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Receptor Kinase 6 Mice Deficient for G Protein-Coupled Increased Acute Inflammation, Leukotriene B 4-Induced Chemotaxis, and Signaling in Mice Deficient for G Protein-Coupled Receptor Kinase 6 This information is current as of September 29, 2021. Annemieke Kavelaars, Anne Vroon, Roel P. Raatgever, Alan M. Fong, Richard T. Premont, Dhavalkumar D. Patel, Robert J. Lefkowitz and Cobi J. Heijnen J Immunol 2003; 171:6128-6134; ; doi: 10.4049/jimmunol.171.11.6128 Downloaded from http://www.jimmunol.org/content/171/11/6128 References This article cites 32 articles, 22 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/171/11/6128.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 29, 2021 • Fast Publication! 4 weeks from acceptance to publication *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 © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Increased Acute Inflammation, Leukotriene B4-Induced Chemotaxis, and Signaling in Mice Deficient for G Protein-Coupled Receptor Kinase 6 Annemieke Kavelaars,1* Anne Vroon,1* Roel P. Raatgever,* Alan M. Fong,† Richard T. Premont,† Dhavalkumar D. Patel,† Robert J. Lefkowitz,†‡ and Cobi J. Heijnen2* Directed migration of polymorphonuclear neutrophils (PMN) is required for adequate host defense against invading organisms and leukotriene B4 (LTB4) is one of the most potent PMN chemoattractants. LTB4 exerts its action via binding to BLT1, a G protein-coupled receptor. G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases (GRK) in an agonist-dependent manner, resulting in receptor desensitization. Recently, it has been shown that the human BLT1 is a substrate for GRK6. To investigate the physiological importance of GRK6 for inflammation and LTB4 signaling in PMN, we used GRK6- Downloaded from deficient mice. The acute inflammatory response (ear swelling and influx of PMN into the ear) after topical application of arachidonic acid was significantly increased in GRK6؊/؊ mice. In vitro, GRK6؊/؊ PMN showed increased chemokinetic and ؊/؊ chemotactic responses to LTB4. GRK6 PMN respond to LTB4 with a prolonged increase in intracellular calcium and prolonged actin polymerization, suggesting impaired LTB4 receptor desensitization in the absence of GRK6. However, pre-exposure to LTB4 ؊/؊ renders both GRK6 as well as wild-type PMN refractory to restimulation with LTB4, indicating that the presence of GRK6 is not required for this process to occur. In conclusion, GRK6 deficiency leads to prolonged BLT1 signaling and increased http://www.jimmunol.org/ neutrophil migration. The Journal of Immunology, 2003, 171: 6128–6134. olymorphonuclear neutrophils (PMN)3 play a central role promotes the binding of proteins from the arrestin family, which in defense against infection. PMN migrate from the circu- can prevent further G protein activation and enhance receptor in- P lation to inflammatory sites, where they kill invading ternalization (4–6). More recently, it has been shown that arrestins pathogens. A large number of chemoattractants and chemokines can also serve as scaffolding proteins and are involved directly in are involved in the regulation of PMN recruitment. Although che- activation of certain signaling pathways (7). GPCR are also sen- motactic factors can be lipids, proteins, and peptides, most of them sitive to phosphorylation by second messenger-activated protein by guest on September 29, 2021 exert their action via binding to G protein-coupled receptors kinases such as protein kinase C and protein kinase A. In general, (GPCR). Little is known about the cellular mechanisms that are phosphorylation and desensitization of GPCR via second messen- responsible for the directed migration of PMN toward chemoat- ger-dependent protein kinases are slower processes than GRK-de- tractants. It has been suggested that receptor desensitization, in- pendent desensitization and do not require agonist binding to the ternalization, and re-expression play an important role in the di- receptor (6). rected migration of cells, although there are also reports indicating The family of GRK consists of seven members, GRK1–7. GRK that receptor recycling is not required per se for chemotaxis to 2, 3, 5, and 6 are expressed at high levels in cells of the immune occur (1–3). system (8, 9). Interestingly, the level of expression of GRK2, 3, Several kinases play a role in the desensitization and internal- and 6 in leukocytes is known to be tightly regulated (10). For ization of GPCR upon agonist activation. GPCR kinases (GRK) example, we have shown that in patients with rheumatoid arthritis are involved in the rapid, agonist-dependent uncoupling of the re- or in rats after development of adjuvant arthritis or experimental ceptor from G proteins in a process called homologous desensiti- autoimmune encephalomyelitis, the level of GRK2, 3, and 6 is zation. Phosphorylation of agonist-stimulated receptors by GRK reduced by 50–80% in leukocytes from the blood or spleen (11– 13). In addition, there is evidence that cytokines as well as oxygen radicals can regulate the level of expression of GRK in leukocytes *Laboratory for Psychoneuroimmunology, University Medical Center Utrecht, Utrecht, The Netherlands; and †Department of Medicine and ‡Howard Hughes Med- and other cell types (11, 14, 15). Moreover, differentiation of the ical Institute, Duke University Medical Center, Durham, NC 27710 pro-myelocytic cell line HL-60 toward the neutrophil lineage re- Received for publication April 11, 2003. Accepted for publication September sults in increased expression of GRK6, suggesting a functional role 22, 2003. for this GRK in PMN (9). The costs of publication of this article were defrayed in part by the payment of page Leukotriene (LT) B4 is one of the most potent chemoattractants charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. for PMN and is known to stimulate adhesion of leukocytes to 1 A.K. and A.V. contributed equally to this study. endothelium, chemotaxis, and PMN degranulation (16, 17). LTB4 has been implicated in the pathology of chronic inflammatory dis- 2 Address correspondence and reprint requests to Prof. Dr. Cobi J. Heijnen, University Medical Center, Room KC03.068.0, Lundlaan 6, 3584 EA, Utrecht, The Netherlands. eases like asthma and rheumatoid arthritis as well as in acute in- E-mail address: [email protected] flammation. High levels of LTB4 have been found in acute and 3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; GPCR, G chronic inflammatory lesions. In animal models for arthritis, skin in- protein-coupled receptor; GRK, GPCR kinase; LT, leukotriene; AA, arachidonic acid; MPO, myeloperoxidase; TEM, transendothelial migration; WT, wild type; SDF-1, flammation, and peritonitis, LTB4 antagonists reduce clinical symp- stromal cell-derived factor 1; hBLT1, human BLT1. toms (18–20). Moreover, in mice deficient for the LTB4 receptor Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 6129 BLT1, the inflammatory response in a peritonitis model is signifi- Transendothelial migration (TEM) assays were performed as previously described (26). Briefly, 105 Ea.hy 926 endothelial cells were plated onto cantly reduced, suggesting an important role for LTB4 in PMN che- motaxis in vivo (21). 24-well Transwells and incubated for 3 days. Monolayer integrity was determined by assessing diffusion of [14C]mannitol (Amersham, Arlington Recently, it has been shown in overexpression systems that the Heights, IL) from the top chamber to the bottom chamber within the Trans- 14 human receptor for LTB4 (hBLT1) can be phosphorylated by well, and those Transwells in which the amount of C cpm detected in the GRK6 (22). In cells overexpressing the hBLT1, overexpression of bottom well was Ͻ35% of control were used. The medium at the bottom GRK6 results in reduced LTB receptor signaling, demonstrating was replaced with medium with or without LTB4 and neutrophils were 4 added to the top chamber. Following4hofincubation at 37oC, cells in the that GRK6 can desensitize this receptor. Threonine 308 within the lower chamber were harvested and stained with anti-mouse Gr-1 conju- cytoplasmic tail of hBLT1 was shown to be crucial for LTB4- gated to PE (BD PharMingen, San Diego, CA). The number of migrated induced desensitization of the receptor (22). To investigate the PMN was determined by flow cytometry. possible role of GRK6 in inflammation and in LTB4 reactivity of PMN, we used mice with a targeted deletion of the GRK6 gene. Calcium mobilization The cytoplasmic tail of the murine BLT1 also contains a threonine Cells were loaded with Fluo-3-AM (Molecular Probes, Eugene, OR) for 30 residue at position 308 in a region that is highly homologous to min at room temperature. Subsequently, cells were stimulated in assay hBLT1(23). We demonstrate that in the absence of GRK6, the in buffer (145 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 1 mM CaCl2, 0.5 mM MgSO4.7H2O, 5 mM glucose, and 10 mM HEPES, pH 7.4) and fluores- vivo acute inflammatory response to topical application of arachi- cence data for PMN were collected by flow cytometry. Data represent donic acid (AA) is increased. Moreover, we show that the in vitro mean fluorescence intensity for 20–40 cells collected in 1-s periods. In chemotactic response of PMN toward LTB4 is increased in the some experiments, cells were stimulated for 5 min with LTB4 (100 nM) absence of GRK6.
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