October 2015
Volume 56, Issue 11
Free
Retinal Cell Biology  |   October 2015
Effects of Inflammasome Activation on Secretion of Inflammatory Cytokines and Vascular Endothelial Growth Factor by Retinal Pigment Epithelial Cells
Author Affiliations & Notes
  • Lena K. M. Mohr
    Department of Ophthalmology University of Bonn, Bonn, Germany
  • Andrea V. Hoffmann
    Department of Ophthalmology University of Bonn, Bonn, Germany
  • Carolina Brandstetter
    Department of Ophthalmology University of Bonn, Bonn, Germany
  • Frank G. Holz
    Department of Ophthalmology University of Bonn, Bonn, Germany
  • Tim U. Krohne
    Department of Ophthalmology University of Bonn, Bonn, Germany
  • Correspondence: Tim U. Krohne, Department of Ophthalmology, University of Bonn, Ernst-Abbe-Str. 2, 53127 Bonn, Germany; krohne@uni-bonn.de
Investigative Ophthalmology & Visual Science October 2015, Vol.56, 6404-6413. doi:10.1167/iovs.15-16898
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      Lena K. M. Mohr, Andrea V. Hoffmann, Carolina Brandstetter, Frank G. Holz, Tim U. Krohne; Effects of Inflammasome Activation on Secretion of Inflammatory Cytokines and Vascular Endothelial Growth Factor by Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(11):6404-6413. doi: 10.1167/iovs.15-16898.

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

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Abstract

Purpose: Activation of the NLRP3 inflammasome has been implicated in the pathogenesis of AMD. Lipofuscin phototoxicity activates the inflammasome in RPE cells by inducing lysosomal membrane permeabilization (LMP). We investigated the effects of LMP-induced inflammasome activation on the secretion of inflammation-related cytokines and VEGF by RPE cells.

Methods: In primary human RPE cells and ARPE-19 cells, the inflammasome was activated by L-leucyl-L-leucine methyl ester (Leu-Leu-OMe)- or lipofuscin phototoxicity-induced LMP. Cytokine secretion was measured by protein dot blot and enzyme-linked immunosorbent assays. The polarization of cytokine secretion was assessed in RPE monolayers on permeable membranes. We analyzed the chemotactic and angiogenic effects of secreted cytokines on murine embryonic stem cell–derived microglia cells and human umbilical vascular endothelial cells, respectively.

Results: Inflammasome activation in RPE cells was associated with caspase-1–dependent secretion of IL-1β, IL-6, IL-18, GM-CSF, and GRO (CXCL1/2/3), whereas constitutive secretion of VEGF was reduced. Secretion of IL-1β and IL-18 was highly polarized to the apical cell side. Incubation with conditioned media of inflammasome-activated RPE cells induced directed migration of microglia cells (11.0-fold increase) and diminished vascular endothelial cells proliferation (39.0% reduction) and migration (69.3% reduction) as compared with conditioned media of untreated control RPE cells.

Conclusions: Lysosomal membrane permeabilization–induced activation of the NLRP3 inflammasome in RPE cells results in apical secretion of inflammatory cytokines with chemotactic effects on microglia cells and reduced constitutive secretion of VEGF. Via these mechanisms, lipofuscin phototoxicity may contribute to local immune processes in the outer retina as observed in AMD.

In the developed world, AMD is the most common cause for severe visual loss and legal blindness.1 Age-related macular degeneration is a disease of the central retina that typically affects elderly people. Age-related macular degeneration is characterized by progressive degeneration of the RPE and secondary loss of photoreceptors in the macula leading to loss of central vision. Two late manifestations of AMD can be distinguished, atrophic AMD and neovascular AMD. Atrophic AMD causes slowly progressive central visual decline by RPE cell degeneration known as geographic atrophy.2 In contrast, neovascular AMD is characterized by rapid visual loss secondary to VEGF-mediated ingrowth of choroidal neovascularizations (CNV).3 Anti-VEGF treatment has proven highly effective in neovascular AMD and is now widely used clinically. In contrast, there is still no treatment available for atrophic AMD. 
Age-related macular degeneration is associated with oxidative and photooxidative damage of the RPE that is believed to be mediated at least in part by the phototoxic properties of lipofuscin that progressively accumulates in the RPE over a life-time.4,5 Further characteristics of the disease include the formation of extracellular deposits called drusen,6,7 and a chronic low-grade immune processes including complement activation in the sub-RPE space.8,9 Thus, chronic innate immune activation plays a crucial role in AMD pathogenesis. 
Recent studies have shown that the NLRP3 inflammasome, a key mediator of the innate immune system, is activated in the RPE of patients with atrophic and neovascular AMD.10,11 Clinical studies demonstrated increased intravitreal and systemic levels of the inflammasome-controlled cytokines IL-1β and IL-18 in AMD patients.12,13 Based on these findings, a role of the NLRP3 inflammasome in AMD pathogenesis has been hypothesized. The NLRP3 inflammasome is a multiprotein complex, which induces caspase-1 activation resulting in secretion of inflammatory cytokines IL-1β and IL-18.14,15 An initial priming signal in combination with a subsequent activation signal, such as reactive oxygen species or lysosomal membrane permeabilization (LMP), leads to assembly of NLRP3, ASC, and procaspase-1 to the active inflammasome. We previously have identified photooxidative damage, intensified by accumulated lipofuscin, with secondary LMP and enzyme leakage as a mechanism of NLRP3 inflammasome activation in human RPE cells.16 
Currently, there is only little data available regarding the profile of secreted cytokines in RPE cells following inflammasome activation as well on their paracrine effects. Doyle and coworkers17 reported that incubation with inflammasome-regulated IL-18 reduces secretion of VEGF in RPE cells and suggested a protective effect against CNV formation. Consistently, they demonstrated increased laser-induced CNV formation in NLRP3 and IL-18 knockout mice.17 In contrast, another inflammasome-regulated interleukin, IL-1β, has been shown to induce VEGF secretion in RPE cells18 and to promote laser-induced CNV formation in mice.19 To elucidate the effects of inflammasome activation on pathologic processes in the outer retina, we induced inflammasome activation by LMP in human RPE cells and investigated the resulting secretion profile of inflammatory cytokines and VEGF as well as their secondary effects on microglial and vascular endothelial cells in vitro. 
Materials and Methods
Cell Culture
Human primary retinal pigment epithelial (pRPE) cells (H-RPE; Lonza, Cologne, Germany) were cultured as recommended by the manufacturer and used in experiments for a maximum of five cell culture passages. The human nontransformed RPE cell line ARPE-19 (ATCC CRL-2302; ATCC, Rockville, MD, USA) was cultured as previously reported.20 Murine embryonic stem cell–derived microglia cells were a generous gift by Harald Neumann (Institute of Reconstructive Neurobiology, University of Bonn, Germany) and had been generated and cultured as described.21 Human umbilical vein endothelial cells (HUVEC; Provitro, Berlin, Germany) were cultured as recommended by the manufacturer and used for experiments at culture passages 5 to 10. 
Inflammasome Activation by L-leucyl-L-leucine methyl ester
For inflammasome activation, pRPE cells and ARPE-19 cells were primed with 4 ng/mL IL-1α (R&D Systems, Wiesbaden, Germany) for 48 hours as described by Tseng and coworkers.10 Subsequently, cells were treated with 1 mM L-leucyl-L-leucine methyl ester (Leu-Leu-OMe; Bachem, Bubendorf, Switzerland) for 1.5 (pRPE) or 3 hours (ARPE-19) to induce LMP. 
Lysosomal membrane permeabilization was assessed by acridine orange staining as previously described.10,16 Briefly, cells were incubated with 5 μM acridine orange (AO; Sigma-Aldrich, Munich, Germany) for 30 minutes and washed with PBS immediately before beginning of the Leu-Leu-OMe treatment. For documentation of AO staining by fluorescence microscopy the rhodamine filter set (excitation 550 nm, emission 650 nm) and fluorescein filter set (excitation 502 nm; emission 526 nm) of an IX71 fluorescence microscope (Olympus, Hamburg, Germany) was used to detect intact lysosomes and nuclei, respectively. 
For inhibition experiments, RPE cells were treated with 20 μM of specific caspase-1 inhibitor Z-YVAD-FMK (BioVision, Munich, Germany) for 60 minutes or with 50 μM of specific cathepsin B inhibitor CA-074 (Calbiochem, Darmstadt, Germany) for 30 minutes prior to Leu-Leu-OMe treatment. 
Inflammasome Activation by Lipofuscin Phototoxicity
The cell culture model used to induce LMP by lipofuscin phototoxicity has been described in detail.16 Briefly, isolated porcine photoreceptor outer segments (POS) were incubated with 4-hydroxynonenal (HNE) to generate covalently modified POS (HNE-POS) that are stabilized against lysosomal degradation by RPE cells.20,22 Cells were incubated with unmodified POS or HNE-POS (concentration equivalent to 4 μg total POS protein per cm2 cell growth area) daily for 7 days to induce lipofuscinogenesis. For inflammasome priming, 4 ng/mL IL-1α (R&D Systems, Wiesbaden, Germany) was added to the media for the last 48 hours of POS incubation. Lipofuscin-loaded and primed cells were then irradiated with blue light-emitting diode (LED) light (wavelength, 455–460 nm; irradiance in our experimental setting, 0.8mW/cm2; XLamp XP-E royal blue; Cree, Durham, NC, USA) for 6 hours to induced LMP and inflammasome activation. Inhibition of caspase-1 or cathepsin B was performed immediately before blue light irradiation as described above. 
Cytokine ELISA Analysis
For quantification of cytokine secretion, we employed specific ELISA assays against human IL-1β (BD OptEIA Human IL-1β ELISA Kit II; BD Biosciences, Heidelberg, Germany), human IL-18 (MBL Human IL-18 ELISA Kit; R&D Systems, Minneapolis, USA), and human VEGF (Human VEGF Quantikine ELISA Kit; R&D Systems) according to the manufacturers' instructions. The lower limit of detection of the IL-1β ELISA as determined by the manufacturer is 0.8 pg/mL, and thus well below the concentrations measured in treated cells in our experiments. 
For collection of media for cytokine analysis, cell culture media was replaced before the experimental treatment (Leu-Leu-OMe or blue light irradiation) and collected thereafter. For VEGF measurements, the new media contained 1% fetal bovine serum as recommended by the ELISA manufacturer. In control groups, untreated cells were incubated for a time corresponding to the respective experimental treatment before media were collected. 
Cytokine Dot Blot Analysis
Confluent ARPE-19 cells in 6-well plates were primed with IL-1α and subsequently incubated with Leu-Leu-OMe in 1000 μL serum-free media. Conditioned media were collected immediately after treatment, and concentrations of 42 inflammation- and angiogenesis-related cytokines were measured using a protein dot blot assay (RayBio Human Cytokine Antibody Array 3; RayBiotech, Norcross, GA, USA) according to the manufacturer's instructions. Appropriate films (Kodak BioMax Light Film; Sigma-Aldrich, Munich, Germany) were exposed to the membranes, and developed films were scanned (Perfection V700 Photo Scanner; Epson, Meerbusch, Germany) before image analysis by ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). 
Analysis of Polarization of Cytokine Secretion
Polarized RPE cell monolayers on permeable membranes were generated as described before.23 Briefly, permeable membrane cell culture inserts (polyester membrane; diameter, 12 mm; pore size, 0.4 μm; Transwell-Clear; Corning, Kaiserslautern, Germany) were coated with 1.8 μg/cm2 laminin (Sigma-Aldrich) for 2 hours at 37°C according to the manufacturer's recommendation. Postconfluent, stationary pRPE or ARPE-19 cells were seeded onto the membranes at a confluent density of 1.66 × 105 cells/cm2. Cells were cultured for another 4 weeks before use in experiments. Cell priming and Leu-Leu-OMe treatment were performed as described above with addition of the Leu-Leu-OMe to both the apical and the basolateral media. Paracellular permeability of the RPE cell monolayer after Leu-Leu-OMe treatment was analyzed by means of marker dye leakage from the apical to the basolateral compartment using FITC-dextran (molecular weight, 20 kDa; Sigma-Aldrich) as described.23 
Microglia Chemotaxis Assay
To obtain conditioned media, ARPE-19 cells were priming with IL-1α and then treated with Leu-Leu-OMe for 1 hour. Cells were washed to remove Leu-Leu-OMe and new media without Leu-Leu-OMe were added for another 2 hours. Control cells were primed with IL-1α but not treated with Leu-Leu-OMe and likewise incubated for 2 hours. Subsequently, conditioned media of treated and control cells were collected for use in the following experiments. 
For assessment of the chemotactic effects of secreted cytokines on microglia cells, 7 × 104 microglia cells in serum-free medium were added to the upper compartment of a permeable membrane cell culture insert (polycarbonate membrane; diameter, 24 mm; pore size, 8 μm; Transwell; Corning) while the RPE-conditioned media was applied to the lower compartment of the insert. In positive control experiments, unconditioned medium containing 1 μM chemotactic peptide N-formyl-methionine-leucine-phenylalanine (fMLP; Sigma-Aldrich) was applied to the lower compartment. For coculture experiments, ARPE-19 cells were cultured in the lower compartment, primed with IL-1α, and treated with Leu-Leu-OMe for 1 hour, before medium was changed and the upper compartment of the cell culture insert containing the microglia cells was added. 
All experimental groups were incubated for 6 hours to allow for migration of the microglia cells. Subsequently, cells adherent to the upper side of the permeable membrane were mechanically removed using a cotton swab. Cells that had migrated through the membrane onto its lower surface were stained with crystal violet as described.24 The dye was then eluted from the cells using 1% SDS. The number of transmigrated microglia cells was assessed by photometric quantification of the eluted dye. 
Vascular Endothelial Cell Proliferation and Migration
Retinal pigment epithelium conditioned media were generated as described above for the microglia chemotaxis assay. The effect of conditioned media on vascular endothelial cell proliferation was analyzed by BrdU cell proliferation assay (Merck, Darmstadt, Germany). Human umbilical vein endothelial cells were seeded onto 96-well plates at a subconfluent density of 1.6 × 104 cells/cm2 and incubated for 6 hours to allow for cellular attachment. Subsequently, media was replaced by RPE cell conditioned media containing the BrdU label and incubated for another 24 hours. BrdU assay analysis was performed as recommended by the manufacturer. 
Vascular endothelial cell migration was assessed by scratch assay as described elsewhere.25 In brief, confluent HUVEC monolayers were scraped with a 200-μL pipet tip to create a scratch of defined width. Cells were washed to remove detached cells before incubation with RPE conditioned media for 24 hours. Cell migration was documented by light microscopy (Olympus CKX41 microscope; Olympus) and the number of cells within the scratch area was counted. In each experiment, five wells were analyzed per treatment group. 
Paracrine Cytokine Effects on VEGF Secretion
Conditioned media were collected from ARPE-19 cells after 3 hours of incubation with either Leu-Leu-OMe or medium alone. In addition, conditioned media were collected of lipofuscin-loaded ARPE-19 cells following irradiation with blue light for 6 hours or following incubation without irradiation for 6 hours. Vascular endothelial growth factor content of conditioned media was measured by ELISA. Untreated ARPE-19 cells were incubated with conditioned media for 24 hours, and VEGF concentration was quantified again. The initial VEGF concentration was subtracted from the final VEGF concentration to allow for specific assessment of VEGF secretion during the 24-hours incubation time. 
Statistical Analysis
Experiments were performed in duplicates (see Figs. 1B, 2, 4F) as recommended by the assay manufacturers, triplicates (see Figs. 3, 4A–E), or quintuplicates (see Fig. 4G), and results are presented as mean ± SD. For statistical analysis, we employed 2-tailed unpaired Student's t-test, and P less than 0.05 was considered statistically significant. 
Figure 1
 
Leu-Leu-OMe induces LMP and inflammasome activation in RPE cells. (A) Acridine orange staining visualizes LMP in pRPE cells and ARPE-19 cells by loss of red lysosomal staining after incubation with Leu-Leu-OMe. Nuclei are labeled green by acridine orange. Scale bar: 200 μm. (B) Interleukin-1β and IL-18 secretion was analyzed by ELISA in IL-1α–primed RPE cells following Leu-Leu-OMe treatment. Caspase-1 and cathepsin B were inhibited in Leu-Leu-OMe–treated cells by Z-YVAD-FMK and CA-074, respectively.
Figure 1
 
Leu-Leu-OMe induces LMP and inflammasome activation in RPE cells. (A) Acridine orange staining visualizes LMP in pRPE cells and ARPE-19 cells by loss of red lysosomal staining after incubation with Leu-Leu-OMe. Nuclei are labeled green by acridine orange. Scale bar: 200 μm. (B) Interleukin-1β and IL-18 secretion was analyzed by ELISA in IL-1α–primed RPE cells following Leu-Leu-OMe treatment. Caspase-1 and cathepsin B were inhibited in Leu-Leu-OMe–treated cells by Z-YVAD-FMK and CA-074, respectively.
Figure 2
 
Profile of secreted cytokines following LMP-induced inflammasome activation in RPE cells. Conditioned media of Leu-Leu-OMe–treated ARPE-19 cells was screened for 42 inflammation- and angiogenesis-related cytokines by dot blot analysis. Results for detected cytokines are shown. Significantly increased secretion was observed for GM-CSF, GRO (CXCL1/2/3), and IL-6. Caspase-1 inhibition by Z-YVAD-FMK partially suppressed this effect. The inset shows consistent results from a second, independent experiment.
Figure 2
 
Profile of secreted cytokines following LMP-induced inflammasome activation in RPE cells. Conditioned media of Leu-Leu-OMe–treated ARPE-19 cells was screened for 42 inflammation- and angiogenesis-related cytokines by dot blot analysis. Results for detected cytokines are shown. Significantly increased secretion was observed for GM-CSF, GRO (CXCL1/2/3), and IL-6. Caspase-1 inhibition by Z-YVAD-FMK partially suppressed this effect. The inset shows consistent results from a second, independent experiment.
Figure 3
 
Polarization of inflammasome-related cytokine secretion in RPE cells and chemotactic effects on microglia cells. (A) The effect of Leu-Leu-OMe on barrier function of ARPE-19 monolayers was assessed by permeability assay. For this, a marker dye (FITC-dextran, 20 kDa) with a molecular weight corresponding to that of IL-1β (17 kDa) was added to the apical compartment only, and dye leakage into the basolateral compartment was measured following Leu-Leu-OMe treatment. (B) Polarized monolayers of pRPE cells and ARPE-19 cells on permeable membranes were treated with Leu-Leu-OMe for 1.5 and 3 hours, respectively. Subsequently, separate analysis of IL-1β in the apical and basolateral media revealed that the cytokine was predominantly secreted to the apical cell side. Induction of cytokine secretion by Leu-Leu-OMe was dose-dependent, and IL-1β secretion to the basolateral side was detectable only at higher concentrations of Leu-Leu-OMe. (C) Similarly, secretion of IL-18 in ARPE-19 cells occurred predominantly toward the apical side. (D) To assess chemotactic effects of the released cytokines, murine embryonic stem cell–derived microglia cells were placed in the upper compartment of a permeable membrane cell culture insert. Migration of microglia cells across the membrane into the lower compartment was assessed following application of the following substances or cells to the lower compartment for 6 hours: conditioned media of untreated control ARPE-19 cells (Co), conditioned media of untreated ARPE-19 cells for the first 3 hours and conditioned media of LeuLeuOMe-treated ARPE-19 cells for the last 3 hours (Le3), conditioned media of LeuLeuOMe-treated ARPE-19 cells for the entire 6 hours (Le6), Leu-Leu-OMe–treated ARPE-19 cells (RPE), and unconditioned media containing the chemotactic peptide fMLP as positive control (fMLP). Migration of microglia cells onto the lower side of the permeable membrane was quantified by crystal violet assay and documented by light microscopy. Scale bar: 100 μm. Significance levels as compared with the respective controls are indicated *P < 0.05, **P < 0.01, and ***P < 0.001.
Figure 3
 
Polarization of inflammasome-related cytokine secretion in RPE cells and chemotactic effects on microglia cells. (A) The effect of Leu-Leu-OMe on barrier function of ARPE-19 monolayers was assessed by permeability assay. For this, a marker dye (FITC-dextran, 20 kDa) with a molecular weight corresponding to that of IL-1β (17 kDa) was added to the apical compartment only, and dye leakage into the basolateral compartment was measured following Leu-Leu-OMe treatment. (B) Polarized monolayers of pRPE cells and ARPE-19 cells on permeable membranes were treated with Leu-Leu-OMe for 1.5 and 3 hours, respectively. Subsequently, separate analysis of IL-1β in the apical and basolateral media revealed that the cytokine was predominantly secreted to the apical cell side. Induction of cytokine secretion by Leu-Leu-OMe was dose-dependent, and IL-1β secretion to the basolateral side was detectable only at higher concentrations of Leu-Leu-OMe. (C) Similarly, secretion of IL-18 in ARPE-19 cells occurred predominantly toward the apical side. (D) To assess chemotactic effects of the released cytokines, murine embryonic stem cell–derived microglia cells were placed in the upper compartment of a permeable membrane cell culture insert. Migration of microglia cells across the membrane into the lower compartment was assessed following application of the following substances or cells to the lower compartment for 6 hours: conditioned media of untreated control ARPE-19 cells (Co), conditioned media of untreated ARPE-19 cells for the first 3 hours and conditioned media of LeuLeuOMe-treated ARPE-19 cells for the last 3 hours (Le3), conditioned media of LeuLeuOMe-treated ARPE-19 cells for the entire 6 hours (Le6), Leu-Leu-OMe–treated ARPE-19 cells (RPE), and unconditioned media containing the chemotactic peptide fMLP as positive control (fMLP). Migration of microglia cells onto the lower side of the permeable membrane was quantified by crystal violet assay and documented by light microscopy. Scale bar: 100 μm. Significance levels as compared with the respective controls are indicated *P < 0.05, **P < 0.01, and ***P < 0.001.
Figure 4
 
Effect of inflammasome activation on RPE cell VEGF secretion and vascular endothelial cell proliferation and migration. For induction of inflammasome activation by LMP, (A) pRPE cells and (B) ARPE-19 cells were treated with Leu-Leu-OMe alone (Le) or Leu-Leu-OMe and the caspase-1 inhibitor Z-YVAD-FMK (L+Z). Control cells (Co) were left untreated. In both cell types, inflammasome activation resulted in a significant reduction of constitutive VEGF secretion that was dependent on caspase-1 activity. (C) Lysosomal membrane permeabilization induced by lipofuscin-mediated photo-oxidative damage similarly reduced VEGF secretion in ARPE-19 cells. For this, low or high lipofuscin accumulation was induced by incubation with native photoreceptor outer segments (POS) and HNE-modified POS (HNE-POS), respectively, and lipofuscin-loaded cells were irradiated with blue light for 6 hours. Incubation of untreated ARPE-19 cells with conditioned media of ARPE-19 cells subjected to either (D) Leu-Leu-OMe treatment or (E) lipofuscin phototoxicity did not result in a significant reduction of VEGF secretion, suggesting a direct effect of inflammasome activation on VEGF secretion rather than an indirect effect mediated by the secreted interleukins. The effect of inflammasome-induced cytokines on human vascular endothelial cell proliferation and migration was assessed by (F) BrdU and (G) scratch assays, respectively. Consistent with the observed reduction in VEGF secretion, incubation with conditioned media of Leu-Leu-OMe–treated ARPE-19 cells resulted in a significant reduction of vascular endothelial cell proliferation and migration as compared with conditioned media of untreated control cells. Scale bar: 250 μm.
Figure 4
 
Effect of inflammasome activation on RPE cell VEGF secretion and vascular endothelial cell proliferation and migration. For induction of inflammasome activation by LMP, (A) pRPE cells and (B) ARPE-19 cells were treated with Leu-Leu-OMe alone (Le) or Leu-Leu-OMe and the caspase-1 inhibitor Z-YVAD-FMK (L+Z). Control cells (Co) were left untreated. In both cell types, inflammasome activation resulted in a significant reduction of constitutive VEGF secretion that was dependent on caspase-1 activity. (C) Lysosomal membrane permeabilization induced by lipofuscin-mediated photo-oxidative damage similarly reduced VEGF secretion in ARPE-19 cells. For this, low or high lipofuscin accumulation was induced by incubation with native photoreceptor outer segments (POS) and HNE-modified POS (HNE-POS), respectively, and lipofuscin-loaded cells were irradiated with blue light for 6 hours. Incubation of untreated ARPE-19 cells with conditioned media of ARPE-19 cells subjected to either (D) Leu-Leu-OMe treatment or (E) lipofuscin phototoxicity did not result in a significant reduction of VEGF secretion, suggesting a direct effect of inflammasome activation on VEGF secretion rather than an indirect effect mediated by the secreted interleukins. The effect of inflammasome-induced cytokines on human vascular endothelial cell proliferation and migration was assessed by (F) BrdU and (G) scratch assays, respectively. Consistent with the observed reduction in VEGF secretion, incubation with conditioned media of Leu-Leu-OMe–treated ARPE-19 cells resulted in a significant reduction of vascular endothelial cell proliferation and migration as compared with conditioned media of untreated control cells. Scale bar: 250 μm.
Results
Leu-Leu-OMe Induces LMP and Inflammasome Activation in ARPE-19 and pRPE Cells
We have demonstrated that blue light irradiation of lipofuscin-loaded RPE cells activates the NLRP3 inflammasome secondary to induction of LMP and cytosolic leakage of lysosomal enzymes.16 In the current study, we employed a model of chemically induced LMP by Leu-Leu-OMe that has been demonstrated to likewise result in inflammasome activation in ARPE-19 cells.10 To assess lysosomal membrane integrity, cells were labeled with acridine orange, a fluorescent dye that both intercalates into DNA (green fluorescence) and stains intact lysosomes (red fluorescence). In both ARPE-19 cells and pRPE cells, we observed a marked loss of lysosomal staining after incubation with Leu-Leu-OMe compared with untreated controls, indicating effective induction of LMP by Leu-Leu-OMe in both cell types (Fig. 1A). 
When RPE cells were primed with IL-1α as described by Tseng and coworkers,10 LMP induction by Leu-Leu-OMe resulted in inflammasome activation with significantly increased secretion of inflammatory cytokines IL-1β and IL-18 in both ARPE-19 and pRPE cells (Fig. 1B). Inhibition of the inflammasome component caspase-1 by Z-YVAD-FMK suppressed the release of IL-1β and IL-18. Leakage of lysosomal enzymes, particularly cathepsin B, has been described to be involved in NRLP3 inflammasome activation secondary to LMP.10,26 Indeed, inhibition of cathepsin B by CA-074 also suppressed IL-1β and IL-18 release. These findings demonstrate that Leu-Leu-OMe-induced LMP induces caspase-1- and cathepsin B–dependent inflammasome activation in both ARPE-19 and pRPE cells. 
Profile of Secreted Cytokines Secondary to Inflammasome Activation in RPE Cells
After detecting an increased secretion of the inflammasome-regulated cytokines IL-1β and IL-18, we performed a screening analysis to identify potential additional cytokines induced by inflammasome activation in RPE cells. For this, protein levels of 42 inflammation- and angiogenesis-related cytokines were analyzed in conditioned media of IL-1α–primed and Leu-Leu-OMe–treated ARPE-19 cells by dot blot analysis. Leu-Leu-OMe treatment resulted in significantly increased secretion of GM-CSF (2.8-fold increase, P = 0.027), GRO (CXCL1/2/3; 5.2-fold, P = 0.020), and IL-6 (13.6-fold, P = 0.031) compared with an IL-1α–primed but not Leu-Leu-OMe–treated control group (Fig. 2). These results were found consistently in two independent experiments. Inflammasome inhibition by incubation with the caspase-1–specific inhibitor Z-YVAD-FMK partially suppressed the Leu-Leu-OMe–induced increase in secretion of GM-CSF, GRO, and IL-6. In summary, Leu-Leu-OMe–induced LMP in RPE cells resulted in a caspase-1–dependent release of IL-1β, IL-18, GM-CSF, GRO, and IL-6. 
Polarization of Cytokine Secretion by RPE Cells and Chemotactic Effects on Microglia Cells
The effects of secreted inflammatory cytokines on adjacent cells and tissues may differ considerably depending on whether the secretion is directed to the apical (neuroretinal) or basolateral (choroidal) side of the RPE monolayer. To identify the predominant direction of cytokine secretion after inflammasome activation we cultured ARPE-19 and pRPE cells on permeable membranes. We demonstrated previously that the culture conditions employed result in the formation of a polarized cell monolayer with apical microvilli and intercellular tight junctions.23 To verify that Leu-Leu-OMe treatment does not compromise RPE barrier function, we incubated RPE cells with the marker dye FITC-dextran of a molecular weight of 20 kDa, similar to that of IL-1β (17 kDa). FITC-dextran was added in the apical cell culture compartment, and dye leakage into the basolateral compartment was monitored during treatment with Leu-Leu-OMe. The assay demonstrated that the ARPE-19 cell monolayers almost completely prevented leakage between the two compartments and that effects of Leu-Leu-OMe treatment on this barrier function were minimal (Fig. 3A). 
We then treated polarized monolayers of pRPE cells and ARPE-19 cells on permeable membranes with Leu-Leu-OMe added to both the apical and basolateral media to induce cytokine secretion. Unlike in Figure 1, secretion levels in Figure 3 are displayed as total cytokine mass instead of cytokine concentration to account for different volumes of the apical and basolateral compartments of the cell culture insert. The cells exhibited a significant increase in IL-1β secretion that was predominantly directed to the apical side (Fig. 3B). Apical secretion of IL-1β in pRPE cells and ARPE-19 cells accounted for at least 69% and 75% of total IL-1β secretion, respectively. The lower IL-1β levels in pRPE cells compared with ARPE-19 cells may correspond to the different durations of Leu-Leu-OMe treatment of 1.5 and 3 hours, respectively. Similarly to IL-1β, IL-18 in ARPE-19 cells was predominantly secreted to the apical side, with at least 92% of total secreted IL-18 detectable in the apical medium (Fig. 3C). 
Of note, IL-1β and IL-18 in the basolateral medium were almost exclusively detectable in the group treated with 1.0 mM Leu-Leu-OMe that was also the only group that demonstrated some compromise of RPE barrier function in FITC-dextran leakage assay. In contrast, the 0.5 and 0.75 mM Leu-Leu-OMe–treatment groups exhibited both intact barrier function and near-complete lack of cytokines in the basolateral medium. It thus appears possible that cytokines in the 1.0 mM Leu-Leu-OMe–treatment group may have reached the basolateral medium by transcellular leakage rather than basolateral secretion and that IL-1β and IL-18 release after Leu-Leu-OMe treatment occurs in fact almost entirely to the apical side. 
As apical secretion of inflammatory cytokines in vivo may affect retinal microglia activation and recruitment, we assessed the effects of secreted cytokines on microglia migration in vitro. For this assay, we placed murine embryonic stem cell–derived microglia cells in the upper compartment of a permeable membrane cell culture insert. The number of microglia cells that transmigrated through the membrane was significantly increased (11.0-fold, P = 0.0003) when conditioned media of Leu-Leu-OMe–treated ARPE-19 cells was applied to the lower compartment as compared with conditioned media of untreated control cells (Fig. 3D). Cocultures of Leu-Leu-OMe–treated ARPE-19 cells in the lower compartment likewise resulted in a chemotactic effect on microglia cells in the upper compartment. The observed effects were comparable to that of the chemotactic peptide fMLP that was used as a positive control. 
Together, these results suggest that secretion of inflammatory cytokines by RPE cells secondary to inflammasome activation is directed predominantly to the apical side, corresponding to the neuroretinal side in vivo, and that the secreted cytokines exert a chemotactic effect on microglial cells. 
Effects of Inflammasome Activation on VEGF Secretion by RPE Cells and Secondary Effects on Vascular Endothelial Cells
Vascular endothelial growth factor plays a central role in CNV formation in neovascular AMD. Inflammasome activation has been suggested to be involved in this process, although the reported effects of different inflammasome-controlled cytokines on VEGF secretion and angiogenesis are divergent.1719 Therefore, we investigated the effects of LMP-induced inflammasome activation on VEGF secretion in RPE cells. Interestingly, the constitutive secretion of VEGF was significantly reduced in both pRPE and ARPE-19 cells following inflammasome activation by Leu-Leu-OMe (Figs. 4A, 4B). Inhibition of caspase-1 by Z-YVAD-FMK reversed this effect. When LMP was induced in ARPE-19 cells by lipofuscin-mediated photooxidative damage as described,16 we likewise detected significantly reduced VEGF secretion (Fig. 4C). These findings demonstrate that inflammasome activation in RPE cells secondary to LMP, both by Leu-Leu-OMe and by lipofuscin phototoxicity, results in a reduction of constitutive VEGF secretion. 
Inflammasome-regulated cytokine IL-18 has been shown to reduce VEGF secretion in RPE cells.17 We therefore sought to investigate whether the reduction of VEGF secretion observed in our experiments was a direct effect of inflammasome activation or rather a secondary effects mediated by secreted IL-18. To test for this, we incubated untreated RPE cells with conditioned media of Leu-Leu-OMe–treated RPE cells or conditioned media of untreated control cells (Fig. 4D). Vascular endothelial growth factor content of conditioned media before incubation was subtracted from VEGF content after incubation to allow for a selective quantification of VEGF secretion by the incubated RPE cells. With this experimental design, we did not detect a significant effect of conditioned media on VEGF secretion. When LMP was induced by lipofuscin phototoxicity instead of Leu-Leu-OMe, we likewise did not find VEGF secretion to be significantly affected by conditioned media (Fig. 4E). These results suggest the reduced VEGF secretion observed in our experiments to be a direct effect of inflammasome activation, rather than a secondary effect mediated by released cytokines such as IL-18. 
Inflammasome-regulated IL-1β has been reported to exert angiogenic effects.19,27 Although our experiments demonstrated that inflammasome-induced release of IL-1β by RPE cells occurs predominantly toward the apical side, we also detected a significant increase in basolateral secretion (Fig. 3B) that could contribute to choroidal angiogenesis in AMD. To determine the prevailing effect on angiogenesis of combined increased IL-1β release and reduced VEGF secretion as observed after inflammasome activation in RPE cells, we incubated HUVEC with conditioned media of Leu-Leu-OMe–treated RPE cells. Measurements by BrdU assay revealed that incubation with conditioned media of Leu-Leu-OMe–treated cells reduced vascular endothelial cell proliferation by 61% compared with conditioned media of untreated control cells (P = 0.0013; Fig. 4F). Likewise, analysis by scratch assay demonstrated a reduction of vascular endothelial cell migration by 31% (P = 0.0009; Fig. 4G). Thus, conditioned media of RPE cells after inflammasome activation reduced vascular endothelial cell proliferation and migration compared with conditioned media of untreated control RPE cells, consistent with the observed reduction in constitutive VEGF secretion. 
Discussion
Various lines of evidence indicate that the chronic innate immune response in the sub-RPE space that is detectable as both local deposition and increased systemic levels of activated complement components represents a key pathogenetic factor in AMD.8,9 In a study examining AMD patients with the AMD risk polymorphism of the complement factor H (CFH) gene, increased systemic concentrations have also been reported for the cytokine IL-18, which represents a product of inflammasome activation.28 Inflammasome activation in the RPE has been detected in patients with both atrophic and neovascular AMD,10,11 and increased intravitreal and systemic levels of the inflammasome-controlled cytokines IL-1β and IL-18 in AMD patients have been reported.12,13 However, clinical data regarding inflammasome activation in AMD is still sparse, and more research is needed on the potential contribution of the inflammasome to AMD pathogenesis. Also, the signal for inflammasome activation in AMD is yet unknown, and substances that have been suggested to provide this signal include drusen components such as C1q17 and amyloid-β,29 Alu RNA accumulating secondary to DICER1 deficiency,11 the lipofuscin component N-retinylidene-N-retinyl-ethanolamine (A2E),30 and the lipid peroxidation product 4-hydroxynonenal (HNE).31 
A well-established mechanism of inflammasome activation in various cell types is induction of LMP, for example, by viruses, bacteriotoxins, and phagocytosed crystalline substances.15 Inflammasome activation by LMP has also been reported to occur in RPE cells.10 In the aging RPE, lipofuscin accumulates within the lysosomal compartment, and we and others have demonstrated that photoreactive properties of lipofuscin induce LMP in RPE cells.32,33 We also demonstrated that lipofuscin phototoxicity, via induction of LMP and cytosolic leakage of lysosomal enzymes, results in inflammasome activation in RPE cells.16 These results make it conceivable that with age, progressive accumulation of lipofuscin together with a declining defensive capacity against photooxidative stress may trigger inflammasome activation in the RPE that contributes to the development of AMD. 
In contrast to the mechanisms of inflammasome activation in the RPE, the consequences of this mechanism have been investigated less intensively so far, in particular with regard to AMD pathogenesis. In this study, we addressed this question by analyzing the profile of cytokines that are secreted by RPE cells following inflammasome activation. For this, we used an established model for LMP-induced inflammasome activation that employs Leu-Leu-OMe for chemical destabilization of lysosomes.10,26 Tseng and coworkers10 demonstrated that incubation of ARPE-19 cells with Leu-Leu-OMe as also employed in our study induces characteristic features of inflammasome activation such as caspase-1, maturation and release of IL-1β, and cell death by pyroptosis.10 
Using the same model in pRPE and ARPE-19 cells, we observed a significant and caspase-1–dependent increase in secretion of the inflammation-related cytokines IL-1β, IL-6, IL-18, GM-CSF, and GRO family cytokines (CXCL1, CXCL2, CXCL3) secondary to LMP-induced inflammasome activation in RPE cells. The cytokines IL-1β and IL-18 are directly regulated by the inflammasome, and thus their increased secretion is most likely a direct result of inflammasome activation. In contrast, IL-6, GM-CSF, and GRO are not considered to be controlled by the inflammasome but are rather upregulated secondary to IL-1β or IL-18 activity. In particular, IL-1β has been demonstrated to induce secretion of IL-6, GM-CSF, and GRO in RPE cells.3437 
The IL-1β concentrations measured in inflammasome-activated RPE cells in our experiments are in the range of those determined by previous studies.16,17,30 Likewise, the concentrations of IL-18 measured in our study correspond to the range of previously reported levels.11,16,31 Secretion of IL-1β was significantly higher in polarized RPE cells (Fig. 3B) compared with unpolarized cells (Fig. 1B). The observation that polarization increases cytokine expression in RPE cells has been described before. For example, secretion of pigment epithelium-derived factor (PEDF) and VEGF has been reported to be increased 34-fold and 6-fold, respectively, in polarized compared with unpolarized RPE cells.38 Polarization may thus likewise be the cause for the increased secretion of IL-1β observed in our polarized RPE cell experiments. 
The identified cytokines may contribute to RPE cell pathology in AMD in various ways. IL-18 has been reported to exert a direct cytotoxic effect on RPE cells in vivo,11 although this observation has been questioned by others.39 In vitro, no cytotoxic effects on RPE cells have been observed for IL-18 concentrations of up to 10 μg/mL.39 This concentration is five orders of magnitude higher than the IL-18 concentrations measured in conditioned media in our experiments (Fig. 1B). Therefore, cytotoxic effects of IL-18 in our experiments are not to be expected. Similar to IL-18, IL-6 was described as capable of inducing degeneration of RPE cells.40 In addition to their direct effects on RPE cells, the identified cytokines could induce activation and recruitment of microglia cells, monocytes, and macrophages, which may exert secondary effects on the RPE. 
We found that secretion of IL-1β and IL-18 was predominantly directed toward the apical side of the RPE monolayer, corresponding to the neuroretinal side in vivo. This is in accordance with the results of previous studies that likewise reported a predominantly apical secretion of inflammatory cytokines by RPE cells following induction by various stimuli.4143 Retinal cell populations that may be affected by the observed apical cytokine secretion include resident microglia cells and infiltrating macrophages. Indeed, activation of these cell types and their migration into the subretinal space has been observed in AMD patients and animal models and have been discussed as a factor contributing to AMD pathogenesis.4446 Our experiments demonstrated a significant chemotactic effect of conditioned media from Leu-Leu-OMe–treated RPE cells on microglia cells, supporting a role of inflammasome-related cytokines in microglia recruitment. Via the apically polarized secretion of inflammatory cytokines and the chemotactic effects on microglia cells, LMP-mediated inflammasome activation in the RPE as observed secondary to lipofuscin phototoxicity may contribute to microglia/macrophages activation and recruitment in vivo in retinal pathologies such as AMD. 
For the evaluation of the inflammasome as a potential new therapeutic target in neovascular AMD, the knowledge of downstream effects of inflammasome activation is crucial. However, previous reports on the effects of inflammasome activation on CNV formation are ambiguous. The two classical inflammasome-induced cytokines, IL-1β and IL-18, have been reported to induce and inhibit VEGF secretion by RPE cells, respectively.17,18 Similarly, IL-1β and IL-18 have been demonstrated to promote and suppress CNV formation in mice, respectively,17,19 whereas others did not find CNV formation to be affected by IL-18.47 We sought to elucidate the angiogenesis-related effects of inflammasome activation in the RPE. Interestingly, we found that LMP-induced inflammasome activation in RPE cells does not result in an increase but rather in a significant reduction of the cells' constitutive secretion of VEGF. Consistently, conditioned media of RPE cells following inflammasome activation reduced proliferation and migration of vascular endothelial cells compared with conditioned media of untreated control cells. In addition to the reduction in VEGF secretion, the released IL-18 may add to this effect due to its suggested antiangiogenic properties.17 While our experiments demonstrate reduced VEGF secretion by RPE cells following inflammasome activation, inflammatory cytokines released by RPE cells after inflammasome activation in vivo may exert indirect angiogenic effects via other cell types such as recruited microglia cells.44 
Both a priming signal and an activation signal are required for inflammasome activation. In IL-1α–primed RPE cells, we found that LMP induced by lipofuscin phototoxicity results in activation of the NLRP3 inflammasome,16 and in the current study this mechanism was associated with a significant reduction in constitutive VEGF secretion. In contrast, the isolated lipofuscin component A2E has been shown to induce VEGF secretion in unprimed ARPE-19 cells.48 After priming of ARPE-19 cells with IL-1α, A2E treatment resulted in both inflammasome activation and induction of VEGF secretion.30 Similar to lipofuscin, phototoxicity of A2E has been shown to induce LMP,32 and blue light irradiation of unprimed A2E-loaded ARPE-19 cells induced an increase in VEGF secretion.49 Whether A2E phototoxicity in primed cells results in a reduction of VEGF secretion similar to our results for lipofuscin phototoxicity still needs to be investigated. 
Following up on our previous investigation describing the mechanism of LMP-induced inflammasome activation induced by lipofuscin-mediated photooxidative damage in RPE cells, our current study identifies the cytokine profile secreted by RPE cells following LMP-induced inflammasome activation. While secretion of VEGF is suppressed after inflammasome activation, several inflammatory cytokines are significantly induced and secreted predominantly to the apical RPE side. Via this mechanism, photooxidative damage to the RPE may trigger local immune processes such as activation and recruitment of retinal microglia/macrophages, and thus contribute to the chronic innate immune response in AMD. 
Acknowledgments
The authors thank Claudine Strack for expert technical assistance and Harald Neumann (Institute of Reconstructive Neurobiology, University of Bonn, Bonn, Germany) for providing the murine embryonic stem cell–derived microglia cells. 
Supported by German Research Foundation (DFG), Bonn, Germany, Grant KR 2863/7-1; Pro Retina Foundation, Bonn, Germany; University of Bonn, Bonn, Germany, BONFOR and SciMed Programs; and the Dr. Eberhard and Hilde Rüdiger Foundation, Bonn, Germany (all to TUK). 
Disclosure: L.K.M. Mohr, None; A.V. Hoffmann, None; C. Brandstetter, None; F.G. Holz, Acucela (F, C, R), Alcon (F, C, R), Allergan (F, C, R), Bayer (F, C, R), Carl Zeiss Meditec (F), Genentech (F, C, R), Heidelberg Engineering (F, C, R), Novartis (F, C, R), Optos (F), Roche (C, R); T.U. Krohne, Alcon (F), Novartis (F), Bayer (C, R), Heidelberg Engineering (C, R) 
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Figure 1
 
Leu-Leu-OMe induces LMP and inflammasome activation in RPE cells. (A) Acridine orange staining visualizes LMP in pRPE cells and ARPE-19 cells by loss of red lysosomal staining after incubation with Leu-Leu-OMe. Nuclei are labeled green by acridine orange. Scale bar: 200 μm. (B) Interleukin-1β and IL-18 secretion was analyzed by ELISA in IL-1α–primed RPE cells following Leu-Leu-OMe treatment. Caspase-1 and cathepsin B were inhibited in Leu-Leu-OMe–treated cells by Z-YVAD-FMK and CA-074, respectively.
Figure 1
 
Leu-Leu-OMe induces LMP and inflammasome activation in RPE cells. (A) Acridine orange staining visualizes LMP in pRPE cells and ARPE-19 cells by loss of red lysosomal staining after incubation with Leu-Leu-OMe. Nuclei are labeled green by acridine orange. Scale bar: 200 μm. (B) Interleukin-1β and IL-18 secretion was analyzed by ELISA in IL-1α–primed RPE cells following Leu-Leu-OMe treatment. Caspase-1 and cathepsin B were inhibited in Leu-Leu-OMe–treated cells by Z-YVAD-FMK and CA-074, respectively.
Figure 2
 
Profile of secreted cytokines following LMP-induced inflammasome activation in RPE cells. Conditioned media of Leu-Leu-OMe–treated ARPE-19 cells was screened for 42 inflammation- and angiogenesis-related cytokines by dot blot analysis. Results for detected cytokines are shown. Significantly increased secretion was observed for GM-CSF, GRO (CXCL1/2/3), and IL-6. Caspase-1 inhibition by Z-YVAD-FMK partially suppressed this effect. The inset shows consistent results from a second, independent experiment.
Figure 2
 
Profile of secreted cytokines following LMP-induced inflammasome activation in RPE cells. Conditioned media of Leu-Leu-OMe–treated ARPE-19 cells was screened for 42 inflammation- and angiogenesis-related cytokines by dot blot analysis. Results for detected cytokines are shown. Significantly increased secretion was observed for GM-CSF, GRO (CXCL1/2/3), and IL-6. Caspase-1 inhibition by Z-YVAD-FMK partially suppressed this effect. The inset shows consistent results from a second, independent experiment.
Figure 3
 
Polarization of inflammasome-related cytokine secretion in RPE cells and chemotactic effects on microglia cells. (A) The effect of Leu-Leu-OMe on barrier function of ARPE-19 monolayers was assessed by permeability assay. For this, a marker dye (FITC-dextran, 20 kDa) with a molecular weight corresponding to that of IL-1β (17 kDa) was added to the apical compartment only, and dye leakage into the basolateral compartment was measured following Leu-Leu-OMe treatment. (B) Polarized monolayers of pRPE cells and ARPE-19 cells on permeable membranes were treated with Leu-Leu-OMe for 1.5 and 3 hours, respectively. Subsequently, separate analysis of IL-1β in the apical and basolateral media revealed that the cytokine was predominantly secreted to the apical cell side. Induction of cytokine secretion by Leu-Leu-OMe was dose-dependent, and IL-1β secretion to the basolateral side was detectable only at higher concentrations of Leu-Leu-OMe. (C) Similarly, secretion of IL-18 in ARPE-19 cells occurred predominantly toward the apical side. (D) To assess chemotactic effects of the released cytokines, murine embryonic stem cell–derived microglia cells were placed in the upper compartment of a permeable membrane cell culture insert. Migration of microglia cells across the membrane into the lower compartment was assessed following application of the following substances or cells to the lower compartment for 6 hours: conditioned media of untreated control ARPE-19 cells (Co), conditioned media of untreated ARPE-19 cells for the first 3 hours and conditioned media of LeuLeuOMe-treated ARPE-19 cells for the last 3 hours (Le3), conditioned media of LeuLeuOMe-treated ARPE-19 cells for the entire 6 hours (Le6), Leu-Leu-OMe–treated ARPE-19 cells (RPE), and unconditioned media containing the chemotactic peptide fMLP as positive control (fMLP). Migration of microglia cells onto the lower side of the permeable membrane was quantified by crystal violet assay and documented by light microscopy. Scale bar: 100 μm. Significance levels as compared with the respective controls are indicated *P < 0.05, **P < 0.01, and ***P < 0.001.
Figure 3
 
Polarization of inflammasome-related cytokine secretion in RPE cells and chemotactic effects on microglia cells. (A) The effect of Leu-Leu-OMe on barrier function of ARPE-19 monolayers was assessed by permeability assay. For this, a marker dye (FITC-dextran, 20 kDa) with a molecular weight corresponding to that of IL-1β (17 kDa) was added to the apical compartment only, and dye leakage into the basolateral compartment was measured following Leu-Leu-OMe treatment. (B) Polarized monolayers of pRPE cells and ARPE-19 cells on permeable membranes were treated with Leu-Leu-OMe for 1.5 and 3 hours, respectively. Subsequently, separate analysis of IL-1β in the apical and basolateral media revealed that the cytokine was predominantly secreted to the apical cell side. Induction of cytokine secretion by Leu-Leu-OMe was dose-dependent, and IL-1β secretion to the basolateral side was detectable only at higher concentrations of Leu-Leu-OMe. (C) Similarly, secretion of IL-18 in ARPE-19 cells occurred predominantly toward the apical side. (D) To assess chemotactic effects of the released cytokines, murine embryonic stem cell–derived microglia cells were placed in the upper compartment of a permeable membrane cell culture insert. Migration of microglia cells across the membrane into the lower compartment was assessed following application of the following substances or cells to the lower compartment for 6 hours: conditioned media of untreated control ARPE-19 cells (Co), conditioned media of untreated ARPE-19 cells for the first 3 hours and conditioned media of LeuLeuOMe-treated ARPE-19 cells for the last 3 hours (Le3), conditioned media of LeuLeuOMe-treated ARPE-19 cells for the entire 6 hours (Le6), Leu-Leu-OMe–treated ARPE-19 cells (RPE), and unconditioned media containing the chemotactic peptide fMLP as positive control (fMLP). Migration of microglia cells onto the lower side of the permeable membrane was quantified by crystal violet assay and documented by light microscopy. Scale bar: 100 μm. Significance levels as compared with the respective controls are indicated *P < 0.05, **P < 0.01, and ***P < 0.001.
Figure 4
 
Effect of inflammasome activation on RPE cell VEGF secretion and vascular endothelial cell proliferation and migration. For induction of inflammasome activation by LMP, (A) pRPE cells and (B) ARPE-19 cells were treated with Leu-Leu-OMe alone (Le) or Leu-Leu-OMe and the caspase-1 inhibitor Z-YVAD-FMK (L+Z). Control cells (Co) were left untreated. In both cell types, inflammasome activation resulted in a significant reduction of constitutive VEGF secretion that was dependent on caspase-1 activity. (C) Lysosomal membrane permeabilization induced by lipofuscin-mediated photo-oxidative damage similarly reduced VEGF secretion in ARPE-19 cells. For this, low or high lipofuscin accumulation was induced by incubation with native photoreceptor outer segments (POS) and HNE-modified POS (HNE-POS), respectively, and lipofuscin-loaded cells were irradiated with blue light for 6 hours. Incubation of untreated ARPE-19 cells with conditioned media of ARPE-19 cells subjected to either (D) Leu-Leu-OMe treatment or (E) lipofuscin phototoxicity did not result in a significant reduction of VEGF secretion, suggesting a direct effect of inflammasome activation on VEGF secretion rather than an indirect effect mediated by the secreted interleukins. The effect of inflammasome-induced cytokines on human vascular endothelial cell proliferation and migration was assessed by (F) BrdU and (G) scratch assays, respectively. Consistent with the observed reduction in VEGF secretion, incubation with conditioned media of Leu-Leu-OMe–treated ARPE-19 cells resulted in a significant reduction of vascular endothelial cell proliferation and migration as compared with conditioned media of untreated control cells. Scale bar: 250 μm.
Figure 4
 
Effect of inflammasome activation on RPE cell VEGF secretion and vascular endothelial cell proliferation and migration. For induction of inflammasome activation by LMP, (A) pRPE cells and (B) ARPE-19 cells were treated with Leu-Leu-OMe alone (Le) or Leu-Leu-OMe and the caspase-1 inhibitor Z-YVAD-FMK (L+Z). Control cells (Co) were left untreated. In both cell types, inflammasome activation resulted in a significant reduction of constitutive VEGF secretion that was dependent on caspase-1 activity. (C) Lysosomal membrane permeabilization induced by lipofuscin-mediated photo-oxidative damage similarly reduced VEGF secretion in ARPE-19 cells. For this, low or high lipofuscin accumulation was induced by incubation with native photoreceptor outer segments (POS) and HNE-modified POS (HNE-POS), respectively, and lipofuscin-loaded cells were irradiated with blue light for 6 hours. Incubation of untreated ARPE-19 cells with conditioned media of ARPE-19 cells subjected to either (D) Leu-Leu-OMe treatment or (E) lipofuscin phototoxicity did not result in a significant reduction of VEGF secretion, suggesting a direct effect of inflammasome activation on VEGF secretion rather than an indirect effect mediated by the secreted interleukins. The effect of inflammasome-induced cytokines on human vascular endothelial cell proliferation and migration was assessed by (F) BrdU and (G) scratch assays, respectively. Consistent with the observed reduction in VEGF secretion, incubation with conditioned media of Leu-Leu-OMe–treated ARPE-19 cells resulted in a significant reduction of vascular endothelial cell proliferation and migration as compared with conditioned media of untreated control cells. Scale bar: 250 μm.
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