Murine Ccl2/Cx3cr1 Deficiency Results in Retinal
Lesions Mimicking Human Age-Related
Jingsheng Tuo,1Christine M. Bojanowski,1,2Min Zhou,1,2Defen Shen,1,2Robert J. Ross,1
Kevin I. Rosenberg,1D. Joshua Cameron,3Chunyue Yin,4Jeffrey A. Kowalak,5
Zhengping Zhuang,4Kang Zhang,3and Chi-Chao Chan1
PURPOSE. Senescent Ccl2?/?mice are reported to develop
cardinal features of human age-related macular degeneration
within CX3CR1 are also found to be associated with AMD. The
authors generated Ccl2?/?/Cx3cr1?/?mice to establish a
more characteristic and reproducible AMD model.
METHODS. Single Ccl2- and Cx3cr1-deficient mice were cross-
bred to obtain Ccl2?/?/Cx3cr1?/?mice. Funduscopy, histo-
pathology, retinal A2E quantification, proteomics, RT-PCR
gene expression assay, immunochemistry, and Western blot-
ting were used to examine the retina and to evaluate gene
expression within the retinal tissue.
RESULTS. By 6 weeks of age, all Ccl2?/?/Cx3cr1?/?mice de-
veloped AMD-like retinal lesions, including drusen, retinal pig-
ment epithelium alteration, and photoreceptor degeneration.
Furthermore, choroidal neovascularization occurred in 15% of
the mice. These degenerative lesions progressed with age. A2E,
a major lipofuscin fluorophore that accumulated during AMD
Cx3cr1?/?retina than in the wild-type retina. Complement
cofactor was higher in the Ccl2?/?/Cx3cr1?/?RPE. Proteom-
ics data indicated that four proteins were differentially ex-
pressed in Ccl2?/?/Cx3cr1?/?retina compared with control.
One of these proteins, ERp29, an endoplasmic reticulum pro-
tein, functions as an escort chaperone and in protein folding.
CONCLUSIONS. The authors concluded that Ccl2?/?/Cx3cr1?/?
mice develop a broad spectrum of AMD abnormalities with
early onset and high penetrance. These observations implicate
certain chemokines and endoplasmic reticulum proteins in
AMD pathogenesis. Similar to the mechanism of neurodegen-
eration caused by dysfunction of endoplasmic reticulum pro-
teins, decreased chaperoning may cause misfolded protein
accumulation, leading to drusen formation and retinal
degeneration. (Invest Ophthalmol Vis Sci. 2007;48:3827–3836)
Considerable efforts have been made to establish AMD animal
models.2–9Even though mice have no macula, existing mouse
AMD models have been shown to develop cardinal pathologic
features of AMD.3,8–10Strong evidence indicates the involve-
ment of immunologic processes in AMD.11Additionally, sev-
eral studies have successfully demonstrated associations be-
tween AMD and various single-nucleotide polymorphisms
(SNPs). Many of these SNPs are within genes encoding immu-
CX3CR1, the specific receptor for CX3CL1/fractalkine che-
mokine, is expressed on leukocytes (e.g., lymphocytes, mac-
rophages, NK cells, mass cells), dendritic cells,24,25brain mi-
croglia, and astrocytes.26–28CX3CR1 expression is also
reported in the eye,29iris, ciliary body,30retinal microglia, RPE,
and Mu ¨ller cell.31,32In a functional study, two CX3CR1 SNPs
resulted in a decreased number of CX3CL1 binding sites and
reduced ligand-binding affinity on peripheral blood mononu-
clear cells.33We have reported that these SNPs are associated
with AMD.22Furthermore, we have demonstrated a decreased
number of CX3CR1 transcripts and protein in AMD maculae
compared with the maculae of normal eyes.22,32However, no
ocular abnormalities have been reported in young adult
In addition to CX3CR1, CCL2 (MCP-1, a CC chemokine) is
thought to play a homeostatic, immunoregulatory role in AMD
pathogenesis.34Aged mice with deficient Ccl2 or Ccr2, the
corresponding receptor, develop many cardinal features of
AMD including drusen formation, RPE accumulation of lipofus-
cin and complement factors, and choroidal neovasculariza-
tion.8In addition, CCL2 may function as an antiapoptotic
factor, as reported in in vitro systems.35,36
In the present study, we examined the hypothesis that
deficiencies in both Cx3cr1 and Ccl2 may induce typical patho-
logic features of AMD in mice more consistently and at an
earlier age of onset than in existing animal models. We found
that AMD features in the mice were highly characteristic and
reproducible. In addition to inflammatory molecules, we also
showed evidence for a potential role of endoplasmic reticulum
proteins (ERp) in AMD.
ge-related macular degeneration (AMD) is the leading
cause of blindness in the elderly in Western countries.1
MATERIALS AND METHODS
Ccl2-deficient and Cx3cr1-deficient mice (obtained from Bao Lu and
Barrett J. Rollins of Children’s Hospital, Harvard Medical School,37and
From the1Laboratory of Immunology, National Eye Institute, the
4Surgical Neurology Branch, National Institute of Neurological Disor-
ders and Stroke, and the
Institute of Mental Health, National Institutes of Health, Bethesda,
Maryland; and the3John Moran Eye Center, University of Utah, Salt
Lake City, Utah.
2These authors contributed equally to the work presented here
and should therefore be regarded as equivalent authors.
Supported by the Intramural Research Program of the National
Eye Institute, National Institutes of Health.
Submitted for publication January 16, 2007; revised February 27,
March 21, and March 27, 2007; accepted May 11, 2007.
Disclosure: J. Tuo, None; C.M. Bojanowski, None; M. Zhou,
None; D. Shen, None; R.J. Ross, None; K.I. Rosenberg, None; D.J.
Cameron, None; C. Yin, None; J.A. Kowalak, None; Z. Zhuang,
None; K. Zhang, None; C.-C. Chan, None
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertise-
ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Chi-Chao Chan, Laboratory of Immunol-
ogy, 10/10N103, National Institutes of Health/National Eye Institute,
10 Center Drive, Bethesda, MD 20892-1857; firstname.lastname@example.org.
5Laboratory of Neurotoxicology, National
Investigative Ophthalmology & Visual Science, August 2007, Vol. 48, No. 8
Copyright © Association for Research in Vision and Ophthalmology
Philip Murphy of the National Institute of Allergy and Infectious Dis-
eases/National Institutes of Health [NIAID/NIH],38respectively) were
used as the founder generation (F0) and were crossbred to obtain
animals that were heterozygous (F1) for Ccl2 and Cx3cr1 alleles.
Heterozygous animals were intercrossed to obtain homozygous Ccl2?/
?/Cx3cr1?/?mice (F2). Wild-type (WT) mice were of C57BL/6 back-
ground. The study was conducted in compliance with ARVO Statement
for the Use of Animals in Ophthalmic and Vision Research, and all
animal experiments were performed under the protocols approved by
the National Eye Institute Institutional Animal Care and Use Commit-
Funduscopic examinations were performed every three weeks from 3
to 5 weeks of age until 10 months with the use of a hand-held fundus
camera (Kowa Optimed, Torrance, CA) and a specialized lens (90D;
Volk Optical, Mentor, OH) after intraperitoneal injection of ketamine
(1.4 mg/mouse) and xylazine (0.12 mg/mouse) for systemic anesthesia
and topical administration of 1% tropicamide ophthalmic solution
(Alcon Inc., Fort Worth, TX) for pupil dilation.
Eyes, brains, livers, spleens, and lungs were harvested after the mice
were humanely killed. Tissues were fixed in 10% formalin for at least
24 hours. All tissues were then embedded in methacrylate. Seventy-
two eyes (60 of the Ccl2?/?/Cx3cr1?/?mice and 12 of the WT mice)
were subjected to histopathology. The eyes were serially sectioned in
the pupillary–optic nerve plane. Each eye was cut into six sections.
Other organs were sectioned routinely. All sections were stained with
hematoxylin and eosin. If an ocular lesion was found, another 6 to 12
sections were cut through the lesion. These slides were also stained
with periodic acid Schiff (PAS) to highlight the Bruch membrane and
small neovascular vessels. This study with human ocular tissues was
approved by the National Eye Institute institutional review board and
was conducted in accordance with the principles expressed in the
Declaration of Helsinki.
Transmission Electron Microscopy
Electron microscopy was performed on 4% glutaraldehyde formalin-
fixed tissue. The fixed neuroretina-RPE-choroid tissue was embedded
in epoxy resin (LX-112; LADD Research Industries, Burlington, VT).
Six-micrometer-thick sections stained with toluidine blue were exam-
ined under light microscopy. Ultrathin sections were stained with
uranyl acetate and lead citrate for examination under a microscope
(JEM-100B; JEOL, Tokyo, Japan). Two eyes in each of three age groups
(12–20, 21–35, and 36–48 weeks, respectively) of the Ccl2?/?/
Cx3cr1?/?mice and two eyes of the WT mice were used for trans-
mission electron microscopic study.
After enucleation, mouse eyes were snap-frozen and embedded in OCT
compound (Sakura Finetek, Inc., Torrance, CA). Three eyes of the
Ccl2?/?/Cx3cr1?/?mice and two eyes of the control mice were used
for immunohistochemistry, as described previously.39Frozen sections
4-?m–thick were fixed in acetone for 7 to 10 minutes and rinsed with
Tris-buffered saline, 0.05 M, pH 7.4. The slides were immersed in 5%
normal serum specifically to block potential background from the
secondary antibody. Because microglia and complement system acti-
vation were suggested to play a role in retina housekeeping and
correlated with AMD development,12,40CD11b, a marker for microglia,
and CD46, the ligand of complement factors C3b and C4b and a
complement regulatory protein,41were measured. For the detection of
microglia, rat-anti–mouse CD11b antibody (Harlan Sera-Laboratory,
Loughborough, UK) was used as the primary antibody; for the detec-
tion of CD46, rabbit-anti–mouse CD46 (H-294: sc-9098; Santa Cruz
Biotechnology, Inc., Santa Cruz, CA) was used as the primary antibody.
Secondary antibodies were biotin-labeled goat anti–rat or anti–rabbit
IgG (Vector Laboratories, Burlingame, CA), respectively. For the detec-
tion of ERp29, rabbit polyclonal antibody against ERp29 (Abcam Inc.,
Cambridge, MA) was used as the primary antibody, followed by incu-
bation with secondary antibody (biotin-labeled goat anti–rabbit IgG;
Vector Laboratories). Sections were treated with the avidin-biotin-
immunoperoxidase system and 3,3?-diaminobenzidine as the substrate
and were counterstained with methyl green. Each staining assay for a
particular primary antibody was repeated at least once. Exposure times
for immunohistochemistry images were matched to ensure appropri-
ate control. Staining was quantified and graded based on the positive
number of cells and the color (black) intensity of the stained cells. Cells
that were more than 50% blackish were graded as having intense
immunoreactivity; in contrast, cells that were less than 20% grayish
were graded as having poor immunoreactivity.
Immunohistochemistry analyzing five human ocular sections (two
wet AMD, two dry AMD, and one normal eye) was conducted in
conformance with the policies and principles stated in the Federal
Policy for the Protection of Human Subjects (US Office of Science and
Technology Policy) and in the Declaration of Helsinki. Formalin-fixed
human ocular sections were deparaffinized.32,42Detection of ERp29 on
human sections was carried out as described and was repeated at least
once in each eye.
A2E Extraction and Quantification
Mice were kept in the dark for more than 12 hours. RPE cells and
neural retina were isolated from the eyecups of 15-, 20-, and 24-week-
old WT and Ccl2?/?/Cx3cr1?/?mice. RPE cells were then dissected
after removal of the neural retina, in a dark room under dim red
light.43,44Six eyes were pooled from each group. Each analysis was
trimethyl-1-cyclohexen-1-yl) 1E,3E,5E,7E-hexatrienyl]-pyridinium (A2E)
was extracted as previously described.3Detection and quantification
were performed with the use of HPLC electrospray ionization mass
spectrometry (ESI-MS; Thermo Electron, San Jose, CA). A gradient of
75% to 96% acetonitrile was used to separate A2E over a period of 40
minutes at a flow rate of 1 mL/min. A2E was quantified according to
external standards, and a standard was used to identify and determine
The mouse retina, including the RPE and excluding the choroid, was
dissolved into extraction buffer II (Bio-Rad Laboratories, Hercules, CA).
Sample preparation—including two-dimensional gel electrophoresis
image analysis (Proteomweaver; Definiens, Munich, Germany) accord-
ing to the manufacture’s protocol, in-gel digestion followed by liquid
chromatography (LC)–MS/MS on an LC/MS system (ProteomeX; Ther-
moElectron Corp., San Jose, CA), and protein identification—was con-
ducted according to Okamoto et al.45Protein identification was ac-
cepted when MS/MS spectra of at least two peptides from the same
protein exhibited at a minimum the default Xcorr versus charge values
set by the program (for Z ? 1,1.50; for Z ? 2, 2.00; for Z ? 3, 2.50).
Six eyes in each group were pooled as one sample. Three independent
experiments (18 Ccl2?/?/Cx3cr1?/?and 18 WT mice) were per-
formed. All samples were run in duplicate to guarantee greater than
90% identity before further analysis was performed.
Western Blot Analysis
Mouse retina, including the RPE, was homogenized with an equal
volume of 2? lysis buffer (RIPA; Upstate Biotechnology, Lake Placid,
NY). The protein was separated by SDS-PAGE under reducing condi-
tions and was transferred to transfer membrane (Immobilon-P; Milli-
pore, Bedford, MA). The membrane was processed with an immuno-
3828Tuo et al.
IOVS, August 2007, Vol. 48, No. 8
detection kit (WesternBreeze; Invitrogen, Carlsbad, CA). Briefly, the
membrane was first incubated with 1:500 diluted anti–ERp29 antibody
(Abcam, Cambridge, MA), followed by incubation with 1:2000 diluted
alkaline phosphatase-labeled secondary antibody (Vector). Signals
were visualized by immersing the membrane in chromogenic sub-
strate. Two independent experiments were performed with two eyes
pooled for each strain in each run.
Detection of Ccl2, Cx3cr1, and ERp29 Transcripts
Five micrograms RNA from mouse retina/RPE was used for cDNA
synthesis (Superscript II RNase H?Reverse Transcriptase; Invitrogen,
Grand Island, NY). Real-time PCR was performed (Stratagene Mx3000
Real-Time PCR System and Brilliant SYBR Green QPCR Master Mix;
Stratagene, La Jolla, CA). Primers for Ccl2 and Cx3cr1 were synthesized
by and supplied as a gene expression assay kit (RT2Real-Time; Super-
Array Bioscience Corp., Frederick, MD). For the internal control, ?-ac-
tin was amplified using primers 5?-CCCAGCACAATGAAGATCAA-3?
and 5?-ACATCTGCTGGAAGGTGGAC-3?. To determine ERp29 tran-
script in mouse ocular tissue, validated and inventoried ERp29 and
GAPDH gene expression kits (TaqMan; Applied Biosystems, Foster
City, CA) were used according to the manufacturer’s instruction. The
comparative Ctmethod was used to establish relative quantification of
the fold change in gene expression (User Bulletin 2; ABI Prism 7700
Sequence Detection System, PE Applied Biosystems, Foster City, CA;
1997). Fold changes were normalized first by the level of GAPDH. The
average fold change resulting from gene manipulation was again nor-
malized to the transcript level of WT mouse and presented graphically.
Four biological samples were analyzed from each group. Each sample
was analyzed at least twice.
Systemic Manifestation of
Among the 400 F2 offspring genotyped, 12 animals were
Ccl2?/?/Cx3cr1?/?, indicating an abnormal Mendelian segre-
gation (1 in 16 expected). Ccl2?/?/Cx3cr1?/?mice had nor-
mal body weight compared with the controls. Ccl2?/?/
Cx3cr1?/?mice were less prolific when maintained as a
separate lineage, with an average of four pups per litter com-
pared with eight per litter in the WT controls. Twenty percent
of the Ccl2?/?/Cx3cr1?/?mice had progressive patchy skin
depigmentation, primarily on the face and upper extremities
(data not shown).
Ocular Features in Ccl2?/?/Cx3cr1?/?Mice
Ophthalmic examination findings on 103 Ccl2?/?/Cx3cr1?/?
mice appeared normal except the retina and choroid. Sequential
funduscopic examinations were performed on 76 Ccl2?/?/
Cx3cr1?/?mice and 27 age-matched WT mice every 3 weeks
beginning at 3 weeks of age. Unlike WT mice that were normal
at all ages (Fig. 1A), all 6- to 9-week-old Ccl2?/?/Cx3cr1?/?
mice spontaneously developed drusenlike lesions character-
ized by heterogenous, round or domed-shaped, soft-bordered,
yellowish deposits within the subretina (Fig. 1B). With aging,
these lesions enlarged or flattened and became confluent (Fig.
1C). Some of the lesions progressed to form chorioretinal scars
and depigmented atrophic areas (Fig. 1D). In comparison,
none of the single knockout Ccl2?/?or Cx3cr1?/?mice de-
veloped retinal lesions at such young ages (Ross RJ, et al. IOVS
2007;48:ARVO E-Abstract 2355).8
A normal retina is illustrated in a 21-
week-old WT mouse. (B) Multiple sub-
retinal lesions mimic drusen formation
(arrow) in a 9-week-old Ccl2?/?/
Cx3cr1?/?mouse. (C) Enlarged and
confluent subretinal drusenlike le-
sions (arrow) in the same eye of
the Ccl2?/?/Cx3cr1?/?mouse at
18 weeks. (D) Scarring and atrophic
retinal lesions (arrow) indicate dis-
ease progression in the same eye of
this mouse at 33 weeks.
Fundus photographs. (A)
IOVS, August 2007, Vol. 48, No. 8
Murine Ccl2/Cx3cr1 Deficiency 3829
Histologic Features in Ccl2?/?/Cx3cr1?/?Retina
Histopathologic examination was conducted on the eyes of 60
Ccl2?/?/Cx3cr1?/?mice (20 mice 8–15 weeks of age, 20
mice 15–21 weeks of age, and 20 mice 21–60 weeks of age)
and 12 WT mice (four younger than 12 weeks of age, four
12–24 weeks of age, and four older than 60 weeks of age). Eyes
of the age-matched WT mice were entirely normal and lacked
drusen formation, neovascularization, photoreceptor degener-
ation, and RPE atrophy (Fig. 2A is a representative image). All
Ccl2?/?/Cc3cr1?/?eyes showed focal thickening of the
Bruch membrane. Drusen (dome-shaped areas of hyaline ex-
crescences within Bruch membrane) was found in some eyes
(Fig. 2B); most druse were small (5–15 ?m). Local RPE hypop-
igmentation and vacuolation (Fig. 2C), photoreceptor outer
segment disorganization, and photoreceptor atrophy were
commonly observed. These local changes could be present at
various severities in the same eye or in different eyes. With
careful examination of the series of consecutive sections, cho-
roidal neovascularization was found in 15% of Ccl2?/?/
Cx3cr1?/?mouse eyes; earliest onset was at 12 weeks of age.
A few of the fragile, small choroidal neovascular vessels that
penetrated Bruch membrane and entered the outer retinal
layers were surrounded by hyperplastic RPE cells or atrophic
RPE areas (Fig. 2D).
Ultrastructure of Ccl2?/?/Cx3cr1?/?Retina
Transmission electron microscopic examinations were per-
formed on the mice at different ages. Retinas of the Ccl2?/?/
Cx3cr1?/?mice showed a decrease in melanosomes and an
increase in lipofuscin within the RPE (hypopigmentation; Figs.
3A, 3B), thickened Bruch membrane (500–1000 nm compared
with 240–350 nm in the normal areas of the Ccl2?/?/
Cx3cr1?/?and WT mice; Fig. 3E) with amorphous granular
and heterogeneous material deposits (Fig. 3C), and disorga-
nization or atrophy of the photoreceptors (Fig. 3D). In
addition, loss of tight junctions and cellular membrane fold-
ing were noted in some RPE cells. These ultrastructural
findings indicated degeneration of the RPE and photorecep-
tors and are reminiscent of the ultrastructural changes ob-
served in human AMD cases.46,47Moreover, these abnormal-
ities were more severe in the older mice, indicating a
progressive degenerative process during aging. In contrast,
none of the abnormalities were found in the eyes of the
age-matched WT mice.
Accumulation of A2E in Ccl2?/?/Cx3cr1?/?RPE
Accumulation of lipofuscin and the fluorophore A2E is an early
pathologic feature observed in human AMD.48The stable A2E,
a pyridinium bis-retinoid derived from all-trans retinal and
phosphatidyl-ethanolamine, is toxic to RPE.49A2E levels were
measured within the RPE with the use of HPLC/ESI-MS, and a
significant increase of more than threefold was found in 15-
week-old and older Ccl2?/?/Cx3cr1?/?mice (3.4 pmol of A2E
per eye) compared with the age-matched WT (approximately 1
pmol; Fig. 4).
Complement Cofactor and Microglia in
CD46 (membrane cofactor protein [MCP]) immunoreactivity
was detected on the entire RPE of the Ccl2?/?/Cx3cr1?/?but
not the apical RPE of the WT mice (Figs. 5A, 5C), indicating
possible enhanced complement (C3b and C4b) activation. No
difference in CD46 expression pattern was noted in the cho-
roids of the Ccl2?/?/Cx3cr1?/?and WT mice, an expected
finding because CD46 is widely distributed on vascular endo-
thelial cells and fibroblasts in the choroid. The infiltration of
microglia (CD11b?cells) was detected in the retinal lesions of
the Ccl2?/?/Cx3cr1?/?mice (Figs. 5B, 5D) but not of the WT
mice. Immunostaining results for CD46 and CD11b were con-
sistent within the strain.
Lower Expression of ERp29 in
Proteomic analysis of retinal lysate using two-dimensional gels
uncovered differential expression of four proteins between the
(A) Retina, RPE, Bruch membrane, and
choroidal capillaries are normal in a
6-month-old WT mouse. (B) Drusen
deposits (arrows) are shown as small
dome-shaped hyaline material within
the Bruch membrane of two Ccl2?/?/
Cx3cr1?/?mice. (C) Hypopigmenta-
tion and lacy degeneration (arrow) of
the RPE are observed in a Ccl2?/?/
Cx3cr1?/?mouse. (D) Choroidal neo-
vascularization is identified by small
patent vessels (arrows) from the cho-
roid piercing Bruch membrane and
RPE into the retina of two Ccl2?/?/
Cx3cr1?/?mice. GCL, ganglion cell
toreceptor outer and inner segments;
CH, choroids. Hematoxylin and eosin
staining. Scale bar, 100 ?m.
3830Tuo et al.
IOVS, August 2007, Vol. 48, No. 8
Ccl2?/?/Cx3cr1?/?and WT control mice (Fig. 6). Three
independent experiments resulted in a consistent pattern of
protein distribution. Three of the four proteins successfully
identified by LC/MS/MS were ERp29 precursor, calcium-
binding 140k protein, and RIKEN cDNA 2210010C04.
ERp29, an endoplasmic reticulum protein, functions in pro-
tein folding and is associated with various degenerative
diseases.50,51We therefore investigated the expression of
ERp29 in the retinas of WT and Ccl2?/?/Cx3cr1?/?mice.
Immunostaining, Western blotting, and RT-PCR data demon-
strated significantly reduced expression of ERp29 in the retinas
of Ccl2?/?/Cx3cr1?/?mice compared with those in WT con-
trols (Fig. 7).
Lower Expression of ERp29 in Human Archived
Decreased expression of ERp29 protein was found by immu-
nohistochemical staining in the neuroretina and RPE cells of
human maculae with AMD compared with normal human
maculae. A representative image is shown in Figure 8.
This study presents a mouse strain, created by knocking out a
chemokine (Ccl2) and a chemokine receptor (Cx3cr1), that
exhibits pathologic features of human AMD. Ccl2?/?/
Cx3cr1?/?mice are shown to spontaneously develop retinal
degenerative lesions, including choroidal neovascularization.
In addition, elevations of A2E in the RPE and enhanced expres-
sion of complement regulatory protein (CD46) and microglia
are also observed in the Ccl2?/?/Cx3cr1?/?mice. These find-
ings are compatible with human AMD eyes in which A2E
accumulates in RPE cells49and CD46 localizes on RPE cells
adjacent to and overlying drusen.52These ocular manifesta-
tions observed in the Ccl2?/?/Cx3cr1?/?mice implicate im-
portant roles of the immune system and specific chemokines
and ligands in the pathogenesis of resultant lesions. Because of
the high penetrance and early presentation of many AMD-like
features these mice display, this phenotype makes the model
an alternative for studying the genetics and pathologic mech-
anisms of AMD and may aptly aid in the evaluation of various
therapies for this blinding disease.
Researchers have linked the immune system and inflamma-
tory processes to the pathophysiology of human AMD.53,54
Moreover, under normal conditions, a dynamic balance is
struck between the generation of macular deposition stimu-
lated by various internal and external factors and the elimina-
tion of these deposits by inflammatory cells attracted to the site
by chemokines. It is hypothesized that the absence of adequate
macrophage recruitment is involved in AMD development.8,22
We have found lower expression of CX3CR1 transcripts in the
maculae than in the perimacular retina within AMD eyes. In
contrast, similar levels of CX3CR1 transcript expression were
detected in the maculae and perimaculae of subjects with
normal eyes.22We have also reported exacerbated retinal de-
generation and choroidal neovascularization after the injection
of subretinal basement membrane preparation (Matrigel; BD
Biosciences, San Jose, CA) in Ccl2-deficient mice.55Many che-
mokines, including CCL2, exhibit protective effects against
neuronal apoptosis.36,56,57In a toxic model of Parkinson dis-
ease and a model of genetic motor neuron disease, Cx3cr1?/?
mice showed more extensive neuronal cell loss than did
Activated microglia are associated with AMD.40,59We ob-
served microglia in the retinal lesions of Ccl2/Cx3cr1-deficient
mice but not of WT mice, suggesting the activation of retinal
microglia that may cause adjacent photoreceptor death.60,61
CD46 is a membrane-bound complement regulator that facili-
tates inactivation of the activated complement component C3b
and C4b.62Similar to the finding of CD46 in the RPE of the
senescent Ccl2, Ccr2, or Sod1 (Cu, Zn-superoxide dismutase)
knockout mice,8,9our Ccl2/Cx3cr1 deficient mice also dem-
onstrated the existence of this complement regulatory protein
in the eye. The data are parallel to those for human eyes with
Significantly lower ERp29 protein and transcript expression
was detected in the ocular tissue of Ccl2?/?/Cx3cr1?/?mice
than of WT controls. Immunohistochemistry also illustrated
decreased ERp29 in human AMD maculae compared with con-
trols. Expression of ERp29 is controlled primarily through the
somes are markedly decreased and lipofuscin is present in the RPE
cells (arrow) of a Ccl2?/?/Cx3cr1?/?mouse. (B) Lipofuscin (ar-
row) and lipid droplets (open arrow) deposits are shown as the
homogeneous electron dense-cytoplasmic inclusion in the RPE of
another Ccl2?/?/Cx3cr1?/?. (C) Thickened Bruch membrane and
amorphous deposits (arrow) are depicted in a Ccl2?/?/Cx3cr1?/?
mouse. (D) Disorganized and atrophic photoreceptors (asterisk) and
lipofuscin (arrow) in the RPE are observed. (E) Normal RPE, Bruch
membrane, and choroid are illustrated in a WT mouse. Scale bars,
500 nm (A, B, D); 2 ?m (C, E).
Retinal transmission electron micrographs. (A) Melano-
IOVS, August 2007, Vol. 48, No. 8
Murine Ccl2/Cx3cr1 Deficiency3831
XBP-1/IRE-1 pathway.63IRE-1 is differentially expressed in
the inflammatory state,64which might account for the lower
ERp29 expression in this chemokine-deficient mouse. Neu-
rodegenerative diseases are known to involve cell death
initiated by endoplasmic reticulum (ER) stress and are thus
regarded as ER stress-associated diseases or conformational
The ER is a central organelle in lipid synthesis, protein
folding, and protein maturation. All newly synthesized mem-
branes and secretory proteins are folded and processed in the
ER. Cells need correctly folded and processed membrane pro-
teins for function, and when proteins are unfolded or mis-
folded, they tend to form toxic aggregates (e.g., lipofuscin in
the RPE) that are harmful to the cells. Conditions of ER mal-
function are called ER stress. ER stress is induced by the
accumulation of unfolded protein aggregates, called the un-
folded protein response. In ER stress, transcription factors are
activated to induce the expression of ER-resident chaperones
to deal with accumulated protein aggregates. ERp29 is one
such ER-resident chaperone that prevents protein aggregation
by keeping the unfolded proteins in a folding-competent state
and that functions as a component of the ER-specific protein-
degrading apparatus to eliminate denatured proteins.50,63,68,69
Decreased chaperoning may cause misfolded protein accumu-
lation. Moreover, ERp29 acts as an escort chaperone that
brings proteins to different locations in the cell.63AMD is a
deposit accumulation disease. In our model, substantial accu-
mulation of lipofuscin could result from the inability of ERp29
with normal controls. Chromato-
grams represent absorbance at 440
nm. A2E peaks elute at 25.5 minutes.
months: A2E approximately 3.4 pmol
(black). Normal RPE at 4 months:
A2E approximately 1.1 pmol (gray).
Each represents a 200-?L injection of
six RPE extracts.
Quantification of A2E in
(A, C) CD46 (arrows, black) staining
in entire RPE and small drusen of a
with none in the retina of a WT mouse
(open arrow, RPE with brown pig-
ment). (B, D) Microglia (CD11b?cells,
of a Ccl2?/?/Cx3cr1?/?mouse; none
are found in a WT mouse (open ar-
row, RPE). Insets: higher magnifica-
tions of the RPE cells in two strains.
(A, B: WT mice; C, D: Ccl2?/?/
Cx3cr1?/?mice). INL, inner nuclear
layer; ONL, outer nuclear layer. Scale
bar, 100 ?m.
3832Tuo et al.
IOVS, August 2007, Vol. 48, No. 8
to escort. The involvement of ER distress and ERp29 protein in
AMD pathogenesis has been reported.70–73Furthermore, de-
creases of retinal ERp29 level have been reported with age.51
In a recent proteomic analysis of human AMD eyes, Ethen et
al.73report a 33% decrease of ERp29 in the macula with the onset
stage of AMD. We are investigating a possible mechanistic role
of ERp29 in the phenotypic development in this mouse strain.
In another recent study, Azfer et al.74reports activation of
a cluster of ER stress-related genes, including ERp29, during
the development of myocardiac deterioration and dysfunction
in the heart of Ccl2 transgenic mice, which protected the
cardiomyocytes from the adverse effect of stress in the early
stage. However, with chronic inflammation, these efforts
failed, and the cells died to the death-inducing processes. In
our model, CCL2 and CX3CR1 levels were low, which might
have resulted in inadequate ER stress protein production and
ER dysfunction. Under conditions of ER impairment, unfolded
proteins accumulated in the ER lumen, a signal responsible for
activation of the unfolded protein response.
In summary, Ccl2?/?/Cx3cr1?/?mice developed early-onset
and progressive retinal degenerative disease with broad-spectrum
pathologic features mimicking human AMD. The phenotype is
highly penetrant, reliable, and reproducible. Data from proteom-
ics, immunohistochemistry, Western blot, and RT-PCR indicate
that the ERp29 protein is involved in this model, a finding that
provides new insight into AMD pathogenesis. The observations
made in this study implicate certain chemokines and ER proteins
as having important roles in the development of AMD.
Cx3cr1?/?mice by two-dimensional
PAGE analysis. (A) Representative
two-dimensional PAGE image from
one WT retina. (B) Representative
two-dimensional PAGE image from
one Ccl2?/?/Cx3cr1?/?retina. Four
silver-stained protein spots are iden-
tified as differentially expressed be-
tween the two groups, as indicated
by circles. The three proteins from
those four spots were successfully
identified by LC/MS/MS. Repetition of
the analysis reveals consistent protein
patterns for each group. The three
identified proteins were (1) calcium
binding 140k protein, (2) ERp29 pre-
Proteomics of retinal ly-
from WT and
retina. (A) Photomicrographs of mice
retinal sections show less ERp29 re-
activity (black, arrows) and pres-
ence of retinal lesions (asterisks) in a
Ccl2?/?/Cx3cr1?/?mouse than in
a WT mouse (avidin-biotin-immuno-
peroxidase staining). Scale bar, 100
?m. (B) Western blotting of retina
lysate shows less density of ERp29
in a Ccl2?/?/Cx3cr1?/?mouse than
in a WT mouse. (C) Lower ocular
ERp29 mRNA detected by quantita-
tive RT-PCR in the ocular tissue of
Ccl2?/?/Cx3cr1?/?mice than in WT
ERp29 expression in the
IOVS, August 2007, Vol. 48, No. 8
Murine Ccl2/Cx3cr1 Deficiency3833
The authors thank Bao Lu and Barrett J. Rollins of Children’s Hospital,
Harvard Medical School, and Philip Murphy of the NIAID/NIH for
providing Ccl2?/?and Cx3cr1?/?founder generations. They also
thank P. Bhosale for help with HPLC analysis, Rachel Caspi of the
National Eye Institute, and Craig Gerard of Children’s Hospital, Harvard
Medical School, for critical scientific discussion.
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