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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: 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.

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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Erythrocytes can be coated with antibodies, protein ligands, or aminophospholipids targeting specific phagocytic receptors. Binding and uptake of erythrocytes by macrophages can then be assessed quantitatively. (A) Cytofluorographs demonstrate the efficient coating of erythrocytes with antibodies, protein ligands, or different forms of PS. Cy3-conjugated antibodies were used to detect either biotinylated antibodies or biotinylated proteins on the surface of erythrocytes. Phycoerythrin–annexin V was used to detect various forms of PS on the surface of erythrocytes. Histogram in black demonstrates unstained erythrocytes. (B) Light microscopy was used to accurately assess the binding or uptake of Ebab by HMDM. Erythrocytes coated with either an irrelevant IgM isotype control (anti-TNP), anti-CD32, or anti-CD36 were incubated with HMDM for 30 min. After washing away nonadherent Ebab, distilled H2O was used to lyse the Ebab bound but not engulfed by the HMDM. This figure illustrates the high level of both tethering and engulfment of Ebab–anti-CD32 by HMDM, whereas Ebab–anti-CD36 were mostly tethered but not engulfed.
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fig1: Erythrocytes can be coated with antibodies, protein ligands, or aminophospholipids targeting specific phagocytic receptors. Binding and uptake of erythrocytes by macrophages can then be assessed quantitatively. (A) Cytofluorographs demonstrate the efficient coating of erythrocytes with antibodies, protein ligands, or different forms of PS. Cy3-conjugated antibodies were used to detect either biotinylated antibodies or biotinylated proteins on the surface of erythrocytes. Phycoerythrin–annexin V was used to detect various forms of PS on the surface of erythrocytes. Histogram in black demonstrates unstained erythrocytes. (B) Light microscopy was used to accurately assess the binding or uptake of Ebab by HMDM. Erythrocytes coated with either an irrelevant IgM isotype control (anti-TNP), anti-CD32, or anti-CD36 were incubated with HMDM for 30 min. After washing away nonadherent Ebab, distilled H2O was used to lyse the Ebab bound but not engulfed by the HMDM. This figure illustrates the high level of both tethering and engulfment of Ebab–anti-CD32 by HMDM, whereas Ebab–anti-CD36 were mostly tethered but not engulfed.

Mentions: The efficient coating of erythrocytes with antibody, protein ligand, or various forms of PS was verified by flow cytometry (Fig. 1 A). The Ebab were then incubated with human monocyte–derived macrophages (HMDM), murine thioglycolate–elicited peritoneal macrophages (MPM), or other phagocytic cells. The use of FACS®-based uptake assays was explored (Licht et al., 1999; Albert et al., 2000), but was not useful in distinguishing between Ebab that had attached to the outside of the HMDM and those that had been engulfed. Therefore, light microscopy was used to accurately quantitate attachment and engulfment of Ebab (Fig. 1 B). As expected, ligation of the Fcγ receptor IIA (CD32) with Ebab–anti-CD32 induced high levels of both binding and uptake, representing the phagocytic potential of the HMDM under these conditions. The use of antibodies for adequate triggering of uptake was further supported by the observation that Ebab–anti-C3bi receptor (anti-CD18) also demonstrated high levels of binding and uptake (unpublished data). Isotype control IgG or IgM on the erythrocytes induced neither binding nor uptake.


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)

Erythrocytes can be coated with antibodies, protein ligands, or aminophospholipids targeting specific phagocytic receptors. Binding and uptake of erythrocytes by macrophages can then be assessed quantitatively. (A) Cytofluorographs demonstrate the efficient coating of erythrocytes with antibodies, protein ligands, or different forms of PS. Cy3-conjugated antibodies were used to detect either biotinylated antibodies or biotinylated proteins on the surface of erythrocytes. Phycoerythrin–annexin V was used to detect various forms of PS on the surface of erythrocytes. Histogram in black demonstrates unstained erythrocytes. (B) Light microscopy was used to accurately assess the binding or uptake of Ebab by HMDM. Erythrocytes coated with either an irrelevant IgM isotype control (anti-TNP), anti-CD32, or anti-CD36 were incubated with HMDM for 30 min. After washing away nonadherent Ebab, distilled H2O was used to lyse the Ebab bound but not engulfed by the HMDM. This figure illustrates the high level of both tethering and engulfment of Ebab–anti-CD32 by HMDM, whereas Ebab–anti-CD36 were mostly tethered but not engulfed.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198875&req=5

fig1: Erythrocytes can be coated with antibodies, protein ligands, or aminophospholipids targeting specific phagocytic receptors. Binding and uptake of erythrocytes by macrophages can then be assessed quantitatively. (A) Cytofluorographs demonstrate the efficient coating of erythrocytes with antibodies, protein ligands, or different forms of PS. Cy3-conjugated antibodies were used to detect either biotinylated antibodies or biotinylated proteins on the surface of erythrocytes. Phycoerythrin–annexin V was used to detect various forms of PS on the surface of erythrocytes. Histogram in black demonstrates unstained erythrocytes. (B) Light microscopy was used to accurately assess the binding or uptake of Ebab by HMDM. Erythrocytes coated with either an irrelevant IgM isotype control (anti-TNP), anti-CD32, or anti-CD36 were incubated with HMDM for 30 min. After washing away nonadherent Ebab, distilled H2O was used to lyse the Ebab bound but not engulfed by the HMDM. This figure illustrates the high level of both tethering and engulfment of Ebab–anti-CD32 by HMDM, whereas Ebab–anti-CD36 were mostly tethered but not engulfed.
Mentions: The efficient coating of erythrocytes with antibody, protein ligand, or various forms of PS was verified by flow cytometry (Fig. 1 A). The Ebab were then incubated with human monocyte–derived macrophages (HMDM), murine thioglycolate–elicited peritoneal macrophages (MPM), or other phagocytic cells. The use of FACS®-based uptake assays was explored (Licht et al., 1999; Albert et al., 2000), but was not useful in distinguishing between Ebab that had attached to the outside of the HMDM and those that had been engulfed. Therefore, light microscopy was used to accurately quantitate attachment and engulfment of Ebab (Fig. 1 B). As expected, ligation of the Fcγ receptor IIA (CD32) with Ebab–anti-CD32 induced high levels of both binding and uptake, representing the phagocytic potential of the HMDM under these conditions. The use of antibodies for adequate triggering of uptake was further supported by the observation that Ebab–anti-C3bi receptor (anti-CD18) also demonstrated high levels of binding and uptake (unpublished data). Isotype control IgG or IgM on the erythrocytes induced neither binding nor uptake.

Bottom Line: 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.

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