Upregulation of Phosphodiesterase-4 in the Lung of Allergic Rats
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美国呼吸和危急护理医学 2005年第4期
Zhejiang Respiratory Drugs Research Laboratory of State Food and Drugs Administration of China
Zhejiang University School of Medicine, Hangzhou, China
Schering-Plough Research Institute, Kenilworth, New Jersey
ABSTRACT
Inhibitors of phosphodiesterase-4 (PDE4) are efficacious for allergic asthma in animal models and have shown some efficacy in human asthma. Regulation of PDE4 in allergy and asthma has been widely investigated in blood leukocytes, with discrepant results. This study investigated PDE4 regulation in the lung in a rat model of allergic asthma. Ovalbumin sensitization and challenge significantly increased pulmonary resistance and lung interleukin (IL)-4 production. The increases in pulmonary resistance and IL-4 production were both suppressed by the PDE4-selective inhibitor rolipram or the corticosteroid drug dexamethasone. Furthermore, cAMP-PDE enzyme activity in the lung was also significantly increased by the sensitization and challenge. mRNA analysis confirmed that PDE4 gene expression was increased in the lung of the allergic rats. A highly significant correlation was observed between the increases in PDE activity and IL-4 production. Our data suggest, for the first time, that PDE4 may be upregulated in the lung and play a role in the pathogenesis of allergic asthma.
Key Words: asthma cAMP PDE4 phosphodiesterase-4 rat
The phosphodiesterase (PDE) superfamily comprises 11 biochemically and pharmacologically distinct enzyme families (PDEs 1eC11) that hydrolyze cAMP and/or cGMP. PDEs represent the only cellular pathways for the degradation of the ubiquitous intracellular second messengers, underscoring their critical role in regulation of cellular functions (1eC6). Most PDE families contain multiple genes (subtypes), and most of these genes give rise to multiple variants by alternative splicing among the 5'eCend exons and/or the use of different transcription initiation sites. Despite the large number of PDE enzymes with distinct biochemical and pharmacologic properties, they share some structural features. In particular, they contain a conserved catalytic domain of approximately 270 to 300 amino acids located toward the C termini.
PDE4 is specific for cAMP and comprises four subtypes (A, B, C, and D). It is predominantly expressed and plays an important role in regulation of cellular functions in inflammatory and immune cells. There has been significant interest in PDE4 inhibitors as a potential therapy for inflammatory diseases, such as allergy and asthma. In fact, PDE4 inhibitors have exhibited efficacy for asthma, allergic rhinitis, and several other diseases in the clinic (7, 8).
The regulation of PDE4 in allergy and asthma has been widely investigated in human blood leukocytes. Interleukin (IL)-4 plays a crucial role in allergic airway diseases, including asthma (9eC11). Furthermore, IL-4 significantly increased PDE4 activity in human monocytes (12). However, whether or not PDE4 is upregulated in blood leukocytes in asthma and/or allergy remains controversial. Some investigators reported a significant increase in PDE4 activity in patients with asthma or allergy as compared with healthy individuals (13eC17). It is noteworthy that when there was an increase in PDE4 activity, the increase was correlated with an increased IL-4 production (18). However, others observed no significant change of PDE4 activity in such patients (19eC22). There has been no reported study on PDE4 regulation in the lung in any animal model of asthma or in patients with asthma.
This study investigated lung PDE4 regulation in an antigen-challenged rat model. In our model, there were significantly increased pulmonary resistance and lung IL-4 production. The increases in pulmonary resistance and IL-4 production were both suppressed by the PDE4-selective inhibitor rolipram or the corticosteroid drug dexamethasone. Furthermore, in the lung, there was also a significant increase in cAMP-PDE enzyme activity as well as increases in PDE4 subtype mRNAs. Our data suggest, for the first time, that PDE4 may be upregulated in the lung and play a role in the pathogenesis of allergic asthma.
METHODS
Animal Handling, Sensitization, and Challenge
Male Sprague-Dawley rats (140eC160 g; Laboratory Animal Center of Zhejiang University School of Medicine, Hangzhou, China) were used, and were maintained under a 24-hour light/dark cycle with food and water allowed ad libitum. Animals were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animal experiments were approved by the Zhejiang Medical Laboratory Animal Administration Committee.
Rats were sensitized with a subcutaneous injection (1 ml) of a saline suspension containing 0.2% ovalbumin (OVA; Sigma, St. Louis, MO) and 10% aluminum hydroxide at their footpad, neck, back, groin, and abdomen on Day 0. In addition, each animal was primed with an intramuscular injection of 2 x 1010 heat-killed Bordatella pertussis organisms. From Day 14 after sensitization, animals were challenged once a day for 7 days by exposure for 20 minutes to aerosolized 1% OVA in saline generated by a jet nebulizer (Master; Pari GmbH, Starnberg, Germany; medium diameter of produced droplets: 1eC5 e). Control animals were exposed to an aerosol of saline. For drug treatment, rolipram (Sigma) or dexamethasone (Changzhou Second Pharmaceuticals Co., Changzhou, China) was given orally or intravenously, respectively, once a day at Days 14 through 20, 30 minutes before challenge. At Day 21, animals were killed, and lung tissues were immediately removed, frozen in liquid nitrogen, and then stored at eC80°C until analysis.
Measurement of Lung Functions
Tracheostomy was performed under urethane anesthesia (1 g/kg, injected intraperitoneally). The rat was then placed in a whole body plethysmograph, and lung functions were measured as described previously (23).
Assay for cAMP-PDE Activity
Frozen lungs were thawed and cut into small cubes. Twenty-five milligrams of lung tissues were homogenized in 100 e of ice-cold 30 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (pH 7.4) containing 0.1% Trion X-100 (Sigma). The cAMP-PDE assay mixture (200 e) in phosphate-buffered saline (pH 7.4) contained 137 mM NaCl, 2.7 mM KCl, 8.8 mM Na2HPO4, 1.5 mM KH2PO4, 1 mM CaCl2, 1 mM MgCl2, 1 e cAMP (Sigma), and lung homogenate. The PDE reaction was started by the addition of 10 e lung homogenate and was performed at 37°C for 10 minutes. The reaction was stopped by boiling the mixture for 3 minutes. The assay mixture was cooled on ice, followed by centrifugation at 12,850 x g for 30 minutes at 4°C. The amount of cAMP present in the supernatant was determined by HPLC (Hypersil ODS 4.0 x 250 mm; Hewlett-Packard, Palo Alto, CA) using a standard curve of cAMP.
Quantification of Lung IL-4
Lungs were homogenized at a ratio of 1 g to 10 ml in 50 mM potassium phosphate buffer (pH 6.0) containing 0.05% NaN3 and 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHARPS). The homogenates were centrifuged twice at 19,851 x g, each for 30 minutes. IL-4 in the supernatants was quantified using a rat IL-4eCspecific ELISA kit (Jingmei BioTech, Shenzhen, China).
Assay for Total Protein
Total protein amounts were determined by the Bradford method (24) using bovine serum albumin as the standard.
RNA Isolation and Complementary DNA Synthesis
Total RNA was isolated from each tissue using Trizol reagent (Invitrogen, Carlsbad, CA), The preparation of first-strand complementary DNA (cDNA) from total RNA was performed using a first-strand cDNA synthesis kit (Shanghai Sangon Biological Engineering Technology and Service [Sangon], Shanghai, China).
Semiquantitative Reverse Transcription-Polymerase Chain Reaction Analysis for Rat PDE4 Subtype mRNAs
Sequences of polymerase chain reaction (PCR) primer sets used for PDE4A, PDE4B, PDE4D (25), and PDE4C (26) were as reported previously. Each of the PCR primer sets was able to detect all known variants derived from the appropriate PDE4 gene (2). PCR amplification was performed in a PCR buffer (10 mM Tris-HCl, pH 9.0; 100 mM KCl; 80 mM [NH4]2SO4; and 0.1% Nonidet P-40) containing 0.2 mM of each nucleotide, 1.5 mM of MgCl2, 500 nM of each primer, and 1 U of Taq DNA polymerase (Sangon) in a total volume of 25 e for 30 cycles, with the following cycle parameters: denaturing, 94°C for 45 seconds; annealing, 58°C for 70 seconds; and extension, 72°C for 2 minutes. After PCR amplification, 8 e of each reaction mixture were resolved by electrophoresis on a 1.5% agarose gel containing ethidium bromide, and the PCR product bands were quantified by using the UVP Gel Documentation system (UVP, Upland, CA). The levels of PDE4 mRNAs were calculated relative to -actin. It was confirmed that under these conditions the PCR product accumulation did not reach plateau levels (data not shown).
Measurement of Tracheal Tissue Contraction and Relaxation
Trachea free of connective tissue was isolated in oxygenated Krebs-Henseleit buffer. Each trachea was cut into six 1-cm-long sections without damage to the epithelium. Three sections were stitched together as one tracheal preparation. Tracheal preparations were equilibrated at 0.5 g of initial tension in the Krebs-Henseleit buffer at 37°C in an atmosphere of 5% CO2/95% O2 for 30 minutes. To measure contractile response, a cumulative concentrationeCresponse curve was obtained with methacholine (Sigma). To measure relaxation response, tracheal preparations were precontracted with methacholine at 1 e ( the median effective concentration value in this assay). Changes in isometric tension were measured with a force transducer (JZ100; Xinhang Machine and Equipment, Gaobeidian, China) coupled to the MedLab Biological Signal Collection System (Medease Science and Technology, Nanjing, China). After the methacholine-induced precontraction reached a plateau level, salbutamol (Sigma) was added to obtain a cumulative concentrationeCresponse curve.
Statistical Analysis
Data are expressed as means ± SD. Statistical analysis was performed with a one-way analysis of variance to evaluate cAMP-PDE activity and IL-4 level, a nonparametric test to evaluate pulmonary functions, and a two-tailed t test to evaluate PDE4 mRNA expression. Correlation was evaluated by multivariate analysis. Tracheal tissue contraction and relaxation were analyzed by factorial design analysis of variance. Differences with p values of less than 0.05 were considered statistically significant.
RESULTS
Antigen-induced Changes of Lung Function in Allergic Rats
In our rat model of allergic asthma, OVA sensitization caused a significant decrease in dynamic lung compliance (Cdyn; Figure 1). On the other hand, sensitization and challenge increased pulmonary resistance (RL) to a level significantly different from that of normal animals. Seven-day treatment with rolipram orally at 0.1, 0.3, or 1 mg/kg/day significantly improved lung function in terms of both Cdyn and RL caused by sensitization and challenge. The effect of rolipram on Cdyn was dose-dependent, with an ED50 of approximately 0.1 mg/kg. Rolipram was even more potent on RL, with complete inhibition at 0.1 mg/kg. Effects of the corticosteroid drug dexamethasone were examined by the same dosing regime, except that it was dosed intravenously. At a dose of 0.1 mg/kg, there was a significant improvement with RL. At a dose of 0.5 mg/kg, both Cdyn and RL were significantly improved.
Antigen-induced IL-4 Production in the Lung of Allergic Rats
Because IL-4 plays a critical role in the pathogenesis of allergic airway diseases, including asthma (9eC11), IL-4 levels in the lungs of rats from the various groups were measured (Figure 2). OVA sensitization did not increase IL-4 production. However, OVA sensitization and challenge significantly increased IL-4 production. The 7-day treatment, once a day, with rolipram at 0.1, 0.3, or 1 mg/kg, or with dexamethasone at 0.1 or 0.5 mg/kg, completely suppressed the increased IL-4 production and, furthermore, gave rise to IL-4 levels lower than that of normal animals.
Allergen-induced cAMP-PDE Activity Elevation in the Lung of Allergic Rats
Regarding PDE4 regulation in the lung tissues, OVA sensitization and challenge significantly elevated the lung cAMP-PDE activity, resulting in a level 2.5-fold higher than that of normal animals (Figure 3). Rolipram-sensitive (100 e) cAMP-PDE activity (i.e., PDE4 activity) was approximately twofold higher in OVA-sensitized and challenged rats than in normal rats. PDE3 is another cAMP-PDE known to play a role in the regulation of airway contraction (27). However, relative PDE3 activity (determined as the activity sensitive to the PDE3-selective inhibitor siguazodan) in total cAMP-PDE activity was conversely lower in OVA-sensitized and challenged rats than in normal rats (Figure 4). PDE5, a cGMP-specific PDE, is also abundantly present in lung (1, 4, 6). However, the PDE5-selective inhibitor zaprinast did not significantly affect the cAMP-PDE activity in either normal or OVA-sensitized and challenged rats. These results suggest that the increase in total cAMP-PDE activity by OVA sensitization and challenge might be primarily caused by upregulation of PDE4. Furthermore, like IL-4 production, the elevation of cAMP-PDE activity was also suppressed by the 7-day treatment with rolipram or dexamethasone. The inhibition by rolipram was dose-dependent, with an ED50 of approximately 0.1 mg/kg.
Because in the lung of allergic rats both cAMP-PDE activity and IL-4 levels were increased, we examined the correlation between the levels of these two biologically important proteins. By multivariate analysis, a highly significant (p < 0.01) correlation was observed (correlation coefficient: r = 0.563; Figure 5).
Allergen-induced PDE4 mRNA Elevation in the Lung of Allergic Rats
To investigate whether or not the cAMP-PDE activity elevation was caused by enhanced PDE4 gene expression, we examined lung mRNA levels of various PDE4 subtypes in normal and allergic rats. As shown in Figure 6, OVA sensitization and challenge significantly increased the levels of PDE4A, PDE4C, and PDE4D mRNAs, as compared with normal rats (p < 0.05 or p < 0.01). This result strongly suggests that the increased cAMP-PDE activity (Figure 3) might be, at least in part, caused by enhanced PDE4 gene expression by antigen sensitization and challenge.
Allergen-induced Increase in Ex Vivo Tracheal Tension
Tracheal tissues from OVA-sensitized and challenged rats had significantly increased tensions as compared with those from normal rats on stimulation with methacholine (0.01 eeC1 mM; Figure 7A). Salbutamol reversed the methacholine-induced contraction in a concentration-dependent manner. There was an increased relaxation of methacholine-induced contraction by salbutamol in normal tracheal tissues as compared with tracheal tissues from OVA-sensitized and challenged rats (Figure 7B).
DISCUSSION
The regulation of PDE4 in allergy and asthma has been investigated by many groups but only in human blood leukocytes, and the data have been discrepant. Thus, some groups have observed a significant increase in PDE4 activity in patients with asthma or allergy (13eC17), whereas others have found no significant change of the enzyme activity in such patients (19eC22) as compared with healthy individuals. The present study investigated PDE4 regulation in the lung of allergic rats. In this rat model of allergic asthma, antigen sensitization and challenge decreased lung function and increased IL-4 production. Moreover, these changes were suppressed by rolipram or dexamethasone. These results show that our model closely reflected several important features of human asthma. Furthermore, regarding PDE4 regulation in the lung, our study demonstrates that lung PDE4 is upregulated in allergic rats. To our knowledge, this study represents the first investigation on PDE4 regulation in the lung in an animal model of asthma. In this model, there was significant infiltration of inflammatory cells in the lung after OVA sensitization and challenge, as observed by bronchoalveolar lavage as well as by histologic examination (data not shown). Whether or not the increased PDE4 activity in allergic rat lungs was mainly a result of the presence of infiltrated inflammatory cells remains to be elucidated.
The upregulation of PDE4 mRNAs demonstrates that the increased lung cAMP-PDE activity was, at least in part, caused by increased PDE4 gene expression. However, nothing is known about the mechanism for the upregulated PDE4 gene expression. Nor is it known why expression of various PDE4 subtype genes was regulated differentially in the lung of allergic rats, although it is known that different PDE4 subtype genes can undergo distinct regulation (28). It is well established that cAMP induces expression of various PDE4 subtype genes, including 4B (2). The only other significant PDE4 gene inducer known thus far is LPS, which specifically activates the PDE4B gene in monocytes/macrophages (28, 29). Rolipram might be expected to enhance, but not prevent (as observed in this study), the upregulation of PDE4 by increasing cAMP levels. In fact, upregulation of PDE4 has been reported in vivo in rats treated chronically with the effective cAMP-elevating agent albuterol (30). However, in the lung of allergic rats, expression of PDE4A, PDE4C, and PDE4D genes, but not the PDE4B gene was upregulated, suggesting that the expression of the genes might be induced by a mechanism or mechanisms distinct from the cAMP or LPS mechanism. Thus, rolipram might prevent the PDE4 upregulation by a secondary mechanism or mechanisms (discussed later). Furthermore, in a cigarette smokingeCinduced mouse model of chronic obstructive pulmonary disease, which shares several important pathophysiologic features with asthma, increased PDE4D gene expression in the lung was observed (31).
It is well known that PDE4 also undergoes activation by phosphorylation (2, 5). PDE4 was activated by IL-4 (12), IFN- (12), IL-3 (32), or IL-2 (14), presumably by post-translational modification, such as phosphorylation. All these cytokines are induced at significant levels in the lungs of allergic animals (33, 34). It is conceivable that in our model the increased lung cAMP-PDE activity could also be caused in part by PDE4 activation by various cytokines. Although it is not clear exactly how dexamethasone or rolipram attenuated the PDE4 enzyme activity upregulation, one possibility for both the drugs was that they might attenuate PDE4 gene expression and/or activation through their extensive antiinflammatory effects. For instance, corticosteroid drugs (35) and PDE4 inhibitors (27) inhibit production of the various cytokines, which could result in the downregulation of PDE4 activity observed in this study. Dexamethasone also might directly inhibit PDE4 gene expression as suggested by a recent report (36). However, the possibility that ex vivo PDE4 inhibition by residual rolipram in lung tissue also contributed to the observed cAMP-PDE attenuation by rolipram treatment (Figure 3) cannot be completely ruled out.
Although it is not clear whether the upregulation of lung cAMP-PDE may be a consequence or a cause of asthma, upregulated cAMP-PDE should contribute to the pathogenesis of this disease. Increased cAMP-PDE activity should lower intracellular cAMP levels and consequently facilitate the progression of airway inflammation and exacerbation of asthmatic symptoms. For instance, it is known that 2-adrenoceptor agonists increase PDE4 enzyme activity and gene expression in the lung and that prolonged treatment with these drugs results in exacerbation of symptoms and airway hyperresponsiveness (37). Our result that rolipram prevented in vivo the enhanced PDE4 activity in the lung of allergic rats supports the therapeutic use of PDE4 inhibitors in asthma. Furthermore, in the smoking model mentioned previously, the increased airway hyperresponsiveness was not associated with increased airway leukocyte migration or mucus production but was causally related to decreased cAMP level and increased PDE4 activity, as well as the increased PDE4D gene expression, in the lung (31).
Asthma is characterized by airway inflammation, tissue injury and remodeling, bronchoconstriction, and mucus hypersecretion involving multiple tissues, such as leukocytes from circulating blood and airway sensory neurons, smooth muscle cells, and epithelial cells. PDE4 inhibitors are efficacious for asthma in animal models and in the clinic (7, 8), probably because they are able to treat all these components of the disease (27, 38, 39). Thus, if PDE4 does play a role in the pathogenesis of asthma, lung PDE4 may be as important as, if not more important than, leukocyte PDE4. Various PDE4 subtypes are known to be expressed in different tissues and play distinct biological roles. For example, PDE4B, but not PDE4D, is the predominant PDE4 subtype in monocytes (29) involved in the regulation of tumor necrosis factor production (40). On the other hand, in lung tissues, PDE4D, but not PDE4B, is the dominant PDE4 subtype, playing a critical role in the control of airway smooth muscle contraction (41). However, major PDE4 subtypes regulating the functions of airway sensory and epithelial cells remain to be elucidated. On the basis of their upregulation in the lung of allergic rats, PDEs 4A and 4C, like 4B and 4D, may also play a role in the pathogenesis of asthma in distinct tissues.
Acknowledgments
The authors thank Drs. Kenneth Adler and Robert Egan for helpful comments on the manuscript.
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Zhejiang University School of Medicine, Hangzhou, China
Schering-Plough Research Institute, Kenilworth, New Jersey
ABSTRACT
Inhibitors of phosphodiesterase-4 (PDE4) are efficacious for allergic asthma in animal models and have shown some efficacy in human asthma. Regulation of PDE4 in allergy and asthma has been widely investigated in blood leukocytes, with discrepant results. This study investigated PDE4 regulation in the lung in a rat model of allergic asthma. Ovalbumin sensitization and challenge significantly increased pulmonary resistance and lung interleukin (IL)-4 production. The increases in pulmonary resistance and IL-4 production were both suppressed by the PDE4-selective inhibitor rolipram or the corticosteroid drug dexamethasone. Furthermore, cAMP-PDE enzyme activity in the lung was also significantly increased by the sensitization and challenge. mRNA analysis confirmed that PDE4 gene expression was increased in the lung of the allergic rats. A highly significant correlation was observed between the increases in PDE activity and IL-4 production. Our data suggest, for the first time, that PDE4 may be upregulated in the lung and play a role in the pathogenesis of allergic asthma.
Key Words: asthma cAMP PDE4 phosphodiesterase-4 rat
The phosphodiesterase (PDE) superfamily comprises 11 biochemically and pharmacologically distinct enzyme families (PDEs 1eC11) that hydrolyze cAMP and/or cGMP. PDEs represent the only cellular pathways for the degradation of the ubiquitous intracellular second messengers, underscoring their critical role in regulation of cellular functions (1eC6). Most PDE families contain multiple genes (subtypes), and most of these genes give rise to multiple variants by alternative splicing among the 5'eCend exons and/or the use of different transcription initiation sites. Despite the large number of PDE enzymes with distinct biochemical and pharmacologic properties, they share some structural features. In particular, they contain a conserved catalytic domain of approximately 270 to 300 amino acids located toward the C termini.
PDE4 is specific for cAMP and comprises four subtypes (A, B, C, and D). It is predominantly expressed and plays an important role in regulation of cellular functions in inflammatory and immune cells. There has been significant interest in PDE4 inhibitors as a potential therapy for inflammatory diseases, such as allergy and asthma. In fact, PDE4 inhibitors have exhibited efficacy for asthma, allergic rhinitis, and several other diseases in the clinic (7, 8).
The regulation of PDE4 in allergy and asthma has been widely investigated in human blood leukocytes. Interleukin (IL)-4 plays a crucial role in allergic airway diseases, including asthma (9eC11). Furthermore, IL-4 significantly increased PDE4 activity in human monocytes (12). However, whether or not PDE4 is upregulated in blood leukocytes in asthma and/or allergy remains controversial. Some investigators reported a significant increase in PDE4 activity in patients with asthma or allergy as compared with healthy individuals (13eC17). It is noteworthy that when there was an increase in PDE4 activity, the increase was correlated with an increased IL-4 production (18). However, others observed no significant change of PDE4 activity in such patients (19eC22). There has been no reported study on PDE4 regulation in the lung in any animal model of asthma or in patients with asthma.
This study investigated lung PDE4 regulation in an antigen-challenged rat model. In our model, there were significantly increased pulmonary resistance and lung IL-4 production. The increases in pulmonary resistance and IL-4 production were both suppressed by the PDE4-selective inhibitor rolipram or the corticosteroid drug dexamethasone. Furthermore, in the lung, there was also a significant increase in cAMP-PDE enzyme activity as well as increases in PDE4 subtype mRNAs. Our data suggest, for the first time, that PDE4 may be upregulated in the lung and play a role in the pathogenesis of allergic asthma.
METHODS
Animal Handling, Sensitization, and Challenge
Male Sprague-Dawley rats (140eC160 g; Laboratory Animal Center of Zhejiang University School of Medicine, Hangzhou, China) were used, and were maintained under a 24-hour light/dark cycle with food and water allowed ad libitum. Animals were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animal experiments were approved by the Zhejiang Medical Laboratory Animal Administration Committee.
Rats were sensitized with a subcutaneous injection (1 ml) of a saline suspension containing 0.2% ovalbumin (OVA; Sigma, St. Louis, MO) and 10% aluminum hydroxide at their footpad, neck, back, groin, and abdomen on Day 0. In addition, each animal was primed with an intramuscular injection of 2 x 1010 heat-killed Bordatella pertussis organisms. From Day 14 after sensitization, animals were challenged once a day for 7 days by exposure for 20 minutes to aerosolized 1% OVA in saline generated by a jet nebulizer (Master; Pari GmbH, Starnberg, Germany; medium diameter of produced droplets: 1eC5 e). Control animals were exposed to an aerosol of saline. For drug treatment, rolipram (Sigma) or dexamethasone (Changzhou Second Pharmaceuticals Co., Changzhou, China) was given orally or intravenously, respectively, once a day at Days 14 through 20, 30 minutes before challenge. At Day 21, animals were killed, and lung tissues were immediately removed, frozen in liquid nitrogen, and then stored at eC80°C until analysis.
Measurement of Lung Functions
Tracheostomy was performed under urethane anesthesia (1 g/kg, injected intraperitoneally). The rat was then placed in a whole body plethysmograph, and lung functions were measured as described previously (23).
Assay for cAMP-PDE Activity
Frozen lungs were thawed and cut into small cubes. Twenty-five milligrams of lung tissues were homogenized in 100 e of ice-cold 30 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (pH 7.4) containing 0.1% Trion X-100 (Sigma). The cAMP-PDE assay mixture (200 e) in phosphate-buffered saline (pH 7.4) contained 137 mM NaCl, 2.7 mM KCl, 8.8 mM Na2HPO4, 1.5 mM KH2PO4, 1 mM CaCl2, 1 mM MgCl2, 1 e cAMP (Sigma), and lung homogenate. The PDE reaction was started by the addition of 10 e lung homogenate and was performed at 37°C for 10 minutes. The reaction was stopped by boiling the mixture for 3 minutes. The assay mixture was cooled on ice, followed by centrifugation at 12,850 x g for 30 minutes at 4°C. The amount of cAMP present in the supernatant was determined by HPLC (Hypersil ODS 4.0 x 250 mm; Hewlett-Packard, Palo Alto, CA) using a standard curve of cAMP.
Quantification of Lung IL-4
Lungs were homogenized at a ratio of 1 g to 10 ml in 50 mM potassium phosphate buffer (pH 6.0) containing 0.05% NaN3 and 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHARPS). The homogenates were centrifuged twice at 19,851 x g, each for 30 minutes. IL-4 in the supernatants was quantified using a rat IL-4eCspecific ELISA kit (Jingmei BioTech, Shenzhen, China).
Assay for Total Protein
Total protein amounts were determined by the Bradford method (24) using bovine serum albumin as the standard.
RNA Isolation and Complementary DNA Synthesis
Total RNA was isolated from each tissue using Trizol reagent (Invitrogen, Carlsbad, CA), The preparation of first-strand complementary DNA (cDNA) from total RNA was performed using a first-strand cDNA synthesis kit (Shanghai Sangon Biological Engineering Technology and Service [Sangon], Shanghai, China).
Semiquantitative Reverse Transcription-Polymerase Chain Reaction Analysis for Rat PDE4 Subtype mRNAs
Sequences of polymerase chain reaction (PCR) primer sets used for PDE4A, PDE4B, PDE4D (25), and PDE4C (26) were as reported previously. Each of the PCR primer sets was able to detect all known variants derived from the appropriate PDE4 gene (2). PCR amplification was performed in a PCR buffer (10 mM Tris-HCl, pH 9.0; 100 mM KCl; 80 mM [NH4]2SO4; and 0.1% Nonidet P-40) containing 0.2 mM of each nucleotide, 1.5 mM of MgCl2, 500 nM of each primer, and 1 U of Taq DNA polymerase (Sangon) in a total volume of 25 e for 30 cycles, with the following cycle parameters: denaturing, 94°C for 45 seconds; annealing, 58°C for 70 seconds; and extension, 72°C for 2 minutes. After PCR amplification, 8 e of each reaction mixture were resolved by electrophoresis on a 1.5% agarose gel containing ethidium bromide, and the PCR product bands were quantified by using the UVP Gel Documentation system (UVP, Upland, CA). The levels of PDE4 mRNAs were calculated relative to -actin. It was confirmed that under these conditions the PCR product accumulation did not reach plateau levels (data not shown).
Measurement of Tracheal Tissue Contraction and Relaxation
Trachea free of connective tissue was isolated in oxygenated Krebs-Henseleit buffer. Each trachea was cut into six 1-cm-long sections without damage to the epithelium. Three sections were stitched together as one tracheal preparation. Tracheal preparations were equilibrated at 0.5 g of initial tension in the Krebs-Henseleit buffer at 37°C in an atmosphere of 5% CO2/95% O2 for 30 minutes. To measure contractile response, a cumulative concentrationeCresponse curve was obtained with methacholine (Sigma). To measure relaxation response, tracheal preparations were precontracted with methacholine at 1 e ( the median effective concentration value in this assay). Changes in isometric tension were measured with a force transducer (JZ100; Xinhang Machine and Equipment, Gaobeidian, China) coupled to the MedLab Biological Signal Collection System (Medease Science and Technology, Nanjing, China). After the methacholine-induced precontraction reached a plateau level, salbutamol (Sigma) was added to obtain a cumulative concentrationeCresponse curve.
Statistical Analysis
Data are expressed as means ± SD. Statistical analysis was performed with a one-way analysis of variance to evaluate cAMP-PDE activity and IL-4 level, a nonparametric test to evaluate pulmonary functions, and a two-tailed t test to evaluate PDE4 mRNA expression. Correlation was evaluated by multivariate analysis. Tracheal tissue contraction and relaxation were analyzed by factorial design analysis of variance. Differences with p values of less than 0.05 were considered statistically significant.
RESULTS
Antigen-induced Changes of Lung Function in Allergic Rats
In our rat model of allergic asthma, OVA sensitization caused a significant decrease in dynamic lung compliance (Cdyn; Figure 1). On the other hand, sensitization and challenge increased pulmonary resistance (RL) to a level significantly different from that of normal animals. Seven-day treatment with rolipram orally at 0.1, 0.3, or 1 mg/kg/day significantly improved lung function in terms of both Cdyn and RL caused by sensitization and challenge. The effect of rolipram on Cdyn was dose-dependent, with an ED50 of approximately 0.1 mg/kg. Rolipram was even more potent on RL, with complete inhibition at 0.1 mg/kg. Effects of the corticosteroid drug dexamethasone were examined by the same dosing regime, except that it was dosed intravenously. At a dose of 0.1 mg/kg, there was a significant improvement with RL. At a dose of 0.5 mg/kg, both Cdyn and RL were significantly improved.
Antigen-induced IL-4 Production in the Lung of Allergic Rats
Because IL-4 plays a critical role in the pathogenesis of allergic airway diseases, including asthma (9eC11), IL-4 levels in the lungs of rats from the various groups were measured (Figure 2). OVA sensitization did not increase IL-4 production. However, OVA sensitization and challenge significantly increased IL-4 production. The 7-day treatment, once a day, with rolipram at 0.1, 0.3, or 1 mg/kg, or with dexamethasone at 0.1 or 0.5 mg/kg, completely suppressed the increased IL-4 production and, furthermore, gave rise to IL-4 levels lower than that of normal animals.
Allergen-induced cAMP-PDE Activity Elevation in the Lung of Allergic Rats
Regarding PDE4 regulation in the lung tissues, OVA sensitization and challenge significantly elevated the lung cAMP-PDE activity, resulting in a level 2.5-fold higher than that of normal animals (Figure 3). Rolipram-sensitive (100 e) cAMP-PDE activity (i.e., PDE4 activity) was approximately twofold higher in OVA-sensitized and challenged rats than in normal rats. PDE3 is another cAMP-PDE known to play a role in the regulation of airway contraction (27). However, relative PDE3 activity (determined as the activity sensitive to the PDE3-selective inhibitor siguazodan) in total cAMP-PDE activity was conversely lower in OVA-sensitized and challenged rats than in normal rats (Figure 4). PDE5, a cGMP-specific PDE, is also abundantly present in lung (1, 4, 6). However, the PDE5-selective inhibitor zaprinast did not significantly affect the cAMP-PDE activity in either normal or OVA-sensitized and challenged rats. These results suggest that the increase in total cAMP-PDE activity by OVA sensitization and challenge might be primarily caused by upregulation of PDE4. Furthermore, like IL-4 production, the elevation of cAMP-PDE activity was also suppressed by the 7-day treatment with rolipram or dexamethasone. The inhibition by rolipram was dose-dependent, with an ED50 of approximately 0.1 mg/kg.
Because in the lung of allergic rats both cAMP-PDE activity and IL-4 levels were increased, we examined the correlation between the levels of these two biologically important proteins. By multivariate analysis, a highly significant (p < 0.01) correlation was observed (correlation coefficient: r = 0.563; Figure 5).
Allergen-induced PDE4 mRNA Elevation in the Lung of Allergic Rats
To investigate whether or not the cAMP-PDE activity elevation was caused by enhanced PDE4 gene expression, we examined lung mRNA levels of various PDE4 subtypes in normal and allergic rats. As shown in Figure 6, OVA sensitization and challenge significantly increased the levels of PDE4A, PDE4C, and PDE4D mRNAs, as compared with normal rats (p < 0.05 or p < 0.01). This result strongly suggests that the increased cAMP-PDE activity (Figure 3) might be, at least in part, caused by enhanced PDE4 gene expression by antigen sensitization and challenge.
Allergen-induced Increase in Ex Vivo Tracheal Tension
Tracheal tissues from OVA-sensitized and challenged rats had significantly increased tensions as compared with those from normal rats on stimulation with methacholine (0.01 eeC1 mM; Figure 7A). Salbutamol reversed the methacholine-induced contraction in a concentration-dependent manner. There was an increased relaxation of methacholine-induced contraction by salbutamol in normal tracheal tissues as compared with tracheal tissues from OVA-sensitized and challenged rats (Figure 7B).
DISCUSSION
The regulation of PDE4 in allergy and asthma has been investigated by many groups but only in human blood leukocytes, and the data have been discrepant. Thus, some groups have observed a significant increase in PDE4 activity in patients with asthma or allergy (13eC17), whereas others have found no significant change of the enzyme activity in such patients (19eC22) as compared with healthy individuals. The present study investigated PDE4 regulation in the lung of allergic rats. In this rat model of allergic asthma, antigen sensitization and challenge decreased lung function and increased IL-4 production. Moreover, these changes were suppressed by rolipram or dexamethasone. These results show that our model closely reflected several important features of human asthma. Furthermore, regarding PDE4 regulation in the lung, our study demonstrates that lung PDE4 is upregulated in allergic rats. To our knowledge, this study represents the first investigation on PDE4 regulation in the lung in an animal model of asthma. In this model, there was significant infiltration of inflammatory cells in the lung after OVA sensitization and challenge, as observed by bronchoalveolar lavage as well as by histologic examination (data not shown). Whether or not the increased PDE4 activity in allergic rat lungs was mainly a result of the presence of infiltrated inflammatory cells remains to be elucidated.
The upregulation of PDE4 mRNAs demonstrates that the increased lung cAMP-PDE activity was, at least in part, caused by increased PDE4 gene expression. However, nothing is known about the mechanism for the upregulated PDE4 gene expression. Nor is it known why expression of various PDE4 subtype genes was regulated differentially in the lung of allergic rats, although it is known that different PDE4 subtype genes can undergo distinct regulation (28). It is well established that cAMP induces expression of various PDE4 subtype genes, including 4B (2). The only other significant PDE4 gene inducer known thus far is LPS, which specifically activates the PDE4B gene in monocytes/macrophages (28, 29). Rolipram might be expected to enhance, but not prevent (as observed in this study), the upregulation of PDE4 by increasing cAMP levels. In fact, upregulation of PDE4 has been reported in vivo in rats treated chronically with the effective cAMP-elevating agent albuterol (30). However, in the lung of allergic rats, expression of PDE4A, PDE4C, and PDE4D genes, but not the PDE4B gene was upregulated, suggesting that the expression of the genes might be induced by a mechanism or mechanisms distinct from the cAMP or LPS mechanism. Thus, rolipram might prevent the PDE4 upregulation by a secondary mechanism or mechanisms (discussed later). Furthermore, in a cigarette smokingeCinduced mouse model of chronic obstructive pulmonary disease, which shares several important pathophysiologic features with asthma, increased PDE4D gene expression in the lung was observed (31).
It is well known that PDE4 also undergoes activation by phosphorylation (2, 5). PDE4 was activated by IL-4 (12), IFN- (12), IL-3 (32), or IL-2 (14), presumably by post-translational modification, such as phosphorylation. All these cytokines are induced at significant levels in the lungs of allergic animals (33, 34). It is conceivable that in our model the increased lung cAMP-PDE activity could also be caused in part by PDE4 activation by various cytokines. Although it is not clear exactly how dexamethasone or rolipram attenuated the PDE4 enzyme activity upregulation, one possibility for both the drugs was that they might attenuate PDE4 gene expression and/or activation through their extensive antiinflammatory effects. For instance, corticosteroid drugs (35) and PDE4 inhibitors (27) inhibit production of the various cytokines, which could result in the downregulation of PDE4 activity observed in this study. Dexamethasone also might directly inhibit PDE4 gene expression as suggested by a recent report (36). However, the possibility that ex vivo PDE4 inhibition by residual rolipram in lung tissue also contributed to the observed cAMP-PDE attenuation by rolipram treatment (Figure 3) cannot be completely ruled out.
Although it is not clear whether the upregulation of lung cAMP-PDE may be a consequence or a cause of asthma, upregulated cAMP-PDE should contribute to the pathogenesis of this disease. Increased cAMP-PDE activity should lower intracellular cAMP levels and consequently facilitate the progression of airway inflammation and exacerbation of asthmatic symptoms. For instance, it is known that 2-adrenoceptor agonists increase PDE4 enzyme activity and gene expression in the lung and that prolonged treatment with these drugs results in exacerbation of symptoms and airway hyperresponsiveness (37). Our result that rolipram prevented in vivo the enhanced PDE4 activity in the lung of allergic rats supports the therapeutic use of PDE4 inhibitors in asthma. Furthermore, in the smoking model mentioned previously, the increased airway hyperresponsiveness was not associated with increased airway leukocyte migration or mucus production but was causally related to decreased cAMP level and increased PDE4 activity, as well as the increased PDE4D gene expression, in the lung (31).
Asthma is characterized by airway inflammation, tissue injury and remodeling, bronchoconstriction, and mucus hypersecretion involving multiple tissues, such as leukocytes from circulating blood and airway sensory neurons, smooth muscle cells, and epithelial cells. PDE4 inhibitors are efficacious for asthma in animal models and in the clinic (7, 8), probably because they are able to treat all these components of the disease (27, 38, 39). Thus, if PDE4 does play a role in the pathogenesis of asthma, lung PDE4 may be as important as, if not more important than, leukocyte PDE4. Various PDE4 subtypes are known to be expressed in different tissues and play distinct biological roles. For example, PDE4B, but not PDE4D, is the predominant PDE4 subtype in monocytes (29) involved in the regulation of tumor necrosis factor production (40). On the other hand, in lung tissues, PDE4D, but not PDE4B, is the dominant PDE4 subtype, playing a critical role in the control of airway smooth muscle contraction (41). However, major PDE4 subtypes regulating the functions of airway sensory and epithelial cells remain to be elucidated. On the basis of their upregulation in the lung of allergic rats, PDEs 4A and 4C, like 4B and 4D, may also play a role in the pathogenesis of asthma in distinct tissues.
Acknowledgments
The authors thank Drs. Kenneth Adler and Robert Egan for helpful comments on the manuscript.
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