Phagocytic removal of apoptotic cells occurs efficiently in vivo such that even in tissues with significant apoptosis, very few apoptotic cells are detectable 1 . This is thought to be due to the release of find-me signals by apoptotic cells that recruit motile phagocytes such as monocytes, macrophages, and dendritic cells, leading to the prompt clearance of the dying cells 2 . However, the identity and in vivo relevance of such find-me signals are not well understood. Here, through several lines of evidence, we identify extracellular nucleotides as a critical apoptotic cell find-me signal. We demonstrate the caspase-dependent release of ATP and UTP (in equimolar quantities) during the early stages of apoptosis by primary thymocytes and cell lines. Purified nucleotides at these concentrations were sufficient to induce monocyte recruitment comparable to apoptotic cell supernatants. Enzymatic removal of ATP and UTP (by apyrase or ectopic CD39 expression)Correspondence and requests for materials should be addressed to K.S.R. (ravi@virginia.edu). Supplementary Information is linked to the online version of the paper at www.nature.com/nature.Author Information Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing interests.Author Contributions M.R.E. designed, performed and analyzed most of the experiments in this study with input from K.S.R. F.B.C. performed ATP quantitation experiments. P.T.C. helped with in vivo thymic apoptosis experiments. E.R.L. carried out HPLC analysis of supernatants. S.F.W. generated the CD39 expression plasmid and stable Jurkat cell lines. D.P. conducted phagocytosis experiments. A.K. and N.L. carried out the MS analysis and provided critical support in establishing the air-pouch model system. R.I.W. and J.J.L. carried out immunohistochemical detection of apoptotic cells in the thymus. M.O. and P.S. assisted with the BMDM generation and macrophage chemotaxis experiments. T.K.H. provided critical intellectual input in the preparation of the manuscript. K.S.R. provided overall coordination with respect to conception, design and supervision of the study. K.S.R. and M.R.E. wrote the manuscript with comments from co-authors. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 April 8. Most developing thymocytes (95%) undergo apoptosis; yet in steady-state only 1-2% are detectable as apoptotic 4,5 . It is hypothesized that dying thymocytes secrete soluble factors that attract resident phagocytes to promote prompt clearance 2,6 . To determine if apoptotic thymocytes release such factors, cell-free supernatants after apoptosis induction (by antiFas/CD95 crosslinking) were assessed for their ability to attract THP-1 monocytesor primary human monocytes in a transwell migration assay ( Figure 1a and Supplemental Figure S2). Apoptotic supernatants caused a 3-fold increase in monocyte migration compared to supernatants of live thymocytes. Such release of chemotactic factors was also seen with Jurkat cells (...

Engulfment and subsequent degradation of apoptotic cells is an essential step that occurs throughout life in all multicellular organisms [1][2][3] . ELMO/Dock180/Rac proteins are a conserved signalling module for promoting the internalization of apoptotic cell corpses 4,5 ; ELMO and Dock180 function together as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac, and thereby regulate the phagocyte actin cytoskeleton during engulfment [4][5][6] . However, the receptor(s) upstream of the ELMO/ Dock180/Rac module are still unknown. Here we identify brainspecific angiogenesis inhibitor 1 (BAI1) as a receptor upstream of ELMO and as a receptor that can bind phosphatidylserine on apoptotic cells. BAI1 is a seven-transmembrane protein belonging to the adhesion-type G-protein-coupled receptor family, with an extended extracellular region 7-9 and no known ligands. We show that BAI1 functions as an engulfment receptor in both the recognition and subsequent internalization of apoptotic cells. Through multiple lines of investigation, we identify phosphatidylserine, a key 'eat-me' signal exposed on apoptotic cells 10-13 , as a ligand for BAI1. The thrombospondin type 1 repeats within the extracellular region of BAI1 mediate direct binding to phosphatidylserine. As with intracellular signalling, BAI1 forms a trimeric complex with ELMO and Dock180, and functional studies suggest that BAI1 cooperates with ELMO/Dock180/Rac to promote maximal engulfment of apoptotic cells. Last, decreased BAI1 expression or interference with BAI1 function inhibits the engulfment of apoptotic targets ex vivo and in vivo. Thus, BAI1 is a phosphatidylserine recognition receptor that can directly recruit a Rac-GEF complex to mediate the uptake of apoptotic cells.Previous studies revealed two 'functional' regions within ELMO1 and its Caenorhabditis elegans homologue CED-12 during phagocytosis 5,14-17 . The amino-terminal 558 amino-acid residues (N-term) were necessary for targeting of the ELMO-Dock180 complex to the membrane 14,17 , whereas the carboxy-terminal 196 residues (C-term) were necessary for binding Dock180 and for optimal Rac activation 15,16 . Because the receptor(s) upstream of ELMO1 during engulfment were not known, we performed a yeast two-hybrid screen, with N-term as bait. After screening more than 1.1 3 10 7 colonies from a mouse embryo library, followed by several subscreens for specificity, we identified a single membrane protein, BAI1.BAI1 belongs to subgroup VII of the adhesion-type G-proteincoupled receptor (GPCR) family 7-9 , with extended extracellular termini containing multiple domains and motifs that are thought to function in cell-cell or cell-matrix interactions 9 . BAI1 (1,584 residues) has an 943-residue extracellular region, a seven-transmembrane

Rapid and efficient removal of apoptotic cells by phagocytes plays a key role during development, tissue homeostasis, and in controlling immune responses1–5. An important feature of efficient clearance is the capacity of a single phagocyte to ingest multiple apoptotic cells successively, and to process the increased load of corpse-derived cellular material6–9. However, factors that influence sustained phagocytic capacity or how they in turn influence continued clearance by phagocytes are not known. Here we identify that the ability of a phagocyte to control its mitochondrial membrane potential is a critical factor in the capacity of a phagocyte to engulf apoptotic cells. Changing the phagocyte mitochondrial membrane potential (genetically or pharmacologically) significantly affected phagocytosis, with lower potential enhancing engulfment and higher membrane potential inhibiting uptake. We then identified that Ucp2, a mitochondrial membrane protein that acts to lower the mitochondrial membrane potential10–12, is upregulated in phagocytes engulfing apoptotic cells (but not synthetic targets, bacteria, or yeast). Loss of Ucp2 limited the capacity of phagocytes to continually ingest apoptotic cells, while overexpression of Ucp2 increased the capacity for engulfment and the ability to engulf multiple apoptotic cells. Mutational and pharmacological inhibition of Ucp2 uncoupling activity reversed the positive effect of Ucp2 on engulfment capacity, suggesting a direct role for Ucp2-mediated mitochondrial function in phagocytosis. Macrophages from Ucp2-deficient mice13, 14 were impaired in their capacity to engulf apoptotic cells in vitro, and Ucp2-deficient mice displayed profound in vivo defects in clearing dying cells in the thymus and the testes. Collectively, these data suggest that phagocytes alter the mitochondrial membrane potential during engulfment to regulate uptake of sequential apoptotic cells, and that Ucp2 is a key molecular determinant of this step in vivo. Since Ucp2 function has also been linked to metabolic diseases and atherosclerosis14–16, these data identifying a new role for Ucp2 in regulating apoptotic cell clearance may provide additional insights toward understanding the complex etiology and pathogenesis of these diseases.

Apoptosis and the subsequent clearance of these dying cells occur throughout development and adult life in many tissues. Failure to promptly clear apoptotic cells has been linked to many diseases1-3. ELMO1 is an evolutionarily conserved cytoplasmic engulfment protein that functions downstream of the phosphatidylserine receptor BAI1, and, along with Dock180 and Rac1, promotes internalization of the dying cells4-7. Here, we generated ELMO1-deficient mice, and unexpectedly found them to be viable and grossly normal. However, ELMO1-deficient mice had a striking testicular pathology, with disrupted seminiferous epithelium, multi-nucleated giant cells, uncleared apoptotic germ cells, and decreased sperm output. Subsequent in vitro and in vivo analyses revealed a crucial role for ELMO1 in the phagocytic clearance of apoptotic germ cells by Sertoli cells lining the seminiferous epithelium. The engulfment receptor BAI1 and the GTPase Rac (upstream and downstream of ELMO1, respectively) were also important for Sertoli cell-mediated engulfment. Collectively, these findings uncover a selective requirement for ELMO1 in Sertoli cell-mediated removal of apoptotic germ cells and make a compelling case for a relationship between engulfment and tissue homeostasis in vivo.