1. Beirne B. Carter Center for Immunology Research,
2. Center for Cell Clearance,
3. Department of Pharmacology,
4. Robert M. Berne Cardiovascular Research Center,
5. Department of Urology,
6. Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908, USA
7. Department of Medicine,
8. Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA

Correspondence to: Kodi S. Ravichandran^1,2,6 Correspondence and requests for materials should be addressed to K.S.R. (Email: ravi@virginia.edu).

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.

1. Beirne B. Carter Center for Immunology Research,
2. Center for Cell Clearance,
3. Department of Pharmacology,
4. Robert M. Berne Cardiovascular Research Center,
5. Department of Urology,
6. Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908, USA
7. Department of Medicine,
8. Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA

Correspondence to: Kodi S. Ravichandran^1,2,6 Correspondence and requests for materials should be addressed to K.S.R. (Email: ravi@virginia.edu).

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