Choroidal neovascularization (CNV) tissue was excised from patients with age-related macular degeneration (AMD) who had no prior treatment for CNV. Retinal fibrosis tissue was excised from patients with a diagnosis of epiretinal membrane formation. Donor eyes from patients with atrophic AMD without CNV and patients without AMD were obtained from eye banks. Eyes with choroidal melanoma were obtained by surgical enucleation. The study followed the guidelines of the Declaration of Helsinki. Institutional review boards granted approval for allocation and histological analysis of specimens.
All animal experiments were in accordance with the guidelines of the University of Kentucky IACUC and the Association for Research in Vision and Ophthalmology. C57BL/6J and KitW-v
mice were purchased from The Jackson Laboratory. Ccr3−/−
, and Δdbl GATA mice have been previously described11,12,25,26
mice were generated by interbreeding “single knockout” mice described previously32
Rat IgG2a neutralizing antibody against mouse CCR3 (R&D Systems), control rat IgG2a (Serotec), goat neutralizing antibody against mouse CCL11 (1 μg; R&D Systems), goat neutralizing antibody against mouse CCL24 (5 μg; R&D Systems), control goat IgG (Jackson Immunoresearch), or (S)-Methyl-2-naphthoylamino-3-(4-nitrophenyl)propionate (SB328437; Calbiochem) dissolved in DMSO were injected into the vitreous humor of mice using a 33-gauge double-calibre needle (Ito Corporation) once, immediately after laser injury as previously described22
Rat antibody against mouse CCR3 (1:250; Santa Cruz) coupled with PE-donkey antibody against rat IgG (1:250; Jackson Immunoresearch) or AlexaFluor647-conjugated rat antibody against mouse CCR3 (10 μg/ml; BD Biosciences) were used to quantify cell surface receptor expression on choroidal endothelial cells, defined by CD31+ VEGFR-2+ expression, gated by FITC-conjugated rat antibody against mouse CD31 (20 μg/ml; BD Biosciences) and PE-conjugated rat antibody against mouse VEGFR-2 (20 μg/ml; BD Biosciences). Macrophages, neutrophils, eosinophils and mast cells were defined as F4/80+CD11c−, Gr-1+F4/80−, CCR3hiCD3−CD117intCD49d+ and CCR3intCD3− CD117hiCD49d+ cells, respectively. DNA content for cell cycle was analyzed after incubation with propidium iodide (0.05 mg/ml; Molecular Probes) containing 0.1% Triton X-100 and RNase A (0.1 mg/ml; Roche). Samples were analyzed on a LSRII (Becton Dickinson).
Immunofluorescent staining was performed with antibodies against human CCR3 (rat monoclonal, R&D Systems) or human CD31 (mouse monoclonal, Dako) and identified with Alexa 488 (Molecular Probes) or Cy3 secondary antibodies (Jackson ImmunoResearch). Immunohistochemical staining with the primary antibodies specific for human eotaxins-1, 2 and 3 (mouse monoclonal, R&D Systems) was performed using horseradish peroxidase. Laser injured mouse eye sections were stained with antibodies against mouse CCL11 or CCL24 (both R&D Systems) along with antibody against mouse CD31 (BD Biosciences) and visualized with FITC or Cy3 secondary antibodies. Images were obtained using Leica SP5 or Zeiss Axio Observer Z1 microscopes.
Tube formation assay
96-well plates were coated with Growth Factor Reduced Matrigel (BD Biosciences) mixed with rat neutralizing antibody against human CCR3 (20 μg/ml, R&D Systems) or control rat IgG2a (Invitrogen) and allowed to solidify in the incubator at 37 °C for 45 min. Human choroidal endothelial cells (CECs)44-47
were plated on top of the Matrigel at 2.25 × 104
in EBM-2 basal media (Cambrex) containing 1% FBS with CCR3 antibody or rat IgG2a at the above concentrations and allowed to grow overnight. Tube formation was analyzed by counting the number of cell junctions per mm2
Human CECs were synchronized for cell cycle state by first cultivating them in EGM2-MV media (Lonza) supplemented with 10% FBS (Gibco) to achieve complete confluence and then by overnight serum starvation in MCDB131 media (Gibco) with 0.1% FBS. They were passaged to 96-well plates at a density of 5,000 cells per well, followed by stimulation for 24 h with eotaxin-1, 2 or 3 (10 ng, 100 ng and 2 μg per ml, respectively; Peprotech) in MCDB131 media with 0.1% FBS. After 24 h, cell viability was measured with BrdU ELISA (Chemicon) according to manufacturer's instructions.
F-actin Polymerization Assay
Human CECs were seeded in black-walled 96-well plates and grown to 70-80% confluence in fully supplemented EGM-2MV. Cultures were serum starved overnight in basal media and then stimulated with recombinant human eotaxin-1 (10 ng/ml), eotaxin-2 (100 ng/ml), eotaxin-3 (2 μg/ml) (Peprotech), or vehicle control (PBS). At 0, 10, 30, 60, or 120 sec time-points, cells were fixed in 3.7% paraformaldehyde for 10 min, washed, permeabilized in PBS with 0.1% Triton-X, and then stained with rhodamine labelled Phalloidin (1:200, Invitrogen) per manufacturer's recommendations. Plates were analyzed on a fluorescent plate reader (Synergy 4, Biotek) followed by fluorescent microscopy (Nikon E800).
Eotaxins-1, -2, -3 were reconstituted in 0.1% bovine serum albumin (BSA) and then mixed with Matrigel diluted 1:1 with serum free endothelial basal media (EBM-2; Lanza). 500 μl of EBM-2 was added to each well of a 24-well plate followed by a 6.5 mm diameter Transwell insert (8 μm pores; Corning). Human CECs in EBM-2 were prestained with Vybrant DiO (Invitrogen) for 30 min at 37 °C and seeded into the inserts at 50,000 cells per 200 μl of serum free EBM-2 media. The plates were allowed to incubate for 16 h at 37 °C, 5% CO2. The migrated cells were imaged with an Olympus CK40 microscope and Olympus DP71 camera.
Human CECs were cultured in EGM-2 MV containing 5% FBS. Prior to starting the assay, cells were serum starved with basal medium (MCDB131) supplemented with 1% FBS overnight. Cells were stimulated for designated times with Eotaxin-1, 2 and 3 (10 ng/ml, 100 ng/ml and 2 μg/ml respectively). Equal amounts of lysates (500 μg) were incubated with GST-Pak1-PBD agarose beads (Upstate) to pull-down active GTP-bound Rac-1 at 4 °C for 1 h with rotation. The samples were subsequently analyzed for bound Rac-1 by western blot analysis using an anti-Rac-1 antibody (Upstate).
Mice were dark adapted overnight and then anesthetized. Both eyes were positioned within a ColorBurst Ganzfeld stimulator (Diagnosys). Espion software (Diagnosys) was used to program a fully automated flash intensity series from which retinal responses were recorded.