In this study, we report that trypsinization of RPE cells challenged with isolated POS results in a release of bound species. To verify that all FITC fluorescence measured by flow cytometry is in fact due to internalized POS, samples were analyzed, treated with Trypan blue, and analyzed again. No change in percent positive or mean fluorescence intensity of phagocytes was observed. All fluorescence of the POS alone population was completely quenched (See Supplementary Material and Supplementary Fig. S2, http://www.iovs.org/content/53/10/6282/suppl/DC1
). When analyzed using flow cytometry, therefore, we can detect three populations: a set of cells that has not internalized POS, a set of cells that has internalized POS (and median fluorescence intensity [MFI] or brightness values representing amount of POS ingested), and POS that have attached to the cells but were released by proteolysis, referred to throughout the text as “freed” POS (see A for a schematic depiction of the assay). When analyzed cumulatively, we provide a more complete depiction of the amount of POS bound by RPE cells and the amount ingested than that obtained using conventional methods. Utilizing flow cytometry, we have developed a quick, higher throughput method of analysis that provides data on both parameters of phagocytosis: binding and internalization. Previous studies using flow cytometry were limited to measures of internalization only30
or require pH specific dyes.31,32
(See B, C for electron micrographs of bound and internalized POS).
(A) Schematic illustrating the flow cytometry–based phagocytosis assay. (B) Electron micrograph showing bound POS at the surface of a murine primary RPE cell (arrow); scale bar = 200 nm. (C) Image showing internalized POS in murine RPE (arrow (more ...)
We then challenged confluent RPE cultures with FITC-labeled porcine POS for 5 hours, trypsinized the cells, and then labeled them with DRAQ5, a fluorescent nuclear marker that labels all cells prior to analyses. Using this method we can easily distinguish cells and isolated POS (See Supplementary Material and Supplementary Fig. S3, http://www.iovs.org/content/53/10/6282/suppl/DC1
). The percentage of cells phagocytosing POS and those that are not can be quantified by gating signals from unfed cells (based on background readings in the FITC channel). This percentage increases to 76.9% after POS challenge indicating active RPE phagocytosis (See Supplementary Material and Supplementary Fig. S3, http://www.iovs.org/content/53/10/6282/suppl/DC1
). After comparing both techniques in parallel, we determined that analyzing freed POS after trypsinization was just as effective and much simpler than performing the extra Trypan blue quenching step.
To establish the assay and to determine an optimal concentration of POS to use in our assays, we exposed ARPE-19 cells to POS for 5 hours to elicit maximal internalization. At 1 μg/cm2 nearly all POS available were internalized by 5 hours (A–C). At 4 μg/cm2, 100% of the cells had internalized some amount of POS by 5 hours, but a small amount was bound but not internalized (6.4%), suggesting that this is an appropriate dose for future experiments (A). When more POS is made available, RPE continued to phagocytose in a linear manner, as detected by increasing MFI (D, E). At higher concentrations (7–10 μg/cm2) the binding sites become saturated and the percentage of freed POS plateaus (C).
Establishment of the flow cytometry–based assay in ARPE-19 cells. (A) Maximum phagocytosis is reached with 4 ug/cm2 outer segments at 5 hours. (B) Gating the freed POS reveals that binding is increased at 7 ug/cm2 but plateaus at higher concentrations (more ...)
Since the initial steps of phagocytosis are controlled by cell surface receptors, we decided to alter the densities of the receptors to examine the effects and to validate that changes in the dynamics of phagocytosis could be determined using the flow cytometry-based assay. A low phagocytic percentage of control ARPE-19 cells nucleofected with GFP was observed (likely since only a few days passed after trypsinization), however, those transfected with CD36, or MerTK began phagocytosing at normal levels and αvβ5 integrin transfected cells began phagocytosing above normal levels (F). In fact, the MFI brightness values in αvβ5 integrin transfected cells were greater than 8-fold higher than in untransfected cells and 4- and 2.5-fold above CD36 or MerTK transfected cells, respectively (F). From these assays, we determined that inducing remodeling of the phagocytosis surface receptors can modulate the dynamics of binding and internalization, and these effects can be monitored using flow cytometry–based techniques.
After determining that the assay was valid, we next examined the dynamics of RPE phagocytosis over time in iPS-RPE, hfRPE, and ARPE-19. Previous studies show that maximal binding is achieved after 2 hours and maximal internalization occurs after 5 hours.19
When exposed to 4 μg/cm2
POS, we also observe maximal binding (percent binding) at 2 hours but maximal internalization occurs after as early as 3 hours (A). For comparison, we examined hfRPE and 1F-iPS–RPE treated with the same concentration of POS and at the same time points. hfRPE are slightly slower than ARPE-19 and reach maximal binding at 3 hours and internalization at 3 to 5 hours (B). 1F-iPS–RPE reach both maximum binding and internalization at 4 to 5 hours (C). Overall, binding and internalization is lower for both hfRPE and 1F-iPS–RPE as compared with ARPE-19 (A–C).
Binding and internalization time course comparison. (A) ARPE19 reaches maximum binding at 2 hours and internalization at 3 hours. For all the graphs the percent binding (dashed line) and the percent internalization (phagocytosis; solid line) are shown. (more ...)
We hypothesized that the variation observed in RPE phagocytosis between different human cell types could be due to divergent phagocytosis receptor densities of αvβ5 integrin, CD36, and MerTK receptors. To test this theory we labeled the cells after release from plates coated with temperature sensitive adhesion matrices with fluorescently conjugated antibodies, and quantified the percentage of single cells expressing the receptors at levels above background fluorescence. Receptor density can be compared by examining MFI brightness values. Using this technique, we can quantify the expression of the receptors on the surface of 10,000+ single cells per experiment. In ARPE-19 cells, in which the percentage of binding and phagocytosis is highest, αvβ5 integrin, CD36, and MerTK all are detected in relatively 40% to 60% of cells. αvβ5 integrin receptor density is very high (> 2-fold higher) as compared with either hfRPE or 1F-iPS–RPE (A). In comparison, very distinct patterns in hfRPE and 1F-iPS–RPE cells were observed along with different binding and internalization dynamics. The phagocytosis dynamics in 1F-iPS–RPE and hfRPE cells are more similar to one another, however (A). When directly compared, hfRPE and 1F-iPS–RPE have similar expression patterns of αvβ5 integrins, although the receptor density in 1F-iPS–RPE is higher (A). The percentage of cells expressing CD36 and receptor density on the individual cells, however, is nearly identical. Conversely, hfRPE express higher levels of MerTK (percentage of positive cells and intrinsic densities; A).
(A–B) Levels of receptor expression on each cell type correlate with their ability to bind and internalize outer segments. Panel A shows percentage of both cells positive (bars; left: y-axis) for each receptor as well as overall brightness of (more ...)
Various RPE cells (seeded on the same day and treated in exactly the same manner) were challenged as those examined above with 4 μg/cm2
POS for 30 minutes, 2 and 5 hours and were compared with naïve cells (B). The results of these analyses show that ARPE-19 cells display the highest percentage of internalization and the highest MFI. The dynamics of hfRPE and 1F-iPS–RPE are quite similar, although the percentage of phagocytosis in 1F-iPS–RPE cells is higher (B). Since αv
integrin receptor density is highest in ARPE-19 and 1F-iPS-RPE, these data suggest that αv
integrin expression may have the greatest impact on the rate of phagocytosis compared with CD36 and MerTK. Since the 1F-iPS–RPE phagocytosis rates were more efficient in this experiment (B compared with C), we hypothesized that differentiation status may also be important since several weeks had passed between experiments. To test this idea, we examined the receptor density of the phagocytosis receptors and phagocytosis dynamics of a cell line that we recently generated (EiPS-RPE)18
at distinct time points. As the RPE cells became more heavily pigmented and developed more typical hexagonal morphologies (A–C), dynamic surface receptor remodeling occurred (D), and the RPE cells became more efficient at phagocytosing POS (E). Therefore, we conclude that as RPE cells in culture become more differentiated, the receptor density of the phagocytosis receptors changes in a fashion that favors more efficient POS phagocytosis.
The status of surface receptor density of phagocytosis receptors and binding and internalization kinetics during RPE differentiation in EiPS-RPE is dynamic. (A–C) Images of EiPS-RPE after 10 weeks of directed differentiation (A), 14 weeks (B), (more ...)