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

Apoptosis occurs in essentially all tissues as part of normal development and homeostasis. Yet even in tissues with high cellular turnover, apoptotic cells are rarely seen; this has been attributed to the ability of apoptotic cells to advertise their presence via release of 'find-me' signals to recruit phagocytes and initiate prompt clearance. It has been unclear, however, what type of find-me signals are released by apoptotic cells and how these are sensed by phagocytes. In this paper apoptotic cells are shown to release ATP and UTP that act as a 'find me ' signal and chemoattractant for phagocytes expressing the P2Y2 ATP/UTP receptor. The efficient removal of apoptotic cells in vivo is thought to be due to the release of 'find-me' signals by apoptotic cells that recruit motile phagocytes. Here, the caspase-dependent release of ATP and UTP during the early stages of apoptosis is demonstrated. ATP and UTP are found to act as chemoattractants in a process mediated through the ATP/UTP receptor P2Y2, which is present on monocytes and macrophages. 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.