Interleukin-6 Receptor-Mediated Activation of Signal Transducer and Activator of Transcription-3 (STAT3) Promotes Choroidal Neovascularization
2007; Elsevier BV; Volume: 170; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2007.061018
ISSN1525-2191
AutoresKanako Izumi‐Nagai, Norihiro Nagai, Yoko Ozawa, Masahiko Mihara, Yoshiyuki Ohsugi, Toshihide Kurihara, Takashi Koto, Shingo Satofuka, Makoto Inoue, Kazuo Tsubota, Hideyuki Okano, Yuichi Oike, Susumu Ishida,
Tópico(s)Glaucoma and retinal disorders
ResumoInterleukin (IL)-6, a potent proinflammatory cytokine, is suggested to be a risk factor for choroidal neovascularization (CNV) because of its increased levels in the serum of patients with age-related macular degeneration; however, the role of IL-6 in CNV has not been defined. The present study reveals the critical contribution of IL-6 signaling and its downstream STAT3 pathway to the murine model of laser-induced CNV. CNV induction by laser treatment stimulated IL-6 expression in the retinal pigment epithelium-choroid complex, and antibody-based blockade of IL-6 receptor or genetic ablation of IL-6 led to significant suppression of CNV. CNV generation was accompanied by STAT3 activation in choroidal endothelial cells and macrophages, and IL-6 receptor blockade resulted in selectively inhibited phosphorylation of STAT3 but not extracellular signal-regulated kinase 1/2. Consistently, pharmacological blockade of STAT3 pathway also suppressed CNV. In addition, IL-6 receptor neutralization led to significant inhibition of the in vivo and in vitro expression of inflammation-related molecules including monocyte chemotactic protein, intercellular adhesion molecule-1, and vascular endothelial growth factor, and of macrophage infiltration into CNV. These results indicate the significant involvement of IL-6 receptor-mediated activation of STAT3 inflammatory pathway in CNV generation, suggesting the possibility of IL-6 receptor blockade as a therapeutic strategy to suppress CNV associated with age-related macular degeneration. Interleukin (IL)-6, a potent proinflammatory cytokine, is suggested to be a risk factor for choroidal neovascularization (CNV) because of its increased levels in the serum of patients with age-related macular degeneration; however, the role of IL-6 in CNV has not been defined. The present study reveals the critical contribution of IL-6 signaling and its downstream STAT3 pathway to the murine model of laser-induced CNV. CNV induction by laser treatment stimulated IL-6 expression in the retinal pigment epithelium-choroid complex, and antibody-based blockade of IL-6 receptor or genetic ablation of IL-6 led to significant suppression of CNV. CNV generation was accompanied by STAT3 activation in choroidal endothelial cells and macrophages, and IL-6 receptor blockade resulted in selectively inhibited phosphorylation of STAT3 but not extracellular signal-regulated kinase 1/2. Consistently, pharmacological blockade of STAT3 pathway also suppressed CNV. In addition, IL-6 receptor neutralization led to significant inhibition of the in vivo and in vitro expression of inflammation-related molecules including monocyte chemotactic protein, intercellular adhesion molecule-1, and vascular endothelial growth factor, and of macrophage infiltration into CNV. These results indicate the significant involvement of IL-6 receptor-mediated activation of STAT3 inflammatory pathway in CNV generation, suggesting the possibility of IL-6 receptor blockade as a therapeutic strategy to suppress CNV associated with age-related macular degeneration. Age-related macular degeneration (AMD) is the most common cause of blindness in developed countries.1Klein R Wang Q Klein BE Moss SE Meuer SM The relationship of age-related maculopathy, cataract, and glaucoma to visual acuity.Invest Ophthalmol Vis Sci. 1995; 36: 182-191PubMed Google Scholar The macula is located at the center of the retina, and the visual acuity depends on the function of the macula where cone photoreceptors are abundant. AMD is complicated by choroidal neovascularization (CNV), leading to severe vision loss and blindness. During CNV generation, new vessels from the choroid invade the subretinal space through Bruch's membrane, resulting in the formation of the neovascular tissue including vascular endothelial cells, retinal pigment epithelial cells, fibroblasts, and macrophages.2Lopez PF Grossniklaus HE Lambert HM Aaberg TM Capone Jr, A Sternberg Jr, P L'Hernault N Pathologic features of surgically excised subretinal neovascular membranes in age-related macular degeneration.Am J Ophthalmol. 1991; 112: 647-656Abstract Full Text PDF PubMed Scopus (227) Google Scholar Subretinal hemorrhage and lipid exudation develop from the immature vessels in the proliferative tissue, causing injury to the photoreceptors. Molecular and cellular mechanisms underlying CNV are not fully elucidated. CNV seen in AMD develops with chronic inflammation adjacent to the retinal pigment epithelium (RPE), Bruch's membrane, and choriocapillaris. Recent experimental and clinical studies have indicated vascular endothelial growth factor (VEGF) as a critical factor for promoting CNV.3Krzystolik MG Afshari MA Adamis AP Gaudreault J Gragoudas ES Michaud NA Li W Connolly E O'Neill CA Miller JW Prevention of experimental choroidal neovascularization with intravitreal anti-vascular endothelial growth factor antibody fragment.Arch Ophthalmol. 2002; 120: 338-346Crossref PubMed Scopus (550) Google Scholar, 4Rosenfeld PJ Brown DM Heier JS Boyer DS Kaiser PK Chung CY Kim RY MARINA Study Group Ranibizumab for neovascular age-related macular degeneration.N Engl J Med. 2006; 355: 1419-1431Crossref PubMed Scopus (4700) Google Scholar CNV formation is associated with the influx of inflammatory cells including macrophages, which are shown to be a rich source of VEGF. Pharmacological depletion of macrophages, present in both human and murine CNV tissues,2Lopez PF Grossniklaus HE Lambert HM Aaberg TM Capone Jr, A Sternberg Jr, P L'Hernault N Pathologic features of surgically excised subretinal neovascular membranes in age-related macular degeneration.Am J Ophthalmol. 1991; 112: 647-656Abstract Full Text PDF PubMed Scopus (227) Google Scholar, 5Sakurai E Anand A Ambati BK van Rooijen N Ambati J Macrophage depletion inhibits experimental choroidal neovascularization.Invest Ophthalmol Vis Sci. 2003; 44: 3578-3585Crossref PubMed Scopus (405) Google Scholar, 6Tsutsumi C Sonoda K Egashira K Qiao H Hisatomi T Nakao S Ishibashi M Charo IF Sakamoto T Murata T Ishibashi T The critical role of ocular-infiltrating macrophages in the development of choroidal neovascularization.J Leukoc Biol. 2003; 74: 25-32Crossref PubMed Scopus (236) Google Scholar, 7Espinosa-Heidmann DG Suner IJ Hernandez EP Monroy D Csaky KG Cousins SW Macrophage depletion diminishes lesion size and severity in experimental choroidal neovascularization.Invest Ophthalmol Vis Sci. 2003; 44: 3586-3592Crossref PubMed Scopus (330) Google Scholar results in significant suppression of murine CNV.5Sakurai E Anand A Ambati BK van Rooijen N Ambati J Macrophage depletion inhibits experimental choroidal neovascularization.Invest Ophthalmol Vis Sci. 2003; 44: 3578-3585Crossref PubMed Scopus (405) Google Scholar, 7Espinosa-Heidmann DG Suner IJ Hernandez EP Monroy D Csaky KG Cousins SW Macrophage depletion diminishes lesion size and severity in experimental choroidal neovascularization.Invest Ophthalmol Vis Sci. 2003; 44: 3586-3592Crossref PubMed Scopus (330) Google Scholar CNV tissues from both human surgical samples and the rodent laser-induced model express inflammation-related molecules including intercellular adhesion molecule (ICAM)-1.8Yeh DC Bula DV Miller JW Gragoudas ES Arroyo JG Expression of leukocyte adhesion molecules in human subfoveal choroidal neovascular membranes treated with and without photodynamic therapy.Invest Ophthalmol Vis Sci. 2004; 45: 2368-2673Crossref PubMed Scopus (43) Google Scholar, 9Sakurai E Taguchi H Anand A Ambati BK Gragoudas ES Miller JW Adamis AP Ambati J Targeted disruption of the CD18 or ICAM-1 gene inhibits choroidal neovascularization.Invest Ophthalmol Vis Sci. 2003; 44: 2743-2749Crossref PubMed Scopus (117) Google Scholar Genetic ablation of ICAM-1 or CC chemokine receptor-2, a receptor for monocyte chemotactic protein (MCP)-1, inhibited CNV in the murine model.6Tsutsumi C Sonoda K Egashira K Qiao H Hisatomi T Nakao S Ishibashi M Charo IF Sakamoto T Murata T Ishibashi T The critical role of ocular-infiltrating macrophages in the development of choroidal neovascularization.J Leukoc Biol. 2003; 74: 25-32Crossref PubMed Scopus (236) Google Scholar, 9Sakurai E Taguchi H Anand A Ambati BK Gragoudas ES Miller JW Adamis AP Ambati J Targeted disruption of the CD18 or ICAM-1 gene inhibits choroidal neovascularization.Invest Ophthalmol Vis Sci. 2003; 44: 2743-2749Crossref PubMed Scopus (117) Google Scholar Interleukin (IL)-6 is a potent proinflammatory cytokine that binds to its receptor IL-6R, and the complex of IL-6 and IL-6R interacts with gp130 on the cell surface, leading to dimerization of gp130 that initiates IL-6-mediated signaling in target cells.10Hibi M Murakami M Saito M Hirano T Taga T Kishimoto T Molecular cloning and expression of an IL-6 signal transducer, gp130.Cell. 1990; 63: 1149-1157Abstract Full Text PDF PubMed Scopus (1091) Google Scholar, 11Murakami M Hibi M Nakagawa N Nakagawa T Yasukawa K Yamanishi K Taga T Kishimoto T IL-6-induced homodimerization of gp130 and associated activation of a tyrosine kinase.Science. 1993; 260: 1808-1810Crossref PubMed Scopus (638) Google Scholar Because of the soluble, diffusible form of IL-6R in addition to membrane-bound IL-6R, the complex of IL-6 and soluble IL-6R is capable of inducing IL-6-mediated signal transduction even in IL-6R-negative cells, if only they express gp130.11Murakami M Hibi M Nakagawa N Nakagawa T Yasukawa K Yamanishi K Taga T Kishimoto T IL-6-induced homodimerization of gp130 and associated activation of a tyrosine kinase.Science. 1993; 260: 1808-1810Crossref PubMed Scopus (638) Google Scholar Downstream pathways following gp130 dimerization include the activation of STAT3 (signal transducer and activator of transcription 3), a known transcription factor that induces inflammation,12Alonzi T Fattori E Cappelletti M Ciliberto G Poli V Impaired Stat3 activation following localized inflammatory stimulus in IL-6-deficient mice.Cytokine. 1998; 10: 13-18Crossref PubMed Scopus (44) Google Scholar, 13Ripperger J Fritz S Richter K Dreier B Schneider K Lochner K Marschalek R Hocke G Lottspeich F Fey GH Isolation of two interleukin-6 response element binding proteins from acute phase rat livers.Ann NY Acad Sci. 1995; 762: 252-260Crossref PubMed Scopus (15) Google Scholar and ERK (extracellular signal-regulated kinase) MAP (mitogen-activated protein) kinase cascade, which mainly promotes cell proliferation.14Ogata A Chauhan D Teoh G Treon SP Urashima M Schlossman RL Anderson KC IL-6 triggers cell growth via the Ras-dependent mitogen-activated protein kinase cascade.J Immunol. 1997; 159: 2212-2221PubMed Google Scholar, 15Iankov I Praskova M Kalenderova S Tencheva Z Mitov I Mitev V The effect of chemical blockade of PKC with Go6976 and Go6983 on proliferation and MAPK activity in IL-6-dependent plasmacytoma cells.Leuk Res. 2002; 26: 363-368Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar Recently, IL-6 has been suggested to play a role in the pathogenesis of ocular diseases. Vitreous aspirates from patients with proliferative diabetic retinopathy, another vision-threatening disease characterized by retinal neovascularization, exhibit the parallel increases in IL-6 and VEGF.16Funatsu H Yamashita H Noma H Mimura T Nakamura S Sakata K Hori S Aqueous humor levels of cytokines are related to vitreous levels and progression of diabetic retinopathy in diabetic patients.Graefes Arch Clin Exp Ophthalmol. 2005; 243: 3-8Crossref PubMed Scopus (241) Google Scholar Interestingly, increased serum levels of IL-6 and C-reactive protein have recently proven to be related with progression of AMD.17Seddon JM George S Rosner B Rifai N Progression of age-related macular degeneration: prospective assessment of C-reactive protein, interleukin 6, and other cardiovascular biomarkers.Arch Ophthalmol. 2005; 123: 774-782Crossref PubMed Scopus (290) Google Scholar No data have been reported, however, showing the direct evidence of the pathogenic role of IL-6 signaling in CNV generation. Here, we report the first evidence of the in vivo effect of IL-6R blockade on the murine model of CNV, together with underlying molecular and cellular mechanisms. Male C57BL/6J mice (CLEA, Tokyo, Japan) at the age of 7 to 10 weeks and age- and sex-matched IL-6-deficient homozygous mice raised on C57BL/6J background (Jackson Laboratories, Bar Harbor, ME) were used. All animal experiments were conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Laser-induced CNV is widely used as an animal model for neovascular AMD and reflects the pathogenesis of choroidal inflammation and neovascularization seen in AMD. In this model, new vessels from the choroid invade the subretinal space after photocoagulation. Laser photocoagulation was performed at five spots per eye around the optic disk with the wavelength of 532 nm, the power of 200 mW, the duration of 100 ms, and the spot size of 75 μm, using a slit-lamp delivery system (Novus Spectra; Lumenis, Tokyo, Japan), as described previously.5Sakurai E Anand A Ambati BK van Rooijen N Ambati J Macrophage depletion inhibits experimental choroidal neovascularization.Invest Ophthalmol Vis Sci. 2003; 44: 3578-3585Crossref PubMed Scopus (405) Google Scholar Animals were treated with an intraperitoneal injection of a rat anti-mouse IL-6R monoclonal antibody MR16-1 (Chugai Pharmaceutical Co. Ltd., Tokyo, Japan), prepared as described previously,18Tamura T Udagawa N Takahashi N Miyaura C Tanaka S Yamada Y Koishihara Y Ohsugi Y Kumaki K Taga T Kishimoto T Suda T Soluble interleukin-6 receptor triggers osteoclast formation by interleukin 6.Proc Natl Acad Sci USA. 1993; 90: 11924-11928Crossref PubMed Scopus (767) Google Scholar or a purified rat nonimmune isotype IgG (MP Biomedicals, Solon, OH) immediately after photocoagulation. MR16-1 has been shown to bind to murine IL-6R and suppress IL-6-induced cellular responses in a dose-dependent manner.19Okazaki M Yamada Y Nishimoto N Yoshizaki K Mihara M Characterization of anti-mouse interleukin-6 receptor antibody.Immunol Lett. 2002; 84: 231-240Crossref PubMed Scopus (89) Google Scholar Other basic properties of this antibody have been described in previously published reports.18Tamura T Udagawa N Takahashi N Miyaura C Tanaka S Yamada Y Koishihara Y Ohsugi Y Kumaki K Taga T Kishimoto T Suda T Soluble interleukin-6 receptor triggers osteoclast formation by interleukin 6.Proc Natl Acad Sci USA. 1993; 90: 11924-11928Crossref PubMed Scopus (767) Google Scholar, 19Okazaki M Yamada Y Nishimoto N Yoshizaki K Mihara M Characterization of anti-mouse interleukin-6 receptor antibody.Immunol Lett. 2002; 84: 231-240Crossref PubMed Scopus (89) Google Scholar MR16-1 was injected into mice with the dose of 1, 10, or 100 μg/g body weight. Animals were also treated with a JAK2 tyrosine kinase inhibitor AG490 (Calbiochem, La Jolla, CA) or phosphate-buffered saline (PBS) containing 1% dimethyl sulfoxide as vehicle daily for 3 days after photocoagulation. AG490 was shown to inhibit JAK/STAT pathway and suppress the growth of various cancers.20Meydan N Grunberger T Dadi H Shahar M Arpaia E Lapidot Z Leeder JS Freedman M Cohen A Gazit A Levitzki A Roifman CM Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor.Nature. 1996; 379: 645-648Crossref PubMed Scopus (847) Google Scholar, 21De Vos J Jourdan M Tarte K Jasmin C Klein B JAK2 tyrosine kinase inhibitor tyrphostin AG490 downregulates the mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription (STAT) pathways and induces apoptosis in myeloma cells.Br J Haematol. 2000; 109: 823-828Crossref PubMed Scopus (160) Google Scholar AG490 was intraperitoneally administered to mice with the dose of 0.1 or 1 μg/g body weight. One week after laser injury, eyes were enucleated and fixed with 4% paraformaldehyde. Eye cups obtained by removing anterior segments were incubated with 0.5% fluorescein isothiocyanate-isolectin B4 (Vector, Burlingame, CA). CNV was visualized with blue argon laser wavelength (488 nm) using a scanning laser confocal microscope (FV1000; Olympus, Tokyo, Japan). Horizontal optical sections of CNV were obtained every 1-μm step from the surface to the deepest focal plane. The area of CNV-related fluorescence was measured by National Institutes of Health Image (Bethesda, MD). The summation of whole fluorescent area was used as the index of CNV volume, as described previously.5Sakurai E Anand A Ambati BK van Rooijen N Ambati J Macrophage depletion inhibits experimental choroidal neovascularization.Invest Ophthalmol Vis Sci. 2003; 44: 3578-3585Crossref PubMed Scopus (405) Google Scholar Immunohistochemical experiments were performed for murine CNV. Murine eyes enucleated 72 hours after photocoagulation were fixed with acetone at 4°C and embedded in paraffin. After blocking nonspecific binding in PBS containing 1% bovine serum albumin for 30 minutes at room temperature, paraffin sections were incubated overnight at 4°C with a rabbit anti-phosphorylated STAT3 antibody (1:100; Cell Signaling Technology, Beverly, MA) together with a rat polyclonal antibody against F4/80 (1:100; Serotec, Oxford, UK) or 0.5% fluorescein isothiocyanate-isolectin B4. Avidin-Alexa 488- and avidin-Alexa 546-tagged secondary antibodies (1:200; Molecular Probes, Eugene, OR) were then applied for 2 hours at room temperature. For nuclear staining, the specimens were treated with TOTO-3 (1:500; Molecular Probes) at room temperature for 30 minutes. After two washes, the samples were viewed with the scanning laser confocal microscope. Protein extracts were obtained from the homogenized RPE-choroid complex 1 day after photocoagulation. The choroid was carefully isolated and placed into 100 μl of lysis buffer (0.02 mol/L HEPES, 10% glycerol, 10 mmol/L Na4P2O7, 100 μmol/L Na3VO4, 1% Triton X-100, 100 mmol/L NaF, and 4 mmol/L ethylenediaminetetraacetic acid, pH 8.0) supplemented with protease inhibitors (2 mg/L aprotinin, 100 μmol/L phenylmethyl sulfonyl fluoride, 10 μmol/L leupeptin, and 2.5 μmol/L pepstatin A) and sonicated. The lysate was centrifuged, and the supernatant was collected. Each sample containing 30 μg of total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted to polyvinylidene fluoride membrane (ATTO, Tokyo, Japan). After blocking nonspecific binding with 5% skim milk, the membranes were incubated with a rabbit polyclonal antibody against STAT3 (1:1000), ERK1/2 (1:2000), phosphorylated forms of STAT3 or ERK1/2 (1:1000; Cell Signaling Technology), or an anti-α-tubulin antibody (1:2000; Sigma, St. Louis, MO) at 4°C overnight, followed by incubation with a horseradish peroxidase-conjugated goat antibody against rabbit IgG (1:5000; BioSource, Camarillo, CA). The signals were visualized with an enhanced chemiluminescence kit (GE Health Care, Buckinghamshire, UK) according to the manufacturer's protocol. Total RNA was isolated from the RPE-choroid complex 3 days after photocoagulation using an extraction reagent (TRIzol; Invitrogen, Carlsbad, CA) and reverse-transcribed with a cDNA synthesis kit (First-Strand; GE Health Care). PCR was performed using TaqDNA polymerase (Takara Bio, Ohtu, Japan) in a thermal controller (Gene Amp PCR system; Applied Biosystems, Foster, CA). The primer sequences and the expected size of amplified cDNA fragments are as follows: 5′-TTCCTCTCTGCAAGAGACT-3′ (sense) and 5′-TGTATCTCTCTGAAGGACT-3′ (anti-sense) (430 bp) for IL-6, 5′-GTGTCGAGCTTTGGGATGGTA-3′ (sense) and 5′-CTGGGCTTGGAGACTCAGTG-3′ (anti-sense) (505 bp) for ICAM-1, 5′-CCCCACTCACCTGCTGCTACT-3′ (sense) and 5′-GGCATCACAGTCCGAGTCACA-3′ (anti-sense) (380 bp) for MCP-1, 5′-GAAGTCCCATGAAGTGATCCAG-3′ (sense) and 5′-TCACCGCCTTGGCTTGTCA-3′ (anti-sense) (319 bp and 451 bp) for VEGF120 and VEGF164, respectively, and 5′-ATGTGGCACCACACCTTCTACAATGAGCTGCG-3′ (sense) and 5′-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3′ (anti-sense) (837 bp) for β-actin. The RPE-choroid complex was carefully isolated from the eyes 3 days after photocoagulation and placed into 100 μl of lysis buffer supplemented with protease inhibitors and sonicated. The lysate was centrifuged at 15,000 rpm for 15 minutes at 4°C, and the levels of IL-6, ICAM-1, MCP-1, and VEGF were determined with the mouse IL-6, ICAM-1, MCP-1, and VEGF ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer's protocols. Three days after laser injury, eyes were enucleated and whole-mount choroid-sclera complexes were incubated overnight at 4°C with a goat polyclonal antibody against mouse PECAM-1 (CD31) and a rat polyclonal antibody against F4/80 (Serotec). Avidin-Alexa 488- and avidin-Alexa 546-tagged secondary antibodies were then applied for 2 hours at room temperature, and CNV was viewed with the scanning laser confocal microscope. PECAM-1-stained area of CNV was measured as fluorescent pixels, the number of F4/80-positive macrophages was counted in every 5-μm step of CNV, and area-adjusted number of macrophages per 10,000-μm2 area of CNV was calculated. Murine brain-derived capillary endothelial cell line (b-End3) and murine macrophages (RAW264.7) were cultured with Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal bovine serum at 37°C in a 95% air-5% CO2 atmosphere. Lipopolysaccharide (LPS; Sigma) was used as a potent inducer of IL-6. Twelve hours before the experiments with b-End3 cells, the culture medium was changed to serum-free Dulbecco's modified Eagle's medium. After a 2-hour incubation with LPS (200 ng/ml) plus MR16-1 (1 or 10 μg/ml) or LPS plus control rat IgG (10 μg/ml), the cell lysate was processed for Western blot analyses for total and phosphorylated STAT3, and total cellular RNA was processed for RT-PCR analyses for ICAM-1 and MCP-1. For protein analyses, the supernatant and cell lysate were collected after a 6-hour incubation and then the concentration of MCP-1 in the supernatant and ICAM-1 in the cell lysate were measured by the ELISA kits (R&D Systems). RAW264.7 cells were treated with Dulbecco's modified Eagle's medium containing LPS (200 ng/ml) plus MR16-1 (1 or 10 μg/ml) or LPS plus control rat IgG (10 μg/ml). After a 2-hour incubation, the cell lysate was collected for Western blot analyses for STAT3. After a 6-hour incubation, the supernatant and total cellular RNA were processed for ELISA and RT-PCR analyses for VEGF. All results were expressed as mean ± SD. The values were processed for statistical analyses (Mann-Whitney test). Differences were considered statistically significant when P < 0.05. The RPE-choroid complex was subjected to RT-PCR and Western blot analyses to detect the expression of IL-6 mRNA (Figure 1A) and protein (Figure 1B), respectively. mRNA expression of IL-6 was higher in the RPE-choroid complex of mice 1 and 3 days after photocoagulation than in age-matched normal controls (Figure 1A). mRNA expression of IL-6 in the RPE-choroid complex was returned to the normal level 7 and 10 days after photocoagulation. Similarly, IL-6 protein levels at 3 days after photocoagulation were significantly increased by inducing CNV (P < 0.01) (Figure 1B). The index of CNV volume was measured to evaluate the effects of the IL-6R signaling on the development of CNV. CNV was significantly suppressed by blocking IL-6R signaling with MR16-1. MR16-1-treated mice at the dose of 10 or 100 μg/g showed a significant (P < 0.001) decrease in the index of CNV volume (312,076 ± 87,973 μm3 for 10 μg/g, 349,720 ± 104,395 μm3 for 100 μg/g) compared with control IgG-treated mice (508,423 ± 136,303 μm3) (Figure 2, A and B). In addition, CNV was also significantly (P < 0.001) attenuated in IL-6-deficient mice (359,878 ± 110,767 μm3) to the similar levels of IL-6R blockade, compared with wild-type animals (510,630 ± 153,019 μm3) (Figure 2, C and D). To examine the expression and tissue localization of phosphorylated STAT3 in murine CNV, CNV tissues were stained with an antibody against phosphorylated STAT3 together with isolectin B4 or an anti-F4/80 antibody, markers for vascular endothelial cells or macrophages, respectively. The immunohistochemical analyses for murine CNV showed phosphorylated STAT3 staining on isolectin B4-positive endothelial cells (Figure 3A) and F4/80-positive macrophages (Figure 3B). STAT3 and ERK MAP kinase signaling cascades are two major pathways activated by IL-6/IL-6R via gp130. To define the signaling pathway involved in the treatment with MR16-1, we analyzed the ratios of protein levels of phosphorylated forms of STAT3 and ERK1/2 to total STAT3 and ERK1/2 in the RPE-choroid complex. STAT3 and ERK1/2 were significantly activated in the RPE-choroid complex by inducing CNV (P < 0.05; Figure 4, A–C). IL-6R signaling blockade by MR16-1 significantly suppressed STAT3, but not ERK1/2, phosphorylation in the RPE-choroid complex (P < 0.05; Figure 4, A–C). Importantly, CNV was significantly suppressed by blocking JAK/STAT pathway with a JAK2 inhibitor AG490. AG490-treated mice at the dose of 0.1 or 1 μg/g showed a significant decrease in the index of CNV volume (404,976 ± 114,524 μm3 for 0.1 μg/g, 309,966 ± 57,126 μm3 for 1 μg/g) compared with vehicle-treated mice (505,750 ± 144,701 μm3) (Figure 4, D and E). To determine whether IL-6R signaling blockade affects inflammatory and angiogenic molecules related to the pathogenesis of CNV, mRNA expression of ICAM-1, MCP-1, and VEGF in the RPE-choroid complex was analyzed by semiquantitative RT-PCR (Figure 5A). mRNA expression of ICAM-1, MCP-1, and VEGF in the RPE-choroid complex was up-regulated by inducing CNV. IL-6R signaling blockade by systemic administration of MR16-1 substantially reduced mRNA expression of ICAM-1, MCP-1, and VEGF (both 164 and 120 isoforms). In addition, protein levels of ICAM-1, MCP-1, and VEGF in the RPE-choroid complex were higher in mice with CNV than in age-matched normal controls (Figure 5, B–D). IL-6R signaling blockade by MR16-1 significantly suppressed protein levels of ICAM-1 (P < 0.01), MCP-1 (P < 0.05), and VEGF (P < 0.01). To confirm the MR16-1-induced suppression of STAT3 phosphorylation in vivo (Figure 4) and choroidal expression of various inflammatory and angiogenic molecules (Figure 5), we further performed in vitro analyses (Figure 6). IL-6 production was markedly induced by the treatment with LPS (data not shown). The ratios of phosphorylated to total STAT-3, significantly (P < 0.01) elevated by LPS application, were significantly (P < 0.01) suppressed by the application of MR16-1 in both b-End3 cells (Figure 6, A and B) and RAW264.7 macrophages (Figure 6, F and G). We analyzed mRNA (Figure 6, C and H) and protein (Figure 6, D, E, and I) levels of ICAM-1 and MCP-1 in b-End3 vascular endothelial cells (Figure 6, C–E) and VEGF in RAW264.7 macrophages (Figure 6, H and I). In b-End3 cells, mRNA (Figure 6C) and protein levels (Figure 6, D and E) of ICAM-1 and MCP-1, induced by the exposure to LPS, were significantly suppressed by the treatment with MR16-1 (P < 0.05). In RAW264.7 macrophages, mRNA (Figure 6H) and protein (Figure 6I) levels of VEGF, induced by LPS stimulation, were significantly suppressed by the treatment with MR16-1 (P < 0.01). As the cellular mechanism in the pathogenesis of CNV, infiltration of inflammatory cells including macrophages plays a critical role in its growth. We compared the area-adjusted number of macrophages, which was adjusted by the area of CNV, between mice treated with MR16-1 versus control IgG (Figure 7, A and B). MR16-1-treated mice at the dose of 10 or 100 μg/g showed a significant decrease in the number of F4/80-positive macrophages, compared with control IgG-treated animals (P < 0.01). The present study reveals, for the first time to our knowledge, several important findings concerning the role of IL-6 signaling in the development of CNV. First, CNV induction by laser treatment stimulated IL-6 expression in the RPE-choroid complex (Figure 1), and antibody-based blockade of IL-6R or genetic ablation of IL-6 led to significant suppression of CNV (Figure 2). Second, CNV generation was accompanied by STAT3 activation in choroidal endothelial cells and macrophages (Figure 3), and IL-6R blockade resulted in selective inhibition of STAT3, but not ERK1/2, phosphorylation (Figure 4). Consistently, pharmacological blockade of STAT3 pathway suppressed CNV (Figure 4). Third, the molecular and cellular mechanisms in the IL-6 signaling blockade included the inhibitory effects on inflammation-related molecules in the RPE-choroid complex (Figure 5) and in cultured endothelial cells and macrophages (Figure 6) and on macrophage infiltration into CNV (Figure 7). Our current data demonstrate the critical role of IL-6 signaling in CNV, the pathogenesis of which has proven to be mediated by inflammation. This is supported by the previous reports showing the pathogenic role of IL-6 in inflammatory disease models including collagen-induced murine arthritis22Takagi N Mihara M Moriya Y Nishimoto N Yoshizaki K Kishimoto T Takeda Y Ohsugi Y Blockage of interleukin-6 receptor ameliorates joint disease in murine collagen-induced arthritis.Arthritis Rheum. 1998; 41: 2117-2121Crossref PubMed Scopus (246) Google Scholar and Th1 cell-mediated murine colitis.23Yamamoto M Yoshizaki K Kishimoto T Ito H IL-6 is required for the development of Th1 cell-mediated murine colitis.J Immunol. 2000; 164: 4878-4882PubMed Google Scholar IL-6/IL-6R binding-mediated gp130 dimerization is known to cause the activation of STAT3 and ERK MAP kinase pathways. Genetically altered mice with a point mutation in gp130, capable of activating STAT3, but not ERK MAP kinase, exhibited joint inflammation mimicking human rheumatoid arthritis,24Naka T Kishimoto T Joint disease caused by defective gp130-mediated STAT signaling.Arthritis Res. 2002; 4: 154-156Crossref PubMed Scopus (16) Google Scholar suggest
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