August 2011
Volume 52, Issue 9
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Biochemistry and Molecular Biology  |   August 2011
The Effect of Photo-oxidative Stress and Inflammatory Cytokine on Complement Factor H Expression in Retinal Pigment Epithelial Cells
Author Affiliations & Notes
  • Ling-Ing Lau
    From the Institute of Clinical Medicine and
    the Departments of Ophthalmology and
    Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan; and
  • Shih-Hwa Chiou
    From the Institute of Clinical Medicine and
    the Departments of Ophthalmology and
    Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan; and
  • Catherine Jui-Ling Liu
    the Departments of Ophthalmology and
    Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan; and
  • May-Yung Yen
    the Departments of Ophthalmology and
    Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan; and
  • Yau-Huei Wei
    From the Institute of Clinical Medicine and
    Biochemistry and Molecular Biology, School of Medicine, National Yang-Ming University, Taipei, Taiwan;
    Department of Medicine, Mackay Medical College, Taipei, Taiwan.
Investigative Ophthalmology & Visual Science August 2011, Vol.52, 6832-6841. doi:10.1167/iovs.11-7815
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      Ling-Ing Lau, Shih-Hwa Chiou, Catherine Jui-Ling Liu, May-Yung Yen, Yau-Huei Wei; The Effect of Photo-oxidative Stress and Inflammatory Cytokine on Complement Factor H Expression in Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(9):6832-6841. doi: 10.1167/iovs.11-7815.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Genetic variation in complement factor H (CFH) has been implicated as a major risk factor for age-related macular degeneration (AMD). The reduction in CFH amount or its complement-modulating activity may lead to inadequate control of complement-driven inflammation at the outer retina. We explored the effect of photo-oxidative stress and inflammatory cytokine on the expression of CFH in retinal pigment epithelial (RPE) cells.

Methods.: Cultured human RPE cells were exposed to blue light in the presence of interferon-γ (IFN-γ). CFH expression in cell lysate was examined by Western blot and the secretory CFH in culture medium was analyzed by ELISA. RPE cells were treated with vitamin C and exogenous superoxide dismutase mimetic (Tempol) before photo-oxidative treatments. The intracellular reactive oxygen species were examined by flow cytometry.

Results.: IFN-γ increased CFH expression in RPE and the expression was suppressed significantly under concomitant blue light illumination. The secretory CFH level also decreased significantly under blue light illumination, which was related to the decreased intracellular mRNA and protein expressions of CFH. The suppression was mediated through an oxidative mechanism, and was particularly related to superoxide anion generation. The suppression of CFH expression in RPE under blue light illumination was abrogated by vitamin C and Tempol.

Conclusions.: Photo-oxidative stress reduces the ability of IFN-γ to increase CFH expression in RPE. Apart from reducing the oxidative damage, vitamin C reduces the suppression of CFH under photo-oxidative stress. These results suggest a new perspective of the interaction between oxidative stress and inflammation, and provide a potential novel treatment strategy for age-related macular degeneration.

Age-related macular degeneration (AMD) is one of the leading causes of blindness among patients aged 50 years or older in developed countries. 1 3 It consists of a spectrum of disease phenotypes including drusen deposits along the Bruch's membrane, geographic atrophy, and choroidal neovascularization. 4  
Recent studies showed that chronic inflammation and complement activation play crucial roles in AMD pathogenesis. 5 There are increased inflammatory cells infiltration at outer retina of macula with advanced AMD. 6 8 Immune-related proteins such as C-reactive protein, immunoglobulin G, vitronectin, and some terminal complement components accumulate in the cytoplasm of retinal pigment epithelium (RPE) in eyes with drusen. 6,9,10 Further identification of the composition of drusen by immunohistochemistry and proteomic studies also showed acute phase reactants, immunoglobulins, activated complement components, and immune-regulatory proteins such as vitronectin and complement factor H (CFH). 5,6,11 13 CFH is a major inhibitor of the alternative complement activation pathway that plays a critical role in driving the inflammatory responses in outer retina and is associated with AMD pathogenesis. 14 16 A number of genetic variants of CFH are associated with the risk of AMD in various ethnic groups. 11,17 21 It is suggested that the reduction of CFH amount or its complement-modulating activity may lead to inadequate control of complement-driven inflammation at the outer retina, which contributes to the pathogenesis of AMD. 22 24  
It was reported that cumulative oxidative stress also plays a significant role in the pathogenesis of AMD. 25 Apart from aging and smoking, the two major risk factors of AMD that are associated with oxidative stress, higher cumulative lifetime exposure to sunlight is also found to increase the risk of AMD. 26 30 Individuals with low levels of serum antioxidants and more blue light exposure are at higher risk of developing neovascular AMD. 31  
RPE defect and loss has been implicated as the key factor of the initiation and progression of AMD. 32 34 Being located in the microenvironment that is constantly exposed to photo-oxidative stress and inflammatory cytokine stimulation, factors that lead to RPE dysfunction can be complicated. In vivo and in vitro studies showed that blue light illumination imposes significant stress on RPE. 32 35 Inflammatory cytokines and leukocyte infiltration can lead to RPE apoptosis. 36,37 However, the effect of simultaneous exposure to blue light-induced photo-oxidative stress and inflammatory cytokine on RPE remain unclear. RPE is an important local source of CFH in outer retina. 11,38 40 Abundant transcripts and proteins of CFH were found in freshly isolated RPE cells. 11,38 40 The decrease in CFH level may lead to excessive complement activation and retinal degeneration. 41 Previous studies showed that phagocytosis of oxidized photoreceptor outer segment 40 and exposure to chemical oxidants 42 reduced the production of CFH in RPE. In the present study, we investigated the effect of inflammatory cytokine and blue light illumination on CFH expression in RPE, which, to the best of our knowledge, has never been determined before. We also investigated the possible mechanisms involved in the modulation of CFH expression under photo-oxidative damage and inflammatory cytokine treatment. 
Methods
Cell Culture
Human adult RPE (ARPE-19; American Type Culture Collection, Manassas, VA) cells were used in this study. The cells were grown in T75 flasks and cultured in Dulbecco modified Eagle medium (DMEM)-F12 medium supplemented with 10% fetal calf serum and penicillin and streptomycin at 37°C in humidified 5% CO2/95% air. All experiments were carried out using RPE cells between 25 and 30 passages. 
Illumination and Treatments
Approximately 5 × 105 cells were seeded in a 6-cm dish for 24 hours before illumination and treatments. The cells were treated with various concentrations (10, 50, and 100 ng/mL) of recombinant human interferon-γ (IFN-γ; Abcam, Cambridge, UK) for 24 hours, after which the cells were lysed to determine the CFH protein level and mRNA expression. To study the effect of concomitant exposure of inflammatory cytokine and photo-oxidative stress on the CFH expression in RPE, the cells were treated with 50 ng/mL IFN-γ and illuminated with blue light. The illumination was delivered by the Super actinic/03 (TLD 15W/03) fluorescent lamp (Philips Lighting Company, Somerset, NJ) with a peak wavelength of 420 nm and a narrow band width in which 90% spectrum distribution was within 400 to 450 nm. 43 The cells were irradiated at 8 mW/cm2 measured at the peak spectrum of 420 nm (OPHIR PD300; Ophir Optronics Inc., Logan, UT) for 30, 45, and 60 minutes, respectively, from the bottom of the culture dish to avoid interference from the red color medium. For some experiments, cells were treated concomitantly with 0.5 mM vitamin C (L-ascorbic acid; Sigma-Aldrich, St. Louis, MO) or 20 hours ahead with 1 mM 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (Tempol; Alfa Aesar, Ward Hill, MA). 
Assay of Cell Viability
Cell viability at 24 hours after treatment was assessed by alamarBlue assay (AbD Serotec, Oxford, UK). Cells were incubated with fresh medium containing alamarBlue cell viability assay reagent at 37°C for 40 minutes. The fluorescence intensity was measured 2 (Victor 1420 Multilabel Counter; PerkinElmer Life and Analytical Sciences, Waltham, MA) with the excitation wavelength at 538 nm and emission wavelength at 590 nm, respectively. 
Western Blot Analysis of CFH
RPE cells were lysed at 24 hours after IFN-γ incubation and blue light illumination. The cell lysate was subjected to Western blot analysis of CFH. Cells were washed twice with phosphate-buffered saline (PBS), lysed on ice with buffer (50 mM HEPES [pH 7.4], 4 mM EDTA [pH 8.0], 2 mM EGTA [pH 8.0], 1% Triton X-100) containing protease inhibitors (Sigma-Aldrich). Cell extracts were then centrifuged at 15,000g for 5 minutes at 4°C, and the supernatant was collected. Protein content in the supernatant was determined using a protein assay reagent (Bradford; Bio-Rad Laboratories, California). An equal amount of protein from each sample was resolved on reducing 10% SDS-PAGE. Western blot analysis was carried out using sheep polyclonal anti-CFH antibody (1:500 dilution; Abcam). Protein bands were detected using an enhanced chemiluminescence kit (Millipore, Billerica, MA) according to the manufacturer's protocol. 
Measurement of Secretory CFH Protein
RPE was seeded at a 5 × 105 cells per mL density in a 6-cm dish. After 24 hours, the cells were gently washed with serum-free medium. The medium was replaced with Dulbecco modified Eagle medium without addition of serum before treatments and illumination. Twenty-four hours after treatments, the serum-free culture medium was collected and concentrated (Amicon Ultra Centrifugal Filter Unit with Ultracel-30 membrane; Millipore). Semiquantitative analysis of the secretory CFH protein in the culture medium was performed by Western blotting. Protein concentrations were quantified by Bradford protein assay and adjusted to allow loading of equal amounts of total proteins on the gel. Quantitative analysis of the secretory CFH concentration in the serum-free culture medium was performed using a commercialized ELISA kit for human CFH (Hycult Biotech, Uden, The Netherlands) according to the manufacturer's instructions. The detection range is 3.9–250 ng/mL. To normalize the secretory CFH in the culture medium with the cell number in each dish, total cellular protein of each dish was determined. Cells in each culture plate were lysed on ice with buffer (50 mM HEPES [pH 7.4], 4 mM EDTA [pH 8.0], 2 mM EGTA [pH 8.0], 1% Triton X-100) containing protease inhibitors (Sigma-Aldrich). Cell extract was then centrifuged at 15,000g for 5 minutes at 4°C, and the supernatant was collected. The protein content in the supernatant was determined using the Bradford protein assay reagent (Bio-Rad Laboratories). The amount of secretory CFH in the culture medium was normalized with the amount of total cellular proteins, expressed as ng/μg (secretory CFH/cellular protein). 
Determination of CFH mRNA Expression by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
The relative expression of CFH mRNA in RPE cells before and after photo-oxidative stress was determined by real-time quantitative PCR. The data were normalized with an internal control, the large P0 subunit of human ribosomal protein. Total RNA was extracted (TRI Reagent; Invitrogen, Carlsbad, CA). Reverse transcription was performed at 42°C for 1 hour using 5 μg of total RNA with RT-PCR beads (Ready-to-Go; GE Health Care, Buckinghamshire, UK). Each 20-μL PCR reaction mixture was prepared (LightCycler FastStart DNA MasterPLUS SYBR Green I kit; Roche Diagnostics, Mannheim, Germany; and DyNAmo ™ Capillary SYBR® Green qPCR Kit; Finnzymes, Vantaa, Finland). The sequences of the primer pair used for the determination of CFH gene expression were 5′-TTGCACACAAGATGGATGGT-3′ and 5′-GGATGCATCTGGGAGTAGGA-3′, which were designed to hybridize in two different exons to prevent amplification of any remaining genomic DNA. 41 The sequences of the primer pair used to amplify the internal control were 5′-CGACCTGGAAGTCCAACTAC-3′ and 5′-ATCTGCTGCATCTGCTTG-3′. 42 The amplification conditions were: denaturation at 95°C for 10 seconds, annealing at 56°C for 15 seconds, and extension at 72°C for 20 seconds for a total of 40 cycles. Sequencing of the PCR products was performed to confirm the amplification of CFH mRNA. 
Determination of Intracellular Reactive Oxygen Species (ROS)
For measurement of the intracellular levels of hydrogen peroxide (H2O2) and superoxide anion (O2 -. ), cells were incubated in a medium containing 20 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCF; Invitrogen) and 10 μg/mL hydroethidine (HE; Invitrogen), respectively, at 37°C in the dark for 10 minutes. Cells were then resuspended in PBS, and the fluorescence intensity of 10,000 cells was recorded on a flow cytometer (model EPICS XL-MCL; Beckman, Brea, CA) with the excitation wavelength at 488 nm and emission wavelengths at 535 nm and 580 nm for the measurement of H2O2 and O2 -. , respectively. 
Immunofluorescent Staining
The cells were seeded in a culture dish with coverslips at 24 hours before treatment. Twenty-four hours after various treatments, coverslips with RPE cells were washed three times with cold PBS. The cells were fixed for 10 minutes at room temperature with 4% paraformaldehyde-PBS, and permeabilized for 5 minutes with 0.2% Triton X-100 PBS. After blocking with 5% BSA, the cells were incubated with the primary antibody (sheep anti-human CFH polyclonal antibody; Abcam) at 1:250 dilution in Tris-buffered saline (TBS) for 90 minutes at room temperature, followed by the secondary antibody (Cy3-conjugated donkey anti-sheep IgG; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) at 1:400 dilution in TBS for another hour. Each step was preceded by a three-time wash in PBS. To stain the nucleus, cells were incubated with 40 μg/mL 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 20 minutes at room temperature. The coverslips were then mounted on glass slides and examined at room temperature with a confocal laser scanning biological microscope (FV1000; Olympus, Tokyo, Japan). 
Statistical Analysis
Statistical analysis was performed (SPSS version 18; SPSS Inc.,Chicago, IL). The data were presented as mean ± SD of the results from three independent experiments. The significance level was determined by one-way analysis of variance (ANOVA) with Bonferroni correction and Dunnett's correction for multiple comparisons. A difference was considered statistically significant at P < 0.05. 
Results
CFH Expression in RPE under Concomitant Exposure of IFN-γ and Blue Light Illumination
The morphology and viability of RPE cells did not change under different concentrations of IFN-γ (Figs. 1A and 1B). Western blotting of the RPE cell lysates after 24 hours of incubation with IFN-γ revealed the presence of a CFH immunoreactive band at 155 kDa. The expression of CFH in RPE cell lysate was significantly upregulated by various concentrations of IFN-γ, and the maximal effect achieved at 50 ng/mL (Figs. 1C and 1D). Furthermore, to study the effect of photo-oxidative stress and inflammatory cytokine on the expression of CFH, RPE was incubated in 50 ng/mL IFN-γ and exposed concomitantly to 30, 45, and 60 minutes of 8 mW/cm2 blue light, respectively. RPE cells that were exposed to 30 minutes blue light illumination did not show significant change in morphology with mild decrease in cell viability; while cells that were exposed to 45 and 60 minutes blue light illumination showed marked decrease in viability with vacuolization and loss of normal epithelioid cell structure (Figs. 2A and 2B). Western blotting of the RPE cell lyastes at 24 hours after blue light illumination showed a dose-dependent suppression of CFH expression (Figs. 2C and 2D). Semiquantitative analysis of the secretory CFH in RPE culture medium by Western blotting also showed a dose-dependent suppression under blue light illumination and 50 ng/mL IFN-γ incubation (Fig. 2E). Blue light illumination had the same suppressive effect on IFN–γ-stimulated CFH expression in both serum-free and serum-containing media (Fig. 2F). These results suggest that blue light illumination reduced the ability of IFN-γ to increase CFH expression in RPE cells in a dose-dependent manner. Both the cellular and secreted CFH protein levels decreased significantly under blue light illumination. 
Figure 1.
 
The effect of recombinant human IFN-γ on the expression of CFH in RPE. Cells were incubated in 0, 10, 50, or 100 ng/mL IFN-γ, respectively. Twenty-four hours later, RPE lysates were used in Western blot analysis for CFH and GAPDH. (A and B) Cell morphology and viability did not change under various concentrations of IFN-γ incubation. Bars represent 50 μm. (C and D) CFH expression was significantly upregulated by various concentrations of IFN-γ with a maximal effect at 50 ng/mL, as shown in the blots and their relative intensities (* P < 0.001, compared with the untreated control by ANOVA with Dunnett's correction for multiple comparisons). All data were obtained from three independent experiments and expressed as mean ± SD.
Figure 1.
 
The effect of recombinant human IFN-γ on the expression of CFH in RPE. Cells were incubated in 0, 10, 50, or 100 ng/mL IFN-γ, respectively. Twenty-four hours later, RPE lysates were used in Western blot analysis for CFH and GAPDH. (A and B) Cell morphology and viability did not change under various concentrations of IFN-γ incubation. Bars represent 50 μm. (C and D) CFH expression was significantly upregulated by various concentrations of IFN-γ with a maximal effect at 50 ng/mL, as shown in the blots and their relative intensities (* P < 0.001, compared with the untreated control by ANOVA with Dunnett's correction for multiple comparisons). All data were obtained from three independent experiments and expressed as mean ± SD.
Figure 2.
 
Blue light illumination reduced IFN–γ-stimulated CFH expression in a dose-dependent effect. RPE cells were incubated with 50 ng/mL IFN-γ alone or illuminated concomitantly with 420 nm blue light with an intensity of 8 mW/cm2 for 30, 45, and 60 minutes, respectively. Western blotting and cell viability and morphology evaluations were performed after 24 hours. (A) The morphology of the RPE cells exposed to 30 minutes blue light illumination did not change significantly at 24 hours. RPE cells exposed to 45 and 60 minutes blue light illumination showed significant cell shrinkage. Bars represent 50 μm. (B) There was a dose-dependent decrease in RPE cell viability at 24 hours after blue light illumination (* P < 0.001, compared with the IFN–γ-treated RPE cells by ANOVA with Dunnett's correction for multiple comparisons). (C and D) There was a dose-dependent suppression of the CFH expression in RPE cells after different durations of blue light illumination, as shown in the blots and their relative intensities (* P < 0.001, compared with the IFN–γ-treated RPE cells by ANOVA with Dunnett's correction for multiple comparisons). (E) Semiquantitative analysis of the secretory CFH in RPE culture medium by Western blotting also showed a dose-dependent suppression by blue light illumination. (F) Western blot for CFH in RPE incubated with 50 ng/mL IFN-γ in serum-free or serum-containing media and illuminated concomitantly with 420 nm blue light with an intensity of 8 mW/cm2 for 30 minutes. Blue light illumination had the same suppressive effect on IFN-γ-stimulated CFH expression in both serum-free and serum-containing media. All data were obtained from three independent experiments and expressed as mean ± SD.
Figure 2.
 
Blue light illumination reduced IFN–γ-stimulated CFH expression in a dose-dependent effect. RPE cells were incubated with 50 ng/mL IFN-γ alone or illuminated concomitantly with 420 nm blue light with an intensity of 8 mW/cm2 for 30, 45, and 60 minutes, respectively. Western blotting and cell viability and morphology evaluations were performed after 24 hours. (A) The morphology of the RPE cells exposed to 30 minutes blue light illumination did not change significantly at 24 hours. RPE cells exposed to 45 and 60 minutes blue light illumination showed significant cell shrinkage. Bars represent 50 μm. (B) There was a dose-dependent decrease in RPE cell viability at 24 hours after blue light illumination (* P < 0.001, compared with the IFN–γ-treated RPE cells by ANOVA with Dunnett's correction for multiple comparisons). (C and D) There was a dose-dependent suppression of the CFH expression in RPE cells after different durations of blue light illumination, as shown in the blots and their relative intensities (* P < 0.001, compared with the IFN–γ-treated RPE cells by ANOVA with Dunnett's correction for multiple comparisons). (E) Semiquantitative analysis of the secretory CFH in RPE culture medium by Western blotting also showed a dose-dependent suppression by blue light illumination. (F) Western blot for CFH in RPE incubated with 50 ng/mL IFN-γ in serum-free or serum-containing media and illuminated concomitantly with 420 nm blue light with an intensity of 8 mW/cm2 for 30 minutes. Blue light illumination had the same suppressive effect on IFN-γ-stimulated CFH expression in both serum-free and serum-containing media. All data were obtained from three independent experiments and expressed as mean ± SD.
Effect of Vitamin C on the Blue Light-Induced CFH Suppression in RPE
To further investigate the role of oxidative stress in the suppression of CFH under blue light illumination, 0.5 mM vitamin C was added into the medium before illumination. The addition of vitamin C restored the cell viability under blue light illumination (Fig. 3A). Cellular and secretory protein levels of CFH decreased significantly under blue light illumination and the addition of vitamin C restored the protein levels (Figs. 3B, 3C, and 3D). The CFH mRNA expression in RPE cells also decreased significantly under blue light illumination and the suppression was reduced by adding vitamin C (Fig. 3E). These data indicate that blue light-induced photo-oxidative stress suppressed mRNA and protein expression of CFH in RPE cells, and the suppression was mediated through an oxidative mechanism because it was abrogated by vitamin C, which is a major antioxidant and free-radical scavenger in the human blood stream. 44  
Figure 3.
 
Vitamin C abrogated the blue light suppression of CFH in RPE incubated with IFN-γ. RPE cells were treated with 0.5 mM vitamin C and 50 ng/mL IFN-γ, and exposed to 30 minutes 8-mW/cm2 blue light illumination. (A) Cell viability of the blue-light irradiated RPE at 24 hours later showed significant reduction compared with the non-irradiated RPE cells. Vitamin C coincubation had a significant rescue effect on the cell viability suppression under blue light illumination (* P < 0.001, compared with the IFN–γ-treated RPE cells by ANOVA with Bonferroni correction for multiple comparisons; ** P < 0.05, compared with the IFN-γ treated RPE cells exposed to 30 minutes blue light illumination by ANOVA with Bonferroni correction for multiple comparisons). (B and C) Western blotting of CFH in RPE cell lysates at 24 hours showed a significant suppression of CFH under blue light illumination and the suppression was significantly abrogated by 0.5 mM vitamin C coincubation (* P < 0.001, ANOVA with Bonferroni correction for multiple comparisons). (D) Quantitative analysis of the secretory CFH in RPE culture medium by ELISA also showed a significant suppression under blue light illumination. The suppression was reduced significantly by 0.5 mM vitamin C coincubation (* P < 0.001, ANOVA with Bonferroni correction for multiple comparisons). (E) The changes in CFH mRNA expression at 24 hours later were analyzed by real-time-PCR. There was a significant upregulation of CFH mRNA expression in RPE under IFN-γ incubation. Concomitant blue light illumination downregulated the CFH mRNA expression and the suppression was abrogated by 0.5 mM vitamin C coincubation (* P < 0.001, ANOVA with Bonferroni correction for multiple comparisons). All data were obtained from three independent experiments, expressed as mean ± SD.
Figure 3.
 
Vitamin C abrogated the blue light suppression of CFH in RPE incubated with IFN-γ. RPE cells were treated with 0.5 mM vitamin C and 50 ng/mL IFN-γ, and exposed to 30 minutes 8-mW/cm2 blue light illumination. (A) Cell viability of the blue-light irradiated RPE at 24 hours later showed significant reduction compared with the non-irradiated RPE cells. Vitamin C coincubation had a significant rescue effect on the cell viability suppression under blue light illumination (* P < 0.001, compared with the IFN–γ-treated RPE cells by ANOVA with Bonferroni correction for multiple comparisons; ** P < 0.05, compared with the IFN-γ treated RPE cells exposed to 30 minutes blue light illumination by ANOVA with Bonferroni correction for multiple comparisons). (B and C) Western blotting of CFH in RPE cell lysates at 24 hours showed a significant suppression of CFH under blue light illumination and the suppression was significantly abrogated by 0.5 mM vitamin C coincubation (* P < 0.001, ANOVA with Bonferroni correction for multiple comparisons). (D) Quantitative analysis of the secretory CFH in RPE culture medium by ELISA also showed a significant suppression under blue light illumination. The suppression was reduced significantly by 0.5 mM vitamin C coincubation (* P < 0.001, ANOVA with Bonferroni correction for multiple comparisons). (E) The changes in CFH mRNA expression at 24 hours later were analyzed by real-time-PCR. There was a significant upregulation of CFH mRNA expression in RPE under IFN-γ incubation. Concomitant blue light illumination downregulated the CFH mRNA expression and the suppression was abrogated by 0.5 mM vitamin C coincubation (* P < 0.001, ANOVA with Bonferroni correction for multiple comparisons). All data were obtained from three independent experiments, expressed as mean ± SD.
The Role of ROS in the Blue Light-Induced CFH Suppression in RPE
We further investigated the ROS primarily involved in the blue light-induced CFH suppression in IFN–γ-treated RPE. Intracellular ROS levels were determined at 3 and 6 hours after IFN-γ incubation and blue light illumination. The O2 -. level, detected by HE stain, increased significantly under blue light illumination and the level reduced with vitamin C coincubation (Fig. 4A). However, the level of H2O2 did not change under different treatments (Fig. 4B). Similar results of HE and DCF staining of intracellular ROS were obtained at 6 hours after treatments (data not shown). The role of O2 -. in the suppression of CFH was further confirmed by the addition of Tempol (Alfa Aesar), which can catalyze the conversion of O2 -. to H2O2 that can be turned into water by catalase or the glutathione peroxidase system. 45 Pretreatment of the RPE cells with this membrane-permeable free radical scavenger significantly reduced the CFH suppression under blue light illumination (Figs. 5A and 5B), and the effect was related to the reduction of intracellular O2 . anions (Fig. 5C). The H2O2 level did not change significantly under different treatments (Fig. 5D). These results suggest that the suppression of CFH under blue light illumination was primarily mediated through the increased generation of intracellular superoxide anions. 
Figure 4.
 
Vitamin C abrogated the blue light suppression of CFH expression in RPE cells incubated with 50 ng/mL IFN-γ through superoxide anion quenching effect. Three hours after 30 minutes 8 mW/cm2 blue light illumination and 50 ng/mL IFN-γ incubation, with or without 0.5 mM vitamin C coincubation, RPE cells were stained with 10 μg/mL HE and 20 μM DCF to detect the intracellular O2 -. and H2O2 levels, respectively, by flow cytometry. RPE cells without any drug or light treatment served as the control. The fluorescence intensity was expressed as the percentage of control. (A) Histogram of the HE stain showed prominent right shift of the fluorescence under blue light illumination. Coincubation with 0.5 mM vitamin C decreased the fluorescent intensity markedly with significant left shift of the fluorescence. (B) The fluorescence level under DCF stain did not change by various treatments. Bar charts showed the mean fluorescent intensity from three independent experiments, expressed as mean ± SD. * P < 0.001 by ANOVA with Bonferroni correction for multiple comparisons.
Figure 4.
 
Vitamin C abrogated the blue light suppression of CFH expression in RPE cells incubated with 50 ng/mL IFN-γ through superoxide anion quenching effect. Three hours after 30 minutes 8 mW/cm2 blue light illumination and 50 ng/mL IFN-γ incubation, with or without 0.5 mM vitamin C coincubation, RPE cells were stained with 10 μg/mL HE and 20 μM DCF to detect the intracellular O2 -. and H2O2 levels, respectively, by flow cytometry. RPE cells without any drug or light treatment served as the control. The fluorescence intensity was expressed as the percentage of control. (A) Histogram of the HE stain showed prominent right shift of the fluorescence under blue light illumination. Coincubation with 0.5 mM vitamin C decreased the fluorescent intensity markedly with significant left shift of the fluorescence. (B) The fluorescence level under DCF stain did not change by various treatments. Bar charts showed the mean fluorescent intensity from three independent experiments, expressed as mean ± SD. * P < 0.001 by ANOVA with Bonferroni correction for multiple comparisons.
Figure 5.
 
Tempol (Alfa Aesar), a membrane-permeable free-radical scavenger, reversed the blue light-induced CFH suppression in IFN–γ-treated RPE mainly through the superoxide anion (O2 -. ) quenching effect. RPE was incubated with 1 mM Tempol for 20 hours before being treated with 50 ng/mL IFN-γ and 30 minutes 8-mW/cm2 blue light illumination. (A and B) Western blotting of RPE lysates at 24 hours later showed a significant suppression of IFN-γ-stimulated CFH expression under blue light illumination, and the suppression was significantly reduced by pretreatment with Tempol (* P < 0.001 by ANOVA with Bonferroni correction for multiple comparisons). (C) RPE cells were stained with 10 μg/mL HE to detect the intracellular O2 -. level by flow cytometry. RPE without any drug or light treatment served as the control. The fluorescence intensity was expressed as the percentage of control. The fluorescence intensity of HE stain increased significantly under blue light illumination, and pretreatment with 1 mM Tempol reduced the fluorescence intensity markedly (* P < 0.001 by ANOVA with Bonferroni correction for multiple comparisons). (D) RPE cells were stained with 20 μM DCF to detect the intracellular H2O2 levels by flow cytometry. The fluorescence level under DCF stain did not change significantly under various treatments. All data were obtained from three independent experiments, expressed as mean ± SD.
Figure 5.
 
Tempol (Alfa Aesar), a membrane-permeable free-radical scavenger, reversed the blue light-induced CFH suppression in IFN–γ-treated RPE mainly through the superoxide anion (O2 -. ) quenching effect. RPE was incubated with 1 mM Tempol for 20 hours before being treated with 50 ng/mL IFN-γ and 30 minutes 8-mW/cm2 blue light illumination. (A and B) Western blotting of RPE lysates at 24 hours later showed a significant suppression of IFN-γ-stimulated CFH expression under blue light illumination, and the suppression was significantly reduced by pretreatment with Tempol (* P < 0.001 by ANOVA with Bonferroni correction for multiple comparisons). (C) RPE cells were stained with 10 μg/mL HE to detect the intracellular O2 -. level by flow cytometry. RPE without any drug or light treatment served as the control. The fluorescence intensity was expressed as the percentage of control. The fluorescence intensity of HE stain increased significantly under blue light illumination, and pretreatment with 1 mM Tempol reduced the fluorescence intensity markedly (* P < 0.001 by ANOVA with Bonferroni correction for multiple comparisons). (D) RPE cells were stained with 20 μM DCF to detect the intracellular H2O2 levels by flow cytometry. The fluorescence level under DCF stain did not change significantly under various treatments. All data were obtained from three independent experiments, expressed as mean ± SD.
Immunofluorescent Staining of CFH
We further studied the distribution and the change of intracellular CFH under IFN-γ treatment and blue light illumination. The constitutive CFH protein expression in RPE cells was undetectable with immunofluorescent staining (Fig. 6B), and the expression increased significantly after treatment with 50 ng/mL IFN-γ (Fig. 6C). The protein was located mainly around the perinuclear area and distributed in a granular pattern, suggesting encapsulation with secretory vacuoles. The intracellular CFH expression decreased markedly under blue light illumination, and the suppression was reduced significantly by vitamin C coincubation (Figs. 6D and 6E). Taking together, these findings suggest that the decrease in the secretory CFH under blue light illumination was related to the reduction of intracellular CFH mRNA and protein expression, and was not related to the interference of the secretory process of the protein. 
Figure 6.
 
Immunolocalization of CFH in RPE cells at 24 hours after various treatments. The cells were stained for CFH (red) and nucleus (DAPI; blue) and observed by confocal microscope. (A) RPE cells that were not stained with primary antibody for CFH served as the negative control. (B) The constitutive CFH expression in cultured human RPE cells was barely detectable by immunofluorescent stain. (C) The CFH expression in RPE increased markedly under the treatment of 50 ng/mL IFN-γ. The protein was distributed in a granular pattern in the cytoplasm, mainly around the perinuclear area. (D) Concomitant 30 minutes 8-mW/cm2 blue light illumination significantly reduced the IFN–γ-stimulated CFH expression in RPE. (E) Coincubation of 0.5 mM vitamin C abrogated the suppression of CFH under blue light illumination.
Figure 6.
 
Immunolocalization of CFH in RPE cells at 24 hours after various treatments. The cells were stained for CFH (red) and nucleus (DAPI; blue) and observed by confocal microscope. (A) RPE cells that were not stained with primary antibody for CFH served as the negative control. (B) The constitutive CFH expression in cultured human RPE cells was barely detectable by immunofluorescent stain. (C) The CFH expression in RPE increased markedly under the treatment of 50 ng/mL IFN-γ. The protein was distributed in a granular pattern in the cytoplasm, mainly around the perinuclear area. (D) Concomitant 30 minutes 8-mW/cm2 blue light illumination significantly reduced the IFN–γ-stimulated CFH expression in RPE. (E) Coincubation of 0.5 mM vitamin C abrogated the suppression of CFH under blue light illumination.
Discussion
Blue light-induced photo-oxidative damage and inflammation have been implicated to impose significant stress on RPE and lead to RPE dysfunction, which is a significant component of the initiation and progression of AMD. 32 37 To our knowledge, the interaction between these two factors and their effects on RPE have not been explored before. Our study showed that blue light-induced photo-oxidative stress reduced the ability of IFN-γ to increase CFH expression in RPE. Both the secretory and cellular CFH protein levels decreased significantly under blue light illumination. The suppression was related to the decrease in CFH mRNA expression and was mediated through superoxide anion generation under blue light illumination. Vitamin C and Tempol (Alfa Aesar) abrogated the suppression of CFH under photo-oxidative stress. To the best of our knowledge, this study is the first to explore the effect of photo-oxidative stress and inflammatory cytokine on the expression of CFH in RPE. 
IFN-γ is a proinflammatory cytokine that plays important roles in both innate and adaptive immunity. 46 It is secreted by various immune-related cells including the dendritic cell that is strongly associated with drusen biogenesis. 12 The immunomodulating effect of IFN-γ at the outer retina is complex and delicate. It alters the antigenicity of RPE cells, activates and recruits leukocytes, and magnifies local inflammatory response in the outer retina. 47 49 On the other hand, IFN-γ may also play a protective role in the immune response by upregulating the expression of CFH to protect RPE from aberrant complement activation. 39,42 Wu et al. 42 and our study showed that IFN-γ upregulates CFH expression in cultured human RPE cells. Kim et al. 39 found similar results in primary culture cell lines of human RPE. RPE is regarded an important local source of CFH in the outer retina due to the abundant mRNA and protein expressions of CFH detected in freshly isolated RPE and primary RPE cell lines. 38 40 The constitutive CFH expression of the cultured human RPE cell lines that was used in our study and the study of Wu et al. 42 is low compared with the primary cell lines derived from human RPE. 38 40 However, the response to IFN-γ incubation is similar in these two types of human RPE cell lines. The IFN-γ-induced CFH upregulation may protect RPE from the inflammatory damage and complement-mediated lysis of cells. 
RPE is constantly exposed to tremendous oxidative stress due to its high metabolic rate and the high cumulative irradiation at the outer retina. 50 52 Both in vivo and in vitro studies showed that blue light illumination imposes significant stress on RPE and leads to RPE dysfunction. 32 34 Apart from causing direct damage to RPE, our study showed that blue light illumination reduced the ability of IFN-γ to increase CFH expression in RPE cells. Other sources of oxidative stress, such as phagocytosis of oxidized photoreceptor outer segment 40 and exposure to chemical oxidants 42 also reduce the production of CFH in RPE. The decrease in CFH expression may lead to aberrant complement activation and RPE dysfunction. 5,41 The compromised RPE may recruit and activate dendritic cells in choroid which activate the immune system and contribute to drusen biogenesis. 5,6,53  
Previous studies revealed that CFH is encapsulated and secreted from the apical portion of RPE, and IFN-γ promotes the secretion. 39 The apical secretion of CFH may promote a CFH gradient that is highest in the subretinal space where it may protect photoreceptors from inappropriate complement activation. 39 Our study showed that the CFH secreted from RPE decreased significantly under photo-oxidative stress, and the decrease was related to the reduced intracellular CFH mRNA and protein expression under photo-oxidative stress but not related to the interference of the secretory process of the protein. This may lead to aberrant complement activation and result in significant retinal degeneration. 41 Animal study showed that the damage caused by photo-oxidative stress on retina is mediated through the alternative complement activation pathway. 54 As an important negative regulator of the alternative pathway, the decrease in CFH expression under photo-oxidative stress may aggravate the light-induced damage on retina. 
Human retina is protected from ultraviolet light by cornea and lens, but can be damaged by visible light, especially by the relatively high energy blue light spectrum. 55,56 The chromophores in RPE formed by rhodopsin intermediates in the photoreceptor outer segments have been regarded as the major source of free radicals under blue light illumination. 30,57 However, blue light illumination can also damage the lipofuscin-free RPE by inducing the production of ROS in mitochondria. 32,35 In addition to causing direct damage to RPE, our study showed that the ROS generated under blue light illumination downregulated the expression of CFH in RPE. STAT1 is the transcription factor that mediates the IFN-γ-induced CFH upregulation in RPE cells. 58 It was shown that when RPE cells were treated with H2O2, FOXO3 of the Forkhead transcription factor family was acetylated and had increased affinity to the promoter region of CFH. 42 Increased binding of the acetylated FOXO3 displaced STAT1 from the promoter region and suppressed CFH transcription. 42 However, our study showed that intracellular superoxide anion generation was directly associated with CFH suppression under blue light illumination and the intracellular H2O2 level did not change throughout the experiments. Further study is warranted to establish the detail mechanism of blue light-induced CFH suppression in IFN–γ-treated RPE cells. Our study results further showed that the suppression of CFH expression under blue light illumination was abrogated by vitamin C and Tempol (Alfa Aesar). Large epidemiologic studies showed that dietary vitamin C supplementation together with other antioxidants have a protective role against AMD. 59,60 Animal studies also showed that vitamin C and Tempol have protective roles against light-induced retinal damage. 61,62 This may be related to the modulation of CFH expression in RPE because previous study showed that alternative complement activation plays a crucial role in generating light-induced retinal damage. 54 These results suggested that in addition to its antioxidative effect, vitamin C could modify the inflammatory response in outer retina through the modulation of CFH expression in RPE. 
In conclusion, our study showed that blue light-induced photo-oxidative stress reduced the ability of IFN-γ to upregulate CFH expression in RPE. The decrease in CFH expression may aggravate the immune-mediated damage of RPE under photo-oxidative stress. In addition to its antioxidative effect, vitamin C may have a protective role against inflammatory damage due to its modulating effect on CFH expression in RPE. These results show a new perspective of the mutual influence of oxidative stress and inflammation, and provide a potential novel treatment strategy for AMD. 
Footnotes
 Supported by Grants VGH V99B2-001 from Taipei Veterans General Hospital, NSC 96-2314-B-075-050-MY2 and NSC97-2320-B-010-013-MY3 from National Science Council, Taiwan.
Footnotes
 Disclosure: L.-I. Lau, None; S.-H. Chiou, None; C.J. Liu, None; M.-Y. Yen, None; Y.-H. Wei, None
The authors thank Sian-Min Tong and Jia-Sin Yu for laboratory assistance. 
References
Kocur I Resnikoff S . Visual impairment and blindness in Europe and their prevention. Br J Ophthalmol. 2002;86:716–722. [CrossRef] [PubMed]
Friedman DS O'Colmain BJ Munoz B . Prevalence of age-related macular degeneration in the United States. Arch Ophthalmol. 2004;122:564–572. [CrossRef] [PubMed]
Chen SJ Cheng CY Peng KL . Prevalence and associated risk factors of age-related macular degeneration in an elderly Chinese population in Taiwan: the Shihpai Eye Study. Invest Ophthalmol Vis Sci. 2008;49:3126–3133. [CrossRef] [PubMed]
Gass JDM . Stereoscopic Atlas of Macular Diseases: Diagnosis and Management. 4th ed. St. Louis, MO: Mosby-Year Book Inc; 1997:70–105.
Donoso LA Kim D Frost A Callahan A Hageman G . The role of inflammation in the pathogenesis of age-related macular degeneration. Surv Ophthalmol. 2006;51:137–152. [CrossRef] [PubMed]
Anderson DH Mullins RF Hageman GS Johnson LV . A role for local inflammation in the formation of drusen in the aging eye. Am J Ophthalmol. 2002;134:411–431. [CrossRef] [PubMed]
Dastgheib K Green WR . Granulomatous reaction to Bruch's membrane in age-related macular degeneration. Arch Ophthalmol. 1994;112:813–818. [CrossRef] [PubMed]
Penfold PL Killingsworth MC Sarks SH . Senile macular degeneration: the involvement of immunocompetent cells. Graefes Arch Clin Exp Ophthalmol. 1985;223:69–76. [CrossRef] [PubMed]
Anderson DH Ozaki S Nealon M . Local cellular sources of apolipoprotein E in the human retina and retinal pigmented epithelium: implications for the process of drusen formation. Am J Ophthalmol. 2001;131:767–781. [CrossRef] [PubMed]
Johnson LV Ozaki S Staples MK Erickson PA Anderson DH . A potential role for immune complex pathogenesis in drusen formation. Exp Eye Res. 2000;70:441–449. [CrossRef] [PubMed]
Hageman GS Anderson DH Johnson LV . A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration. Proc Natl Acad Sci USA. 2005;102:7227–7232. [CrossRef] [PubMed]
Hageman GS Luthert PJ Victor Chong NH Johnson LV Anderson DH Mullins RF . An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch's membrane interface in aging and age-related macular degeneration. Prog Retin Eye Res. 2001;20:705–732. [CrossRef] [PubMed]
Crabb JW Miyagi M Gu X . Drusen proteome analysis: an approach to the etiology of age-related macular degeneration. Proc Natl Acad Sci U S A. 2002;99:14682–14687. [CrossRef] [PubMed]
de Córdoba SR Esparza-Gordillo J de Jorge EG Lopez-Trascasa M Sánchez-Corral P . The human complement factor H: functional roles, genetic variations and disease associations. Mol Immunol. 2004;41:355–367. [CrossRef] [PubMed]
Bora NS Kaliappan S Jha P . Complement activation via alternative pathway is critical in the development of laser-induced choroidal neovascularization: role of factor B and factor H. J Immunol. 2006;177:1872–1878. [CrossRef] [PubMed]
Rohrer B Long Q Coughlin B . A targeted inhibitor of the alternative complement pathway reduces angiogenesis in a mouse model of age-related macular degeneration. Invest Ophthalmol Vis Sci. 2009;50:3056–3064. [CrossRef] [PubMed]
Klein RJ Zeiss C Chew EY . Complement factor H polymorphism in age-related macular degeneration. Science. 2005;308:385–389. [CrossRef] [PubMed]
Edwards AO Ritter R3rd Abel KJ Manning A Panhuysen C Farrer LA . Complement factor H polymorphism and age-related macular degeneration. Science. 2005;308:421–424. [CrossRef] [PubMed]
Haines JL Hauser MA Schmidt S . Complement factor H variant increases the risk of age-related macular degeneration. Science. 2005;308:419–421. [CrossRef] [PubMed]
Lau LI Chen SJ Cheng CY . Association of the Y402H polymorphism in complement factor H gene and neovascular age-related macular degeneration in Chinese patients. Invest Ophthalmol Vis Sci. 2006;47:3242–3246. [CrossRef] [PubMed]
Kim NR Kang JH Kwon OW Lee SJ Oh JH Chin HS . Association between complement factor H gene polymorphisms and neovascular age-related macular degeneration in Koreans. Invest Ophthalmol Vis Sci. 2008;49:2071–2076. [CrossRef] [PubMed]
Skerka C Lauer N Weinberger AA . Defective complement control of factor H (Y402H) and FHL-1 in age-related macular degeneration. Mol Immunol. 2007;44:3398–3406. [CrossRef] [PubMed]
Ormsby RJ Ranganathan S Tong JC . Functional and structural implications of the complement factor H Y402H polymorphism associated with age-related macular degeneration. Invest Ophthalmol Vis Sci. 2008;49:1763–1770. [CrossRef] [PubMed]
Laine M Jarva H Seitsonen S . Y402H polymorphism of complement factor H affects binding affinity to C-reactive protein. J Immunol. 2007;178:3831–3836. [CrossRef] [PubMed]
Beatty S Koh H Phil M Henson D Boulton M . The role of oxidative stress in the pathogenesis of age-related macular degeneration. Surv Ophthalmol. 2000;45:115–134. [CrossRef] [PubMed]
Cruickshanks KJ Klein R Klein BE . Sunlight and age-related macular degeneration. The Beaver Dam Eye Study. Arch Ophthalmol. 1993;111:514–518. [CrossRef] [PubMed]
Darzins P Mitchell P Heller RF . Sun exposure and age-related macular degeneration. An Australian case-control study. Ophthalmology. 1997;104:770–776. [CrossRef] [PubMed]
Taylor HR Munoz B West S . Visible light and risk of age-related macular degeneration. Trans Am Ophthalmol Soc. 1989;88:163–173.
Taylor HR West S Munoz B . The long-term effects of visible light on the eye. Arch Ophthalmol. 1992;110:99–104. [CrossRef] [PubMed]
Cruickshanks KJ Klein R Klein BE Nondahl DM . Sunlight and the 5-year incidence of early age-related maculopathy: the beaver dam eye study. Arch Ophthalmol. 2001;119:246–250. [PubMed]
Fletcher AE Bentham GC Agnew M . Sunlight exposure, antioxidants, and age-related macular degeneration. Arch Ophthalmol. 2008;126:1396–1403. [CrossRef] [PubMed]
Godley BF Shamsi FA Liang FQ Jarrett SG Davies S Boulton M . Blue light induces mitochondrial DNA damage and free radical production in epithelial cells. J Biol Chem. 2005;280:21061–21066. [CrossRef] [PubMed]
Espinosa-Heidmann DG Sall J Hernandez EP Cousins SW . Basal laminar deposit formation in APO B100 transgenic mice: complex interactions between dietary fat, blue light, and vitamin E. Invest Ophthalmol Vis Sci. 2004;45:260–266. [CrossRef] [PubMed]
Sparrow JR Zhou J Ben-Shabat S Vollmer H Itagaki Y Nakanishi K . Involvement of oxidative mechanisms in blue-light-induced damage to A2E-laden RPE. Invest Ophthalmol Vis Sci. 2002;43:1222–1227. [PubMed]
King A Gottlieb E Brooks DG Murphy MP Dunaief JL . Mitochondria-derived reactive oxygen species mediate blue light-induced death of retinal pigment epithelial cells. Photochem Photobiol. 2004;79:470–475. [CrossRef] [PubMed]
Yang D Elner SG Lin LR Reddy VN Petty HR Elner VM . Association of superoxide anions with retinal pigment epithelial cell apoptosis induced by mononuclear phagocytes. Invest Ophthalmol Vis Sci. 2009;50:4998–5005. [CrossRef] [PubMed]
Yoshida A Elner SG Bian ZM Kindezelskii AL Petty HR Elner VM . Activated monocytes induce human retinal pigment epithelial cell apoptosis through caspase-3 activation. Lab Invest. 2003;83:1117–1129. [CrossRef] [PubMed]
Mandal MN Ayyagari R . Complement factor H: spatial and temporal expression and localization in the eye. Invest Ophthalmol Vis Sci. 2006;47:4091–4097. [CrossRef] [PubMed]
Kim YH He S Kase S Kitamura M Ryan SJ Hinton DR . Regulated secretion of complement factor H by RPE and its role in RPE migration. Graefes Arch Clin Exp Ophthalmol. 2009;247:651–659. [CrossRef] [PubMed]
Chen M Forrester JV Xu H . Synthesis of complement factor H by retinal pigment epithelial cells is down-regulated by oxidized photoreceptor outer segments. Exp Eye Res. 2007;84:635–645. [CrossRef] [PubMed]
Coffey PJ Gias C McDermott CJ . Complement factor H deficiency in aged mice causes retinal abnormalities and visual dysfunction. Proc Natl Acad Sci U S A. 2007;104:16651–16656. [CrossRef] [PubMed]
Wu Z Lauer TW Sick A Hackett SF Campochiaro PA . Oxidative stress modulates complement factor H expression in retinal pigmented epithelial cells by acetylation of FOXO3. J Biol Chem. 2007;282:22414–22425. [CrossRef] [PubMed]
Tanito M Kaidzu S Anderson RE . Protective effects of soft acrylic yellow filter against blue light-induced retinal damage in rats. Exp Eye Res. 2006;83:1493–1504. [CrossRef] [PubMed]
Frei B England L Ames BN . Ascorbate is an outstanding antioxidant in human blood plasma. Proc Natl Acad Sci U S A. 1989;86:6377–6381. [CrossRef] [PubMed]
Mitchell JB Samuni A Krishna MC . Biologically active metal-independent superoxide dismutase mimics. Biochemistry. 1990;29:2802–2807. [CrossRef] [PubMed]
Schroder K Hertzog PJ Ravasi T Hume DA . Interferon-gamma: an overview of signals, mechanisms and functions. J Leukoc Biol. 2004;75:163–189. [CrossRef] [PubMed]
Nagineni CN Kutty RK Detrick B Hooks JJ . Inflammatory cytokines induce intercellular adhesion molecule-1 (ICAM-1) mRNA synthesis and protein secretion by human retinal pigment epithelial cell cultures. Cytokine. 1996;8:622–630. [CrossRef] [PubMed]
Hollborn M Kohen L Wiedemann P Enzmann V . The influence of pro-inflammatory cytokines on human retinal pigment epithelium cell receptors. Graefes Arch Clin Exp Ophthalmol. 2001;239:294–301. [CrossRef] [PubMed]
Gabrielian K Osusky R Sippy BD Ryan SJ Hinton DR . Effect of TGF-beta on interferon-gamma-induced HLA-DR expression in human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 1994;35:4253–4259. [PubMed]
Tate DJJr Miceli MV Newsome DA . Phagocytosis and H2O2 induce catalase and metallothionein gene expression in human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 1995;36:1271–1279. [PubMed]
Gaillard ER Atherton SJ Eldred G . Photophysical studies on human retinal lipofuscin. Photochem Photobiol. 1995;61:448–453. [CrossRef] [PubMed]
Rozanowska M Jarvis-Evans J Korytowski W . Blue light induced reactivity of retinal age pigment. In vitro generation of oxygen-reactive species. J Biol Chem. 1995;270:18825–18830. [CrossRef] [PubMed]
Ishibashi T Patterson R Ohnishi Y Inomata H Ryan SJ . Formation of drusen in the human eye. Am J Ophthalmol. 1986;101:342–353. [CrossRef] [PubMed]
Rohrer B Guo Y Kunchithapautham K Gilkeson GS . Eliminating complement factor D reduces photoreceptor susceptibility to light-induced damage. Invest Ophthalmol Vis Sci. 2007;48:5282–5289. [CrossRef] [PubMed]
Boulton M Rozanowska M Rozanowski B . Retinal photodamage. J Photochem Photobiol B. 2001;64:144–161. [CrossRef] [PubMed]
Wenzel A Grimm C Samardzija M Remé CE . Molecular mechanisms of light-induced photoreceptor apoptosis and neuroprotection for retinal degeneration. Prog Retin Eye Res. 2005;24:275–306. [CrossRef] [PubMed]
Sparrow JR Nakanishi K Parish CA . The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells. Invest Ophthalmol Vis Sci. 2000;41:1981–1989. [PubMed]
Platanias LC . Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol. 2005;5:375–386. [CrossRef] [PubMed]
Goldberg J Flowerdew G Smith E Brody JA Tso MO . Factors associated with age-related macular degeneration. An analysis of data from the first National Health and Nutrition Examination Survey. Am J Epidemiol. 1988;128:700–710. [PubMed]
Age-Related Eye Disease Study Research Group. A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss: AREDS report no. 8. Arch Ophthalmol. 2001;119:1417–1436. [CrossRef] [PubMed]
Organisciak DT Wang HM Li ZY Tso MO . The protective effect of ascorbate in retinal light damage of rats. Invest Ophthalmol Vis Sci. 1985;26:1580–1588. [PubMed]
Tanito M Li F Elliott MH Dittmar M Anderson RE . Protective effect of TEMPOL derivatives against light-induced retinal damage in rats. Invest Ophthalmol Vis Sci. 2007;48:1900–1905. [CrossRef] [PubMed]
Figure 1.
 
The effect of recombinant human IFN-γ on the expression of CFH in RPE. Cells were incubated in 0, 10, 50, or 100 ng/mL IFN-γ, respectively. Twenty-four hours later, RPE lysates were used in Western blot analysis for CFH and GAPDH. (A and B) Cell morphology and viability did not change under various concentrations of IFN-γ incubation. Bars represent 50 μm. (C and D) CFH expression was significantly upregulated by various concentrations of IFN-γ with a maximal effect at 50 ng/mL, as shown in the blots and their relative intensities (* P < 0.001, compared with the untreated control by ANOVA with Dunnett's correction for multiple comparisons). All data were obtained from three independent experiments and expressed as mean ± SD.
Figure 1.
 
The effect of recombinant human IFN-γ on the expression of CFH in RPE. Cells were incubated in 0, 10, 50, or 100 ng/mL IFN-γ, respectively. Twenty-four hours later, RPE lysates were used in Western blot analysis for CFH and GAPDH. (A and B) Cell morphology and viability did not change under various concentrations of IFN-γ incubation. Bars represent 50 μm. (C and D) CFH expression was significantly upregulated by various concentrations of IFN-γ with a maximal effect at 50 ng/mL, as shown in the blots and their relative intensities (* P < 0.001, compared with the untreated control by ANOVA with Dunnett's correction for multiple comparisons). All data were obtained from three independent experiments and expressed as mean ± SD.
Figure 2.
 
Blue light illumination reduced IFN–γ-stimulated CFH expression in a dose-dependent effect. RPE cells were incubated with 50 ng/mL IFN-γ alone or illuminated concomitantly with 420 nm blue light with an intensity of 8 mW/cm2 for 30, 45, and 60 minutes, respectively. Western blotting and cell viability and morphology evaluations were performed after 24 hours. (A) The morphology of the RPE cells exposed to 30 minutes blue light illumination did not change significantly at 24 hours. RPE cells exposed to 45 and 60 minutes blue light illumination showed significant cell shrinkage. Bars represent 50 μm. (B) There was a dose-dependent decrease in RPE cell viability at 24 hours after blue light illumination (* P < 0.001, compared with the IFN–γ-treated RPE cells by ANOVA with Dunnett's correction for multiple comparisons). (C and D) There was a dose-dependent suppression of the CFH expression in RPE cells after different durations of blue light illumination, as shown in the blots and their relative intensities (* P < 0.001, compared with the IFN–γ-treated RPE cells by ANOVA with Dunnett's correction for multiple comparisons). (E) Semiquantitative analysis of the secretory CFH in RPE culture medium by Western blotting also showed a dose-dependent suppression by blue light illumination. (F) Western blot for CFH in RPE incubated with 50 ng/mL IFN-γ in serum-free or serum-containing media and illuminated concomitantly with 420 nm blue light with an intensity of 8 mW/cm2 for 30 minutes. Blue light illumination had the same suppressive effect on IFN-γ-stimulated CFH expression in both serum-free and serum-containing media. All data were obtained from three independent experiments and expressed as mean ± SD.
Figure 2.
 
Blue light illumination reduced IFN–γ-stimulated CFH expression in a dose-dependent effect. RPE cells were incubated with 50 ng/mL IFN-γ alone or illuminated concomitantly with 420 nm blue light with an intensity of 8 mW/cm2 for 30, 45, and 60 minutes, respectively. Western blotting and cell viability and morphology evaluations were performed after 24 hours. (A) The morphology of the RPE cells exposed to 30 minutes blue light illumination did not change significantly at 24 hours. RPE cells exposed to 45 and 60 minutes blue light illumination showed significant cell shrinkage. Bars represent 50 μm. (B) There was a dose-dependent decrease in RPE cell viability at 24 hours after blue light illumination (* P < 0.001, compared with the IFN–γ-treated RPE cells by ANOVA with Dunnett's correction for multiple comparisons). (C and D) There was a dose-dependent suppression of the CFH expression in RPE cells after different durations of blue light illumination, as shown in the blots and their relative intensities (* P < 0.001, compared with the IFN–γ-treated RPE cells by ANOVA with Dunnett's correction for multiple comparisons). (E) Semiquantitative analysis of the secretory CFH in RPE culture medium by Western blotting also showed a dose-dependent suppression by blue light illumination. (F) Western blot for CFH in RPE incubated with 50 ng/mL IFN-γ in serum-free or serum-containing media and illuminated concomitantly with 420 nm blue light with an intensity of 8 mW/cm2 for 30 minutes. Blue light illumination had the same suppressive effect on IFN-γ-stimulated CFH expression in both serum-free and serum-containing media. All data were obtained from three independent experiments and expressed as mean ± SD.
Figure 3.
 
Vitamin C abrogated the blue light suppression of CFH in RPE incubated with IFN-γ. RPE cells were treated with 0.5 mM vitamin C and 50 ng/mL IFN-γ, and exposed to 30 minutes 8-mW/cm2 blue light illumination. (A) Cell viability of the blue-light irradiated RPE at 24 hours later showed significant reduction compared with the non-irradiated RPE cells. Vitamin C coincubation had a significant rescue effect on the cell viability suppression under blue light illumination (* P < 0.001, compared with the IFN–γ-treated RPE cells by ANOVA with Bonferroni correction for multiple comparisons; ** P < 0.05, compared with the IFN-γ treated RPE cells exposed to 30 minutes blue light illumination by ANOVA with Bonferroni correction for multiple comparisons). (B and C) Western blotting of CFH in RPE cell lysates at 24 hours showed a significant suppression of CFH under blue light illumination and the suppression was significantly abrogated by 0.5 mM vitamin C coincubation (* P < 0.001, ANOVA with Bonferroni correction for multiple comparisons). (D) Quantitative analysis of the secretory CFH in RPE culture medium by ELISA also showed a significant suppression under blue light illumination. The suppression was reduced significantly by 0.5 mM vitamin C coincubation (* P < 0.001, ANOVA with Bonferroni correction for multiple comparisons). (E) The changes in CFH mRNA expression at 24 hours later were analyzed by real-time-PCR. There was a significant upregulation of CFH mRNA expression in RPE under IFN-γ incubation. Concomitant blue light illumination downregulated the CFH mRNA expression and the suppression was abrogated by 0.5 mM vitamin C coincubation (* P < 0.001, ANOVA with Bonferroni correction for multiple comparisons). All data were obtained from three independent experiments, expressed as mean ± SD.
Figure 3.
 
Vitamin C abrogated the blue light suppression of CFH in RPE incubated with IFN-γ. RPE cells were treated with 0.5 mM vitamin C and 50 ng/mL IFN-γ, and exposed to 30 minutes 8-mW/cm2 blue light illumination. (A) Cell viability of the blue-light irradiated RPE at 24 hours later showed significant reduction compared with the non-irradiated RPE cells. Vitamin C coincubation had a significant rescue effect on the cell viability suppression under blue light illumination (* P < 0.001, compared with the IFN–γ-treated RPE cells by ANOVA with Bonferroni correction for multiple comparisons; ** P < 0.05, compared with the IFN-γ treated RPE cells exposed to 30 minutes blue light illumination by ANOVA with Bonferroni correction for multiple comparisons). (B and C) Western blotting of CFH in RPE cell lysates at 24 hours showed a significant suppression of CFH under blue light illumination and the suppression was significantly abrogated by 0.5 mM vitamin C coincubation (* P < 0.001, ANOVA with Bonferroni correction for multiple comparisons). (D) Quantitative analysis of the secretory CFH in RPE culture medium by ELISA also showed a significant suppression under blue light illumination. The suppression was reduced significantly by 0.5 mM vitamin C coincubation (* P < 0.001, ANOVA with Bonferroni correction for multiple comparisons). (E) The changes in CFH mRNA expression at 24 hours later were analyzed by real-time-PCR. There was a significant upregulation of CFH mRNA expression in RPE under IFN-γ incubation. Concomitant blue light illumination downregulated the CFH mRNA expression and the suppression was abrogated by 0.5 mM vitamin C coincubation (* P < 0.001, ANOVA with Bonferroni correction for multiple comparisons). All data were obtained from three independent experiments, expressed as mean ± SD.
Figure 4.
 
Vitamin C abrogated the blue light suppression of CFH expression in RPE cells incubated with 50 ng/mL IFN-γ through superoxide anion quenching effect. Three hours after 30 minutes 8 mW/cm2 blue light illumination and 50 ng/mL IFN-γ incubation, with or without 0.5 mM vitamin C coincubation, RPE cells were stained with 10 μg/mL HE and 20 μM DCF to detect the intracellular O2 -. and H2O2 levels, respectively, by flow cytometry. RPE cells without any drug or light treatment served as the control. The fluorescence intensity was expressed as the percentage of control. (A) Histogram of the HE stain showed prominent right shift of the fluorescence under blue light illumination. Coincubation with 0.5 mM vitamin C decreased the fluorescent intensity markedly with significant left shift of the fluorescence. (B) The fluorescence level under DCF stain did not change by various treatments. Bar charts showed the mean fluorescent intensity from three independent experiments, expressed as mean ± SD. * P < 0.001 by ANOVA with Bonferroni correction for multiple comparisons.
Figure 4.
 
Vitamin C abrogated the blue light suppression of CFH expression in RPE cells incubated with 50 ng/mL IFN-γ through superoxide anion quenching effect. Three hours after 30 minutes 8 mW/cm2 blue light illumination and 50 ng/mL IFN-γ incubation, with or without 0.5 mM vitamin C coincubation, RPE cells were stained with 10 μg/mL HE and 20 μM DCF to detect the intracellular O2 -. and H2O2 levels, respectively, by flow cytometry. RPE cells without any drug or light treatment served as the control. The fluorescence intensity was expressed as the percentage of control. (A) Histogram of the HE stain showed prominent right shift of the fluorescence under blue light illumination. Coincubation with 0.5 mM vitamin C decreased the fluorescent intensity markedly with significant left shift of the fluorescence. (B) The fluorescence level under DCF stain did not change by various treatments. Bar charts showed the mean fluorescent intensity from three independent experiments, expressed as mean ± SD. * P < 0.001 by ANOVA with Bonferroni correction for multiple comparisons.
Figure 5.
 
Tempol (Alfa Aesar), a membrane-permeable free-radical scavenger, reversed the blue light-induced CFH suppression in IFN–γ-treated RPE mainly through the superoxide anion (O2 -. ) quenching effect. RPE was incubated with 1 mM Tempol for 20 hours before being treated with 50 ng/mL IFN-γ and 30 minutes 8-mW/cm2 blue light illumination. (A and B) Western blotting of RPE lysates at 24 hours later showed a significant suppression of IFN-γ-stimulated CFH expression under blue light illumination, and the suppression was significantly reduced by pretreatment with Tempol (* P < 0.001 by ANOVA with Bonferroni correction for multiple comparisons). (C) RPE cells were stained with 10 μg/mL HE to detect the intracellular O2 -. level by flow cytometry. RPE without any drug or light treatment served as the control. The fluorescence intensity was expressed as the percentage of control. The fluorescence intensity of HE stain increased significantly under blue light illumination, and pretreatment with 1 mM Tempol reduced the fluorescence intensity markedly (* P < 0.001 by ANOVA with Bonferroni correction for multiple comparisons). (D) RPE cells were stained with 20 μM DCF to detect the intracellular H2O2 levels by flow cytometry. The fluorescence level under DCF stain did not change significantly under various treatments. All data were obtained from three independent experiments, expressed as mean ± SD.
Figure 5.
 
Tempol (Alfa Aesar), a membrane-permeable free-radical scavenger, reversed the blue light-induced CFH suppression in IFN–γ-treated RPE mainly through the superoxide anion (O2 -. ) quenching effect. RPE was incubated with 1 mM Tempol for 20 hours before being treated with 50 ng/mL IFN-γ and 30 minutes 8-mW/cm2 blue light illumination. (A and B) Western blotting of RPE lysates at 24 hours later showed a significant suppression of IFN-γ-stimulated CFH expression under blue light illumination, and the suppression was significantly reduced by pretreatment with Tempol (* P < 0.001 by ANOVA with Bonferroni correction for multiple comparisons). (C) RPE cells were stained with 10 μg/mL HE to detect the intracellular O2 -. level by flow cytometry. RPE without any drug or light treatment served as the control. The fluorescence intensity was expressed as the percentage of control. The fluorescence intensity of HE stain increased significantly under blue light illumination, and pretreatment with 1 mM Tempol reduced the fluorescence intensity markedly (* P < 0.001 by ANOVA with Bonferroni correction for multiple comparisons). (D) RPE cells were stained with 20 μM DCF to detect the intracellular H2O2 levels by flow cytometry. The fluorescence level under DCF stain did not change significantly under various treatments. All data were obtained from three independent experiments, expressed as mean ± SD.
Figure 6.
 
Immunolocalization of CFH in RPE cells at 24 hours after various treatments. The cells were stained for CFH (red) and nucleus (DAPI; blue) and observed by confocal microscope. (A) RPE cells that were not stained with primary antibody for CFH served as the negative control. (B) The constitutive CFH expression in cultured human RPE cells was barely detectable by immunofluorescent stain. (C) The CFH expression in RPE increased markedly under the treatment of 50 ng/mL IFN-γ. The protein was distributed in a granular pattern in the cytoplasm, mainly around the perinuclear area. (D) Concomitant 30 minutes 8-mW/cm2 blue light illumination significantly reduced the IFN–γ-stimulated CFH expression in RPE. (E) Coincubation of 0.5 mM vitamin C abrogated the suppression of CFH under blue light illumination.
Figure 6.
 
Immunolocalization of CFH in RPE cells at 24 hours after various treatments. The cells were stained for CFH (red) and nucleus (DAPI; blue) and observed by confocal microscope. (A) RPE cells that were not stained with primary antibody for CFH served as the negative control. (B) The constitutive CFH expression in cultured human RPE cells was barely detectable by immunofluorescent stain. (C) The CFH expression in RPE increased markedly under the treatment of 50 ng/mL IFN-γ. The protein was distributed in a granular pattern in the cytoplasm, mainly around the perinuclear area. (D) Concomitant 30 minutes 8-mW/cm2 blue light illumination significantly reduced the IFN–γ-stimulated CFH expression in RPE. (E) Coincubation of 0.5 mM vitamin C abrogated the suppression of CFH under blue light illumination.
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