Here we show that retinal MCs all and invariably expressed CX3CR1, that these cells accumulated subretinally in AMD, and that the T280M polymorphism of CX3CR1 was associated with both impaired cell migration and AMD. MCs accumulated under the retinas of CX3CR1-deficient mice with age, after laser injury, or with albino background. This accumulation elicited photoreceptor degeneration and exacerbated neovascularization. Moreover, the resulting prolonged contact of MC with lipid-rich OS was associated with intracellular lipid accumulation in the MCs. Surprisingly, these subretinal microglial foam cells turned out to be the origin of drusen-like deposits in mice. Our results show that accumulation of MCs in the subretinal space may be a driving force in the pathogenesis of AMD, rather than a secondary occurrence.
Our study of patients with AMD showed that homozygosity for the M280 allele was consistently more frequent in groups with AMD than in studies of other populations and confirmed a previous report (10
) of a dominant association between the CX3CR1 M280 allele and AMD risk. Subjects in the control studies were not checked for AMD, and some certainly had or will develop the disease. The effect of this bias, however, should be an underestimation of the impact of CX3CR1 polymorphisms on AMD risk. These data indicate that the chemokine receptor CX3CR1 may play an important role in AMD pathogenesis.
To understand how CX3CR1 polymorphisms contribute to AMD pathogenesis, it is essential to identify the types of cells expressing CX3CR1 in the retina. Our immunohistochemical analysis in humans showed that CX3CR1 was expressed on all retinal MCs, which were identified by their morphology and CD18 expression. In addition, experiments using CX3CR1+/GFP
mice and the murine MC markers CD11b, F4/80, and 5D4 confirmed that only MCs express CX3CR1. Using both techniques in 2 different species, we found no CX3CR1 expression in neurons, astrocytes or glial cells: as a previous report showed for the CNS (14
), MCs are the only cells in the retina that express CX3CR1. Contrary to a previous report (10
), when we used the CX3CR1-specific antibody revealed by the fast red system to circumvent autofluorescence interference and conducted experiments with a reporter gene, we failed to find significant CX3CR1 expression in RPE cells. In AMD, CX3CR1-positive MCs accumulated subretinally in affected areas of the macula. Our data are consistent with the infiltration of the subretinal space by activated MCs previously described in AMD (15
) and indicate that a specific inflammatory environment characterized by production of specific cytokines and chemokines may determine particular pathological conditions.
Cells of the CNS and of the retina express CX3CL1 abundantly (26
). On the other hand, RPE cells under oxidative stress express other monocyte chemoattractant chemokines such as CCL2 (28
), which may be responsible for the recruitment of MCs in the subretinal space. To evaluate the effect of the M280 polymorphism on cell migration in a CX3CL1-rich environment, we tested M280 CX3CR1 monocyte migration through a CX3CL1-coated porous membrane in response to CCL2. Previous studies show that the M280 polymorphism provokes loss of chemotaxis (17
) or increases adherence to its ligand (19
). Here, the presence of bound CX3CL1 significantly inhibited the CCL2-dependent chemotaxis of monocytes expressing the CX3CR1 M280 variant (Figure ). If similar alterations occur in vivo to retinal MCs, the M280 polymorphism may cause excessive MC adherence to membrane-anchored CX3CL1 and reduce migration in response to other inflammatory chemoattractants. Over the years, in subjects with the M280 polymorphism, clearance of MCs from the subretinal space (in response to soluble CX3CL1 or other chemoattractants) would thereby be inhibited, and SrMC accumulation might occur.
To simulate CX3CR1 dysfunction in an animal model, we used 2 independently generated CX3CR1 knockout mouse strains. Our data demonstrated strong and persistent SrMC accumulation in CX3CR1-deficient mice in 3 experimental conditions: laser injury, aging, and albino animals. In contrast, control mice with laser injury only transiently amassed ramified F4/80- and GFP-positive cells, and aging and albino control animals slowly or steadily accumulated SrMCs. We and others have previously demonstrated that CX3CR1 deficiency reduces macrophage aggregation in atherosclerotic lesions (20
), probably by limiting monocyte recruitment from the blood. In view of the role of CX3CR1 in monocyte recruitment, the excessive number of MCs observed in CX3CR1-deficient mice is unlikely to be the result of an excessive influx of monocytes.
Unlike the monocytes in atherosclerotic lesions, MCs can proliferate in situ (30
). Their accumulation may be the result of excessive proliferation or inhibition of their clearance from the subretinal space. CX3CL1, however, has been shown to facilitate microglia proliferation (31
), and excessive proliferation is unlikely to occur in CX3CR1 deficiency. On the other hand, SrMC clearance by apoptosis or migration (egression) may be affected by abrogation of CX3CR1. This question is currently under investigation in our laboratory.
Interestingly, CCL2 and CCR2 signaling did not appear to be involved in retinal MC distribution. Although CCL2–/–
) mice have previously been shown to develop signs of AMD secondary to a macrophage recruitment deficit (11
), they did not develop significant SrMC accumulation (1.7 ± 0.2 SrMCs/mm2
) at 12 months of age in the present study. The macrophage-dependent debris clearance (11
) may play a role only at a more advanced stage of the disease.
In CX3CR1-deficient mice, the resulting prolonged presence of MCs in the subretinal space was associated with excessive OS phagocytosis by SrMCs, which subsequently accumulated intracellular lipids. These subretinal microglial foam cells were the origin of the drusen-like deposits in mice. Similarly, in humans, we found CX3CR1-positive bloated SrMCs in the eyes of individuals with AMD (Figure B), and others have previously shown that clearance of photoreceptor debris by activated MCs causes bloated SrMCs (15
). It is tempting to speculate that SrMCs contribute to some extent to the drusen formation in AMD and that the rounded contours and steeply sloping sides characteristic of drusen (5
) may derive in part from degenerating bloated MCs that are subsequently covered by RPE cells. Supporting this theory is the fact that drusen contain numerous degenerating organelles, the origin of which may be retinal MCs (32
). Moreover, CX3CR1 deposits were found in drusen (Figure F). Similar drusen deposits have previously been reported to contain apolipoprotein E, complement factors, MHC, and amyloid oligomers, among others (5
). When activated, MCs can express ApoE (37
), complement factors (38
), MHC (39
), and the β-amyloid precursor protein (40
) as well as CX3CR1. MCs can be a major source of oxidative stress through respiratory bursts (41
) that can cause the oxidative protein and lipid modifications associated with drusen. The inflammatory proteins found in drusen may therefore be of retinal MC origin, at least in part.
Another consequence of the prolonged presence of MCs in the subretinal space may be photoreceptor cell death. In CX3CR1–/–
mice, retinal degeneration was observed in senescent mice, in albino mice, and in the vicinity of subretinal neovascularization. The retinal degeneration in CX3CR1–/–
mice was always preceded by SrMC accumulation and could be averted by limiting MC accumulation in the subretinal space, as seen in CX3CR1–/–
albino mice raised in the dark (Figure ). SrMC accumulation was observed as early as 6 months of age in pigmented and 1 month in albino CX3CR1-deficient mice, before any significant retinal degeneration (data not shown). There are no naturally occurring mutations known to cause retinal degeneration in BALB/c mice, and the time course of the degeneration, starting at 2 months, did not match any known retinal degeneration caused by photoreceptor or RPE gene defects. We further note that neither photoreceptors nor RPE cells contained express CX3CR1. Activated MCs participate in ganglion cell death in vivo (42
) and can induce photoreceptor cell death in vitro (43
). Neuronal cell toxicity caused by the prolonged presence of MCs (CX3CR1GFP/GFP
) in the brain has previously been described as a mechanism of neurodegenerative diseases (14
), and similar mechanisms may cause the degeneration observed in the CX3CR1–/–
BALB/c mice, in the senescent CX3CR1–/–
C57BL/6 mice, and in the vicinity of subretinal neovascularization after laser injury. To recapitulate, SrMC accumulation in CX3CR1-deficient mice is associated with photoreceptor degeneration in the absence of any primary photoreceptor or RPE disease. To our knowledge, no such mechanism of retinal degeneration has previously been described.
Furthermore, we showed that activated SrMCs adjacent to the laser injury site express VEGF. In CX3CR1-deficient mice, the increased accumulation of SrMCs and subsequent secretion of VEGF may be responsible for the development of the exaggerated neovascularization. This is consistent with our previous work, which showed that MCs can develop proangiogenic properties in the retina (44
). Moreover, CX3CR1-positive MCs were found in human CNV, which indicates that SrMCs are a possible cause or aggravating factor for CNV in AMD.
In summary, our results suggest that MC accumulation in the subretinal space may be a driving force in the pathogenesis of AMD and not a mere consequence of primary RPE or photoreceptor disease. Our study challenges the long-standing paradigm of drusen genesis as the gradual accumulation of extracellular debris from the RPE and choroid. We propose that SrMC accumulation, resulting from a migratory defect associated with CX3CR1, plays a key role in drusen formation, CNV, and retinal degeneration, the main features of AMD.