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Immunology and Microbiology  |   May 2013
Mature Dendritic Cell Suppression by IL-1 Receptor Antagonist on Retinal Pigment Epithelium Cells
Author Affiliations & Notes
  • Sunao Sugita
    Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University Graduate School of Medicine and Dental Sciences, Tokyo, Japan
    Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Kobe, Japan
  • Yuko Kawazoe
    Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University Graduate School of Medicine and Dental Sciences, Tokyo, Japan
  • Ayano Imai
    Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University Graduate School of Medicine and Dental Sciences, Tokyo, Japan
  • Yoshihiko Usui
    Department of Ophthalmology, Tokyo Medical University, Tokyo, Japan
  • Yoichiro Iwakura
    Research Institute for Biomedical Sciences, Tokyo University of Science, Chiba, Japan
  • Kikuo Isoda
    Department of Internal Medicine-I, National Defense Medical College, Saitama, Japan
  • Masataka Ito
    Department of Developmental Anatomy and Regenerative Biology, National Defense Medical College, Saitama, Japan
  • Manabu Mochizuki
    Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University Graduate School of Medicine and Dental Sciences, Tokyo, Japan
  • Correspondence: Sunao Sugita, Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan; sunaoph@cdb.riken.jp
Investigative Ophthalmology & Visual Science May 2013, Vol.54, 3240-3249. doi:10.1167/iovs.12-11483
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      Sunao Sugita, Yuko Kawazoe, Ayano Imai, Yoshihiko Usui, Yoichiro Iwakura, Kikuo Isoda, Masataka Ito, Manabu Mochizuki; Mature Dendritic Cell Suppression by IL-1 Receptor Antagonist on Retinal Pigment Epithelium Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(5):3240-3249. doi: 10.1167/iovs.12-11483.

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

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Abstract

Purpose.: To determine whether retinal pigment epithelial (RPE) cells can inhibit mature dendritic cells (mDCs).

Methods.: Cultured RPE cells were established from C57BL/6 mice. DCs were established from bone marrow cells of normal mice, and mDCc were induced by culture in medium containing granulocyte macrophage–colony-stimulating factor (GM-CSF) and IL-4 in the presence of lipopolysaccharide and TNF-α. Activation of mDCs was assessed by a proliferation assay and ELISA to measure the production of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-12p40). Expression of major histocompatibility complex (MHC) class II, CD11c, and costimulatory molecules such as CD80, CD86, programmed cell death 1 ligand 1 (PD-L1), and PD-L2 on mDCs or RPE-exposed mDCs was evaluated by immune staining and flow cytometry. Production of IL-1 receptor antagonist (IL-1Ra) by RPE cells was evaluated by oligonucleotide microarray or ELISA. Anti–IL-1Ra neutralizing antibodies or RPE cells from IL-1Ra knockout donors were used for the assay.

Results.: Cultured RPE cells greatly suppressed the activation of mDCs, especially the production of pro-inflammatory cytokines, and the expression of cell–surface molecules. Moreover, RPE cells significantly suppressed mixed lymphocyte reactions by mDCs. In an examination of immunoregulatory candidate molecules, RPE cells expressed much higher levels of IL-1Ra as compared with control cells, and RPE cells pretreated with recombinant TNF-α and/or IL-1β produced high levels of IL-1Ra. RPE cells in the presence of anti–IL-1Ra antibodies, but not other candidate factors, failed to suppress activation by mDCs. In addition, RPE cells from IL-1Ra null donors failed to suppress mDC activation.

Conclusions.: Our results suggest that ocular resident cells can produce pro-inflammatory cytokine antagonist that suppresses antigen-presenting cell activation.

Introduction
Dendritic cells (DCs) are crucial regulators of the induction of immune responses. 15 Immature DCs can be converted into mature DCs (mDCs) when stimulated with microorganism components such as lipopolysaccharides (LPSs) or pro-inflammatory cytokines such as TNF-α. mDCs express high levels of major histocompatibility complex (MHC) class II, CD11c, and costimulatory molecules such as CD80 (B7-1), CD86 (B7-2), programmed cell death 1 ligand 1 (PD-L1: B7-H1), and PD-L2 (B7-DC) on their surface. DCs are potent stimulators of naïve T cells and key inducers of primary immune responses. DCs are the most potent professional antigen presenting cells, and they play an important role in inducing antigen-specific activation of T cells and B cells. During migration from the periphery to the lymph nodes, DCs undergo a maturation process that upregulates the expression of MHC class II and costimulatory molecules. 15 DC maturation amplifies the autoimmune responses in various autoimmune disorders including ocular inflammatory diseases. 69 Thus, DCs, especially mDCs, are the most important cells during inflammation. 
RPE contributes to immune tolerance in the eye, especially the posterior segment of the eye, which includes the subretinal space. The immunosuppressive properties of intraocular cells create immune tolerance, which is necessary to avoid adverse consequences of intraocular inflammation, such as blindness. Cultured RPE cells suppress T cell activation via the production of immunosuppressive factor(s). 1016 Cultured RPE cells established from normal mice suppress Th1 and Th17 inflammatory effector T cells in vitro. 11,14 RPE cells can also convert T cells into regulatory T cells. 1719 Moreover, cultured RPE cells can suppress the activation of B cells 20 and macrophages. 21 However, the mechanisms by which resident retinal cells can suppress DCs have not yet been elucidated. 
Therefore, we designed experiments to investigate whether cultured ocular resident cells can suppress the activation of mDCs in vitro. We used primary cultured RPE cells from the retina, which have powerful immunosuppressive properties and create immune tolerance in the posterior segment of the eye. mDCs were generated from bone marrow cells by using stimulators, such as recombinant TNF-α and LPS in the presence of recombinant proteins and medium containing IL-4 and granulocyte macrophage–colony-stimulating factor (GM-CSF). 
Materials and Methods
Mice
Adult C57BL/6 mice (CLEA Japan, Tokyo, Japan) were used as donors for ocular pigment epithelial cells. RPE cells were also established from IL-1Ra knockout donors (C57BL/6 background) or wild-type control mice. 22,23  
Establishment of Primary Cultured RPE Cells
RPE cells were cultivated as described previously. 1012 The RPE tissues were resuspended in Dulbecco's modified eagle medium (DMEM; Sigma-Aldrich, St. Louis, MO), placed in 24-well plates and incubated for 2 weeks. DMEM containing 20% fetal bovine serum was used for the primary cultures of RPE. 1012 As determined by flow cytometry, the primary RPE cultures were greater than 98% cytokeratin positive. 1012  
We also prepared other ocular PE cells, iris pigment epithelium (IPE), ciliary body pigment epithelium (CBPE), and CE cells, as described previously. 10,24 This research was approved by the institutional review board of Tokyo Medical and Dental University. All animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Isolation of mDCs
The generation of mDCs was performed according to the previous reports. 25,26 Briefly, mDCs were generated by culturing bone marrow cells with mouse GM-CSF (20 ng/mL; PeproTech, Rocky Hill, NJ) and recombinant mouse IL-4 (200 U/mL; eBioscience, San Diego, CA) in a bacteriologic Petri dish (BIO-BIK, Tokyo, Japan) for 7 days. Nonadherent cells were collected and isolated by CD11c beads and columns (MACS System; Miltenyi Biotec, Auburn, CA). Purified CD11c-positive cells were stimulated with LPS (1 μg/mL; Sigma-Aldrich) and recombinant mouse TNF-α (20 ng/mL; R&D Systems, Minneapolis, MN) for 3 days. These DC preparations typically had a purity of greater than 90% as estimated by anti–I-A/I-E or anti-CD11c monoclonal antibody staining and contained less than 0.1% erythrocytes, T cells, B cells, monocytes or macrophages, natural killer cells, and neutrophils as determined by flow cytometry. 26 Cultured RPE cells (0.5–1.0 × 105 cells/well) were added to mDCs at the initiation of TNF-α and LPS stimulation in the presence of medium containing GM-CSF and IL-4. 
Evaluation of mDC Activation
DCs were labeled with carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) as described in previous reports. 11,20 Briefly, 5 × 106 bone marrow–derived DCs were diluted in 1 mL of serum-free Hanks' Balanced salt solution (HBSS; Sigma-Aldrich), 1 μM CFSE was added, and the cell suspension was incubated for 8 minutes at room temperature. Purified CD11c-positive DCs labeled with CFSE were added (1.5 × 106/well in 24-well plates) to wells containing TNF-α, LPS, GM-CSF, and IL-4 in the presence or absence of RPE. Unlabeled DCs were used as a control, and some wells contained only RPE cells without DCs. After 96 hours, the DCs were washed and analyzed by flow cytometry. The amounts of pro-inflammatory cytokines such as TNF-α (R&D Systems), IL-1β (R&D Systems), and IL-12p40 (Thermo Scientific, Rockford, IL) in the supernatants were measured by ELISA. Anti-mouse TNF-α antibody (10 μg/mL, eBioscience), anti-mouse IL-1β antibody (10 μg/mL, eBioscience), or isotype control antibody (10 μg/mL, rat Immunoglobulin G [IgG]) were also used in mDCs-RPE cultures. 
Mixed Leukocyte Reactions (MLRs)
After depletion of red blood cells by treatment with red blood cell lysis buffer (ACK lysing buffer; Lonza, Tokyo, Japan), splenic T cells from BALB/c mice were purified by CD3 beads and columns (MACS system). The purity of BALB/c T cells was analyzed by flow cytometry (CD3 > 94%+). Then, T cells (1 × 105/well) were cocultured with mDCs (1 × 104/well: C57BL/6 donors) in a total volume of 200 μL in U-bottom 96-well culture plates for 72 hours (allogeneic reactive MLRs). mDCs were pretreated with 50 μg/mL mitomycin C (Sigma-Aldrich, Tokyo, Japan) for 1 hour at 37°C. T cell proliferation was assessed by CFSE flow cytometry or BrdU proliferation ELISA (Roche, Rotkreuz, Switzerland). The cytokine production of T cells was also assessed by IFN-γ ELISA (R&D Systems). 
Flow Cytometry
Flow cytometry was used to analyze the expression of surface molecules such as MHC class II, CD11c, CD80 (B7-1), CD86 (B7-2), PD-L1 (B7-H1), and PD-L2 (B7-DC) on DCs. The following antibodies were used to stain the RPE-exposed mDCs and mDCs without RPE: FITC-conjugated anti-mouse MHC class II antibody (eBioscience), PE-conjugated anti-mouse CD11c (BioLegend, San Diego, CA), PE-conjugated anti-mouse CD80 (B7-1, eBioscience), PE-conjugated anti-mouse CD86 (B7-2, eBioscience), PE-conjugated anti-mouse PD-L1 (B7-H1, BioLegend), and PE-conjugated anti-mouse PD-L2 (B7-DC, BioLegend). FITC- or PE-conjugated rat IgG and PE-conjugated goat IgG were used as isotype control antibodies. After 72 hours of activation with TNF-α, LPS, GM-CSF, and IL-4, DC cultures were harvested, washed twice, and then stained with antibodies. Before staining, the cocultured cells were incubated with mouse Fc block (Fcγ III/II Receptor, Clone 2.4G2; BD Biosciences, San Diego, CA) for 15 minutes. Cells (0.5 × 106) were stained for 30 minutes at 4°C in the dark and then analyzed by using a FACSCalibur cytometer (BD Biosciences, Bedford, MA). 
Immunohistochemistry
Cultured RPE cells were grown on a 4-well cell culture slide (BD Biosciences). mDCs exposed to RPE were also prepared. After washing with PBS, the RPE cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. The cells were incubated for 3 hours with monoclonal antibodies against mouse MHC class II (I-A/I-E, 1:100, eBioscience) or control rat IgG (1:100) as the isotype control. Subsequently, the cells were washed with PBS followed by a 1 hour incubation with fluorescence-labeled secondary antibody, Alexa Fluor 488 (anti-rat abs; Life Technologies, Tokyo, Japan). Fluorescence signals were detected by confocal microscopy (Radiance 2000; Bio-Rad Laboratories, Tokyo, Japan). 
Assay for IL-1Ra
Production of IL-1Ra by mDCs was evaluated by ELISA (R&D Systems). For the assay, we prepared supernatants of two RPE cell cultures (RPE-1 and RPE-2) as well as cultures of IPE, CBPE, or control cells (mouse splenic T cells). We also collected the supernatants from DCs exposed to RPE cells treated with rTNF-α or rIL-1β. 
Anti-mouse IL-1Ra neutralizing antibodies (10 μg/mL; R&D Systems) or an isotype control (10 μg/mL; R&D Systems) was used for the RPE-DC assay. As controls, 10 μg/mL of the following abs were used: anti-mouse TGF-β neutralizing antibodies (R&D Systems), anti-mouse CTLA-2α neutralizing antibodies, 17,18 anti-mouse PD-L1 neutralizing antibodies, 13 or isotype rabbit control. After incubation for 2 hours, the ab-pretreated RPE cells were rinsed once with PBS and added to wells containing the stimulated mDCs. Recombinant mouse IL-1Ra/IL-1F3 proteins (1 or 10 ng/mL; R&D Systems) were also used in another experiment. 
GeneChip Analysis
Transcription of the IL-1Ra gene in mDCs was also evaluated by oligonucleotide microarray. Total RNA in RPE cells was isolated by using TRIzol reagent (Life Technologies) as described previously, 13,27 and in accordance with the manufacturer's instructions. RNA was purified by using a Nucleospin RNA II kit (Macherey Nagel, Inc., Düren, Germany). Experimental procedures for the GeneChip were performed according to the Affymetrix GeneChip Expression Analysis Technical Manual. 13,27 The cRNA was hybridized to an oligonucleotide microarray (Mouse Genome 430 2.0). The microarray data are deposited in the Gene Expression Omnibus (GEO) public database under accession number GSE5134. 
Statistical Evaluation
Each experiment was repeated at least twice with similar results. Parametric data were analyzed with Student's t-tests. Statistical significance was defined as P less than 0.05. 
Results
Suppression of Activation of mDCs by Cultured RPE
We first assessed whether RPE cells could suppress mDC activation. Primary RPE cells were cocultured in 24-well plates and added to purified bone marrow–derived mDCs stimulated with TNF-α and LPS in medium containing IL-4 and GM-CSF. As revealed in Figure 1A, the production of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-12p40, by mDCs was significantly suppressed when RPE cells were cocultured with mDCs, whereas mDCs without RPE produced high levels of these cytokines (Fig. 1A). 
Figure 1
 
Suppression of mature dendritic cells (mDCs) by RPE cells. (A) In the presence of LPS, TNF-α, IL-4, and GM-CSF, purified DCs were cocultured with primary RPE cells for 72 hours. Then, supernatants of mDCs were harvested and measured for pro-inflammatory cytokines, TNF-α, IL-1β, and IL-12p40. Data are the mean ± SEM of three ELISA determinations. Open bars indicate RPE cells only as a control. Black bars indicate mDCs with or without RPE. *P < 0.05, **P < 0.005. (B) mDCs in the absence or presence of RPE cells were labeled with CFSE for flow cytometric analysis. After 72 hours, the mDCs were harvested for flow cytometry. The graph indicates the number of CFSE-positive cells. **P < 0.005. (C) mDCs were cocultured with IPE, CBPE, and CE cells. *P < 0.05, compared with positive control (mDCs alone).
Figure 1
 
Suppression of mature dendritic cells (mDCs) by RPE cells. (A) In the presence of LPS, TNF-α, IL-4, and GM-CSF, purified DCs were cocultured with primary RPE cells for 72 hours. Then, supernatants of mDCs were harvested and measured for pro-inflammatory cytokines, TNF-α, IL-1β, and IL-12p40. Data are the mean ± SEM of three ELISA determinations. Open bars indicate RPE cells only as a control. Black bars indicate mDCs with or without RPE. *P < 0.05, **P < 0.005. (B) mDCs in the absence or presence of RPE cells were labeled with CFSE for flow cytometric analysis. After 72 hours, the mDCs were harvested for flow cytometry. The graph indicates the number of CFSE-positive cells. **P < 0.005. (C) mDCs were cocultured with IPE, CBPE, and CE cells. *P < 0.05, compared with positive control (mDCs alone).
As another measure of mDC activation, we examined the suppressive effect of RPE cells on the dilution of CFSE in mDCs. mDCs were labeled with CFSE and stimulated with TNF-α, LPS, IL-4, and GM-CSF in the presence of RPE cells. The mDCs were harvested after 96 hours, and flow cytometry was used to evaluate the extent of progressive cell division. As shown in Figure 1B, a significant amount of cell division was evident in positive control cultures without the RPE cells (mDCs alone). When RPE cells were present, the division of mDCs was significantly suppressed. 
We next investigated whether other ocular resident cells located on the anterior segment in the eye, IPE, CBPE, and CE cells, could suppress the activation of mDCs. As expected, both types of PE cells (IPE and CBPE) and CE cells significantly suppressed TNF-α production, similar to RPE cells (Fig. 1C). 
Expression of Costimulatory Molecules by mDCs Exposed to RPE Cells
mDCs express high levels of MHC class II and costimulatory molecules. We evaluated the expression of costimulatory molecules on mDCs activated by TNF-α, LPS, IL-4, and GM-CSF in the presence of RPE. mDCs in the presence or absence of RPE cells were stained with anti-mouse MHC class II, CD80, CD86, PD-L1 (B7-H1), and PD-L2 (B7-DC) mAbs and analyzed by flow cytometry. The mDCs expressed high levels of MHC class II, CD80, CD86, PD-L1, and PD-L2 (Fig. 2A). In contrast, mDCs exposed to RPE cells had low expression levels as compared with the control cultures, indicating that expression was downregulated when RPE cells were cocultured with mDCs. Immunohistochemical staining of MHC class II molecules revealed that the expression was upregulated when mDCs were simulated by TNF-α, LPS, IL-4, and GM-CSF (Fig. 2B). In contrast, RPE-exposed mDCs failed to express MHC class II on their surface. Thus, mDCs exposed to RPE cells were converted into inactivated cells with downregulated expression of MHC class II and costimulatory molecules. 
Figure 2
 
Expression of costimulatory molecules by mDCs exposed to RPE. (A) Histograms represent the expression of costimulatory molecules on mDCs activated by LPS, TNF-α, IL-4, and GM-CSF in the presence (red line histogram) or absence of RPE (white histogram). Cells were stained with anti-mouse MHC class II FITC, anti-CD11c PE, anti-CD80 PE (B7-1), anti-CD86 PE (B7-2), anti-PD-L1 PE (B7-H1), or anti-PD-L2 PE (B7-DC) and analyzed by flow cytometry. (B) mDCs were stained with anti-mouse MHC class II antibodies and then examined by fluorescence confocal microscopy. In the upper figure, MHC class II (green) is expressed by bone marrow–derived mDCs. The expression of MHC class II on RPE-exposed mDCs is not seen in the lower figures. Arrows indicate DCs exposed to RPE cells (pigmented large cells). Bar: 60 μm.
Figure 2
 
Expression of costimulatory molecules by mDCs exposed to RPE. (A) Histograms represent the expression of costimulatory molecules on mDCs activated by LPS, TNF-α, IL-4, and GM-CSF in the presence (red line histogram) or absence of RPE (white histogram). Cells were stained with anti-mouse MHC class II FITC, anti-CD11c PE, anti-CD80 PE (B7-1), anti-CD86 PE (B7-2), anti-PD-L1 PE (B7-H1), or anti-PD-L2 PE (B7-DC) and analyzed by flow cytometry. (B) mDCs were stained with anti-mouse MHC class II antibodies and then examined by fluorescence confocal microscopy. In the upper figure, MHC class II (green) is expressed by bone marrow–derived mDCs. The expression of MHC class II on RPE-exposed mDCs is not seen in the lower figures. Arrows indicate DCs exposed to RPE cells (pigmented large cells). Bar: 60 μm.
Suppression of MLRs of mDCs by RPE
mDCs can activate naïve T cells by MLRs. For the assay, T cells from BALB/c mice were cocultured with mitomycin C-pretreated mDCs from C57BL/6 mice. T cell proliferation was assessed by CFSE flow cytometry analysis or BrdU proliferation ELISA. T cell production of IFN-γ was also assessed by ELISA. As revealed in Figure 3A, CFSE-labeled T cells cocultured with mDCs proliferated more than T cells without mDCs. By contrast, T cells cocultured with mDCs plus RPE cells failed to proliferate, indicating that cultured RPE cells suppress MLRs by mDCs. Similarly, BrdU-labeled T cells cocultured with mDCs proliferated, but proliferation was significantly suppressed in the presence of RPE cells (Fig. 3B). T cells cocultured with mDCs produced high levels of IFN-γ, whereas T cells cocultured with mDCs and RPE cells produced less IFN-γ (Fig. 3C). 
Figure 3
 
Suppression of DC-derived MLRs by RPE. Purified BALB/c T cells were cocultured with mitomycin C-pretreated mDCs for 72 hours. (A) T-cell proliferation was assessed by CFSE flow cytometry analysis. The M1 gate indicates the number of CFSE-positive cells. (B) T-cell proliferation was also assessed by BrdU proliferation ELISA. BrdU-labeled T cells were cocultured with or without mDCs ± RPE cells. Data are the mean ± SEM of three ELISA determinations. *P < 0.05. (C) Cytokine production of T cells was also assessed by IFN-γ ELISA. Data are the mean ± SEM of three ELISA determinations. **P < 0.005.
Figure 3
 
Suppression of DC-derived MLRs by RPE. Purified BALB/c T cells were cocultured with mitomycin C-pretreated mDCs for 72 hours. (A) T-cell proliferation was assessed by CFSE flow cytometry analysis. The M1 gate indicates the number of CFSE-positive cells. (B) T-cell proliferation was also assessed by BrdU proliferation ELISA. BrdU-labeled T cells were cocultured with or without mDCs ± RPE cells. Data are the mean ± SEM of three ELISA determinations. *P < 0.05. (C) Cytokine production of T cells was also assessed by IFN-γ ELISA. Data are the mean ± SEM of three ELISA determinations. **P < 0.005.
Expression of IL-1Ra by RPE Cells
RPE cells express several immunoregulatory molecules including IL-1Ra, 28,29 which can block inflammatory cytokines IL-1 and TNF-α. 30 We used oligonucleotide microarray and ELISA to evaluate the production of IL-1Ra by RPE cells. We first summarized the gene expression of cytokine antagonists and inhibitory cytokines by primary cultured RPE as determined by GeneChip analysis (Table). Among the representative cytokine antagonists and inhibitory cytokines, RPE cells expressed mRNA for IL-1Ra, TNF-RI, TNF-RII, and TGFβ1, 2, 3. The GeneChip analysis showed that cultured RPE cells as well as other ocular PE cells (IPE and CBPE) expressed high levels of IL-1Ra genes (Fig. 4A). Similarly, supernatants of primary cultured RPE cells contained significant levels of IL-1Ra proteins (Fig. 4B). Importantly, as compared with RPE cells alone, RPE cells exposed to mDCs produced significant amounts of IL-1Ra (Fig. 4C). RPE cells pretreated with TNF-α and/or IL-1β produced significant amounts of IL-1Ra (Fig. 4D). In addition, RPE cells exposed to mDCs in the presence of blocking antibodies to TNF-α and/or IL-1β significantly reduced the IL-1Ra production as compared with the results of an isotype control (Fig. 4E). These results suggest that RPE cells produce the inhibitory antagonist to protect against inflammation. 
Figure 4
 
Detection of IL-1 receptor antagonist (IL-1Ra) on RPE cells. (A) Expression of IL-1Ra genes in cultured RPE cells as assessed from the GeneChip results (n = 2, RPE-1, and RPE-2). The y-axis indicates the signal in RPE and control cells (IPE, CBPE, and T cells as a control). (B) Production of IL-1Ra proteins in supernatants of RPE cells. The supernatants of RPE cells (RPE-1 and RPE-2) and other cells were subjected to IL-1Ra ELISA. (C) The supernatants of RPE cells exposed to mDCs were subjected to IL-1Ra ELISA. Data are the mean ± SEM of three ELISA determinations. **P < 0.005, compared with two groups. (D) The supernatants of recombinant mouse TNF-α and/or IL-1β–pretreated RPE cells were subjected to IL-1Ra ELISA. We used both recombinant proteins (20 ng/mL) in vitro. Data are the mean ± SEM of three ELISA determinations. **P < 0.005, ***P < 0.0005, compared with RPE control cells (RPE alone: open bar). (E) The supernatants of RPE cells exposed to mDCs in the presence of anti-TNF-α, anti–IL-1β, or isotype control were subjected to IL-1Ra ELISA. Data are the mean ± SEM of three ELISA determinations. *P < 0.05, **P < 0.005, compared with mDCs plus RPE (open bar).
Figure 4
 
Detection of IL-1 receptor antagonist (IL-1Ra) on RPE cells. (A) Expression of IL-1Ra genes in cultured RPE cells as assessed from the GeneChip results (n = 2, RPE-1, and RPE-2). The y-axis indicates the signal in RPE and control cells (IPE, CBPE, and T cells as a control). (B) Production of IL-1Ra proteins in supernatants of RPE cells. The supernatants of RPE cells (RPE-1 and RPE-2) and other cells were subjected to IL-1Ra ELISA. (C) The supernatants of RPE cells exposed to mDCs were subjected to IL-1Ra ELISA. Data are the mean ± SEM of three ELISA determinations. **P < 0.005, compared with two groups. (D) The supernatants of recombinant mouse TNF-α and/or IL-1β–pretreated RPE cells were subjected to IL-1Ra ELISA. We used both recombinant proteins (20 ng/mL) in vitro. Data are the mean ± SEM of three ELISA determinations. **P < 0.005, ***P < 0.0005, compared with RPE control cells (RPE alone: open bar). (E) The supernatants of RPE cells exposed to mDCs in the presence of anti-TNF-α, anti–IL-1β, or isotype control were subjected to IL-1Ra ELISA. Data are the mean ± SEM of three ELISA determinations. *P < 0.05, **P < 0.005, compared with mDCs plus RPE (open bar).
Table
 
Summary of Gene Expression of Cytokine Antagonists and Inhibitory Cytokines in Primary Cultured RPE by GeneChip Analysis
Table
 
Summary of Gene Expression of Cytokine Antagonists and Inhibitory Cytokines in Primary Cultured RPE by GeneChip Analysis
Probe Set Accession Number Gene Signal in GeneChip
RPE-1 RPE-2 CBPE IPE T Cells (Control)
1451798_at M57525 IL-1F3/IL-1Ra 2027 2627 1374 2750 ND
1421370_a_at NM_019451 IL-1F5/IL-36Ra ND ND ND ND ND
1449864_at NM_021283 IL-4 ND ND ND ND 99
1450330_at NM_010548 IL-10 ND ND ND ND 141
1450564_x_at NM_010502 IFN-a ND ND ND ND ND
1417291_at I_26349 TNF-RI 716 1544 603 1107 132
1418099_at M50469 TNF-RII 181 243 110 245 109
1420653_at NM_011577 TGFβ1 283 507 147 201 563
1450922_a_at BF144658 TGFβ2 192 102 179 623 ND
1417455_at BC014690 TGFβ3 637 854 402 357 ND
Functional Analysis of RPE-Dependent Suppression of mDCs Via IL-1Ra
Next, we confirmed that IL-1Ra plays a critical role in the RPE-dependent suppression of mDCs by using anti-mouse IL-1Ra neutralizing antibodies or RPE cells from IL-1Ra knockout donors. As shown in Figure 5A, mDCs were cocultured with RPE cells in the presence of blocking anti–IL-1Ra abs or control abs. As experimental controls, we used blocking abs against TGF-β, PD-L1 (B7-H1), and cytotoxic T lymphocyte antigen-2α (CTLA-2α) because primary cultured RPE cells produce and secrete these molecules, which are critical mediators of immunosuppression. 12,13,17,18 The mDCs did not produce TNF-α in the presence of RPE cells treated with control abs (TGF-β, CTLA-2α, PD-L1, or isotype control). In contrast, mDCs produced significant amounts of TNF-α if the RPE cells were pretreated with anti–IL-1Ra (Fig. 5A). 
Figure 5
 
Effect of neutralizing antibodies or RPE cells from IL-1Ra knockout mice in RPE-dependent mDC suppression. (A) For the in vitro assay, mDCs were cocultured with RPE for 72 hours. Neutralizing abs, anti-mouse IL-1Ra, anti-mouse TGF-β, anti-mouse CTLA-2α, anti-mouse PD-L1, or isotype rabbit IgG were added to some of the wells. Bars indicate percent inhibition of TNF-α production of mDCs by RPE in vitro. abs (–), mDCs + RPE without abs (open bar). (B) mDCs from normal mice were cocultured with RPE from IL-1Ra knockout (KO) or wild-type (WT) donors. Supernatants of mDCs were harvested for measurement of TNF-α. Data are the mean ± SEM of three ELISA determinations. *P < 0.05, compared with two groups. n.s., not significant. (C) mDCs were cocultured with recombinant mouse IL-1Ra proteins (1 or 10 ng/mL) for 72 hours. Supernatants of mDCs were harvested for measurement of TNF-α mDCs without IL-1Ra proteins (open bar). Data are the mean ± SEM of three ELISA determinations. **P < 0.005, compared with control culture (mDCs alone: open bar).
Figure 5
 
Effect of neutralizing antibodies or RPE cells from IL-1Ra knockout mice in RPE-dependent mDC suppression. (A) For the in vitro assay, mDCs were cocultured with RPE for 72 hours. Neutralizing abs, anti-mouse IL-1Ra, anti-mouse TGF-β, anti-mouse CTLA-2α, anti-mouse PD-L1, or isotype rabbit IgG were added to some of the wells. Bars indicate percent inhibition of TNF-α production of mDCs by RPE in vitro. abs (–), mDCs + RPE without abs (open bar). (B) mDCs from normal mice were cocultured with RPE from IL-1Ra knockout (KO) or wild-type (WT) donors. Supernatants of mDCs were harvested for measurement of TNF-α. Data are the mean ± SEM of three ELISA determinations. *P < 0.05, compared with two groups. n.s., not significant. (C) mDCs were cocultured with recombinant mouse IL-1Ra proteins (1 or 10 ng/mL) for 72 hours. Supernatants of mDCs were harvested for measurement of TNF-α mDCs without IL-1Ra proteins (open bar). Data are the mean ± SEM of three ELISA determinations. **P < 0.005, compared with control culture (mDCs alone: open bar).
We also examined if TGF-β produced by RPE cells is important for the suppression of mDCs, because TGF-β is a powerful mediator that suppresses immune cells. Unexpectedly, recombinant porcine TGF-β2 failed to suppress the activation of mDCs (data not shown). Moreover, TGF-β siRNA–transfected RPE cells (TGF-β downregulation) did not suppress TNF-α production by mDCs (data not shown). Together, these results suggest that TGF-β produced by RPE cells appears to be irrelevant for the suppression of mDCs. 
Finally, we examined the capacity of RPE cells established from IL-1Ra knockout donors to suppress TNF-α production by mDCs. RPE cells from wild-type mice were used as a control. RPE cells from wild-type donors significantly suppressed TNF-α production by mDCs (Fig. 5B), but RPE cells from IL-1Ra knockout donors did not suppress the activation of mDCs. We also examined whether recombinant mouse IL-1Ra to administer to mDCs (without RPE) still suppresses the production of TNF-α. As expected, recombinant mouse IL-1Ra significantly suppressed the production of TNF-α by mDCs (Fig. 5C). Taken together, these results imply that RPE cells produce IL-1Ra, a pro-inflammatory antagonist, and that IL-1Ra is a critical mediator of immunosuppression by RPE cells. 
Discussion
IL-1Ra is widely expressed on fibroblast cells, epithelial cells, and immunocytes such as monocytes, neutrophil, and dendritic cells. 31 In ocular studies, the antagonist is constitutively expressed on healthy, human RPE cells 28,29 and corneal tissues. 32 Yamada et al. demonstrated that IL-1Ra treatment suppressed allosensitization in corneal transplantation. 33 Pro-inflammatory cytokines including IL-1, IL-6, and TNF-α are potent multifunctional cell activators that stimulate RPE cells. 28 In reaction to these cytokines, RPE cells produce high levels of immunomodulatory molecules. 28,34 To prevent retinal tissue damage during inflammation in the eye, the stimulation of RPE by these cytokines should, therefore, be precisely regulated and require tight physiologic control. One mechanism to regulate the effect of IL-1 is via its naturally occurring antagonist, IL-1Ra, produced by ocular resident cells. Therefore, we hypothesize that pro-inflammatory cytokines induce RPE cells to express IL-1Ra, which suppresses DC activation by competition with pro-inflammatory cytokines. 
DCs are bone marrow–derived leukocytes, and the maturity of DCs is reflected in the expression of increased levels of cell–surface molecules associated with inflammation (e.g., MHC class II and costimulatory molecules) and altered cytokine production. Stimulation with the components of micro-organisms such as LPS and pro-inflammatory cytokines such as TNF-α is required for DC maturation. GM-CSF is necessary to prepare a sufficient number of DCs from bone marrow cell culture. In addition, IL-4 is necessary to prevent monocytes from converting into macrophages. Therefore, we used TNF-α and IL-4 to induce mDCs in vitro. CD11c+ mDCs express high levels of MHC class II and costimulatory molecules and produce pro-inflammatory cytokines. 15 They are highly specialized antigen-presenting cells that respond to the presence of pathogens and inflammation at peripheral tissues including the eye, migrating to secondary lymphoid organs where the DCs present antigens to T cells, leading to cell proliferation and differentiation into effector cells. In animal models of inflammation, MHC class II-positive DCs and macrophages are present in the iris, ciliary body, and retina. 3537 Recently, HLA class IIhigh CD86high DCs were detected in patients with active uveitis, suggesting that the activated DCs, as well as other immune cells, play an important role in the generation of inflammatory responses during inflammatory ocular diseases. 9 Therefore, we designed experiments to investigate whether cultured ocular cells can suppress DCs in vitro. 
We found that primary RPE cells constitutively expressed IL-1Ra and that this molecule blocked the effect of pro-inflammatory cytokines. The production of this antagonist was induced in RPE cells pretreated with pro-inflammatory cytokines or exposed to mDCs. In antigen-dependent immune responses, DCs, which can efficiently stimulate T cells, function as antigen-presenting cells. mDCs produce large amounts of pro-inflammatory cytokines. 15 As shown in this study, RPE cells can suppress DC functions and interfere with DC maturation, indicating that RPE cells are able to suppress the expression of costimulatory molecules such as CD80, CD86, PD-L1, and PD-L2 in activated mDCs. In addition, IL-1Ra plays a critical role in mDC suppression, as shown by experiments with blocking antibody and RPE cells from IL-1Ra knockout mice. Thus, the naturally occurring antagonist is a common denominator in various suppressive mechanisms elicited by RPE. 
The eye possesses inherent immune privilege, and inflammation is self-regulated to preserve vision. 38 The immunosuppressive intraocular microenvironment and eye-derived immunologic tolerance (anterior chamber-associated immune deviation) also contribute to ocular immune privilege. However, innate and adaptive immune cells and molecules can still access the eye, as the anatomical barriers, or eye-derived immunological tolerance, are not absolute. The eye contains soluble and cell–surface immunomodulatory factors that suppress cells and molecules that mediate innate and adaptive immune inflammation. This intraocular milieu is called the immunosuppressive microenvironment. The functions of the cells and factors that manage natural immunity and acquired immunity are inhibited by factors that are expressed in the anterior segment, including corneal endothelium, iris–ciliary body, and aqueous humor. 39,40 Among these factors, α-melanocyte–stimulating hormone, vasoactive intestinal peptide, calcitonin gene-related peptide, TGFβ, and thrombospondin-1 (TSP-1) regulate the functions of T cells, B cells, macrophages, and DCs. In the posterior segment in the eye, CTLA-2α, 17,18 TGF-β, 12,14 TSP-1, 27 and retinoic acid 41 are produced by RPE. These immunosuppressive molecules that suppress inflammatory cells may create the immune-suppressive microenvironment. In addition to the immunoregulatory molecules, IL-1Ra expressed by ocular resident cells and tissues might include the candidate molecules. 
Recently, the application of cell-based therapy including DCs for sight threatening ocular inflammation has been considered. Immature bone marrow–derived DCs have been effective in reducing inflammation in experimental autoimmune uveitis (EAU). 42 Immature DCs that were treated with immunosuppressive cytokines such as TGF-β and IL-10, but still required LPS activation for full effect significantly suppressed intraocular inflammation in EAU models. 26 In EAU, tolerogenic immature DCs were more effective when pulsed with specific antigen, but there was also some effect with nonantigen pulsed DCs. 42 There is broad agreement that tolerogenic DCs mediate their suppressive function through the induction of regulatory T cells (Tregs). 43,44 Although we do not have evidence for RPE-induced suppressor DCs like RPE-induced Tregs 1719 and RPE-induced myeloid-derived suppressor cells, 45 suppressor DCs may be used to treat severe intraocular inflammation in the near future. 
In conclusion, RPE cells, through naturally occurring antagonist, inhibit mature DCs that have been simultaneously activated. The cytokine antagonist pathway, which is constitutively expressed within the eye, 28,29,32 is an important immunomodulatory mechanisms by which infiltrating antigen-presenting cells and ocular resident antigen-presenting cells are suppressed in the eye during inflammation. 
Acknowledgments
The authors thank Ikuyo Yamamoto for the expert technical assistance. 
Supported by grants from the Takeda Science Foundation, Japan. 
Disclosure: S. Sugita, None; Y. Kawazoe, None; A. Imai, None; Y. Usui, None; Y. Iwakura, None; K. Isoda, None; M. Ito, None; M. Mochizuki, None 
References
Cella M Engering A Pinet V Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells. Nature . 1997; 388: 782–787. [CrossRef] [PubMed]
Romani N Reider D Heuer M Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability. J Immunol Methods . 1996; 196: 137–151. [CrossRef] [PubMed]
Ni K O'Neill HC. The role of dendritic cells in T cell activation. Immunol Cell Biol . 1997; 75: 223–230. [CrossRef] [PubMed]
Banchereau J Steinman RM. Dendritic cells and the control of immunity. Nature . 1998; 392: 245–252. [CrossRef] [PubMed]
Banchereau J Briere F Caux C Immunobiology of dendritic cells. Annu Rev Immunol . 2000; 18: 767–811. [CrossRef] [PubMed]
Banchereau J Pascual V. Type I interferon in systemic lupus erythematosus and other autoimmune diseases. Immunity . 2006; 25: 383–392. [CrossRef] [PubMed]
Banchereau J Pascual V Palucka AK. Autoimmunity through cytokine-induced dendritic cell activation. Immunity . 2004; 20: 539–550. [CrossRef] [PubMed]
Melli K Friedman RS Martin AE Amplification of autoimmune response through induction of dendritic cell maturation in inflamed tissues. J Immunol . 2009; 182: 2590–2600. [CrossRef] [PubMed]
Kim TW Kang JS Kong JM Maturation profiles of peripheral blood dendritic cells in patients with endogenous uveitis. Immunol Lett . 2012; 142: 14–19. [CrossRef] [PubMed]
Sugita S Streilein JW. Iris pigment epithelium expressing CD86 (B7-2) directly suppresses T cell activation in vitro via binding to cytotoxic T lymphocyte-associated antigen 4. J Exp Med . 2003; 198: 161–171. [CrossRef] [PubMed]
Sugita S Ng TF Schwartzkopff J CTLA-4+CD8+ T cells that encounter B7-2+ iris pigment epithelial cells express their own B7-2 to achieve global suppression of T cell activation. J Immunol . 2004; 172: 4184–4194. [CrossRef] [PubMed]
Sugita S Futagami Y Smith SB Retinal and ciliary body pigment epithelium suppress activation of T lymphocytes via transforming growth factor beta. Exp Eye Res . 2006; 83: 1459–1471. [CrossRef] [PubMed]
Sugita S Usui Y Horie S T-cell suppression by programmed cell death 1 ligand 1 on retinal pigment epithelium during inflammatory conditions. Invest Ophthalmol Vis Sci . 2009; 50: 2862–2870. [CrossRef] [PubMed]
Sugita S Horie S Yamada Y Suppression of interleukin-17–producing T-helper 17 cells by retinal pigment epithelial cells. Jpn J Ophthalmol . 2011; 55: 565–575. [CrossRef] [PubMed]
Ishida K Panjwani N Cao Z Participation of pigment epithelium in ocular immune privilege. 3. Epithelia cultured from iris, ciliary body, and retina suppress T-cell activation by partially non-overlapping mechanisms. Ocul Immunol Inflamm . 2003; 11: 91–105. [CrossRef] [PubMed]
Liversidge J McKay D Mullen G Retinal pigment epithelial cells modulate lymphocyte function at the blood-retina barrier by autocrine PGE2 and membrane-bound mechanisms. Cell Immunol . 1993; 149: 315–330. [CrossRef] [PubMed]
Sugita S Horie S Nakamura O Retinal pigment epithelium-derived CTLA-2alpha induces TGFbeta-producing T regulatory cells. J Immunol . 2008; 181: 7525–7536. [CrossRef] [PubMed]
Sugita S Horie S Nakamura O Acquisition of T regulatory function in cathepsin L-inhibited T cells by eye-derived CTLA-2α during inflammatory conditions. J Immunol . 2009; 183: 5013–5022. [CrossRef] [PubMed]
Vega JL Saban D Carrier Y Retinal pigment epithelial cells induce foxp3(+) regulatory T cells via membrane-bound TGF-beta. Ocul Immunol Inflamm . 2010; 18: 459–469. [CrossRef] [PubMed]
Sugita S Horie S Yamada Y Inhibition of B cell activation by retinal pigment epithelium. Invest Ophthalmol Vis Sci . 2010; 51: 5783–5798. [CrossRef] [PubMed]
Zamiri P Masli S Streilein JW Taylor AW. Pigment epithelial growth factor suppresses inflammation by modulating macrophage activation. Invest Ophthalmol Vis Sci . 2006; 47: 3912–3918. [CrossRef] [PubMed]
Horai R Asano M Sudo K Production of mice deficient in genes for interleukin (IL)-1alpha, IL-1beta, IL-1alpha/beta, and IL-1 receptor antagonist shows that IL-1beta is crucial in turpentine-induced fever development and glucocorticoid secretion. J Exp Med . 1998; 187: 1463–1475. [CrossRef] [PubMed]
Isoda K Shiigai M Ishigami N Deficiency of interleukin-1 receptor antagonist promotes neointimal formation after injury. Circulation . 2003; 108: 516–518. [CrossRef] [PubMed]
Sugita S Yamada Y Horie S Induction of T regulatory cells by cytotoxic T-lymphocyte antigen-2 alpha on corneal endothelial cells. Invest Ophthalmol Vis Sci . 2011; 52: 2598–2605. [CrossRef] [PubMed]
Shen L Barabino S Taylor AW Dana MR. Effect of the ocular microenvironment in regulating corneal dendritic cell maturation. Arch Ophthalmol . 2007; 125: 908–915. [CrossRef] [PubMed]
Usui Y Takeuchi M Hattori T Suppression of experimental autoimmune uveoretinitis by regulatory dendritic cells in mice. Arch Ophthalmol . 2009; 127: 514–519. [CrossRef] [PubMed]
Futagami Y Sugita S Vega J Role of thrombospondin-1 in T cell response to ocular pigment epithelial cells. J Immunol . 2007; 178: 6994–7005. [CrossRef] [PubMed]
Jaffe GJ Van Le L Valea F Expression of interleukin-1 alpha, interleukin-1 beta, and an interleukin-1 receptor antagonist in human retinal pigment epithelial cells. Exp Eye Res . 1992; 55: 325–335. [CrossRef] [PubMed]
Holtkamp GM de Vos AF Kijlstra A Peek R. Expression of multiple forms of IL-1 receptor antagonist (IL-1ra) by human retinal pigment epithelial cells: identification of a new IL-1ra exon. Eur J Immunol . 1999; 29: 215–224. [CrossRef] [PubMed]
Arend WP Malyak M Bigler CF The biological role of naturally-occurring cytokine inhibitors. Br J Rheumatol . 1991; 30: 49–52. [PubMed]
Arend WP. Interleukin-1 receptor antagonist. Adv Immunol . 1993; 54: 167–227. [PubMed]
Heur M Chaurasia SS Wilson SE. Expression of interleukin-1 receptor antagonist in human cornea. Exp Eye Res . 2009; 88: 992–994. [CrossRef] [PubMed]
Yamada J Dana MR Zhu SN Interleukin 1 receptor antagonist suppresses allosensitization in corneal transplantation. Arch Ophthalmol . 1998; 116: 1351–1357. [CrossRef] [PubMed]
Benson MT Shepherd L Rees RC Rennie IG. Production of interleukin-6 by human retinal pigment epithelium in vitro and its regulation by other cytokines. Curr Eye Res . 1992; 11: 173–179. [CrossRef] [PubMed]
McMenamin PG Crewe J Morrison S Holt PG. Immunomorphologic studies of macrophages and MHC class II-positive dendritic cells in the iris and ciliary body of the rat, mouse, and human eye. Invest Ophthalmol Vis Sci . 1994; 35: 3234–3250. [PubMed]
McMenamin PG Crewe J. Endotoxin-induced uveitis. Kinetics and phenotype of the inflammatory cell infiltrate and the response of the resident tissue macrophages and dendritic cells in the iris and ciliary body. Invest Ophthalmol Vis Sci . 1995; 36: 1949–1959. [PubMed]
Jiang HR Lumsden L Forrester JV. Macrophages and dendritic cells in IRBP-induced experimental autoimmune uveoretinitis in B10RIII mice. Invest Ophthalmol Vis Sci . 1999; 40: 3177–3185. [PubMed]
Streilein JW. Ocular immune privilege: therapeutic opportunities from an experiment of nature. Nat Rev Immunol . 2003; 3: 879–889. [CrossRef] [PubMed]
Taylor AW. Ocular immunosuppressive microenvironment. Chem Immunol Allergy . 2007; 92: 71–85. [PubMed]
Granstein RD Staszewski R Knisely TL Aqueous humor contains transforming growth factor-beta and a small (less than 3500 daltons) inhibitor of thymocyte proliferation. J Immunol . 1990; 144: 3021–3027. [PubMed]
Kawazoe Y Sugita S Keino H Retinoic acid from retinal pigment epithelium induces T regulatory cells. Exp Eye Res . 2012; 94: 32–40. [CrossRef] [PubMed]
Jiang HR Muckersie E Robertson M Forrester JV. Antigen-specific inhibition of experimental autoimmune uveoretinitis by bone marrow-derived immature dendritic cells. Invest Ophthalmol Vis Sci . 2003; 44: 1598–607. [CrossRef] [PubMed]
Huang H Dawicki W Zhang X Tolerogenic dendritic cells induce CD4+CD25hiFoxp3+ regulatory T cell differentiation from CD4+CD25-/loFoxp3- effector T cells. J Immunol . 2010; 185: 5003–5010. [CrossRef] [PubMed]
Maldonado RA von Andrian UH. How tolerogenic dendritic cells induce regulatory T cells. Adv Immunol . 2010; 108: 111–165. [PubMed]
Tu Z Li Y Smith D Myeloid suppressor cells induced by retinal pigment epithelial cells inhibit autoreactive T-cell responses that lead to experimental autoimmune uveitis. Invest Ophthalmol Vis Sci . 2012; 53: 959–966. [CrossRef] [PubMed]
Figure 1
 
Suppression of mature dendritic cells (mDCs) by RPE cells. (A) In the presence of LPS, TNF-α, IL-4, and GM-CSF, purified DCs were cocultured with primary RPE cells for 72 hours. Then, supernatants of mDCs were harvested and measured for pro-inflammatory cytokines, TNF-α, IL-1β, and IL-12p40. Data are the mean ± SEM of three ELISA determinations. Open bars indicate RPE cells only as a control. Black bars indicate mDCs with or without RPE. *P < 0.05, **P < 0.005. (B) mDCs in the absence or presence of RPE cells were labeled with CFSE for flow cytometric analysis. After 72 hours, the mDCs were harvested for flow cytometry. The graph indicates the number of CFSE-positive cells. **P < 0.005. (C) mDCs were cocultured with IPE, CBPE, and CE cells. *P < 0.05, compared with positive control (mDCs alone).
Figure 1
 
Suppression of mature dendritic cells (mDCs) by RPE cells. (A) In the presence of LPS, TNF-α, IL-4, and GM-CSF, purified DCs were cocultured with primary RPE cells for 72 hours. Then, supernatants of mDCs were harvested and measured for pro-inflammatory cytokines, TNF-α, IL-1β, and IL-12p40. Data are the mean ± SEM of three ELISA determinations. Open bars indicate RPE cells only as a control. Black bars indicate mDCs with or without RPE. *P < 0.05, **P < 0.005. (B) mDCs in the absence or presence of RPE cells were labeled with CFSE for flow cytometric analysis. After 72 hours, the mDCs were harvested for flow cytometry. The graph indicates the number of CFSE-positive cells. **P < 0.005. (C) mDCs were cocultured with IPE, CBPE, and CE cells. *P < 0.05, compared with positive control (mDCs alone).
Figure 2
 
Expression of costimulatory molecules by mDCs exposed to RPE. (A) Histograms represent the expression of costimulatory molecules on mDCs activated by LPS, TNF-α, IL-4, and GM-CSF in the presence (red line histogram) or absence of RPE (white histogram). Cells were stained with anti-mouse MHC class II FITC, anti-CD11c PE, anti-CD80 PE (B7-1), anti-CD86 PE (B7-2), anti-PD-L1 PE (B7-H1), or anti-PD-L2 PE (B7-DC) and analyzed by flow cytometry. (B) mDCs were stained with anti-mouse MHC class II antibodies and then examined by fluorescence confocal microscopy. In the upper figure, MHC class II (green) is expressed by bone marrow–derived mDCs. The expression of MHC class II on RPE-exposed mDCs is not seen in the lower figures. Arrows indicate DCs exposed to RPE cells (pigmented large cells). Bar: 60 μm.
Figure 2
 
Expression of costimulatory molecules by mDCs exposed to RPE. (A) Histograms represent the expression of costimulatory molecules on mDCs activated by LPS, TNF-α, IL-4, and GM-CSF in the presence (red line histogram) or absence of RPE (white histogram). Cells were stained with anti-mouse MHC class II FITC, anti-CD11c PE, anti-CD80 PE (B7-1), anti-CD86 PE (B7-2), anti-PD-L1 PE (B7-H1), or anti-PD-L2 PE (B7-DC) and analyzed by flow cytometry. (B) mDCs were stained with anti-mouse MHC class II antibodies and then examined by fluorescence confocal microscopy. In the upper figure, MHC class II (green) is expressed by bone marrow–derived mDCs. The expression of MHC class II on RPE-exposed mDCs is not seen in the lower figures. Arrows indicate DCs exposed to RPE cells (pigmented large cells). Bar: 60 μm.
Figure 3
 
Suppression of DC-derived MLRs by RPE. Purified BALB/c T cells were cocultured with mitomycin C-pretreated mDCs for 72 hours. (A) T-cell proliferation was assessed by CFSE flow cytometry analysis. The M1 gate indicates the number of CFSE-positive cells. (B) T-cell proliferation was also assessed by BrdU proliferation ELISA. BrdU-labeled T cells were cocultured with or without mDCs ± RPE cells. Data are the mean ± SEM of three ELISA determinations. *P < 0.05. (C) Cytokine production of T cells was also assessed by IFN-γ ELISA. Data are the mean ± SEM of three ELISA determinations. **P < 0.005.
Figure 3
 
Suppression of DC-derived MLRs by RPE. Purified BALB/c T cells were cocultured with mitomycin C-pretreated mDCs for 72 hours. (A) T-cell proliferation was assessed by CFSE flow cytometry analysis. The M1 gate indicates the number of CFSE-positive cells. (B) T-cell proliferation was also assessed by BrdU proliferation ELISA. BrdU-labeled T cells were cocultured with or without mDCs ± RPE cells. Data are the mean ± SEM of three ELISA determinations. *P < 0.05. (C) Cytokine production of T cells was also assessed by IFN-γ ELISA. Data are the mean ± SEM of three ELISA determinations. **P < 0.005.
Figure 4
 
Detection of IL-1 receptor antagonist (IL-1Ra) on RPE cells. (A) Expression of IL-1Ra genes in cultured RPE cells as assessed from the GeneChip results (n = 2, RPE-1, and RPE-2). The y-axis indicates the signal in RPE and control cells (IPE, CBPE, and T cells as a control). (B) Production of IL-1Ra proteins in supernatants of RPE cells. The supernatants of RPE cells (RPE-1 and RPE-2) and other cells were subjected to IL-1Ra ELISA. (C) The supernatants of RPE cells exposed to mDCs were subjected to IL-1Ra ELISA. Data are the mean ± SEM of three ELISA determinations. **P < 0.005, compared with two groups. (D) The supernatants of recombinant mouse TNF-α and/or IL-1β–pretreated RPE cells were subjected to IL-1Ra ELISA. We used both recombinant proteins (20 ng/mL) in vitro. Data are the mean ± SEM of three ELISA determinations. **P < 0.005, ***P < 0.0005, compared with RPE control cells (RPE alone: open bar). (E) The supernatants of RPE cells exposed to mDCs in the presence of anti-TNF-α, anti–IL-1β, or isotype control were subjected to IL-1Ra ELISA. Data are the mean ± SEM of three ELISA determinations. *P < 0.05, **P < 0.005, compared with mDCs plus RPE (open bar).
Figure 4
 
Detection of IL-1 receptor antagonist (IL-1Ra) on RPE cells. (A) Expression of IL-1Ra genes in cultured RPE cells as assessed from the GeneChip results (n = 2, RPE-1, and RPE-2). The y-axis indicates the signal in RPE and control cells (IPE, CBPE, and T cells as a control). (B) Production of IL-1Ra proteins in supernatants of RPE cells. The supernatants of RPE cells (RPE-1 and RPE-2) and other cells were subjected to IL-1Ra ELISA. (C) The supernatants of RPE cells exposed to mDCs were subjected to IL-1Ra ELISA. Data are the mean ± SEM of three ELISA determinations. **P < 0.005, compared with two groups. (D) The supernatants of recombinant mouse TNF-α and/or IL-1β–pretreated RPE cells were subjected to IL-1Ra ELISA. We used both recombinant proteins (20 ng/mL) in vitro. Data are the mean ± SEM of three ELISA determinations. **P < 0.005, ***P < 0.0005, compared with RPE control cells (RPE alone: open bar). (E) The supernatants of RPE cells exposed to mDCs in the presence of anti-TNF-α, anti–IL-1β, or isotype control were subjected to IL-1Ra ELISA. Data are the mean ± SEM of three ELISA determinations. *P < 0.05, **P < 0.005, compared with mDCs plus RPE (open bar).
Figure 5
 
Effect of neutralizing antibodies or RPE cells from IL-1Ra knockout mice in RPE-dependent mDC suppression. (A) For the in vitro assay, mDCs were cocultured with RPE for 72 hours. Neutralizing abs, anti-mouse IL-1Ra, anti-mouse TGF-β, anti-mouse CTLA-2α, anti-mouse PD-L1, or isotype rabbit IgG were added to some of the wells. Bars indicate percent inhibition of TNF-α production of mDCs by RPE in vitro. abs (–), mDCs + RPE without abs (open bar). (B) mDCs from normal mice were cocultured with RPE from IL-1Ra knockout (KO) or wild-type (WT) donors. Supernatants of mDCs were harvested for measurement of TNF-α. Data are the mean ± SEM of three ELISA determinations. *P < 0.05, compared with two groups. n.s., not significant. (C) mDCs were cocultured with recombinant mouse IL-1Ra proteins (1 or 10 ng/mL) for 72 hours. Supernatants of mDCs were harvested for measurement of TNF-α mDCs without IL-1Ra proteins (open bar). Data are the mean ± SEM of three ELISA determinations. **P < 0.005, compared with control culture (mDCs alone: open bar).
Figure 5
 
Effect of neutralizing antibodies or RPE cells from IL-1Ra knockout mice in RPE-dependent mDC suppression. (A) For the in vitro assay, mDCs were cocultured with RPE for 72 hours. Neutralizing abs, anti-mouse IL-1Ra, anti-mouse TGF-β, anti-mouse CTLA-2α, anti-mouse PD-L1, or isotype rabbit IgG were added to some of the wells. Bars indicate percent inhibition of TNF-α production of mDCs by RPE in vitro. abs (–), mDCs + RPE without abs (open bar). (B) mDCs from normal mice were cocultured with RPE from IL-1Ra knockout (KO) or wild-type (WT) donors. Supernatants of mDCs were harvested for measurement of TNF-α. Data are the mean ± SEM of three ELISA determinations. *P < 0.05, compared with two groups. n.s., not significant. (C) mDCs were cocultured with recombinant mouse IL-1Ra proteins (1 or 10 ng/mL) for 72 hours. Supernatants of mDCs were harvested for measurement of TNF-α mDCs without IL-1Ra proteins (open bar). Data are the mean ± SEM of three ELISA determinations. **P < 0.005, compared with control culture (mDCs alone: open bar).
Table
 
Summary of Gene Expression of Cytokine Antagonists and Inhibitory Cytokines in Primary Cultured RPE by GeneChip Analysis
Table
 
Summary of Gene Expression of Cytokine Antagonists and Inhibitory Cytokines in Primary Cultured RPE by GeneChip Analysis
Probe Set Accession Number Gene Signal in GeneChip
RPE-1 RPE-2 CBPE IPE T Cells (Control)
1451798_at M57525 IL-1F3/IL-1Ra 2027 2627 1374 2750 ND
1421370_a_at NM_019451 IL-1F5/IL-36Ra ND ND ND ND ND
1449864_at NM_021283 IL-4 ND ND ND ND 99
1450330_at NM_010548 IL-10 ND ND ND ND 141
1450564_x_at NM_010502 IFN-a ND ND ND ND ND
1417291_at I_26349 TNF-RI 716 1544 603 1107 132
1418099_at M50469 TNF-RII 181 243 110 245 109
1420653_at NM_011577 TGFβ1 283 507 147 201 563
1450922_a_at BF144658 TGFβ2 192 102 179 623 ND
1417455_at BC014690 TGFβ3 637 854 402 357 ND
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