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Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis and promotes clearance of apoptotic cells.

Hoffmann PR, deCathelineau AM, Ogden CA, Leverrier Y, Bratton DL, Daleke DL, Ridley AJ, Fadok VA, Henson PM - J. Cell Biol. (2001)

Bottom Line: Our results suggest that regardless of the receptors engaged on the phagocyte, ingestion does not occur in the absence of phosphatidylserine (PS).Further, recognition of PS was found to be dependent on the presence of the PS receptor (PSR).We propose that the phagocytosis of apoptotic cells requires two events: tethering followed by PS-stimulated, PSR-mediated macropinocytosis.

View Article: PubMed Central - PubMed

Affiliation: Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206, USA.

ABSTRACT
Efficient phagocytosis of apoptotic cells is important for normal tissue development, homeostasis, and the resolution of inflammation. Although many receptors have been implicated in the clearance of apoptotic cells, the roles of these receptors in the engulfment process have not been well defined. We developed a novel system to distinguish between receptors involved in tethering of apoptotic cells versus those inducing their uptake. Our results suggest that regardless of the receptors engaged on the phagocyte, ingestion does not occur in the absence of phosphatidylserine (PS). Further, recognition of PS was found to be dependent on the presence of the PS receptor (PSR). Both PS and anti-PSR antibodies stimulated membrane ruffling, vesicle formation, and "bystander" uptake of cells bound to the surface of the phagocyte. We propose that the phagocytosis of apoptotic cells requires two events: tethering followed by PS-stimulated, PSR-mediated macropinocytosis.

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TGF-β secretion and PS-dependent engulfment of targeted erythrocytes and apoptotic cells is mediated by PSR. (A) Transfection of NIH 3T3 fibroblasts with vectors containing PSR antisense DNA resulted in a decrease of PSR expression to 25% of control levels, as measured by flow cytometry. Antisense transfection did not alter the levels of other phagocytic receptors, shown here by flow cytometric analysis of CD51 (αv integrin) expression. Transfection with sense PSR (unpublished data) or empty vector controls did not alter levels of PSR expression. □, PSR expression; ▪, αv expression. (B) The decreased expression of PSR induced by antisense transfection (Fig. 3 A) resulted in a decrease of apoptotic cell engulfment compared with control levels. However, antisense PSR DNA transfection did not alter the ability of these cells to engulf latex beads. Data were normalized to control (unmanipulated) cells. All results represent mean ± SEM from at least three experiments. □, Apoptotic cell uptake; ▪, latex bead uptake. (C) PSR is essential for TGF-β release by phagocytes during uptake of apoptotic cells. NIH 3T3 fibroblasts showed decreased production of TGF-β after an overnight feeding of apoptotic cells, as compared with unmanipulated fibroblasts and fibroblasts transfected with empty vector controls. All results represent mean ± SEM from at least two experiments.
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fig4: TGF-β secretion and PS-dependent engulfment of targeted erythrocytes and apoptotic cells is mediated by PSR. (A) Transfection of NIH 3T3 fibroblasts with vectors containing PSR antisense DNA resulted in a decrease of PSR expression to 25% of control levels, as measured by flow cytometry. Antisense transfection did not alter the levels of other phagocytic receptors, shown here by flow cytometric analysis of CD51 (αv integrin) expression. Transfection with sense PSR (unpublished data) or empty vector controls did not alter levels of PSR expression. □, PSR expression; ▪, αv expression. (B) The decreased expression of PSR induced by antisense transfection (Fig. 3 A) resulted in a decrease of apoptotic cell engulfment compared with control levels. However, antisense PSR DNA transfection did not alter the ability of these cells to engulf latex beads. Data were normalized to control (unmanipulated) cells. All results represent mean ± SEM from at least three experiments. □, Apoptotic cell uptake; ▪, latex bead uptake. (C) PSR is essential for TGF-β release by phagocytes during uptake of apoptotic cells. NIH 3T3 fibroblasts showed decreased production of TGF-β after an overnight feeding of apoptotic cells, as compared with unmanipulated fibroblasts and fibroblasts transfected with empty vector controls. All results represent mean ± SEM from at least two experiments.

Mentions: To directly address the requirement for the PSR in the uptake of apoptotic cells, NIH 3T3 fibroblasts, which express the PSR, were transfected with a PSR antisense DNA construct, a sense construct, or an empty vector control. Apoptotic Jurkat cells were fed to transfected fibroblasts and their uptake was determined by light microscopy (see Materials and methods for induction and characterization of Jurkat apoptosis). When expression of the PSR was reduced with antisense transfection to below 25% of control levels (Fig. 4 A), the uptake of apoptotic cells was diminished by a nearly equivalent amount (Fig. 4 B). Transfection did not affect the phagocyte membrane or phagocytosis in a general manner, as both expression of the α chain of the VnR and uptake of latex beads were unaffected.


Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis and promotes clearance of apoptotic cells.

Hoffmann PR, deCathelineau AM, Ogden CA, Leverrier Y, Bratton DL, Daleke DL, Ridley AJ, Fadok VA, Henson PM - J. Cell Biol. (2001)

TGF-β secretion and PS-dependent engulfment of targeted erythrocytes and apoptotic cells is mediated by PSR. (A) Transfection of NIH 3T3 fibroblasts with vectors containing PSR antisense DNA resulted in a decrease of PSR expression to 25% of control levels, as measured by flow cytometry. Antisense transfection did not alter the levels of other phagocytic receptors, shown here by flow cytometric analysis of CD51 (αv integrin) expression. Transfection with sense PSR (unpublished data) or empty vector controls did not alter levels of PSR expression. □, PSR expression; ▪, αv expression. (B) The decreased expression of PSR induced by antisense transfection (Fig. 3 A) resulted in a decrease of apoptotic cell engulfment compared with control levels. However, antisense PSR DNA transfection did not alter the ability of these cells to engulf latex beads. Data were normalized to control (unmanipulated) cells. All results represent mean ± SEM from at least three experiments. □, Apoptotic cell uptake; ▪, latex bead uptake. (C) PSR is essential for TGF-β release by phagocytes during uptake of apoptotic cells. NIH 3T3 fibroblasts showed decreased production of TGF-β after an overnight feeding of apoptotic cells, as compared with unmanipulated fibroblasts and fibroblasts transfected with empty vector controls. All results represent mean ± SEM from at least two experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198875&req=5

fig4: TGF-β secretion and PS-dependent engulfment of targeted erythrocytes and apoptotic cells is mediated by PSR. (A) Transfection of NIH 3T3 fibroblasts with vectors containing PSR antisense DNA resulted in a decrease of PSR expression to 25% of control levels, as measured by flow cytometry. Antisense transfection did not alter the levels of other phagocytic receptors, shown here by flow cytometric analysis of CD51 (αv integrin) expression. Transfection with sense PSR (unpublished data) or empty vector controls did not alter levels of PSR expression. □, PSR expression; ▪, αv expression. (B) The decreased expression of PSR induced by antisense transfection (Fig. 3 A) resulted in a decrease of apoptotic cell engulfment compared with control levels. However, antisense PSR DNA transfection did not alter the ability of these cells to engulf latex beads. Data were normalized to control (unmanipulated) cells. All results represent mean ± SEM from at least three experiments. □, Apoptotic cell uptake; ▪, latex bead uptake. (C) PSR is essential for TGF-β release by phagocytes during uptake of apoptotic cells. NIH 3T3 fibroblasts showed decreased production of TGF-β after an overnight feeding of apoptotic cells, as compared with unmanipulated fibroblasts and fibroblasts transfected with empty vector controls. All results represent mean ± SEM from at least two experiments.
Mentions: To directly address the requirement for the PSR in the uptake of apoptotic cells, NIH 3T3 fibroblasts, which express the PSR, were transfected with a PSR antisense DNA construct, a sense construct, or an empty vector control. Apoptotic Jurkat cells were fed to transfected fibroblasts and their uptake was determined by light microscopy (see Materials and methods for induction and characterization of Jurkat apoptosis). When expression of the PSR was reduced with antisense transfection to below 25% of control levels (Fig. 4 A), the uptake of apoptotic cells was diminished by a nearly equivalent amount (Fig. 4 B). Transfection did not affect the phagocyte membrane or phagocytosis in a general manner, as both expression of the α chain of the VnR and uptake of latex beads were unaffected.

Bottom Line: Our results suggest that regardless of the receptors engaged on the phagocyte, ingestion does not occur in the absence of phosphatidylserine (PS).Further, recognition of PS was found to be dependent on the presence of the PS receptor (PSR).We propose that the phagocytosis of apoptotic cells requires two events: tethering followed by PS-stimulated, PSR-mediated macropinocytosis.

View Article: PubMed Central - PubMed

Affiliation: Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206, USA.

ABSTRACT
Efficient phagocytosis of apoptotic cells is important for normal tissue development, homeostasis, and the resolution of inflammation. Although many receptors have been implicated in the clearance of apoptotic cells, the roles of these receptors in the engulfment process have not been well defined. We developed a novel system to distinguish between receptors involved in tethering of apoptotic cells versus those inducing their uptake. Our results suggest that regardless of the receptors engaged on the phagocyte, ingestion does not occur in the absence of phosphatidylserine (PS). Further, recognition of PS was found to be dependent on the presence of the PS receptor (PSR). Both PS and anti-PSR antibodies stimulated membrane ruffling, vesicle formation, and "bystander" uptake of cells bound to the surface of the phagocyte. We propose that the phagocytosis of apoptotic cells requires two events: tethering followed by PS-stimulated, PSR-mediated macropinocytosis.

Show MeSH
Related in: MedlinePlus