Clearance of Apoptotic Cells Different Axl/Mertk/Tyro3 Receptors In

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

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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 © 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 autoantibody 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. a negative control. Cells were then washed and analyzed by ﬂow cytometry to determine uptake of albumin or dextran. Materials and Methods Ligand binding assay and Western blotting

Animals Downloaded from Axl, Mertk, or Tyro3 extracellular domain-human IgG1 Fc chimeras were All mice were housed in the specific pathogen-free Division of Laboratory purchased from R&D Systems. Ten nanograms of chimera was added to Animal Medicine facilities in accordance with Institutional Animal Care 100 ␮l of normal mouse serum (Vector Laboratories) or PBS at room and Use Committee regulations. Male mice 8–10 wk old were used in temperature for 1 h with gentle rotation. Protein A Sepharose (Amersham studies and wild-type mice are C57BL/6 (The Jackson Laboratory; and Biosciences) was added to chimera/serum mixture and incubated for an bred in house). mertkkd, axlϪ/Ϫ, and tyro3Ϫ/Ϫ mice were backcrossed onto additional 30 min at room temperature with gentle rotation. the C57BL/6 background for six generations. Previously designated merkd For phosphorylation experiments, thioglycollate-elicited peritoneal cells kd 7 mice are now designated mertk in this report, because Mertk is the proper were plated at 1 ϫ 10 cells per 100-mm petri dish. After 2 h, nonadherent http://www.jimmunol.org/ nomenclature. These mertkkd mice show no detectable protein by Western cells were washed off and macrophages were rested for 7 days. Apoptotic blot or flow cytometry (our unpublished results), therefore they have a cells were added at 10:1, thymocytes:macrophage, ratio for 15 min and functionally null mutation. then macrophages were washed three times with PBS. Macrophages were lysed using immunoprecipitation (IP) lysis buffer (TBS, 1% Nonidet P-40 Macrophage in vitro phagocytosis (Pierce), 0.1 mg of aprotinin, 0.1 mg of ␣-1-antitrypsin, 0.1 mg of leupep- Peritoneal macrophages were isolated and phagocytosis assay were per- tin (Sigma-Aldrich), 1 mM PMSF (Sigma-Aldrich), 2 mM sodium or- formed as previously described (12) with the following modifications. Peri- thovanadate (Sigma-Aldrich), and phosphatase inhibitor mixtures I and II toneal exudate cells were elicited by i.p. injection with 3 ml of 3% thio- (Sigma-Aldrich)) and lysates cleared by centrifugation. Nonspecific pro- glycollate (Fisher Scientific) for 72 h followed by lavaging the peritoneum teins bound to Sepharose were removed by incubating with protein G Sepharose (Amersham Biosciences) alone for 30 min at 4°C followed by with 3 ml of PBS. Resident macrophages were obtained by lavaging the ␮ by guest on September 28, 2021 peritoneum of naive mice with PBS. Peritoneal exudate cells were plated centrifugation. Anti-Mer Ab (0.5 g) (R&D Systems) was added to cleared at 1.5 ϫ 105 cells/well of a 24-well plate. After 2 h, nonadherent cells were lysate and incubated 30 min at 4°C with gentle rotation. Protein G Sepha- washed off with PBS and fresh macrophage medium (RPMI 1640 (Life rose was added and lysates were incubated for an additional 30 min at 4°C Technologies) supplemented with 5% heat-inactivated FBS (Gemina Bio- with gentle rotation. Proteins bound to Sepharose were isolated by washing Products), 50 U penicillin G, and 50 ␮gmlϪ1 streptomycin sulfate (In- three times in IP lysis buffer. After a final wash, SDS sample-loading buffer vitrogen Life Technologies), sodium pyruvate (Sigma-Aldrich), and 2-ME was added, and the sample was boiled and resolved on a SDS-PAGE gel. (Invitrogen Life Technologies)) was added. The resulting adherent perito- Proteins were transferred onto polyvinylidene difluoride membrane (Mil- lipore) and blotted in 5% milk-TBS with 0.1% Tween 20 (TBS-T) for 30 neal macrophages were then rested overnight at 37°C with 5% CO2.To generate apoptotic thymocytes, we collected thymus from naive wild-type min. Primary Ab, anti-protein S (Santa Cruz Biotechnology), anti- animals and dissociated the tissue using forceps. The single-cell suspension phosphotyrosine (Cell Signaling), or anti-Mer (R&D Systems) was added was then washed one time in PBS and resuspended in macrophage medium at 1:1,000 overnight at 4°C. Blots were washed in TBS-T and incubated (described above) with 2 ␮M dexamethasone (Sigma-Aldrich). After5hof in anti-goat-HRP (Vector Laboratories) or anti-mouse-HRP (Vector incubation at 37°C, 2 ␮M Cell Tracker Green or Cell Tracker Orange Laboratories) for phosphotyrosine blots at 1:10,000 for 2 h followed by (Molecular Probes) was added to thymocytes and incubated for 30 min, further washing in TBS-T. Blots were incubated with ECL-Plus (Am- cells were washed with PBS and then rested an additional 30 min in me- ersham Biosciences) and visualized using Maximum Resolution film dium. Annexin V-FITC (Trevigen) and VAD-FMK-FITC staining and (Kodak). analysis by flow cytometry revealed that thymocytes were 70–80% apo- RT-PCR of GAS6 and protein S ptotic. To generate opsonized thymocytes, freshly isolated thymocytes were incubated with murine anti-murine CD3 Ab (Serotec) for 30 min at Reverse transcriptase was performed mostly as described previously (25). 4°C with gentle rotation. After staining, cells were washed three times with Briefly, RNA was isolated from cells using the TRIzol (Invitrogen Life PBS and then resuspended in medium. Addition of fluorochrome-conju- Technologies) reagent and 15 ␮g of crude RNA was treated with DNase gated anti-mouse secondary Ab revealed Ͼ80% of the thymocytes were (Promega). After isolation, cDNA was synthesized for 60 min at 42°C labeled with CD3 Ab by flow cytometry. For phagocytosis assays, thymo- using 5 ␮g of RNA with 150 ng of random hexamers and Moloney murine cytes were added at a 10:1 thymocyte:macrophage ratio for indicated time leukemia virus reverse transcriptase (Invitrogen Life Technologies). PCR points in macrophage medium. Macrophages were then washed extensively was then performed using 50 ng of cDNA and primers specific for GAS6 with PBS and fixed with 1% paraformaldehyde (Sigma-Aldrich). Phago- (forward, 5Ј-ACAGGCTCAACTACACCCGAACAT-3Ј and reverse, 5Ј- cytosis was determined by first counting macrophages in brightfield and TGACGGGTGCAGAAATCACCGATA-3Ј), protein S (forward, 5Ј-CG then overlaying image of fluorescent apoptotic cells to determine the num- CCGTCTTGGACAAAGCAATGAA-3Ј and reverse, 5Ј-TGCCAGCTGG ber of macrophages with ingested apoptotic cells. Only apoptotic cells that TGATAGGAATGTGA-3Ј), and GAPDH (forward, 5Ј-CTACACTGAG were completely within boundaries of macrophage were scored as phago- GACCAGGTTGTCT-3Ј and reverse, 5Ј-GCGAACTTTATTGATGGTAT cytized. Two hundred macrophages per time point in triplicate were TCAA-3Ј). The PCR mixture contained 0.2 mM dNTPs and 1.5 mM counted for each genotype. MgCl2. The PCR products were resolved on a 1.5% agarose gel using 100-bp m.w. standards (Invitrogen Life Technologies). DC in vitro phagocytosis In vivo clearance of apoptotic cells and immunohistochemistry Bone marrow-derived DCs (BMDCs) were isolated by harvesting femurs from mice and flushing out bone marrow with PBS. Bone marrow cells Adult mice were injected i.p. with 0.2 mg/25 g of dexamethasone (Sigma- were then layered on a density gradient of lymphocyte separation medium Aldrich) as described previously (12). At indicated time points, the thymus The Journal of Immunology 5637 was removed and half was fixed in 10% buffered formalin overnight at 4°C, followed by processing and embedding in paraffin. Sections (5 ␮m) were cut and stained using the TUNEL method. Briefly, sections were deparaffinized with Histoclear (National Diagnostics) and graded ethanol TBS. Sections were then permeabilized in TBS ϩ 0.1% Triton X-100 for 5 min and nuclear material was exposed using Proteinase K. After washing out detergents and enzyme, sections were incubated with dUTP-FITC (Roche) and TdT enzyme (Invitrogen Life Technologies) for 30 min at 37°C. The other half of the thymus was dissociated into a single-cell suspension and stained with VAD-FMK-FITC (Promega) to detect active caspase 3-positive apoptotic cells. These cells were analyzed on a BD Biosciences FACScan cytometer using Summit software. To examine the histopathology of the retina, eyes were removed from adult mice and fixed in 4% paraformaldehyde overnight at 4°C. The tissues were then embedded in paraffin and 5-␮m sections were cut and stained with H&E as described previously (26).

Statistical analysis Statistics were calculated using JMP IN 4.0 software (SAS Institute). Tukey-Kramer HSD one-way ANOVA was performed at an alpha level of 0.01–0.05 to compare all variants in a data set. Downloaded from

Results Mertk is critical for phagocytosis of apoptotic cells by macrophages (12), but the role of Axl and Tyro3 has not been adequately studied. Primary peritoneal macrophages were isolated and ex-

posed to apoptotic thymocytes. Greater than 40% of the macro- http://www.jimmunol.org/ phages from wild-type mice ingested apoptotic cells. In contrast, macrophages from mertkkd mice had a dramatic deficit in clearance of apoptotic cells in vitro. Although not as complete, macrophages from axlϪ/Ϫ, tyro3Ϫ/Ϫ, and axl/tyro3Ϫ/Ϫ mice were significantly less able (by ϳ50%) to phagocytose apoptotic cells when compared with macrophages from wild-type mice (Fig. 1A). To determine differences in the rate of ingestion, phagocytosis of apoptotic cells by macrophages was quantified over a time course. At 60 and 90 min, macrophages from mice lacking axl/ by guest on September 28, 2021 tyro3Ϫ/Ϫ had significantly lower levels of phagocytosis than wild- type macrophages (Fig. 1B). Macrophages lacking Mertk remained incompetent even at 90 min. To ensure the phagocytosis was not affected by the thioglycollate used to elicit the macrophages, we isolated resident peritoneal macrophages and compared phagocytosis. Resident macrophages from wild-type mice initiated phago- FIGURE 1. Macrophages lacking Axl and/or Tyro3 have a deficiency in clearance of apoptotic cells. A, In vitro phagocytosis assay with thiogly- cytosis of apoptotic cells readily at 30 min and then declined there- collate-elicited peritoneal exudate cells collected at 60 min after nonapop- after (Fig. 1C). Similar to thioglycollate-elicited macrophages, Ⅺ f B Ϫ/Ϫ totic (UT, ) or apoptotic (AT, ) thymocytes were added. , Thiogly- resident peritoneal macrophages from axl/tyro3 mice had sig- collate-elicited macrophages from wild-type (ࡗ), mertkkd (f), or axl/ nificant defects in phagocytosis at 60 and 90 min, although they tyro3Ϫ/Ϫ(Œ) mice or resident macrophages from wild-type (ࡗ), mertkkd exhibited an intermediate response between macrophages from (f), or axl/tyro3Ϫ/Ϫ(Œ) mice (C) were fed apoptotic thymocytes for indi- wild-type and mertkkd mice (Fig. 1C). cated time points in minutes. D, Thioglycollate-elicited macrophages were Other mechanisms of phagocytosis were examined to determine fed 2 ␮M latex beads (LB, f) or CD3 opsonized nonapoptotic thymocytes whether Axl and Tyro3 were involved. Macrophages from wild- (OP, Ⅺ) for 60 min. E, Thioglycollate-elicited macrophages were fed type or mutant mice were given latex beads or anti-CD3 Ab op- nonapoptotic (NAC) or apoptotic cells (AC) for 60 minutes with 2 uM ϩ sonized thymocytes. There was no difference in phagocytosis by Cytocholasin D (AC CytoD) or incubated at 4 C (AC 4C). Bound AC kd were incubated with Cytocholasin D for 30 min. Error bars are SE; n Ͼ 3. Ͻ ءء Ͻ ء , macrophages from wild-type mice vs macrophages from mertk Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ , p 0.05 and , p 0.01 compared with wild-type. These data are axl , tyro3 ,oraxl/tyro3 mice (Fig. 1D). This finding representative of four independent experiments. indicated that the defect in phagocytosis observed in mertkkd and axl/tyro3Ϫ/Ϫ macrophages was specific for apoptotic cells. To ensure that the phagocytosis assay was measuring ingested tic cells bound to the macrophages from wild-type, mertkkd, and cells as opposed to bound/noningested cells, macrophages from axl/tyro3Ϫ/Ϫ (Fig. 1E, cross-hatched bars). wild-type, mertkkd, and axl/tyro3Ϫ/Ϫ mice were treated with apo- The mechanism for the phagocytosis of apoptotic cells by DCs ptotic cells at 4°C or with 2 ␮M cytochalasin D, which prevents is not known; however, it does not require Mertk (24). We postu- phagocytosis and ingestion. After extensive washing, there were lated that Axl or Tyro3 may function as receptors important in the no ingested apoptotic cells within macrophages after incubation at recognition and phagocytosis of apoptotic cells. Bone marrow 4°C or with cytochalasin D (Fig. 1E, p and f). To ensure that from femurs of wild-type and knockout mice was cultured and day binding was not different among the genotypes, macrophages that 7 immature DCs were produced. Characterization of these were not washed extensively after incubation with apoptotic cells CD11Cϩ BMDCs by flow cytometry showed no difference in co- were examined. There was no difference in the number of apopto- stimulatory molecules CD80, CD86, or MHC class II expression 5638 PHAGOCYTES USE Mertk, Axl, AND Tyro3 DIFFERENTLY

between DCs from wild-type vs those from mertkkd and axl/ tyro3Ϫ/Ϫ mice (Fig. 2A). To determine whether Axl or Tyro3 plays a role in phagocytosis of apoptotic cells by DCs, immature BMDCs were cocultured with fresh or apoptotic thymocytes and the amount of phagocytosis was quantified. BMDCs from wild- type, and mertkkd mice cleared apoptotic cell with equal efficiency In contrast, a significant inhibition in phagocytosis was observed in DCs from axlϪ/Ϫ, tyro3Ϫ/Ϫ, and axl/tyro3Ϫ/Ϫ mice (Fig. 2B). Fur- thermore, whereas BMDCs from axl/tyro3Ϫ/Ϫ mice never reached levels comparable with BMDCs from wild-type or mertkkd mice, BMDCs from mertkkd mice continued to ingest apoptotic cells up to 12 h after being fed (Fig. 2C). To ensure that the BMDCs were indicative of those found in vivo, splenic DCs were isolated and cocultured with apoptotic cells. Although the CD11cϩCD8ϩ population is thought to be the primary phagocytic DC in the spleen, we examined the number of these cells in mice from wild-type, mertkkd, and axl/tyro3Ϫ/Ϫ and found no significant differences (mean fluorescence intensity

(MFI) of 11.72, 14.39, and 13.99, respectively). Similar to BMDCs Downloaded from in Fig. 2B, splenic DCs from axl/tyro3Ϫ/Ϫ mice had a signiﬁcant decrease in phagocytosis at 6 h. In contrast, there was no signiﬁ- cant difference between splenic DCs from wild-type and mertkkd mice (Fig. 2D). A major characteristic ascribed to DCs is pinocytosis, where microparticles are continuously taken up from the

environment for Ag presentation. To determine whether this pro- http://www.jimmunol.org/ cess was altered in mertkkd or axl/tyro3Ϫ/Ϫ mice, we incubated DCs with FITC-conjugated albumin or FITC-conjugated dextran at 37°C or as a negative control at 4°C. There was no significant difference in the uptake of dextran or albumin in wild-type BMDCs vs BMDCS from mertkkd and axl/tyro3Ϫ/Ϫ mice. This result indicated that pinocytosis is normal in BMDCs from mertkkd and axl/tyro3Ϫ/Ϫ mice (Fig. 2E). To demonstrate our quantification of ingested apoptotic cells can discriminate apoptotic cells merely bound on the cell surface by guest on September 28, 2021 of phagocytes, BMDCs from wild-type, mertkkd, and axl/tyro3Ϫ/Ϫ mice were incubated with apoptotic cells in the presence of 2 ␮M cytochalasin D to inhibit actin rearrangements. Only very low levels of phagocytosis in the presence of cytochalasin D was observed, suggesting that our phagocytosis assays are measuring ingested and not just externally bound apoptotic cells (Fig. 2F, u). In addition, there was no difference in binding between genotypes when BMDCs incubated with apoptotic cells were not extensively washed (Fig. 2F, cross-hatched bars). These data suggest that macrophages and DCs have a fundamental difference in their ability to phagocytize apoptotic cells, and this mechanism is mediated by different combinations of the Axl/Mertk/Tyro3 receptor family. Preferential use of the three receptor tyrosine kinases by macrophages and DCs may be dictated by the presence of specific ligands. Due to conflicting reports in the literature about whether or not same species Tyro3 and protein S are binding partners (22), we examined whether murine Tyro3 extracellular domain interacts with murine serum-derived protein S. In these experiments, only Tyro3 extracellular domain interacted with murine protein S, whereas Mertk and Axl had undetectable amounts of protein FIGURE 2. DCs lacking Axl and/or Tyro3 have a deficiency in clearance bound (Fig. 3A). The selective usage of Axl/Mertk/Tyro3 receptors of apoptotic cells. A, BMDCs were stained for CD11c-PE and MHC class II-FITC, CD80-FITC, or CD86-FITC and analyzed by flow cytometry. Data are gated on CD11cϩ cells. These data are representative of three independent experiments. BMDCs (B and C) or splenic CD11cϩ cells (D) from wild-type (WT) or knockout mice were fed nonapoptotic thymocytes (UT, Ⅺ) or apo- albumin at 4°C as a control or 37°C. F, BMDC’s were fed non-apoptotic ptotic thymocytes (AT, f)for6h(B and D) for indicated time points in hours (NAC) or apoptotic cells (AC) for 6 hours in the presence of 2 ␮M Cy- (C) and then plated on coverslips. BMDCs are indicated as wild-type (ࡗ), tocholasin D (ACϩCytoD). Bound AC were fed to BMDCs incubated in mertkkd (f), or axl/tyro3Ϫ/Ϫ(Œ) mice. D, BMDCs were treated with FITC- CytoD for 1 hour and BMDCs were not washed before counting. Error bars .p Ͻ 0.01 compared with wild-type ,ءء p Ͻ 0.05 and ,ء .conjugated dextran or FITC-conjugated albumin at 4°C as a control or 37°C. are SE; n Ͼ 3 E, BMDCs were treated with FITC-conjugated dextran or FITC-conjugated These data are representative of three independent experiments. The Journal of Immunology 5639 Downloaded from

FIGURE 3. Binding of GAS6 and protein S to Axl, and Tyro-3 and expression by phagocytes. A, Mertk, Axl, and Tyro3 extracellular domains fused with human IgG1 Fc domains (R&D Systems) were incubated with or without normal mouse serum (NMS) and Western (WB) blotted with Abs specific for protein S. B, Detection of GAS6 and protein S by RT-PCR. cDNA was isolated from lung as a positive control, and immature BMDCs http://www.jimmunol.org/ (iBMDC), macrophages (M⌽), or a monocyte cell line 32D were isolated as a negative control. Absence of Taq polymerase (ϪTaq) is negative con- FIGURE 4. Mertk is responsible for clearance of apoptotic cells in the trol for PCR and lack of reverse transcriptase (ϪRT) is negative control for thymus. Mice were injected with dexamethasone and each thymus har- cDNA preparation. Primers specific for GAS6, protein S, and GAPDH vested was halved: one half was used for in situ TUNEL staining at 24 h were used to examine expression by these cell types. Molecular weight is (A) and the other half was dissociated into single-cell suspension and la- a 100-bp ladder. C–E, Thioglycollate-elicited peritoneal cells from wild- beled with VAD-FMK to quantify remaining apoptotic cells at 8 (B)or type mice were isolated and stained with F4/80-FITC (BD Pharmingen) 24h(C). Dexamethasone-treated mice are represented by f or PBS-treated and Mertk (R&D Systems), Axl (Santa Cruz Technology), and Rse mice in Ⅺ. Data acquired from cytometry was gated on the whole/live cells p Ͻ 0.05 compared by guest on September 28, 2021 ,ء .Tyro-3; Santa Cruz Technology) Abs followed by anti-goat-PE (Caltag in the suspension. Error bars represent SE; n Ͼ 3) Laboratories). Histograms are gated on F4/80-positive cells. F–H, BMDCs with wild-type. These data are representative of four independent from wild-type mice on day 7 were harvested and stained for CD11c (BD experiments. Pharmingen) and receptors as indicated above. Histograms are gated on CD11c-positive cells. Shaded sample is isotype control. Histograms are representative of at least three independent cell preparations. Tyro3 on macrophages, in contrast, is consistent with their greater efficiency in recognition, binding, and rate of ingestion of apopto- by phagocytes prompted an examination of whether DCs or mac- tic cells. rophages express different ligands. Using RT-PCR, we found that To determine whether Axl and Tyro3 are necessary for phago- macrophages express both GAS6 and protein S, whereas immature cytosis in other cell types and in vivo, we examined the thymus, an BMDCs only express protein S (Fig. 3B). The differential expres- organ in which continuous cell death occurs as a consequence of sion of GAS6 and protein S by these phagocytes may partly explain maturation and negative selection of thymocytes. Administration why selected receptors are playing a role in phagocytosis. Thus, of the glucocorticoid dexamethasone to mice induces thymo- the expression of both protein S and GAS6 by macrophages may cyte death and provides a model to experimentally monitor the afford engagement of all three receptors, whereas the expression of clearance of apoptotic cells. Previously, our group has shown only protein S by DCs allows preferential interaction of only that mertkkd mice are unable to clear an in vivo dexamethasone- Tyro3. induced apoptotic cell burden in the thymus (12). To determine The efficiency with which DCs ingest apoptotic cells is mark- whether Axl and Tyro3 function as cophagocytic receptors in the edly slower than macrophages. The maximum percentage of thymus, we injected wild-type, mertkkd, axlϪ/Ϫ, tyro3Ϫ/Ϫ, and axl/ phagocytic DCs require 6–9 h (Fig. 2C), whereas macrophages tyro3Ϫ/Ϫ mice with dexamethasone and monitored apoptotic cell reach optimum numbers by 60 min (Fig. 1B). One plausible ex- accumulation in the thymus as exemplified in Fig. 4A. The max- planation is the relative cell surface expression of Axl, Mertk, and imum level of apoptosis is typically observed at 8 h in the thymus. Tyro3 on these phagocytes. Macrophages have high levels of At this time point, all animals had similar numbers of apoptotic Mertk, Axl, and Tyro3 on their surface; MFIs of 22.43, 15.38, and cells in the thymus as measured by flow cytometry staining and 11.44, respectively (Fig. 3, C–E). In contrast, DCs have low levels TUNEL staining in tissues (Fig. 4B and data not shown). This of surface Mertk, Axl, and Tyro3; MFI of 2.74, 7.51, and 4.74 result indicated that the induction of apoptotic cells by dexameth- compared with macrophages (Fig. 3, F–H). The low levels of asone was similar in all genotypes. Mertk, Axl, and Tyro3 expression on the surface of DCs suggest At 24 h, as previously shown, mertkkd mice were defective in that these phagocytes may be less efficient at recognizing, binding, clearance and therefore had an abundance of noningested apoptotic and ingesting apoptotic cells; hence, this could correlate with their cells in the thymus as evidenced by TUNELϩ staining (Fig. 4A). slower rate of phagocytosis. The high level of Axl, Mertk, and In contrast, axlϪ/Ϫ, tyro3Ϫ/Ϫ, and axl/tyro3Ϫ/Ϫ animals cleared 5640 PHAGOCYTES USE Mertk, Axl, AND Tyro3 DIFFERENTLY

FIGURE 6. Axl and Tyro3 are required for efﬁcient Mertk phosphorylation. Macrophages from wild-type (WT) and knockout mice were incubated with apoptotic cells (AC) for 15 min and then immunoprecipitated for Mertk. Proteins were resolved and blotted with Abs speciﬁc for phosphotyrosine, stripped, and reprobed for total Mer.

lation. In contrast, macrophages lacking Axl and Tyro3 had markedly diminished Mertk phosphorylation (Fig. 6). These results suggest that in macrophages (and perhaps other cells), Axl and Tyro3, which have higher affinity for ligand, may be required for efficient FIGURE 5. Mertk is responsible for retina homeostasis. Eyes were re- Mertk tyrosine phosphorylation. Axl and/or Tyro3 may bind li- Downloaded from moved from 8-wk-old mice, fixed in 4% paraformaldehyde, and embedded gand, then activate Mertk by heterodimerization similar to ligand- in paraffin. Sections were stained with H&E to examine morphology. ONL dependent heterodimers found in other receptor families (e.g., epi- or PRL is missing from retina in mertkkd mice, and arrowhead indicates retinal pigment epithelium. Scale bar, 50 ␮m. dermal growth factor receptor family). Whether this is true in other cells (e.g., retinal epithelial cells) in which Mertk seems to function well in the absence of Axl and Tyro3 remains to be seen.

apoptotic cells similar to wild-type. Apoptotic cell number in each http://www.jimmunol.org/ genotype was confirmed by dissociating the thymus and staining Discussion for the active caspase-3 marker VAD-FMK-FITC by flow cytom- The efficient clearance of apoptotic cells is paramount for main- etry (Fig. 4C). The fact that the axlϪ/Ϫ, tyro3Ϫ/Ϫ, and axl/tyro3Ϫ/Ϫ taining tissue homeostasis. Patients and animal models of systemic and wild-type mice showed no difference in the number of apo- lupus erythematosus including, New Zealand Black/White, MRL, ptotic cells indicated that Axl and Tyro3 were not required for the and lpr mice have phagocytes that are unable to clear apoptotic clearance of a large burden of apoptotic cells in the thymus. Mertk debris (29–31). Many genes have been implicated in apoptotic cell was both necessary and sufficient in this context. clearance, and a subset of these genes (such as mfg-e8 and mertk), Recently, GAS6 has been implicated in the efficient removal of when deleted, lead to autoimmune phenotypes (9, 12). Thus, the outer segment debris from photoreceptors in the retina, suggesting relationship between apoptotic cell phagocytosis and immune ho- by guest on September 28, 2021 that the cognate receptors Axl, Mertk, and Tyro3 may be partici- meostasis is important, and the exact mechanism by which differ- pating in the clearance of outer segments by retinal pigment epi- ent phagocytes recognize and ingest apoptotic debris merits further thelial cells (27). In addition, mutations in mertk have been linked study. Mertk is emerging as a pivotal cell surface receptor that with retinitis pigmentosa, indicating a role for Mertk in maintain- bridges innate immune responses and regulation of autoimmune ing normal retinal apoptotic material clearance and suggesting that disease, but mice lacking all three Axl/Mertk/Tyro3 receptors have Mertk functions in epithelial cells as well as in macrophages (12, an enhanced phenotype compared with mice lacking Mertk alone, 28). Retinal pigment epithelial cells lacking Mertk cannot clear e.g., autoantibody production, splenomegaly, and lymphocyte ac- apoptotic-like outer segment debris. This results in retinal degen- tivation. This finding prompted further evaluation of the role that eration in the photoreceptor layer (PRL) and destruction of the Axl and Tyro3 play. outer nuclear layer (ONL) by 6–7 wk of age (28). To determine In this study, we used single and double knockout mice for axl whether Axl and Tyro3, which have a higher affinity for GAS6 and tyro3 to help elucidate their function in clearance of apoptotic than Mertk, also contribute to retina homeostasis (as they do in cells. Because different organs require different cell types to engulf macrophages; see Fig. 2), we examined the retina of young and old apoptotic debris and maintain tissue homeostasis, we first chose adult axlϪ/Ϫ, tyro3Ϫ/Ϫ, and axl/tyro3Ϫ/Ϫ knockout mice. At 2 mo two phagocytes that also serve in Ag presentation and regulate of age, there was no retinal degeneration in normal mice or in adaptive immune responses, macrophages, and DCs. Our original axlϪ/Ϫ, tyro3Ϫ/Ϫ,oraxl/tyro3Ϫ/Ϫ mice (Fig. 5). The retina of report suggested that Mertk was critical for the phagocytosis of axlϪ/Ϫ, tyro3Ϫ/Ϫ, and axl/tyro3Ϫ/Ϫ mice had an intact PRL and an apoptotic cells by macrophages (12), and we find in this study that ONL of between 12 and 14 nuclei similar to wild-type mice. Fur- Axl and Tyro3 function as phagocytic receptor. However, macro- thermore, older axlϪ/Ϫ, tyro3Ϫ/Ϫ, and axl/tyro3Ϫ/Ϫ mice were phages lacking axl, tyro3, or both axl/tyro3 were less efficient in without evidence of retinal degeneration at 6 mo of age (data not clearing apoptotic cells compared with wild-type macrophages shown). In contrast, and as previously reported, mertkkd mice had (Fig. 1, A–C). In contrast, whereas DCs from mice lacking mertk complete degeneration of the photoreceptors and ONL at this age had no defect in clearance of apoptotic cells as previously noted (Fig. 5). Thus, the homeostasis of the retina and the clearance of (24), DCs from mice lacking axl or tyro3 had a dramatic deficit. outer segment debris by retinal pigment epithelial cells does not Thus, Axl and Tyro3 are the two receptor tyrosine kinase family require Axl or Tyro3 and appears to rely solely on functional members that are critical for the ingestion of apoptotic cells Mertk. by DCs. To further examine the cell type specificity, we studied Mertk It is unclear why receptor usage among these two cell types phosphorylation in macrophages stimulated with apoptotic cells. differs so dramatically, but perhaps these discrepancies are corre- Macrophages from wild-type mice possessing Axl and Tyro3 were lated with their different functions as efficient phagocytes vs pro- able to phosphorylate Mertk at 15 min after apoptotic cell stimu- fessional APCs. In our studies, apoptotic cell phagocytosis by The Journal of Immunology 5641 macrophages took only 60 min; DCs took 6 h. Although the notion a need for at least two family members to trigger Mertk tyrosine of macrophages as more efficient phagocytes than DCs has been phosphorylation. Binding of apoptotic cells to macrophages only observed for many particles, one plausible explanation for this results in robust phosphorylation of Mertk in the presence of Axl slower ingestion of apoptotic cells was the lower expression of and/or Tyro3. We are currently investigating whether receptor ag- Axl, Mertk, and Tyro3 in DCs, compared with macrophages (Fig. gregation is different in macrophages vs DCs. Alternative experi- 3, C–H). Thus, the lower levels of phagocytic receptors on DCs ments to identify interaction among the Axl/Mertk/Tyro3 family of may prevent adequate receptor activation. Alternatively, Axl and receptors have been difficult due to cross-reactivity of current Abs; Tyro3, which are critical on DCs, may be less efficient at triggering however, clues that these receptors act in combination have been phagocytosis. Furthermore, later time points revealed that DCs demonstrated in triple axl/mertk/tyro3 knockout mice (17) and in from mice lacking mertk had prolonged or extended phagocytic recent studies using platelets, which also express all three receptors activity (Fig. 2C), perhaps suggesting that Mertk may be playing a and appear to require heterodimerization for receptor tyrosine role to down-regulate ingestion and become a nonphagocytic DC. phosphorylation (37). Nonetheless, our report is the first to dem- We also examined ligand expression for this family of receptors onstrate that multiple members of this receptor family are needed as a potential control point that regulates phagocytosis. We found for macrophage ingestion of apoptotic cells. that whereas macrophages express both protein S and GAS6, DCs Many questions regarding Axl/Mertk/Tyro3 receptors and how only express protein S (Fig. 3B and Ref. 32). This finding could they function differently on different phagocytes remain. Low lev- suggest that DCs are more restricted and reliant on Tyro3 for els of these receptors on DCs have made detection of protein and phagocytosis, because Tyro3 was the only receptor shown to bind phosphorylation difficult; therefore, we cannot assess the require- Downloaded from mouse protein S (Fig. 3A). The physiologic role of protein S, ment of Axl and Tyro3 for Mertk activation in DCs. MFG-E8 is an which is present in the circulation, is known to serve in the natural intermediate protein that facilitates clearance of apoptotic cells in coagulation system and binds to phosphatidylserine on apoptotic the spleen and is secreted by macrophages and DCs and interacts cells, platelets, and endothelial cells (33). In vivo, it is certainly ␣ ␤ with the integrin V 5 (38). Other reports have shown that Mertk possible that the microenvironment of phagocytes contacting ap- may also require the integrin ␣ ␤ for efficient signaling (39). optotic cells and the selective local production of protein S and V 5

Therefore, it remains controversial which combination of recep- http://www.jimmunol.org/ GAS6 ligand may dictate engagement of specific receptors and tors, ligands, and signaling is required for proper induction of partly regulate rates of phagocytosis. phagocytosis of apoptotic bodies. In our report, we cannot exclude In tissues such as the thymus, where phagocytes and apoptotic the possibility that integrins may be cooperating with Axl, Mertk, cells are present, we examined whether Axl and Tyro3 affect clear- and Tyro3 to facilitate this process as IPs to demonstrate that inter- ance of apoptotic cells. Mertk was critical for clearance of an abun- actions have been difficult with these cell types and with potential dant apoptotic cell burden in the thymus, but Axl and Tyro3 were cross-reactive Abs. Secondly, Axl and Tyro3 appear to have different not required. Mice lacking axl, tyro3, or both axl/tyro3 cleared the ligand affinity and yet they both are required for efficient phagocytosis burden of apoptotic cells as efficiently as wild-type mice (Fig. 4, A kd by macrophages and most importantly by DCs. Axl is known to be and C). It appears that mertk mice can clear apoptotic cells re- by guest on September 28, 2021 cleaved by ADAM10 and is thought to exist primarily as a cleaved sulting from the physiologic negative and positive selection of thy- soluble form in murine DCs (32). This soluble form of Axl (sAxl) has mocytes, because the untreated Mertk mouse does not exhibit a been found in complex with GAS6 in the serum of mice (32) to the large excess of apoptotic cells. It is possible that Axl and Tyro3 extent that no free GAS6 was detected. These studies further indicated may be compensating for the lack of Mer in mertkkd mice in nor- that sAxl might enhance GAS6 expression and stability. Therefore, a mal homeostasis; however, our data suggest that Mertk is the primary molecule on thymic phagocytes critical for clearance of syn- more complex relationship between sAxl/GAS6, Tyro3, and Mertk chronous, large burden of apoptotic cells (Fig. 4, A and C). Even may exist, and an understanding of how this receptor family may in the genetic absence of Axl and Tyro3, Mertk is sufficient to interact is an area currently under investigation. allow ingestion of large amounts of apoptotic cells and maintain In summary, our report documents a novel role for Axl and normal tissue homeostasis (Fig. 4C). When all three receptors are Tyro3 receptors in the clearance of apoptotic cells, and the com- missing and apoptotic cell removal is therefore hindered severely, binatorial usage by macrophages and DCs may provide clues for multiple organs are affected including brain, testes, liver, and lym- their biologic relevance. For macrophages, Mertk remains a key phoid tissue (17, 34) above that seen in mertkkd mice alone. receptor; however, Axl and Tyro3 participate to a lesser extent and In the retina, we also show that Mertk is critical for the main- appear to be important for phosphorylation of Mertk, an event tenance of the retinal tissues, whereas deletion of axl, tyro3,or thought to be necessary for rapid activation of phagocytosis. In both axl/tyro3 did not result in retinal degeneration (Fig. 5). Fur- contrast, DCs that express Mertk but do not require Mertk for the thermore, this observation is consistent with the literature that sug- phagocytosis apoptotic cells are mostly dependent on Axl and gests a mutation in mertk is one primary cause of retinal degen- Tyro3. The different engagement of this receptor family by mac- eration (28, 35). These data suggest that degeneration of the retina rophages vs DCs may be partly dictated by the GAS6 or protein S reported in the triple mutant axl/mertk/tyro3Ϫ/Ϫ mice is primarily expression profile and the level of expression of these receptors on due to the absence of Mertk (34). Currently, it is not known phagocytes. DCs express lower levels of Axl/Mertk/Tyro3 and this whether retinal epithelial cells express Axl or Tyro3; however, our may result in slower ingestion rates. Nonetheless, we suggest that data suggest that these two receptor family members are not crit- the reliance of DCs on Axl and Tyro3 for the clearance of apo- ical for normal maintenance of the retina. ptotic cells may be correlated to the induction of autoimmunity in We have previously shown that GAS6 triggers Mertk tyrosine these knockout mice. In vivo, we have also demonstrated that phosphorylation and downstream activation of Vav1, Rac1, and Mertk compensates for the lack of Axl and Tyro3 in the thymus Cdc42 (36). The complexity and, perhaps, the cell type specificity and retina, making Mertk most important for these organs in main- are emphasized by our findings regarding Mertk tyrosine phos- taining homeostasis. Lastly, the cooperation among this receptor phorylation in monocytes. We show in this report that, in macro- family may be important for efficient signaling because the differ- phages that exhibit a role for Mertk, Axl, and Tyro3, there may be ent receptors have a different ligand preference and affinity. 5642 PHAGOCYTES USE Mertk, Axl, AND Tyro3 DIFFERENTLY

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