Each cigarette was puffed 15 times for 3 min at a rate of 5 puffs·min−1.一支烟的意思画35毫升的CS 50毫升syringe, and then blowing this CS, which was diluted to 4–5% with air, into the apparatus. Fresh air inhalation was performed for 1 min after every 3 min of CS exposure. In the chronic phase experiment, animals were repeatedly exposed to the smoke of 10 cigarettes·day−1, 5 consecutive days·week−1, for 4 weeks under the same conditions as described above. Animals placed into the same type of apparatus and exposed to fresh air instead of CS were used as the fresh air control.
Histopathological evaluation
In the chronic model, animals were sacrificed 2–3 h after the last exposure, the lungs were then removed and fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned at 2 μm, and stained with haematoxylin and eosin. Histopathological assessment in the bronchus and the parenchyma was performed in a blind fashion.
Measurement of airway resistance
In the chronic model, at 2–4 h after CS exposure, specific airway resistance (sRaw) was measured using a two-chambered double-flow plethysmograph16.简而言之,一个有意识的动物的neck extending through the partition of a two-chambered box, and sRawwas measured using a noninvasive airway mechanics analyser (BioSystem XA software; Buxco Electronics, Sharon, CT, USA). A bias flow of air (25 mL·s−1) was maintained through the nasal chamber to ensure a constant supply of fresh air to the animal. sRawdata were logged at 5-s intervals; the mean of 12 consecutive interval averages was calculated as the measurement for time point.
Brochoalveolar lavage
The guinea pigs were sacrificed under urethane anaesthesia (1.2 g·kg−1i.p.), after which their tracheas were cannulated. The lungs were lavaged with 5 mL of ice-cold saline containing heparin (1 U·mL−1) five timesviathe cannula. Heparinised saline was used to prevent the coagulation of recovered cells. The BALF was centrifuged at 400×g10分钟4°C. The cell pellet was resuspended in 2 mL of ice-cold heparinised saline for measuring the total cell count, and the supernatant was stored at -80°C until used. BAL was performed 3, 24 and 48 h after CS exposure in the acute experiment and 2–3 h after the last exposure in the chronic experiment. The total number of leukocytes in the BALF was counted using an automated cell counter (Celltac-α; Nihon Kohden, Tokyo, Japan). The differential cell count was performed using a cytospin preparation stained with Diff-Quik (International Reagent Corp., Kobe, Japan). A minimum of 300 cells were identified and differentiated as mononuclear cells, neutrophils or eosinophils using the standard morphological criteria. The total protein content in the BALF supernatant was measured using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA).
Extraction of total RNA
The lungs were removed 2–3 h after CS exposure in both the acute and chronic experiments. Total RNA was extracted from the lung with ISOGEN reagent (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. The amount of extracted RNA was quantified by measuring the absorbance at 260 nm.
Measurement of cytokine and chemokine mRNA expression using quantitative RT-PCR
The cytokines and chemokines that were analysed in this study were selected based on the following reasons: 1) they were implicated in inflammatory conditions such as COPD and asthma; and 2) their sequences in guinea pigs were already published. PCR primers and TaqMan® probes were designed using Primer Express 1.5 Software (Applied Biosystems, Foster City, CA, USA) based on the sequence entries in GenBank. The sequences of primers and probes used in this experiment are summarised in table 1⇓.All primers and probes were obtained from Proligo Japan (Kyoto, Japan). In this study, the sequence of guinea pig neutrophil attractant protein-1 was used as IL-8, as described in a previous report10.RT-PCR was performed using the ABI PRISM 7700 sequence detection system (Applied Biosystems) with a one-step RT-PCR kit (TaqMan® EZ RT-PCR Core Reagents; Applied Biosystems) in accordance with the manufacturer's instructions. In brief, final concentrations used in the 25 μL reaction mix were: 50 ng (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), IL-1β, IL-2, IL-5, eotaxin, GM-CSF and TGF-β) or 500 ng (TNF-α, IL-8 and MCP-1) of total RNA; 200 nM of each primer; 100 nM of probe; 2.5 U rtenzyme; 300 μM deoxynucleotide triphosphates; 3 mM manganese acetate; and 1×EZ buffer. Thermal cycling conditions were as follows: 55°C for 50 min, 60°C for 10 min, and 95°C for 2 min, followed by 40 cycles of 95°C for 20 s and 58°C for 90 s. The expression levels of each gene were normalised to GAPDH levels and displayed as the fold change relative to normal or fresh air controls.
Gelatin zymography
The BALF supernatants were concentrated five-fold with Centricon YM-3 filters (Millipore Corp., Bedford, MA, USA). The concentrated samples and positive controls (purified human MMP-2 and -9; Chemicon, Temecula, CA, USA) were loaded onto 8% sodium dodecyl sulfate (SDS)-polyacrylamide gels containing 1 mg·mL−1gelatin under nonreducing conditions. After electrophoresis, the gel was washed in 50 mM Tris-HCl (pH 7.5) containing 0.1 M NaCl and 2.5% Triton X-100 for 2 h to remove the SDS. The gel was then rinsed in water and incubated overnight at 37°C in development buffer, 50 mM Tris-HCl (pH 7.5) and 20 mM CaCl2.After development, the gel was stained with staining solution, 0.25% Coomassie blue R250 in 45% methanol/10% acetic acid/45% H2O, and subsequently washed with destaining solution, 30% methanol/10% acetic acid/60% H2O. Gelatinolytic activities were detected as clear bands of gelatin lysis against a blue background stain.
Statistical analysis
Data were expressed as the means±sem.The statistical significance of differences between groups was determined using unpaired t-tests or one-way ANOVA with Dunnett's multiple range test, and p<0.05 were considered significant.
RESULTS
A single exposure to cigarette smoke (acute phase model)
Analysis of bronchoalveolar lavage fluid
Under the conditions used in this experiment, a single exposure of the guinea pigs to CS (10 cigarettes) caused a significant increase in the BALF protein level, which was detectable 3 h after exposure and was sustained at the same level for up to 24 h after (fig. 1a⇓). However, significant changes were not detected in cell number (fig. 1b⇓) or gelatinolytic activity (data not shown) in the BALF of the CS-exposed animals compared with normal control (age-matched untreated) animals at the given time points (3, 24 and 48 h). These results indicate that a single exposure to CS can elicit an acute and significant increase of airway permeability without obvious inflammatory cell accumulation in the airways.
Cytokine and chemokine mRNA expression in the lungs of the acute phase model
The sequences of the PCR primers and TaqMan® probes used for the detection of cytokine and chemokine mRNA were all based on previously published guinea pig sequences (table 1⇑). In the lungs harvested from animals 2–3 h after CS exposure, significant increases in the mRNA expression of TNF-α (3.5-fold), IL-1β (70-fold), IL-8 (4.0-fold) and MCP-1 (3.4-fold), as well as significant decreases in IL-5 (0.55-fold) and GM-CSF (0.51-fold) were observed compared with normal control animals (fig. 2⇓). Among the nine genes examined, the increase in IL-1β mRNA was the most remarkable. All these alterations of mRNA expressions returned to almost normal levels 24 h after exposure, except IL-5, which remained at the lower level (data not shown).
Repeated exposure to cigarette smoke (chronic phase model)
Body weight gain and airway resistance
The body weight of guinea pigs repeatedly exposed to CS (10 cigarettes·day−1, 5 days·week−1, for 4 weeks) was consistently lower than that of the fresh air control animals during the experimental period. The average weight of CS-treated animals in the fourth week of exposure was 630±10.0 g, which was significantly decreased compared with fresh air control animals (664±7.1 g; fig. 3a⇓). Since there was no apparent difference in body weight between the fresh air control and the age-matched normal animals (data not shown), restraint stress was deemed to have had no effect on the results.
The basal level of airway resistance was measured with a whole-body, double-chamber plethysmograph in a conscious and restrained state. In animals repeatedly exposed to CS, airway resistance did not change during the first week (after 4 consecutive days) of CS exposure, which would have been similar to the acute phase model. However, the airway resistance in animals repeatedly exposed to CS was slightly, but significantly, elevated compared with the control animals in weeks 2 and 4 of exposure (fig. 3b⇑).
Histological evaluation
In the lungs from guinea pigs repeatedly exposed to CS for 4 weeks, the epithelium of the bronchus showed desquamative and squamous metaplastic changes (fig. 4a⇓versusb). In addition, the alveolar walls of the lungs collected from CS-exposed animals were mildly thickened. Marked infiltration of macrophages into the alveoli and neutrophils into the alveoli and small airways were also noted. The control animals showed none of these signs (fig. 4c⇓versusd). Emphysematous changes, which are characterised by obvious enlargement of the alveolar space, did not develop during the 4-week treatment.
Analysis of bronchoalveolar lavage fluid
The repeated exposure of guinea pigs to CS for 4 weeks caused a significant increase in the BALF protein level (fig. 5a⇓). Differing from the single exposure model, the repeated CS exposure also induced a marked increase in the cell counts in the BALF compared with the control animals. Differential cell count analysis revealed that the increased cells were composed mainly of mononuclear cells (especially macrophages) and a small amount of neutrophils, but not eosinophils (fig. 5b⇓). The BALF supernatants from animals repeatedly exposed to CS exhibited gelatinolytic activity in the bands of 92 and 220 kD, which were regarded as MMP-9 and the homodimer17, but no such activity was observed in the BALF from control animals. In contrast, the 72 kD bands, which were regarded as MMP-2, were observed at the same levels in the BALF of the repeated CS-exposed and the control animals (fig. 5c⇓).
Cytokine and chemokine mRNA expression in the lungs of the chronic phase model
In the lungs from guinea pigs 2–3 h after the last exposure, significant increases in the mRNA expression of IL-1β (6.4-fold) and MCP-1 (2.7-fold) were observed compared with the control animals. Although not statistically significant, the level of IL-8 in repeated CS-exposed animals showed a tendency to increase (1.7-fold, p = 0.12). Unlike in the acute phase, there was no difference in the level of TNF-α between the CS-exposed and the control groups. In addition, significant decreases in the mRNA expression of IL-2 (0.57-fold), IL-5 (0.47-fold), TGF-β (0.68-fold) and eotaxin (0.27-fold) were also observed in the diseased lungs (fig. 6⇓). The decreased mRNA levels of IL-2, TGF-β and eotaxin were characteristic events observed only in the chronic phase.
DISCUSSION
The guinea pig model of CS-induced lung injury is a well-defined COPD model. Previous studies using this model have provided much valuable information on the histological and physiological aspects of smoke-associated lung disease. However, except for an acute exposure study that included IL-810, the pulmonary mRNA expression levels of inflammatory mediators in this model have not been reported. In this study, the pulmonary expression profiles of cytokines and chemokines in response to acute and chronic CS exposure was comprehensively investigated.
Under the current study's experimental conditions, a single CS exposure induced a significant increase in the BALF protein level, but obvious increased numbers of the BALF total cells or neutrophils (data not shown), which had been reported previously9,10,12, were not apparent. However, in some animals, increased myeloperoxidase activities in the BALF could be detected (data not shown), which is a sensitive neutrophil marker. These results suggest that mild neutrophil infiltration, which could not significantly affect the BALF total cell counts, was induced in the current acute phase model. The smoking conditions used in the present study seem to be relatively milder than those described in previous reports.
In this acute phase model, significant increases in the mRNA expression of proinflammatory cytokines and chemokines,e.g.TNF-α, IL-1β, IL-8 and MCP-1, in the lung were observed. Among them, the upregulation of IL-1β mRNA (70-fold) was especially prominent when compared with those of TNF-α, IL-8 and MCP-1 mRNA (p<0.01, Dunnett's multiple range test). However, in the chronic phase model, in which many clinical features of COPD were reproduced, the upregulation of IL-1β mRNA (6.4-fold) was not as prominent as observed in the acute phase (p<0.01). These results seem to correlate well with the IL-1β expression levels that were observed in healthy smokers and COPD patients. That is, the concentration of IL-1β is significantly increased in the BALF of healthy smokers compared with nonsmokers18, whereas there are few reports that suggest the active participation of IL-1β in patients with COPD. Although the importance of IL-1β in COPD is still unknown, IL-1β has been shown to mediate trafficking of inflammatory cells to airway epithelial cells through the upregulation of adhesion molecules such as intercellular adhesion molecule (ICAM)-119.Thus, IL-1β may be a crucial cytokine especially in the acute/induction phase of CS-associated lung disease, with the ability to trigger leukocyte trafficking in the lung.
Marked accumulation of macrophages and neutrophils in various parts of the lung is one of the most remarkable features of COPD, and the levels of MCP-1 and IL-8, which are major chemoattractants for macrophages and neutrophils, respectively, are elevated in the BALF20, lung biopsy21and induced sputum22慢性阻塞性肺病患者。在慢性阶段模式l, an accumulation of macrophages and neutrophils in the lung was observed, as well as a significant elevation of MCP-1 mRNA and an increasing tendency for IL-8 mRNA to be present, which suggested that the chemokine expression patterns are similar to those in human COPD. In addition, the expression patterns of chemokines are well correlated with the selective accumulation of their target cells.
It is noteworthy to mention that a concurrent downregulated expression of IL-5 and eotaxin mRNA was observed in the chronic phase. IL-5 and eotaxin have both been shown to be important in the differentiation, maturation and activation of eosinophils23,24.In contrast to asthma, eosinophils do not seem to be prominent in COPD, except during exacerbation or when patients have concomitant asthma2.Consistent with this, eosinophilia was not observed in this nonasthmatic chronic CS model.
In the chronic phase, a decreased level of IL-2 mRNA was also observed. IL-2 is produced primarily by T-cells, especially activated CD4+ T-cells, and plays important roles in regulating T-cell functions25.Previously, Hagiwaraet al.26had demonstrated that there were no cells spontaneously secreting IL-2 in the BALF of smokers, whereas most nonsmokers had detectable IL-2-secreting cells. They suggested that cigarette smoking can disturb the T-cell immune balance, which may explain the susceptibility of smokers to certain airway disease conditions such as viral or microbial infections. The current results seem to correlate well with these observations in humans.
TNF-α is one of the most extensively studied cytokines in COPD. An increased level of TNF-α has been observed in lung biopsy27, induced sputum22and serum28慢性阻塞性肺病患者。Although TNF-α elevation in plasma has been reported in a previous guinea pig CS study12, expression profiles in the lung have not been demonstrated. In the present guinea pig model, a significant increase of TNF-α mRNA was observed in the lung during the acute phase, but not the chronic phase. There is no convincing reason for the lack of elevation in TNF-α levels in the chronic phase. However, Soleret al.29reported a positive correlation between TNF-α levels in the BALF and the presence of pathogenic microbial infection in COPD patients. Additionally, there is evidence that the TNF-α level was significantly higher in COPD patients experiencing exacerbation than in stable patients30.The specific contribution of TNF-α in the chronic phase model may depend on additional disease conditions.
A significant decrease of GM-CSF mRNA was observed only in the acute phase. At present, it is difficult to explain the physiological implications of this phenomenon because there is no evidence supporting this result.
In the histological examination of the chronic phase model, lung structural alterations, such as desquamative and squamous metaplastic changes of airway epithelium, as well as alveolar wall thickening. These changes are commonly observed characteristics of COPD were observed31.However, emphysematous airspace enlargement in the parenchyma was not observed. The most accepted hypothesis for the development of emphysema is the protease–antiprotease imbalance theory2,32,33.Although no emphysematous lesions were observed, a marked increase of BALF MMP-9 activity could be detected in the chronic phase model. In addition, an increase in MMP-8 mRNA (1.3-fold) and a decrease in TIMP-2 mRNA (0.68-fold) were also observed in this phase (data not shown). These findings indicate that the protease-antiprotease imbalance had already occurred in this 4-week CS exposure model. Furthermore, the mRNA expression of TGF-β, which is considered to play a protective role in the development of emphysema34,35, was significantly decreased in this model. Therefore, the extension of the smoking period would lead to emphysematous pathophysiology, as described in previous reports12–15.
A decrease of weight gain in the repeated CS-exposed animals was also observed. Although body weight loss is a common feature in COPD patients, its exact mechanism and what role repeated CS exposure plays in it are not known at present.
In summary, this study investigated the pulmonary expression profiles of inflammatory mediators in the acute and chronic phases of the well-defined guinea pig cigarette smoke model using quantitative RT-PCR technology. The current results showed that the guinea pig cigarette smoke model had many similarities to those reported in smokers and/or chronic obstructive pulmonary disease patients not only histologically and physiologically, but also with regard to inflammatory mediator changes. Recently, the number of available DNA sequences for guinea pigs has been rapidly increasing, so more elaborate expression analysis will be useful for further understanding of the complex pathology of chronic obstructive pulmonary disease.
Acknowledgments
The authors would like to thank T. Yamada and J. Ishikawa (Inflammation Research, Pharmacology Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd, Ibaraki, Japan) for their invaluable suggestions and S. Kuromitsu for technical advice with the real-time PCR studies.
- ReceivedApril 10, 2005.
- AcceptedAugust 14, 2005.
- © ERS Journals Ltd