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Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance

Nature volume 461, pages 282–286 (2009)Cite this article

Abstract

Phagocytic removal of apoptotic cells occurs efficiently in vivo such that even in tissues with significant apoptosis, very few apoptotic cells are detectable1. 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 cells2. 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 that of apoptotic cell supernatants. Enzymatic removal of ATP and UTP (by apyrase or the expression of ectopic CD39) abrogated the ability of apoptotic cell supernatants to recruit monocytes in vitro and in vivo. We then identified the ATP/UTP receptor P2Y2 as a critical sensor of nucleotides released by apoptotic cells using RNA interference-mediated depletion studies in monocytes, and macrophages from P2Y2-null mice3. The relevance of nucleotides in apoptotic cell clearance in vivo was revealed by two approaches. First, in a murine air-pouch model, apoptotic cell supernatants induced a threefold greater recruitment of monocytes and macrophages than supernatants from healthy cells did; this recruitment was abolished by depletion of nucleotides and was significantly decreased in P2Y2-/- (also known as P2ry2-/-) mice. Second, clearance of apoptotic thymocytes was significantly impaired by either depletion of nucleotides or interference with P2Y receptor function (by pharmacological inhibition or in P2Y2-/- mice). These results identify nucleotides as a critical find-me cue released by apoptotic cells to promote P2Y2-dependent recruitment of phagocytes, and provide evidence for a clear relationship between a find-me signal and efficient corpse clearance in vivo.

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Figure 1: Chemotactic factor released by apoptotic cells attracts monocytes in vitro and in vivo.
Figure 2: Regulated release of ATP and UTP as chemoattractants by apoptotic cells.
Figure 3: P2Y 2 receptor on monocytes and macrophages as a sensor of ATP and UTP released by apoptotic cells.
Figure 4: Interference with the nucleotide find-me signal or its sensing impairs the clearance of apoptotic cells in the thymus.

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Acknowledgements

We thank K. Rock, C. Borowski and members of the Ravichandran laboratory for helpful suggestions; I. Juncadella for lung epithelial cells; K. Lauber and S. Wesselborg for providing MCF-7/caspase-3 cells; and R. Tacke for assistance with primary monocyte experiments. This work was supported by Public Health Service grants from the National Institutes of Health (to K.S.R. and N.L.), the American Cancer Society (to M.R.E.) and the University of Virginia Farrow Fellowship (to M.R.E.).

Author Contributions M.R.E. designed, performed and analysed most of the experiments in this study, with input from K.S.R. ATP quantification experiments were performed by F.B.C., and P.T.C. assisted with in vivo thymic apoptosis experiments. E.R.L. performed the high-performance liquid chromatography 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. performed the mass spectrometry analysis and provided critical support in establishing the air-pouch model system. R.I.W. and J.J.L. conducted 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.

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Authors and Affiliations

  1. Beirne B. Carter Center for Immunology Research,,

    Michael R. Elliott, Faraaz B. Chekeni, Paul C. Trampont, Scott F. Walk, Daeho Park & Kodi S. Ravichandran

  2. Center for Cell Clearance,,

    Michael R. Elliott, Paul C. Trampont, Scott F. Walk, Daeho Park & Kodi S. Ravichandran

  3. Department of Pharmacology,,

    Faraaz B. Chekeni & Norbert Leitinger

  4. Robert M. Berne Cardiovascular Research Center,,

    Alexandra Kadl, Marina Ostankovich, Poonam Sharma & Norbert Leitinger

  5. Department of Urology,,

    Robin I. Woodson & Jeffrey J. Lysiak

  6. Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908, USA,

    Kodi S. Ravichandran

  7. Department of Medicine,,

    Eduardo R. Lazarowski

  8. Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA,

    T. Kendall Harden

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Correspondence to Kodi S. Ravichandran.

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Elliott, M., Chekeni, F., Trampont, P. et al. Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance. Nature 461, 282–286 (2009). https://doi.org/10.1038/nature08296

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