Macrophages and Dendritic Cells Use Different Axl/Mertk/Tyro3 Receptors in Clearance of Apoptotic Cells This information is current as Heather M. Seitz, Todd D. Camenisch, Greg Lemke, H. of September 28, 2021. Shelton Earp and Glenn K. Matsushima J Immunol 2007; 178:5635-5642; ; doi: 10.4049/jimmunol.178.9.5635 http://www.jimmunol.org/content/178/9/5635 Downloaded from References This article cites 37 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/178/9/5635.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 28, 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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Macrophages and Dendritic Cells Use Different Axl/Mertk/Tyro3 Receptors in Clearance of Apoptotic Cells1 Heather M. Seitz,* Todd D. Camenisch,† Greg Lemke,‡ H. Shelton Earp,§ and Glenn K. Matsushima2*¶ʈ The clearance of apoptotic cells is important for regulating tissue homeostasis, inflammation, and autoimmune responses. The absence of receptor tyrosine kinases (Axl, Mertk, and Tyro3) results in widespread accumulation of apoptotic cells and autoan- tibody production in mice. In this report, we examine the function of the three family members in apoptotic cell clearance by different phagocytic cell types. Mertk elimination nearly abolished macrophage apoptotic cell phagocytosis; elimination of Axl, Tyro3, or both, reduced macrophage phagocytosis by approximately half, indicating that these also play a role. In contrast, apoptotic cell clearance in splenic and bone marrow-derived dendritic cells (DCs) is prolonged compared with macrophages and relied primarily on Axl and Tyro3. The slower ingestion may be due to lower DC expression of Axl and Tyro3 or absence of GAS6 Downloaded from expression, a known ligand for this receptor family. In vivo, phagocytosis of apoptotic material by retinal epithelial cells required Mertk. Unlike macrophages, there did not appear to be any role for Axl or Tyro3 in retinal homeostasis. Likewise, clearance of apoptotic thymocytes in vivo was dramatically reduced in mertkkd mice, but was normal in axl/tyro3؊/؊ mice. Thus, cell and organ type specificity is clearly delineated, with DCs relying on Axl and Tyro3, retina and thymus requiring Mertk, and macrophages exhibiting an interaction that involves all three family members. Surprisingly, in macrophages, tyrosine phosphorylation of Mertk /in response to apoptotic cells is markedly diminished from axl/tyro3؊/؊ mice, suggesting that the interactions of these receptors http://www.jimmunol.org by heterodimerization may be important in some cells. The Journal of Immunology, 2007, 178: 5635–5642. he phagocytosis of apoptotic cells is important during de- and Axl/Mertk/Tyro3 receptor tyrosine kinase family (3–13). velopment, lymphocyte maturation, and normal cell turn- However, only knockout mice lacking C1q, MFG-E8, and Axl/ T over. Without efficient apoptotic cell clearance, dying Mertk/Tyro3 receptor tyrosine kinases generate spontaneous cells accumulate, undergo secondary necrosis, and release intra- autoimmunity (9, 12, 13). cellular and nuclear contents into the extracellular environment. Mertk (also known as Eyk, Nyk, and Tyro-12) belongs to a Released self-molecules may become antigenic and cause lympho- family of receptor tyrosine kinases that include Axl (also known as by guest on September 28, 2021 cyte activation and autoantibody production (1). The phagocytosis ARK, Ufo, Tyro-7) and Tyro3 (also known as Rse, Sky, Brt, Tif, of apoptotic cells has become an area of intense study, and many Dtk) (14). Each member of the Axl/Mertk/Tyro3 receptor family surface receptors have been implicated in the process of apoptotic shares a similar extracellular domain structure and a signature cell recognition and engulfment (2). Knockout mice have been KWAIAES sequence in the cytoplasmic kinase domain. Mertk, generated, deleting many of the implicated receptors and inter- Axl, and Tyro3 are widely expressed in adult tissues although their mediate-bridging molecules including the following: CD14, C4, function in many of these tissues remains unclear (15). Mice lack- scavenger receptor A, phosphatidylserine receptor, ABCA1, ing Mertk, or mertkkd (previously known as merkd) mice show 3 CD93, milk-fat globule (MFG )-E8, C1q, vitronectin receptor, spontaneous autoantibody production (12, 15), splenomegaly, and enhanced TNF-␣ production in response to LPS (16). Furthermore, mice lacking all three receptors display a hyperimmune phenotype *Department of Microbiology and Immunology, University of North Carolina-Chapel Hill, Chapel Hill, NC 27599; †Department of Pharmacology and Toxicology, Uni- illustrated by enhanced autoantibody production, splenomegaly, versity of Arizona, Tucson, AZ 85721; ‡Molecular Neurobiology Laboratory, The and lymphocyte activation greater than that seen in mice lacking Salk Institute for Biological Sciences, La Jolla, CA 92037; and §Lineberger Com- prehensive Cancer Center, ¶Neuroscience Center, and ʈProgram for Molecular Mertk alone (17). One ligand for these receptors is growth arrest- Biology and Biotechnology, University of North Carolina-Chapel Hill, Chapel specific gene 6 (GAS6), which has the highest affinity for Axl, Hill, NC 27599 followed by Tyro3 and then Mertk (Kd of 0.4, 2.9, and 29 nM, Received for publication August 25, 2006. Accepted for publication February respectively) (18). GAS6 binds to phosphatidylserine on the outer 13, 2007. leaflet of an apoptotic cell and may serve as a bridging molecule 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 between Axl/Mertk/Tyro3 family receptors on phagocytes and the with 18 U.S.C. Section 1734 solely to indicate this fact. apoptotic cell (19). However, affinity of the GAS6 phosphatidyl- 1 This work was supported in part by National Institute for Allergy and Infectious serine complex for Axl, Mertk, and Tyro3 has not been measured. Disease Grant 5-31702. Another potential ligand is protein S, a serum protein involved in 2 Address correspondence and reprint requests to Dr. Glenn K. Matsushima, Neuro- the coagulation cascade (20), but its role as a ligand for this family science Center, 105 Mason Farm Road, Chapel Hill, NC 27599. E-mail address: [email protected] is still unclear, because Tyro3 is the only family member that binds to protein S (21). This finding was made using Tyro3 and protein 3 Abbreviations used in this paper: MFG, milk-fat globule; DC, dendritic cell; BMDC, bone marrow-derived DC; IP, immunoprecipitation; MFI, mean fluores- S from different species, and, until now, binding of murine Tyro3 cence intensity; PRL, photoreceptor layer; ONL, outer nuclear layer; sAxl, soluble to murine protein S has yet to be reported (22). However, protein form of Axl. GAS6, growth arrest specific gene. S binds phosphatidylserine on apoptotic cells and can enhance Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 phagocytosis, similar to GAS6 (23). www.jimmunol.org 5636 PHAGOCYTES USE Mertk, Axl, AND Tyro3 DIFFERENTLY The lack of apoptotic cell clearance in the mertkkd mouse re- (MP) and centrifuged 500 ϫ g for 15 min. The monocyte layer was col- sulting in autoantibodies coupled with the enhanced autoimmunity lected, washed in PBS, and resuspended in RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% FBS, 50 U penicillin-G, and 50 ␮g observed in the triple knockout mouse led us to investigate Ϫ ml 1 streptomycin sulfate (Invitrogen Life Technologies), 10 ng/ml GM- whether Axl and Tyro3 are also involved in clearance of apoptotic CSF, and 10 ng/ml IL-4 (PeproTech). Monocytes were plated on low clus- cells in different phagocytes (17, 18). In this report, we demon- ter 6-well plates (Costar). On day 3, the medium was doubled and cyto- strate that Axl and Tyro3 do function in apoptotic cell phagocy- kines were readjusted to 10 ng/ml. On day 7, immature DCs were washed 6 tosis but are more important in dendritic cells (DCs) and to a lesser once in PBS and plated in low cluster 24-well plates at 10 cells/well. Splenic DCs were isolated using anti-CD11c Ab bound to microbeads fol- extent in macrophages. In contrast, Mertk is the primary phago- lowing the manufacturer’s protocol (Miltenyi Biotec). Splenic CD11c-pos- cytic receptor for apoptotic cells in macrophages, but it does not itive cells were used immediately for phagocytosis. Apoptotic thymocytes have this function in DCs as published previously (12, 24). We were generated as described above and added to DCs at a 10:1 ratio. At also demonstrate that Axl and Tyro3 preferentially bind the ligands indicated time points, the cell suspension was plated on a glass coverslip GAS6 and protein S, respectively, and, surprisingly, that in the and DCs were adhered for 1 h. Noningested thymocytes were washed off and DCs were stained with anti-CD11c-FITC Ab (BD Pharmingen). Cells absence of Axl and Tyro3, Mertk phosphorylation is markedly that were positive for both CD11c and ingested apoptotic cells were scored. reduced in response to apoptotic cells. However, we show that in To analyze pinocytosis, 1 mg/ml albumin-FITC and 0.5 mg/ml dextran- vivo Mertk, even in the absence of Axl and Tyro3, is fully com- FITC (Sigma- Aldrich) were added to DCs for 30 min at 37°C or 4°C as petent to clear apoptotic cell/material in the thymus and the retina.