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

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 anti-Fas/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 (a mature T cell line) induced to undergo Fas- or ultraviolet (UV)-mediated apoptosis (Figure 1b). There was no detectable increase in membrane permeability or leakage of cytoplasmic markers when the supernatants were collected (Supplemental Figure S1a-e and Figure 2g). Moreover, supernatants from cells pretreated with the caspase inhibitor zVAD-fmk prior to the induction of apoptosis failed to induce monocyte migration (Figure 1a, c), suggesting caspase-dependent and regulated chemoattractant release. In a time-course, chemotactic factor release correlated with the onset and progression of apoptosis (assessed by annexin V exposure and caspase 3/7 activation, Figure 1b, Supplemental Figure S1b,c). Lastly, the chemotactic factor(s) was soluble and heat-stable, since high-speed centrifugation or boiling of the supernatants did not affect the chemotactic potential (Supplemental Figure S3f).

We next tested whether find-me signal(s) in apoptotic cell supernatants could attract phagocytesin vivo. We used a murine dorsal air-pouch model (Figure 1d) where the supernatants from apoptotic or healthy cells were injected into sterile, subcutaneous air-pouches⁷. When cells in the air-pouch were recovered by lavage after 24 hr, apoptotic cell supernatants caused a 3-fold increase in the number of CD45+ leukocytes recruited compared to live cell supernatants or medium alone (Figure 1e, n=8, p=0.02). The total number of monocytes and macrophages (CD11b+/Gr-l^low) in the lavage were increased about 3-fold compared to neutrophils (Gr-1^high cells) (Figure 1e). By contrast, bacterial lipopolysaccharide (LPS) injection induced the recruitment of mostly Gr-1^high neutrophils (Figure 1f). This is consistent with previous studies on preferential recruitment of monocyte/macrophages over inflammatory neutrophils to cells undergoing apoptosis^8,9. F4/80+ macrophages recruited to the pouch could also engulf apoptotic Jurkat cells injected into the pouch (not shown). These data revealed the release of find-me signal(s) by apoptotic lymphocytes that attract monocytes in vitro and in vivo.

We then sought to determine the nature of the chemoattractant. Based on in vitro studies, the lipid lysophosphatidylcholine (LPC) was implicated as a find-me signal released by apoptotic MCF-7 cancer cells¹⁰. However, we did not observe monocyte migration to purified LPC over a range of concentrations (0.1–100 μM, data not shown); moreover, treatment of apoptotic cell supernatants with phospholipase D (PLD), an enzyme that promotes hydrolysis of LPC (see Supplemental Figure S3a,b), did not affect chemotactic activity of supernatants from apoptotic thymocytes, Jurkat cells or MCF-7 cells (Figure 1a, j, k). CX₃CL1 (fractalkine) released by apoptotic Burkitt-lymphoma B cells can also act as a find-me signal¹¹; however, THP-1 monocytes used here failed to show migration toward purified CX₃CL1, and the anti-fractalkine depleting antibody did not block migration in our assays (data not shown). Thus, the find-me signal released by apoptotic primary thymocytes and Jurkat cells appeared to be distinct from those previously reported.

Subsequently, several lines of evidence suggested a role for extracellular nucleotides as a possible find-me signal. Treatment of apoptotic cell supernatants with recombinant apyrase, an enzyme that hydrolyzes nucleoside triphosphates and diphosphates to nucleoside monophosphates (e.g. ATP → ADP→ AMP), abolished the monocyte chemoattractant activity of apoptotic thymocytes, Jurkat and MCF-7 cells at all time points (Figure 1a, g, j, k, and Supplemental Figure S3e). Apyrase did not affect monocyte migration to chemokines CCL2 or CXCL12 (Figure 1g and data not shown). Importantly, treatment of apoptotic cell supernatants (but not CCL2) with apyrase prior to injection into the dorsal air-pouch also inhibited the attraction of leukocytes in vivo (Figure 1h, i). As another approach, we expressed in Jurkat cells the transmembrane protein CD39 (NTPDase-l), the primary mammalian ecto-apyrase responsible for NTP degradation by immune cells in vivo¹² (see Supplemental Figure S4b); CD39 expression abrogated the chemoattractant activity in the supernatants of apoptotic Jurkat (Figure 1l). Neither apyrase treatment nor CD39 overexpression impaired the induction of apoptosis (Supplemental Figure S4a and Figure 4g). Heat-inactivation of the recombinant apyrase (prior to addition to apoptotic cell supernatants) abolished its effect (Figure 1a and Supplemental Figure S3d), suggesting a need for intact enzymatic activity. Thus, induction of apoptosis led to accumulation of extracellular nucleotides, capable of monocyte chemoattraction in vitro and in vivo.

Among the four naturally occurring extracellular nucleotides (ATP, ADP, UTP and UDP) ATP and UTP induced strong chemotactic activity in THP-1 (Figure 2a); in contrast, ADP and UDP exhibited partial activity at the highest concentrations tested (Figure 2a), but lower than NTPs. The migration was also stimulated by non-hydrolyzable ATPγS, but not adenosine (Supplemental Figure S5a), suggesting attraction primarily toward triphosphate nucleotides. When ATP and UTP levels in apoptotic cell supernatants were directly quantified (see methods), higher ATP and UTP could be detected as early as 2 hr after apoptosis induction, with further increase by 4 hours (Figure 2b). The concentration of ATP and UTP at the time point when the apoptotic supernatants induced maximal monocyte migration correlated well with the concentration at which pure ATP and UTP caused maximal migration (~100 nM) (Figure 2a, b and Figure 1b). Adding pure ATP and UTP to the upper chamber of the transwell, to disrupt the gradient, blocked the migration of monocytes to the lower chamber containing apoptotic cell supernatants (Supplemental Figure S6 a-c). Although ATP can promote chemokinesis/random migration of neutrophils¹³, addition of pure ATP or UTP only to the upper chamber did not induce migration of THP-1 cells to the lower chamber, indicating that the migration induced is not chemokinesis, rather chemotaxis (Supplemental Figure S6d). Furthermore, the nucleotide release from apoptotic cells is caspase-dependent, occurs after different types of apoptosis induction (DNA damage, receptor-mediated, and steroid-induced), occurred in primary thymocytes, Jurkat and epithelial cells undergoing apoptosis, and this ATP release correlates well with the induction of apoptosis (Figure 2c-e and Supplemental Figures S1b-d and S8). The release of ATP during apoptosis was not due to leakage of cellular contents or mechanical stress during handling of cells¹⁴ (Figure 2f, g). Collectively, these data strongly suggested a role for ATP and UTP as find-me signals important for phagocyte chemoattraction by apoptotic cells.

We then addressed how the phagocytes may ‘sense’ the extracellular nucleotides as a find-me cue. Leukocyte migration toward nucleotides has been shown to be dependent on members of the P2Y family of G protein-coupled receptors^15,16. We tested the role of P2Y receptors on monocytes and macrophages in migration toward apoptotic cell supernatants. First, pretreatment of THP-l cells with suramin, a nonselective inhibitor of P2 family members, showed dose-dependent inhibition of migration to apoptotic supernatants, but not CCL2 (Figure 3a and Supplemental Figure S5c). After evaluating several P2Y family members through known P2Y antagonists to inhibit migration toward apoptotic cell supernatants, we focused on P2Y₂ based on its known affinities for ATP and UTP (since both are released by apoptotic cells), and P2Y₂ expression on monocytes and macrophages¹⁶. Although P2Y₄ also fit the profile for ATP and UTP binding, THP-1 cells express no detectable P2Y₄, yet strongly express P2Y₂ mRNA (ref. ¹⁷ and data not shown). siRNA-mediated knockdown of P2Y₂ in THP-1 monocytes led to a 60% decrease in P2Y₂mRNA, and also partially inhibited migration to apoptotic cell supernatants (10.5% ± 0.8 versus 16.3% ± 2.0 for control siRNA, n=6, p=0.003), but did not affect migration to CCL2 or CXCL12 (Figure 3b and data not shown). Bone marrow derived macrophages (BMDM) from P2Y₂^−/− mice³ showed impaired migration to apoptotic supernatants, but their migration to CXCL12 was intact (Figure 3c). Importantly, when apoptotic cell supernatants were injected into the air-pouch of P2Y₂-deficientmic e, there was a strong reduction in the recruitment of monocytes and macrophages to the pouch, indicating in vivo relevance of this receptor in sensing the find-me signal (Figure 3d). Although P2Y₆ was shown to play a role in UDP-dependent leukocyte migration^15,18 and phagocytosis by microglial cells¹⁹, neither RNAi toward P2Y_6, nor the P2Y₆ antagonist MRS2578 showed specific inhibition of migration (Supplemental Figure S5b and data not shown) Finally, addition of antagonists to the adenosine receptors A1, A_2aand A3 ^13,20, or the A2 receptor agonistCGS21860 did not significantly affect the migration of monocytes to the apoptotic cell supernatants (Figure 3e and Supplemental Figure S5d). Moreover, adenosine itself did not induce migration of THP-1 cells and addition of exogenous adenosine did not affect migration to apoptotic cell supernatants (Supplemental Figure S5a, d). Together, these results identify the P2Y₂ receptor on monocytes and macrophages as a critical sensor of the find-me signal released by apoptotic cells.

To further test the importance of nucleotides as a find-me signal in an in vivo model of apoptosis, we used intraperitoneal (i.p.) injection of dexamethasone (Dex), where a large fraction of immature thymocytes undergo relatively synchronous apoptosis and phagocytic clearance^5,21. Dex injection induced thymic apoptosis, with decline in thymus size and cellularity within 4 hrs, and by 8 hrs was less than half that of control-treated mice (Figure 4a). Treatment of thymocytes with Dex in vitro also induces apoptosis in a large fraction of the cells by 4 hrs (>40%) and 6 hrs (>60%) (Figure 4g). We asked whether apyrase-mediated destruction of nucleotides in vivo could influence Dex-mediated thymocyte apoptosis and clearance. Injection of apyrase significantly reversed the decline in thymus size and cellularity due to Dex treatment (especially at 6 hrs) (Figure 4a). This was not due to an effect of apyrase on the apoptotic process itself, as the fraction of cells undergoing apoptosis due to Dex was unchanged upon apyrase treatment in vivo or in vitro (Figure 4e,g). Interestingly, the total number of apoptotic cells remaining at 6 hr and 8 hr were increased in mice treated with apyrase+Dex, compared to Dex alone (Figure 4b). Since apyrase had no effect on the phagocytic capacity of macrophages (Supplemental Figure S9), apyrase-mediated destruction of the nucleotide find-me signal appeared to affect phagocyte recruitment, and in turn delay clearance.

In a complementary set of studies, we tested how disrupting the‘ sensing’ of the find-me signal affects apoptotic cell clearance. Injection of the P2Y inhibitor suramin prior to Dex reversed the diminution of the thymic cellularity and organ size seen with Dex alone (Figure 4c, d). The total number of apoptotic thymocytes also increased in suramin+Dex conditions (Figure 4d). As was the case with apyrase, suramin itself did not alter the induction of thymocyte apoptosis (Figure 4f, g). When we assessed the presence of apoptotic cells in the native thymic architecture (by immunohistochemistry using anti-ssDNA antibody), there were increased numbers of uncleared apoptotic cells in Dex+suramin treated conditions compared to Dex alone (Supplemental Figure S10). We also assessed whether genetic disruption of the putative find-me signal receptor P2Y₂would affect apoptotic cell clearance in vivo. Following Dex injection, the number of apoptotic thymocytes in the thymi of P2Y₂^−/− mice was significantly increased compared to control mice (Figure 4h). Taken together, the disruption of a find-me signal circuit at the level of nucleotides or the sensing receptor significantly impairs the clearance of apoptotic thymocytes, without an apparent effect on the induction of apoptosis or engulfment.

The data presented in this report provide new insights on particular aspects of the programmed cell death process. First, this work identifies ATP and UTP as critical and non-redundant find-me signal released by apoptotic cells. To our knowledge, this is the first documentation of regulated and caspase-dependent release of nucleotides from apoptotic cells, with a functional secondary consequence. Since nucleotide release is seen in primary cells and cell lines (after different types of apoptotic stimuli), nucleotides may be a broadly used find-me signal. However, these data do not rule out other chemotactic factors that work alone or together with nucleotides. Second, these data establish a clear relationship between a find-me signal and efficient apoptotic cell clearance in vivo; the disruption of the ‘find-me signal’ circuit at the level of ATP/UTP or the receptors (P2Y) impaired the clearance of apoptotic thymocytes. Although we focused here on motile monocytes/macrophages, genetic studies in C. elegans, where healthy cells engulf the dying neighbors, have revealed a link between apoptosis and engulfment ^22,23. How nucleotides might regulate engulfment by neighboring non-professional phagocytes^6,24 remains to be determined.

Extracellular nucleotides at higher concentrations are considered pro-inflammatory²⁵ (>1 μM, e.g. necrotic cells, Supplemental Figure S7), but nucleotides can also induce an anti-inflammatory response²⁶. Besides serving as a find-me signal, whether nucleotides participate in anti-inflammatory signaling during engulfment remains to be determined. Bournazou et al recently showed that lactoferrin released by apoptotic cells inhibit neutrophil migration²⁷. How nucleotides and lactoferrin concurrently promote monocyte migration while inhibiting neutrophil migration remains to be seen. Since failure to promptly clear dying cells can lead to autoimmunity and chronic inflammatory diseases⁹, phagocyte chemoattraction to apoptotic cells via nucleotides may have implications for human disease states related to failed clearance.