Abstract
Retinal pigment epithelial (RPE) cells can undergo different forms of cell death, including autophagy-associated cell death during age-related macular degeneration (AMD). Failure of macrophages or dendritic cells (DCs) to engulf the different dying cells in the retina may result in the accumulation of debris and progression of AMD. ARPE-19 and primary human RPE cells undergo autophagy-associated cell death upon serum depletion and oxidative stress induced by hydrogen peroxide (H2O2). Autophagy was revealed by elevated light-chain-3 II (LC3-II) expression and electron microscopy, while autophagic flux was confirmed by blocking the autophago-lysosomal fusion using chloroquine (CQ) in these cells. The autophagy-associated dying RPE cells were engulfed by human macrophages, DCs and living RPE cells in an increasing and time-dependent manner. Inhibition of autophagy by 3-methyladenine (3-MA) decreased the engulfment of the autophagy-associated dying cells by macrophages, whereas sorting out the GFP-LC3-positive/autophagic cell population or treatment by the glucocorticoid triamcinolone (TC) enhanced it. Increased amounts of IL-6 and IL-8 were released when autophagy-associated dying RPEs were engulfed by macrophages. Our data suggest that cells undergoing autophagy-associated cell death engage in clearance mechanisms guided by professional and non-professional phagocytes, which is accompanied by inflammation as part of an in vitro modeling of AMD pathogenesis.
Highlights
The human retina is under constant remodeling throughout the lifetime, with various forms of cell death occurring in its 10 anatomical layers including the outermost – retinal pigment epithelium (RPE).[1]
Phagocytosis of apoptotic, autophagyassociated dying and necrotic cells has been extensively studied in other organ systems, and we have previously shown the clearance dynamics of apoptotic/anoikic RPE cells in vitro,[41] no data exist on how autophagy-associated dying cells get removed from the retina
Our data suggest that the clearance of autophagyassociated dying RPE cells by macrophages leads to a Retinal cells can undergo a wide range of cell death modalities including apoptosis, anoikis, autophagy and necrosis throughout their lifetime
Summary
The human retina is under constant remodeling throughout the lifetime, with various forms of cell death occurring in its 10 anatomical layers including the outermost – retinal pigment epithelium (RPE).[1]. The final fate of dead cells in the body depends upon the clearance mechanisms posed by macrophages and dendritic cells (DCs) both acting as professional phagocytes and/or antigen-presenting cells.[24] These cells are capable of engulfing apoptotic and necrotic cells without causing inflammation, respectively,[25] while autophagy-associated dying cells are capable of inducing inflammation.[26,27,28] During embryonic development, clearance of a large number of apoptotic cells takes place; clearance of apoptotic granulocytes occurs during inflammation, and daily clearance of Abbreviations: RPE, retinal pigment epithelium; AMD, age-related macular degeneration; DC, dendritic cell; H2O2, hydrogen peroxide; LC3, light-chain 3; CQ, chloroquine; 3-MA, 3-methyladenine; TC, triamcinolone; CNV, choroidal neovascularization; AV, autophagic vacuole; TEM, transmission electron microscopy; GFP, green fluorescent protein; FACS, fluorescence-activated cell sorter; AnxV, annexin V; PI, propidium iodide; GMCSF, granulocyte-macrophage colony-stimulating factor; PS, phosphatidylserine; DMEM, Dulbecco’s modified Eagle’s medium; CFDA-SE, carboxyfluorescein-diacetate-succinimidyl ester; CMTMR, 5-(and-6)-(((4-chloromethyl) benzoyl) amino)tetramethylrhodamine; PEI, polyethylenimine; DAPI, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride; MCSF, macrophage colonystimulating factor; IMDM, Iscove's Modified Dulbecco's Medium. Received 17.1.16; revised 01.4.16; accepted 04.4.16; Edited by GM Fimia photoreceptor outer segments occurs throughout the lifetime[29,30] and intensifies during aging.[31,32] Many different cell types are equipped with machinery to engulf, including epithelial cells and RPEs, which can act as non-professional phagocytes.[33]
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