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

Abstract

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 (...

Molecular mechanisms of ATP secretion during immunogenic cell death

“…45 The administration of cumate to tPANX1-transfected, but not to control, MCA205 cells rendered them permeable to the small fluorescent dye YO-PRO-1, but not to the vital dye DAPI (Figure 3c), in line with the previously reported capacity of tPANX1 to promote a selective permeabilization of the plasma membrane. [32][33][34] The expression of tPANX1 drove a YO-PRO-1 influx coupled to an ATP efflux that could be inhibited in a dose-dependent manner by monensin and DIDS but not by Y-27632, blebbistatin and Z-VAD-fmk (Figures 3d and e), confirming the specificity of this panel of inhibitors.…”

“…[32][33][34] Surprisingly, however, the Figure 7 Lysosomes and autophago(lyso)somes participate in the trafficking and/or in the maintenance of the intracellular ATP pool. (a-c) Human osteosarcoma U2OS cells were transfected with a non-targeting siRNA (siUNR) or with the indicated siRNAs for 48 h, and then maintained in control conditions (Co) or treated with 4 mM mitoxantrone (MTX) or 300 mM oxaliplatin (OXA) for additional 18 h. Finally, cells were processed for the fluorescence microscopy-assisted visualization of nuclei (with Hoechst 33342, blue fluorescence), ATP-containing vesicles (with quinacrine, green fluorescence) or LAMP1 (revealed with an secondary antibody emitting in red) (a and b), and ATP levels in culture supernatants assessed by a luciferase-based test (c).…”

“…31 In line with this notion, the pharmacological inhibition of caspases, the knockout of Panx1, the depletion of PANX1 as well as the substitution of PANX1 by a non-cleavable variant all abolish apoptosis-associated ATP release. [32][33][34] Moreover, caspase activation and PANX1 expression are both required for the partial, apoptotic increase in the permeability of the plasma membrane to small solutes that precedes the complete, post-apoptotic (necrotic) plasma membrane breakdown. [32][33][34] In a cell death-unrelated setting, astrocytes have been reported to secrete ATP by lysosomal exocytosis, a process in which lysosomes fuse with the plasma membrane to release their luminal content into the extracellular space.…”

“…[32][33][34] Moreover, caspase activation and PANX1 expression are both required for the partial, apoptotic increase in the permeability of the plasma membrane to small solutes that precedes the complete, post-apoptotic (necrotic) plasma membrane breakdown. [32][33][34] In a cell death-unrelated setting, astrocytes have been reported to secrete ATP by lysosomal exocytosis, a process in which lysosomes fuse with the plasma membrane to release their luminal content into the extracellular space. 35 Driven by these premises and incognita, we decided to characterize the mechanisms through which cancer cells extrude ATP in response to chemotherapeutic agents.…”

Adenosine Production by Biomaterial‐Supported Mesenchymal Stromal Cells Reduces the Innate Inflammatory Response in Myocardial Ischemia/Reperfusion Injury

“…This cycle of cell recruitment is often self‐amplified and sustained in MI/R and leads to further cell necrosis, scar formation, and cardiac dysfunction 6, 7, 8, 9, 10, 11. Adenine nucleotides such as ATP and AMP are potent danger‐associated molecular patterns that trigger chemotactic migration, vessel wall adhesion, and pro‐inflammatory cytokine production by early infiltrating leukocytes such as neutrophils and macrophages 12, 13, 14, 15. However, these adenine nucleotides can be dephosphorylated to adenosine.…”