Abstract
Purpose :
Retinal pigment epithelial (RPE) cells phagocytose photoreceptor outer segment fragments (POS) using a molecular mechanism that overlaps with pathways employed by other cells to phagocytose apoptotic debris. In vitro POS challenge is thus commonly used to assess phagocytic capacity by cells in culture. However, variability of POS characteristics, assay conditions, and quantification methods has hindered direct comparison of phagocytic activities across cell types or studies. Here, we optimize fluorescent beads bound with ligand proteins for known RPE phagocytic receptors to confirm their utility in RPE phagocytosis assays and define molecular characteristics of Müller cell clearance phagocytosis.
Methods :
1 μm-diameter fluorospheres were irreversibly bound with MERTK-ligand (PROS1) and/or αv integrin-ligand MFG-E8 (MFG), or albumin. RPE or Müller primary cells, ARPE-19 and MIO-M1 Müller cell lines were challenged with POS or beads, followed by immunoblotting, immunofluorescence microscopy, and fluorescence scanning quantification. αv integrin inhibitory peptide was added to select assays. Integrin antibodies were used to live label integrin surface receptors. Experiments were performed at least 3 times independently. Differences were deemed significant if p<0.05 by ANOVA or Student’s t-test.
Results :
RPE cells phagocytosed ligand-bound beads like POS, with strong preference for combination of MFG+PROS1 over albumin or PROS1 alone. Integrin inhibition abolished POS uptake as expected, and similarly diminished MFG+PROS1-bead uptake by RPE (p<0.0001). RPE cells phagocytosed similar numbers of beads even if ligand-coated beads were stored at 4oC for up to 2 months. We observed both αvβ3 and αvβ5 integrins at the surface of MIO-M1 Müller glia, while RPE cells expressed only αvβ5 at their apical, phagocytic surface, as established previously. POS and bead uptake by MIO-M1 Müller cells was significantly increased by MFG+PROS1 as compared to albumin control (p<0.005). Integrin inhibition significantly reduced ligand-bead uptake (p<0.005). MFG+PROS ligand and integrin receptor dependence was shared by primary Müller glia.
Conclusions :
Ligand-bound fluorospheres have utility as stable phagocytic particles that permit standardization across cell types and studies. Using these novel reagents, we found that primary and immortalized Müller glia in culture employ αv integrin receptors for clearance phagocytosis
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.