Recruitment to the Site of Inflammation Phosphodiesterases 4D and 4B In

Nonredundant Function of Phosphodiesterases 4D and 4B in Neutrophil Recruitment to the Site of Inflammation

This information is current as Miyako Ariga, Barbara Neitzert, Susumu Nakae, Genevieve of September 27, 2021. Mottin, Claude Bertrand, Marie Pierre Pruniaux, S.-L. Catherine Jin and Marco Conti J Immunol 2004; 173:7531-7538; ; doi: 10.4049/jimmunol.173.12.7531

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Nonredundant Function of Phosphodiesterases 4D and 4B in Neutrophil Recruitment to the Site of Inﬂammation1

Miyako Ariga,* Barbara Neitzert,* Susumu Nakae,† Genevieve Mottin,‡ Claude Bertrand,‡ Marie Pierre Pruniaux,‡ S.-L. Catherine Jin,* and Marco Conti2*

Neutrophils have been implicated in the pathogenesis of many inflammatory lung diseases, including chronic obstructive pulmonary disease and asthma. With this study, we investigated how disruption of cAMP signaling impacts the function of neutrophil recruitment to the lung. Four genes code for type 4 phosphodiesterases (PDE4s), enzymes critical for regulation of cAMP levels and cell signaling. Ablation of two of these genes, PDE4B and PDE4D, but not PDE4A, has profound effects on neutrophil function. In a paradigm of mouse lung injury induced by endotoxin inhalation, the number of neutrophils recovered in the bronchoalveolar lavage was markedly decreased in PDE4D؊/؊ and PDE4B؊/؊ mice 4 and 24 h after exposure to LPS. Acute PDE4 inhibition with Downloaded from rolipram had additional inhibitory effects on neutrophil migration in PDE4B؊/؊ and, to a lesser extent, PDE4D؊/؊ mice. This decreased neutrophil recruitment occurred without major changes in chemokine accumulation in bronchoalveolar lavage, sug- gesting a dysfunction intrinsic to neutrophils. This hypothesis was confirmed by investigating the expression of adhesion molecules on the surface of neutrophils and chemotaxis in vitro. CD18 expression was decreased after ablation of both PDE4B and PDE4D, whereas CD11 expression was not significantly affected. Chemotaxis in response to KC and macrophage inflammatory protein-2 was markedly reduced in PDE4B؊/؊ and PDE4D؊/؊ neutrophils. The effect of PDE4 ablation on chemotaxis was comparable, but http://www.jimmunol.org/ not additive, to the effects of acute PDE4 inhibition with rolipram. These data demonstrate that PDE4B and PDE4D play complementary, but not redundant, roles in the control of neutrophil function. The Journal of Immunology, 2004, 173: 7531–7538.

yclic nucleotides play an important modulatory function Despite the overwhelming quantity of data in support of the in virtually all cell types involved in the pathogenesis of importance of this pathway, it is still uncertain how cAMP signal- C asthma and other chronic inﬂammatory diseases of the ing contributes to the orchestration of the inﬂammatory responses airway. Activation of cAMP signaling and protein kinase A under physiological conditions as well as in pathological states, (PKA)3 has a negative impact on T cell activation and proliferation including those associated with the airway hyper-reactivity and

(1, 2), production of cytokines and chemokines (3), and eosinophil airway remodeling found in asthmatic patients. Most of the obser- by guest on September 27, 2021 recruitment to the site of inflammation (3). Furthermore, monocyte vations pertinent to cAMP signaling have been based on in vitro adhesion and migration are inhibited by high cAMP levels (4–6). pharmacological manipulations of inflammatory cell models, Pharmacological activation of cAMP signaling suppresses several which sometimes produced conflicting results (3). In this study we neutrophil responses, including degranulation (7, 8), superoxide investigated the role of cAMP signaling in proinflammatory cells anion generation (9, 10), release of IL-8 (11), and phagocytosis in vivo using a genetic approach. By inactivating genes coding for (10). In addition, an increase in cAMP impacts the expression of three isoenzymes of type 4 phosphodiesterases (PDE4), the en- adhesion molecules (CD11b/CD18/L-selectin) and adhesion to zymes that degrade cAMP expressed in inflammatory cells, we other cells, and disrupts chemokine-induced chemotaxis (12, 13). have been able to test the effect of altered, but not interrupted, Finally, smooth muscle contractility in the airway is also regulated cAMP signaling on neutrophil function. by cAMP signaling (14). In a few instances it has been reported that signals that activate inflammatory cells also produce an increase in cAMP (15). How- ever, the significance of the activation of cAMP signaling on the overall responses of inflammatory cells is largely unknown. Con- *Division of Reproductive Biology, Department of Obstetrics and Gynecology, and sistent with a negative role of cAMP in inflammatory cell activa- † Department of Pathology, Stanford University School of Medicine, Stanford, CA tion, activation of T cells (3, 16), T cell lines (17), monocytes 94305; and ‡Pfizer Global Research and Development, Fresnes, France (18–20), and macrophages (18) is associated with an increase in Received for publication April 8, 2004. Accepted for publication October 4, 2004. PDE activity. This regulation is viewed as a positive feedback The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance necessary to remove a cAMP negative constraint that prevents in- with 18 U.S.C. Section 1734 solely to indicate this fact. flammatory cell activation. PDEs are a large superfamily of en- 1 This work was supported in part by a grant from Pfizer France, The Sandler Foun- zymes encoded by at least 25 genes subdivided into 11 families dation for Asthma Research, and the Specialized Center of Research (National Insti- (21). The expression of PDE1, -3, -4, -5, -7, and -8 in inflammatory tutes of Health Grant HL67674). S.N. was supported by National Institutes of Health Grants 2R37AI23990-16, 9RO1CA72074-18, 1P01HL67674-02, and RO1AI50209. cells is inferred by mRNA detection, whereas the presence and 2 Address correspondence and reprint requests to Dr. Marco Conti, Division of Re- function of corresponding proteins have been difficult to deter- productive Biology, Department of Obstetrics and Gynecology, Stanford University mine. PDE3, -4, -7, and -8 are regulated during T cell activation School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5317. E-mail address: (16, 22). Of the four PDE4 genes present in the human genome, [email protected] three are expressed ubiquitously in most inflammatory cells at least 3 Abbreviations used in this paper: PKA, protein kinase A; BAL, bronchoalveolar lavage; PDE, phosphodiesterase; PEG, polyethylene glycol; MIP-2, macrophage- as mRNAs (3, 23–25). In some cases PDE4 activity has been char- inflammatory protein-2. acterized (26, 27), but few attempts have been made to determine

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 7532 ROLE OF PDE4s IN NEUTROPHILS the contribution of each of the isoenzymes to the overall PDE ity from the total activity. The protein concentration was determined by the activity. This PDE4 family of enzymes has received attention be- Bradford method. cause PDE4 inhibitors have important anti-inflammatory effects Immunoprecipitation (3). However, the side effects of nonselective PDE4 inhibitors have hindered the development of useful drugs (28, 29). In addition, Mice were treated with LPS for 1 h, and BAL fluid was collected after 24 h PDE4 target validation has been hampered by the fact that PDE4A, as described above. Neutrophils were isolated using a Percoll gradient (Amersham Biosciences, Piscataway, NJ) as described previously (32). PDE4B, and PDE4D are expressed in most inflammatory cells, Approximately 95% of the recovered cells were neutrophils, as judged by rendering it difficult to define the role of individual PDE4s in these morphology and Gr-1 expression. Crude extracts were prepared from these and other cells. neutrophils and assayed for PDE activity as described above. For immu- ϫ Using a model of lung inflammation, this study investigated the noprecipitation, cell extracts were clarified by centrifugation at 20,000 g for 15 min, and supernatants were incubated with rabbit preimmune serum, function of each PDE4 gene in neutrophils in vivo by deleting PDE4D-specific (M3S1), or PDE4B-specific Abs (K118) that were prein- individual PDE4 genes. We provide evidence that PDE4D and cubated with protein G-Sepharose. After 1-h incubation with Abs, samples PDE4B, but not PDE4A, are necessary for neutrophil recruitment were centrifuged, and pellets were washed, resuspended in PBS, and as- to the lung after exposure to endotoxin. Ablation of each of these sayed for PDE activity. genes produces impaired neutrophil function with altered chemo- Measurement of adhesion molecule expression in the neutrophils taxis and, possibly, adhesion. The functions of the two PDE4 genes from BAL fluid overlap, but are not completely redundant, and one gene is unable to compensate for inactivation of the other. BAL fluids were collected from the mice 4 h after LPS inhalation. Cells recovered from the BAL fluid were washed once with a FACS buffer (2% Downloaded from FBS and 0.01% sodium azide in PBS) and then incubated with mouse Fc Materials and Methods block (BD Pharmingen) on ice for 15 min before staining with FITC anti- Mice mouse Gr-1 Ab plus either PE anti-mouse CD18 or PE anti-mouse CD11b Ab (eBioscience, San Diego, CA) for 40 min on ice. For the detection of Generation of PDE4B- and PDE4D-deficient mice has been described pre- CD11a, PE Ly-6G Ab plus either FITC anti-mouse CD11a Ab or isotype viously (18, 30). Following a similar strategy, generation of PDE4A knock- control FITC rat IgG2a,␬ was used. As an isotype control for anti-CD18 out mice will be reported elsewhere. In all cases the effect of the null PDE4 Ab, PE-rat IgG2a,␬ (eBioscience) was used. PE-rat IgG2b,␬ (eBioscience) allele was tested on mice of a mixed background (C57BL/6 ϫ 129/Ola) was used as a control for the anti-CD11b Ab. The adhesion molecule ex- http://www.jimmunol.org/ using littermates as controls. Colonies were maintained by out-breeding pression on neutrophils was then quantitated using a FACScan flow cy- heterozygous pairs and genotyping the offspring. All mice used in these tometer (BD Biosciences, Mountain View, CA). experiments were Ͼ2 mo old. To generate the compound heterozygous ϩ Ϫ ϩ Ϫ PDE4D / /PDE4B / , homozygous null PDE4D and PDE4B mice were Measurement of phagocytic activity in bone marrow-derived mated. The compound heterozygous mice thus obtained were mated again ϩ Ϫ Ϫ Ϫ Ϫ Ϫ ϩ Ϫ neutrophils to produce PDE4D / /PDE4B / or PDE4D / /PDE4B / mice. All procedures were approved by the administrative panel on laboratory animal Bone marrow cells collected from the femurs of wild-type PDE4BϪ/Ϫ and care at Stanford University. PDE4DϪ/Ϫ mice were incubated for 5 days at a density of 0.7–1 ϫ 106 cells/ml in RPMI 1640 medium containing 10% FBS, 100 U/ml penicillin, LPS exposure and bronchoalveolar lavage (BAL) and 100 ␮g/ml streptomycin. The culture was supplemented with 20 ng/ml G-CSF to induce neutrophil differentiation. For phagocytosis assay, ali- by guest on September 27, 2021 Mice were exposed to saline or 100 ␮g/ml LPS aerosols in PBS for1hin quots of 1 ϫ 106 cells in 1 ml of medium were incubated for1hinthe a container connected to a SPAG-2 system (ICN Pharmaceuticals, Costa absence or the presence of 10 ␮l of FITC-latex bead suspension (Sigma- Mesa, CA). At 1, 2, 4, or 24 h after LPS exposure, mice were overdosed Aldrich). As a negative control, 1 ϫ 106 splenocytes were prepared from with an i.p. injection of 6% ketamine and 2% xylazine (v/v) in PBS, and wild-type mouse spleen and incubated with FITC-latex beads for 1 h. To a partial tracheotomy was performed. Lungs were flushed with PBS six terminate phagocytosis, the cells were pelleted and washed once with times through a cannula, and a total of 1.6–1.7 ml of lavage fluid was FACS buffer, followed by incubation with anti-CD16/CD32 mAb (2.4G2; recovered. Total cells in the BAL fluid were counted using a hemocytom- BD Pharmingen) on ice for 15 min. A PE anti-mouse Gr1 mAb (BD eter. Cells present in the BAL (1.6 ϫ 104 cells) also were prepared on Pharmingen) then was used to stain the cells on ice for 40 min. After slides by cytospin centrifugation (Thermo Shandon, Pittsburgh, PA) and washing, cells were resuspended in FACS buffer and subsequently added to stained by May-Gru¨nwald and Giemsa stain (Sigma-Aldrich, St. Louis, an equal volume of FACS buffer containing 1 ␮g/ml 7-aminoactinomycin MO). After staining, percentages of neutrophils, macrophages, and lym- D (Sigma-Aldrich). Green fluorescence in neutrophils was quantitated us- phocytes were determined by counting at least 200 cells. The levels of ing a FACScan flow cytometer as described above. TNF-␣, KC, and MIP-2 in the supernatant of BAL fluid were measured ␣ using a mouse TNF- immunoassay kit (BD Pharmingen, San Diego, CA), Chemotaxis assay a mouse KC immunoassay, and a mouse MIP-2 immunoassay (R&D Sys- tems, Minneapolis, MN). All migration assays were performed in 24-well plates with 3-␮m pore size polycarbonate filters (Corning, Corning, NY) using RPMI 1640 medium Rolipram injection supplemented with 10% FBS (Life Technologies, Auckland, New Zea- ϫ 5 ␮ Mice were injected i.p. with either polyethylene glycol (PEG; m.w., 200) land). Unfractionated splenocytes (8 10 cells) in 100 l of medium or rolipram (3 mg/kg mouse) dissolved in PEG 45 min before the exposure were placed in the upper chamber, and increasing concentrations of re- to LPS. Four hours later, BAL fluid was obtained, and total cells and combinant murine KC (PeproTech, Rocky Hill, NJ) or recombinant murine neutrophils were determined as described above. MIP-2 (R&D Systems) were added in the lower chamber. In the total splenocytes derived from wild-type, PDE4B-deficient, and PDE4D-defi- PDE assay cient mice, the percentages of neutrophils were 2.4 Ϯ 0.4, 2.1 Ϯ 1.1, and 2.2 Ϯ 0.1, respectively. After 2-h incubation at 37°C, upper chambers were Mice were treated with LPS for 1 h, and BAL fluid was collected 4 h later removed, 20 ␮l of polystyrene beads (Polysciences, Warrington, PA) were as described above. Cells from the BAL fluid were recovered by centrif- added to each well, and the suspension of cells/beads was transferred to ugation, and pellets were lysed in a buffer containing 1% Nonidet P-40, 50 tubes. Wells were rinsed with 5 mM PBS containing 5 mM EDTA to mM Tris-HCl (pH 7.5), 250 mM NaCl, 5% glycerol, 1 mM EDTA, 0.2 mM detach remaining cells, and the washes were combined. The migrated cells

EGTA, 10 mM NaF, 10 mM sodium pyrophosphate, 1 mM Na3VO4,1mM were counted according to the method of Campbell et al. (33). Briefly, cells 4-(2-aminoethyl)benzenesulfonyl fluoride (Roche, Mannheim, Germany), were stained with FITC-anti-mouse Gr-1 Ab, and the stained cell/bead and 1 tablet/10 ml of protease inhibitor mixture (Roche). After sonication, suspension was loaded onto a FACScan flow cytometer. Because beads aliquots of the cell extract were assayed for total PDE activity and could be distinguished from cells in the plot of side and forward scatter, the rolipram-insensitive PDE activity in the presence of 10 ␮M rolipram. The ratio of cells to beads was calculated to give the number of migrated Gr- PDE assay was performed according to the method of Thompson and 1-positive cells. To assess the effect of acute PDE inhibition on chemotaxis, Appleman (31) as detailed previously (30). The rolipram-sensitive activity cells were pretreated with vehicle (DMSO) or rolipram (10 ␮M) for 1 h, (PDE4 activity) was derived by subtracting the rolipram-insensitive activ- then transferred to the upper chamber. Either vehicle (DMSO) or rolipram The Journal of Immunology 7533

Table I. Inﬂammatory cell number in wild-type, PDE4BϪ/Ϫ, and PDE4DϪ/Ϫ micea

Cells in BAL/Mouse (ϫ104)

Total Macrophages Neutrophils Lymphocytes Eosinophils

Saline Wild type (n ϭ 18) 21.28 Ϯ 2.50 20.33 Ϯ 3.71 0.08 Ϯ 0.02 0.63 Ϯ 0.11 0.13 Ϯ 0.02 PDE4BϪ/Ϫ (n ϭ 6) 27.25 Ϯ 11.25 25.88 Ϯ 10.68 0.27 Ϯ 0.11 0.27 Ϯ 0.11 0.55 Ϯ 0.23 PDE4DϪ/Ϫ (n ϭ 11) 35.18 Ϯ 10.01 34.31 Ϯ 9.76 0.13 Ϯ 0.04 0.74 Ϯ 0.21 n/d LPS Wild type (n ϭ 26) 174.4 Ϯ 13.58 23.89 Ϯ 2.53 149.8 Ϯ 11.66 1.06 Ϯ 0.11 0.73 Ϯ 0.08 PDE4BϪ/Ϫ (n ϭ 10) 124.8 Ϯ 17.61 25.90 Ϯ 3.66 104.4 Ϯ 14.74* 1.22 Ϯ 0.17 0.21 Ϯ 0.03 PDE4DϪ/Ϫ (n ϭ 16) 103.9 Ϯ 11.02** 20.06 Ϯ 2.13 77.9 Ϯ 8.26** 0.80 Ϯ 0.08 n/d

a Total and differential cell counts (macrophages, neutrophils, lymphocytes, and eosinophils) in BAL ﬂuid from mice 24 h after exposure to LPS (100 ng/ml) or vehicle (saline). Values represent the mean Ϯ SEM of the cells per sample. .p Ͻ 0.01) between wild-type and PDE4BϪ/Ϫ or PDE4DϪ/Ϫ is indicated. n/d; not detectable ,ءء p Ͻ 0.05 and ,ء) Signiﬁcant difference

(10 ␮M) was added to the lower chamber containing increasing concen- course study demonstrated that the decreased neutrophil recruit- trations of KC (0 or 100 ng/ml). ment in the lungs after LPS inhalation was evident 4 h after treatment and was maintained for at least 20 h (Fig. 1). The reduced Results number of neutrophils in the BAL fluid of PDE4-null mice was not Downloaded from Neutrophil recruitment to the lung is impaired in due to a decreased production of cells by bone marrow, because PDE4-deficient mice the number of circulating neutrophils was not different among the Several animal models recapitulate the pathological changes ob- three genotypes (wild-type, 0.27 Ϯ 0.02 ϫ 103 cells/␮l blood (n ϭ served in chronic obstructive pulmonary disease, nonallergic 8); PDE4D-deficient mice, 0.29 Ϯ 0.04 ϫ 103 cells/␮l blood (n ϭ asthma, and other chronic inflammatory disorders, including air- 5); PDE4B-deficient mice, 0.22 Ϯ 0.03 ϫ 103 cells/␮l blood way neutrophilia and enhanced cytokine production (29). One in (n ϭ 5)). http://www.jimmunol.org/ vivo mouse model developed to assess neutrophil function is the ␣ airway infiltration that follows LPS administration by aerosol. To TNF- and chemokine production in PDE4-deficient mice investigate the effect of PDE4 ablation on neutrophil recruitment, To investigate the cause of reduction in neutrophil recruitment to wild-type and PDE4A-, PDE4B-, and PDE4D-deficient mice were the airway, cytokine and chemokine accumulation was measured exposed to LPS for 1 h, and the number of cells recruited in BAL at different times after LPS inhalation in wild-type and PDE4- was scored 24 h from the end of the treatment. The neutrophil deficient mice. TNF-␣ accumulation in BAL fluid was not signif- number recovered in BAL was reduced by 31 and 48% in PDE4B- icantly different between wild-type and PDE4-deficient mice 1 h and PDE4D-deficient mice, respectively (Table I). Conversely, no after LPS inhalation (Fig. 2); however, the levels of TNF-␣ were

significant differences were observed between wild-type and significantly reduced in PDE4D-deficient mice compared with by guest on September 27, 2021 PDE4A-deficient mice (PDE4Aϩ/ϩ, 15.85 Ϯ 2.73 ϫ 105 neutro- wild-type mice at 2 h and remained lower for up to 4 h. There was phils/mouse (n ϭ 4); PDE4AϪ/Ϫ, 16.65 Ϯ 5.41 ϫ 105 neutrophils/ a trend toward a similar decrease in TNF-␣ accumulation in mouse (n ϭ 4)). In addition, the number of cells recruited to the PDE4B-deficient mice, although it did not reach statistical signif- lung after LPS was not different in all genotypes (Table I). Con- icance. Conversely, no significant difference in either KC or MIP-2 sistent with the decreased neutrophil cell counts, myeloperoxidase activity was significantly decreased in the lungs of PDE4-deficient mice after LPS inhalation (data not shown). A more detailed time-

FIGURE 1. Time course of leukocyte and neutrophil recruitment to the lung after LPS inhalation in wild-type (WT), PDE4BϪ/Ϫ, and PDE4DϪ/Ϫ FIGURE 2. Concentrations of TNF-␣, KC, and MIP-2 in BAL fluid of mice. Mice were exposed to an aerosol of LPS (100 ␮g/ml) for 1 h, and wild-type (WT), PDE4BϪ/Ϫ, and PDE4DϪ/Ϫ mice at different times after BAL fluid was collected at the end of the treatment (time zero) or at 1, 2, LPS inhalation. Mice were exposed to an aerosol of LPS (100 ␮g/ml) for 4, or 24 h after treatment. Total cells (A) and neutrophils (B) in BAL were 1 h, then the BAL fluid was collected at 1, 2, or 4 h after treatment. Levels counted. Results are expressed as the mean Ϯ SEM of 3 to 26 mice/point. of TNF-␣, KC, and MIP-2 in the supernatant of the BAL fluid were mea- .(p Ͻ 0.05 (compared with WT mice ,ء .p Ͻ 0.01 (compared with WT mice). sured by ELISA ,ءء ;p Ͻ 0.05 ,ء 7534 ROLE OF PDE4s IN NEUTROPHILS accumulation was observed in the three genotypes at any time and PDE4-deficient mice. Rolipram should have no additional ef- point investigated. Thus, the decreased neutrophil recruitment ob- fect on neutrophil recruitment in the PDE4-deficient mice if the served in the two mouse lines is not associated with major changes functions of the two genes are redundant. If, instead, PDE4D and in chemokine production. PDE4B have distinct functions, rolipram by itself would have a more pronounced effect than the genetic inactivation of each in- PDE4D and PDE4B are expressed at different levels in dividual PDE4 and should further decrease neutrophil recruitment PDE4-deficient mice when tested in the PDE4-deficient mice. Rolipram at 3 mg/kg ad- To investigate the levels of PDE4 expression in the neutrophils, ministered 45 min before LPS inhalation produced a decrease in PDE4 activity was assessed in cells recovered from the BAL of the neutrophil recruitment in wild-type mice more pronounced wild-type and PDE4-deficient mice. A major decrease in PDE4 than that obtained with inactivation of either PDE4D or PDE4B activity was observed in PDE4D-deficient neutrophils (Fig. 3A). (Fig. 4). In addition, rolipram pretreatment significantly decreased Similar results were obtained with neutrophils purified from BAL neutrophil recruitment in PDE4B-deficient mice and, to a lesser of PDE4D-deficient mice (data not shown). Furthermore, a small, extent, in PDE4D-deficient mice. A combination of rolipram plus but significant, decrease in PDE4 activity was observed in the inactivation of one of the PDE4 genes decreased neutrophil re- PDE4B-deficient neutrophils (Fig. 3B). To confirm this pattern of cruitment to levels similar to those observed with rolipram in expression, PDE4 activity in wild-type BAL cells after LPS inha- wild-type mice. lation was measured after immunoprecipitation of the cell extracts A similar conclusion about the additive effects of PDE4 inacti- with PDE4D- and PDE4B-specific Abs. This alternative approach vation was reached using an alternative genetic approach, i.e., by confirmed that PDE4D is expressed at much higher levels than measuring neutrophil recruitment in mice heterozygous for one Downloaded from PDE4B in BAL neutrophils (Fig. 3C). These data demonstrate that PDE4 and homozygous null for the other. Mice homozygous null both PDE4s are expressed in these cells, with PDE4D being the for both PDE4B and PDE4D could not be used in this study be- Ϫ Ϫ ϩ Ϫ predominant form. No significant compensatory increase in rolip- cause they do not survive after birth. In PDE4B / /PDE4D / ϩ Ϫ Ϫ Ϫ ram-insensitive PDE activity was observed in any cell preparation. and PDE4B / /PDE4D / mice, neutrophil recruitment was decreased more than when a single PDE4 was inactivated (Fig. 5).

Nonredundant roles of PDE4D and PDE4B in neutrophil These experiments suggest that PDE4D and PDE4B have comple- http://www.jimmunol.org/ function mentary roles in the control of neutrophil recruitment. The functions of the two PDE4s in neutrophil recruitment were Adhesion is disrupted in PDE4-deficient mice further assessed by comparing the effects of rolipram on wild-type The above data suggest that dysfunction of the neutrophils them- selves rather than a disruption of chemotactic cues is the primary cause of the reduced recruitment of neutrophils. Neutrophil recruit- ␤ ment is dependent on expression of 2 integrins upon activation. To investigate whether the expression of integrin subunits is one of the causes of decreased recruitment of neutrophils, the expression by guest on September 27, 2021 ␣ ␤ of the two subunits, CD11a and CD11b, and the 2 subunit, CD18, was assessed in neutrophils recovered from BAL after LPS treatment. Whereas no major differences were detected in the expression of either CD11a or CD11b (data not shown), the expression of CD18 was significantly reduced in PDE4B- and PDE4D- deficient neutrophils compared with that in wild-type cells (Fig. 6).

FIGURE 3. PDE activities in BAL leukocytes after LPS inhalation. Cells derived from BAL fluid of PDE4Dϩ/ϩ and PDE4DϪ/Ϫ (A) and PDE4Bϩ/ϩ and PDE4BϪ/Ϫ mice (B) were homogenized as detailed in Ma- terials and Methods, and PDE activity in the homogenate was measured in the absence (Total) or the presence (R-insensitive) of 10 ␮M rolipram. PDE4 activity (R-sensitive) was obtained by subtracting the rolipram-insensitive activity from the total activity. C, BAL was obtained from wild- type (WT) mice, and neutrophils were purified by a Percoll gradient FIGURE 4. Effects of rolipram on total leukocytes and neutrophil re- method and homogenized as detailed in Materials and Methods. After im- cruitment after LPS inhalation. Mice were injected i.p. with rolipram (Rol munoprecipitation of the cell extract with preimmune serum (PS) or spe- ϩ; 3 mg/kg) 45 min before LPS inhalation. Control mice were injected cific Abs against PDE4B and PDE4D, PDE4 activity recovered in the with vehicle only (PEG200). BAL fluid was collected 4 h after LPS inha- immunoprecipitation pellet was measured as described above. Results are lation, and total leukocytes (A) and neutrophils (B) were counted. Results ,ءءء ;p Ͻ 0.05 ,ء .p Ͻ are expressed as the mean Ϯ SEM of four mice per group ,ءء ;p Ͻ 0.05 ,ء .expressed as the mean Ϯ SEM of at least three mice 0.01 (compared with WT mice). p Ͻ 0.0001 (compared with vehicle-injected mice). The Journal of Immunology 7535

FIGURE 5. Leukocyte recruitment in BAL of mice deﬁcient in both Ϫ/Ϫ Ϫ/Ϫ

PDE4D and PDE4B. Wild-type (WT), PDE4B , PDE4B / Downloaded from PDE4Dϩ/Ϫ, PDE4DϪ/Ϫ, and PDE4DϪ/Ϫ/PDE4Bϩ/Ϫ mice were treated with LPS for 1 hr, and after 24 h, BAL fluid was collected. The numbers of total leukocytes (A) and neutrophils (B) in BAL were determined. Each FIGURE 6. Adhesion molecule expression on BAL neutrophils from p Ͻ Ϫ Ϫ Ϫ Ϫ ,ءء ;p Ͻ 0.05 ,ء .bar represents the mean Ϯ SEM of at least three mice PDE4B / and PDE4D / mice. Cells from BAL collected 4 h after LPS 0.01 (compared with each single gene-deficient mouse). treatment were obtained as described in Materials and Methods. Cells were then prepared for FACS analysis by incubation with an FITC-conjugated Gr1 (neutrophil marker) Ab and a PE-conjugated CD18 Ab. Representative http://www.jimmunol.org/ It should be pointed out that the decrease in integrin expression histograms of CD18 (A) are shown. The open area shows the fluorescence of the expression of CD18 in wild-type (WT; upper panel), PDE4BϪ/Ϫ observed in neutrophils should be considered a minimal estimate, Ϫ Ϫ (middle panel), and PDE4D / (lower panel) neutrophils. The shaded area because the neutrophils used in the assay have successfully mi- corresponds to the fluorescence of neutrophils incubated with control IgGs grated across the epithelium. We inferred that a more dramatic instead of primary Abs. B, Summary of CD18 expression determined by reduction in integrin expression would occur in cells that failed to FACS. The levels are expressed as a percentage of the neutrophils that migrate. express CD18 and are within the region M1 marked in A, Each bar rep- p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .resents the mean Ϯ SEM of at least seven mice

Chemotaxis is disrupted in PDE4-deficient neutrophils (compared with WT mice). by guest on September 27, 2021 To further assess the possibility that neutrophils derived from PDE4-deficient mice have intrinsic dysfunction in migration, neutrophils were obtained from the spleen of wild-type and PDE4- in vivo. Because cytokine and chemokine accumulation is not deficient mice and used in a Boyden two-chamber chemotactic greatly affected in the two mouse strains after LPS exposure, we assay. In the presence of KC, wild-type neutrophils exhibited a surmise that this loss of function is intrinsic to neutrophils. Indeed, concentration-dependent transmigration with an EC50 of 10–15 this reduction in recruitment to the lung in vivo is associated with ng/ml, values comparable to those previously reported (34). Con- an altered neutrophil expression of adhesion molecules and re- versely, the responses of both PDE4D- and PDE4B-deficient neu- duced chemotaxis measured in isolated cells in vitro. Both genetic trophils were significantly reduced (Fig. 7A). Similar results were and pharmacological evidence indicates that the functions of the obtained with MIP-2 (data not shown). When the wild-type cells two PDE4 genes are nonredundant and complementary, even were exposed to either 10 or 50 ␮M rolipram, a significant reduc- though PDE4B has an impact on neutrophil function greater than tion in KC-induced neutrophil migration was observed (Fig. 7B), that expected from its level of expression in these cells. These and the decrease was comparable to that observed in the two findings underscore the important role of cAMP homeostasis in PDE4-deficient neutrophils. This latter finding is in agreement neutrophil activation and open new avenues for pharmacological with the hypothesis that PDE4 activity is necessary for efficient intervention. Thus, PDE4 subtype-selective inhibitors may be use- chemotaxis in response to chemokines. ful in the treatment of chronic inflammatory diseases of the lung. To exclude the possibility that the disruption of chemotaxis is Both immunological data and activity measurements in PDE4- due to nonspecific generalized loss of cell function, neutrophils deficient cells show that PDE4D and PDE4B are expressed in neu- from wild-type and PDE4-deficient mice were tested for their trophils, although at different levels. This finding is consistent with phagocytic activity. Fig. 8 shows that phagocytosis of FITC-latex several reports on expression of PDE4 mRNAs in these cells from beads was identical in the three genotypes. This finding confirms rodents and humans (19, 26, 35). PDE4 inhibitors also have been that inactivation of PDE4 does not compromise all functions of used to indirectly assess the presence of PDE4 in neutrophils (3, neutrophils, and only chemokine-stimulated migration is affected. 26, 36). PDE4A may be expressed in these cells, albeit at very low levels, and its ablation does not produce significant effects on neu- Discussion trophil recruitment. Despite the complex pattern of PDE expres- Using a combination of pharmacological and genetic strategies, we sion in neutrophils, we have been able to dissect the function of provide evidence that the two PDE4B and PDE4D genes play a each individual PDE4 expressed with selective ablation of PDE4 nonredundant function in polymorphonuclear neutrophil recruit- genes. Although we have measured major differences in the ex- ment to the lung. Ablation of each of the two genes has a signif- pression of PDE4D and PDE4B in neutrophils, the phenotypes icant impact on the neutrophil ability for transepithelial migration associated with the ablation of the two genes are comparable. 7536 ROLE OF PDE4s IN NEUTROPHILS

FIGURE 7. Chemotactic responses to KC stimulation of neutrophils from the spleens of wild-type (WT), PDE4BϪ/Ϫ, and PDE4DϪ/Ϫ mice and effect of rolipram on the chemotaxis of WT neutrophils. A, Splenocytes 5 (8 ϫ 10 ) were added to the top chamber of the 24-transwell plate. The Downloaded from bottom chamber contained various concentrations of KC (0, 1, 3, 10, 30, or

100 ng/ml). After a 2-h incubation at 37°C in 5% CO2, the number of the neutrophils (Gr1-positive cells) that had migrated to the lower chamber was determined by a ﬂow cytometry. The percentage of migrated neutro- FIGURE 8. Phagocytosis of FITC-latex beads by neutrophils from phils represents the fraction of total neutrophils applied to the top chamber. Ϫ Ϫ Ϫ Ϫ wild-type (WT), PDE4B / , and PDE4D / mice. Bone marrow-derived p Ͻ 0.01 (compared with WT neutrophils). B, Cells from Ϫ/Ϫ Ϫ/Ϫ ,ءء ;p Ͻ 0.05 ,ء

neutrophils from WT, PDE4B , and PDE4D mice were obtained as http://www.jimmunol.org/ the spleens of WT mice were preincubated with vehicle (DMSO) or roli- described in Materials and Methods. Cells were incubated in the absence pram (10 and 50 ␮M). After preincubation, the chemotatic assay was per- or the presence of FITC-latex beads for 1 h, then processed for FACS formed in the presence of vehicle or rolipram, which was added to the analysis by labeling with a PE-conjugated Gr1 Ab. Representative histo- bottom chamber. Each bar represents the mean Ϯ SEM of at least three grams of FITC-latex beads are shown in A. The open area corresponds to .(p Ͻ 0.05 (as compared with vehicle-treated samples ,ء .mice the fluorescence of neutrophils that have phagocyte-labeled beads. The shaded area corresponds to cells that were incubated without latex beads. CD4ϩ T cells from mouse spleen were used as a negative control of phago- There are several possibilities that should be considered for the cytosis. The fluorescence intensity determined by FACS was analyzed and interpretation of these findings. First, PDE activity was measured summarized in B. The data are expressed as the percentage of neutrophils in activated neutrophils that had emigrated from the vasculature with phagocytic activity. Each bar represents the mean Ϯ SD of at least five by guest on September 27, 2021 and were recovered in the BAL. It is possible that before LPS mice. No significant difference (NS) was observed compared with stimulation, the proportion of PDE4D/PDE4B is different from WT mice. that in activated cells. Indeed, PDE4B expression is induced by LPS exposure in monocytes/macrophages (18) even though it has been reported that the expression of PDE4B is constitutive in hu- Although we were not able to measure cAMP levels in neutrophils man neutrophils (19). Additional studies are required to address deficient in the two PDE4 genes, it is unlikely that major changes the possibility of changes in the pattern of PDE expression in neu- in cAMP levels would be observed. This view is supported by trophils during their recruitment. In addition, that the level of PDE observations made in macrophages, where no detectable changes expression may not be a predictor of its impact on cell function in cAMP were observed after PDE4 ablation (S.-L. C. Jin, L. Lan, should be considered. In several instances we observed that min- M. Zoudilova, and M. Conti, manuscript in preparation). PDE4 imal changes in PDE activity have profound effects on cell func- inactivation most likely has subtle and no generalized disruptive tion with no detected changes in overall cAMP levels (S.-L. C. Jin, effects on cAMP signaling. It has been shown that cAMP has a L. Lan, M. Zoudilova, and M. Conti, manuscript in preparation). biphasic effect on permeabilized neutrophil migration because it is To explain these findings, we hypothesize that PDE4D and PDE4B stimulatory at low concentrations, but inhibitory at high concen- control two different pools of cAMP in neutrophils, and disruption trations (15, 38). Thus, it is possible that ablation of PDE4 disrupts of either of these pools affects their ability to migrate, possibly by the fine balance required for chemotaxis that involves movement independent mechanisms. Because of the scarcity of cells available of signaling molecules between compartments (39). In human neu- with the murine models, we have not been able to define whether trophils, a cAMP analog abrogated the response to chemoattrac- the loss of neutrophil function in PDE4D- and PDE4B-deficient tants and caused inhibition of integrin-mediated adhesion through mice is indeed due to separate or overlapping mechanisms. Cer- down-regulation of Rho activity (40). Moreover, integrin-mediated tainly, ablation of the activity of PDE4 is required for these effects adhesion is inhibited by activation of PKA and requires A-kinase because acute inhibition of PDE4 with rolipram produces a loss of anchor protein binding (41). In addition, PDE4D and PKA are function comparable to that produced by PDE4 genetic ablation. found in complex with AKAPs (42). It is then likely that the effect These findings also indicate that rolipram pharmacological effects of PDE4 ablation leads to activation of PKA and interference with on neutrophil recruitment are due to inhibition of both PDE4D and integrin expression and signaling. The formation of complexes of PDE4B, but not PDE4A. the chemokine receptor with ␤-arrestin-2 has been observed in From numerous studies of neutrophils as well as other inflam- response to a chemoattractant (43, 44). Fong et al. (45) showed that matory cells, it is generally agreed that activation of the cAMP in mouse neutrophils in which ␤-arrestin-2 has been depleted, the pathway has a negative impact on activation of these cells. How- chemotactic response to KC is defective. It is possible, then, that ever, the mechanistic details of cAMP inhibition are unclear (37). the function of ␤-arrestin-2 is also affected by PDE4 ablation in The Journal of Immunology 7537 view of the observation that ␤-arrestins are in complex with PDE4 References (46, 47). 1. Kammer, G. M. 1988. The adenylate cyclase-cAMP-protein kinase A pathway Both PDE4B- and PDE4D-deficient neutrophils display a re- and regulation of the immune response. Immunol. Today 9:222. 2. Skalhegg, B. S., B. F. Landmark, S. O. Doskeland, V. Hansson, T. Lea, and duced expression of CD18. As discussed above, we believe that T. Jahnsen. 1992. Cyclic AMP-dependent protein kinase type I mediates the the reduction detected is more profound in neutrophils that failed inhibitory effects of 3Ј,5Ј-cyclic adenosine monophosphate on cell replication in to transmigrate. Whether this reduction in integrin is the major human T lymphocytes. J. Biol. Chem. 267:15707. 3. Torphy, T. J. 1998. Phosphodiesterase isozymes: molecular targets for novel cause of the inability to transmigrate remains to be determined. antiasthma agents. Am. J. Respir. Crit. Care Med. 157:351. Conflicting results have been reported on the effect of CD18 ab- 4. Tannenbaum, C. S., and T. A. Hamilton. 1989. Lipopolysaccharide-induced gene lation on neutrophil recruitment to the lung (48). It is also unclear expression in murine peritoneal macrophages is selectively suppressed by agents that elevate intracellular cAMP. J. Immunol. 142:1274. whether deficits in the vasculature or endothelial cells contribute to 5. Parhami, F., Z. T. Fang, A. M. Fogelman, A. Andalibi, M. C. Territo, and the phenotype. This is a possibility that should be investigated; J. A. Berliner. 1993. Minimally modified low density lipoprotein-induced inflam- however, in other paradigms of recruitment of inflammatory cells matory responses in endothelial cells are mediated by cyclic adenosine monophosphate. J. Clin. Invest. 92:471. to the lung, no major deficits in eosinophil recruitment were ob- 6. Fine, J. S., H. D. Byrnes, P. J. Zavodny, and R. W. Hipkin. 2001. Evaluation of served in PDE4-null mice, arguing against a defect in the vascu- signal transduction pathways in chemoattractant-induced human monocyte che- lature (49). motaxis. Inflammation 25:61. 7. Barnette, M. S., J. O. Bartus, M. Burman, S. B. Christensen, L. B. Cieslinski, The effects of inactivation of the two PDE genes do not com- K. M. Esser, U. S. Prabhakar, J. A. Rush, and T. J. Torphy. 1996. Association of pletely overlap with the effects of acute inhibition of PDE4 with the anti-inflammatory activity of phosphodiesterase 4 (PDE4) inhibitors with either inhibition of PDE4 catalytic activity or competition for [3H]rolipram bind- rolipram. This discrepancy is most prominent for cytokine and ing. Biochem. Pharmacol. 51:949. chemokine production. With the caveat that considerable variation 8. Barnette, M. S., S. B. Christensen, D. M. Essayan, M. Grous, U. Prabhakar, Downloaded from among these mice was observed, possibly due to the mixed back- J. A. Rush, A. Kagey-Sobotka, and T. J. Torphy. 1998. SB 207499 (Ariflo), a potent and selective second-generation phosphodiesterase 4 inhibitor: in vitro ground and outbreeding of the colony, there was no major differ- anti-inflammatory actions. J. Pharmacol. Exp. Ther. 284:420. ence among the three groups in TNF-␣ accumulation in the BAL 9. Nielson, C. P., R. E. Vestal, R. J. Sturm, and R. Heaslip. 1990. Effects of selective fluid 1 h after LPS. A trend toward a decrease was observed at 2 phosphodiesterase inhibitors on the polymorphonuclear leukocyte respiratory burst. J. Allergy Clin. Immunol. 86:801. and 4 h with both knockouts, even though statistical significance 10. Bessler, H., R. Gilgal, M. Djaldetti, and I. Zahavi. 1986. Effect of pentoxifylline was reached in only one case. On the contrary, PDE4 inhibitors on the phagocytic activity, cAMP levels, and superoxide anion production by http://www.jimmunol.org/ have been shown to markedly decrease LPS-induced TNF-␣ pro- monocytes and polymorphonuclear cells. J. Leukocyte Biol. 40:747. 11. Au, B. T., M. M. Teixeira, P. D. Collins, and T. J. Williams. 1998. Effect of PDE4 duction in BAL (8, 50–53). There are several explanations that inhibitors on zymosan-induced IL-8 release from human neutrophils: synergism could reconcile these differences. The first possibility is that non- with prostanoids and salbutamol. Br. J. Pharmacol. 123:1260. 12. Derian, C. K., R. J. Santulli, P. E. Rao, H. F. Solomon, and J. A. Barrett. 1995. selective PDE4 inhibitors have an effect because they block all Inhibition of chemotactic peptide-induced neutrophil adhesion to vascular endo- PDE4s, whereas ablation of a single PDE4 is not sufficient to affect thelium by cAMP modulators. J. Immunol. 154:308. TNF-␣ accumulation. However, we have shown that ablation of 13. Harvath, L., J. D. Robbins, A. A. Russell, and K. B. Seamon. 1991. cAMP and ␣ human neutrophil chemotaxis. Elevation of cAMP differentially affects chemo- PDE4B in leukocytes is sufficient to markedly suppress TNF- tactic responsiveness. J. Immunol. 146:224. production (18). Thus, it appears more likely that although inhi- 14. Torphy, T. J. 1994. ␤-Adrenoceptors, cAMP and airway smooth muscle relax- bition of a single PDE4 may be sufficient to block TNF-␣ produc- ation: challenges to the dogma. Trends Pharmacol. Sci. 15:370. by guest on September 27, 2021 15. VanUffelen, B. E., B. M. de Koster, and J. G. Elferink. 1998. Interaction of cyclic tion in a given cell, a variety of cell types contributes to overall GMP and cyclic AMP during neutrophil migration: involvement of phosphodi- cytokine production in vivo. In the airway, TNF-␣ derives from esterase type III. Biochem. Pharmacol. 56:1061. monocytes/macrophages, airway epithelial cells, and perhaps neu- 16. Li, L., C. Yee, and J. A. Beavo. 1999. CD3- and CD28-dependent induction of PDE7 required for T cell activation. Science 283:848. trophils (54–57). In our experimental conditions, it is possible that 17. Glavas, N. A., C. Ostenson, J. B. Schaefer, V. Vasta, and J. A. Beavo. 2001. T the major producers of TNF-␣ are perhaps epithelial cells or mac- cell activation up-regulates cyclic nucleotide phosphodiesterases 8A1 and 7A3. rophages, and inactivation of a single PDE is not sufficient. Proc. Natl. Acad. Sci. USA 98:6319. 18. Jin, S. L., and M. Conti. 2002. Induction of the cyclic nucleotide phosphodies- From a pharmacological standpoint, we propose that inhibitors terase PDE4B is essential for LPS-activated TNF-␣ responses. Proc. Natl. Acad. with increased selectivity toward one PDE4 may have advantages Sci. USA 99:7628. over nonselective PDE4 inhibitors because they avoid potential 19. Wang, P., P. Wu, K. M. Ohleth, R. W. Egan, and M. M. Billah. 1999. Phospho- diesterase 4B2 is the predominant phosphodiesterase species and undergoes dif- side effects. Robichaud et al. (58) indicate that PDE4D, but not ferential regulation of gene expression in human monocytes and neutrophils. Mol. PDE4B, may be responsible for the emetic side effects often ob- Pharmacol. 56:170. 20. Seldon, P. M., and M. A. Giembycz. 2001. Suppression of granulocyte/macro- served with PDE4 inhibitors. Thus, a compound that inhibits phage colony-stimulating factor release from human monocytes by cyclic AMP- PDE4B preferentially should retain many of the beneficial prop- elevating drugs: role of interleukin-10. Br. J. Pharmacol. 134:58. erties of PDE4 inhibition (blockade of neutrophil adhesion and 21. Conti, M., and S. L. Jin. 1999. The molecular biology of cyclic nucleotide phosphodiesterases. Prog. Nucleic Acid Res. Mol. Biol. 63:1. chemotaxis); however, it could probably be used at higher con- 22. Seybold, J., R. Newton, L. Wright, P. A. Finney, N. Suttorp, P. J. Barnes, centrations because of the decreased emetic effects. I. M. Adcock, and M. A. Giembycz. 1998. Induction of phosphodiesterases 3B, In conclusion, we determined that PDE4B and PDE4D are both 4A4, 4D1, 4D2, and 4D3 in Jurkat T-cells and in human peripheral blood T- lymphocytes by 8-bromo-cAMP and Gs-coupled receptor agonists: potential role important for neutrophil recruitment during lung inflammation ␤ in 2-adrenoreceptor desensitization. J. Biol. Chem. 273:20575. caused by LPS. The two PDEs have complementary roles, and 23. Engels, P., K. Fichtel, and H. Lubbert. 1994. Expression and regulation of human their ablation affects both the expression of adhesion molecules and rat phosphodiesterase type IV isogenes. FEBS Lett. 350:291. 24. Houslay, M. D., M. Sullivan, and G. B. Bolger. 1998. The multienzyme PDE4 and chemotaxis. Additional studies are required to determine the cyclic adenosine monophosphate-specific phosphodiesterase family: intracellular signaling pathways affected by the two PDEs. Understanding their targeting, regulation, and selective inhibition by compounds exerting anti- exact function would certainly provide a rational basis for more inflammatory and antidepressant actions. Adv. Pharmacol. 44:225. 25. Giembycz, M. A. 2000. Phosphodiesterase 4 inhibitors and the treatment of asth- selective pharmacological strategies. ma: where are we now and where do we go from here? Drugs 59:193. 26. Gantner, F., H. Tenor, V. Gekeler, C. Schudt, A. Wendel, and A. Hatzelmann. 1997. Phosphodiesterase profiles of highly purified human peripheral blood leu- Acknowledgments kocyte populations from normal and atopic individuals: a comparative study. We thank Dr. Stephen J. Galli and his colleagues for advice and support J. Allergy Clin. Immunol. 100:527. and for sharing the FACScan equipment, Drs. Brent Johnston and 27. Tenor, H., A. Hatzelmann, A. Wendel, and C. Schudt. 1995. Identification of phosphodiesterase IV activity and its cyclic adenosine monophosphate-dependent Eugene Butcher for advice on the chemotaxis assay, and Maria Zoudilova up-regulation in a human keratinocyte cell line (HaCaT). J. Invest. Dermatol. for the genotyping of the mice. 105:70. 7538 ROLE OF PDE4s IN NEUTROPHILS

28. Howell, R. E., W. T. Muehsam, and W. J. Kinnier. 1990. Mechanism for the 46. Perry, S. J., G. S. Baillie, T. A. Kohout, I. McPhee, M. M. Magiera, K. L. Ang, emetic side effect of xanthine bronchodilators. Life Sci. 46:563. W. E. Miller, A. J. McLean, M. Conti, M. D. Houslay, et al. 2002. Targeting of ␤ ␤ 29. Souness, J. E., D. Aldous, and C. Sargent. 2000. Immunosuppressive and anti- cyclic AMP degradation to 2-adrenergic receptors by -arrestins. Science inflammatory effects of cyclic AMP phosphodiesterase (PDE) type 4 inhibitors. 298:834. Immunopharmacology 47:127. 47. Baillie, G. S., A. Sood, I. McPhee, I. Gall, S. J. Perry, R. J. Lefkowitz, and 30. Jin, S. L., F. J. Richard, W. P. Kuo, A. J. D’Ercole, and M. Conti. 1999. Impaired M. D. Houslay. 2003. ␤-Arrestin-mediated PDE4 cAMP phosphodiesterase re- ␤ growth and fertility of cAMP-specific phosphodiesterase PDE4D-deficient mice. cruitment regulates -adrenoceptor switching from Gs to Gi. Proc. Natl. Acad. Proc. Natl. Acad. Sci. USA 96:11998. Sci. USA 100:940. 31. Thompson, W. J., and M. M. Appleman. 1971. Multiple cyclic nucleotide phos- 48. Mizgerd, J. P., H. Kubo, G. J. Kutkoski, S. D. Bhagwan, K. Scharffetter-Kochanek, phodiesterase activities from rat brain. Biochemistry 10:311. A. L. Beaudet, and C. M. Doerschuk. 1997. Neutrophil emigration in the skin, 32. Devi, S., J. Laning, Y. Luo, and M. E. Dorf. 1995. Biologic activities of the lungs, and peritoneum: different requirements for CD11/CD18 revealed by ␤-chemokine TCA3 on neutrophils and macrophages. J. Immunol. 154:5376. CD18-deficient mice. J. Exp. Med. 186:1357. 33. Campbell, J. J., E. F. Foxman, and E. C. Butcher. 1997. Chemoattractant receptor 49. Hansen, G., S. Jin, D. T. Umetsu, and M. Conti. 2000. Absence of muscarinic cross talk as a regulatory mechanism in leukocyte adhesion and migration. Eur. cholinergic airway responses in mice deficient in the cyclic nucleotide phospho- J. Immunol. 27:2571. diesterase PDE4D. Proc. Natl. Acad. Sci. USA 97:6751. 34. Frevert, C. W., S. Huang, H. Danaee, J. D. Paulauskis, and L. Kobzik. 1995. 50. Corbel, M., N. Germain, J. Lanchou, S. Molet, P. M. R. e Silva, M. A. Martins, Functional characterization of the rat chemokine KC and its importance in neu- E. Boichot, and V. Lagente. 2002. The selective phosphodiesterase 4 inhibitor RP trophil recruitment in a rat model of pulmonary inflammation. J. Immunol. 73–401 reduced matrix metalloproteinase 9 activity and transforming growth 154:335. factor-␤ release during acute lung injury in mice: the role of the balance between 35. Dasi, F. J., J. L. Ortiz, J. Cortijo, and E. J. Morcillo. 2000. Histamine up-regulates tumor necrosis factor-␣ and interleukin-10. J. Pharmacol. Exp. Ther. 301:258. phosphodiesterase 4 activity and reduces prostaglandin E2-inhibitory effects in 51. Goncalves de Moraes, V. L., M. Singer, B. B. Vargaftig, and M. Chignard. 1998. human neutrophils. Inflamm. Res. 49:600. Effects of rolipram on cyclic AMP levels in alveolar macrophages and lipopo- 36. Teixeira, M. M., R. W. Gristwood, N. Cooper, and P. G. Hellewell. 1997. Phos- lysaccharide-induced inflammation in mouse lung. Br. J. Pharmacol. 123:631. phodiesterase (PDE)4 inhibitors: anti-inflammatory drugs of the future? Trends 52. Trifilieff, A., D. Wyss, C. Walker, L. Mazzoni, and R. Hersperger. 2002. Phar- Pharmacol. Sci. 18:164. macological profile of a novel phosphodiesterase 4 inhibitor, 4-(8- 37. Tasken, K., and E. M. Aandahl. 2004. Localized effects of cAMP mediated by benzo[1,2,5]oxadiazol-5-yl-[1,7]naphthyridin-6-yl)-benzoic acid (NVP- Downloaded from distinct routes of protein kinase A. Physiol. Rev. 84:137. ABE171), a 1,7-naphthyridine derivative, with anti-inflammatory activities. 38. Rivkin, I., and E. L. Becker. 1976. Effect of exogenous cyclic AMP and other J. Pharmacol. Exp. Ther. 301:241. adenine nucleotides on neutrophil chemotaxis and motility. Int. Arch. Allergy 53. Haddad, E. B., M. Birrell, K. McCluskie, A. Ling, S. E. Webber, M. L. Foster, Appl. Immunol. 50:95. and M. G. Belvisi. 2001. Role of p38 MAP kinase in LPS-induced airway in- 39. Servant, G., O. D. Weiner, P. Herzmark, T. Balla, J. W. Sedat, and H. R. Bourne. flammation in the rat. Br. J. Pharmacol. 132:1715. 2000. Polarization of chemoattractant receptor signaling during neutrophil che- 54. Profita, M., G. Chiappara, F. Mirabella, R. Di Giorgi, L. Chimenti, G. Costanzo, motaxis. Science 287:1037. L. Riccobono, V. Bellia, J. Bousquet, and A. M. Vignola. 2003. Effect of cilo- ␣ 40. Laudanna, C., J. J. Campbell, and E. C. Butcher. 1997. Elevation of intracellular milast (Ariflo) on TNF- , IL-8, and GM-CSF release by airway cells of patients http://www.jimmunol.org/ cAMP inhibits RhoA activation and integrin-dependent leukocyte adhesion in- with COPD. Thorax 58:573. duced by chemoattractants. J. Biol. Chem. 272:24141. 55. Haddad, J. J., S. C. Land, W. O. Tarnow-Mordi, M. Zembala, D. Kowalczyk, and ␤ 41. Jones, S. L. 2002. Protein kinase A regulates 2 integrin avidity in neutrophils. R. Lauterbach. 2002. Immunopharmacological potential of selective phosphodi- J. Leukocyte Biol. 71:1042. esterase inhibition. I. Differential regulation of lipopolysaccharide-mediated 42. Tasken, K. A., P. Collas, W. A. Kemmner, O. Witczak, M. Conti, and K. Tasken. proinflammatory cytokine (interleukin-6 and tumor necrosis factor-␣) biosynthe- 2001. Phosphodiesterase 4D and protein kinase a type II constitute a signaling sis in alveolar epithelial cells. J. Pharmacol. Exp. Ther. 300:559. unit in the centrosomal area. J. Biol. Chem. 276:21999. 56. Dubravec, D. B., D. R. Spriggs, J. A. Mannick, and M. L. Rodrick. 1990. Cir- 43. Barlic, J., J. D. Andrews, A. A. Kelvin, S. E. Bosinger, M. E. DeVries, L. Xu, culating human peripheral blood granulocytes synthesize and secrete tumor ne- T. Dobransky, R. D. Feldman, S. S. Ferguson, and D. J. Kelvin. 2000. Regulation crosis factor ␣. Proc. Natl. Acad. Sci. USA 87:6758. of tyrosine kinase activation and granule release through ␤-arrestin by CXCRI. 57. Ackerman, V., M. Marini, E. Vittori, A. Bellini, G. Vassali, and S. Mattoli. 1994. Nat. Immunol. 1:227. Detection of cytokines and their cell sources in bronchial biopsy specimens from

44. Luttrell, L. M., and R. J. Lefkowitz. 2002. The role of ␤-arrestins in the termi- asthmatic patients: relationship to atopic status, symptoms, and level of airway by guest on September 27, 2021 nation and transduction of G-protein-coupled receptor signals. J. Cell Sci. hyperresponsiveness. Chest 105:687. 115:455. 58. Robichaud, A., P. B. Stamatiou, S. L. Jin, N. Lachance, D. MacDonald, 45. Fong, A. M., R. T. Premont, R. M. Richardson, Y. R. Yu, R. J. Lefkowitz, and F. Laliberte, S. Liu, Z. Huang, M. Conti, and C. C. Chan. 2002. Deletion of ␤ ␣ D. D. Patel. 2002. Defective lymphocyte chemotaxis in -arrestin2- and GRK6- phosphodiesterase 4D in mice shortens 2-adrenoceptor-mediated anesthesia, a deﬁcient mice. Proc. Natl. Acad. Sci. USA 99:7478. behavioral correlate of emesis. J. Clin. Invest. 110:1045.