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. 2009 Jul 9;460(7252):225-30.
doi: 10.1038/nature08151. Epub 2009 Jun 14.

CCR3 is a target for age-related macular degeneration diagnosis and therapy

Atsunobu Takeda  1 , Judit Z Baffi, Mark E Kleinman, Won Gil Cho, Miho Nozaki, Kiyoshi Yamada, Hiroki Kaneko, Romulo J C Albuquerque, Sami Dridi, Kuniharu Saito, Brian J Raisler, Steven J Budd, Pete Geisen, Ariel Munitz, Balamurali K Ambati, Martha G Green, Tatsuro Ishibashi, John D Wright, Alison A Humbles, Craig J Gerard, Yuichiro Ogura, Yuzhen Pan, Justine R Smith, Salvatore Grisanti, M Elizabeth Hartnett, Marc E Rothenberg, Jayakrishna Ambati
Affiliations

CCR3 is a target for age-related macular degeneration diagnosis and therapy

Atsunobu Takeda et al. Nature. .

Abstract

Age-related macular degeneration (AMD), a leading cause of blindness worldwide, is as prevalent as cancer in industrialized nations. Most blindness in AMD results from invasion of the retina by choroidal neovascularisation (CNV). Here we show that the eosinophil/mast cell chemokine receptor CCR3 is specifically expressed in choroidal neovascular endothelial cells in humans with AMD, and that despite the expression of its ligands eotaxin-1, -2 and -3, neither eosinophils nor mast cells are present in human CNV. Genetic or pharmacological targeting of CCR3 or eotaxins inhibited injury-induced CNV in mice. CNV suppression by CCR3 blockade was due to direct inhibition of endothelial cell proliferation, and was uncoupled from inflammation because it occurred in mice lacking eosinophils or mast cells, and was independent of macrophage and neutrophil recruitment. CCR3 blockade was more effective at reducing CNV than vascular endothelial growth factor A (VEGF-A) neutralization, which is in clinical use at present, and, unlike VEGF-A blockade, is not toxic to the mouse retina. In vivo imaging with CCR3-targeting quantum dots located spontaneous CNV invisible to standard fluorescein angiography in mice before retinal invasion. CCR3 targeting might reduce vision loss due to AMD through early detection and therapeutic angioinhibition.

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Conflict of interest statement

Competing Interests Statement The authors declare competing financial interests.

Figures

Fig. 1
Fig. 1
CCR3 and eotaxins are expressed in choroidal neovascularization. a,b, Immunofluorescence shows that CCR3 (green) receptor expression colocalizes with CD31+ (red) expressing blood vessels in surgically excised human age-related macular degeneration (AMD) choroidal neovascular (CNV) tissue. Nuclei stained blue by DAPI. b, Specificity of CCR3 staining is confirmed by absence of staining with isotype control IgG (a). Individual red and green fluorescence channels are shown in Supplementary Fig. S1. c,d, CCR3 is not immunolocalized in CD31+ (red) blood vessels (white arrowheads) in the choroid of patients with atrophic AMD who do not have CNV (c) or in aged patients without AMD (d). Autofluorescence of retinal pigmented epithelium (white arrow) and Bruch's membrane (asterisks) overlying choroid is seen (c,d). e,f, CCR3 is not expressed in surgically excised avascular retinal fibrosis tissue (e) or in blood vessel of choroidal melanoma (f). g-j, Immunohistochemistry (golden brown reaction product) shows expression of CCL11 (g), CCL24 (h), and CCL26 (i) in surgically excised AMD CNV tissue, primarily in the stroma (red arrowheads) but also in the blood vessels (yellow arrows). Specificity of staining is confirmed by absence of staining with isotype control IgG (j). Scale bars, 10 μm.
Fig. 2
Fig. 2
CCR3 activation promotes angiogenesis. a, Tube formation of primary human choroidal endothelial cells (CECs) in Matrigel in vitro was reduced by neutralizing anti-human CCR3 antibodies (Ab) compared to isotype IgG. n = 6, * P < 0.05 compared to isotype IgG. b, Fraction of CD31+VEGFR2+ gated mouse CECs in vivo in proliferative state (S phase) was increased 5 days after laser injury in wild-type mouse eyes compared to control (uninjured eyes), and was reduced by intraocular administration of neutralizing anti-mouse CCR3 Ab compared to isotype IgG. n = 6–10, * P < 0.05 compared to IgG treatment. c, Stimulation with eotaxins for 24 h induced human CEC proliferation. n = 4, * P < 0.05 compared to bovine serum albumin (BSA) treatment. d,e, Stimulation with eotaxins, but not PBS, induced actin polymerization in human CECs. Relative F-actin content is expressed as the ratio of the mean channel fluorescence between eotaxin- and media alone-stimulated cells (d). Rhodamine-phalloidin staining (red) shows F-actin fibre formation in eotaxin-stimulated cells (e). Nuclei stained blue by DAPI. Data representative of 3–4 independent experiments are shown. c,e, CCL11 (10 ng/ml), CCL24 (100 ng/ml), CCL26 (2 μg/ml). f, Stimulation with eotaxins for 16 hours induces dose-dependent migration of human CECs across 8 μm pore size Transwells. n = 5–10, * P < 0.05 compared to BSA treatment. (a–c, f) Significance by Mann-Whitney U test. Error bars depict s.e.m.
Fig. 3
Fig. 3
CNV reduced by CCR3 or eotaxin ablation or blockade independent of leukocyte modulation. a,b, Laser-induced CNV in wild-type mice was reduced by neutralizing anti-mouse CCR3 Ab compared to isotype IgG (a) and by the CCR3 receptor antagonist (RA) SB328437 ((S)-Methyl-2-naphthoylamino-3-(4-nitrophenyl)propionate) compared to vehicle (PBS/DMSO) (b) in a dose-dependent fashion. n = 8–12, * P < 0.05 compared to no antibody or receptor antagonist. c, Representative examples of CNV in drug-treated mice. d, Laser-induced CNV was reduced in Ccr3−/− mice compared to wild-type mice. n = 9, * P < 0.05 compared to wild-type mice. e, Eotaxin-1 (Ccl-11) and eotaxin-2 (Ccl-24) protein levels, measured by ELISA, were increased following laser injury in wild-type mice. n = 6, * P < 0.05, # P < 0.01 compared to 0 h baseline. f, Ccl-11 and Ccl-24 immunofluorescence (green) was localized in the retinal pigmented epithelial cell layer (arrows) adjacent to CD31+ (red) choroidal endothelial cells (arrowheads) on day 1 after laser injury in wild-type mice. Nuclei stained blue by DAPI. No specific immunofluorescence was detected with isotype control IgGs. Images representative of 3 independent experiments are shown. g, Laser-induced CNV was reduced in Ccl11−/− and in Ccl24−/− mice compared to wild-type mice. n = 8–10, * P < 0.05 compared to wild-type mice. CNV is further reduced in Ccl11−/− × Ccl24−/− mice compared to single null mice. # P < 0.05 compared to single null mice. h, Laser-induced CNV in wild-type mice was reduced by neutralizing antibodies against mouse CCL11 or CCL24 compared to isotype IgG. n = 7–10, * P < 0.05 compared to no injection (control) or IgG. i, Representative examples of CNV in eotaxin-neutralized mice. j, Neutralizing anti-CCR3 antibodies (Ab) reduced laser-induced CNV in mice deficient in eosinophils (Δdbl GATA) or mast cells (Kitw-v). n = 6–9, * P < 0.05 compared to IgG. Scale bars, (c,i), 100 μm; f, 20 μm. Error bars depict s.e.m.
Fig. 4
Fig. 4
CCR3-targeting quantum dots detect subretinal choroidal neovascularization (CNV). a, Images of the fundus taken after intravenous injection of sodium fluorescein in wild-type and Ccl2−/− × Ccr2−/− mice showed normal retinal vascular filling but no areas of hyperfluorescence indicative of CNV. b, After intravenous injection of QDot-CCR3 Fab in the same Ccl2−/− × Ccr2−/− mouse shown in (a), focal branching choroidal hyperfluorescence was visualized (arrow) at 1 h in the same area that was not hyperfluorescent during fluorescein angiography (arrowhead in a). The intensity of this hyperfluorescence (shown in red pseudocolour in the inset) increased, attaining a peak at 4 h, and then declined in intensity but still persisted at 12 h. Corresponding images of QDot-Isotype Fab angiography showed no hyperfluorescence. c–e, The region corresponding to the area of hyperfluorescence seen on QDot-CCR3 Fab angiography in (b) contained multiple CD31+ blood vessels in the choroid (Ch) that were proliferating (Ki67+; arrows) and had not invaded the retina (Ret). Individual red (CD31+, c), and green (Ki67+, d), and merged (e) fluorescence channel images are shown. Arrows point to proliferating endothelial cells. Inset shows Ki67+ CD31+ cells in higher magnification. f, QDot-CCR3 Fab hyperfluorescent areas were localized to areas of subretinal CNV with CCR3+ endothelial cells. g, The QDot label was visualized within CD31+ vasculature of subretinal CNV lesions. Images representative of 6 independent experiments. Scale bars, (c–e), 10 μm.
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