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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 http://www.jimmunol.org/content/173/12/7531 Downloaded from References This article cites 58 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/173/12/7531.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 pulmo- nary 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 com- plementary, 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
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