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Originally published online as doi:10.1189/jlb.0308166 on June 24, 2008

Published online before print June 24, 2008
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(Journal of Leukocyte Biology. 2008;84:721-729.)
© 2008 by Society for Leukocyte Biology

Differential turnover rates of monocyte-derived cells in varied ocular tissue microenvironments

Jelena Kezic and Paul G. McMenamin1

School of Anatomy and Human Biology, The University of Western Australia, Perth, Western Australia

1 Correspondence: School of Anatomy and Human Biology, The University of Western Australia, Crawley (Perth), 6009, Western Australia. E-mail: mcmenamin{at}anhb.uwa.edu.au


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ABSTRACT
 
Monocytes of bone marrow (BM) origin are circulating precursors that replenish dendritic cells and macrophage populations in peripheral tissues during homeostasis. The eye provides a unique range of varying tissue microenvironments in which to compare the different turnover rates of monocyte-derived cells. This was investigated in the present study using radiation chimeras, whereby BM from Cx3cr1+/gfp mice was used to rescue myeloablated wild-type (WT) BALB/c mice (conventional chimeras). The use of Cx3cr1+/gfp mice as BM donors allowed the clear visualization of newly recruited monocyte-derived cells. Following BM reconstitution, mice were killed at 2, 4, 6, and 8 weeks, and wholemount ocular tissues were processed for immunohistochemistry and confocal microscopy. "Reverse" chimeras (WT into Cx3cr1+/gfp) were also created to act as a further method of cross-referencing cell turnover rates. In conventional chimeras, Cx3cr1+/gfp cells began repopulating the uveal tract (iris, ciliary body, choroid) 2 weeks post-transplantation with close to complete replenishment by 8 weeks. By contrast, the earliest recruitment of Cx3cr1+/gfp cells into the host retina occurred at 4 weeks. In reverse chimeras, a steady accumulation of host Cx3cr1+/gfp macrophages in the subretinal space of Cx3cr1+/gfp adult mice suggests that these cells arise from long-term resident microglia and not newly recruited WT donor cells. In summary, chimeric mouse models, in which lineage-specific cells carry a fluorescent reporter, have been used in the present study to visualize the turnover of monocyte-derived cells in different tissue compartments of the eye. These data provide valuable insights into differential monocyte turnover rates within a single complex organ.

Key Words: macrophages • microglia • choroid • iris • retina • replenishment


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INTRODUCTION
 
The uveal tract, the vascularized middle coat of the eye, consisting of the iris, ciliary body, and choroid, contains rich networks of immune cells that reside and traffic through the eye [1 2 3 4 5 ]. These populations include large numbers of macrophages and immature dendritic cells (DCs), which have been implicated in the maintenance of local immunological homeostasis as well as in inflammatory processes and immune-mediated diseases, including endotoxin-induced uveitis [6 , 7 ] and experimental autoimmune uveoretinitis [8 9 10 11 ]. Recent attention has focused on the role of choroidal macrophages in age-related macular degeneration (AMD), with these cells proposed as key players in the removal of age-related accumulation of extracellular debris at the choroidal-retinal interface [12 , 13 ]. In addition to the potential role of choroidal macrophages, the discovery of age-dependent accumulation of subretinal microglia has recently implicated this population of cells as potential initiators of neovascularization and photoreceptor damage [14 , 15 ].

In the rodent retina, it has been well established that at least two distinct populations of monocyte-derived cells exist, namely, resident retinal microglia and perivascular macrophages [16 , 17 ]. More recently, a novel population of bone marrow (BM)-derived MHC Class II+ 33D1+ DCs has been identified, of which small numbers reside in the peripheral margin and juxtapapillary regions [18 ]. It is well accepted that resting microglia play various roles in host defense, immunoregulation, and tissue repair [19 20 21 ] and rapidly increase in numbers in response to various insults in the CNS [22 , 23 ], including the retina [24 , 25 ]. Whether an increase in microglial numbers in disease and microgliosis reflects proliferation of resident microglia or the influx of new blood-borne monocytic cells is still controversial [26 , 27 ].

Monocytes of BM origin have been well recognized as the circulating precursors for tissue DCs and macrophages and are believed to maintain or replenish populations in the peripheral tissues during homeostasis [28 , 29 ], with over half of circulating monocytes leaving the circulation in 22 h [30 ]. Interruption of the supply of DCs and macrophage precursors by myeloablation and subsequent monitoring of the ensuing population decline are well-recognized means of estimating population kinetics [31 ]. BM rescue prevents death of host animals, and with the availability of congenic and transgenic (Tg) strains of mice, especially those in which GFP has been "knocked in" to lineage-specific markers such as Cx3cr1, it is possible to precisely monitor and quantify the replenishment of monocyte-derived cells [28 ].

The eye, with its closely related but widely different tissue microenvironments such as the vascular connective tissue of the uveal tract and the neural parenchyma of the retina, offers a unique opportunity to investigate turnover rates and function of monocyte-derived cells in functionally differing environments within a single, albeit paired, complex organ. Studies of this nature are greatly aided by the layered arrangement of the ocular tissues, which allows for the examination of entire immune cell populations in tissue wholemounts [32 ], thus eliminating sampling errors associated with conventional histological or frozen sections. With the increasing use of BM mouse chimeras in the study of various disease models [33 34 35 36 ], it is important to establish normal turnover rates of immune cells to accurately determine recruitment in experimentally induced disease conditions. The aim of this study was to take advantage of the unique conditions of the eye to contrast and compare the different rates of turnover of monocyte-derived cell populations in a range of tissue microenvironments within a single organ.

In the present study, conventional [Cx3cr1+/gfp into wild-type (WT)] and "reverse" chimeric mice (WT into Cx3cr1+/gfp) allowed us to visualize the replenishment of monocyte-derived cells in the uveal and retinal tissues in the normal adult mouse eye. We were able to show that although cells of monocyte lineage begin to repopulate the uveal tract as early as 2 weeks post-transplantation with near complete replenishment by 8 weeks, the earliest recruitment in the retina occurs at 4 weeks. Our data also demonstrate the steady accumulation of a unique population of subretinal microglia in the normal host Cx3cr1+/gfp adult mouse in reverse chimeras. The cytoplasmic enhanced GFP+ (eGFP+) expression in these cells strongly suggests they arise from displaced host-resident, radio-resistent Cx3cr1+ microglia and not newly arriving donor WT monocyte-derived cells.


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MATERIALS AND METHODS
 
Animals
Female BALB/c mice, aged 6 weeks, and female Tg Cx3cr1+/gfp mice on a BALB/c background, aged between 6 and 8 weeks, were used in the present study. BALB/c Cx3cr1gfp/gfp Tg mice contain an eGFP-encoding cassette knocked into the Cx3cr1 gene that disrupts its expression but facilitates GFP expression under the control of the Cx3cr1 promoter. Heterozygous Tg (Cx3cr1+/gfp) mice were generated by crossing BALB/c Cx3cr1gfp/gfp mice to WT BALB/c mice and were used as BM donors or recipients. The BALB/c Cx3cr1gfp/gfp Tg mice were established from a primary colony provided by Dr. Steffen Jung (Weizmann Institute of Science, Rehovot, Israel) [37 ]. All procedures conformed to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by The University of Western Australia Animal Ethics Committee.

Creation of chimeras for turnover studies
Recipient BALB/c WT mice were irradiated with two doses of 5.5 Gy 14 h apart. Donor Cx3cr1+/gfp mice were euthanized, and femurs and tibia were harvested. Following removal of the proximal and distal ends of the bone, the shafts were centrifuged at 10,000 rpm for 30 s at 4°C. The pellet was resuspended in RPMI media (N6396, Sigma Chemical Co., St. Louis, MO, USA), centrifuged at 1200 rpm for 5 min at room temperature, and resuspended again, and live cells were counted by trypan blue exclusion. Cells were resuspended and diluted as appropriate. Recipient mice received an injection of 3–5 x 106 BM cells (in 150 µl) into the lateral tail vein (2–3 h after the second dose of irradiation). Antibiotics were given to recipient mice (Neomycin trisulfate salt hydrate, Sigma Chemical Co.) for 7 days before and 2 weeks after irradiation. Chimeric animals were killed at 1, 2, 4, 6, and 8 weeks post-transplantation (n=6 per time-point) for collection of ocular tissues to examine the recruitment of donor Cx3cr1+/gfp cells into WT host ocular tissue. As a cross-check for possible overt changes in resident cells following irradiation, as well as to confirm and compare the turnover of monocyte-derived cells, as determined using the conventional chimeras, reverse chimera studies were performed. In these reverse chimeras, WT BALB/c BM was used to rescue myeloablated Cx3cr1+/gfp mice. Animals were killed at Weeks 4, 6, 8, and 12 postreconstitution (n=3 per time-point) to quantify the rate of diminution of resident eGFP+ Cx3cr1+ cells.

Tissue collection and processing for immunohistochemistry
Mice were killed and perfusion-fixed using 4% paraformaldehyde, and from each eye, the iris, ciliary body, choroids and retina were dissected free and cut into quadrants as documented previously [32 ]. Tissue pieces were washed in PBS, incubated in 20 mM EDTA tetrasodium at 37°C for 30 min, and then incubated with a 0.2% solution of Triton-X in PBS with 2% BSA at room temperature for 30 min to assist antibody penetration. Tissues were treated at 4°C overnight with rabbit anti-GFP (1/200, Chemicon, El Segundo, CA, USA) and washed with PBS prior to incubation at room temperature for 60 min with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1/200, Molecular Probes, Eugene, OR, USA). Further washes with PBS were followed by overnight incubation (4°C) with a range of mAb, including anti-MHC class II (M5/114; 1/200, BD PharMingen, San Jose, CA, USA), anti-CD169 (Ser4; 1/100, Serotec, UK), anti-CD45 (1/100, Serotec), and anti-CD11b (1/100, BD PharMingen), as well as isotype controls (IgG2a and IgG2b, 1/100, BD PharMingen). Tissues were then treated with biotinylated goat anti-rat IgG (1/200, Amersham Biosciences, Piscataway, NJ, USA) at room temperature for 60 min, washed with PBS, and incubated with streptavidin-Cy3 (1/100, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at room temperature for 60 min. 4',6-Diamidine-2'-phenylindole dihydrochloride (Roche Diagnostics, Germany) was added at room temperature for 7 min as a nuclear stain. Stained wholemount tissues were mounted onto slides (retinae were mounted with the vitreous side face-up) using an aqueous mounting medium (Immunomount, Thermo Shandon, Waltham, MA, USA) and coverslipped.

Examination of wholemount tissue
Stained specimens were examined by conventional epifluorescence microscopy (Olympus, Tokyo, Japan; DMRBE, Leica, Australia) and confocal microscopy (Leica TCS SP2) to visualize repopulating eGFP+ cells in the iris/ciliary body, choroids and retina. Images of the entire tissue wholemount were produced by performing Z-stacks of the tissue from the internal to external aspect at increments ranging from 0.4 to 0.8 µm. Adobe Photoshop (Version 7.0) was used to perform final image processing.

Quantitative analysis of turnover of monocyte lineage cells in the uveal tract of the normal mouse eye
Donor monocyte lineage eGFP+ cells that began to repopulate host WT ocular tissues were counted in a 375-µm x 375-µm frame in a minimum of six randomly selected areas of each tissue. The mean cell density (per mm2) was calculated (Image ProPlus, Version 5.1), and the turnover of monocyte lineage cells in the iris and choroid at each time-point was determined by calculating the density of donor eGFP+ cells as a percentage of the normal density of monocyte lineage cells in Cx3cr1+/gfp mice. This was based on the assumption that at Week 0, the normal cell density in BALB/c WT mice was equivalent to that previously calculated in BALB/c Cx3cr1+/gfp mice [38 ]. In reverse chimera studies, the density of resident host eGFP+ Cx3cr1+ cells was calculated as a percentage of the normal density of cells in age-matched, untreated Cx3cr1+/gfp mice.


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RESULTS
 
Monocyte-derived eGFP+ cells begin to repopulate the uveal tract as early as 2 weeks postreconstitution
The percentage turnover of monocyte-derived cells in the iris and choroid was calculated from the density of donor eGFP+ cells at 2, 4, 6, and 8 weeks post-transplantation (Table 1 ). No donor eGFP+ cells were observed in any of the uveal tract tissues in WT hosts at 1 week following BM reconstitution.


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  		Table 1. Donor eGFP+ Cell Density/mm2 and Percentage Turnover in Conventional Chimeras (Cx3cr1+/gfp into WT)

The repopulation of the iris stroma (Fig. 1A 1B 1C 1D ), posterior iris (Fig. 1E 1F 1G 1H) , ciliary body stroma (Fig. 1I 1J 1K 1L) , ciliary epithelium (Fig. 1M 1N 1O 1P) , and choroid (Fig. 1Q 1R 1S 1T) by donor eGFP+ cells at 2, 4, 6, and 8 weeks post-BM transplantation was visualized using confocal microscopy. Donor eGFP+ cells were present in the iris stroma (Fig. 1A) and posterior iris (Fig. 1E) as early as 2 weeks post-transplantation. A steady increase in the number of eGFP+ cells was evident at 4 (Fig. 1B and 1F) and 6 (Fig. 1C and 1G) weeks, with almost complete turnover (anterior stroma, 96%; posterior iris, 94%) of iris cells by Week 8 (Fig. 1D and 1H) . A similar pattern of donor BM-derived cell repopulation of the iris stroma was noted in the ciliary body stroma, with the number of eGFP+ cells increasing from Week 2 to Week 8 (Fig. 1I 1J 1K 1L) post-transplantation. Interestingly, repopulation of the ciliary epithelium appeared to occur at a slower rate than that of the ciliary body stroma, with few eGFP+ cells being present at Week 2 (Fig. 1M) . The number of donor cells increased steadily by Week 4 (Fig. 1N) , Week 6 (Fig. 1O) , and Week 8 (Fig. 1P) post-transplantation.


Figure 1
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  		Figure 1. Donor eGFP+ cell entry into the uveal tract tissues in conventional chimeras. Donor eGFP+ cells began repopulating the iris stroma (A) and posterior iris (E) at 2 weeks post-transplantation. A steady increase in the number of eGFP+ cells was evident at Week 4 (B and F), Week 6 (C and G), and Week 8 (D and H). In the ciliary body stroma, eGFP+ cells increased in number from Week 2 to Week 8 (I–L) post-transplantation. Few donor eGFP+ cells were present in the ciliary epithelium at Week 2 (M). This number increased at Weeks 4 (N), 6 (O), and 8 (P). There were few donor eGFP+ cells in the choroid at 2 weeks (Q) but had entered the tissue by 4 weeks (R) and were at high density at 6 (S) and 8 (T) weeks post-transplantation.

Although a few eGFP+ donor cells were evident in the choroid at 2 weeks (Fig. 1Q) , greater numbers of eGFP+ cells began to migrate into the tissue by 4 weeks (Fig. 1R) . A mixture of round and dendriform donor cells was identifiable, the former decreasing by Week 6, as the number of dendriform donor cells continued to increase (Fig. 1S) . By Week 8 post-transplantation, repopulating cells had established a regular dense network of eGFP+ cells with almost complete turnover of monocyte-derived cells in the choroid (99%; Fig. 1T ).

Chimeric studies confirm a slow rate of renewal of microglia in the retina
At 1 and 2 weeks following BM reconstitution, no eGFP+ donor cells were detectable in host retinal wholemounts (data not shown). The earliest sign of monocytic cell recruitment into the retina was at 4 weeks, evident at the juxtapapillary margin (Fig. 2A 2B 2C 2D ). These donor-derived, monocytic eGFP+ Cx3cr1+ cells appeared to first enter the retina at the vitread aspect adjacent to the optic disc (Fig. 2A and 2C) . These amoeboid-like donor cells then migrated deeper into the retinal layers (Fig. 2B and 2D) , where some displayed a ramified appearance typical of normal resident host microglia (Fig. 2D) . By Week 6 post-transplantation, larger numbers of donor eGFP+ cells had migrated radially from the optic disc region (Fig. 2E) in the inner retina, evident as perivascular cells with an elongated and dendriform morphology (Fig. 2F) , and in the deeper retinal layers, where they displayed a more typical microglia morphology (Fig. 2G) . By Week 8, the number of donor monocyte-derived cells had increased further (Fig. 2I) , and the majority of these donor eGFP+ Cx3cr1+ cells surrounding the optic disc displayed a ramified morphology (Fig. 2J) . By 6 weeks post-transplantation, donor eGFP+ cells had also begun to migrate into the retina from the peripheral zone, close to the ciliary body junction (Fig. 2H) . Their number only marginally increased between Week 6 and Week 8 (Fig. 2L) . The regular network of retinal microglia (CD11b+) in these WT hosts was present throughout the retina, except where newly repopulating donor eGFP+ cells were more evident in the regions of the juxtapapillary margin and peripheral retina (Fig. 2K and 2L) .


Figure 2
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  		Figure 2. Donor eGFP+ cells repopulating the retina in conventional chimeras. Donor eGFP+ cells were present at the juxtapapillary margin by 4 weeks (A–D). These cells entered at the vitread aspect (A and C) to then migrate to deeper retinal layers (B and D), where some cells displayed a ramified morphology (D). At 6 weeks, eGFP+ cell numbers had increased and appeared to be migrating outwards from the optic disc region (E). Some perivascular cells were present along vessels (F). eGFP+ cells in the deep retina displayed ramified morphology (G). Donor cells were evident at the periphery by 6 weeks (H). Note, eGFP+ cells in the ciliary body (CB) are visible (H). By 8 weeks post-transplantation, the number of donor eGFP+ cells at the juxtapapillary region had increased further (I), and cells in the deeper retinal layers displayed a ramified morphology (J). Resident microglia were viewed using anti-CD11b (K and L, red). Donor eGFP+ cells in peripheral retina did not increase greatly in number from 6 to 8 weeks (L), and resident CD11b+ microglia were clearly visible in this region.

Reverse chimeras confirm the steady turnover of monocyte lineage cells in the uveal tract
Reverse chimeras, namely, a chimera produced by injecting BM from WT BALB/c mice into irradiated Cx3cr1+/gfp mice, acted as a valuable experimental means of confirming the turnover of BM-derived cells in ocular tissues as described above in conventional chimeras. The data from reverse chimera experiments (Table 2 ) closely corroborated with the pattern and general kinetics of turnover of monocyte lineage cells in the iris stroma, posterior iris, and choroid, described in the conventional chimeras (Table 1) .


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  		Table 2. Resident eGFP+ Cell Density/mm2 and Percentage of Resident Cells in Reverse Chimeras (WT into Cx3cr1+/gfp)

Confocal microscopy of reverse chimera tissues stained with a variety of markers, including CD11b and MHC class II, confirmed the gradual replenishment of host eGFP+ Cx3cr1+ cells in the anterior iris (Fig. 3A 3B 3C 3D ), posterior iris (Fig. 3E 3F 3G 3H) , ciliary body (data not shown), and choroid (Fig. 3I 3J 3K 3L) at the various time-points following irradiation and BM reconstitution.


Figure 3
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  		Figure 3. Resident eGFP+ cells in reverse chimeras (BALB/c WT BM into BALB/c Cx3cr1+/gfp mice). There was a clear decrease in the number of resident eGFP+ cells in the anterior (A–D) and posterior (E–H) iris and choroid (I–L). There was a corresponding increase in number of donor cells as visualized using the mAb for MHC Class II (red) from Weeks 4 (A, E, I), 6 (B, F, J), and 8 (C, G, K) to Week 12 (D, H, L). In the subretinal space, resident eGFP+ subretinal macrophages appeared to increase in number from Weeks 6 (M) and 8 (N) to Week 12 (O and P). Some subretinal macrophages appeared to have accumulated photoreceptor debris (red autofluorescent) at 8 (not shown) and 12 (O and P) weeks post-transplantation. Note, the photoreceptor debris or lipofuscin is contained within phagosomes, and the eGFP is intracytoplasmic (arrows in O and P). Tissues are counterstained with CD11b (membrane-localized); hence, the microglia appear yellow (O and P; compare with M and N).

The number of host microglia/macrophages in the photoreceptor layer and subretinal space increases in chimeric mice
In normal aging WT and Cx3cr1gfp/gfp mice, the photoreceptor layer and subretinal space contain a population of macrophages, which have recently been shown to accumulate photoreceptor debris as part of the aging process [14 , 15 ]. These cells have the immunophenotype of microglia, which are not normally present in this outer region of the retina [38 39 40 41 ]. In the reverse chimeras generated in the present study, host eGFP+ CD11b+ subretinal macrophages, unlike other host microglia, appeared to increase in number from Weeks 6 to 12 (Fig. 3M 3N 3O 3P) . Those located amongst the photoreceptors, although ramified (Fig. 3N and 3O) , were less dendriform than microglia in the inner retina (Fig. 2J and 2K) . Some of these host microglia/macrophages contained phagocytosed, autofluorescent photoreceptor debris at 8 (data not shown) and 12 (Fig. 3P) weeks post-transplantation. This particular subset generally had a less-ramified morphology (arrows in Fig. 3O and 3P ). No donor-derived CD11b+ microglia were detected in the photoreceptor layer or subretinal space in these reverse chimeras.


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DISCUSSION
 
The eye consists of a diverse range of tissues, which contain unique networks of resident monocyte-derived DCs and tissue macrophages specific to the differing microenvironments. These cell populations have been shown to play various roles in a range of ocular inflammatory conditions that pose significant threats to normal vision, such as uveitis [8 , 10 ] and AMD [12 , 42 ]. Mouse chimera models are increasingly being used as tools to differentiate between resident and blood-borne cells in a range of research fields. This is largely based on the well-accepted belief that DCs and macrophages in peripheral tissues are continuously replenished by blood-borne precursors [29 ]; however, the steady-state turnover rates of these cells in a full range of tissue microenvironments have not been investigated widely. The eye, with its diverse range of closely related microenvironments, ranging from the dense connective tissue of the cornea, the rich vascular connective tissue beds of the uveal tract, to the neural parenchyma of the retina, offers a unique opportunity to compare turnover rates of monocyte-derived cells within close proximity in a single organ. In the present study, we took advantage of the unique morphological organization of the eye, together with Tg Cx3cr1+/gfp knock-in mice in BM chimera models to study the recruitment and migration of eGFP+ monocyte-derived cells in a range of host ocular tissues. This model offers advantages over previous turnover studies that have mostly relied on donor BM harvested from conventional eGFP animals, in which all cells express GFP (under the chicken β-actin promotor; H-2Kb promotor) [39 , 43 , 44 ] in that the eGFP expression is under the control of a promoter (Cx3cr1), which restricts expression of the protein to cells of monocyte lineage. The experimental approach of the present study also has advantages over the use of congenic mouse strains, such as Ly5.1 and Ly5.2, as these models rely heavily on the quality of immunostaining for the differentiation of donor-derived versus resident host cells.

Previous studies of turnover of immune cells in the eye have been limited. Several years ago, our lab [45 ] reported that rat iris DC and macrophages had half-lives of 3 days and 10–12 days, respectively. The rapid turnover of DCs was quite surprising considering the previously recognized "immune privileged" nature of the internal ocular environment, as well as the limited exposure to exogenous, antigenic stimulation of this part of the uveal tract, behind the blood-ocular barrier [46 ]. By comparison, the DC population of the epidermis, Langerhans cells (LC), has been reported to take up to 18 months for complete turnover, unless activated otherwise [47 ], although in different chimeric models, much lower rates of LC turnover have been documented [48 , 49 ]. Indeed, a body of evidence is emerging, partly through the use of parabiotic mouse models, which suggests LC progenitors populate the epidermis at birth and replicate in situ throughout life [47 ]. Similar evidence is emerging that CNS microglia may also be replenished from tissue-dwelling progenitors [27 ].

The only previous study of turnover rates of BM-derived cells in the mouse uveal tract relied on immunolocalization to identify donor-derived cells and did not provide quantitative analysis [43 ]. In the present study, the quantification of monocyte-lineage donor cells, from Cx3cr1gfp Tg or knock-in mice, entering ocular tissue, using confocal microscopic analysis of entire tissue wholemounts ex vivo, revealed almost complete turnover of host BM-derived cells by 8 weeks in the iris stroma and posterior iris surface. The first BM-derived eGFP+ CD45+ cells were observed entering the tissue at 2 weeks post-transplantation. Qualitative and quantitative confirmation of this observation in reverse chimeras reassured us of the accuracy of the data. Although cell replenishment rates in the uveal tract were similar in the choroid, iris, and ciliary body stroma, which all consist of a rich vascular connective tissue, DCs in the ciliary epithelium appeared to be replenished at a slightly slower rate. This slower replenishment of intraepithelial DC may relate to the presence of the blood-ocular barrier at the level of the ciliary epithelia [50 ], which is closely homologous to the choroid plexus of the brain [51 ].

The presence of an intact blood-brain barrier (BBB) and blood-retinal barrier (BRB) appears to play an important role in determining the turnover of BM-derived cells in neural parenchymal environments. Rodent studies have revealed much slower turnover rates of CNS and retinal microglia compared with other resident tissue macrophages [29 ], with some studies suggesting that complete replenishment takes up to 1 year [40 , 52 ]. A recent study of the turnover of CNS microglia by Ajami and colleagues [27 ], using the elegant parabiotic mouse model, which obviates the need for irradiation and BM transfer, suggested microglia progenitors exist within the brain and act as a reservoir for replenishment. The authors proposed that the turnover seen in radiation chimeras may in fact be a result of a surge in BM-derived progenitors in the irradiated host following BM transplantation. Interestingly, Ajami et al. [27 ] found no evidence that radiation-induced alteration in the BBB properties on its own was sufficient to cause increased recruitment of donor monocyte-derived cells into the brain parenchyma.

Conventional BM chimera studies of the microglia cell population in the retina have demonstrated only minimal self-renewal under steady-state conditions [39 ]. This concurs with our own data, which suggest that the turnover of microglia within the retinal microenvironment occurs at a much slower rate than other peripheral tissue macrophages, even their close cousins located in the choroid, and relies mainly on circulating monocyte precursors. Xu et al. [39 ] demonstrated complete turnover of retinal microglia by 6 months post-BM reconstitution in C57Bl/6 mice, and the earliest eGFP+ donor cells appeared in the juxtapapillary and peripheral retinal regions at approximately 8 weeks post-transplantation. This pattern of microglial cell entry into the eye and their subsequent radial and sclerad migration are consistent with that described in the developing human and quail retinae [41 , 53 ]. The present study confirmed the optic nerve head as an important site of entry of donor cells, with some observed as early as 4 weeks postreconstitution. The earlier appearance of donor cells in the present study than that described by Xu et al. [39 ] may reflect a difference in chimera models (eGFP mice vs. Cx3cr1+/gfp mice in the present study) or in BRB integrity between mouse strains, as has been suggested previously by these authors [54 ]. The direct effects of radiation on the BRB, although suspected as a possible reason for variations in precursor cell entry in CNS models [27 ], can be ruled out here, as Xu et al. [39 ] and our own study used identical radiation dosages.

The reverse BM chimeras (WT into Cx3cr1+/gfp) performed in the present study provided us with a powerful means of cross-referencing the data obtained in conventional chimeras (Cx3cr1+/gfp into WT). The reverse chimera data revealed a gradual diminution of the resident Cx3cr1+/gfp cell populations in the iris and choroid, which was inversely proportional to the turnover rates of donor-derived cells repopulating the uveal tract observed in the conventional chimeras. We found no overt changes to the morphology of BM-derived cells, consistent with previous studies that have ruled out possible local effects of X-irradiation on monocyte-derived population dynamics by comparing full body-irradiated and head-shielded animals [45 ]. Similarly, a recent investigation of retinal microglia turnover compared the effects of various irradiation doses and ruled out possible retinal damage that may have contributed to microglial replenishment in irradiated tissues [39 ].

Interest in the role of microglia and choroidal macrophages in human ocular disease has heightened following the publication of genetic linkage studies, suggesting the pathogenesis of AMD may have an inflammatory component, with various haplotypes of complement factor H increasing the risk of AMD development [55 56 57 ]. The potential role of macrophages in AMD was also highlighted by observations in aging Ccr-2–/– or Ccl-2–/– mice, which indicate that defective clearance or scavenging mechanism, by resident choroidal macrophages, may, in part, be responsible for the presence of drusen deposits at the choroidal-retinal interface [13 ]. In a recent study, microglia accumulation in the subretinal space of the mouse eye was found to be associated with not only age of mice and exposure to light but also the presence of the chemokine receptor CX3CR1 [15 ]. A more marked accumulation of lipofuscin-laden macrophages/microglia was observed in Cx3cr1gfp/gfp mice when compared with Cx3cr1+/gfp mice and appeared to correlate with the severity of retinal degeneration in these mice [15 ]. In the present study, we noted the abnormal accumulation of eGFP+ CX3CR1+ host microglia in the photoreceptor layer and subretinal space of reverse chimera animals. Some of these contained lipofuscin-like inclusions, as has been described previously [14 , 15 ], and were less ramified than microglia in the inner retina. These findings support previous suggestions that retinal microglia respond to photoreceptor light-induced injury or degeneration by migration from the inner retinal layers toward the photoreceptor layer and subretinal space, where they appear to phagocytose photoreceptor debris and remain for prolonged periods [58 , 59 ]. In the present study, we did not observe donor eGFP– CD11b+ microglia in the photoreceptor layer or subretinal space in reverse chimeras; interestingly, nor did we observe donor eGFP+ CX3CR1+ cells in the photoreceptor layer or subretinal space at any of the time-points in the conventional chimeras (Cx3cr1+/gfp into WT). Taken together, these and other recently published data [14 , 15 ] suggest that resident host microglia migrating in a sclerad direction from the inner retina are the principal source of the phagocytic and less-ramified microglia that accumulate in the photoreceptor layer and subretinal space during aging or retinal degeneration, rather than newly recruited BM-derived cells from the circulation.

Macrophages and DCs are key players in a number of clinically important ocular inflammatory conditions. Knowledge of the location, phenotype, and steady-state turnover of these cells residing in the uveal tract and retina is crucial to the exploration of their potential role in the pathogenesis of various ocular diseases, where their role is still uncertain. The chimeric mouse model used in the present study allowed for the specific visualization of monocyte-derived cells in the various tissue microenvironments of the eye. The results of this study have allowed us to establish essential baseline data critical to ongoing studies exploring the role of resident retinal microglia and uveal tract macrophages and DCs versus hematogenous cells in models of ocular inflammatory disease.


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ACKNOWLEDGEMENTS
 
This work was funded by a University of Western Australia (UWA) Small Grant. All confocal microscopy was carried out using facilities at the Centre for Microscopy, Characterization and Analysis, UWA, which are supported by university, state, and federal government funding. The authors thank Prof. Wally Langdon (Faculty of Medicine, Dentistry and Health Sciences, UWA) for his assistance and advice in setting up BM chimeras. Rajin Nathan and Karen Waldock performed all mouse irradiations in the Department of Radiation Oncology, QEII Medical Centre, UWA. Valentina Voigt (Lions Eye Institute) provided technical assistance with tail vein injections for creation of BM chimeras. Holly Chinnery assisted with BM preparation and tail vein injections. Byron Scott and Vinay Menon carried out some retinal wholemount staining as part of a 4th-year medical project at UWA. Jelena Kezic is supported by a UWA postgraduate award.

Received March 7, 2008; revised April 24, 2008; accepted April 24, 2008.


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