Two-step engulfment of apoptotic cells - PubMed (original) (raw)

Two-step engulfment of apoptotic cells

Satoshi Toda et al. Mol Cell Biol. 2012 Jan.

Abstract

Apoptotic cells expose phosphatidylserine on their surface as an "eat me" signal, and macrophages respond by engulfing them. Although several molecules that specifically bind phosphatidylserine have been identified, the molecular mechanism that triggers engulfment remains elusive. Here, using a mouse pro-B cell line, Ba/F3, that grows in suspension, we reconstituted the engulfment of apoptotic cells. The parental Ba/F3 cells did not engulf apoptotic cells. Ba/F3 transformants expressing T cell immunoglobulin- and mucin-domain-containing molecule 4 (Tim4), a type I membrane protein that specifically binds phosphatidylserine, efficiently bound apoptotic cells in a phosphatidylserine-dependent manner but did not engulf them. However, Ba/F3 transformants expressing both Tim4 and the integrin α(v)β(3) complex bound to and engulfed apoptotic cells in the presence of milk fat globule epidermal growth factor factor VIII (MFG-E8), a secreted protein that can bind phosphatidylserine and integrin α(v)β(3). These results indicate that the engulfment of apoptotic cells proceeds in two steps: Tim4 tethers apoptotic cells, and the integrin α(v)β(3) complex mediates engulfment in coordination with MFG-E8. A similar two-step engulfment of apoptotic cells was observed with mouse resident peritoneal macrophages. Furthermore, the Tim4/integrin-mediated engulfment by the Ba/F3 cells was enhanced in cells expressing Rac1 and Rab5, suggesting that this system well reproduces the engulfment of apoptotic cells by macrophages.

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Figures

Fig 1

Fig 1

Effect of Tim4 on the engulfment of apoptotic cells by NIH 3T3 cells. (A) Expression of Tim4 in mouse NIH 3T3 cells. NIH 3T3 cells and transformants expressing mouse Tim4 were stained with a biotinylated anti-Tim4 MAb, followed by APC-conjugated streptavidin. The staining profile was analyzed by flow cytometry using a FACSAria apparatus. (B and C) Tim4-dependent engulfment of apoptotic cells. (B) The pHrodo-labeled apoptotic thymocytes were incubated for 90 min with NIH 3T3 cells or NIH 3T3 transformants expressing Tim4 and analyzed by flow cytometry. (C) The engulfment of pHrodo-labeled apoptotic cells was performed in a Lab-Tek chamber and observed by fluorescence microscopy.

Fig 2

Fig 2

Effect of Tim4 on the engulfment of apoptotic cells by Ba/F3 cells. (A) Expression of Tim4 in mouse Ba/F3 cells. The Ba/F3 cells were transformed with an expression vector for GFP and Rac1 (BaF/GFP/Rac) or with a vector for GFP, Rac1, and Tim4 (BaF/GFP/Rac/Tim4). The stable transformants were stained with a biotinylated anti-Tim4 MAb, followed by PE-conjugated streptavidin. (B and C) The BaF/GFP/Rac and BaF/GFP/Rac/Tim4 cells were incubated with pHrodo-labeled apoptotic thymocytes at 37°C for 120 min and subjected to flow cytometry. (B) The pHrodo profile of the GFP-positive population is shown. The numbers indicate the percentage of pHrodo-positive cells in the GFP-positive population. (C) The BaF/GFP/Rac and BaF/GFP/Rac/Tim4 cells were incubated with pHrodo-labeled apoptotic thymocytes at 37°C for 120 min. The cell mixture was transferred to a Lab-Tek chamber and observed by fluorescence microscopy.

Fig 3

Fig 3

Tim4-dependent binding of apoptotic cells to Ba/F3 cells. (A) FACS analysis of the binding of apoptotic cells to Ba/F3 cells. Ba/F3 cells expressing GFP and Rac1 (BaF/GFP/Rac) or GFP, Rac1, and Tim4 (BaF/GFP/Rac/Tim4) were incubated at 37°C for 30 min with CellTracker Orange-labeled apoptotic thymocytes and subjected to flow cytometry for GFP and CellTracker Orange. The numbers indicate the percentage of the CellTracker-positive cells in the GFP-positive population. (B) Microscopic analysis of the binding of apoptotic cells. The BaF/GFP/Rac and BaF/GFP/Rac/Tim4 cells were incubated for 25 min at room temperature with CellTracker Orange-labeled living or apoptotic thymocytes in a Lab-Tek chamber and observed by fluorescence microscopy. (C) Time course of the binding of apoptotic cells to Tim4-expressing cells. The BaF/GFP/Rac and BaF/GFP/Rac/Tim4 cells were incubated with CellTracker Orange-labeled living or apoptotic thymocytes at 37°C for the indicated periods of time. The percentage of GFP-positive Ba/F3 cells that bound apoptotic cells was determined by flow cytometry as described above. The experiments were done in triplicate, and the average values are plotted (bars indicate SDs). (D) Phosphatidylserine-dependent binding of apoptotic cells to Tim4-expressing Ba/F3 cells. The BaF/GFP/Rac and BaF/GFP/Rac/Tim4 cells were incubated with CellTracker Orange-labeled living or apoptotic thymocytes at 37°C for 30 min in the presence of the indicated amount of MFG-E8 D89E. The cell mixture was subjected to FACS analysis, and the percentage of GFP-positive Ba/F3 cells that bound apoptotic cells was determined as described above. The experiments were done in triplicate, and the average values are plotted (bars indicate SDs).

Fig 4

Fig 4

Effect of integrins/MFG-E8 and Tim4 on the engulfment of apoptotic cells by Ba/F3 cells. (A) Effect of integrin αvβ3/MFG-E8 and Tim4 on the engulfment of apoptotic cells by Ba/F3 cells. BaF/GFP/Rac, BaF/GFP/Rac/αβ, BaF/GFP/Rac/Tim4, or BaF/GFP/Rac/Tim4/αβ cells were incubated at 37°C for 120 min with pHrodo-labeled apoptotic thymocytes in the absence or presence of 0.1 μg/ml mouse MFG-E8. The cell mixture was subjected to flow cytometry for GFP and pHrodo. The pHrodo staining profile of the GFP-positive population is shown. The experiment was performed three times, and representative data are shown. (B) Microscopic observation. BaF/GFP/Rac/αβ or BaF/GFP/Rac/Tim4/αβ cells were incubated as described above with pHrodo-labeled apoptotic thymocytes in the presence of 0.1 μg/ml mouse MFG-E8. The cells were transferred to a Lab-Tek chamber and observed by fluorescence microscopy. (C) Enhanced engulfment of apoptotic cells by Tim4 and MFG-E8. BaF/GFP/Rac (−), BaF/GFP/Rac/αβ (αβ), BaF/GFP/Rac/Tim4 (Tim4), or BaF/GFP/Rac/Tim4/αβ (Tim/αβ) cells were incubated at 37°C for 120 min with pHrodo-labeled apoptotic thymocytes in the absence (−) or in the presence (+) of 0.1 μg/ml mouse MFG-E8. The cell mixture was transferred to a Lab-Tek chamber and observed by fluorescence microscopy. The phagocytosis index was determined for at least 3,500 Ba/F3 cells in 10 fields and is plotted. (D) No effect of integrin or MFG-E8 on the binding of apoptotic cells to Ba/F3 cells. BaF/GFP, BaF/GFP/Tim4, or BaF/GFP/αβ cells were incubated at 37°C with CellTracker Orange-labeled apoptotic thymocytes in the absence or presence of 0.1 μg/ml mouse MFG-E8 for the indicated periods of time. The percentage of GFP-positive Ba/F3 cells that bound apoptotic cells was determined by flow cytometry. The experiments were done in triplicate, and the average values are plotted with SDs (bars). (E) Tim4-dependent tethering and uptake of apoptotic cells by mouse resident peritoneal macrophages. (Left) Peritoneal cells from the wild-type and Tim4−/− mice were incubated with pHrodo-labeled apoptotic thymocytes. The cells were stained with APC-conjugated anti-mouse Mac1 and subjected to flow cytometry for APC and pHrodo. The engulfment was determined as the percentage of pHrodo-positive cells in the Mac1-positive population. The experiment was carried out in triplicate, and the average values were plotted with SDs (bars). (Right) Peritoneal cells from the wild-type and Tim4−/− mice were incubated at room temperature with CellTracker-labeled apoptotic thymocytes for the indicated periods of time. After staining with APC-labeled anti-Mac1, the percentage of Mac1-positive cells that bound apoptotic cells was determined by flow cytometry. The experiments were done in triplicate, and the average values are plotted with SDs (bars). (F) Requirement of Ca2+ for the uptake but not binding of apoptotic cells to peritoneal macrophages. (Left) Peritoneal cells from the wild-type mice were incubated at 37°C for 60 min with pHrodo-labeled apoptotic thymocytes in PBS containing 2% FCS in the absence or presence of 0.4 mM Ca2+. The cells were stained with APC-labeled anti-Mac and analyzed by flow cytometry as described above. The experiments were done in triplicate, and the average values are plotted with SDs (bars). (Right) Peritoneal cells from the wild-type mice were incubated at room temperature in PBS containing 2% FCS in the absence or presence of 0.4 mM Ca2+ with CellTracker-labeled apoptotic thymocytes for the indicated periods of time and analyzed by flow cytometry as described above. The experiments were done in triplicate, and the average values are plotted with SDs (bars).

Fig 5

Fig 5

Effect of Rac1 and Rab5 on the engulfment of apoptotic cells by Ba/F3 cells. BaF/GFP/Tim4/αβ cells (Tim4/αβ) were transformed with Rac1 (Tim4/αβ/Rac) or Rac1 and Rab5 (Tim4/αβ/Rac/Rab) and incubated at 37°C for 120 min with pHrodo-labeled apoptotic thymocytes in the absence (open bars) or presence (filled bars) of 0.1 μg/ml mouse MFG-E8. Ba/F3 cells were sorted by FACS, transferred to a Lab-Tek chamber, and observed by fluorescence microscopy. (Bottom) The phagocytosis index for more than 4,500 Ba/F3 cells in 10 to 12 fields is plotted.

Fig 6

Fig 6

The tether-and-uptake model for the engulfment of apoptotic cells. In the tethering step, Tim4 catches apoptotic cells by recognizing phosphatidylserine. A putative coreceptor that associates with Tim4 then activates the integrin αvβ3 complex to initiate uptake, in which MFG-E8 binds to phosphatidylserine on apoptotic cells and to the activated integrin αvβ3 complex on the phagocytes. The signal from the integrin αvβ3 complex then activates the macrophages' internalization of apoptotic cells into lysosomes. This final step is mediated by Rac1, Rab5, and other, as-yet-unidentified molecules.

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