Abstract
Purpose: :
To study the effect of different intravitreal treatments on the phagocytosis of dying retinal pigment epithelial cells and the inflammatory response associated with it.
Methods: :
Two different death patterns were induced in vitro in ARPE-19 cells: death through detachment from the extracellular matrix on polyHEMA coated surfaces known as anoikis and UV-induced apoptosis. Two-colored phagocytic assays were carried out where the phagocytes (human monocyte-derived macrophages) engulfed the dying cells under different treatment modalities (triamcinolone (1uM), bevacizumab (312.5ug/mL), pegaptanib (75ug/mL) and ranibizumab (125ug/mL)). Flow cytometric analysis (FACS Calibur) was used to quantify the phagocytic process as well as measure the released amounts of IL-1β, IL-6, IL-8, IL-10 and TNFalpha using a Cytokine Bead Array.
Results: :
Macrophages engulfed the dying anoikic and apoptotic ARPE-19 cells at a similar and increasing rate over 8 hours of co-incubation (11.2+/-1.7% and 10.5+/-1.2% at 8 hours, respectively). Their phagocytic capacity increased by 2.1+/-0.3 times during engulfment of both types of dying cells under triamcinolone treatment, but remained unchanged for all other treatments. In the case of UV-induced dying ARPE-19 cells, probably due to presence of secondary necrosis, increased IL-6 and IL-1beta levels were detected which could be suppressed by triamcinolone, but not the other three treatments.
Conclusions: :
The macrophage mediated clearance of different dying ARPE-19 cells can serve as a good in vitro model for studying wet AMD and for testing different pharmacological and inflammatory aspects of this process.
Keywords: retina • phagocytosis and killing • inflammation