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. 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. 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 P2Y(2) as a critical sensor of nucleotides released by apoptotic cells using RNA interference-mediated depletion studies in monocytes, and macrophages from P2Y(2)-null mice. 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 P2Y(2)(-/-) (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 P2Y(2)(-/-) mice). These results identify nucleotides as a critical find-me cue released by apoptotic cells to promote P2Y(2)-dependent recruitment of phagocytes, and provide evidence for a clear relationship between a find-me signal and efficient corpse clearance in vivo.

Figure 1. Chemotactic factor released by apoptotic cells attracts monocytes in vitro and in vivo

a, Migration of THP-1 monocytes through transwell(5 μm pore) to supernatants from control (‘live’) or Fas-induced apoptotic murine thymocytes, thymocytes pre-treated with caspase inhibitor (zVAD-fmk), apoptotic cell supernatants with apyrase, heat-inactivated apyrase or phospholipase D. The fraction of input monocytes that migrated to the lower chamber is shown. b, Attraction of monocytesby Jurkat T cell supernatants collected at the indicated times after apotosis induction via UV or anti-Fas for the indicated times. c, Monocyte attraction was inhibited by pre-treatment of Jurkat cells with zVAD-fmk prior to UV or anti-Fas treatment. d, Schematic for testing recruitment of leukocytes by apoptotic cell supernatants in the mouse air-pouch model. e, Recruitment to the air-pouch of macrophages and monocytes (CD11b+/Gr-1^low) or neutrophils (CD11b+/Gr-1^high) 24 hrs after injection of apoptotic cell supernatants. Eight mice per group, *p=0.02. f, Monocyte/macrophage and neutrophil populations recruited to the air-pouch 24 hrs after injection of LPS (1 μg) or apoptotic supernatants. Results are the average of six (LPS) and nine (apoptotic supernatant) mice. g, h, and i, Treatment of apoptotic cell supernatants with apyrase inhibits attraction of monocytes in vitro (g) or in the air-pouch model in vivo (h, i), but does not affect monocyte migration to the chemokine CCL2 (250 ng). Five (h) or three (i) mice per group, *p=0.005. j, k, Migration of monocytes to supernatants from apoptotic Jurkat or MCF-7/caspase-3 cells, supernatants being treated with apyrase or PLD. l, (right) CD39 surface expression on transfected Jurkat cells, and (left) monocyte migration to supernatants from CD39-overexpressing cells after UV treatment. Error bars indicate s.e.m.

Figure 2. Regulated release of ATP and UTP as chemoattractants by apoptotic cells

a, Migration of THP-1 monocytes to purified nucleotides at the indicated concentrations. b, Quantitation of ATP and UTP in supernatants of control and apoptotic Jurkat cells at 2 and 4hr after apoptosis induction. c, ATP level in supernatants of apoptotic Jurkat cells at indicated times and the inhibition by zVAD-fmk. d, ATP levels in supernatants of control or anti-Fas treated thymocytes for the indicated times. e, ATP levels in supernatants of thymocytes treated with zVAD-fmk before anti-Fas treatment. f, Left, Schematic of supernatant collection without disturbing the cells. Right, Quantitation of ATP that has diffused through the 0.4 μm filter into the medium from untreated (live) or anti-Fas treated Jurkat cells. g, Integrity of the cell membrane is retained when apoptotic cell supernatants are collected, as determined by ATP release but not leakage of cytoplasmic protease activity. Error bars indicate s.e.m.

Figure 3. P2Y₂ receptor on monocytes and macrophages as a sensor of ATP/UTP released by apoptotic cells

a, Effect of pretreatment of monocytes with P2Y receptor antagonist suramin (100 μM) on migration to apoptotic cell supernatants or the chemokine CCL2 (50 ng/mL). n=3, *p=0.003. b, Migration of THP-1 transfected with siRNA specific for P2Y₂ receptoror control siRNA. n=6, *p=0.03. Right, qPCR and agarose gel electrophoresis (inset, inverted image) analysis of P2Y₂ receptor mRNA levels in siRNA transfected THP -1 cells. c, BMDM from P2Y₂^+/+ or P2Y₂^−/− mice were assessed for transwell migration to apoptotic Jurkat supernatant or CXCL12 (50ng/mL). d, Recruitment of CD45+ cells (left) and CD11b+/Gr-1^low monocytes and macrophages (right) to the air-pouch of mice with the indicated P2Y₂genotypes 24hr after injection of apoptotic Jurkat supernatants. Five (P2Y₂^+/+) and seven (P2Y₂^−/−) mice per group, *p≤0.03. e, THP-1 cells pretreated with antagonists targeting adenosine receptors A1, A2a and A3, apyrase or suramin prior to migration assay. Error bars indicate s.e.m.

Figure 4. Interference with the nucleotide find-me signal or its sensing impairs clearance of apoptotic cells in the thymus

a, b, C57BL/6 mice (4–6 wk) were injected i.p. with 250μg dexamethasone (Dex) for the indicated times, with or without apyrase, and the cellularity (a) or number of apoptotic cells per thymus (b) was determined (annexin V positive/propidium iodide-negative populations). Data in a were normalized to untreated animals within the same experimental group (4, 6 or 8hr). Representative thymus from each group is shown below. Data shown are representative of 2–4 experiments per time point using at least three mice/group, *p=0.03. c, d, Same as in a, b, except mice were injected with 6mg of suramin 1hr prior to Dex injection (6hr). Representative thymus from each group is shown below c. Four mice per group, *p=0.03. e, f, Effect of apyrase or suramin on percentage of apoptotic cells in vivo in the thymi of Dex treated mice (from a-d above). g, Apyrase (0.05U/mL) and suramin (100μM) do not affect Dex-induced thymocyte apoptosis in vitro. zVAD was included as a control. Percentage of apoptotic cells is shown. h, Left, Paraffin sections from thymi of wild-type mice and P2Y₂^−/− mice treated with Dex for 6hr (Apostain, brown. Hematoxylin, blue). Right, mean number of Apostain positive nuclei per field from 10–16 random fields per section per mouse, *p=0.001. Error bars indicate s.e.m.