Photobleaching of Melanosomes From Retinal Pigment Epithelium: II. Effects on the Response of Living

Aug 11, 04:00 AM

Current Headlines: By Zareba, Mariusz Sarna, Tadeusz; Szewczyk, Grzegorz; Burke, Janice M

ABSTRACT Melanosomes of the retinal pigment epithelium (RPE) are long lived organelles that may undergo photobleaching with aging, which can diminish the antioxidant efficiency of melanin. Here, isolated porcine RPE melanosomes were experimentally photobleached with visible light to simulate aging and compared with untreated granules or control particles (black latex beads) for their effects on the survival of photically stressed ARPE-19 cultures. Particles were delivered to cultures for uptake by phagocytosis then cells were exposed to violet light and analyzed by a new live cell imaging method to identify the time of apoptotic blebbing as a dynamic measure of reduced cell survival. Results indicated that untreated melanosomes did not decrease photic injury to ARPE-19 cells when compared with cells lacking particles or with ceils containing control particles, as might be expected if melanin performed an antioxidant function. Instead cells with untreated melanosomes showed reduced survival indicated by an earlier onset of blebbing and a lower fraction of surviving cells after photic stress. Ceil survival was reduced even further in stressed cells containing melanosomes that were photobleached, and survival decreased with increasing photobleaching time. Photobleaching of RPE melanosomes therefore makes cells containing them more sensitive to light- induced cytotoxicity. This observation raises the possibility that aged melanosomes increase RPE cell photic stress in situ, perhaps contributing to reduced tissue function and to degeneration of the adjacent retina that the RPE supports. How melanosomes (photobleached or not) interact with their local subcellular environment to modify RPE cell survival is poorly understood and is likely determined by the physicochemical state of the granule and its constituent melanin. The live cell imaging method introduced here, which permitted detection of a graded effect of photobleaching, provides a sensitive bioassay for probing the effects of melanosome modifications.

INTRODUCTION

In cells of the retinal pigment epithelium (RPE) melanosome biogenesis occurs largely during development and the pigment granules show little turnover thereafter (1,2). This melanosome longevity makes RPE pigment granules susceptible to a lifetime of modifications. One modification appears to be photo-oxidative bleaching of melanin, which produces a decline in the melanin content of human RPE cells as a function of donor age (3).

The biological properties of intact melanin are complex and include the potential to act as both an anti- and a pro-oxidant (4), and photobleaching appears to decrease the antioxidant potential of melanin (5,6). However, it is not clear precisely what functions melanosomes perform within pigmented cells such as the RPE as most information about the biological properties of melanin comes from studies of synthetic melanin or suspensions of isolated melanosomes. Whether melanosome behavior in cells changes with photobleaching is also an unanswered question.

One of the reasons it is not straightforward to analyze the anti- or pro-oxidant behavior of melanosomes within cells is that the melanosome is a granule. Conventional methods to analyze putative antioxidants in oxidatively stressed cells are designed for soluble factors, not insoluble particles. We (7) and others (8-10) have employed a variety of commonly-used methods to determine whether melanin/melanosomes perform an antioxidant function in cultured RPE ceils subjected to photic stress and the results have been mixed, perhaps predictably. In our study of ARPE-19 cultures containing phagocytized porcine melanosomes we identified cell-cell heterogeneity in stress susceptibility and in particle uptake as impediments to detecting a melanosome effect (7). This heterogeneity reduces the likelihood of seeing particle effects when population- based assays are used to quantify cell survival, especially if the effect is small and limited to subcellular regions, as predicted for granules like melanosomes. Here we report the development and use of a live cell imaging method to analyze the effects of phagocytized particles within ARPE-19 cultures. The technique permits selection of cells for analysis so individual cells containing comparable numbers of internalized particles can be compared. Further, the dynamic nature of the assay permits quantification of the timing of cell death as an additional sensitive measure of survival. Using this strategy, the cellular effects of internalized control particles, untreated or photobleached melanosomes were compared in ARPE-19 cells that were photically stressed by irradiation with violet light.

MATERIALS AND METHODS

Isolation and photobleaching of melanosomes. Melanosomes were purified from the RPE of porcine eyes and photobleached as previously described (6) with the additional modifications reported in the companion paper (II). Briefly, purified granules were incubated in Laemmli (12) electrophoresis buffer containing protease inhibitors to remove contaminating materials and proteins or membranes associated with the granule surface. Melanosomes were photobleached by suspending them in pH 7.2 phosphate buffer at 1 x 10^sup 9^ granules mL^sup -1^ followed by irradiation for intervals to 24 h with 190 m W cm^sup -2^ ultraviolet-free visible light using a ThermoOriel Solar Simulator. Bleaching produced decreases in absorbance from ~ 1 5-50% as determined by measurements of aliquots of melanosome suspensions sollubilized in Soluene 350.

Cell culture and particle delivery. ARPE19 cells (a spontaneously immortalized human RPE cell line) were maintained for all protocols in Minimum Essential Medium containing 10% fetal bovine serum. For particle delivery, cells were plated at subconfluent density, then 24 h after plating cultures were fed untreated or photobleached melanosomes by published methods (7) using 2.5 x 10^sup 7^ particles cm^sup -2^ of culture substrate. Using the same protocol, control cultures were fed black latex beads (1 [mu]m, Interfacial Microspheres & Nanospheres, Eugene, OR) as a control particle with similar light absorbance properties to untreated melanosomes. Cultures were allowed to phagocytize and internalize particles for 24 h, then cells were re-plated at low density in eight-chamber glass slides. Live cell imaging experiments were typically performed 3-4 days later.

Live cell imaging. Chamber slides containing particle-loaded cells were mounted on the stage of a Nikon Eclipse TE2000U microscope outfitted with a motorized, computer-controlled stage and a CoolSnap ES digital camera. The stage was equipped with a Live Cell 3 environmental chamber (Pathology Devices, Westminster, MD) to control temperature, humidity, and CO2 levels. Image collection and data analysis were performed using Premier MetaMorph software.

For image collection, a 10 x objective was used and microscope fields containing ~20 cells were selected. Using the epi- illumination port of the microscope with a 100-W mercury lamp and interference filters (Chroma Technology. Rockingham. VT). cultures were exposed to 400-410 nm visible light at 4 m W mm^sup -2^. Cells were illuminated for 30 s every 4 min over a total time course of 180 min. At the end of each 30 s light treatment, a phase contrast image was captured. During each 4 min interval, images were captured sequentially from each of the eight culture wells of the chamber slide using the computer-driven motorized stage, which permitted comparison of cells with different particle complements within the same experiment.

For data analysis, phase contrast images collected from each field were assembled to generate time lapse movies. Frame-by-frame analysis was conducted and the time was recorded when each cell initiated blebbing, a marker of apoptosis (13) that was used here as an indicator of cell death. Data were plotted as the percentage of the pre-selected cell population that underwent blebbing over time and plots of the particle groups were compared by fitting with a polynomial, cubic function at the 0.05 significance level using the fit comparison feature of Origin 7.5 software. All comparisons among cells containing different particle types were made within experiments by using replicate culture wells from the same multi- chamber slide. Three to ten independent experiments of each type were performed. Additional information regarding cell selection criteria and image analysis is provided with the Results.

Preliminary experiments were conducted in which 100 [mu]M propidium iodide (Sigma, St. Louis, MO) was added to the culture medium to quantify the timing of nuclear staining during illumination. Propidium iodide fluorescence was detected using a 555/ 617 nm excitation/emission filter set; fluorescence images were captured immediately after phase contrast image capture. For propidium iodide experiments the illumination time was extended beyond 180 min.

Photoconsumption of oxygen. To determine whether control particles exhibit photoreactivity, photo-induced changes in the concentration of oxygen were measured in suspensions of the control black latex beads and compared to untreated melanosomes using electron spin resonance (ESR) oxymetry as described previously (14). Samples containing particles (1.5 x 10^sup 9^ mL^sup -1^ in phosphate buffer) and the spin probe mHCTPO (0.1 mM) were illuminated in the resonant cavity of a Bruker ESP300E ESR spectrometer using blue light (400-490 nm) and an irradiance at the sample surface of 25 mW cm^sup -2^. RESULTS

Control latex beads, untreated melanosomes or photobleached melanosomes were delivered to A RPE- 19 cells for uptake by phagocytosis to analyze their effects in living cells. Similar to our previous observations (7), particle uptake varied among cells in the population, but particle internalization was similar for all particle types permitting identification of cells with equivalent particle numbers (Fig.1).

The live cell imaging method that was developed to analyze photically stressed, particle-containing ARPE19 cells is illustrated in Figs. 2 and 3. Using bright field images to visualize intracellular granules (Fig. 2), cells were pre-selected for analysis that contained equivalent particle numbers (> 50 particles/ cell), similar to the melanosome number estimated for RPE cells in situ (7). After cultures were light treated and phase contrast images captured over 180 min as described in Materials and Methods, time lapse image sequences were generated from which the time of apoptotic blebbing was recorded and used as a cell death marker. In the field shown (Fig. 2), blebbing was initiated at 120 and 170 min for cells 1 and 2, respectively. Blebbing precedes by about an hour the loss of membrane integrity, another marker of cell death, as indicated by nuclear staining with the membrane impermeant nucleic acid stain propidium iodide (Fig. 3). The fluorescent stain was not present during illumination experiments reported below when particle effects were quantified.

Using the live cell imaging protocol, multiple experiments were performed to compare photically stressed cells containing untreated melanosomes to those lacking particles or containing control particles (black latex beads). The results consistently showed an increased cytotoxic effect of light treatment in cells containing the biological granules (Fig. 4); cells with melanosomes showed an earlier onset of cell death and higher fraction of cells that had blebbed at the termination of the experiment (180 min) than particle- free cells or cells with control particles. The timing of onset and extent of cell death after 180 min of illumination varied among experiments, but in all experiments of this type (six out of six) cell death was significantly greater for cells containing untreated melanosomes. Melanosomes differ from control particles in that melanosomes photoconsume oxygen due to the photoformation of superoxide anion, while the black latex beads exhibit virtually no intrinsic photoreactivity (Fig. 5). There was a slight but not significant protective effect of the control particles when compared with particle-free cells (Fig. 4).

Figure 1. Phase contrast micrographs of ARPE-19 cultures showing cells containing equivalent numbers of all particle types. Individual cells containing > 50 particles per cell, as illustrated here, were used for live cell image analysis.

Figure 2. For imaging experiments, bright field images were first collected at time 0 (t = 0) to pre-select cells with > 50 particles per cell; shown here is a field with two cells containing the specified number of control particles. Cultures were exposed to violet light as described in Materials and Methods and phase contrast images were collected a t = 0 and at 4 min intervals over a time course of 180 min to generate time lapse movies. Frames collected at t = 120 and 170 min are shown to illustrate the time of initiation of blebbing (arrows) in cells 1 and 2, respectively.

Figure 3. Phase contrast and fluorescence images captured across an illumination time course for four particle-free ARPE-19 cells to illustrate the relationship between the timing of apoptotic blebbing and nuclear staining with propidium iodide. Frames from the phase contrast sequence (upper row) show the onset of blebbing and frames from the fluorescence sequence (lower row) show propidium iodide staining for each cell in the order of blebbing: cell 1, cells 2 and 3 (~simultaneously), then cell 4. The time of image capture (in minutes) after the onset of illumination is indicated in each frame and the time delay between image capture showing blebbing and nuclear staining is provided with the connecting arrows.

Figure 4. Results of a representative live cell imaging experiment for ARPE-19 cultures illuminated for 180 min comparing cells containing untreated melanosomes (black bars) to control populations that lacked particles (open bars) or contained control particles (black latex beads) (hatched bars). Data represent analysis of about 150 cells per particle group and are expressed as a percentage of the total cells that had undergone blebbing by each indicated time. Cells containing melanosomes showed significantly more blebbing than both particle-free cells and cells with control particles (P < 0.05).

Figure 5. Photo-induced oxygen uptake by suspensions of melanosomes (black symbols) and control particles (black latex beads, open symbols) measured with ESR oxymetry using mHCTPO as the spin probe. Control particles show no photoreactivity.

Figure 6. Results of a representative live cell imaging experiment for ARPE-19 cultures illuminated for 180 min comparing cells containing untreated melanosomes (black bars) to cells containing melanosomes that were photobleached for 24 h (gray bars). Data represent analysis of about 150 cells per particle group and are expressed as a percentage of the total cells that had undergone blebbing by each indicated time. Cells containing photobleached melanosomes showed significantly more blebbing (P < 0.05).

Figure 7. Results of a live cell imaging experiment for ARPE-19 cultures illuminated for 180 min comparing cells that contained untreated melanosomes to cells containing melanosomes that were photobleached for intervals from 16 to 24 h. Data represent analysis of about 150 cells per particle group and are expressed as a percentage of the total cells that had undergone blebbing by each indicated time. Cells showed earlier onset and higher rates of blebbing with increasing photobleaching time. All photobleached granule groups differed significantly from untreated melanosomes (P < 0.05). Additionally, the 16 and 18 h photobleached groups differed significantly from the 24 h group (P < 0.05).

The apparently phototoxic effect of untreated melanosomes was increased even further when melanosomes were photobleached prior to introduction into ARPE-19 cultures. Several independent experiments were performed to compare photically stressed cells containing untreated melanosomes versus granules that had been photobleached for 24 h; a representative experiment is shown in Fig. 6. As for the experiments described above, there was inter-experiment variability in the timing and magnitude of the cytotoxic effect, but in all experiments of this type cell death was significantly greater for cells containing photobleached granules; in eight independent experiments, at 150 min of illumination the fraction of cells containing photobleached melanosomes that had blebbed was 40% higher (mean) (range: 14-76%) than cells in paired cultures containing untreated granules. Further, the photobleaching effect was dose dependent; the granules produced increased cytotoxicity as the duration of photobleaching increased, which was manifest as a time shift to earlier death in cells containing melanosomes photobleached for longer intervals (Fig. 7).

DISCUSSION

Interest in the antioxidant functions of RPE melanin arises from its potential to protect cells from oxidative stress, especially photic stress. However, it has been difficult to obtain strong evidence supporting or refuting a cytoprotective function for melanin. Not only are the properties of melanin complex, but assays to measure antioxidants in cultured cells are designed to quantify the effects of soluble factors, not insoluble granules such as melanosomes. Recently several studies have been conducted to determine whether endogenous or phagocytized melanosomes protect cultured RPE cells from photic stress (7-10). The model systems and methods of analysis varied, and so did the outcomes. ARPE-19 cells that contained phagocytized melanosomes were not protected from photic stress (7,10), although photoprotection could be demonstrated if the cells also contained a photoreactive component of lipofuscin (10). Human fetal RPE showed evidence for photoprotection by melanin that appeared to be related to its aggregation state (8) and which was increased when supplemented by uptake of aggregated squid melanin (9). The type of RPE cell culture, and the type, quantity and organizational state of the melanin could all affect whether and how melanin modulates the cellular response to photic stress. Mechanisms of stress induction are also likely to determine whether melanin acts as a significant antioxidant defense system (15).

A complicating factor in analyzing the effects of melanin within photically stressed RPE cells is the variability among cells in the amount of melanin found in or taken up by the cells (7). This heterogeneity in melanin content reduces the likelihood of detecting small effects of melanin or discriminating effects of melanosome modifications. To circumvent this problem, here we developed a live cell imaging assay that permits selection for analysis of cultured cells that contain similar particle numbers. The dynamic nature of the assay also allows detection of time-dependent shifts in cell stress susceptibility, which provides an improved discrimination of a graded effect. Using this assay we found that intact, untreated melanosomes within ARPE-19 cells increased the cytotoxicity resulting from light treatment, rather than decreasing it as might be expected if melanosomes performed an antioxidant, photoprotective function. Cells containing untreated melanosomes were more susceptible to photic stress than particle-free cells or than cells containing a comparable number of inert control particles with similar light absorbance properties. The use of control particles was intended to control for any particle-loading effects on cell survival and to discriminate the biological effects of the melanosome aside from absorbance. We confirmed here that the control particles, unlike melanosomes, are not photoreactive. In our analyses control particles showed a weak protective effect that was not statistically significant, but intact melanosomes clearly conferred a reproducible increased risk for A RPE-19 cell death from photic stress.

This outcome appears to differ from the results of investigations on fetal RPE cells with high melanin content (8,9). Direct comparison between the studies is difficult, however, because of methodological differences including cell culture type (cell line vs cultures from fetal donors), melanosome type, and how photic stress was induced and measured. The latter issue is a particularly relevant one. In the studies of fetal RPE cells, cultures were continuously illuminated with blue light for 7 days (8,9), unlike the more acute 3 h illumination protocol that was used here, and the different light exposure conditions could produce different outcomes. At low light intensities, the cell's antioxidant systems may be competent to cope with reactive oxygen species generated by the intrinsic photoreactivity of melanin. Under these conditions the ability of melanin to act as an antioxidant by other mechanisms, such as sequestering metal ions (4), may then become the dominant effect and the result would be photoprotection. In contrast, under conditions of high light intensity such as those used here, the flux of reactive oxygen species photogenerated by melanin may exceed cellular antioxidant systems as well as all antioxidant properties of melanin. The result would then be increased photoxicity rather than photoprotection.

Aside from different light exposure conditions, different measures of photic stress were used in our study and the previous reports (8,9). In the previous studies, blue light-induced apoptosis was measured after several days of light treatment by annexin V staining. This method detects an early event in apoptosis (exposure of phosphatidyl serine on membrane surfaces) and is commonly used shortly after stress induction. It is unclear what it means to observe reduced annexin V staining in melanin-rich cells after long periods of light treatment. Perhaps cell death was delayed so this late snapshot provided an appropriate comparative measure of death in cells with low and high melanin content. Another possibility is that cells with high melanin underwent greater cell death during the early stages of light exposure (as we have observed), which could, over time, select for an apoptosis-resistant, melanin-containing subpopulation. Then when annexin V staining was analyzed in the later stages of light treatment, melanin could appear to be protective. Whether this sequence of events occurred is clearly speculative, but analysis of a time course for annexin V staining during chronic light treatment might prove revealing, especially if accompanied by measures of cell number or of cell turnover rates over time. Until identical methods are used to compare RPE cell cultures of different types containing the same type and number of melanosomes, it will not be possible to resolve issues of differing outcome. We are currently developing imaging methods to quantify chronic, sub-lethal levels of photic stress to RPE cultures to determine whether melanosomes confer photoprotection under these conditions. We are also performing experiments using different types of RPE cultures (cell lines and cell cultures from donors of different age) to determine the role that cellular background plays in modifying or revealing the biological properties of melanosomes.

Regardless of how untreated melanosomes affect photically stressed ARPE-19 cells, melanosome photobleaching significantly increases cellular stress susceptibility, and susceptibility increases as a function of photobleaching time. The mechanisms whereby melanosomes, photobleached or not, affect cell survival are not well understood. Blue light-induced RPE cell death is mediated by reactive oxygen species derived from mitochondria, presumably because mitochondrial cytochromes act as a blue light-sensitive endogenous chromophore (16). As melanosomes (14,17), and especially photobleached melanosomes (6), can photogenerate superoxide, reactive species arising from melanosomes may be additive with those from mitochondria thereby increasing stress susceptibility.

Overall the results shown here for photobleached RPE melanosomes support the concept that photobleaching, which appears to accompany aging (3), reduces the likelihood that the granules perform an antioxidant function. As photic stress to the RPE is believed to contribute to diseases such as age-related macular degeneration (18), the results further suggest that aging of melanosomes may contribute to disease risk. Determining how melanosomes function within cells and how their functions change with age therefore becomes increasingly important. Such studies will benefit from new experimental approaches including the imaging method that was introduced here to quantify particle effects in living cells.

Acknowledgements-This work was supported by National Eye Institute grants R01 EY013722 and P30 EY01931 (J.M.B), Poland Ministry of Science and Information Technology grant 3 P04A 009 25 (T.S.), the Posner Foundation and the Coleman Charitable Foundation (Milwaukee, WI), and by an unrestricted grant from Research to Prevent Blindness, Inc. We thank Michele Henry for assistance with preparing melanosome samples.

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Mariusz Zareba1,2, Tadeusz Sarna2, Grzegorz Szewczyk2 and Janice M. Burke*1

1 Department of Ophthalmology, Medical College of Wisconsin, Milwaukee, Wi 2 department of Biophysics, Jagiellonian University, Krakow, Poland

Received 10 January 2007; accepted 20 January 2007; DOI: 10.1111/ J.1751-1097.2007.00080.x

* Corresponding author email: jburke@mcw.edu (Janice M. Burke)

(c) 2007 The Authors. Journal Compilation. The American Society of Photobiology 00318655/07

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