October 2010
Volume 51, Issue 10
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Immunology and Microbiology  |   October 2010
The Monocyte Chemokine Receptor CX3CR1 Does Not Play a Significant Role in the Pathogenesis of Experimental Autoimmune Uveoretinitis
Author Affiliations & Notes
  • Jelena Kezic
    From the Centre for Ophthalmology and Visual Sciences, Lions Eye Institute, The University of Western Australia, Perth, Australia; and
  • Paul G. McMenamin
    From the Centre for Ophthalmology and Visual Sciences, Lions Eye Institute, The University of Western Australia, Perth, Australia; and
    the Department of Anatomy and Developmental Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria, Australia.
  • Corresponding author: Paul G. McMenamin, Department of Anatomy and Developmental Biology, School of Biomedical Sciences, Monash University, Wellington Road, Clayton, Victoria 3800, Australia; paul.mcmenamin@med.monash.edu.au
Investigative Ophthalmology & Visual Science October 2010, Vol.51, 5121-5127. doi:10.1167/iovs.10-5325
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      Jelena Kezic, Paul G. McMenamin; The Monocyte Chemokine Receptor CX3CR1 Does Not Play a Significant Role in the Pathogenesis of Experimental Autoimmune Uveoretinitis. Invest. Ophthalmol. Vis. Sci. 2010;51(10):5121-5127. doi: 10.1167/iovs.10-5325.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To examine the role of the monocyte chemokine receptor CX3CR1 in experimental autoimmune uveoretinitis (EAU).

Methods.: EAU was induced in naive WT, Cx3cr1 gfp/+, and Cx3cr1gfp/gfp C57Bl/6 mice or chimeric mice. Ocular disease severity was graded by histologic analysis of resin sections. In addition, immunohistochemistry and confocal microscopy were performed on retinal whole mounts to characterize the monocytic infiltrate and changes in retinal microglia. To determine the relative roles of resident and blood-borne monocyte-derived cells in the active phase of uveoretinitis, EAU was induced 4 weeks after transplantation in chimeric mice (Cx3cr1gfp/gfp →WT and Cx3cr1 gfp/+→WT), and analysis was performed at days 14, 16, 21, and 28 after immunization.

Results.: After EAU induction, disease scores were not significantly different in WT, Cx3cr1 gfp/+, and Cx3cr1gfp/gfp mice. Chimeric studies revealed both donor- and host-derived monocyte-derived cells in the inner retinal layers during early EAU; however, it was donor monocytic cells that infiltrated the photoreceptors, the site of the target antigen. The absence of CX3CR1 did not impede the ability of monocyte-derived cells from Cx3cr1gfp/gfp donor mice to infiltrate during the peak of EAU.

Conclusions.: The lack of CX3CR1 on monocyte-derived cells does not significantly influence the onset or severity of EAU. In addition, chimeric studies revealed that it is primarily blood-derived monocytes that mediate photoreceptor damage in the effector phase of EAU, and this process is not CX3CR1 dependent.

Experimental autoimmune uveoretinitis (EAU) is an animal model representing human endogenous posterior uveitis, a diverse group of intraocular inflammatory conditions of a presumed autoimmune nature affecting mainly the retina. EAU is a CD4+ T-cell mediated disease that is induced in susceptible rodent strains using antigens derived from the retinal photoreceptors, including S-Ag (arrestin) and interphotoreceptor retinal binding protein (IRBP), or by adoptive transfer of retina-specific CD4+ T cells. 15 Although described as a classical T cell–mediated disease, T cells do not proliferate in the target organ, and it appears that monocytes/macrophages are the primary mediators of the irreversible damage to the rod outer segment photoreceptors. 610 In addition to an influx of blood-derived monocytes/macrophages, retinal microglia are also activated after EAU induction and have been implicated in various stages of the disease. 11 The elucidation of an IFNγ/TNFα-primed phenotype (NO-producing) of retinal infiltrating macrophages at the peak of EAU 7 highlights these cells as potential targets for novel therapeutic applications aimed at either halting disease induction or reducing retinal tissue destruction. 
Current treatment modalities of ocular inflammatory diseases, such as corticosteroids, can serve to slow down the rapid progression of disease, but, like other drug therapies, pose myriad adverse effects on the eye. 12,13 More recent therapeutic medications, including a number of biological agents directed against specific soluble mediators of inflammation such as cellular adhesion molecules, cytokines and chemokines, and their receptors, have been developed. 12,14,15 Consequently, studies using animal models for uveitis and other autoimmune diseases have been largely focused on targeting these inflammatory agents as novel therapeutic applications. 1621 CX3CR1, the sole receptor for CX3CL1, or fractalkine (FKN) has recently been of increasing interest. Fractalkine exists in both a membrane-bound form, sitting atop a mucin-like stalk, and a soluble form after proteolytic cleavage. 22 CX3CR1 is expressed by monocyte-derived cells, including dendritic cells (DCs), natural killer cells, and macrophages. 23,24 Membrane-bound FKN mediates firm adhesion of CX3CR1-bearing cells, whereas soluble fractalkine acts as a chemoattractant directing the migration of CX3CR1-bearing cells in both homeostatic and inflammatory conditions. 2427 CX3CR1 phenotype is a determining feature of monocyte fate: inflammatory macrophages are CX3CR1low CCR2high whereas tissue resident or noninflammatory macrophages are CX3CR1high and CCR2low. 28  
Data have emerged demonstrating a lack of CX3CR1 dependence in homeostatic replenishment of macrophages and DCs in the uveal tract and retina 29 as well as microglial responses to laser injury, 30 yet upregulated expression of both CX3CL1 and CX3CR1 have been demonstrated in murine experimental autoimmune anterior uveitis. 31 Associations between CX3CR1 polymorphisms and an increased risk for age-related macular degeneration (AMD) 32,33 and retinal vasculitis 34 have also emerged. In addition, CX3CR1 has been shown to regulate NK cell migration into the inflamed CNS during mouse experimental autoimmune encephalomyelitis (EAE), resulting in earlier disease onset and increased disease severity in Cx3cr1gfp/gfp mice. 35 This study and the recognized importance of macrophages in mediating target organ damage in EAU 610 led us to investigate the role of CX3CR1 in the development and progression of this model of intraocular autoimmune disease. 
The rodent retina contains at least two distinct resident populations of monocyte-derived cells, retinal microglia and perivascular macrophages, 36,37 with a minor population of major histocompatibility complex (MHC) class II+ 33D1+ DCs in the retinal margin and juxtapapillary regions. 38 Given that microglia and perivascular macrophages 3637,39 and blood-derived macrophages express many of the same immunophenotypic markers, 40,41 particularly when microglia are activated, 42,43 we sought to investigate the roles of these two cell lineages in EAU by inducing the disease 4 weeks after bone marrow reconstitution in chimeric mice. The use of Cx3cr1-gfp mice as cell donors allowed us to discriminate between host (GFP) cells in the retina and blood-borne donor (GFP+) cells. Furthermore, the production of chimeras using either Cx3cr1 gfp/+ (heterozygous) or Cx3cr1gfp/gfp (homozygous) mice as bone marrow (BM) donors allowed us to further address the question of whether this chemokine receptor was critical to monocyte/macrophage infiltration in EAU. 
Our data demonstrate no significant differences in the onset, disease severity, and progression of EAU in wild-type (WT), Cx3cr1 gfp/+, and Cx3cr1gfp/gfp mice at the time points examined. The BM chimera studies revealed that though both host- and donor-derived macrophage cells accumulated in the inner retina during early stages of disease, it was the blood-borne monocyte-lineage cells that infiltrated the retinal photoreceptors during active EAU. This did not differ significantly when the donor cells were CX3CR1-deficient, indicating that other chemokines play a role during active inflammation within the retina. 
Methods
Animals
Female C57BL/6 wild-type (WT), heterozygous (Cx3cr1 gfp/+), and homozygous (Cx3cr1gfp/gfp ) transgenic (Tg) mice aged between 6 to 12 weeks were obtained from the Animal Resources Centre (Murdoch University, Western Australia) and kept under pathogen-free conditions in chaff-lined cages with individual ventilation in 12-hour day/night cycles. Food (Stockfeeders RM2 autoclaved mouse diet; Animal Resources Center, Murdoch, Western Australia, Australia) and water were supplied ad libitum. C57Bl/6 Cx3cr1gfp/gfp Tg mice contain an enhanced green fluorescence protein (GFP)–encoding cassette knocked into the Cx3cr1 gene that disrupts its expression but facilitates GFP expression under the control of the Cx3cr1 promoter. 44 Cx3cr1 gfp/+ Tg mice were generated by crossing C57Bl/6 Cx3cr1gfp/gfp mice to WT C57Bl/6 mice. All procedures conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Creation of Bone Marrow Chimeras
BM radiation chimeras were created as previously described. 45 Briefly, recipient WT mice were irradiated with two doses of 5.5 Gy 14 hours apart and received an injection of 3 to 5 × 106 bone marrow cells (in 150 μL) from donor Cx3cr1 gfp/+ or Cx3cr1gfp/gfp mice into the lateral tail vein (approximately 2–3 hours after the second dose of irradiation). In accordance with local Animal Ethics Guidelines, antibiotics (Neomycin; Sigma-Aldrich, St. Louis, MO) were given to recipient mice for 7 days before and 2 weeks after irradiation. 
Induction of EAU in WT, Cx3cr1 gfp/+, and Cx3cr1gfp/gfp Mice and BM Chimeric Mice
EAU was induced in naive WT, Cx3cr1 gfp/+, and Cx3cr1gfp/gfp mice (n = 98; Table 1), and in BM chimeric mice. Animals were anesthetized by inhalation of oxygen and nitrous oxide (4:1) with 1.0% isopentane. To induce EAU, injections of a total of 100 μL of a 50/50 (1:1 ratio) mix of peptide IRBP1–16 (interphotoreceptor retinoid binding protein, 400 μg; Custom Peptide Synthesis [Auspep; Tullamarine, Victoria, Australia]; sequence GPTHLFQPSLVLDMAK) in complete Freund's adjuvant (CFA; Sigma-Aldrich) supplemented with Mycobacterium tuberculosis (2.5 mg/mL; BD PharMingen, San Diego, CA) was administered at the base of the tail and the right flank. Simultaneous to the IRBP injection, an intraperitoneal injection of pertussis toxin (PTX; 1.5 μg; Sigma-Aldrich) was administered. Control mice were injected with PTX and CFA without IRBP. EAU was induced in BM chimeras (Cx3cr1 gfp/+→WT, n = 40; Cx3cr1gfp/gfp →WT, n = 18) 4 weeks after BM reconstitution. 
Table 1.
 
Numbers of WT, Cx3cr1 gfp/+, and Cx3cr1 gfp/gfp C57Bl/6 Mice Injected with IRBP or CFA and PTX at Each Time Point (for Histologic Analysis)
Table 1.
 
Numbers of WT, Cx3cr1 gfp/+, and Cx3cr1 gfp/gfp C57Bl/6 Mice Injected with IRBP or CFA and PTX at Each Time Point (for Histologic Analysis)
Day WT Cx3cr1 gfp/+ Cx3cr1gfp/gfp
IRBP CFA/PTX IRBP CFA/PTX IRBP CFA/PTX
14 10 4 6 1 6 1
16 8 6 0 0 6 1
21 6 4 0 0 6 1
28 12 6 6 1 6 1
Histologic Processing and Staining of Tissue Sections from IRBP-Injected Mice
WT, Cx3cr1 gfp/+, and Cx3cr1gfp/gfp mice were killed with a single lethal injection of sodium pentobarbitone (100 mg/kg body weight). Cx3cr1 gfp/+ mice were examined at days 14 and 28 because preliminary studies in WT and Cx3cr1gfp/gfp mice indicated that these two time points would most likely display any differences in response. Mice were perfusion-fixed with Karnovsky's fixative (1% glutaraldehyde, 4% paraformaldehyde). 46 Eyes were enucleated, postfixed, and embedded in glycol-methacrylate resin. Tissue sections (5 μm) were cut with a microtome (RM 2255; Leica, Wetzlar, Germany) through the pupillary-optic nerve axis at six different levels, and sections were stained with hematoxylin and eosin. 
Histopathologic Grading of Disease
Five sections from different levels of each eye were examined by light microscopy and scored (masked observer) for disease using the histopathology grading system for mouse EAU. 47 The grading system rates ocular inflammation based on two parameters, cellular infiltration (which encompasses anterior and posterior segment infiltrate) and structural/morphologic changes to the retina. Average infiltrative and structural disease scores per eye were calculated by combining scores from each section, and an average grade per mouse was assigned by averaging the mean score from both eyes. Grades from individual mice were pooled, and a group mean and SEM were calculated for each time point (Prism; Graph Pad Software, San Diego, CA). 
EAU in Chimeric Mice
A selection of BM chimeric mice (Cx3cr1 gfp/+→WT [n = 16] or Cx3cr1gfp/gfp →WT [n = 10]) underwent immunization with IRBP or CFA and pertussis toxin only (controls) at 4 weeks after BM reconstitution. This time point was chosen based on preliminary studies 29 that revealed virtually no baseline recruitment of GFP+ myeloid-derived cells into the neural retina at this time point. Mice were killed at days 14, 16, 21, and 28 after IRBP injections. 
Tissue Collection and Processing for Immunostaining
Tissues destined for immunostaining were obtained from mice perfusion-fixed with 2% paraformaldehyde. The iris, ciliary body, choroid, and retina were dissected free and stained as quadrants. 46 A range of monoclonal antibodies (mAbs) was used, including anti–MHC class II (M5/114; 1/200; BD PharMingen), anti-CD169 (Ser4; 1/100; Serotec), anti-CD45 (1/100; Serotec), and anti-CD11b (1/100; BD PharMingen), as were isotype controls (IgG2a and IgG2b, 1/100; BD PharMingen). Tissues were treated with biotinylated goat anti–rat IgG (1/200; Amersham Biosciences, Piscataway, NJ) at room temperature (RT; 60 minutes), washed, and incubated with streptavidin-Cy3 (1/200; Jackson ImmunoResearch Laboratories, Bar Harbor, ME) (RT; 60 minutes). DAPI (4′,6-diamidine-2′-phenylindole dihydrochloride; Roche Diagnostics, Mannheim, Germany) was added as a nuclear stain. Stained whole mounts were mounted onto slides using aqueous mounting medium. Retinas were mounted with vitread aspect superiorly. 
Examination of Whole Mount Tissue
Stained whole mount specimens were examined by confocal microscopy (TCS SP2; Leica, Wetzlar, Germany). Images of the entire tissue whole mounts were produced by performing Z-stacks of the tissue from the internal to external aspect at increments ranging from 0.4 μm to 0.8 μm. Image editing software (Photoshop, version 7.0; Adobe, San Jose, CA) was used to perform final image processing. 
Statistical Analysis
EAU histologic disease scores in WT C57Bl/6 mice were compared with those in Cx3cr1 gfp/+ and Cx3cr1gfp/gfp mice using unpaired Student's t-test (Prism; Graph Pad Software). 
Results
The Role of CX3CR1 on the Onset and Severity of EAU
EAU was induced in WT, Cx3cr1 gfp/+ (heterozygous), and Cx3cr1gfp/gfp (homozygous) mice, and disease onset and severity were quantitatively assessed using a standard histologic grading system 47 to determine whether CX3CR1 plays a role in the onset or pathogenesis of this disease. Onset of EAU occurred at day 14 and day 16 after IRBP injection in both WT and Cx3cr1gfp/gfp mice (Table 2). Disease incidence rose at day 28 in both strains, with 100% incidence in Cx3cr1gfp/gfp mice compared with 67% incidence in WT mice. Cx3cr1 gfp/+ mice, acting as a further positive control (predicted to respond as WT), had a disease incidence similar to that of WT and homozygous mice (Table 2). At all time points examined, WT, Cx3cr1 gfp/+, and Cx3cr1gfp/gfp controls (injected with CFA and PTX only) did not develop EAU (Fig. 1A). The cardinal qualitative histologic features of disease in C57Bl/6 Cx3cr1 gfp/+ and Cx3cr1gfp/gfp mice were similar to those in WT mice and were consistent with those previously described in this model of EAU 1,3,48,49 (Figs. 1A–C). 
Table 2.
 
Incidence of Disease in IRBP-Injected WT, Cx3cr1 gfp/+ (Ht), and Cx3cr1 gfp/gfp (Hm) C57B1/6 Mice
Table 2.
 
Incidence of Disease in IRBP-Injected WT, Cx3cr1 gfp/+ (Ht), and Cx3cr1 gfp/gfp (Hm) C57B1/6 Mice
Day 14 Day 16 Day 21 Day 28
Strain WT Ht Hm WT Hm WT Hm WT Ht Hm
Incidence 1/10 0/6 2/6 4/8 2/6 3/6 4/6 8/12 6/6 6/6
Figure 1.
 
Histologic analysis of EAU in WT, Cx3cr1 gfp/+, and Cx3cr1gfp/gfp C57Bl/6 mice. Representative images of Cx3cr1gfp/gfp mice. (A) Normal retinal morphology in control mice (immunized with CFA and PTX). (B) EAU in IRBP-treated mice at day 16 after immunization. (C) Severe retinal pathology at day 28 after IRBP immunization (magnification, 200×). (D–F) Average histologic disease scores in WT versus Cx3cr1 gfp/+ (HT) and Cx3cr1gfp/gfp (HM) mice at days 14, 16, 21, and 28 after IRBP injection. Mean score per mouse calculated by combining infiltrative (D) and structural (E) disease scores for both eyes.
Figure 1.
 
Histologic analysis of EAU in WT, Cx3cr1 gfp/+, and Cx3cr1gfp/gfp C57Bl/6 mice. Representative images of Cx3cr1gfp/gfp mice. (A) Normal retinal morphology in control mice (immunized with CFA and PTX). (B) EAU in IRBP-treated mice at day 16 after immunization. (C) Severe retinal pathology at day 28 after IRBP immunization (magnification, 200×). (D–F) Average histologic disease scores in WT versus Cx3cr1 gfp/+ (HT) and Cx3cr1gfp/gfp (HM) mice at days 14, 16, 21, and 28 after IRBP injection. Mean score per mouse calculated by combining infiltrative (D) and structural (E) disease scores for both eyes.
Quantitative analysis of histopathologic changes in WT, Cx3cr1 gfp/+, and Cx3cr1gfp/gfp mice, obtained by comparing the nature and extent of the cellular infiltrative (Fig. 1D; vasculitis, vitreitis) and structural damage (Fig. 1E; photoreceptor loss, retinal folding) at days 14, 16, 21, and 28 after IRBP injection revealed no significant differences in either disease onset or severity between these experimental groups of mice (Figs. 1D–F). Although all Cx3cr1 gfp/+- and Cx3cr1gfp/gfp -immunized groups displayed signs of ocular inflammation at day 28, compared with 8 of 12 immunized WT mice, and there was a slight trend for greater disease severity at the later time points in Cx3cr1 gfp/+ and Cx3cr1gfp/gfp mice compared with WT mice, the data were not statistically significant, leading us to conclude that the absence of CX3CR1 alone does not alter the onset or severity of murine EAU. 
The Phenotype of Monocyte-Derived Cells in the Inner and Outer Retina during Active EAU
Confocal microscopy of immunostained retinal whole mounts from days 21 and 28 EAU in Cx3cr1 gfp/+ (Figs. 2A–C) and Cx3cr1gfp/gfp (Figs. 2D–F) mice revealed that the monocytes/macrophages in the inner retina of both strains of mice constituted Cx3cr1hi CD11b+ cells that were interpreted as representing activated microglia (Fig. 2A). These were more dendriform than the bulk of the rounded CD45+ CD11b+ Cx3cr1low cells at day 21, which were largely perivascular in distribution (CD45; Fig. 2B). This pattern of distribution and phenotype of infiltrating cells was similar at day 28 (Figs. 2D, 2E), though some inflamed retinas demonstrated heavier areas of infiltrate (Fig. 2D) than that noted at day 21. Some immunopositive CD45+ infiltrating cells (Figs. 2B, 2E) were Cx3cr1low or Cx3cr1, possibly representing T cells; however, because the primary focus of the present study was on the nature of cells of monocyte/macrophage lineage, further immunophenotypic analysis of T cells was not pursued. 
Figure 2.
 
Confocal microscopic analysis of retinal whole mounts after EAU induction. Representative images from IRBP-injected mice at day 21 (Cx3cr1 gfp/+ mice; A–C) and day 28 (Cx3cr1gfp/gfp mice; D–F). Green: Cx3cr1 gfp/+ and Cx3cr1gfp/gfp monocytic lineage cells. Red: immunopositive cells (various markers). Inflammatory cells infiltrating the inner retina at day 21 (A, B) and day 28 (D, E) were mostly Cx3cr1+ CD11b+ (A, D) and CD45+ (B, E), and some immunopositive infiltrating cells were Cx3cr1lo or Cx3cr1. Inflammatory cells were evident in the photoreceptor cell layer at day 21 (C) and day 28 (F) after EAU induction and were composed of mainly Cx3cr1+, CD11b+ (C), and CD45+ (F) cells.
Figure 2.
 
Confocal microscopic analysis of retinal whole mounts after EAU induction. Representative images from IRBP-injected mice at day 21 (Cx3cr1 gfp/+ mice; A–C) and day 28 (Cx3cr1gfp/gfp mice; D–F). Green: Cx3cr1 gfp/+ and Cx3cr1gfp/gfp monocytic lineage cells. Red: immunopositive cells (various markers). Inflammatory cells infiltrating the inner retina at day 21 (A, B) and day 28 (D, E) were mostly Cx3cr1+ CD11b+ (A, D) and CD45+ (B, E), and some immunopositive infiltrating cells were Cx3cr1lo or Cx3cr1. Inflammatory cells were evident in the photoreceptor cell layer at day 21 (C) and day 28 (F) after EAU induction and were composed of mainly Cx3cr1+, CD11b+ (C), and CD45+ (F) cells.
In the outer retina (photoreceptors) most of the monocyte-derived cells (GFP+ in both Cx3cr1 gfp/+ and Cx3cr1gfp/gfp mice) at days 21 and 28 were CD45+ CD11b+ (Figs. 2C, 2F), and a subpopulation were MHC class II+ cells (not shown), a phenotypic profile suggestive of activated macrophages. The extent of the changes described was similar in both Cx3cr1 gfp/+ and Cx3cr1gfp/gfp mice. Taken together, these findings concur with histologic grading data and confirm that CX3CR1 does not play a major role in the pathogenesis of EAU. Eyes from control animals (pertussis toxin and CFA alone) revealed none of the inflammatory changes described (data not shown). 
Induction of EAU in Chimeric Mice Demonstrates CX3CR1+ Blood-Borne Monocyte Lineage Cells Infiltrate the Photoreceptors during the Late Stages of EAU
After our observations that CX3CR1 does not play a major role during EAU, we sought to take advantage of Tg-WT chimeric mice to investigate whether the monocyte-derived cells in the effector phase of EAU represent activated host microglia or newly recruited blood-borne Cx3cr1+ monocytes/macrophages. To this end, EAU was induced in both Cx3cr1 gfp/+→WT and Cx3cr1gfp/gfp →WT chimeric mice 4 weeks after myeloablation and BM reconstitution. By comparing both types of chimeras, we also sought to determine whether CX3CR1 was critical to the infiltration of monocyte-derived cells in EAU. Thus retinal whole mounts from these chimeras were studied at days 14, 16, 21, and 28 after IRBP injection. 
At all time points examined, we noted no differences in the pathologic changes in EAU between WT mice that received bone marrow from Cx3cr1 gfp/+ donors (Fig. 3) and those that received bone marrow from CX3CR1-deficient donors (Cx3cr1gfp/gfp ). At day 14, a small number of round donor-derived GFP+ cells infiltrated the retina at the juxtapapillary region in both Cx3cr1 gfp/+ and Cx3cr1gfp/gfp chimeric mice (data not shown). At this time point, host CD45low CD11b+ microglia (GFP) throughout the retinal parenchyma appeared normal. The number of donor (GFP+) cells infiltrating both the inner and the outer retina at the optic disc region increased from day 16 (Fig. 3A) to day 21 (data not shown) and day 28 (Fig. 3B), which concurs with previous turnover studies performed in our laboratory. 45 The infiltrating cells in the remainder of the inner retina were GFP+ (CX3CR1+) CD11b+ donor cells of monocyte origin among the CD11b+ GFP host cells (Fig. 3C). The latter group could be separated into obvious microglia-like cells and rounded CD11b+ GFP cells that likely represent activated host microglia. In eyes stained with anti-CD45 (Fig. 3D), the full extent of the cellular infiltrate (monocytic and lymphocytic) was evident with CD45+ cells either GFP+ (donor-derived) or GFP (host-derived; Fig. 3D), implying a mixture of host microglia and donor monocytes/macrophages are involved in the early stages of EAU. At day 16 CD11b+ Cx3cr1+ cells were present in the juxtapapillary region of the retinal photoreceptor cell layer (Fig. 3E). By day 28 after EAU induction, the extent of cellular infiltrate in the photoreceptors had greatly increased (Fig. 3F). Z-profiles illustrating the full thickness of the retinal whole mounts from the inner retina (top) to the photoreceptor layer (bottom) demonstrated a higher density of cellular infiltrate of both host cell (GFP) and donor cell (GFP+) origin in the inner retinal layers (vitread aspect), whereas infiltrating cells in the retinal photoreceptors were mainly of monocytic donor-derived (GFP+) rather than host origin (Figs. 3F, 3G). 
Figure 3.
 
Confocal microscopic analysis of retinal whole mounts at day 16 (A, C, E) and day 28 (B, D, F, G) after EAU induction in BM chimeras (representative images from Cx3cr1 gfp/+→WT mice). Donor GFP+/Cx3cr1+ cells at the juxtapapillary margin of the retina at day 16 (A) and day 28 (B). Infiltrate of the inner retina composed of both Cx3cr1+ (donor derived) and Cx3cr1 (host derived) cells that were CD11b+ (C), CD45+ (D) and MHC class II+ (not shown). At day 16, inflammatory cells in the photoreceptor cell layer (PCL) were of both host (GFP) and donor (GFP+) origin and were restricted to the juxtapapillary region (E). Infiltrate in the PCL had greatly increased and was largely of donor cell origin at day 28 after EAU induction (F). Z-profile illustrating the full thickness of the retina from the inner retina (top) to the PCL (bottom) (G).
Figure 3.
 
Confocal microscopic analysis of retinal whole mounts at day 16 (A, C, E) and day 28 (B, D, F, G) after EAU induction in BM chimeras (representative images from Cx3cr1 gfp/+→WT mice). Donor GFP+/Cx3cr1+ cells at the juxtapapillary margin of the retina at day 16 (A) and day 28 (B). Infiltrate of the inner retina composed of both Cx3cr1+ (donor derived) and Cx3cr1 (host derived) cells that were CD11b+ (C), CD45+ (D) and MHC class II+ (not shown). At day 16, inflammatory cells in the photoreceptor cell layer (PCL) were of both host (GFP) and donor (GFP+) origin and were restricted to the juxtapapillary region (E). Infiltrate in the PCL had greatly increased and was largely of donor cell origin at day 28 after EAU induction (F). Z-profile illustrating the full thickness of the retina from the inner retina (top) to the PCL (bottom) (G).
Discussion
Uveitis is the third most common cause of blindness in developed countries and though posterior uveitis has a lower frequency of occurrence than anterior uveitis, the consequences in terms of permanent visual loss make this condition a continuing challenge for ophthalmologists. The newer generation of biological therapeutics for the treatment of intraocular inflammation includes a number of agents directed against specific soluble mediators of inflammation, including cellular adhesion molecules, cytokines and chemokines, and their receptors. 12,14,15 Our understanding of the role of these mediators of inflammation has largely arisen from animal models for uveitis, including murine EAU. 1620,50 More recently, the importance of interactions between the chemokine CX3CL1 and its cognate sole receptor, CX3CR1, has been demonstrated in numerous ocular inflammatory conditions, including experimental autoimmune anterior uveitis 31 and putative murine models of age-related macular degeneration. 32,51  
The generation of transgenic mice in which the GFP-encoding cassette is “knocked in” to the CX3CR1 locus, thus disrupting its expression but facilitating GFP expression under the control of the CX3CR1 promoter, 44 has enabled novel insights into the role of monocytic-lineage cells in both homeostatic and inflammatory conditions. Thus, one of the primary aims of the present study was to determine whether the absence of CX3CR1, which is primarily expressed in the retina on microglia, would influence the development of experimentally induced posterior uveitis. Our data reveal no statistically significant differences between those mice deficient in CX3CR1 and WT (Cx3cr1 +/+) mice. This leads us to conclude that the expression of CX3CR1 on resident monocyte-derived cells in the retina itself (microglia), in the uveal tract (tissue macrophages and DCs), or on infiltrating cells is not critical to the initiation of an autoimmune-driven inflammation of the murine retina and choroid. 
The present data differ from those of a similar study in experimental autoimmune encephalomyelitis (EAE), whereby Cx3cr1gfp/gfp mice displayed an earlier onset of disease and significantly greater disease severity than WT and Cx3cr1 gfp/+ mice. 35 The increase in disease severity and the earlier onset of EAE in Cx3cr1gfp/gfp mice was ascribed to an impaired recruitment of CX3CR1 NK cells to the CNS. NK cells appear to play a dual role in different inflammatory conditions by having either a protective or a destructive effect on target tissues. In EAE, NK cells perform an immunomodulatory function by reducing inflammatory damage to the CNS during the course of disease. 35 By contrast, in EAU, the depletion of NK cells resulted in a reduction of tissue damage, suggestive of a proinflammatory role. 52 Although we did not analyze NK cell numbers in the present study, a recent study 53 investigating the role of CX3CR1 in a similar mouse model of EAU has shown that the number of NK cells present in the infiltrate during disease did not differ between Cx3cr1 gfp/+ and Cx3cr1gfp/gfp mice. Dagkalis et al. 53 recently reported significantly higher EAU disease severity by histologic grading of disease in the absence of CX3CR1 at day 23 after immunization; however, this difference was not reflected in the percentage of macrophages present in the retinal infiltrate. Although we did note slightly increased severity of disease in both Cx3cr1 gfp/+ and Cx3cr1gfp/gfp mice, this difference, which was based on overall histologic grade, was not statistically significant. Possible explanations for the observed differences in findings of our own and those of Dagkalis et al. 53 include the different form of IRBP used to immunize animals, which is known to alter the course of EAU. 1  
Although CX3CL1-mediated leukocyte chemotaxis and adhesion have been implicated as key in inflammatory and autoimmune conditions, including atopic dermatitis, 54 collagen-induced arthritis, 55 experimental autoimmune myositis 56 and mouse EAE, 35,57 a clear role for CX3CR1 in the regulation of microglial responses during different ocular inflammatory conditions is yet to be determined. Two recent studies failed to demonstrate a role for CX3CR1 in microglial responses to acute light damage 30 and during proliferative neovascularization in a mouse model of retinopathy of prematurity. 58 Interestingly, the latter group of researchers have also recently demonstrated that the dynamic behavior of retinal microglia in the resting state and microglial migration after laser injury were impaired in the absence of CX3CR1 signaling. 59 Our study of the role of CX3CR1 in EAU is timely in light of emerging evidence that has implicated fractalkine-CX3CR1 interactions in microglial-mediated neuroprotective mechanisms in the brain during episodes of systemic inflammation. 60,61  
The present study used a novel strategy whereby EAU was induced four weeks after BM chimera production in an effort to understand the relative roles of blood-derived macrophages versus activated resident retinal microglia as the key initiators or effector cells in the retina during EAU. Our data reveal no differences in the severity of EAU between Cx3cr1 gfp/+→WT chimeric mice or Cx3cr1gfp/gfp →WT chimeric mice. However, though both donor and host-derived cells were present in the inner retina during early stages of EAU, blood-derived macrophages, rather than host microglia, primarily infiltrated the retinal photoreceptors during active disease at day 28 after EAU induction. These data differ from those of BM chimeric studies in the rat whereby microglial activation and migration to the retinal photoreceptors (day 9 after immunization) occurred before the recruitment of blood-derived macrophages (day12) in EAU. 11 Although the present study assessed retinal whole mounts as early as day 14 after EAU induction, with donor-derived CX3CR1+ cells present in the photoreceptor cell layer from day 16, examining even earlier time points after IRBP immunization might have allowed us to visualize possible earlier infiltration of the photoreceptor layer by resident microglia. 
Collectively, data presented in this study indicate CX3CR1 alone does not play a direct role in the homing of monocyte-derived cells during intraocular inflammation. The induction of EAU in BM chimeras demonstrated that though both host- and donor-derived monocytic inflammatory cells populated the inner retinal layers during early stages of disease, it was blood-borne cells of monocytic origin that infiltrated the retinal photoreceptors during the active stages of murine EAU. It remains unclear whether retinal microglia or myeloid-derived cells in the uveal tract are the key players in the initiation of disease. Despite a large body of literature describing the role of various chemokines and their receptors during different homeostatic and inflammatory conditions, the demonstration of the lack of a significant role for CX3CR1 in this study compared with its proposed role in other retinal and neurodegenerative conditions highlights the need for further studies of these soluble mediators in different disease states and in different tissue microenvironments. 
Footnotes
 Supported by a University Postgraduate Award from the University of Western Australia (JK). Confocal microscopy was performed at the Centre for Microscopy, Characterization and Analysis at University of Western Australia, which is supported by university, state, and federal funding.
Footnotes
 Disclosure: J. Kezic, None; P.G. McMenamin, None
The authors thank Karen Waldock (Department of Radiation Oncology, QEII Medical Centre, University of Western Australia) for performing irradiation and Valentina Voigt and Holly Chinnery (Lions Eye Institute, University of Western Australia) for technical assistance with tail vein injections. 
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Figure 1.
 
Histologic analysis of EAU in WT, Cx3cr1 gfp/+, and Cx3cr1gfp/gfp C57Bl/6 mice. Representative images of Cx3cr1gfp/gfp mice. (A) Normal retinal morphology in control mice (immunized with CFA and PTX). (B) EAU in IRBP-treated mice at day 16 after immunization. (C) Severe retinal pathology at day 28 after IRBP immunization (magnification, 200×). (D–F) Average histologic disease scores in WT versus Cx3cr1 gfp/+ (HT) and Cx3cr1gfp/gfp (HM) mice at days 14, 16, 21, and 28 after IRBP injection. Mean score per mouse calculated by combining infiltrative (D) and structural (E) disease scores for both eyes.
Figure 1.
 
Histologic analysis of EAU in WT, Cx3cr1 gfp/+, and Cx3cr1gfp/gfp C57Bl/6 mice. Representative images of Cx3cr1gfp/gfp mice. (A) Normal retinal morphology in control mice (immunized with CFA and PTX). (B) EAU in IRBP-treated mice at day 16 after immunization. (C) Severe retinal pathology at day 28 after IRBP immunization (magnification, 200×). (D–F) Average histologic disease scores in WT versus Cx3cr1 gfp/+ (HT) and Cx3cr1gfp/gfp (HM) mice at days 14, 16, 21, and 28 after IRBP injection. Mean score per mouse calculated by combining infiltrative (D) and structural (E) disease scores for both eyes.
Figure 2.
 
Confocal microscopic analysis of retinal whole mounts after EAU induction. Representative images from IRBP-injected mice at day 21 (Cx3cr1 gfp/+ mice; A–C) and day 28 (Cx3cr1gfp/gfp mice; D–F). Green: Cx3cr1 gfp/+ and Cx3cr1gfp/gfp monocytic lineage cells. Red: immunopositive cells (various markers). Inflammatory cells infiltrating the inner retina at day 21 (A, B) and day 28 (D, E) were mostly Cx3cr1+ CD11b+ (A, D) and CD45+ (B, E), and some immunopositive infiltrating cells were Cx3cr1lo or Cx3cr1. Inflammatory cells were evident in the photoreceptor cell layer at day 21 (C) and day 28 (F) after EAU induction and were composed of mainly Cx3cr1+, CD11b+ (C), and CD45+ (F) cells.
Figure 2.
 
Confocal microscopic analysis of retinal whole mounts after EAU induction. Representative images from IRBP-injected mice at day 21 (Cx3cr1 gfp/+ mice; A–C) and day 28 (Cx3cr1gfp/gfp mice; D–F). Green: Cx3cr1 gfp/+ and Cx3cr1gfp/gfp monocytic lineage cells. Red: immunopositive cells (various markers). Inflammatory cells infiltrating the inner retina at day 21 (A, B) and day 28 (D, E) were mostly Cx3cr1+ CD11b+ (A, D) and CD45+ (B, E), and some immunopositive infiltrating cells were Cx3cr1lo or Cx3cr1. Inflammatory cells were evident in the photoreceptor cell layer at day 21 (C) and day 28 (F) after EAU induction and were composed of mainly Cx3cr1+, CD11b+ (C), and CD45+ (F) cells.
Figure 3.
 
Confocal microscopic analysis of retinal whole mounts at day 16 (A, C, E) and day 28 (B, D, F, G) after EAU induction in BM chimeras (representative images from Cx3cr1 gfp/+→WT mice). Donor GFP+/Cx3cr1+ cells at the juxtapapillary margin of the retina at day 16 (A) and day 28 (B). Infiltrate of the inner retina composed of both Cx3cr1+ (donor derived) and Cx3cr1 (host derived) cells that were CD11b+ (C), CD45+ (D) and MHC class II+ (not shown). At day 16, inflammatory cells in the photoreceptor cell layer (PCL) were of both host (GFP) and donor (GFP+) origin and were restricted to the juxtapapillary region (E). Infiltrate in the PCL had greatly increased and was largely of donor cell origin at day 28 after EAU induction (F). Z-profile illustrating the full thickness of the retina from the inner retina (top) to the PCL (bottom) (G).
Figure 3.
 
Confocal microscopic analysis of retinal whole mounts at day 16 (A, C, E) and day 28 (B, D, F, G) after EAU induction in BM chimeras (representative images from Cx3cr1 gfp/+→WT mice). Donor GFP+/Cx3cr1+ cells at the juxtapapillary margin of the retina at day 16 (A) and day 28 (B). Infiltrate of the inner retina composed of both Cx3cr1+ (donor derived) and Cx3cr1 (host derived) cells that were CD11b+ (C), CD45+ (D) and MHC class II+ (not shown). At day 16, inflammatory cells in the photoreceptor cell layer (PCL) were of both host (GFP) and donor (GFP+) origin and were restricted to the juxtapapillary region (E). Infiltrate in the PCL had greatly increased and was largely of donor cell origin at day 28 after EAU induction (F). Z-profile illustrating the full thickness of the retina from the inner retina (top) to the PCL (bottom) (G).
Table 1.
 
Numbers of WT, Cx3cr1 gfp/+, and Cx3cr1 gfp/gfp C57Bl/6 Mice Injected with IRBP or CFA and PTX at Each Time Point (for Histologic Analysis)
Table 1.
 
Numbers of WT, Cx3cr1 gfp/+, and Cx3cr1 gfp/gfp C57Bl/6 Mice Injected with IRBP or CFA and PTX at Each Time Point (for Histologic Analysis)
Day WT Cx3cr1 gfp/+ Cx3cr1gfp/gfp
IRBP CFA/PTX IRBP CFA/PTX IRBP CFA/PTX
14 10 4 6 1 6 1
16 8 6 0 0 6 1
21 6 4 0 0 6 1
28 12 6 6 1 6 1
Table 2.
 
Incidence of Disease in IRBP-Injected WT, Cx3cr1 gfp/+ (Ht), and Cx3cr1 gfp/gfp (Hm) C57B1/6 Mice
Table 2.
 
Incidence of Disease in IRBP-Injected WT, Cx3cr1 gfp/+ (Ht), and Cx3cr1 gfp/gfp (Hm) C57B1/6 Mice
Day 14 Day 16 Day 21 Day 28
Strain WT Ht Hm WT Hm WT Hm WT Ht Hm
Incidence 1/10 0/6 2/6 4/8 2/6 3/6 4/6 8/12 6/6 6/6
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