Abstract
The present study characterized two different internalization mechanisms used by macrophages to engulf apoptotic and necrotic cells. Our in vitro phagocytosis assay used a mouse macrophage cell line, and murine L929sAhFas cells that are induced to die in a necrotic way by TNFR1 and heat shock or in an apoptotic way by Fas stimulation. Scanning electron microscopy (SEM) revealed that apoptotic bodies were taken up by macrophages with formation of tight fitting phagosomes, similar to the ‘zipper’-like mechanism of phagocytosis, whereas necrotic cells were internalized by a macropinocytotic mechanism involving formation of multiple ruffles directed towards necrotic debris. Two macropinocytosis markers (Lucifer Yellow (LY) and horseradish peroxidase (HRP)) were excluded from the phagosomes containing apoptotic bodies, but they were present inside the macropinosomes containing necrotic material. Wortmannin (phosphatidylinositol 3′-kinase (PI3K) inhibitor) reduced the uptake of apoptotic cells, but the engulfment of necrotic cells remained unaffected. Our data demonstrate that apoptotic and necrotic cells are internalized differently by macrophages.
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Abbreviations
- PS:
-
phosphatidylserine
- PI:
-
propidium iodide
- FACS:
-
fluorescence activated cell sorter
- SEM:
-
scanning electron microscopy
- TEM:
-
transmission electron microscope
- LY:
-
Lucifer Yellow
- HRP:
-
horseradish peroxidase
- PI3K:
-
phosphatidylinositol 3′-kinase
- mTNF:
-
recombinant murine tumor necrosis factor
- anti-Fas antibodies:
-
agonistic antihuman Fas antibodies
- DAPI:
-
2,6-diamidino-2-fenylindole
- DAB:
-
diaminobenzidine
- PARP:
-
poly(ADP-ribose) polymerase
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Acknowledgements
We thank Dominique Jacobus, Barbara De Bondt, and Hubert Stevens for excellent technical assistance. We are grateful to Amin Bredan for copy editing the manuscript. This study was supported by Ghent University GOA Grant no. 12050502.
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Krysko, D., Denecker, G., Festjens, N. et al. Macrophages use different internalization mechanisms to clear apoptotic and necrotic cells. Cell Death Differ 13, 2011–2022 (2006). https://doi.org/10.1038/sj.cdd.4401900
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DOI: https://doi.org/10.1038/sj.cdd.4401900
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