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1.
Figure 2

Figure 2. Regulated release of ATP and UTP as chemoattractants by apoptotic cells. From: Nucleotides released by apoptotic cells act as a find-me signal for phagocytic clearance.

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.

Michael R. Elliott, et al. Nature. ;461(7261):282-286.
2.
Figure 3

Figure 3. P2Y2 receptor on monocytes and macrophages as a sensor of ATP/UTP released by apoptotic cells. From: Nucleotides released by apoptotic cells act as a find-me signal for phagocytic clearance.

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-1low 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.

Michael R. Elliott, et al. Nature. ;461(7261):282-286.
3.
Figure 4

Figure 4. Interference with the nucleotide find-me signal or its sensing impairs clearance of apoptotic cells in the thymus. From: Nucleotides released by apoptotic cells act as a find-me signal for phagocytic clearance.

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.

Michael R. Elliott, et al. Nature. ;461(7261):282-286.
4.
Figure 1

Figure 1. Chemotactic factor released by apoptotic cells attracts monocytes in vitro and in vivo. From: Nucleotides released by apoptotic cells act as a find-me signal for phagocytic clearance.

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-1low) or neutrophils (CD11b+/Gr-1high) 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.

Michael R. Elliott, et al. Nature. ;461(7261):282-286.

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