Induction of Angiogenic Cytokine Expression in Cultured RPE by Ingestion of Oxidized Photoreceptor Outer Segments

Abstract

purpose. Normal aging is associated with accumulation of lipofuscin pigment in the retinal pigment epithelium (RPE). This may occur as a result of phagocytosis and incomplete degradation of oxidized photoreceptor outer segments (POS). This study was undertaken to determine whether phagocytosis of UV-irradiated POS (artificial lipofuscin) would increase expression in the RPE of various chemotactic and angiogenic cytokines.

methods. ARPE-19 cells were exposed to latex beads (0.76 μm), naïve bovine POS, and UV-irradiated POS (Ox-POS; 2 × 10⁷/mL), and supernatants were collected at 18 and 36 hours. The supernatants were assayed for IL-8, monocyte chemotactic protein-(MCP)-1, and TNF-α by ELISA. Protein synthesis and NFκB activity were inhibited by actinomycin D and SN50, respectively. Phagocytosis and generation of intracellular reactive oxygen species were assessed by flow cytometry. Confocal and electron microscopy studies were also performed to verify phagocytosis and cellular integrity.

results. IL-8 and MCP-1 levels were decreased in the naïve POS group (IL-8: 473.76 ± 66.9 pg/mL, P = 0.0005; MCP-1: 550.1 ± 21.8 pg/mL, P = 0.0001), but were increased in the Ox-POS group (IL-8: 1348.8 ± 164.9 pg/mL; MCP-1: 1772.28 ± 65.19 pg/mL) compared with the control (IL-8: 741.09 ± 39.8 pg/mL; MCP-1: 1413.47 ± 38.4 pg/mL) and latex bead groups (data not shown). TNF-α levels were not affected. At 12 hours (but not at 6 hours), ROS were increased in the Ox-POS group. The cytokine increases observed were dependent on de novo protein synthesis and were NF-κB dependent.

conclusions. Ingestion by RPE of oxidized bovine POS stimulates expression of the chemotactic and angiogenic factors IL-8 and MCP-1 that have the capability to promote angiogenesis directly, or indirectly through the accumulation of immune cells such as macrophages, which themselves may release angiogenic promoters and degrade Bruch’s membrane. This may be of significance in the development of exudative AMD.

Age-related macular degeneration (AMD) is an idiopathic retinal degenerative disease that is the leading cause of blindness in the elderly population in the Western world. ^{1 2 3} The disease is characterized by two stages: The dry or atrophic form accounts for 80% of cases and is characterized by drusen formation at the macula, degeneration of the retinal pigment epithelium (RPE), and photoreceptor death. ⁴ Clinically, it is characterized by slowly progressive central visual loss. The wet or exudative form of AMD accounts for only 20% of cases, but for 80% to 90% of resultant blindness, and is characterized by choroidal neovascularization (CNV) leading to edema beneath the macula and rapidly progressive central visual loss. ⁵ Only 18% of cases of wet AMD are amenable to laser photocoagulation at presentation, and many in of these cases, disease recurs. ⁶  

The first sign of impending disease in AMD is the appearance of RPE mottling and soft drusen. Soft drusen are recognized histologically as a localized thickening of Bruch’s membrane, and the ultrastructure of these thickened areas demonstrate an accumulation of membranous debris in the inner collagenous layer of Bruch’s membrane (basal linear deposits). ^{7 8 9 10 11} The location and composition of the lipids in drusen are consistent with their derivation from RPE and photoreceptor membranes. ¹² Age-related, progressive accumulation of yellow-brown pigments (age pigment or lipofuscin) within RPE is a consistently recognized phenomenon in humans and animals. ^{13 14 15 16} Progressive engorgement of RPE cells with lipofuscin is thought to be associated with the extrusion of aberrant materials that accumulate in Bruch’s membrane and aggregate in the form of drusen and basal laminar deposits. ⁴ One theory of lipofuscinogenesis and the formation of drusen involves its generation within the lysosomal vacuome, because of intralysosomal, iron-catalyzed peroxidation of photoreceptor outer segment (POS) material undergoing phagocytic degradation. ¹⁷ It is thought that if POS are oxidatively damaged before phagocytosis, the RPE lysosomal system may fail to digest them adequately because the aberrant molecular species no longer match active sites on the degradative enzymes. ^{18 19 20} Outer segment discs and photoreceptor membranes are rich in polyunsaturated fatty acids, and because the susceptibility of fatty acids to auto-oxidation is proportional to their degree of unsaturation, they are therefore particularly vulnerable to oxidation. ^{21 22} The rate of lipofuscin accumulation could therefore be influenced by factors such as the level of retinal oxygenation and light exposure, and the antioxidant systems in the retina. ¹⁹  

Lipofuscin-like fluorophores may be generated in vitro from a variety of sources (e.g., lipid peroxidation, polyenic molecules [oxidation or cleavage of retinol or carotenoids, respectively], nucleic acids, glycation/Maillard reactions [advanced glycation end products; AGEs], protein oxidation, and oxidation products of ascorbic acid). Most biomaterials form brown, fluorescent ceroid-lipofuscin-like fluorophores when subjected to oxidative stress, unless protected by antioxidative defense systems. ²³ Exposure of RPE cells in culture to POS preoxidized by exposure to ultraviolet (UV) light has been used as a model of lipofuscin accumulation, because UV-irradiation has been demonstrated to convert POS into a lipofuscin-like structure (Sidikaro Y, Trüb PR, Morse LS, ARVO Abstract 9, 1988). ²⁴  

RPE cells have been shown to secrete chemoattractant and inflammatory cytokines, such as interleukin (IL)-8, monocyte chemotactic protein (MCP)-1, and tumor necrosis factor (TNF)-α, in response to a variety of stimuli. ^{25 26 27 28} IL-8 and MCP-1 are potent chemoattractants for neutrophils and macrophages that have also been shown to have potent proangiogenic properties in vitro and in vivo and to be elevated in the vitreous of patients with retinal neovascularization. ^{25 29 30 31 32} Tumor necrosis factor (TNF)-α is a proinflammatory cytokine that also has proangiogenic properties. ^{33 34}  

In our study, we determined whether ingestion of preoxidized POS (Ox-POS) by RPE cells would increase their production of the cytokines IL-8, MCP-1, and TNF-α compared with cells that had ingested naïve POS or 0.76-μm latex beads. Phagocytosis was confirmed by flow cytometry and by confocal and electron microscopy. We also investigated the role of intracellular oxidative stress and NF-κB activation by measuring generation of intracellular reactive oxygen species (ROS) and activation of NF-κB and by determining the effect of antioxidants and of a specific inhibitor of NF-κB in our model. 

Materials and Methods

Reagents

Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12, phosphate-buffered saline (PBS) without Ca²⁺ and Mg²⁺, fetal calf serum (FCS), penicillin, streptomycin sulfate, amphotericin B, glutamine, and 0.05% trypsin-0.02% EDTA solution were purchased from GibcoBRL (Paisley, Scotland, UK). Sodium orthohydrophosphate (Na₂HPO₄) and potassium dihydro-orthophosphate (KH₂PO₄) were obtained from BDH Laboratory Supplies (Poole, UK). Latex beads (0.76 μm), fluorescein isothiocyanate (FITC), propidium iodide, N-acetyl-cysteine (NAC), and sucrose were obtained from Sigma (St. Louis, MO). Acridine orange and 5-(and 6-)-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate (CM-H₂DCFDA) were obtained from Molecular Probes (Leiden, The Netherlands). Bovine eyes were obtained from a local abattoir. A micro bicinchoninic acid (BCA) protein assay kit was obtained from Pierce (Rockford, IL). ELISA kits for IL-8, MCP-1, and TNFα were obtained from R&D Systems (Abingdon, UK). SN50 was obtained from Calbiochem (Darmstadt, Germany). 

Cell Culture

The human retinal pigment epithelial cell line ARPE19 was obtained from the American Type Culture Collection (Manassas, VA) and cultured in DMEM/Ham’s F12 supplemented with 10% FCS, penicillin (100 U/mL), and streptomycin sulfate (100 μg/mL). Cells were grown at 37°C in a humidified 5% CO₂ condition and split twice a week when approximately 90% confluence was reached. Cells were obtained at passage 19 and used at passages 21 to 28. 

Photoreceptor Outer Segment Isolation

Fresh bovine retinas were isolated under far-red illumination and stored at −70°C. Thawed retinas were homogenized by agitation in 0.73 M sucrose in 0.1 M phosphate buffer (Na₂HPO₄/KH₂PO₄ [pH 6.8]), filtered through a 100-μm nylon mesh, layered on top of a discontinuous sucrose density gradient, and centrifuged for 1 hour at 60,000g. ³⁵ Purified POS were harvested from the interface between the 0.8 and 1.0 M sucrose solutions (in 0.1 M phosphate buffer), diluted in plain buffer solution, and pelleted at 27,000g for 20 minutes. The resuspended POS concentration was reported in micrograms protein per milliliter using the micro-BCA protein assay kit (Pierce). Hemocytometer counts indicate that 50 μg POS protein/mL corresponds to approximately 2 × 10⁷ POS particles/mL. 

UV Irradiation of POS

Isolated POS (∼2 × 10⁸/mL in 2 mL phenol red-free DMEM) were exposed in a six-well tissue culture plate, to a 302-nm light source (Ultraviolet Products, Cambridge, UK) with a fluence of 0.5 mW for 10 hours at 5% CO₂, 37°C, in an adaptation of previously described methods (Sidikaro Y, Trüb PR, Morse LS, ARVO Abstract 9, 1988). ²⁴ At the end of the irradiation period, POS were drawn off and the well was rinsed with PBS and aspirated to ensure all were recovered. The irradiated POS were then pelleted by centrifugation at 12,000g for 20 minutes. At this point, the POS were resuspended in RPE culture medium and made up to their final concentration of 2 × 10⁷/mL. 

Labeling of Isolated POS and Quantification of Phagocytosis

According to an established method, ³⁶ POS and Ox-POS were labeled with 10 μg/mL FITC, pelleted, and rinsed four times in PBS. Cells were challenged with 2 × 10⁷ FITC-POS/mL or FITC-Ox-POS for 12 hours. After POS challenge, cells in each well were rinsed three times in PBS and were then trypsinized and prepared as a cell suspension. Extracellular fluorescence (bound but uningested POS) was quenched by the addition of trypan blue (1 mg/mL in PBS) for 15 minutes. The intracellular fluorescence (λ_excitation = 488 nm, λ_emission = 530 ± 15 nm) of 10,000 of these unfixed cells per well was then assayed immediately on a flow cytometer (FACScan; BD Immunocytometry Systems, San Jose, CA) using a live gate to exclude cell fragments, POS particles, and other unwanted debris. ³⁷ A logarithmic scale of relative fluorescence intensity was used, and POS phagocytosis was calculated by subtracting the geometric mean autofluorescence of control cells from the geometric mean autofluorescence of cells challenged with FITC-POS. The experiment was performed three times. 

Electron Microscopy of Phagocytosing Cells

Cells were cultured on plastic coverslips (Thermanox; Nunc, Rochester, NY) in 24-well plates and challenged with latex beads, naïve POS, and Ox-POS, as described. At 18 and 36 hours, they were washed three times in PBS and fixed by addition of a 2% solution of glutaraldehyde in 0.1 M phosphate buffer with 0.1 M sucrose (pH 7.2) at room temperature. After postfixation for 1 hour in 1% OsO₄ in phosphate buffer, the cells were dehydrated in a graded series of ethanol and embedded in Araldite. Thin sections (50–70 nm) were cut on a microtome (OMU4; Reichert Jung, Vienna, Austria), stained with uranyl acetate and lead citrate, and viewed on an electron microscope (model 100; JEOL, Tokyo, Japan). 

Confocal Microscopy of Phagocytosing Cells

RPE were grown on glass coverslips in 24-well plates until confluent and challenged with FITC-conjugated latex beads, naïve POS, and Ox-POS. At 6, 18, and 36 hours the cells were fixed in 6% paraformaldehyde. The cells were then counterstained and examined on a dimpled slide by confocal microscopy (Zeiss, Thornwood, NY). 

Challenging RPE with Latex Beads, Naïve Bovine POS, and Ox-POS

ARPE-19 were plated on 24-well tissue culture plates (1 × 10⁵ cells/well; BD Immunocytometry Systems) and incubated under the conditions described at 37°C in a humidified 5% CO₂ environment until confluent. They were washed twice with PBS and challenged with latex beads (0.76 μm), naïve bovine POS, and Ox-POS at a concentration of 2 × 10⁷ particles/mL (50 μg/mL POS protein). They were then incubated in the aforementioned conditions for 18 and 36 hours, at which point supernatants were collected and centrifuged at 3000 rpm for 15 minutes at 4°C to remove cells and POS. The supernatant was drawn off and stored at −80°C until assay. 

Quantification of Cytokines in Conditioned Medium

IL-8, MCP-1, and TNFα were assayed in the supernatants using a commercially available sandwich-type ELISA, according to the manufacturer’s protocol (R&D Systems). New protein synthesis was blocked by preincubating the RPE cells with a 1-μg/mL solution of actinomycin D (Sigma) in culture medium followed by continued exposure to actinomycin D, plus treatment according to the manufacturer’s guidelines and previously published studies. ³⁸  

NF-κB Activation

NF-κB activation in the RPE cells was assessed by transfection of ARPE-19 cells with the luciferase reporter plasmid pNF-κB-luciferase vector, with the Renilla luciferase reporter vector as an internal control. After transfection of the target cells with the vector, activated endogenous NF-κB binds to the κB4 on the vector and initiates transcription of luciferase, which can then be detected by addition of its substrate and quantification of the resultant luminescence by luminometer. Briefly, RPE cells were transfected with the pNF-κB-luciferase reporter vector for 24 hours at 37°C in 5% CO₂. At this point the cells were washed twice with PBS, and the cells were then challenged with 2 × 10⁷/mL latex beads, naïve POS, UV-irradiated POS, or control. At 12 hours, the luciferase substrate was added, and mean luminescence was determined. The experiment was performed three times. 

Blocking of NF-κB Activation

Activation and nuclear translocation of the transcription factor NF-κB was inhibited by preincubation of the cells for 30 minutes with 100 μg/mL SN50, a specific NF-κB inhibitor. 

Measurement of Intracellular ROS

The intracellular formation of ROS in RPE was detected by using the fluorescent probe 5-(and 6-)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA; Molecular Probes), as described previously. In this method, RPE cells incorporate the CM-H₂DCFDA, and the diacetate moiety is cleaved to produce the nonfluorescent compound DCFH. The hydrogen peroxide (H₂O₂) and peroxidases generated by activated RPE oxidize the intracellular DCFH to the fluorescent compound 2′,7′-dichlorofluorescein. The green fluorescence produced by the RPE is proportional to the amount of H₂O₂ produced. ⁴⁰ Briefly, the RPE cells were grown to confluence and challenged with latex beads, naïve POS, and Ox-POS at 2 × 10⁷ particles/mL (50 μg/mL POS protein), as described earlier. At 6 and 12 hours the cells were washed twice, trypsinized, and resuspended in PBS. Cells were loaded with 20 μM CM-H₂DCFDA and incubated at 37°C for 10 minutes. The measurement of intracellular ROS was performed on a flow cytometer (BD Immunocytometry Systems), which detects the log of the mean channel fluorescence intensity with an acquisition of FL1. A minimum of 5000 events was recorded and analyzed with the software (Cell Quest; BD Immunocytometry Systems). The experiment was performed three times. 

Effect of Incubation with Antioxidants

The effect of incubation with the antioxidant NAC was assessed. POS were irradiated as discussed previously and then added to the RPE cells, as described. The cells were incubated in medium containing various concentrations (10 and 20 μM) of NAC or untreated control, and supernatants were collected at 18 hours. 

Cellular Viability

RPE cell viability in response to the above stimuli was assessed by incubating RPE cells in 96-well plates at 2 × 10⁴ cells/well under the described conditions. After the challenges described previously, RPE cells were then incubated for 18 hours, at which time the number of viable cells was determined by a cell-viability assay (Cell Titer 96 Aqueous One Solution Assay; Promega, Madison, WI), according to the manufacturer’s protocol. In brief, this modified MTT assay determines the number of viable cells by bioreduction of MTS tetrazolium into a colored formazan product, which is detected by absorbance at 490 nm by a computerized plate reader. Cellular viability is expressed as a percentage of that of control medium. 

Statistical Analysis

All data are presented as the mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA). Differences were judged statistically significant at P < 0.05. 

Results

Quantification and Morphologic Verification of Phagocytosis

At 12 hours after exposure, ARPE-19 readily phagocytosed latex beads, naïve bovine POS, and Ox-POS, as determined by flow cytometry after challenge with FITC-conjugated latex beads, naïve POS, and Ox-POS. Mean cellular fluorescence was increased in RPE cells exposed to FITC-conjugated naïve bovine POS and Ox-POS at 12 hours (n = 3, P = 0.0001), as assessed by flow cytometry (Fig. 1) . This indicated that the cells were actively phagocytosing the POS. Phagocytosis was confirmed by electron microscopy (Fig. 2) and also by confocal microscopy, which revealed the presence of large amounts of fluorescent POS within the cytoplasm of the cells (Fig. 3) . 

Cytokine Levels in the Supernatants

Levels of both IL-8 and MCP-1 (Fig. 4) were decreased in the supernatants from RPE cells that had phagocytosed naïve bovine POS compared with the control at both 18 and 36 hours. However, IL-8 and MCP-1 were significantly increased in supernatants from RPE that had ingested Ox-POS, compared with control and naïve POS groups (n = 5, P = 0.0001). Ingestion of latex beads did not affect the levels of IL-8 or MCP-1 in the supernatants (data not shown). TNF-α levels in the supernatants were minimal, and there were no significant differences between the groups (data not shown). 

Blocking of Protein Synthesis with Actinomycin D

To confirm whether the increases in IL-8 and MCP-1 were due to de novo cytokine protein synthesis, we blocked new protein synthesis in the cells with actinomycin D. Incubation of the RPE cells challenged with Ox-POS with 1 μg/mL actinomycin D completely eliminated the increases in IL-8 and MCP-1 (n = 5, P = 0.0001) that were observed at 18 and 36 hours (Fig. 5) . In fact, the levels of IL-8 and MCP-1 were reduced below control levels, indicating that the control cells constitutively expressed these cytokines. This result indicated that the increases between the groups were dependent on de novo protein synthesis. 

NF-κB Activation

Expression of both IL-8 and MCP-1 have been demonstrated to be dependent on activation of NF-κB. Measurement of luminescence of a luciferase substrate in RPE cells that have been transfected with a luciferase reporter vector provides an indirect measure of NF-κB activation. Our results demonstrated that NF-κB activation was increased to double that of the control group in cells that had ingested Ox-POS (n = 3, P < 0.05). Ingestion of latex beads and naïve POS did not significantly affect activation of NF-κB (Fig. 6) . 

Blocking of NF-κB Activation with SN50

To further confirm that the increases we had demonstrated in expression of IL-8 and MCP-1 by RPE ingesting Ox-POS were dependent on NF-κB activation, we blocked NF-κB with the specific inhibitor SN50. Preincubation of the RPE cells with 100 μg/mL SN50 reduced IL-8 and MCP-1 levels in the supernatants to control levels (n = 4, P < 0.05; Fig. 7 ). This result demonstrates that the increases in IL-8 and MCP-1 in the Ox-POS group were dependent on activation of NF-κB. 

Flow Cytometric Analysis of Generation of Intracellular ROS

Because generation of intracellular ROS has been shown to activate NFκB, we investigated whether they were generated in RPE in this model. Generation of intracellular ROS was significantly increased at 12 hours (Fig. 8) , but not at 6 hours, in RPE challenged with Ox-POS, compared with the control, latex bead, and naïve POS groups (n = 3, P < 0.05). 

Effect of Incubation of RPE Cells with Antioxidants

The antioxidant NAC at a dose of 10 μM reduced by half the expression of IL-8 induced by ingestion of irradiated POS and completely eliminated the response at 20 μM (n = 4, P = 0.0001; Fig. 9 , left). However, neither dose of NAC reduced the OX-POS-induced expression of MCP-1 (Fig. 9 , right). 

Discussion

Oxidative damage has been implicated in the pathogenesis of AMD. ⁴⁰ ROS generated from phagocytosis, lipid peroxidation, ⁴¹ and photic stress, ⁴² together with the high oxygen tension in the macular region, ⁴³ contribute to the particular susceptibility to oxidative stress of the RPE at the macula. Later stages of AMD are characterized by choroidal neovascularization leading to edema beneath the macula and rapidly progressive central visual loss. Authors have hypothesized an immunologic etiology for the progression of AMD to the neovascular form. ⁴⁴ Immune complexes and macrophages have been found in and around drusen in early AMD ^{45 46 47} and macrophages, lymphocytes, and plasma cells have been found in excised choroidal neovascular membranes. ⁴⁸  

In our study, ingestion by RPE cells of outer segments that had been oxidatively altered by UV-irradiation caused them to increase significantly their expression of the cytokines IL-8 and MCP-1. This was mediated through activation of the transcription factor NF-κB, which has been shown to be involved in the expression of these cytokines. ^{25 34 49 50 51 52} Indeed, NFκB-mediated expression of IL-8 has been implicated in intraocular neovascularization. ²⁵ NFκB activation is known to be induced by oxidative stress and in our model, generation of intracellular ROS was increased at 12 hours in RPE that had ingested Ox-POS, whereas they were not significantly increased in cells that had ingested latex beads or untreated POS. Intracellular ROS unexpectedly were not increased at 6 hours, suggesting that the effect may be cumulative, as the cells accumulate oxidized outer segments over time. Untreated POS and latex beads did not produce this effect; therefore, it may be due to accumulation of the indigestible aberrant molecular species previously discussed. Incubation with the antioxidant NAC inhibited expression of IL-8 induced by ingestion of oxidized POS; however, this was not the case for MCP-1, suggesting that there may be a different, as yet undetermined, mechanism involved in induction of MCP-1 that is not directly dependent on generation of ROS. The slight reduction in IL-8 and MCP-1 observed in the RPE cells exposed to untreated POS is consistent with previous studies that have demonstrated a reduction in proinflammatory cytokines in phagocytosing cells. ⁵³  

Our experimental model is an artificial system based on inducing lipid peroxidation in isolated POS and studying the effect of their phagocytosis on RPE cell function in vitro. It is not certain whether this occurs in vivo, but this model has been shown to result in accumulation of lipofuscin-like material in cultured RPE and has been used as a model of lipofuscinogenesis (Sidikaro Y, Trüb PR, Morse LS, ARVO Abstract 9, 1988). ²⁴ Our results raise the possibility that expression of IL-8 and MCP-1 may be induced in RPE ingesting oxidatively damaged outer segment material. If this is the case in vivo, it may explain the accumulation of immune cells observed in areas of drusen formation and in excised choroidal neovascular membranes. Immune cells such as macrophages contain proteinases that degrade anatomic barriers for migrating vascular cells and release angiogenic promoters, which could lead to neovascularization into the subretinal space and exudative AMD. ^{45 54} Alternatively, IL-8 and MCP-1 themselves could be directly proangiogenic; they have both been shown to promote angiogenesis in vitro and in vivo, and NFκB-induced IL-8 has been shown to play a role in retinal neovascularization. ^{25 29 30 31 32} Thus, as well as inducing neovascularization by chemotaxis of immune cells, they could directly promote angiogenesis. 

We report a mechanism by which oxidative damage to photoreceptor outer segments and their subsequent ingestion by RPE may lead through cellular stress and subsequent NFκB-mediated cytokine expression to chemotaxis of immune cells and subsequent CNV. 

 Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2001.

 Supported by a grant from Fighting Blindness, Ireland. GTH is currently the Fighting Blindness Research Fellow at the Departments of Ophthalmology and Academic Surgery, University College, Cork.

 Submitted for publication July 22, 2002; revised September 3, 2002; accepted October 24, 2002.

 Disclosure: G.T. Higgins, None; J.H. Wang, None; P. Dockery, None; P.E. Cleary, None; H.P. Redmond, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Quantification of phagocytosis by RPE of FITC-conjugated POS and Ox-POS by flow cytometry. Data are expressed as the mean ± SEM and are representative of results in three separate experiments (*P = 0.0001 vs. control). MCF, mean channel fluorescence.

Figure 2.

 

Electron microscopy showing detail of RPE phagocytic activity (A). (B) Higher magnification shows RPE cell in process of engulfing outer segment material. Note lamellae of rod-cone debris (arrow). (C) Outer segment material visible within the cell (⋆). Bar: (A) 1 μm; (B, C) 0.5 μm.

Figure 3.

 

Confocal microscopy showing phagocytosis of FITC-labeled POS by cultured RPE. Cell nuclei have been counterstained with acridine orange. Three cross sections, 2.5 mm apart, are represented. (A) Apical limit of the cells with some outer segment material outside the cells. (B) A section 2.5 mm below (A) with outer segment material visible within the cytoplasm of an RPE cell, surrounding the nucleus (arrow). (C) A section 2.5 mm below (B) showing that outer segment material is not present at this level (arrow) indicating that it is confined to the apical portion of the cell. (★) A corresponding point in each of the three micrographs.

Figure 4.

 

Cytokine levels in RPE supernatants indicate a significant increase in IL-8 and MCP-1 after challenge with oxidized POS. Data expressed as mean ± SEM and are representative of five separate experiments (P = 0.0001 *vs. control, ^∧vs. POS).

Figure 5.

 

Blocking de novo protein synthesis with actinomycin D (1 mg/mL) eliminates observed cytokine increases. Data are expressed as the mean ± SEM and are representative of five separate experiments (P < 0.0001 *vs. control, ^∧vs. Ox-POS).

Figure 6.

 

NF-κB activity in RPE is significantly increased at 12 hours after challenge with oxidized POS. Data expressed as relative luminescence units (RLU) of luciferase substrate and expressed as the mean ± SEM, representative three separate experiments (P < 0.05 *vs. control, ^∧vs. latex beads, ^@vs. POS).

Figure 7.

 

IL-8 and MCP-1 increases in RPE supernatants after challenge with oxidized POS were blocked by incubation with the NF-κB inhibitor SN50 (100 mg/mL). Data are expressed as the mean ± SEM and are representative of four separate experiments (P < 0.05, *vs. control, ^∧vs. Ox-POS).

Figure 8.

 

Flow cytometric analysis for intracellular ROS using CM-H₂DCFDA indicates a significant increase after challenge with oxidized POS. The data are expressed as the mean ± SEM and are representative of four separate experiments (P < 0.05 *vs. control, ^∧vs. latex beads, ^∼vs. POS).

Figure 9.

 

Incubation of RPE with the antioxidant NAC after exposure to oxidized POS eliminates the increase in IL-8, but not that of MCP-1. Shown are protein levels in RPE supernatants at 18 hours. Left: IL-8; right: MCP-1. Data are expressed as the mean ± SEM and are representative of four separate experiments (P = 0.0001 *vs. control, ^∧vs. Ox-POS).