Enhanced efferocytosis of aged apoptotic neutrophils is dependent on signaling through macrophage G2A and results in multiple ingestions. A, UV-irradiated apoptotic or aged apoptotic neutrophils were co-cultured with RPMΦ from C57BL/6 mice, and the phagocytic index was determined after 1 h. n = 4; *, p < 0.05 compared with UV-irradiated cells. B, shown are representative images of cytospins from A. Single ingestions are identified by red arrows, and multiple ingestions are identified by green arrows. C, single and multiple (two or more targets/MΦ) ingestions were quantitated for RPMΦ co-cultured with the indicated apoptotic neutrophils generated in the absence or presence of 6.6 μm DPI. n = 3; *, p < 0.05 for multiple ingestions compared with UV-irradiated cells; #, p < 0.05 compared with aged cells. D, WT RPMΦ were pretreated for 30 min with 5 μg/ml normal goat IgG (isotype control) or anti-G2A blocking antibody and then co-cultured with apoptotic neutrophil targets as indicated, and phagocytic indices were determined. n = 5; *, p < 0.05 compared with UV-irradiated cells; #, p < 0.05 compared with isotype control in the same group. E, RPMΦ from WT or G2A^−/− mice were co-cultured with the indicated apoptotic neutrophil targets, and phagocytic indices were determined. n = 5; *, p < 0.05 compared with UV-irradiated cells; #, p < 0.05 compared with WT RPMΦ fed aged apoptotic neutrophils. F, WT or G2A^−/− mice were injected intraperitoneally with 1 mg of zymosan. Differentials and cell counts were determined from cytospins at the indicated times. Apoptotic neutrophils were determined microscopically by nuclear morphology. n = 4; *, p < 0.05 compared with C57BL/6 in the same treatment group; #, p < 0.05 compared with the 18-h C57BL/6 control. PMN, polymorphonuclear neutrophils; Apo, apoptotic; Mac, macrophages. Data represent mean ± S.E.

Exogenous addition of lyso-PS, but not other activators of G2A, enhances efferocytosis in G2A-dependent manner. WT RPMΦ (A–F) were co-cultured with UV-irradiated apoptotic neutrophils in the absence or presence of added lipids as follows, and phagocytic indices were determined after 1 h. A, liposomes containing carrier lipid (PC) or the indicated dose (mol %) of lyso-PS. n = 3; *, p < 0.05 compared with PC control. B, liposomes containing 30 mol % lyso-PS where indicated. Multiple ingestions were quantitated as in Fig. 1C. n = 3–4; *, p < 0.05 compared with UV-irradiated cells. C, PBS (Ctr) or liposomes of 30 mol % lyso-PS, lyso-PC, or PC carrier lipid alone. D, 30 nmol of lyso-PS or lyso-PC delivered in liposomes or on 0.05% albumin as indicated. E, lyso-PS liposomes (30 mol %) or the indicated concentration of 9-HODE, 13-HODE, or 12-HETE delivered on 0.05% albumin. F, lyso-PS liposomes (30 mol %) following pretreatment with isotype control or anti-G2A as in Fig. 1C. *, p < 0.05 compared with UV-irradiated cells; #, p < 0.05 compared with isotype control in the same treatment group. G, RPMΦ from WT and G2A^−/− mice were co-cultured with UV-irradiated apoptotic neutrophils in the presence of lyso-PS liposomes (30 mol %) as indicated. n = 3–5; *, p < 0.05 compared with UV-irradiated cells; #, p < 0.05 compared with C57BL/6 in the same treatment group. H, in vivo phagocytosis by F4/80-positive macrophages was determined by flow cytometry 1 h following intraperitoneal injection of fluorescent carboxylate-modified beads (see “Experimental Procedures”). Shown is the average geometric mean of F4/80-positive macrophages associated with beads. n = 4; *, p < 0.05 compared with WT beads alone; #, p < 0.05 compared with WT following the same treatment. Ctr, control; PI, phagocytic index. Data represent mean ± S.E.

Signaling through G2A enhances efferocytosis via cAMP production and PKA-dependent enhancement of Rac1 activity. A, phagocytic indices were determined as in Fig. 1 using RPMΦ co-cultured with UV-irradiated apoptotic neutrophils in the absence or presence of the indicated concentrations of db-cAMP. n = 4; *, p < 0.05 compared with 0 nm db-cAMP. B, intracellular cAMP was measured at the indicated times in RPMΦ following co-culture with carboxylate-modified beads in the absence or presence of lyso-PS liposomes (30 mol %). Data are displayed as -fold over control. n = 4; *, p = 0.035 compared with beads alone at 4 h. C and D, phagocytic indices were determined for RPMΦ pretreated for 30 min in the absence or presence of 10 μm SQ22563 to inhibit adenylyl cyclase or 10 μm H89 or 2 μm KT5720 to inhibit PKA and then co-cultured with apoptotic cells and lyso-PS liposomes as indicated. n = 3–5; *, p < 0.05 compared with UV-irradiated cells; #, p < 0.05 compared with untreated control for each group, respectively. E, C57BL/6 or G2A^−/− RPMΦ were pretreated with the indicated concentrations of either the Epac agonist or PKA agonist for 15 min and then co-cultured with UV-irradiated cell targets, and phagocytic indices were determined as above. n = 4; *, p < 0.05 compared with respective UV-irradiated cells; #, p < 0.05 compared with WT ingesting UV-irradiated cells + lyso-PS. F, Rac1 activity was determined as in Fig. 4 in RPMΦ that had been pretreated for 30 min in the absence or presence of 2 μm KT5720 prior to addition of UV-irradiated apoptotic cells and lyso-PS or PC (carrier) liposomes. n = 3; *, p < 0.05 compared with macrophages alone; **, p < 0.05 compared with UV-irradiated cells alone; #, p < 0.05 compared with UV-irradiated cells + lyso-PS. Ctr, control. Data represent mean ± S.E.

Enhanced efferocytosis by G2A requires activation of cPLA₂α. A, RPMΦ were pretreated for 30 min in the absence or presence of pyrrolidine (Pyr; 2 μm), a cPLA₂α inhibitor, or bromoenol lactone (BEL; 0.5 μm), a calcium-independent PLA₂ inhibitor, and then co-cultured with lyso-PS liposomes and UV-irradiated apoptotic cells, and phagocytic indices were determined as before. n = 3; *, p < 0.05 compared with UV-irradiated cells; #, p < 0.05 compared with UV-irradiated cells + lyso-PS. B, phagocytic indices were determined in RPMΦ from BALB/c (WT) or cPLA₂α^−/− mice following the addition of UV-irradiated apoptotic cells in the absence or presence of lyso-PS liposomes or 1 nm db-cAMP. n = 3; *, p < 0.05 compared with its own genotype treated with UV-irradiated cells. C, RPMΦ were treated for 30 min in the absence or presence of COX inhibitor indomethacin (Indo; 20 μm) or NS-398 (10 μm) and then co-cultured with either UV-irradiated apoptotic cells with and without lyso-PS liposomes or aged apoptotic neutrophils, and phagocytic indices were determined. n = 3; **, p < 0.05 compared UV-irradiated cells; #, p < 0.05 compared with UV-irradiated cells + lyso-PS; *, p < 0.001 compared with aged cells. D, COX1 and COX2 expression was determined by Western immunoblot analysis in whole cell lysates of RPMΦ left untreated or treated with UV-irradiated apoptotic cells in the absence or presence of 30 mol % lyso-PS liposomes for the indicated times. Pos, positive control for COX2 expression; C, control. Data are representative of three independent experiments. Ctr, control. Data represent mean ± S.E.

G2A activation in inflammatory macrophages enhances phagocytosis via the same proposed pathway. A, thioglycollate-elicited ΜΦ (TGMΦ) were pretreated with either lyso-PS liposomes or the indicated dose of PGE₂ for 15 min and then co-cultured with UV-irradiated apoptotic cells, and phagocytic indices were determined. n = 3; *, p < 0.05 compared with UV-irradiated cells. B, thioglycollate-elicited macrophages were pretreated with a 10 μm concentration of either the EP4 (GW627368X) or EP2 (AH6809) receptor antagonist or 2 μm KT5720 for 30 min and then co-cultured with UV-irradiated apoptotic cells in the absence or presence of lyso-PS liposomes, and phagocytic indices were determined as above. n = 3; *, p < 0.05 compared with UV-irradiated cells; #, p < 0.05 compared with UV-irradiated cells + lyso-PS. Ctr, control. Data represent mean ± S.E.

Lyso-PS, but not other activators of G2A, is generated in NADPH oxidase-dependent manner in neutrophils induced to undergo apoptosis by overnight culture. Lyso-PC was measured by LC/MS/MS in neutrophils undergoing apoptosis by aging in the absence or presence of 6.6 μm DPI and expressed as -fold change from freshly isolated neutrophils (viable control) of the same donor. Lyso-PS was measured by LC/MS/MS and quantitated in neutrophils either freshly isolated, undergoing apoptosis by UV irradiation or aging, or activated by phorbol 12-myristate 13-acetate (PMA) (20 ng/ml; 30 min) in the absence or presence of 6.6 μm DPI to inhibit the NADPH oxidase. Results are presented as -fold increase over viable control from the same donor (n = 5–10; **, p < 0.001; *, p = 0.013 by paired t test). PL, phospholipid; Ctr, control; Cond., condition. Data represent mean ± S.E.

Signaling through G2A augments Rac1 activity. A, Rac1 activity by G-LISA^TM was determined in C57BL/6 RPMΦ treated for the indicated times with UV-irradiated apoptotic neutrophils (UV cells) alone, UV-irradiated cells plus lyso-PS liposomes (30 mol %), or lyso-PS liposomes alone. Data are represented as -fold over control. n = 4; *, p = 0.002 compared with UV-irradiated cells at time 0; **, p = 0.03 compared with UV-irradiated cells alone at 20 min. B, Rac1 activity was determined in C57BL/6 or G2A^−/− RPMΦ after 20 min of co-culture in the absence or presence of UV-irradiated apoptotic neutrophils with or without lyso-PS liposomes. n = 3; *, p < 0.05 compared with MΦ only for each genotype, respectively; **, p < 0.05 compared with its own genotype treated with UV-irradiated cells; #, p < 0.05 compared with C57BL/6 in the same treatment group. ctr, control. Data represent mean ± S.E.

Signaling through G2A augments PGE₂ production. A, RPMΦ were pretreated in the absence or presence of isotype control or anti-G2A blocking antibody for 30 min and then co-cultured with aged apoptotic neutrophils. PGE₂ production was measured in supernatants at the indicated times by LC/MS/MS (see “Experimental Procedures”). n = 4; *, p < 0.05 compared with aged polymorphonuclear neutrophils (PMN). B, C57BL/6 or G2A^−/− RPMΦ were co-cultured with UV-irradiated apoptotic cells in the absence or presence of lyso-PS liposomes, and PGE₂ production was measured in supernatants after 1 h. Data are presented as -fold over WT MΦ control. n = 3; *, p < 0.05 compared with UV-irradiated cells; #, p < 0.05 compared with WT MΦ treated with UV-irradiated cells + lyso-PS. C, RPMΦ were co-cultured with carboxylate-modified beads in the absence or presence of lyso-PS liposomes, and PGE₂ production was measured in the supernatants at the indicated times. n = 3; *, p < 0.05 compared with beads alone. D and E, phagocytic indices of RPMΦ were determined as in Fig. 1 with additional conditions. D, C57BL/6 RPMΦ were treated with lyso-PS or the indicated doses of PGE₂ 15 min before addition of UV-irradiated apoptotic cells. n = 3–4; *, p < 0.05 compared with UV-irradiated cells. E, RPMΦ from C57BL/6 or G2A^−/− mice were treated with the indicated concentrations of PGE₂ or lyso-PS liposomes as described in D, and phagocytic indices were determined as above. n = 3; *, p < 0.05 compared UV-irradiated cells; #, p < 0.05 compared with WT MΦ ingesting UV-irradiated cells + lyso-PS. F, expression of EP2 and EP4 was investigated by Western immunoblot analysis in RPMΦ. Pos, positive control; MΦ, RPMΦ whole cell lysates; Tubulin, loading control for MΦ. Data are representative of three independent experiments. G, RPMΦ were pretreated for 30 min in the absence or presence of either GW627368X (GW; 10 μm), an EP4 antagonist, or AH6809 (AH; 10 μm), an EP2 antagonist, and then co-cultured with UV-irradiated apoptotic cells with and without lyso-PS liposomes and PGE₂ as indicated. n = 3; *, p < 0.05 compared with UV-irradiated cells; #, p < 0.05 compared with untreated UV-irradiated cells + lyso-PS or UV-irradiated cells + PGE₂ for each group, respectively. Ctr, control. Data represent mean ± S.E.

Proposed signaling pathway for G2A-mediated enhancement of apoptotic cell engulfment. PGES, prostaglandin-E synthase; AC, adenylyl cyclase.