Roflumilast Fully Prevents Emphysema in Mice Chronically Exposed to Cigarette Smoke
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美国呼吸和危急护理医学 2005年第10期
Department of Physiopathology and Experimental Medicine, University of Siena, Siena, Italy
ALTANA Pharma, Konstanz, Germany
ABSTRACT
Rationale: There is a need for new agents capable of suppressing the inflammatory response in chronic obstructive pulmonary disease. Objectives: This study evaluated the effects of roflumilast, a phosphodiesterase 4 (PDE4) inhibitor on acute lung inflammation and chronic lung changes in models of cigarette exposure in mice.
Methods: Roflumilast was given orally either at 1 mg/kg (R1) or at 5 mg/kg (R5). In the acute model (five cigarettes for 20 minutes), bronchoalveolar lavage fluid (BALF) changes were investigated at 4 and 24 hours. In the chronic model (three cigarettes/day for 7 months), morphometric and biochemical parameters were assessed at 7 months.
Measurements and Main Results: Acute exposure caused a fivefold increase in BALF neutrophils. Both doses of roflumilast partially prevented (by 30%) this increase. In addition, after smoke exposure, R1 increased BALF interleukin-10 by 79% and R5 by 129%. Chronic smoke exposure caused a 1.8-fold increase in lung macrophage density, emphysema, an increase of the mean linear intercept (+21%), a decrease of the internal surface area (eC13%), and a drop (eC13%) in lung desmosine content. R1 did not have any effect, whereas R5 prevented the increase in lung macrophage density by 70% and fully prevented the other changes. In addition, in the smoke-exposure group, 63% of the mice showed goblet cell metaplasia, and neither of the doses of roflumilast had any effect.
Conclusions: This study shows for the first time that a PDE4 inhibitor partially ameliorates lung inflammation and fully prevents parenchymal destruction induced by cigarette smoke.
Key Words: chronic obstructive pulmonary disease interleukin-10 lung inflammation
Chronic obstructive pulmonary disease (COPD) is a rapidly increasing global health problem (1). It includes emphysema, which is characterized by airspace enlargement and destruction of lung parenchyma, and chronic bronchitis. Recently, COPD has been defined by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) as a disease characterized by progressive, not fully reversible, flow limitation and "associated with an abnormal inflammatory response of the lungs to noxious particles and gases" (2). Thus, a central role has been attributed to the chronic inflammatory response that is present throughout the airways and parenchyma and that participates in the progression and exacerbation of this disease (3). Cigarette smoke has been identified as the most important risk for the development of COPD (4).
The therapeutic approach to COPD is mainly supportive and symptomatic (bronchodilators) (5). In an approach aiming at modulating the chronic inflammatory response, corticosteroids are used. However, these drugs have been found to be largely ineffective in attenuating inflammation in patients with COPD (6, 7). The resistance to corticosteroids may involve an impaired activity of the enzyme histone deacetylase, probably related to oxidative stress. In fact, a blunted activity of this enzyme is associated with a reduced response to corticosteroids and an enhanced expression of inflammatory cytokines (8, 9). Also, a latent adenovirus infection has been reported to induce corticosteroid resistance (10). Thus, as indicated in the GOLD guidelines, there is a pressing need to develop new agents capable of suppressing the inflammatory response in COPD (2). Such compounds may have a beneficial effect in preventing the progression of this disease.
Phosphodiesterases (PDEs) are a large family of intracellular enzymes that degrade cyclic nucleotides. The PDE4 subtype specifically targets cyclic 3',5'-adenosine monophosphate, a second messenger that exerts inhibitory effects on many inflammatory cells. Thus, substances that prevent the degradation of cyclic 3',5'-adenosine monophosphate by inhibiting the activity of PDE4 will potentiate the antiinflammatory action of this second messenger (11). It is therefore postulated that these substances may also limit inflammation in COPD.
Acute and chronic exposures of mice to cigarette smoke lead to lung responses that, at least in part, mimic the lung inflammatory and structural changes observed in COPD (12eC15). The present study was designed to evaluate the effects of roflumilast, a PDE4 inhibitor, on the inflammatory and structural lesions observed in an acute and in a chronic model of cigarette smoke exposure in mice. Some of the results of these studies have been previously reported in the form of an abstract (16).
METHODS
Animals
Six-week-old C57Bl/6J male mice (Charles River, Calco, Italy) were used. Animal experimentation was approved by the Ethical Committee of the University of Siena.
Exposure to Cigarette Smoke
The methodology for acute and chronic smoke exposure has been described (12, 13). In the acute study, the mice were exposed either to room air or to the smoke of five cigarettes (Virginia filter cigarettes: 12 mg of tar and 0.9 mg of nicotine) for 20 minutes. In the chronic study, the mice were exposed to either room air or to the smoke of three cigarettes/day for 5 days/week for 7 months.
Acute Study
Mice were divided into three groups of 40 animals each. These groups were then divided into four subgroups of 10 mice each as follows: (1) no treatment/air-exposed, (2) no treatment/smoke-exposed, (3) 1 mg/kg roflumilast + smoke-exposed, or (4) 5 mg/kg roflumilast + smoke-exposed. In the first group, trolox equivalent antioxidant capacity was assessed at the end of the exposure in bronchoalveolar lavage fluid (BALF) (12, 13). In the second group, cytokines and chemokines (interleukin [IL]-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, regulated on activation, normal T-cell expressed and secreted [RANTES], macrophage inflammatory protein-1, cytokine-induced neutrophil chemoattractant [KC], granulocyte colony-stimulating factor, granulocyte-macrophage colonyeCstimulating factor, tumor necrosis factor- [TNF-], and IFN-) were determined in BALF using a commercial Bio-Plex mouse cytokine 18-plex panel (Bio-Rad, Hercules, CA) (17) at 4 hours; and in the third group, BALF cell count was assessed at 24 hours (12).
Chronic Study
Five groups of animals were used: (1) no treatment/air-exposed (n = 13), (2) 5 mg/kg roflumilast + air-exposed (n = 15), (3) no treatment/smoke-exposed (n = 15), (4) 1 mg/kg roflumilast + smoke-exposed (n = 20), and (5) 5 mg/kg roflumilast + smoke exposed (n = 20).
Seven months after chronic exposure to room air or cigarette smoke, 5 to 12 animals of each group were killed and the lungs fixed intratracheally with formalin (5%) at a pressure of 20 cm H2O. Lung volume was measured by water displacement. Lungs were stained with hematoxylineCeosin and/or periodic acid-Schiff. Assessment of emphysema included mean linear intercept (18) and internal surface area (19). The volume density of macrophages, marked immunohistochemically with antimouse Mac-3 monoclonal antibodies (BD Pharmingen, Buccinasco, Italy), was determined by point counting.
Because mice do not have goblet cells in their bronchi (20), a mouse was considered to have goblet cell metaplasia (GCM) when at least one or more midsize bronchi/lung showed a positive periodic acid-Schiff staining.
For the determination of desmosine, fresh lungs were homogenized, processed, and analyzed by high-pressure liquid chromatography (21).
In both studies, roflumilast (ALTANA Pharma, Konstanz, Germany) was given orally in a volume of 10 ml/kg 60 minutes before air/smoke exposure.
Statistical Analysis
The significance of the differences was calculated using one-way analysis of variance. For goblet cell data, the 2 test was used. A p value of less than 0.05 was considered significant.
RESULTS
Acute Study
Table 1 shows the results of the trolox equivalent antioxidant capacity and of the differential cell count in the various groups. Mice exposed to cigarette smoke had a 23% lower trolox equivalent antioxidant capacity value than control mice exposed to air (p < 0.05). Both doses of roflumilast failed to affect this drop.
Acute smoke exposure caused a large and significant fivefold increase in BALF neutrophil count as assessed at 24 hours (p < 0.001). Both doses of roflumilast, 1 mg/kg and 5 mg/kg, partially but significantly attenuated (by 31 and 32%, respectively; p < 0.001) this increase. BALF macrophage count was significantly increased in the cigarette smokeeCexposed group (+133%, p < 0.001) as compared with the air-exposed control group. Roflumilast at both doses did not affect this increase.
The results of the cytokine/chemokine analysis performed 4 hours after the exposure revealed that at this time interval only the levels of IL-10 showed some changes. This antiinflammatory interleukin was increased in a dose-related fashion in the cigarette smokeeCexposed groups treated with roflumilast (+79% at 1 mg/kg and +129% at 5 mg/kg) as compared with the control (not significant and p < 0.05, respectively) and cigarette smoke groups (p < 0.05 and p < 0.01, respectively), which showed similar values (Figure 1).
Chronic Study
At 7 months, Mac-3eCpositive cells (macrophages) could be detected in the lungs of both air- and smoke-exposed mice (Figures 2AeC2C). Results from the quantitative histology show that chronic cigarette smoke resulted in a 1.8-fold increase (p < 0.01) in lung macrophage density. The low dose of roflumilast did not have any effect on this change, whereas the high dose of roflumilast prevented the increase in lung macrophage density by 70% (p < 0.01; Figure 2D).
Histologically, the lungs of the mice exposed to room air for 7 months showed a well-fixed normal parenchyma with normal airways (Figure 3A). Daily administration of 5 mg/kg roflumilast to air-exposed mice did not influence their lung structure (data not shown). The lungs of mice exposed for 7 months to cigarette smoke showed foci of emphysema disseminated throughout the lung parenchyma (Figure 3B). The lungs of the mice exposed to cigarette smoke and treated with 1 mg/kg roflumilast were practically indistinguishable from the mice exposed to cigarette smoke alone (Figure 3C). On the other hand, the lungs of the mice exposed to cigarette smoke and treated with 5 mg/kg roflumilast had a normal parenchyma and could not be distinguished from the lungs of the air-exposed mice (Figure 3D).
The results of the morphometric investigation of the lungs are given in Table 2. Chronic exposure to cigarette smoke resulted in a significant increase of the mean linear intercept (+21%, p < 0.001) and in a significant decrease of the internal surface area (eC13%, p < 0.05). Treatment with 1 mg/kg roflumilast did not ameliorate these changes. However, treatment with 5 mg/kg roflumilast fully prevented the increase of the mean linear intercept (p < 0.001) and the decrease of the internal surface area (p < 0.05).
The results of the biochemical analysis of lung desmosine content, expressed as micrograms per lung, are also shown in Table 2. In the mice exposed for 7 months to cigarette smoke, lung levels of desmosine were significantly lower (eC13%, p < 0.05) than those of air-exposed animals. Roflumilast doses of 1 mg/kg did not have any effect against the drop in desmosine content, whereas 5 mg/kg roflumilast completely prevented it (p < 0.01).
As expected, no goblet cells were seen in the bronchi and in the bronchioles of air-exposed mice (Figure 4A) and air-exposed mice treated with 5 mg/kg roflumilast. In the smoke-exposed group, five of eight mice (63%) had GCM (Figure 4B). In the smoke-exposed group treated with 1 mg/kg roflumilast, 6 of 12 mice (50%) had GCM (data not shown). In the smoke-exposed group treated with 5 mg/kg roflumilast, 7 of 12 mice (58%) had GCM (Figure 4C). The latter three groups were not statistically different from each other.
DISCUSSION
PDE inhibitors, in view of their antiinflammatory effects, have recently been confirmed as a potential novel therapeutic approach for the treatment of COPD and asthma (22, 23). Roflumilast, a PDE4 inhibitor, has been shown to possess a potent antiinflammatory activity in vitro on a range of inflammatory cell types relevant to COPD and asthma, as well as in a wide range of animal models in vivo (24eC26).
In the present study after acute smoke exposure, the antiinflammatory activity of roflumilast was mirrored by the inhibition of BALF neutrophil influx and was associated with an increase in BALF IL-10. IL-10, produced by CD8+ T cells, is an antiinflammatory cytokine that is constitutively expressed in mice (27). The dose-related increase in BALF IL-10 observed here is probably a drug class effect, because the PDE4 inhibitor rolipram has been shown to have a similar effect in a model of acute lung injury in the rat (28). The elevation in IL-10 may have contributed to the prevention of BALF neutrophil influx in the roflumilast-treated groups. In fact, rolipram has been reported to inhibit BALF neutrophil recruitment in the rat after endotoxin exposure, and this effect was mimicked by exogenous IL-10 (28). In the present study, roflumilast doses of both 1 mg/kg and 5 mg/kg reduced the influx of neutrophil in the BALF by approximately 32%. This result is consistent with recent data reported in abstract form showing that roflumilast administered as an aerosol (10 mg/ml) to Balb/c mice before and after an acute cigarette smoke exposure (five cigarettes), reduced BALF neutrophil count by approximately 40% (29). In the present investigation, acute cigarette smoke produced a modest but significant increase in BALF macrophages, and roflumilast did not affect this increase. Lavage macrophage numbers depend on the number of cigarettes smoked; however, it has been suggested that it is macrophage activation rather than the increase in macrophage numbers that is crucial to initiating the acute inflammatory response (30). One may expect that roflumilast limits macrophage activation by cigarette smoke because this compound has been reported to inhibit in vitro the endotoxin-induced synthesis of TNF- in isolated macrophages (24). In vivo, roflumilast was found to decrease dose-dependently the release of TNF- into the airspaces of challenged rats (25, 26), an effect which may be attributed to an inhibitory effect on the activation of monocytes/macrophages.
In the present study, C57Bl/6J mice, which contrary to other strains are sensitive to oxidative stress by cigarette smoke (13), responded to acute exposure to cigarette smoke with a decline in their BALF antioxidant capacity. A roflumilast dose of 5 mg/kg had only a small statistically insignificant effect on this parameter.
Chronic exposure of untreated mice to cigarette smoke resulted in a 1.8-fold increase in lung macrophage density, in disseminated foci of emphysema, in an increase of the mean linear intercept by +21%, and in a decrease of the internal surface area by eC13%. These morphologic and morphometric changes were associated with a significant decline in lung desmosine content (eC13%). Desmosine is an elastin-specific imino acid, and the assessment of desmosine in the lung is taken as a value for the lung elastin content. The decline in lung desmosine content can be taken as evidence that the emphysematous changes in this model are associated with a proteolytic process and matrix breakdown.
Roflumilast dosages of 1 mg/kg did not affect the increase in the lung density of macrophages, the development of emphysema, or the decline in lung desmosine content. However, 5 mg/kg roflumilast prevented increase in lung macrophage density by 70% and fully prevented both the morphometric changes and the decline in lung desmosine content. The pathogenetic pathway responsible for the development of emphysema is still controversial. A recent hypothesis suggests that cigarette smoke activates nuclear factoreCB, which orchestrates the production of the inflammatory molecules macrophage chemoattractant protein-1 and macrophage inflammatory protein-2. The macrophages are then activated to release matrix metalloprotease-12 (macrophage metalloelastase). This protease then mediates cigarette smoke-induced inflammation by releasing TNF- from macrophages, with subsequent endothelial activation, neutrophil influx, and proteolytic matrix breakdown caused by neutrophil-derived proteases (14). Thus, this hypothesis, elaborated from findings obtained in an acute model of cigarette smoke exposure, suggests that TNF- may be central to the inflammatory events that lead to emphysema and that neutrophil elastase may be the causative factor in this chain of events. In a subsequent study in a chronic model of cigarette smoke exposure, it was postulated that in C57Bl/6J mice TNF-eCdriven processes may account for approximately 70% of emphysema, whereas the remaining 30% of matrix breakdown and emphysema may be related to a different process that drives neutrophil influx independently of the actions of TNF- (31).
The beneficial effect of roflumilast observed here may have been due to an interruption of the chain of inflammatory events as indicated by the significant effect on macrophage volume density observed at 7 months. Roflumilast has been shown to prevent the production of TNF- both in vitro and in vivo (24, 25) and to limit the influx of neutrophils in the airspaces (25, 29, and present data), and these two effects taken together may have played the pivotal role in the protective activity of roflumilast. It is of interest that, in the above-mentioned hypothesis, neutrophil elastase is the last ring in the chain of events that leads to emphysema. However, mice lacking neutrophil elastase are only 59% protected against cigarette smokeeCinduced emphysema (32), and 1-antitrypsin given chronically to mice provided 63% protection against smoke-induced emphysema (15). Both of these results suggest that the final proteolytic attack by neutrophil elastase may be only one factor in the development of emphysema and that other factors may also be involved in the process of parenchymal enlargement. One of these factors may involve the release and the activation of matrix metalloproteases from fibroblasts with consequent matrix destruction (33). Because PDE4 inhibitors, but not PDE3 or PDE5 inhibitors, have been reported to inhibit both the release and activation of these proteases as well as matrix destruction (33), these effects may also play a role in the protective effect of rofumilast against emphysema development.
It is of interest that antiinflammatory steroids were reported to have only a limited effect against pulmonary inflammation in a model of acute smoke exposure and no effect against the increase of the of the mean linear intercept in a model of chronic smoke exposure (34, 35).
In the present study, chronic cigarette smoke also resulted in airway changes (GCM) in more than 60% of the untreated animals. Roflumilast, contrary to its positive effect against the development of emphysema, had no effect against these airway changes. Because the airway changes of the C57Bl/6J mice do not result in morphologic evidence of obstruction (air trapping), they are probably not relevant for the pathogenesis of the parenchymal lesions, and the development of GCM and emphysema most likely rely on two different mechanisms of action. We recently observed a positive immunohistochemical reaction for IL-4 and IL-13 and MUC5A in the airways of C57Bl/6J mice with smoke-induced GCM (36). The IL-4 effects include the recruitment of type 2 helper T cells, which release IL-13. Thus, a chain of events consisting of cigarette smoke, IL-4, IL-13, GCM, and mucin secretion may be at the base of the development of GCM in these mice (36). The present results indicate that roflumilast does not affect this chain of events in this particular experimental setting. However, roflumilast suppressed IL-4 and IL-5 mRNA expression in lung tissue and IL-13 release into the alveolar lumen in a rat model of ovalbumin-induced late allergic pulmonary inflammation (37).
In summary, in these animal models, we have shown for the first time that oral administration of a PDE4 inhibitor (roflumilast) partially ameliorates acute and chronic lung inflammation and fully prevents parenchymal destruction induced by cigarette smoke in mice. This is associated with evidence of a decreased number of acute inflammatory cells in BALF, lower macrophage volume density at 7 months, and a complete prevention of matrix degradation and parenchymal enlargement. This study supports the role of an inflammatory process in the development of cigarette smokeeCinduced matrix destruction and emphysema and indicates that such alterations may be prevented by the administration of a PDE4 inhibitor.
Acknowledgments
The authors thank Ms. Benedetta Lunghi, M.Sc., for treatment and exposure of the mice and for the histologic and immunohistochemical preparation of the samples.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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ALTANA Pharma, Konstanz, Germany
ABSTRACT
Rationale: There is a need for new agents capable of suppressing the inflammatory response in chronic obstructive pulmonary disease. Objectives: This study evaluated the effects of roflumilast, a phosphodiesterase 4 (PDE4) inhibitor on acute lung inflammation and chronic lung changes in models of cigarette exposure in mice.
Methods: Roflumilast was given orally either at 1 mg/kg (R1) or at 5 mg/kg (R5). In the acute model (five cigarettes for 20 minutes), bronchoalveolar lavage fluid (BALF) changes were investigated at 4 and 24 hours. In the chronic model (three cigarettes/day for 7 months), morphometric and biochemical parameters were assessed at 7 months.
Measurements and Main Results: Acute exposure caused a fivefold increase in BALF neutrophils. Both doses of roflumilast partially prevented (by 30%) this increase. In addition, after smoke exposure, R1 increased BALF interleukin-10 by 79% and R5 by 129%. Chronic smoke exposure caused a 1.8-fold increase in lung macrophage density, emphysema, an increase of the mean linear intercept (+21%), a decrease of the internal surface area (eC13%), and a drop (eC13%) in lung desmosine content. R1 did not have any effect, whereas R5 prevented the increase in lung macrophage density by 70% and fully prevented the other changes. In addition, in the smoke-exposure group, 63% of the mice showed goblet cell metaplasia, and neither of the doses of roflumilast had any effect.
Conclusions: This study shows for the first time that a PDE4 inhibitor partially ameliorates lung inflammation and fully prevents parenchymal destruction induced by cigarette smoke.
Key Words: chronic obstructive pulmonary disease interleukin-10 lung inflammation
Chronic obstructive pulmonary disease (COPD) is a rapidly increasing global health problem (1). It includes emphysema, which is characterized by airspace enlargement and destruction of lung parenchyma, and chronic bronchitis. Recently, COPD has been defined by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) as a disease characterized by progressive, not fully reversible, flow limitation and "associated with an abnormal inflammatory response of the lungs to noxious particles and gases" (2). Thus, a central role has been attributed to the chronic inflammatory response that is present throughout the airways and parenchyma and that participates in the progression and exacerbation of this disease (3). Cigarette smoke has been identified as the most important risk for the development of COPD (4).
The therapeutic approach to COPD is mainly supportive and symptomatic (bronchodilators) (5). In an approach aiming at modulating the chronic inflammatory response, corticosteroids are used. However, these drugs have been found to be largely ineffective in attenuating inflammation in patients with COPD (6, 7). The resistance to corticosteroids may involve an impaired activity of the enzyme histone deacetylase, probably related to oxidative stress. In fact, a blunted activity of this enzyme is associated with a reduced response to corticosteroids and an enhanced expression of inflammatory cytokines (8, 9). Also, a latent adenovirus infection has been reported to induce corticosteroid resistance (10). Thus, as indicated in the GOLD guidelines, there is a pressing need to develop new agents capable of suppressing the inflammatory response in COPD (2). Such compounds may have a beneficial effect in preventing the progression of this disease.
Phosphodiesterases (PDEs) are a large family of intracellular enzymes that degrade cyclic nucleotides. The PDE4 subtype specifically targets cyclic 3',5'-adenosine monophosphate, a second messenger that exerts inhibitory effects on many inflammatory cells. Thus, substances that prevent the degradation of cyclic 3',5'-adenosine monophosphate by inhibiting the activity of PDE4 will potentiate the antiinflammatory action of this second messenger (11). It is therefore postulated that these substances may also limit inflammation in COPD.
Acute and chronic exposures of mice to cigarette smoke lead to lung responses that, at least in part, mimic the lung inflammatory and structural changes observed in COPD (12eC15). The present study was designed to evaluate the effects of roflumilast, a PDE4 inhibitor, on the inflammatory and structural lesions observed in an acute and in a chronic model of cigarette smoke exposure in mice. Some of the results of these studies have been previously reported in the form of an abstract (16).
METHODS
Animals
Six-week-old C57Bl/6J male mice (Charles River, Calco, Italy) were used. Animal experimentation was approved by the Ethical Committee of the University of Siena.
Exposure to Cigarette Smoke
The methodology for acute and chronic smoke exposure has been described (12, 13). In the acute study, the mice were exposed either to room air or to the smoke of five cigarettes (Virginia filter cigarettes: 12 mg of tar and 0.9 mg of nicotine) for 20 minutes. In the chronic study, the mice were exposed to either room air or to the smoke of three cigarettes/day for 5 days/week for 7 months.
Acute Study
Mice were divided into three groups of 40 animals each. These groups were then divided into four subgroups of 10 mice each as follows: (1) no treatment/air-exposed, (2) no treatment/smoke-exposed, (3) 1 mg/kg roflumilast + smoke-exposed, or (4) 5 mg/kg roflumilast + smoke-exposed. In the first group, trolox equivalent antioxidant capacity was assessed at the end of the exposure in bronchoalveolar lavage fluid (BALF) (12, 13). In the second group, cytokines and chemokines (interleukin [IL]-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, regulated on activation, normal T-cell expressed and secreted [RANTES], macrophage inflammatory protein-1, cytokine-induced neutrophil chemoattractant [KC], granulocyte colony-stimulating factor, granulocyte-macrophage colonyeCstimulating factor, tumor necrosis factor- [TNF-], and IFN-) were determined in BALF using a commercial Bio-Plex mouse cytokine 18-plex panel (Bio-Rad, Hercules, CA) (17) at 4 hours; and in the third group, BALF cell count was assessed at 24 hours (12).
Chronic Study
Five groups of animals were used: (1) no treatment/air-exposed (n = 13), (2) 5 mg/kg roflumilast + air-exposed (n = 15), (3) no treatment/smoke-exposed (n = 15), (4) 1 mg/kg roflumilast + smoke-exposed (n = 20), and (5) 5 mg/kg roflumilast + smoke exposed (n = 20).
Seven months after chronic exposure to room air or cigarette smoke, 5 to 12 animals of each group were killed and the lungs fixed intratracheally with formalin (5%) at a pressure of 20 cm H2O. Lung volume was measured by water displacement. Lungs were stained with hematoxylineCeosin and/or periodic acid-Schiff. Assessment of emphysema included mean linear intercept (18) and internal surface area (19). The volume density of macrophages, marked immunohistochemically with antimouse Mac-3 monoclonal antibodies (BD Pharmingen, Buccinasco, Italy), was determined by point counting.
Because mice do not have goblet cells in their bronchi (20), a mouse was considered to have goblet cell metaplasia (GCM) when at least one or more midsize bronchi/lung showed a positive periodic acid-Schiff staining.
For the determination of desmosine, fresh lungs were homogenized, processed, and analyzed by high-pressure liquid chromatography (21).
In both studies, roflumilast (ALTANA Pharma, Konstanz, Germany) was given orally in a volume of 10 ml/kg 60 minutes before air/smoke exposure.
Statistical Analysis
The significance of the differences was calculated using one-way analysis of variance. For goblet cell data, the 2 test was used. A p value of less than 0.05 was considered significant.
RESULTS
Acute Study
Table 1 shows the results of the trolox equivalent antioxidant capacity and of the differential cell count in the various groups. Mice exposed to cigarette smoke had a 23% lower trolox equivalent antioxidant capacity value than control mice exposed to air (p < 0.05). Both doses of roflumilast failed to affect this drop.
Acute smoke exposure caused a large and significant fivefold increase in BALF neutrophil count as assessed at 24 hours (p < 0.001). Both doses of roflumilast, 1 mg/kg and 5 mg/kg, partially but significantly attenuated (by 31 and 32%, respectively; p < 0.001) this increase. BALF macrophage count was significantly increased in the cigarette smokeeCexposed group (+133%, p < 0.001) as compared with the air-exposed control group. Roflumilast at both doses did not affect this increase.
The results of the cytokine/chemokine analysis performed 4 hours after the exposure revealed that at this time interval only the levels of IL-10 showed some changes. This antiinflammatory interleukin was increased in a dose-related fashion in the cigarette smokeeCexposed groups treated with roflumilast (+79% at 1 mg/kg and +129% at 5 mg/kg) as compared with the control (not significant and p < 0.05, respectively) and cigarette smoke groups (p < 0.05 and p < 0.01, respectively), which showed similar values (Figure 1).
Chronic Study
At 7 months, Mac-3eCpositive cells (macrophages) could be detected in the lungs of both air- and smoke-exposed mice (Figures 2AeC2C). Results from the quantitative histology show that chronic cigarette smoke resulted in a 1.8-fold increase (p < 0.01) in lung macrophage density. The low dose of roflumilast did not have any effect on this change, whereas the high dose of roflumilast prevented the increase in lung macrophage density by 70% (p < 0.01; Figure 2D).
Histologically, the lungs of the mice exposed to room air for 7 months showed a well-fixed normal parenchyma with normal airways (Figure 3A). Daily administration of 5 mg/kg roflumilast to air-exposed mice did not influence their lung structure (data not shown). The lungs of mice exposed for 7 months to cigarette smoke showed foci of emphysema disseminated throughout the lung parenchyma (Figure 3B). The lungs of the mice exposed to cigarette smoke and treated with 1 mg/kg roflumilast were practically indistinguishable from the mice exposed to cigarette smoke alone (Figure 3C). On the other hand, the lungs of the mice exposed to cigarette smoke and treated with 5 mg/kg roflumilast had a normal parenchyma and could not be distinguished from the lungs of the air-exposed mice (Figure 3D).
The results of the morphometric investigation of the lungs are given in Table 2. Chronic exposure to cigarette smoke resulted in a significant increase of the mean linear intercept (+21%, p < 0.001) and in a significant decrease of the internal surface area (eC13%, p < 0.05). Treatment with 1 mg/kg roflumilast did not ameliorate these changes. However, treatment with 5 mg/kg roflumilast fully prevented the increase of the mean linear intercept (p < 0.001) and the decrease of the internal surface area (p < 0.05).
The results of the biochemical analysis of lung desmosine content, expressed as micrograms per lung, are also shown in Table 2. In the mice exposed for 7 months to cigarette smoke, lung levels of desmosine were significantly lower (eC13%, p < 0.05) than those of air-exposed animals. Roflumilast doses of 1 mg/kg did not have any effect against the drop in desmosine content, whereas 5 mg/kg roflumilast completely prevented it (p < 0.01).
As expected, no goblet cells were seen in the bronchi and in the bronchioles of air-exposed mice (Figure 4A) and air-exposed mice treated with 5 mg/kg roflumilast. In the smoke-exposed group, five of eight mice (63%) had GCM (Figure 4B). In the smoke-exposed group treated with 1 mg/kg roflumilast, 6 of 12 mice (50%) had GCM (data not shown). In the smoke-exposed group treated with 5 mg/kg roflumilast, 7 of 12 mice (58%) had GCM (Figure 4C). The latter three groups were not statistically different from each other.
DISCUSSION
PDE inhibitors, in view of their antiinflammatory effects, have recently been confirmed as a potential novel therapeutic approach for the treatment of COPD and asthma (22, 23). Roflumilast, a PDE4 inhibitor, has been shown to possess a potent antiinflammatory activity in vitro on a range of inflammatory cell types relevant to COPD and asthma, as well as in a wide range of animal models in vivo (24eC26).
In the present study after acute smoke exposure, the antiinflammatory activity of roflumilast was mirrored by the inhibition of BALF neutrophil influx and was associated with an increase in BALF IL-10. IL-10, produced by CD8+ T cells, is an antiinflammatory cytokine that is constitutively expressed in mice (27). The dose-related increase in BALF IL-10 observed here is probably a drug class effect, because the PDE4 inhibitor rolipram has been shown to have a similar effect in a model of acute lung injury in the rat (28). The elevation in IL-10 may have contributed to the prevention of BALF neutrophil influx in the roflumilast-treated groups. In fact, rolipram has been reported to inhibit BALF neutrophil recruitment in the rat after endotoxin exposure, and this effect was mimicked by exogenous IL-10 (28). In the present study, roflumilast doses of both 1 mg/kg and 5 mg/kg reduced the influx of neutrophil in the BALF by approximately 32%. This result is consistent with recent data reported in abstract form showing that roflumilast administered as an aerosol (10 mg/ml) to Balb/c mice before and after an acute cigarette smoke exposure (five cigarettes), reduced BALF neutrophil count by approximately 40% (29). In the present investigation, acute cigarette smoke produced a modest but significant increase in BALF macrophages, and roflumilast did not affect this increase. Lavage macrophage numbers depend on the number of cigarettes smoked; however, it has been suggested that it is macrophage activation rather than the increase in macrophage numbers that is crucial to initiating the acute inflammatory response (30). One may expect that roflumilast limits macrophage activation by cigarette smoke because this compound has been reported to inhibit in vitro the endotoxin-induced synthesis of TNF- in isolated macrophages (24). In vivo, roflumilast was found to decrease dose-dependently the release of TNF- into the airspaces of challenged rats (25, 26), an effect which may be attributed to an inhibitory effect on the activation of monocytes/macrophages.
In the present study, C57Bl/6J mice, which contrary to other strains are sensitive to oxidative stress by cigarette smoke (13), responded to acute exposure to cigarette smoke with a decline in their BALF antioxidant capacity. A roflumilast dose of 5 mg/kg had only a small statistically insignificant effect on this parameter.
Chronic exposure of untreated mice to cigarette smoke resulted in a 1.8-fold increase in lung macrophage density, in disseminated foci of emphysema, in an increase of the mean linear intercept by +21%, and in a decrease of the internal surface area by eC13%. These morphologic and morphometric changes were associated with a significant decline in lung desmosine content (eC13%). Desmosine is an elastin-specific imino acid, and the assessment of desmosine in the lung is taken as a value for the lung elastin content. The decline in lung desmosine content can be taken as evidence that the emphysematous changes in this model are associated with a proteolytic process and matrix breakdown.
Roflumilast dosages of 1 mg/kg did not affect the increase in the lung density of macrophages, the development of emphysema, or the decline in lung desmosine content. However, 5 mg/kg roflumilast prevented increase in lung macrophage density by 70% and fully prevented both the morphometric changes and the decline in lung desmosine content. The pathogenetic pathway responsible for the development of emphysema is still controversial. A recent hypothesis suggests that cigarette smoke activates nuclear factoreCB, which orchestrates the production of the inflammatory molecules macrophage chemoattractant protein-1 and macrophage inflammatory protein-2. The macrophages are then activated to release matrix metalloprotease-12 (macrophage metalloelastase). This protease then mediates cigarette smoke-induced inflammation by releasing TNF- from macrophages, with subsequent endothelial activation, neutrophil influx, and proteolytic matrix breakdown caused by neutrophil-derived proteases (14). Thus, this hypothesis, elaborated from findings obtained in an acute model of cigarette smoke exposure, suggests that TNF- may be central to the inflammatory events that lead to emphysema and that neutrophil elastase may be the causative factor in this chain of events. In a subsequent study in a chronic model of cigarette smoke exposure, it was postulated that in C57Bl/6J mice TNF-eCdriven processes may account for approximately 70% of emphysema, whereas the remaining 30% of matrix breakdown and emphysema may be related to a different process that drives neutrophil influx independently of the actions of TNF- (31).
The beneficial effect of roflumilast observed here may have been due to an interruption of the chain of inflammatory events as indicated by the significant effect on macrophage volume density observed at 7 months. Roflumilast has been shown to prevent the production of TNF- both in vitro and in vivo (24, 25) and to limit the influx of neutrophils in the airspaces (25, 29, and present data), and these two effects taken together may have played the pivotal role in the protective activity of roflumilast. It is of interest that, in the above-mentioned hypothesis, neutrophil elastase is the last ring in the chain of events that leads to emphysema. However, mice lacking neutrophil elastase are only 59% protected against cigarette smokeeCinduced emphysema (32), and 1-antitrypsin given chronically to mice provided 63% protection against smoke-induced emphysema (15). Both of these results suggest that the final proteolytic attack by neutrophil elastase may be only one factor in the development of emphysema and that other factors may also be involved in the process of parenchymal enlargement. One of these factors may involve the release and the activation of matrix metalloproteases from fibroblasts with consequent matrix destruction (33). Because PDE4 inhibitors, but not PDE3 or PDE5 inhibitors, have been reported to inhibit both the release and activation of these proteases as well as matrix destruction (33), these effects may also play a role in the protective effect of rofumilast against emphysema development.
It is of interest that antiinflammatory steroids were reported to have only a limited effect against pulmonary inflammation in a model of acute smoke exposure and no effect against the increase of the of the mean linear intercept in a model of chronic smoke exposure (34, 35).
In the present study, chronic cigarette smoke also resulted in airway changes (GCM) in more than 60% of the untreated animals. Roflumilast, contrary to its positive effect against the development of emphysema, had no effect against these airway changes. Because the airway changes of the C57Bl/6J mice do not result in morphologic evidence of obstruction (air trapping), they are probably not relevant for the pathogenesis of the parenchymal lesions, and the development of GCM and emphysema most likely rely on two different mechanisms of action. We recently observed a positive immunohistochemical reaction for IL-4 and IL-13 and MUC5A in the airways of C57Bl/6J mice with smoke-induced GCM (36). The IL-4 effects include the recruitment of type 2 helper T cells, which release IL-13. Thus, a chain of events consisting of cigarette smoke, IL-4, IL-13, GCM, and mucin secretion may be at the base of the development of GCM in these mice (36). The present results indicate that roflumilast does not affect this chain of events in this particular experimental setting. However, roflumilast suppressed IL-4 and IL-5 mRNA expression in lung tissue and IL-13 release into the alveolar lumen in a rat model of ovalbumin-induced late allergic pulmonary inflammation (37).
In summary, in these animal models, we have shown for the first time that oral administration of a PDE4 inhibitor (roflumilast) partially ameliorates acute and chronic lung inflammation and fully prevents parenchymal destruction induced by cigarette smoke in mice. This is associated with evidence of a decreased number of acute inflammatory cells in BALF, lower macrophage volume density at 7 months, and a complete prevention of matrix degradation and parenchymal enlargement. This study supports the role of an inflammatory process in the development of cigarette smokeeCinduced matrix destruction and emphysema and indicates that such alterations may be prevented by the administration of a PDE4 inhibitor.
Acknowledgments
The authors thank Ms. Benedetta Lunghi, M.Sc., for treatment and exposure of the mice and for the histologic and immunohistochemical preparation of the samples.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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