The Role of PDE4 in Pulmonary Inflammation and Goblet Cell

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Biochimica et Biophysica Acta 1762 (2006) 525–532 http://www.elsevier.com/locate/bba

Review The role of PDE4 in pulmonary inflammation and goblet cell hyperplasia in allergic rats ⁎ Hui-Fang Tang a, Ji-Qiang Chen a, , Qiang-Min Xie a, Xu-Yang Zheng, Yi-Liang Zhu a, ⁎ Ian Adcock b, Xiangdong Wang c,

a Zhejiang Respiratory Drugs Research Laboratory of State Foods and Drugs Administration of China, Medical School of Zhejiang University, Hangzhou 310031, China b Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College Faculty of Medicine, UK c Department of Respiratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China Received 1 December 2005; accepted 16 December 2005 Available online 18 January 2006

Abstract

Phosphodiesterase 4 (PDE4) has been suggested to a critical factor in the pathogenesis of inflammation by metabolizing cAMP in human leukocytes, endothelium and epithelium. The present study aimed at evaluating the PDE4 activity and expression, the relationship between the inflammation and cAMP- activity in the lungs, and potential interventions of PDE inhibitors and antiinflammatory drugs in the reduction of lung inflammation and goblet cell hyperplasia in allergic rats. The total leukocyte number and eosinophil number in bronchoalveolar lavegar fluid and PDE4 activity and expression in lungs significantly increased in OVA-sensitized and challenged allergic rat. Lung histology showed an increased infiltration of inflammatory cells in the perivascular and peribronchial spaces, structure changes and goblet cell hyperplasia in the OVA-sensitized and -challenged allergic rats. A significant correlation was observed between the increases in cAMP-PDE activity and inflammation in the lung. Those OVA-induced changes were prevented by pretreatment with PDE inhibitor in a dose-related patterns and with glucocorticosteroid. We found an increase in the proportion of PDE4 and PDE4 gene expression, while a decrease in the proportion of PDE3 in the lung of allergic rats. Incubation with different PDE inhibitors down-regulated OVA-induced cAMP hydrolysis. Our data suggest that PDE4C may play an important role in the airway inflammation, remodeling and goblet cell hyperplasia after repeated challenge of sensitized rats. © 2006 Elsevier B.V. All rights reserved.

Keywords: Asthma; PDE4s; Piclamilast; Dexamethasone; Indomethacin; Inflammation; Goblet cell hyperplasia; Rat

1. Introduction isoforms of PDE4 have been shown to interact with other proteins and lipids, allowing specific isoforms to be targeted to Cyclic nucleotide phosphodiesterases (PDEs) represent distinct intracellular sites and signaling complexes within cells critical pathways for the degradation of the ubiquitous [3]. Experimental and clinical studies demonstrated that intracellular second messenger 3′,5′-cyclic adenosine mono- inhibition of PDE4 can attenuate cell inflammatory response phosphate (cAMP) and/or 3′,5′-cyclic guanosine monopho- and bronchial hyperresponsiveness [4–8]. It has been suggested sphate (cGMP), involved in regulation of cellular functions [1]. that PDE4 inhibitors may be a potential therapy for chronic PDEs comprise eleven biochemically and pharmacologically airway diseases such as asthma and chronic obstructive lung distinct enzyme families (PDEs 1–11), of which PDE4 is cAMP- disease, since treatment with PDE4 inhibitors showed better specific and encoded by four genes (A, B, C, D) [2]. Various effects of antiinflammation and immunomodulation [2,9]. Although PDE4 activity was noted to be increased in circulating leukocytes in asthmatic or allergic patients [10– ′ ′ Abbreviations: PDE, phosphodiesterase; cAMP, 3 ,5-cyclic adenosine 15], the role of PDE4 activity in the pathogenesis of asthma monophosphate phosphodiesterase; OVA, ovalbumin; BALF, bronchoalveolar lavage fluid; AB/PAS, Alcian Blue-Periodic Acid Schiff and/or allergy remains unclear. While others observed no ⁎ Corresponding authors. significant change of PDE4 activity in such patients [16–18]. E-mail address: [email protected] (X. Wang). Recently, Singh SP et al. reported that prenatal exposure to

0925-4439/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2005.12.009 526 H.-F. Tang et al. / Biochimica et Biophysica Acta 1762 (2006) 525–532 cigarette smoke affected the airway reactivity by modulating levels of cAMP in the lung through changes in PDE-4D activity [19], indicating the importance of altered PDE4 activity. However, there is still a need for understanding the role of lung PDE4 activity in the development of allergic asthma. Our previous study demonstrated that cAMP-PDE activity was elevated in lung tissue harvested from rats sensitized and challenged with ovalbumin (OVA) [20].In order to understand the involvement of PDE4 in antigen- induced airway inflammation and remodeling, the mRNA Fig. 1. Experimental protocol of the sensitization with 0.2% ovalbumin (OVA) expression of PDE4 subtypes and PDE4 activity in the lung and 10% aluminium and repeated inhalation of OA once daily for 7 days from the day 14 after the sensitization. Animals in control group received saline were investigated in a rat model of asthma after intratracheal aerosol. Piclamilast, dexamethasone and indomechacin or vehicle (1% MC) was exposure of OVA. We also evaluate the potential effects of orally administered once a day during the exposure to OVA. cAMP-PDE or cyclooxygenase activity on the development of antigen-induced airway inflammation, goblet cell hyper- 2.4. Bronchoalveolar lavage plasia, and lung damage, by using a selective PDE4 inhibitor piclamilast (RP73401, N-(3,5-dichloropyrid-4-yl)-3-cyclopen- Bronchoalveolar lavage (BAL) was performed by gently instilling D-Hank' tyl-oxy-4-methoxy benzamide) and a non-selective cycloox- solution into the lung via a tracheal catheter followed by withdrawal. Such ygenase inhibitor indomethacin, to compare with those of process was repeated three times with 5 mL D-Hank's as total volume. Total antiinflammatory glucocorticosteroid dexamethasone. BAL cells counts were determined with a hemacytometer. Differential cells were counted after slides were prepared by centrifuged at 1500 rpm for 10 min and staining with Wright stain. The number of total cells for each sample was then 2. Materials and methods determined according to the volume of BAL recovered.

2.1. Animals 2.5. Histology

Male Sprague–Dawley rats, weighing 150–170 g (Shanghai Laboratory The lungs were harvested and fixed in the buffered formalin (10%, pH 7.0) Animal Center, Chinese Academy Sciences, Grade II, certificate No., 22- for 48 h. The samples were embedded in paraffin and dehydrated in 70 to 100% 9601018), were maintained on diets free of OVA. All experimental animals used ethanol/xylene. Sections were cut at 2–4 μm thickness, deparaffinized, and in this study were under a protocol approved by the Zhejiang Medical stained with hematoxylin and eosin. Pathological changes in the lungs were Laboratorial Animal Administration Committee of China. assessed, including the number of eosinophil influx, tissue edema, and epithelial lesion. Inflammatory changes were graded as normal (scale 0), rare (scale 1), 2.2. Drugs mild (scale 2), moderate (scale 3), severe (scale 4), as described previously [21]. To analyze airway remodeling, the thickness of the bronchiolar wall was Piclamilast, Siguazodan (SK and F 94836) and theophylline were kindly measured by a pathologic diagram-writing analyzing system (HPIAS-1000, provided by Beijing Joinn Drug Research Center, Professor Han-Liang Zhou Wuhan Champion Image Technology Corporation Limited, Wuhan, China). (Medical School of Zhejiang University), and MinSheng Pharmaceutical Co Goblet cells containing the mucous were identified by staining with Alcian Ltd. (HangZhou, China). Rolipram, indomethacin, ovalbumin (Grade V), Blue-periodic acid Schiff (AB/PAS). Positive cells with purple/magenta color cAMP, cGMP, phenyl methyl sulfonyl fluoride (PMSF), pepstatin A were were counted by using Olympus CH-20 photomicroscope at ×400 and expressed purchased from Sigma (Shanghai, China). Dexamethasone sodium phosphate as the number of goblet cells per mm of epithelial layer. Histopathological (Dex) was from Zhejiang Xuanju Pharmaceutical Co Ltd., Xuanju, China, assessment was performed blind on randomized sections. methylhydroxylpropyl cellulose (MC) from Zhongmei Huadong Pharmaceutical Co Ltd. (HangZhou, China), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic 2.6. Tissue homogenates acid (HEPES) from Sangon Biological Engineering Technology and Service Co Ltd. (Shanghai, China), Coomassie brilliant blue protein reagents from The lung right lobes were removed and homogenated (DY89-I Homo- Jiancheng Bio-Tek Corporation (Nanjing, China), and methanol from Siyou genater, Xinzhi SCITECH research institute, Linbo, China) in ice-cold Pharmaceutical Co Ltd. (Tianjing, China). hypotonic homogenization buffer and centrifuged (Eppendorf Centrifuge, 5804R, Germany) at 12,850×g for 30 min at 4 °C. The buffer solution 2.3. OVA sensitization and challenge contained 30 mmol/L HEPES, pH 7.4, 1 mmol/L Ethylenediaminetetraacetic Acid (EDTA), 1 mmol/L β-mercaptoethanol, 2 mmol/L PMSF and 10 g/L The procedure of animal sensitization and challenge was modified on the pepstatin A (0.1% Triton X-100). The supernatants were collected and stored at − basis of the previous study [21] and the experimental protocol is shown in Fig. 1. 80 °C until the further assay PDE activity. In short, 0.5 mL of an allergen solution containing 0.2% OVA mixed with 10% aluminium hydroxide gel was injected intraperitoneally, while another 0.5 mL 2.7. Assay of PDE activity was divided nine intracutaneous injection sites in the proximity of lymph nodes in the paws, lumbar regions and the neck on day 0. For the preparation of the The PDE activity was analyzed by a modified method of the previous study drugs suspensions, the solution of 1% MC were used. The solution of piclamilst [20] at RP-HPLC (HP1100, agilent, USA). A 10-μL sample was added to the (0.3, 1, 3 or 10 mg/kg, respectively), dexamethasone (0.3 mg/kg), indomethacin Eppendorf tubes. Each sample has an inactive copy for control. After addition of −1 (3 mg/kg), or 1% MC for same volume was intratracheally administered at the PBS (mmol/L : NaCl 137, KCl 2.7, Na2HPO4 8.8, KH2PO4 1.5, CaCl2 1.0, and volume of 2 mL/100 g (body weight) for 7 days once a day from 14 days and on MgCl2 1.0, pH=7.4), and 10 μmol/L cAMP and cGMP 20 μL, respectively, the after the sensitization of OVA. Thirty minutes after each administration of reaction was allowed to proceed under the incubation at 37 °C for 10 min and pretreatments, rats were placed in an arosol chamber and arosolly exposed to stopped in boiling water for 2 min. The supernatants were collected, and either saline (NS) or 1% OVA, generated in a jet nebulizer (particle size 1–5 μm; centrifuged at 12,850×g for 30 min at 4 °C. The amount of cAMP present in 50 PARI MASTER, Starnberg, Germany) for 20 min. μL supernatant was determined by RP-HPLC analysis (Hypersil ODS 4.0×250 H.-F. Tang et al. / Biochimica et Biophysica Acta 1762 (2006) 525–532 527 mm, Hewlett-Packard, Palo Alto, CA) using a standard curve of cAMP. The changes at the concentrations of 1 and 3 mg/kg with a dose- PDE inhibitors, theophylline (non-selective), rolipram (PDE4-selective) and related pattern. OVA-animals pretreated with dexamethasone siguazodan (PDE3-selective) were used to inhibit the cAMP-PDE activity to analyze the component of cAMP-PDE activity in the lung. Protein was had similar levels to controls, significantly different from determined by the Bradford method with bovine serum albumin as a standard OVA-sensitized and -challenged animals pretreated with MC [22]. The results were expressed as nanomolar of PDE activity per milligram of (Pb0.01, Fig. 3A, B and C). Animals with indomethacin had protein. significantly lower number of infiltrated eosinophils than OVA-animals with MC (Fig. 3A), but still significantly higher 2.8. Analysis of PDE4 subtype mRNAs than controls (P b0.05, respectively). Pretreatment with indomethacin had not effects on OVA-induced lung tissue Total RNA was isolated from the tissue using TRIzol Reagent (Invitrogen, Carlsbad, CA). Preparation of first-strand cDNA from rat was performed edema (Fig. 3B) and epithelial damage (Fig. 3C). using First-strand cDNA Synthesis kit (Shanghai Sangon Biological Levels of bronchial wall thickness and the number of goblet Engineering Technology and Service, Shanghai, China). Sequences of PCR cells were significantly higher in OVA-challenged animals primer sets used for PDE4 were used as reported previously, each of the PCR pretreated with MC, as compared to controls (Pb0.05, Table 1), primer sets was able to detect all known variants derived from the appropriate while pretreatment with dexamethasone significantly prevented PDE4 gene [23,24]. PCR amplification was performed in a PCR buffer [10 from OVA-induced bronchial thickness (Pb0.05, vs. OVA- mM Tris–HCl, pH 9.0, 100 mM KCl, 80 mM (NH4)2SO4 and 0.1% NP-40] containing each dNTP at 0.2 mM, 1.5 mM MgCl2, each primer at 500 nM, challenged rats pretreated with MC), rather than by piclamilast and 1 U of Taq DNA polymerase (Sangon) in a total reaction volume of 25 at the low concentrations and indomethacin. Pretreatment with μL for 30 cycles, with the following cycle parameters: denaturing 94 °C for either piclamilast or dexamethasone significantly prevented 45 s, annealing 56 °C for 70 s and extension 72 °C for 2 min. After PCR μ OVA-induced increased number of goblet cells, as compared to amplification, 8 L of each reaction mixture was resolved by electrophoresis b on a 1.5% agarose gel containing ethidium bromide, and the PCR product OVA-challenged animals pretreated with MC (P 0.05 or 0.01, bands were quantified by using UVP Gel Documentation system (UVP, respectively, Table 1). Upland, CA). The levels of PDE4 mRNAs were calculated relative to β-actin. In the lung histology, increased number of infiltrated Under the condition, the PCR product accumulation did not reach plateau eosinophils was observed in peri-bronchial (Fig. 4B) and peri- levels (data not shown). vascular (Fig. 4F) tissues of OVA-challenged animals pretreated with MC. In OVA-animals with MC, monolayer of epithelial 2.9. Statistics cells on the small airway became multiple and thicker (Fig. 5B) All data were presented as Mean±S.E.M. One-way ANOVA analysis was and goblet cell hyperplasia contained both acid (purple color) used to evaluate the levels of cAMP-PDE activities, bronchial wall thickness and and neutral (magenta) mucins (Fig. 5F). These OVA-induced goblet cell hyperplasia. Statistical Package for the Social Sciences (SPSS) 11.0 histological alterations were obviously prevented by pretreat- b was used to evaluate the histological score. A probability of P 0.05 was ment with dexamethasone (Figs. 4D and H, 5D and H). considered significant. Piclamist at higher doses (3 mg/kg) inhibited the influx of inflammatory cells in the lung tissue (Fig. 4C and G) and 3. Results thickening of the bronchiolar wall in small bronchial (Fig. 5C). Pretreatment with piclamilast at all doses had partially There was no significant difference in histological inhibitory effects on goblet cell hyperplasia (Fig. 5G), while measurements between animals sensitized and challenged indomethacin did not have inhibitory effects on all changes with saline and challenged with OVA or sensitized with OVA (Figs. 4E and I, 5E and I). and challenged with saline (data not shown). Data from those The cAMP-PDE activity significantly increased in the lung animals were pooled as controls. The number of total cells tissue of OVA-sensitized and challenged rats pretreated with (Fig. 2A) and eosinophils (Fig. 2B) in BALF significantly MC, compared with controls (Pb0.01, Fig. 6). Pretreatment increased in OVA-sensitized and challenged rats pretreated with piclamilast showed an inhibitory effects on lung cAMP- with MC, as compared to controls (Pb0.01, respectively). PDE with a dose-dependent patter. The similar effects of Pretreatment with piclamilast and dexamethasone significantly dexamethasone (0.3 mg/kg) to piclamilast at the dose of 3 mg/ prevented from OVA-induced increased number of both total kg on the inhibition of lung cAMP-PDE activities were noted leukocytes and eosinophils in BAL fluid (Pb0.05 or less), as with a significant different from OVA rats with MC (Pb0.05, compared to OVA-sensitized and challenged animals pre- respectively, Fig. 6), but not indomethacin. In order to treated with MC, while indomethacin showed inhibitory investigate the potential effects of PDE inhibitors on OVA- effects only on number of leukocytes (Fig. 2A). Number of induced cAMP-PDE activities, theophylline as a non-selective total leukocytes and eosinophils in BAL fluid of animals PDE inhibitor, rolipram as a PDE4-selective inhibitor and pretreated with piclamilast at the concentrations of 0.3 mg/kg siguazodan as a PDE3-selective inhibitor were incubated with and indomethacin was significantly higher than that in the lung homogenates from controls or OVA-sensitized and controls. Number of infiltrated eosinophils (Fig. 3A), scores challenged rats pretreated with MC. Fig. 7 shows that these of tissue edema (Fig. 3B) and epithelial damage (Fig. 3C) in compounds significantly inhibited cAMP-PDE activity at a the lung tissue significantly increased in OVA-sensitized and concentration-related pattern. Theophylline inhibited the hy- challenged animals pretreated with MC, as compared to drolysis of homogenates in the lung from controls or OVA- controls (Pb0.05, respectively). Pretreatment with piclamilast challenged rats with a parallel potency. The inhibited hydrolysis showed significant inhibitory effects on OVA induced these of cAMP was 69% and 84% by theophylline at the 528 H.-F. Tang et al. / Biochimica et Biophysica Acta 1762 (2006) 525–532

Table 1 Bronchial wall thickness and number of goblet cell in pulmonary tissue of OVA- challenged rats treated with piclamilast, dexamethasone or indomethacin Groups Dose n Bronchial wall Number of Goblet thickness (μm) cell/mm epithelium Controls Saline 4 7 0.4±0.0 4.9±1.7 mL/kg MC-treated OVA Saline 4 7 0.6±0.0+ 38.8±7.1++ rats mL/kg Piclamilast-treated 0.3 mg/kg 6 0.6±0.1 22.9±2.8* OVA rats 1 mg/kg 6 0.5±0.0 18.1±5.3* 3 mg/kg 6 0.4±0.0 23.2±3.8* Dexamethasone- 0.3 mg/kg 6 0.3±0.0* 12.2±1.8** treated OVA rats Indomethacin- 3 mg/kg 7 0.7±0.1 46.1±4.2 treated OVA rats + and ++ stand for the P values less than 0.05 and 0.01, respectively, as compared to the control group. * and ** stand for the P values less than 0.05 and 0.01, respectively, as compared to OVA-challenged animals treated with Fig. 2. The number of total leukocytes (A) and eosinophils (B) in vehicle. bronchoalveolar lavage fluid in OVA- or PBS-sensitized and challenged animals treated with piclamilast (Picl), dexamethasone (Dex) and indomechacin (Indom) or vehicle (1% MC). + and ++ stand for the P values less than 0.05 and sensitized and challenged rats, as shown in Fig. 8A. Expression 0.01, respectively, as compared to the control group. * and ** stand for the P of PDE4C mRNA significantly increased after OVA sensitiza- values less than 0.05 and 0.01, respectively, as compared to OVA-challenged tion and challenge, as compared with controls (Pb0.01, Fig. animals treated with vehicle. 8B). There was a significant correlation between lung cAMP- concentration of 10−3 M(Fig. 7A) in controls and OVA- PDE activity and the number of leukocytes and eosinophils in challenged rats, 40% and 70% by rolipram at 10−4 M(Fig. 7B), BAL fluid (Spearman's Correlation Coefficient=0.836, and 47% and 20% by siguazodan at 10−4 M(Fig. 7C), Pb0.01) and in the lung tissue (Pb0.05). respectively. Fig. 8 demonstrates lung mRNA levels of PDE4 subtypes 4. Discussion measured by semi-quantitative PCR in controls and OVA- sensitized and challenged rats. A moderate elevation of PDE4A Airway inflammation is a critical feature of asthma, and 4B mRNA expression in the lung harvested from OVA- involving mediator release from mast cells and eosinophils and orchestrating by T cells. Eosinophilic infiltration is a prominent feature in the pathophysiology of asthma. In addition, asthma is also characterized by pronounced structural changes of the bronchial wall associated with inflammatory process and termed as airway remodeling, such as damage and shedding of the airway surface epithelium, thickness of the epithelial reticular basement membrane [25,26]. While excess mucus secretion not only obstructs airways but also contributes to airway hyperresponsiveness [27,38]. Potential role of PDE4 in the development of allergical diseases has been investigated only in human blood leukocytes. Our previous study demonstrated that cAMP-PDE activity was significant increased in lung homogenate harvested from antigen-sensitized and challenged rats [20]. The results from the present study furthermore suggested that the increase of cAMP-PDE activity in the lung of OVA-sensitized and -challenged rat may result from the increased activity of PDE4, since the significant increase in PDE4 mRNA expression was noted after antigen challenges. Little has been known about the mechanism by which Fig. 3. Eosinophil number (A), tissue edema score (B), and epithelial damage PDE4 gene expression was up-regulated and about the scores (C) in the lung tissue harvested from OVA- or PBS-sensitized and expression of various PDE4 subtype genes in the lungs after challenged animals treated with piclamilast (Picl), dexamethasone (Dex) and antigen challenges, although it was shown that different PDE4 indomechacin (Indom) or vehicle (1% MC). + and ++ stand for the P values less – than 0.05 and 0.01, respectively, as compared to the control group. * and * stand subtype genes can undergo distinct regulation [28 30]. cAMP for the P values less than 0.05 and 0.01, respectively, as compared to OVA- has been found to induce the expression of various PDE4 challenged animals treated with vehicle. subtype genes including 4D5 [28]. In transiently transfected H.-F. Tang et al. / Biochimica et Biophysica Acta 1762 (2006) 525–532 529

Fig. 4. Histological alterations of the bronchial mucosal layer (A–E) and perivascular inflammation (E–I) in control animals (A and E), OVA-challenged animals treated with vehicle (B and F), with piclamilast at the high dose (C and G), with dexamethasone (D and H), or with indomethacin (E and I). into β 2 adrenoreceptor-expressing CHO-K1 cell line, Houslay gene activation by LPS may constitute a feedback regulation et al. demonstrated that the PDE4D5 promoter up-regulated essential for an efficient immune response. Furthermore, reporter gene expression in response to increased cell cAMP experimental studies also demonstrated that cigarette smoking [3]. Increased cAMP-dependent induction of PDE4D5 tran- induced chronic pulmonary pathophysiological features in script and activity in primary cultured human airway smooth mice, accompanied by an increased PDE4D gene expression muscle cells (hASMs) led to an up-regulation of functional in the lung [31]. It is possible that the expression and activity [28]. The inflammatory mediators, lipopolysaccharide, activation of PDE4 subtypes are highly associated with the specifically activates PDE4B gene and PDE4 activity in pathological situations. Our results showed that challenge with peripheral monocytes and neutrophils harvested from mice antigen could obviously up-regulate expression of the PDE 4C deficient in PDE4 (type 4 cAMP-specific PDE)-B and PDE4D gene, rather than 4A, 4B and 4D genes. It indicates that the [29,30]. Such response was completely absent in mice mechanism by which antigen stimulates these gene expres- deficient in PDE4B but not PDE4D, indicating that PDE4B sions via cAMP signaling might be distinct from other

Fig. 5. Histological alterations of the small airway (A–E) and goblet cells (E–I) in control animals (A and E), OVA-challenged animals treated with vehicle (B and F), with piclamilast at the high dose (C and G), with dexamethasone (D and H), or with indomethacin (E and I). 530 H.-F. Tang et al. / Biochimica et Biophysica Acta 1762 (2006) 525–532

Fig. 6. Levels of cAMP-PDE activity in the lung harvested from OVA- or PBS- sensitized and challenged animals treated with piclamilast (Picl), dexamethasone (Dex) and indomechacin (Indom) or vehicle (1% MC). + and ++ stand for the P values less than 0.05 and 0.01, respectively, as compared to the control Fig. 8. PDE4 subtype mRNA expression (A) and levels (B) in the lungs group. * and * stand for the P values less than 0.05 and 0.01, respectively, as harvested from OVA- or PBS-sensitized and challenged animals treated with compared to OVA-challenged animals treated with vehicle. piclamilast (Picl), dexamethasone (Dex) and indomechacin (Indom) or vehicle (1% MC). ** stands for the P values less than 0.01, as compared to OVA- challenged animals treated with vehicle. challenges. The present study furthermore confirmed the up- regulation of gene expression and overactivation of PDE4 in suggests that selective PDE4 inhibitor can be competitive the lung tissues harvested from antigen challenged rats, to bind the active site of PDE4 and inhibit the activity, but obviously correlated with the development of antigen-induced dexamethasone potently suppressed the PDE activity by lung inflammation. Of its subtypes, PDE4C maybe play an other mechanisms, e.g., inhibition of protein synthesis [35]. important role in PDE4-involved inflammation. Our results also indicate that cAMP-PDE activity plays, at Piclamilast as a selective PDE 4 inhibitor has been shown least partially, an important role in the development of to inhibit eosinophil adhesion and migration in the in vitro antigen-induced goblet cell hyperplasia. Although there is system with a dose-dependent pattern [32], have potent and evidence that COX-2 may be a key element in the long-acting effects on relaxation of human bronchial muscle pathophysiological process of asthma [36,37], the present [33], and modulate tissue remodeling associated with lung study failed to show preventive effects of indomethacin on inflammatory processes including asthma [34]. The results PDE activity and goblet cell hyperplasia. from the present study demonstrated that pretreatment with Our results demonstrate that goblet cell hyperplasia is piclamilast significantly attenuated eosinophilic infiltration associated with the infiltration and activation of eosinophils, and sequential goblet cell hyperplasia induced by OVA in a similar to the previous findings [20]. Furthermore, cAMP-PDE dose-related manner. The inhibitory effect of piclamilast at activity is critical in the development of lung inflammation, the high concentration was equivalent to that of dexameth- injury and remodeling, since PDE inhibition prevented from asone on OVA-induced eosinophil infiltration, while more eosinophil infiltration from the circulation to the lung tissue and potent than dexamethasone on cAMP-PDE activity. It alveolar space, lung edema, and increased airway wall thickness

Fig. 7. Inhibitory effects (%) of theophylline (A), rolipram (B) and SKF94836 (C) on the cAMP-PDE activity in the lung homogenate harvested from OVA- or PBS- sensitized and challenged animals treated with piclamilast (Picl), dexamethasone (Dex) and indomechacin (Indom) or vehicle (1% MC). + and ++ stand for the P values less than 0.05 and 0.01, respectively, as compared to the control group. * and ** stand for the P values less than 0.05 and 0.01, respectively, as compared to OVA-challenged animals treated with vehicle. H.-F. Tang et al. / Biochimica et Biophysica Acta 1762 (2006) 525–532 531 and number of goblet cells. Of PDEs, PDE4 may play a key role [14] S. Mue, T. Ise, S. Shibahara, M. Takahashi, Y. Ono, Leukocyte cyclic- ′ ′ in antigen-induced alterations, evidenced by that PDE4 3 ,5 -nucleotide phosphodiesterase activity in human bronchial asthma, Ann. Allergy 37 (1976) 201–207. comprises a higher proportion of the cAMP hydrolyzing [15] T. Sawai, K. Ikai, M. 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