Steroid-resistant Inflammation in a Rat Model of Chronic Obstructive Pulmonary Disease Is Associated with a Lack of Nuclear Factor-B Pathway
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美国呼吸和危急护理医学 2005年第7期
Respiratory Pharmacology Group, Faculty of Medicine, National Heart and Lung Institute, Imperial College, London, United Kingdom
ABSTRACT
Rationale: Emphysema is one component of chronic obstructive pulmonary disease (COPD), a respiratory disease currently increasing in prevalence worldwide. The mainstay therapy adopted to treat patients with COPD is glucocorticoids; unfortunately, this treatment has limited impact on disease symptoms or underlying airway inflammation. Objective: There is an urgent need to develop therapies that modify both the underlying inflammation, thought to be involved in disease progression, and the structural changes in the emphysematous lung. Methods: We have characterized an elastase-driven model of experimental emphysema in the rat that demonstrates COPD-like airway inflammation and determined the impact of a clinically relevant glucocorticoid. Measurements and main results: We observed an increase in lung neutrophils, lymphomononuclear cells, mucus production, and inflammatory cytokines. Also present were increases in average air space area, which are associated with emphysema-like changes in lung function, such as increased residual volume and decreased flow; these increases in area were maintained for up to 10 weeks. In addition, we observed that elastase-induced airway neutrophilia is steroid resistant. Interestingly, the inflammation observed after elastase administration was found to be temporally associated with a lack of nuclear factor-B pathway activation. This apparent nuclear factor-BeCindependent inflammation may explain why treatment with a glucocorticoid was ineffective in this preclinical model and could suggest parallels in the steroid-resistant human disease. Conclusion: We believe that this model, in addition to its suitability for testing therapies that may modify existing emphysema, could be useful in the search for new therapies to reduce the steroid-resistant airway inflammation evident in COPD.
Key Words: inflammation; lung; rodent; steroid
Pulmonary emphysema is a disease of the parenchyma, which is characterized by the destruction of alveolar septa resulting in abnormal and permanent enlargement of the air spaces (1). This disease, along with chronic bronchitis, is a component of chronic obstructive pulmonary disease (COPD), which is currently the fourth leading cause of death in the United States, with up to 7 million people diagnosed each year (2). It is predicted by the World Health Organization that the prevalence of COPD will increase in the coming years to become the fifth most common cause of morbidity and the third most common chronic disease worldwide. Patients with COPD have progressive airflow limitation leading to disabling dyspnea, with an inability to properly oxygenate the blood. Although the development of COPD is known to be linked to cigarette smoke, little is known about the pathogenesis of the disease. At present there are a number of theories as to how chronic cigarette smoking causes emphysema; these include an altered proteaseeCantiprotease balance (3), increased inflammatory cell burden (4), enhanced oxidative stress levels (5), and raised alveolar septal cell apoptosis (6). Whether some, all, or none of these theories are involved in the breakdown of the lung structure seen in COPD is not known, but it is believed that understanding the pathogenesis of the disease process is central to the development of appropriate new therapies (7).
Current COPD therapy is limited to treatment of symptoms, with the mainstay being drugs that have been developed to treat asthma, long-acting bronchodilators and corticosteroids (8). The use of inhaled corticosteroids to treat COPD is controversial with some studies claiming efficacy and others not (9eC12). A study by Hattotuwa (13) showed that treatment with inhaled fluticasone resulted in small improvements in aspects of COPD including symptom score, exacerbations, and sputum production. However, treatment with corticosteroids has been shown to have no impact on the cellular inflammation or increased protease burden seen in COPD (13, 14). In addition, unlike cytokine release from cells collected from nondiseased airways, the same cells from patients with COPD have been shown to be resistant to steroid treatment (15, 16). There are also no therapies currently available that have been shown to halt the relentless decline in lung function and the progressive damage of the lung parenchyma seen in COPD (17). It is commonly believed that if a therapy can be found to impact on the airway inflammation seen in COPD then that therapy may slow the progression of the disease, although a direct correlation is yet to be established and indeed they may well be dissociated. There is therefore a need for fully characterized, steroid-insensitive, in vivo models of COPD to aid in the understanding of the pathogenesis of the disease and for the discovery of new antiinflammatory treatments.
It has previously been demonstrated that exposing the lungs to porcine pancreatic elastase (PPE), an enzyme that acts predominantly, although not exclusively, on elastin (18), induces acute airway inflammation (19eC21) followed by emphysema (22eC26) and compromised lung function (27, 28). The aim of this study was to perform a comprehensive characterization of the effects of elastase on lung inflammation while confirming the data obtained by others regarding the structural and functional changes. Our findings concur with others in that we have demonstrated that PPE treatment caused acute inflammation followed by increases in average airspace area and that this was maintained over at least a 10-week period and was associated with changes in lung function measurement.
To date there are no publications assessing the impact of corticosteroids on elastase-induced airway inflammation and experimental emphysema. Therefore, a secondary aim of this study was to determine the impact of a glucocorticoid, budesonide, in this model. Some of the results of this study have been reported in the form of an abstract (29).
METHODS
Model Characterization
Male Sprague-Dawley rats (280eC320 g) were purchased from Harlan UK (Bicester, UK). Home Office guidelines for animal welfare, based on the Animals (Scientific Procedures) Act 1986, were strictly observed. Experimental emphysema was induced by instilling one dose of PPE (120 U/kg, freshly prepared in saline, administered intratracheally at 1 ml/kg) directly into the airways; control groups were given saline in the same way at the same time. At various time points (either 2, 6, 24, 48, 72, 96, and 168 hours or 2, 4, 6, 8, and 10 weeks after treatment) the animals were culled and the inflammatory status of the airways was determined. Airway lumen and tissue differential white cell count, histology of lung tissue samples, and protein expression levels were determined by methods detailed in McCluskie and coworkers (30). Matrix metalloproteinase (MMP-2 and MMP-9) levels were determined in the lavage supernatant according to the method of Birrell and coworkers (31). Elastase activity in lavage samples was determined by a fluorogenic assay outlined by Trifilieff and coworkers (32). The level of nuclear factor (NF)-B pathway activation was determined in lung tissue with an Active Motif (Carlsbad, CA) kit, which measures p65:DNA binding, in accordance with the manufacturer's instructions. NF-B:DNA binding was also determined by electrophoretic mobility shift assay (EMSA). The number of mucus-containing cells was determined in the trachea by periodic acideCSchiff staining and manually counting 240-e lengths of airway epithelium. Fifty readings were taken and averaged per trachea.
In satellite groups (4 and 8 weeks after vehicle or PPE administration), changes in air space area were measured by computer-assisted image analysis as described by Belloni and coworkers (33). Quantification was performed with an Olympus BX40 microscope using Zeiss KS300 image-processing software (Imaging Associates, Bicester, UK). Using color-thresholding techniques the total parenchymal air spaces in square micrometers and the number of air spaces were measured from 10 random fields per slide/lobe. From these figures the mean air space area for each field can be calculated. Any fields containing airways or vasculature were excluded.
Lung function was assessed (2, 4, 6, 8, and 10 weeks after vehicle or PPE treatment) in anesthetized rats (ketamine-HCl and xylazine-HCl, 144 and 10 mg/kg, respectively, administered intraperitoneally), using a forced maneuvers system supplied by Buxco Europe (Winchester, UK). Functional residual capacity (FRC) was calculated by the software, using Boyle's law, on the basis of data collected after occluding the airway at the end of expiration and measuring the pressure and thoracic displacement. Residual volume (RV) was calculated by subtracting the vital capacity from the total lung capacity (TLC), determined by the pressureeCvolume maneuver. During the same maneuver, peak compliance was determined, at which point the pressure was recorded:—pressure at maximum compliance (Pmcmax). The forced expiration maneuver determines the amount of air expired during a fast expiration from TLC and is called forced expiratory capacity (FEC). A curve was plotted of expired volume versus flow, resulting in a floweCvolume curve. On this plot, when 25% of the FEC has been expired the instantaneous flow is said to be FEF25. This range of measurements enabled the assessment of the impact of PPE treatment on lung volume, pressure, and flow.
Effect of Budesonide on PPE-induced Inflammation and Emphysema
To determine the impact of budesonide on the early inflammatory response after PPE exposure, vehicle (0.5% methylcellulose and 0.2% Tween 80 in water, 2 ml/kg) or compound was administered (0.1eC3 mg/kg, per os) twice daily before and after PPE. Forty-eight hours after PPE administration the lungs were assessed for cellular infiltration, cytokine content, MMP levels, and goblet cell staining.
Two dosing protocols were used to determine the impact of budesonide on chronic airway changes after PPE. The first protocol was thought of as a "prophylactic" dosing protocol, in which vehicle or compound (0.03eC0.3 mg/kg, per os) was administered throughout the 8-week study; the second, "therapeutic" protocol consisted of dosing starting 4 weeks after PPE insult. Eight weeks after PPE insult the rats had their lung function measured and the average air space area was determined. Two satellite groups were incorporated to establish experimental emphysema 4 weeks after PPE treatment, that is, at the time at which the therapeutic dosing protocol began.
RESULTS
Treatment of the rats with this dose of PPE did not cause mortality in any of the studies performed.
Characterization of the Model: Time Course of the Acute Inflammatory Phase
Treatment of the rats with PPE caused significant increases in neutrophil and lymphomononuclear (LMN; i.e., monocytes, macrophages, and lymphocytes) cell recruitment into the airway lumen and tissue. The peak of inflammatory cell burden in the airway appears between 24 and 48 hours after PPE insult, with levels returning to that of the controls by Day 7 (Figures 1 and 4). The vehicle for elastase appeared to cause an increase in lung tissue LMN cell numbers 4 and 7 days after treatment when compared with the earlier time points (Figure 1D); the reason for this is not known but could be due to day-to-day differences in tissue cell recovery seen with the tissue-digesting technique employed. This increase in inflammatory cell number was accompanied by a significant increase in tumor necrosis factor-, interleukin (IL)-1, monocyte chemoattractant protein (MCP)-1, and cytokine-induced chemoattractant (CINC)-1, CINC-2, and CINC-3 protein levels in lavage fluid and IL-1, MCP-1, CINC-1, and CINC-2 protein levels in lung tissue (Figure 2 and Tables 1 and 2). Levels of MMP-2 and MMP-9 were significantly increased in the lung lavage fluid (Figure 2). Interestingly, there appeared to be an increase in MMP activity before recruitment of the majority of inflammatory cells, which may suggest that the origin was cells resident in the lung. It is possible that this apparent "acute" release could be due to the presence of PPE, or other contaminant, in the airways impacting on the zymography analysis; however, when we spiked a zymography sample with PPE (a 1:20 dilution, in RPMI, of the dose used in this study, which represents the dilution during the lavage) we did not detect any activity at the molecular weight of MMP-2 or MMP-9, nor did it appear to impact on standard MMP activity. The level of elastase activity in the lavage fluid after PPE returned to near normal levels within 24 hours of insult (Table 1). One caveat to these measurements is that the fluorogenic peptide used in the elastase measurements is also a substrate for other enzymes such as proteinase-3, which may have been increased as part of the inflammatory response.
Administration of PPE caused a significant increase in numbers of mucus-containing cells in the trachea and upper airways 48 hours after insult (3.2 ± 0.4 increased to 4.7 ± 0.6, and 1.3 ± 0.3 increased to 3.6 ± 0.5, mucus-containing cells per 250 e, respectively). An example of mucus staining in the trachea of vehicle- and PPE-exposed animals is shown in Figure 4.
Characterization of the Model: Time Course of the Chronic Phase
Assessment of the lungs 2, 4, 6, 8, and 10 weeks after PPE insult revealed significant changes in lung function. The PPE-treated lungs had significantly increased residual volume (FRC and RV; Figure 3), and significantly reduced development pressure and flow (Pmcmax and FEF25; Figure 3). These changes in lung function were temporally associated with changes in the lung structure as indicated by a significant increase in average air space area (3,002 ± 290 e2 increased to 8,415 ± 1081 e2 at 4 weeks; 2,860 ± 184 e2 increased to 8,253 ± 832 e2 at 8 weeks). There does appear to be some apparent differences in saline-treated lung function values over the time course; the reason for this is not clear but could be due to the inherent variation with in vivo studies and the fact that the lung function apparatus was freshly calibrated before each time point. Figure 4 shows histologic preparations of vehicle- and PPE-exposed lung samples.
In this chronic phase of the model we could not measure any differences in airway inflammatory cell number, cytokine levels, MMP levels, or numbers of mucus-containing cells between the two treated groups (data not shown).
Effect of Budesonide on the Acute Inflammatory Response after PPE
To rule out any direct effect of budesonide on PPE activity it was tested in a fluorimetric elastase assay. Budesonide had no effect on PPE activity when tested at 10 e (fluorescence: vehicle, 55,876 ± 528; budesonide, 57,543 ± 585) whereas 10 e elastase inhibitor III (Calbiochem; EMD Biosciences, San Diego, CA) significantly inhibited PPE activity (fluorescence: vehicle, 57,117 ± 1,684; budesonide, 2,091 ± 49).
In this study the insult with PPE caused significant increases in lung lumen and tissue neutrophils and LMN cell number (Figure 5). This was accompanied by a significant increase in MCP-1, IL-1, CINC-3, MMP-2, and MMP-9 levels in the lavage fluid (Table 3) and an increase in average air space area (2,501 ± 280 e2 increased to 6,141 ± 741 e2). These inflammatory indices were associated with a significant increase in goblet cells (Figure 5). There was no effect on the increase in airway neutrophil recruitment or on the increase in lavage biomarker levels with budesonide. However, budesonide treatment caused a significant reduction in tissue LMN cell number in the high-dose saline- and elastase-treated groups and an increase in IL-1 protein expression in the high-dose, saline-treated group (Figure 5 and Table 3). In addition, it did appear that steroid treatment caused a reduction in the number of goblet cells; however, this effect failed to reach significance.
Treatment with the higher doses of budesonide caused a significant loss of body weight (Table 3). Because of this dramatic loss in body weight with the higher doses of budesonide in the acute study it was decided that for the chronic studies a top dose of 0.3 mg/kg would be used.
Effect of Budesonide on Chronic Airway Changes after PPE
Satellite groups of vehicle- or PPE-treated animals had their airways assessed, 4 weeks after insult, to demonstrate that PPE-induced emphysema-like airway changes existed before starting treatment in the therapeutic protocol study. The rats treated with PPE had significantly higher FRC, RV, and average air space area compared with controls (2.5 ± 0.1 versus 3.9 ± 0.3 ml, 1.0 ± 0.2 versus 2.1 ± 0.3 ml, and 2,835 ± 239 versus 7,191 ± 535 e2, respectively). These changes were accompanied by significant reductions in Pmcmax and FEF25 (4.6 ± 0.3 versus 3.0 ± 0.3 cm H2O and 39.3 ± 6.4 versus 24.3 ± 2.7 ml/second, respectively).
Eight weeks after PPE insult the vehicle-treated rats in both the prophylactic (i.e., dosed throughout the 8 weeks) and therapeutic (i.e., dosed from Week 4) dosing protocol had significantly increased FRC, RV, and average air space area and significantly decreased Pmcmax and FEF25 compared with controls (Figure 6, prophylactic dosing, lung function; Figure 7, therapeutic dosing, lung function; and Figure 8, prophylactic and therapeutic dosing, average air space area). Treatment with budesonide caused a reduction in FRC, RV, and body weight in the noneCPPE-insulted group in the prophylactic study but not the therapeutic study. Budesonide treatment failed to impact on any lung function changes caused by PPE insult but did cause a significant dose-related increase in average air space area in the prophylactic study, but not in the therapeutic study.
There were no significant changes in lung tissue MMP-9 levels after prophylactic or therapeutic dosing of budesonide (prophylactic study: vehicle/non-PPE, 643 ± 153; budesonide [0.3 mg/kg]/non-PPE, 475 ± 118; vehicle/PPE, 1,137 ± 313; budesonide [0.3 mg/kg]/PPE, 1,031 ± 196 optical density. Therapeutic study: vehicle/non-PPE, 663 ± 105; budesonide [0.3 mg/kg]/non-PPE, 840 ± 168; vehicle/PPE, 896 ± 109; budesonide [0.3 mg/kg]/PPE, 725 ± 192 optical density).
NF-B Pathway Activation Levels in the Acute Inflammatory Phase
The level of NF-B pathway activation was assessed by measuring the amount of p65 able to bind to DNA, using a commercially available kit. The results show that there appears to be a reduction in NF-B pathway activation after PPE insult (2, 6, and 24 hours) compared with the vehicle controls, which returns to normal levels after 48 hours (Figure 9). It is possible that the PPE, or other contaminants, in the airways may have impacted on the measurement of NF-B pathway activation; however, a spiking experiment in which the assay standard or vehicle was added to the samples in the p65 assay suggests this was not the case. The results were as follows: 2-hour vehicle alone, 0.155 ± 0.015; 2-hour PPE alone, 0.066 ± 0.003; 2-hour vehicle plus assay standard, 0.315 ± 0.026; 2-hour PPE plus assay standard, 0.216 ± 0.012 absorbance value. The data demonstrate that the increase in p65:DNA binding after addition of the assay standard was similar in both treatment groups, suggesting that any contaminants present do not impact on the assay. To confirm the result observed with the plate assay, NF-B:DNA binding was also measured by EMSA. The results concur with the plate assay inasmuch as PPE treatment appeared not to induce NF-B pathway activation (Figure 9). To demonstrate the assays used would show an increase in NF-B pathway activation, data from separate assays on LPS-challenged lung tissue were included (Figure 9). The reason for the apparent difference between the elastase and LPS vehicle groups in the EMSAs is because it was necessary to develop the PPE study samples for a longer time to pick up any signal.
In the vehicle groups from the budesonide study, again there was no increase in p65:DNA binding and drug treatment appeared to cause a small reduction (Figure 10).
DISCUSSION
Instillation of PPE into the airways caused pronounced acute airway inflammation that resolved in about 1 week. This result is similar to that described in previous work (21, 23). PPE induced a significant increase in neutrophils and LMN cells, which were mainly macrophages, in both the lung lavage fluid and tissue. Exactly how PPE treatment causes this airway inflammation is not clear but it has been suggested that it could involve PPE-induced endothelial damage (34) and/or the generation of elastin fragments, which are known to be chemoattractants (35, 36). This acute cellular infiltration was accompanied by an increase in various cytokines, mucus-containing cell number, and proteolytic burden, all of which are also cardinal features observed in animal models of cigarette smoke exposure (37eC43) and in COPD (44, 45). Whereas the airway inflammation resolved after about 1 week, the increase in average air space area measured was maintained for up to 10 weeks. Exactly why some of the changes in average air space appear and are maintained after the inflammation is resolved is not known, but we would suggest that it takes time for changes in the parenchyma to complete after the PPE/inflammation and if it is possible for the rat airway to repair itself, this process takes longer than the length of this study. This damage to the lung parenchyma was associated with an alteration in lung function. PPE treatment increased lung residual volume and decreased lung function as described in similar animal models (27) and patients with COPD (44). It is not clear from these experiments whether PPE-induced parenchymal damage is a direct consequence of the elastolytic activity or an indirect result of PPE-induced inflammation. In a publication using a similar rodent model of experimental emphysema, using human neutrophil elastase as a stimulus, the authors suggest that the experimental emphysema is a result of both the direct and indirect actions of elastase (46).
Budesonide, which had no direct effect on PPE activity, had no impact on PPE-induced acute airway neutrophilia or inflammatory biomarker release. Steroid treatment has been shown to affect selected aspects of airway inflammation in COPD such as sputum production and mast cell number (13) and, indeed, in this model budesonide did reduce aspects of inflammation such as number of mucus-containing cells and airway tissue LMN cell number. However, the reduction in LMN cell number in lung tissue elicited by budesonide treatment was not accompanied by a fall in inflammatory biomarker levels, which could imply that tissue LMN cells were not responsible for the release of these biomarkers. A caveat to this is that we observed a significant fall in noneCPPE-treated tissue LMN cell number after budesonide treatment, which could mean there is a subset of airway tissue LMN cells that are steroid sensitive but not involved in the release of biomarkers in this model.
To attempt to determine the impact of steroid treatment on PPE-induced experimental emphysema we employed two dosing protocols: one was "prophylactic," in which compounds were administered before and continuously throughout the 8-week study. The other was "therapeutic," in which we started dosing the compounds after we had established experimental emphysema (4 weeks after PPE treatment) and then continued dosing for a further 4 weeks. Because of the dramatic loss in body weight observed with budesonide treatment in the acute dosing experiment we reduced the dose used in the chronic studies. Although therapeutic treatment with budesonide did not impact on PPE-induced changes in average air space area or lung function, it was interesting that prophylactic dosing significantly increased air space area. It is unclear why prophylactic but not therapeutic dosing causes this apparent worsening of experimental emphysema. It may be that long-term therapy with a steroid is needed to induce these changes and that the 4 weeks of drug administration was not sufficient. Another possible reason for this enhanced PPE-induced emphysema with prophylactically dosed budesonide could be that the steroid is causing an increase in aspects of the inflammatory phase; indeed, a possible example of this is the increased IL-1 levels in the airway after steroid treatment. However, the same increase in IL-1 levels was also measured in the budesonide-treated, noneCPPE-exposed animals, which did not have increased air space area, and the dose of steroid used in the chronic studies was 10 time less. However, it should be noted that the increase in air space area elicited by elastase in the prophylactic treatment group was reduced compared with the therapeutic treatment group and as such may be more easily modulated by steroid treatment.
How prophylactic treatment with budesonide causes the enhanced PPE-induced experimental emphysema is not known. A report by Choe and coworkers (47) suggests that methylprednisolone causes emphysema-like changes in the lungs of rats as the result of an increase in MMP. They demonstrate, by zymography, an increase in MMP-9 after chronic treatment with methylprednisolone. We feel that, in this model, steroid induction of MMP-9 is not the cause of enhanced air space area because we did not observe an increase in MMP-9 in our lung samples and the budesonide-treated controls did not have experimental emphysema. Another possible reason for the enhanced experimental emphysema with the high dose of budesonide in the prophylactic, and not the therapeutic, dosing protocol is the loss of body weight observed. It is well established that starvation in rats is associated with the development of experimental emphysema (48eC51). Indeed, even in humans, it has been shown that anorexia is associated with emphysema-like changes (52). Although the loss of body weight is not due to starvation, it could be impacting on the lungs. A similar loss of body weight was observed in the intratracheal vehicle-treated, high-dose budesonide group, in which we did not observed any change in average air space area; however, the enhancement could be due to a combination of the body weight loss and PPE.
Although we measured an increase in air space area after prophylactic treatment with budesonide, there was no corresponding change in lung function. This may be because the changes in air space area were too small to impact on lung function or because the two measurements are not related. Another possible reason for the apparent lack of change in lung function could be related to the effect of steroid treatment on body weight. The chronic treatment of control animals with steroid resulted in a reduced residual volume, which may be related to the loss in overall body weight. Therefore, it is possible that no overall change in lung volume measurements was observed after chronic steroid dosing in PPE-treated rats because of the two opposing affects.
The effect of chronic steroid therapy on air space area is of concern when considering the clinical scenario. This result may possibly be mirrored in patients with COPD, in whom although the effectiveness of glucocorticoids is questionable, steroid treatment is still the mainstay chronic therapy. The effectiveness of any therapy in patients with COPD is normally monitored by measuring parameters such as lung function and symptoms and not by histologic lung assessment. Therefore, if long-term steroid therapy in patients with COPD has the same effect on the lung structure as it does in this model then it may possibly be detrimental to the patient. However, extrapolation of these findings in this study to human disease may be overreaching as the mechanism of PPE-induced experimental emphysema is likely to differ from the slowly developing cigarette smokeeCinduced disease in humans. In addition, steroids inhibit alveolarization and as this continues postpartum in rats it may explain the increase in air space observed.
In an attempt to determine why aspects of the COPD-like inflammation were resistant to steroid treatment we measured the levels of NF-B activation by determining the amount of p65 able to bind to DNA. Under normal conditions NF-B is unable to enter the nucleus because it is bound to I-B; however, once the NF-B pathway is stimulated, the inhibitory subunit is removed, allowing NF-B to enter the nucleus and bind to its consensus sequence on DNA. The p65 subunit of NF-B is abundant, which is why we chose it as a marker of NF-B pathway activation. Using this method, our group has shown an increase in NF-B pathway activation in rat lung tissue after exposure to antigen (53) and LPS (54). In this model of COPD, however, after PPE insult the level of p65:DNA binding was actually significantly reduced. To back up this finding we performed EMSA analysis of the same samples. The results showed there was no increase in NF-B pathway activation at the time points measured. Unlike the results of p65:DNA binding, the EMSA results did not show a decrease after PPE treatment. The reason for this could be that the EMSA is less sensitive or that analysis by EMSA measures DNA binding of all Rel family members. This would imply that aspects of the inflammation seen in this preclinical model are NF-B independent. This suggestion is further strengthened by the lack of effect of budesonide, in that although the exact antiinflammatory mechanism of action of steroids is unknown, they are believed to act by somehow impacting on the actions of proinflammatory transcription factors such as NF-B (55). Indeed, the antigen- and LPS-driven airway inflammation in rodent models mentioned above, in which we observed an increase in NF-B pathway activation, were both sensitive to treatment with a steroid. These data would suggest that COPD-like inflammation could be developed without involving NF-B, which may explain the resistance to steroid treatment seen in the clinic. Paradoxically, there have been many reports in which an increase in NF-B:DNA binding has been seen in cigarette smoke-exposed rodent models of COPD and in lung biopsy samples from patients with COPD; see Marwick and coworkers (56) and Caramori and coworkers (57). The current dogma suggests that the normal steroid-induced control of NF-B-induced inflammation is altered in patients with COPD (58). This may suggest that this model does not completely mimic all the aspects of the situation in the clinic, which is often the case with preclinical in vivo models. However, we suggest a possible scenario in which aspects of the inflammation observed in COPD could be NF-B independent, possibly suggesting that the steroid resistance and the increase in NF-B pathway activation reported are due to the multitude of biologically active components in cigarette smoke. Also, the reported increase in NF-B:DNA binding observed in clinical samples taken from patients with COPD could be due to infections and increased bacterial load prevalent in this disease. Caramori and coworkers (57) published clinical data that goes some way to support our proposed theory in that they show no nuclear p65 staining in neutrophils from patients with COPD, which interestingly is the cell type resistant to steroids in this model. In addition, although Di Stefano and coworkers (59) demonstrate an increase in expression of NF-B in the lungs of patients with COPD, they also show an increase in cigarette-smoking nondiseased controls, suggesting the increase may not be related to the disease but to smoking cigarettes.
In summary, we have characterized an elastase-driven rodent model of experimental emphysema, confirming previous findings of acute COPD-like airway inflammation and enlargement of air space area, which is associated with emphysema-like changes in lung function. We have also shown that, as in the inflammation seen in COPD, aspects of PPE-induced airway inflammation are steroid resistant. This inflammation appears to be associated with a lack of NF-B pathway activation, suggesting a possible reason for the lack of effect of steroid therapy in patients with COPD. In addition, we found that chronic treatment with a glucocorticoid actually increased the PPE-induced experimental emphysema. This result is of concern if it translates to patients with COPD, as steroids are the mainstay therapy for the treatment of this disease.
In addition to the suitability of this model for testing therapies that modify existing airway structural and functional parameters in experimental emphysema, we suggest that this model may be useful to understand the steroid-resistant nature of the airway inflammation seen in this model and in COPD to develop new treatment paradigms.
Acknowledgments
The authors thank Mr. S. Bottoms and Dr. R. McAnulty for histologic processing, staining, and quantification of the air space area and number of goblet cells. The authors are also grateful to Miss H. Keen for help with the preparation of this manuscript. The authors thank Buxco Europe Ltd. for the loan of a forced maneuvers machine
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ABSTRACT
Rationale: Emphysema is one component of chronic obstructive pulmonary disease (COPD), a respiratory disease currently increasing in prevalence worldwide. The mainstay therapy adopted to treat patients with COPD is glucocorticoids; unfortunately, this treatment has limited impact on disease symptoms or underlying airway inflammation. Objective: There is an urgent need to develop therapies that modify both the underlying inflammation, thought to be involved in disease progression, and the structural changes in the emphysematous lung. Methods: We have characterized an elastase-driven model of experimental emphysema in the rat that demonstrates COPD-like airway inflammation and determined the impact of a clinically relevant glucocorticoid. Measurements and main results: We observed an increase in lung neutrophils, lymphomononuclear cells, mucus production, and inflammatory cytokines. Also present were increases in average air space area, which are associated with emphysema-like changes in lung function, such as increased residual volume and decreased flow; these increases in area were maintained for up to 10 weeks. In addition, we observed that elastase-induced airway neutrophilia is steroid resistant. Interestingly, the inflammation observed after elastase administration was found to be temporally associated with a lack of nuclear factor-B pathway activation. This apparent nuclear factor-BeCindependent inflammation may explain why treatment with a glucocorticoid was ineffective in this preclinical model and could suggest parallels in the steroid-resistant human disease. Conclusion: We believe that this model, in addition to its suitability for testing therapies that may modify existing emphysema, could be useful in the search for new therapies to reduce the steroid-resistant airway inflammation evident in COPD.
Key Words: inflammation; lung; rodent; steroid
Pulmonary emphysema is a disease of the parenchyma, which is characterized by the destruction of alveolar septa resulting in abnormal and permanent enlargement of the air spaces (1). This disease, along with chronic bronchitis, is a component of chronic obstructive pulmonary disease (COPD), which is currently the fourth leading cause of death in the United States, with up to 7 million people diagnosed each year (2). It is predicted by the World Health Organization that the prevalence of COPD will increase in the coming years to become the fifth most common cause of morbidity and the third most common chronic disease worldwide. Patients with COPD have progressive airflow limitation leading to disabling dyspnea, with an inability to properly oxygenate the blood. Although the development of COPD is known to be linked to cigarette smoke, little is known about the pathogenesis of the disease. At present there are a number of theories as to how chronic cigarette smoking causes emphysema; these include an altered proteaseeCantiprotease balance (3), increased inflammatory cell burden (4), enhanced oxidative stress levels (5), and raised alveolar septal cell apoptosis (6). Whether some, all, or none of these theories are involved in the breakdown of the lung structure seen in COPD is not known, but it is believed that understanding the pathogenesis of the disease process is central to the development of appropriate new therapies (7).
Current COPD therapy is limited to treatment of symptoms, with the mainstay being drugs that have been developed to treat asthma, long-acting bronchodilators and corticosteroids (8). The use of inhaled corticosteroids to treat COPD is controversial with some studies claiming efficacy and others not (9eC12). A study by Hattotuwa (13) showed that treatment with inhaled fluticasone resulted in small improvements in aspects of COPD including symptom score, exacerbations, and sputum production. However, treatment with corticosteroids has been shown to have no impact on the cellular inflammation or increased protease burden seen in COPD (13, 14). In addition, unlike cytokine release from cells collected from nondiseased airways, the same cells from patients with COPD have been shown to be resistant to steroid treatment (15, 16). There are also no therapies currently available that have been shown to halt the relentless decline in lung function and the progressive damage of the lung parenchyma seen in COPD (17). It is commonly believed that if a therapy can be found to impact on the airway inflammation seen in COPD then that therapy may slow the progression of the disease, although a direct correlation is yet to be established and indeed they may well be dissociated. There is therefore a need for fully characterized, steroid-insensitive, in vivo models of COPD to aid in the understanding of the pathogenesis of the disease and for the discovery of new antiinflammatory treatments.
It has previously been demonstrated that exposing the lungs to porcine pancreatic elastase (PPE), an enzyme that acts predominantly, although not exclusively, on elastin (18), induces acute airway inflammation (19eC21) followed by emphysema (22eC26) and compromised lung function (27, 28). The aim of this study was to perform a comprehensive characterization of the effects of elastase on lung inflammation while confirming the data obtained by others regarding the structural and functional changes. Our findings concur with others in that we have demonstrated that PPE treatment caused acute inflammation followed by increases in average airspace area and that this was maintained over at least a 10-week period and was associated with changes in lung function measurement.
To date there are no publications assessing the impact of corticosteroids on elastase-induced airway inflammation and experimental emphysema. Therefore, a secondary aim of this study was to determine the impact of a glucocorticoid, budesonide, in this model. Some of the results of this study have been reported in the form of an abstract (29).
METHODS
Model Characterization
Male Sprague-Dawley rats (280eC320 g) were purchased from Harlan UK (Bicester, UK). Home Office guidelines for animal welfare, based on the Animals (Scientific Procedures) Act 1986, were strictly observed. Experimental emphysema was induced by instilling one dose of PPE (120 U/kg, freshly prepared in saline, administered intratracheally at 1 ml/kg) directly into the airways; control groups were given saline in the same way at the same time. At various time points (either 2, 6, 24, 48, 72, 96, and 168 hours or 2, 4, 6, 8, and 10 weeks after treatment) the animals were culled and the inflammatory status of the airways was determined. Airway lumen and tissue differential white cell count, histology of lung tissue samples, and protein expression levels were determined by methods detailed in McCluskie and coworkers (30). Matrix metalloproteinase (MMP-2 and MMP-9) levels were determined in the lavage supernatant according to the method of Birrell and coworkers (31). Elastase activity in lavage samples was determined by a fluorogenic assay outlined by Trifilieff and coworkers (32). The level of nuclear factor (NF)-B pathway activation was determined in lung tissue with an Active Motif (Carlsbad, CA) kit, which measures p65:DNA binding, in accordance with the manufacturer's instructions. NF-B:DNA binding was also determined by electrophoretic mobility shift assay (EMSA). The number of mucus-containing cells was determined in the trachea by periodic acideCSchiff staining and manually counting 240-e lengths of airway epithelium. Fifty readings were taken and averaged per trachea.
In satellite groups (4 and 8 weeks after vehicle or PPE administration), changes in air space area were measured by computer-assisted image analysis as described by Belloni and coworkers (33). Quantification was performed with an Olympus BX40 microscope using Zeiss KS300 image-processing software (Imaging Associates, Bicester, UK). Using color-thresholding techniques the total parenchymal air spaces in square micrometers and the number of air spaces were measured from 10 random fields per slide/lobe. From these figures the mean air space area for each field can be calculated. Any fields containing airways or vasculature were excluded.
Lung function was assessed (2, 4, 6, 8, and 10 weeks after vehicle or PPE treatment) in anesthetized rats (ketamine-HCl and xylazine-HCl, 144 and 10 mg/kg, respectively, administered intraperitoneally), using a forced maneuvers system supplied by Buxco Europe (Winchester, UK). Functional residual capacity (FRC) was calculated by the software, using Boyle's law, on the basis of data collected after occluding the airway at the end of expiration and measuring the pressure and thoracic displacement. Residual volume (RV) was calculated by subtracting the vital capacity from the total lung capacity (TLC), determined by the pressureeCvolume maneuver. During the same maneuver, peak compliance was determined, at which point the pressure was recorded:—pressure at maximum compliance (Pmcmax). The forced expiration maneuver determines the amount of air expired during a fast expiration from TLC and is called forced expiratory capacity (FEC). A curve was plotted of expired volume versus flow, resulting in a floweCvolume curve. On this plot, when 25% of the FEC has been expired the instantaneous flow is said to be FEF25. This range of measurements enabled the assessment of the impact of PPE treatment on lung volume, pressure, and flow.
Effect of Budesonide on PPE-induced Inflammation and Emphysema
To determine the impact of budesonide on the early inflammatory response after PPE exposure, vehicle (0.5% methylcellulose and 0.2% Tween 80 in water, 2 ml/kg) or compound was administered (0.1eC3 mg/kg, per os) twice daily before and after PPE. Forty-eight hours after PPE administration the lungs were assessed for cellular infiltration, cytokine content, MMP levels, and goblet cell staining.
Two dosing protocols were used to determine the impact of budesonide on chronic airway changes after PPE. The first protocol was thought of as a "prophylactic" dosing protocol, in which vehicle or compound (0.03eC0.3 mg/kg, per os) was administered throughout the 8-week study; the second, "therapeutic" protocol consisted of dosing starting 4 weeks after PPE insult. Eight weeks after PPE insult the rats had their lung function measured and the average air space area was determined. Two satellite groups were incorporated to establish experimental emphysema 4 weeks after PPE treatment, that is, at the time at which the therapeutic dosing protocol began.
RESULTS
Treatment of the rats with this dose of PPE did not cause mortality in any of the studies performed.
Characterization of the Model: Time Course of the Acute Inflammatory Phase
Treatment of the rats with PPE caused significant increases in neutrophil and lymphomononuclear (LMN; i.e., monocytes, macrophages, and lymphocytes) cell recruitment into the airway lumen and tissue. The peak of inflammatory cell burden in the airway appears between 24 and 48 hours after PPE insult, with levels returning to that of the controls by Day 7 (Figures 1 and 4). The vehicle for elastase appeared to cause an increase in lung tissue LMN cell numbers 4 and 7 days after treatment when compared with the earlier time points (Figure 1D); the reason for this is not known but could be due to day-to-day differences in tissue cell recovery seen with the tissue-digesting technique employed. This increase in inflammatory cell number was accompanied by a significant increase in tumor necrosis factor-, interleukin (IL)-1, monocyte chemoattractant protein (MCP)-1, and cytokine-induced chemoattractant (CINC)-1, CINC-2, and CINC-3 protein levels in lavage fluid and IL-1, MCP-1, CINC-1, and CINC-2 protein levels in lung tissue (Figure 2 and Tables 1 and 2). Levels of MMP-2 and MMP-9 were significantly increased in the lung lavage fluid (Figure 2). Interestingly, there appeared to be an increase in MMP activity before recruitment of the majority of inflammatory cells, which may suggest that the origin was cells resident in the lung. It is possible that this apparent "acute" release could be due to the presence of PPE, or other contaminant, in the airways impacting on the zymography analysis; however, when we spiked a zymography sample with PPE (a 1:20 dilution, in RPMI, of the dose used in this study, which represents the dilution during the lavage) we did not detect any activity at the molecular weight of MMP-2 or MMP-9, nor did it appear to impact on standard MMP activity. The level of elastase activity in the lavage fluid after PPE returned to near normal levels within 24 hours of insult (Table 1). One caveat to these measurements is that the fluorogenic peptide used in the elastase measurements is also a substrate for other enzymes such as proteinase-3, which may have been increased as part of the inflammatory response.
Administration of PPE caused a significant increase in numbers of mucus-containing cells in the trachea and upper airways 48 hours after insult (3.2 ± 0.4 increased to 4.7 ± 0.6, and 1.3 ± 0.3 increased to 3.6 ± 0.5, mucus-containing cells per 250 e, respectively). An example of mucus staining in the trachea of vehicle- and PPE-exposed animals is shown in Figure 4.
Characterization of the Model: Time Course of the Chronic Phase
Assessment of the lungs 2, 4, 6, 8, and 10 weeks after PPE insult revealed significant changes in lung function. The PPE-treated lungs had significantly increased residual volume (FRC and RV; Figure 3), and significantly reduced development pressure and flow (Pmcmax and FEF25; Figure 3). These changes in lung function were temporally associated with changes in the lung structure as indicated by a significant increase in average air space area (3,002 ± 290 e2 increased to 8,415 ± 1081 e2 at 4 weeks; 2,860 ± 184 e2 increased to 8,253 ± 832 e2 at 8 weeks). There does appear to be some apparent differences in saline-treated lung function values over the time course; the reason for this is not clear but could be due to the inherent variation with in vivo studies and the fact that the lung function apparatus was freshly calibrated before each time point. Figure 4 shows histologic preparations of vehicle- and PPE-exposed lung samples.
In this chronic phase of the model we could not measure any differences in airway inflammatory cell number, cytokine levels, MMP levels, or numbers of mucus-containing cells between the two treated groups (data not shown).
Effect of Budesonide on the Acute Inflammatory Response after PPE
To rule out any direct effect of budesonide on PPE activity it was tested in a fluorimetric elastase assay. Budesonide had no effect on PPE activity when tested at 10 e (fluorescence: vehicle, 55,876 ± 528; budesonide, 57,543 ± 585) whereas 10 e elastase inhibitor III (Calbiochem; EMD Biosciences, San Diego, CA) significantly inhibited PPE activity (fluorescence: vehicle, 57,117 ± 1,684; budesonide, 2,091 ± 49).
In this study the insult with PPE caused significant increases in lung lumen and tissue neutrophils and LMN cell number (Figure 5). This was accompanied by a significant increase in MCP-1, IL-1, CINC-3, MMP-2, and MMP-9 levels in the lavage fluid (Table 3) and an increase in average air space area (2,501 ± 280 e2 increased to 6,141 ± 741 e2). These inflammatory indices were associated with a significant increase in goblet cells (Figure 5). There was no effect on the increase in airway neutrophil recruitment or on the increase in lavage biomarker levels with budesonide. However, budesonide treatment caused a significant reduction in tissue LMN cell number in the high-dose saline- and elastase-treated groups and an increase in IL-1 protein expression in the high-dose, saline-treated group (Figure 5 and Table 3). In addition, it did appear that steroid treatment caused a reduction in the number of goblet cells; however, this effect failed to reach significance.
Treatment with the higher doses of budesonide caused a significant loss of body weight (Table 3). Because of this dramatic loss in body weight with the higher doses of budesonide in the acute study it was decided that for the chronic studies a top dose of 0.3 mg/kg would be used.
Effect of Budesonide on Chronic Airway Changes after PPE
Satellite groups of vehicle- or PPE-treated animals had their airways assessed, 4 weeks after insult, to demonstrate that PPE-induced emphysema-like airway changes existed before starting treatment in the therapeutic protocol study. The rats treated with PPE had significantly higher FRC, RV, and average air space area compared with controls (2.5 ± 0.1 versus 3.9 ± 0.3 ml, 1.0 ± 0.2 versus 2.1 ± 0.3 ml, and 2,835 ± 239 versus 7,191 ± 535 e2, respectively). These changes were accompanied by significant reductions in Pmcmax and FEF25 (4.6 ± 0.3 versus 3.0 ± 0.3 cm H2O and 39.3 ± 6.4 versus 24.3 ± 2.7 ml/second, respectively).
Eight weeks after PPE insult the vehicle-treated rats in both the prophylactic (i.e., dosed throughout the 8 weeks) and therapeutic (i.e., dosed from Week 4) dosing protocol had significantly increased FRC, RV, and average air space area and significantly decreased Pmcmax and FEF25 compared with controls (Figure 6, prophylactic dosing, lung function; Figure 7, therapeutic dosing, lung function; and Figure 8, prophylactic and therapeutic dosing, average air space area). Treatment with budesonide caused a reduction in FRC, RV, and body weight in the noneCPPE-insulted group in the prophylactic study but not the therapeutic study. Budesonide treatment failed to impact on any lung function changes caused by PPE insult but did cause a significant dose-related increase in average air space area in the prophylactic study, but not in the therapeutic study.
There were no significant changes in lung tissue MMP-9 levels after prophylactic or therapeutic dosing of budesonide (prophylactic study: vehicle/non-PPE, 643 ± 153; budesonide [0.3 mg/kg]/non-PPE, 475 ± 118; vehicle/PPE, 1,137 ± 313; budesonide [0.3 mg/kg]/PPE, 1,031 ± 196 optical density. Therapeutic study: vehicle/non-PPE, 663 ± 105; budesonide [0.3 mg/kg]/non-PPE, 840 ± 168; vehicle/PPE, 896 ± 109; budesonide [0.3 mg/kg]/PPE, 725 ± 192 optical density).
NF-B Pathway Activation Levels in the Acute Inflammatory Phase
The level of NF-B pathway activation was assessed by measuring the amount of p65 able to bind to DNA, using a commercially available kit. The results show that there appears to be a reduction in NF-B pathway activation after PPE insult (2, 6, and 24 hours) compared with the vehicle controls, which returns to normal levels after 48 hours (Figure 9). It is possible that the PPE, or other contaminants, in the airways may have impacted on the measurement of NF-B pathway activation; however, a spiking experiment in which the assay standard or vehicle was added to the samples in the p65 assay suggests this was not the case. The results were as follows: 2-hour vehicle alone, 0.155 ± 0.015; 2-hour PPE alone, 0.066 ± 0.003; 2-hour vehicle plus assay standard, 0.315 ± 0.026; 2-hour PPE plus assay standard, 0.216 ± 0.012 absorbance value. The data demonstrate that the increase in p65:DNA binding after addition of the assay standard was similar in both treatment groups, suggesting that any contaminants present do not impact on the assay. To confirm the result observed with the plate assay, NF-B:DNA binding was also measured by EMSA. The results concur with the plate assay inasmuch as PPE treatment appeared not to induce NF-B pathway activation (Figure 9). To demonstrate the assays used would show an increase in NF-B pathway activation, data from separate assays on LPS-challenged lung tissue were included (Figure 9). The reason for the apparent difference between the elastase and LPS vehicle groups in the EMSAs is because it was necessary to develop the PPE study samples for a longer time to pick up any signal.
In the vehicle groups from the budesonide study, again there was no increase in p65:DNA binding and drug treatment appeared to cause a small reduction (Figure 10).
DISCUSSION
Instillation of PPE into the airways caused pronounced acute airway inflammation that resolved in about 1 week. This result is similar to that described in previous work (21, 23). PPE induced a significant increase in neutrophils and LMN cells, which were mainly macrophages, in both the lung lavage fluid and tissue. Exactly how PPE treatment causes this airway inflammation is not clear but it has been suggested that it could involve PPE-induced endothelial damage (34) and/or the generation of elastin fragments, which are known to be chemoattractants (35, 36). This acute cellular infiltration was accompanied by an increase in various cytokines, mucus-containing cell number, and proteolytic burden, all of which are also cardinal features observed in animal models of cigarette smoke exposure (37eC43) and in COPD (44, 45). Whereas the airway inflammation resolved after about 1 week, the increase in average air space area measured was maintained for up to 10 weeks. Exactly why some of the changes in average air space appear and are maintained after the inflammation is resolved is not known, but we would suggest that it takes time for changes in the parenchyma to complete after the PPE/inflammation and if it is possible for the rat airway to repair itself, this process takes longer than the length of this study. This damage to the lung parenchyma was associated with an alteration in lung function. PPE treatment increased lung residual volume and decreased lung function as described in similar animal models (27) and patients with COPD (44). It is not clear from these experiments whether PPE-induced parenchymal damage is a direct consequence of the elastolytic activity or an indirect result of PPE-induced inflammation. In a publication using a similar rodent model of experimental emphysema, using human neutrophil elastase as a stimulus, the authors suggest that the experimental emphysema is a result of both the direct and indirect actions of elastase (46).
Budesonide, which had no direct effect on PPE activity, had no impact on PPE-induced acute airway neutrophilia or inflammatory biomarker release. Steroid treatment has been shown to affect selected aspects of airway inflammation in COPD such as sputum production and mast cell number (13) and, indeed, in this model budesonide did reduce aspects of inflammation such as number of mucus-containing cells and airway tissue LMN cell number. However, the reduction in LMN cell number in lung tissue elicited by budesonide treatment was not accompanied by a fall in inflammatory biomarker levels, which could imply that tissue LMN cells were not responsible for the release of these biomarkers. A caveat to this is that we observed a significant fall in noneCPPE-treated tissue LMN cell number after budesonide treatment, which could mean there is a subset of airway tissue LMN cells that are steroid sensitive but not involved in the release of biomarkers in this model.
To attempt to determine the impact of steroid treatment on PPE-induced experimental emphysema we employed two dosing protocols: one was "prophylactic," in which compounds were administered before and continuously throughout the 8-week study. The other was "therapeutic," in which we started dosing the compounds after we had established experimental emphysema (4 weeks after PPE treatment) and then continued dosing for a further 4 weeks. Because of the dramatic loss in body weight observed with budesonide treatment in the acute dosing experiment we reduced the dose used in the chronic studies. Although therapeutic treatment with budesonide did not impact on PPE-induced changes in average air space area or lung function, it was interesting that prophylactic dosing significantly increased air space area. It is unclear why prophylactic but not therapeutic dosing causes this apparent worsening of experimental emphysema. It may be that long-term therapy with a steroid is needed to induce these changes and that the 4 weeks of drug administration was not sufficient. Another possible reason for this enhanced PPE-induced emphysema with prophylactically dosed budesonide could be that the steroid is causing an increase in aspects of the inflammatory phase; indeed, a possible example of this is the increased IL-1 levels in the airway after steroid treatment. However, the same increase in IL-1 levels was also measured in the budesonide-treated, noneCPPE-exposed animals, which did not have increased air space area, and the dose of steroid used in the chronic studies was 10 time less. However, it should be noted that the increase in air space area elicited by elastase in the prophylactic treatment group was reduced compared with the therapeutic treatment group and as such may be more easily modulated by steroid treatment.
How prophylactic treatment with budesonide causes the enhanced PPE-induced experimental emphysema is not known. A report by Choe and coworkers (47) suggests that methylprednisolone causes emphysema-like changes in the lungs of rats as the result of an increase in MMP. They demonstrate, by zymography, an increase in MMP-9 after chronic treatment with methylprednisolone. We feel that, in this model, steroid induction of MMP-9 is not the cause of enhanced air space area because we did not observe an increase in MMP-9 in our lung samples and the budesonide-treated controls did not have experimental emphysema. Another possible reason for the enhanced experimental emphysema with the high dose of budesonide in the prophylactic, and not the therapeutic, dosing protocol is the loss of body weight observed. It is well established that starvation in rats is associated with the development of experimental emphysema (48eC51). Indeed, even in humans, it has been shown that anorexia is associated with emphysema-like changes (52). Although the loss of body weight is not due to starvation, it could be impacting on the lungs. A similar loss of body weight was observed in the intratracheal vehicle-treated, high-dose budesonide group, in which we did not observed any change in average air space area; however, the enhancement could be due to a combination of the body weight loss and PPE.
Although we measured an increase in air space area after prophylactic treatment with budesonide, there was no corresponding change in lung function. This may be because the changes in air space area were too small to impact on lung function or because the two measurements are not related. Another possible reason for the apparent lack of change in lung function could be related to the effect of steroid treatment on body weight. The chronic treatment of control animals with steroid resulted in a reduced residual volume, which may be related to the loss in overall body weight. Therefore, it is possible that no overall change in lung volume measurements was observed after chronic steroid dosing in PPE-treated rats because of the two opposing affects.
The effect of chronic steroid therapy on air space area is of concern when considering the clinical scenario. This result may possibly be mirrored in patients with COPD, in whom although the effectiveness of glucocorticoids is questionable, steroid treatment is still the mainstay chronic therapy. The effectiveness of any therapy in patients with COPD is normally monitored by measuring parameters such as lung function and symptoms and not by histologic lung assessment. Therefore, if long-term steroid therapy in patients with COPD has the same effect on the lung structure as it does in this model then it may possibly be detrimental to the patient. However, extrapolation of these findings in this study to human disease may be overreaching as the mechanism of PPE-induced experimental emphysema is likely to differ from the slowly developing cigarette smokeeCinduced disease in humans. In addition, steroids inhibit alveolarization and as this continues postpartum in rats it may explain the increase in air space observed.
In an attempt to determine why aspects of the COPD-like inflammation were resistant to steroid treatment we measured the levels of NF-B activation by determining the amount of p65 able to bind to DNA. Under normal conditions NF-B is unable to enter the nucleus because it is bound to I-B; however, once the NF-B pathway is stimulated, the inhibitory subunit is removed, allowing NF-B to enter the nucleus and bind to its consensus sequence on DNA. The p65 subunit of NF-B is abundant, which is why we chose it as a marker of NF-B pathway activation. Using this method, our group has shown an increase in NF-B pathway activation in rat lung tissue after exposure to antigen (53) and LPS (54). In this model of COPD, however, after PPE insult the level of p65:DNA binding was actually significantly reduced. To back up this finding we performed EMSA analysis of the same samples. The results showed there was no increase in NF-B pathway activation at the time points measured. Unlike the results of p65:DNA binding, the EMSA results did not show a decrease after PPE treatment. The reason for this could be that the EMSA is less sensitive or that analysis by EMSA measures DNA binding of all Rel family members. This would imply that aspects of the inflammation seen in this preclinical model are NF-B independent. This suggestion is further strengthened by the lack of effect of budesonide, in that although the exact antiinflammatory mechanism of action of steroids is unknown, they are believed to act by somehow impacting on the actions of proinflammatory transcription factors such as NF-B (55). Indeed, the antigen- and LPS-driven airway inflammation in rodent models mentioned above, in which we observed an increase in NF-B pathway activation, were both sensitive to treatment with a steroid. These data would suggest that COPD-like inflammation could be developed without involving NF-B, which may explain the resistance to steroid treatment seen in the clinic. Paradoxically, there have been many reports in which an increase in NF-B:DNA binding has been seen in cigarette smoke-exposed rodent models of COPD and in lung biopsy samples from patients with COPD; see Marwick and coworkers (56) and Caramori and coworkers (57). The current dogma suggests that the normal steroid-induced control of NF-B-induced inflammation is altered in patients with COPD (58). This may suggest that this model does not completely mimic all the aspects of the situation in the clinic, which is often the case with preclinical in vivo models. However, we suggest a possible scenario in which aspects of the inflammation observed in COPD could be NF-B independent, possibly suggesting that the steroid resistance and the increase in NF-B pathway activation reported are due to the multitude of biologically active components in cigarette smoke. Also, the reported increase in NF-B:DNA binding observed in clinical samples taken from patients with COPD could be due to infections and increased bacterial load prevalent in this disease. Caramori and coworkers (57) published clinical data that goes some way to support our proposed theory in that they show no nuclear p65 staining in neutrophils from patients with COPD, which interestingly is the cell type resistant to steroids in this model. In addition, although Di Stefano and coworkers (59) demonstrate an increase in expression of NF-B in the lungs of patients with COPD, they also show an increase in cigarette-smoking nondiseased controls, suggesting the increase may not be related to the disease but to smoking cigarettes.
In summary, we have characterized an elastase-driven rodent model of experimental emphysema, confirming previous findings of acute COPD-like airway inflammation and enlargement of air space area, which is associated with emphysema-like changes in lung function. We have also shown that, as in the inflammation seen in COPD, aspects of PPE-induced airway inflammation are steroid resistant. This inflammation appears to be associated with a lack of NF-B pathway activation, suggesting a possible reason for the lack of effect of steroid therapy in patients with COPD. In addition, we found that chronic treatment with a glucocorticoid actually increased the PPE-induced experimental emphysema. This result is of concern if it translates to patients with COPD, as steroids are the mainstay therapy for the treatment of this disease.
In addition to the suitability of this model for testing therapies that modify existing airway structural and functional parameters in experimental emphysema, we suggest that this model may be useful to understand the steroid-resistant nature of the airway inflammation seen in this model and in COPD to develop new treatment paradigms.
Acknowledgments
The authors thank Mr. S. Bottoms and Dr. R. McAnulty for histologic processing, staining, and quantification of the air space area and number of goblet cells. The authors are also grateful to Miss H. Keen for help with the preparation of this manuscript. The authors thank Buxco Europe Ltd. for the loan of a forced maneuvers machine
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