December 2003
Volume 44, Issue 12
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Immunology and Microbiology  |   December 2003
Local Retention of Soluble Antigen by Potential Antigen-Presenting Cells in the Anterior Segment of the Eye
Author Affiliations
  • Serge Camelo
    From the School of Anatomy and Human Biology, The University of Western Australia, Crawley, Western Australia, Australia.
  • Angel S. P. Voon
    From the School of Anatomy and Human Biology, The University of Western Australia, Crawley, Western Australia, Australia.
  • Stuart Bunt
    From the School of Anatomy and Human Biology, The University of Western Australia, Crawley, Western Australia, Australia.
  • Paul G. McMenamin
    From the School of Anatomy and Human Biology, The University of Western Australia, Crawley, Western Australia, Australia.
Investigative Ophthalmology & Visual Science December 2003, Vol.44, 5212-5219. doi:10.1167/iovs.03-0181
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      Serge Camelo, Angel S. P. Voon, Stuart Bunt, Paul G. McMenamin; Local Retention of Soluble Antigen by Potential Antigen-Presenting Cells in the Anterior Segment of the Eye. Invest. Ophthalmol. Vis. Sci. 2003;44(12):5212-5219. doi: 10.1167/iovs.03-0181.

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

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Abstract

purpose. To determine the capacity of bone marrow–derived cells in the anterior segment of the eye to capture a fluorescence-labeled antigen (Ag) injected into the anterior chamber (AC).

methods. Uveal tract and corneoscleral tissues from Lewis rats were cultured in vitro, with or without FITC-dextran (4 μg/mL final concentration), for 48 hours and examined by confocal microscopy. To investigate antigen uptake in vivo 2 μL (20 μg) of Cascade Blue-labeled dextran (CB-Dx) was injected into the right AC of Lewis rats. The density of Ag-positive cells in the iris at 1, 3, 5, or 12 days after injection was examined by in vivo video fluorescence microscopy. The distribution and phenotype of Ag-positive cells in frozen and paraffin-embedded sections of ocular tissues and in iris wholemounts from animals killed at 24 hours and day 7 were analyzed by fluorescence and confocal microscopy.

results. In organ culture conditions numerous cells in the iris, ciliary body, choroid, and corneal limbus were capable of capturing fluorescence-labeled Ag. In vivo observations and microscopic examination of experimental eyes at days 1 and 7 after AC injection revealed Ag-positive cells within the iris, iridocorneal angle, the suprachoroidal space and around limbal-episcleral vessels. Ag-bearing cells in the iris express combinations of macrophage markers but rarely expressed major histocompatibility complex (MHC) class II molecules. A reduced number of Ag-bearing cells were still present in the iris at day 12.

conclusions. Potential antigen-presenting cells (APCs) in the iris and ciliary body are capable of internalizing intracameral Ag. The characteristics of these cells in the iris are consistent with a predominantly macrophage phenotype. These observations also suggest that the Ag leaving the eye through both the conventional and nonconventional aqueous outflow pathways may be captured by potential APCs in the episcleral tissues.

In the well investigated murine model of anterior chamber associated immune deviation (ACAID), 1 2 injection of antigen (Ag) into the anterior chamber (AC) of the eye induces subsequent suppression of delayed-type hypersensitivity (DTH) responses to the same Ag injected subcutaneously. 1 This phenomenon, involves the generation of regulatory CD4+ and CD8+ T cells in the spleen. 3 4 In this murine model, Wilbanks and Streilein 5 have proposed that the Ag-specific ACAID-inducing signal is carried through the blood by F4/80+ antigen-presenting cells (APCs). Moreover, they have reported that resident iris-ciliary body F4/80+ cells and indeed peritoneal cavity–derived F4/80+ cells loaded in vivo or in vitro with Ag induce ACAID when injected intravenously or intracamerally into a second animal. 6 7 Earlier experiments in this murine model by the same group revealed that a small but significant amount of 125I-BSA injected in the AC of one eye remained within that eye for up to 14 days. 8 These observations led to the hypothesis that Ag injected into the mouse AC is taken up and processed by resident iris-ciliary body F4/80+ ocular APCs which then migrate to the spleen through the blood, where they deliver their ACAID-inducing signal. 5 6 9 It has been unclear, however, whether these cells are of the monocyte-macrophage or dendritic cell (DC) phenotype, because these two distinct lineages unfortunately are not distinguished by the mAb F4/80. 10  
This laboratory and other groups have characterized the dense network of macrophages and/or DCs in the uveal tract in different species in normal and pathologic conditions. 11 12 13 14 15 16 17 In the rat, the iris is endowed with predominantly pleomorphic or dendriform DCs at approximately 500 cells/mm2. These cells express major histocompatibility complex (MHC) class II molecules and/or OX62 (anti-α E2-integrin) and have a half-life of approximately 3 days, 18 equivalent to the turnover of DC populations in mucosal tissues. 19 20 By contrast, macrophages present in the iris are positive for the pan-macrophage marker ED2 (anti-scavenger receptor CD163), are generally MHC class II, and exhibit a much longer turnover period, 18 comparable to other resident macrophage populations. 21 22 23 In addition, DC and macrophage populations have also been described in the ciliary body, choroid, and aqueous outflow pathways of the eye, 13 15 all sites that come into contact with Ag within the AC. 24  
Early functional studies of single-cell suspensions or whole preparations of excised iris and ciliary body revealed that they were unable to function as allogeneic APCs 25 and in fact displayed the ability to suppress mixed lymphocyte reaction. 26 27 This immunosuppressive property can be subverted by the intraocular injection of the inflammatory cytokine interferon-γ in vivo 28 or the granulocyte-macrophage–colony-stimulating factor (GM-CSF)–induced maturation of isolated and purified MHC class II+ iris DCs in vitro. 29 We have proposed 29 that the function of uveal tract macrophages may include the induction of secondary immune responses by restimulating previously activated T cells entering the AC, whereas uveal tract DCs may play a more sentinel function and regulate primary ocular immune responses by migrating to the secondary lymphoid organs and presenting ocular-derived Ag to naïve or regulatory T cells. 
The purpose of the present study was to determine whether macrophages and DCs in the anterior segment can internalize Ag injected into the AC. Our data show that 24 hours after an intracameral injection of a fluorescence-labeled Ag, resident cells in the iris, ciliary body, anterior suprachoroidal space, and episcleral connective tissue were observed by in vivo video microscopy to have captured Ag. This was confirmed in vitro. Confocal microscopy of immunostained sections and wholemounts revealed that the Ag-bearing cells are predominantly of the macrophage phenotype. Although the number of these cells diminished with time, a small number of Ag-bearing cells were still detectable in the anterior segment after 12 days. 
Materials and Methods
Animals
Female Lewis rats, 8 to 11 weeks old, were obtained from the Animal Resources Center (Murdoch University, Western Australia) and were kept under pathogen free conditions in chaff-lined cages and housed in 12 hour day–night cycles (Animal House, University of Western Australia, Western Australia). Food (Stockfeeders RM2 Autoclaved rat and mouse diet; Animal Resources Center) and water were supplied ad libitum. All procedures conform to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Internalization of Fluorescent Ag In Vitro
Rats were killed by a lethal intraperitoneal injection of pentobarbitone sodium (100 mg/kg body weight, Rhone Merieux Australia, Pikenba, Queensland, Australia) diluted in cold phosphate-buffered saline (PBS). Irides, choroid, limbus, and corneal buttons were excised and placed into individual wells in a 48-well plate. Tissues were cultured in 500 μL of RPMI 1640 (Invitrogen-Gibco, Auckland, New Zealand) alone (supplemented with 10% fetal calf serum [FCS], penicillin, and streptomycin) and with or without the addition of lysine-fixable fluorescein- labelled (40 kDa) dextran (FITC-Dx; 4 μg/mL final concentration; [Molecular Probes, Eugene, OR]) and incubated at 37°C for 48 hours. Ocular tissues were then mounted intact on slides with mounting medium (Immunomount; Shandon Lipshaw, Pittsburgh, PA) and the presence of FITC-Dx–positive cells was evaluated by confocal microscopy. 
Intracameral Injections
Animals were anesthetized by inhalation of oxygen and nitrous oxide (4:1) and 1.0% halothane (ICI Pharmaceuticals, Melbourne, Australia). The heads of the anesthetized animals were secured in a stereotactic frame. Topical proparacaine (Alcaine 0.5%; Alcon, Frenchs Forest, New South Wales, Australia) was applied to the right eye. After removal of 6 to 8 μL of aqueous humor with a sterile glass microcannula, a second glass microcannula was inserted obliquely through the same corneal wound and 2 μL (20 μg) of lysine-fixable Cascade Blue-Dextran (CB-Dx, 70 kDa; Molecular Probes), or in control experiments, sterile PBS (0.015 M, pH 7.4; Invitrogen-Gibco) was injected. Lysine-fixable dextrans have covalently bound lysine residues that bind to immediately surrounding tissue on aldehyde fixation, thereby preventing their further movement during tissue preparation. Fluorescence-labeled dextrans have been widely used to study the endocytic capacity of DC. 30 The corneal wound was sealed with a small droplet of superglue. Spillage was kept to absolute minimum and any animal that displayed leakage was not included in the study. 
In Vivo Video Fluorescence Microscopy
Animals were anesthetized by intraperitoneal injection of xylazine (0.033 mg/100 g, Xylazil-20; Ilium, Troy Laboratories, Sydney New South Wales, Australia) and ketamine (0.167 mg/100 g, Ketamil; Ilium). The anesthetized animals were kept warm inside an electrically heated plastic tube and placed on the mechanical stage of the microscope. Topical proparacaine 0.5% was administered to the right eye to eliminate reflex blinking. The head was positioned so that the right eye was resting against a drop of PBS on a glass coverslip and aligned with the microscope objective. This provides a perpendicular view of approximately one third to one half of the iris and limbal region. The method is similar to that described by Becker et al. 31 for examination of leukocyte migration in the mouse iris. The illumination of the anterior chamber of the eye was achieved with an HBO mercury lamp (HBO-100/W2; Osram, Berlin, Germany) attached to an inverted epifluorescence microscope (Eclipse TE300; Nikon, Maxwell Corp., Meadowbank, New South Wales, Australia) with built-in UV filter block for Cascade Blue fluorescence (excitation 340 to 380 nm; emission 420 nm). The iris was examined using long-distance ×10 (Plan fluoro, numerical aperture [NA] 0.3) and ×20 (Plan fluoro, NA 0.45; both from Nikon) objective lenses. Real-time videos were recorded during the in vivo examinations, using a black-and-white analog video camera (nonintensified one-half-inch peltier-cooled CCD, PAL-standard; Dage MTI, Michigan City, IN), either coupled to an image intensifier (Geniisys; Dage MTI), or attached to a C-0.6X TV lens adapter (Nikon). Real-time digital images were recorded with a workstation (Pentium III PC) equipped with an ultrafast, ultrawide hard disc (9Gb SCSI) connected to a video board (MiroVideo DC-50; Pinnacle Systems, Mountain View, CA). This system, connected to a 14-inch video monitor (Sony, North Ryde, Australia), allowed direct viewing of captured images during recording. Videos were captured on computer (Premiere 5.1 software; Adobe Systems, Mountain View, CA) at a rate of 25 frames per second and were stored in M-JPEG format at an average data rate of 2.52 MB/s. The images were edited and pieced together as separate layers to construct a montage of overlapping images of the iris. Video recording took between 1 and 2 minutes. No animal was examined on more than three separate occasions, to avoid UV-induced damage. Examples of the videos are available at http://school.anhb.uwa.edu.au/personalpages/mcmenamin/research_project/project1/research_eye/main1.html. 
Tissue Collection, Processing, and Sectioning
At days 1 and 7 after intracameral injection animals were deeply anesthetized by intraperitoneal injection of pentobarbitone sodium (100 mg/kg body weight) diluted in cold PBS. Animals were then perfused by gravity flow with PBS plus heparin (1000 U/L) followed by perfusion with 4% paraformaldehyde (BDH Laboratories Supplies, Poole, UK). This serves to remove the intravascular pool of cells. After perfusion, both eyes were enucleated and prepared for freezing and cryostat sectioning by infiltration with embedding medium (OCT; ProSciTech, Thuringowa, Queensland, Australia), as previously described 11 or processed for paraffin histology. Cryostat or paraffin-embedded sections (H&E; both 8 μm thick) cut in the conventional anteroposterior plane were examined for the presence of FITC-Dx– or CB-Dx–positive cells by conventional epifluorescence microscopy. 
Immunostaining of Iris Wholemounts
At various time points after intracameral injection animals were killed and ocular tissue wholemounts were prepared as previously described. 32 After perfusion, both eyes were enucleated and postfixed in 4% paraformaldehyde. To characterize CB-Dx–containing cells, we chose red and green fluorochromes for immunofluorescence thus allowing triple-stained cells to be observed (red: Alexa Fluor 546–conjugated anti-mouse IgG, emission 572 nm; Molecular Probes; green: FITC-conjugated streptavidin, emission 520 nm; Amersham-Pharmacia, Uppsala, Sweden). Iridial sheets were dissected from the eye according to previously documented methods. 32 Routine single immunofluorescence was performed on the half irides, as previously described. 13 32 Briefly, PBS-washed irides were incubated in EDTA (0.76 g/100 mL; Sigma-Aldrich, St. Louis, MO) at 37°C for 30 minutes. Wholemounts were then blocked with a 0.2% solution of Triton X-100 in PBS plus 1% BSA (wt/vol) and 10% (vol/vol) normal rat serum for 20 minutes at room temperature. 
For single staining, tissue sections were incubated in a range of mouse anti-rat primary monoclonal antibodies (mAb) for 45 minutes (anti-CD68 [clone ED1], anti-scavenger receptor CD163 [clone ED2], anti-sialoadhesin [clone ED3], anti-MHC class II [I-Ab] [clone MRC OX6], or anti-α E2-integrin [clone MRC OX62]; Serotec Ltd., Oxford, UK). The primary mAb was then revealed by incubation at room temperature for 45 minutes with the secondary biotinylated sheep anti-mouse IgG (Amersham-Pharmacia) followed by streptavidin-FITC. For double staining the first mouse anti-rat primary mAb (mAb 1) was revealed by incubation with a secondary anti-mouse antibody directly coupled to Alexa-Fluor 546 for 45 minutes. Tissues were then incubated in the second biotinylated mouse anti-rat primary mAb (mAb 2) (anti-ED1, anti-ED2, and anti-I-Ab) followed by incubation with streptavidin-FITC. Between incubation, samples were thoroughly washed. Therefore, mAb 1 was seen as red and mAb 2 was seen as green in epifluorescence microscopy. Negative controls (no primary mAbs or one mAb omitted) were performed on some iris wholemounts in each experiment. 
Confocal Microscopy
Iris wholemounts were analyzed by laser scanning confocal microscope (MRC-1000/1024 UV; Bio-Rad, Hercules, CA) equipped with a multiline argon laser emitting 351-nm (UV) and 488-nm (red) laser lines, and a green helium/neon laser (543 nm). Separate images were collected sequentially for the three fluorochromes with either a ×10 or ×40 oil immersion objective (NA 1.3; Fluor; Nikon). Images were merged (Confocal Assistant, ver. 4.02; Bio-Rad) to produce a composite multicolor image. Final image processing was performed on computer (Photoshop; Adobe). 
Quantitative Analysis
Because of the topographically unique angioarchitecture of the iris vessels in each rat, the precise region of the iris previously examined by in vivo video fluorescence microscopy could be repeatedly located and examined at different time points. Iris montages were constructed for experimental animals at various time points (day 1, n = 5; days 3, 5, and 10, n = 2). Iris montages were achieved by superimposing a large number of still frames obtained from the digital videos of the in vivo examinations. To ascertain the emigration of CB-Dx–positive cells from the iris in vivo, the number of CB-Dx–positive cells within the iris montages were determined and compared over time. This was achieved by defining a common area of iris using four to six common points between montages obtained from the same animal at different time points. Enumeration of CB-Dx–positive cells in the defined area of each montage was performed (Image-Pro Plus 4.1; MediaCybernetics, Silver Spring, MD), and expressed as cells per square millimeter. Statistical analysis was not appropriate because of the small number of animals at each time point. 
Results
Uptake of Fluorescence-Labeled Ag by Cells in the Anterior Segment of the Eye In Vitro
Iris-ciliary body, anterior choroid, corneal button, and corneal limbus specimens from Lewis rats were excised and cultured in RPMI 1640 alone or in the presence of FITC-Dx. After 48 hours, tissues were mounted on slides and analyzed for the presence of FITC-Dx by confocal microscopy. FITC-Dx–positive cells were not observed in any ocular tissues cultured in the absence of fluorescence labeled Ag. In cultures exposed to FITC-Dx, Ag-positive cells were uniformly distributed in the iris (Fig. 1A) , ciliary body (Fig. 1B) , and choroid (Fig. 1C) . In the corneal limbus FITC-Dx–positive cells were distributed around the episcleral vessels (Fig. 1D) . Ag-positive cells were not observed in the corneal buttons (data not shown). These results indicate that resident potential APCs from both the uveal tract and episcleral limbal region are capable of Ag uptake in vitro. The addition of lipopolysaccharide (LPS) to the organ culture did not appear to influence Ag uptake (data not shown). 
Uptake of Ag by Cells in the Anterior Segment In Vivo
To determine whether cells in the anterior segment of the eye can internalize soluble Ag in vivo, 22 experimental animals received an intracameral injection of either CB-Dx (n = 20) or FITC-Dx (n = 2) and a further 10 animals received sterile PBS (control). Images of the iris, limbus, and cornea were recorded by in vivo video fluorescence microscopy at various time points. Observation of limbal vessels of FITC-Dx–injected animals by in vivo video microscopic analysis revealed large numbers of Ag-positive cells around the episcleral vessels in the perilimbal conjunctiva (Figs. 2A 2B) . Similar results were obtained in CB-Dx–injected rats. Large numbers of FITC-Dx–bearing cells were also observed in the peripheral cornea (Fig. 2B) , and their density decreased progressively from the periphery toward the central cornea. The presence of fluorescent dextran-bearing cells in the eye was also assessed in frozen sections at days 1 and 7 after injection. This technique revealed a large amount of fluorescence within the extracellular space of the ciliary body and extending posteriorly into the suprachoroidal spaces (Figs. 2C 2D) . The Ag appeared to have accumulated in cells, but some fluorescence was also visible in the interstitial space of the iridocorneal angle, suggesting that not all the Ag had been phagocytosed, even by day 7 (Fig. 2C) . CB-Dx also accumulated close to Schlemm’s canal (Fig. 2C) , and sections also confirmed in vivo observations that they lay in close association with episcleral vessels within the loose connective tissue of the limbal conjunctiva and around limbal-episcleral vessels (Figs. 2D 2E) . These observations are consistent with the suggestion that the Ag leaves the eye through both the conventional and nonconventional aqueous outflow pathways. 
In the posterior segment, Ag-positive cells were absent from the retina but were identified in the most anterior choroidal tissue (Fig. 2E) . Frozen and paraffin-embedded sections also confirmed that fluorescent label was absent from the central cornea and lens (Fig. 2F)
No fluorescence was detected in animals injected with PBS (Fig. 3A) or in the uninjected eye of experimental animals at any time point (data not shown). 
In vivo video fluorescence microscopic images of the right iris of CB-Dx–injected animals were recorded at days 1, 3, 5, and 12. On day 1 post injection (PI), signs of mild inflammation, including low-grade flare and cellular infiltrates in the aqueous and iris hyperemia were occasionally observed in both experimental and control animals. A dense but unevenly distributed network of CB-Dx–positive cells, which displayed dendriform or pleomorphic morphology, was observed at day 1 (Fig. 3B) . Fluorescent cells tended to be more concentrated at the iris base, and occasionally focal clumping of Ag-positive cells was noted (Fig. 3C)
The distribution of Ag-positive cells in the iris of individual animals was examined at days 1, 3, 5, and 12 after injection. Video sequences from a control eye and day 1 after CB-Dx injection are available for viewing at the Internet address provided in the Methods section. 
Examination of iris montages from the same animal at days 1 and 3 (Figs. 3C 3D) show a progressive decrease of CB-Dx–positive cells over time. Figures 3E and 3F are from a different animal that was assessed at days 5 and 12. It can be seen from these montages that, in general, the overall decrease of CB-Dx–positive cells in the iris was more apparent at the pupil margin than at the iris base and that significant numbers of CB-Dx–positive cells were still identifiable in the iris and iridocorneal angle region for up to 12 days PI (Fig. 3F) . Quantitative analysis of individual animals confirmed the qualitative impressions we have described and revealed the variance in numbers of CB-Dx–positive cells between experiments (Fig. 4)
Phenotype of CB-Dx–Positive Cells in the Iris
The purpose of this part of the study was to characterize the cells that had captured the fluorescence-labeled dextran. To this end, iris wholemounts from CB-Dx–injected rats (control and experimental eyes) were subjected to single- and double-immunofluorescence staining with a range of mAbs specific for macrophage and DC phenotypic markers and studied by confocal microscopy. The uninjected eyes were devoid of any CB-Dx–bearing cells or extracellular CB-Dx (Fig. 5A) . In the uninjected eyes, observations confirmed our several published studies that extensive networks of ED1+/ED2+ macrophages coexists alongside MHC class II+/ED2 DCs. 14 However, in addition we noted OX62+ (red) ED2+ (green) colabeled cells (Fig. 5A) , a double-staining combination not previously reported. 
In experimental animals, a large number of CB-Dx–positive cells were observed at day 1 (Fig. 5B) . In these irides some of the CB-Dx–positive cells expressed ED1 (Fig. 5C) and ED3 (Fig. 5D) both characteristics of macrophages. However, these cells were mainly MHC class II (OX6; Fig. 5E ). Our previous studies have shown that iris DCs are generally MHC class II+. The pleomorphic and dendriform ED2+ OX62+ cells described above were CB-Dx positive (Fig. 5F) . In summary, the immunostaining data from iris wholemounts indicate that the cells within the iris that had internalized fluorescently labeled dextran injected into the AC that were observed by in vivo video fluorescence microscopy and frozen and paraffin-embedded sections are probably of the macrophage phenotype. Very few cells in the iris of the DC phenotype were found to have internalized labeled dextran. 
Preliminary phenotypic analysis of the Ag+ episcleral cells suggests that these are also predominantly of the macrophage phenotype (data not shown) however further studies are in progress. 
Discussion
In the generally accepted paradigm of AC-associated immune responses, it has been postulated that Ag placed in the AC of the eye is somehow transported by APCs to the spleen, 1 6 the local draining submandibular-cervical lymph nodes, 33 or, as has recently been postulated, the thymus. 34 However, the precise phenotype of the cells involved, evidence that they do indeed trap Ag in the AC, as well as the route by which they leave the eye and travel to lymphoid organs are still unclear. Candidate APCs include the rich networks of both resident tissue macrophages and DCs in the uveal tract (iris, ciliary body, and choroid) and outflow pathways. 11 12 13 14 15 16 17 These two cell types have distinct functional roles in immune defenses (for review, see Ref. 35 ), and therefore it was considered important to establish whether either or both cell types is primarily responsible for internalizing intraocular Ag. In the present study, we investigated this issue using in vivo video microscopy followed by immunophenotypic studies of iris wholemounts and fluorescence microscopy of ocular tissue sections to demonstrate that cells distributed throughout the iris, ciliary body, anterior choroid, conventional and nonconventional aqueous outflow pathways, and limbal episcleral tissue internalize a fluorescence-labeled Ag in vitro and in vivo. The data suggest that the Ag-bearing cells in the uveal tract are macrophages. Furthermore, in vivo studies reveal that although the number of these Ag-bearing cells decreases over time, some cell-associated Ag persists within the iris for up to 12 days PI. A recent study by Becker et al., 36 which has close parallels to our own, has shown the uptake of fluorescently labeled ovalbumin by cells with the phenotypic characteristics of both macrophages and DCs in the mouse iris after intracameral injection. This study differs from our present investigation in that it examined the iris at two short-term time points (6 and 24 hours) and phenotypic analysis was performed by direct injection of fluorescein-labeled mAbs into the AC. It is possible that our choice to avoid detailed phenotypic analysis of early time points, due to the compounding factor of the mild flare that follows any intracameral injection, may have meant we did not sample early DC-related, Ag-trapping events. 
The choice of dextran as a “mock” Ag was based on its extensive use as a marker of fluid phase endocytotic activity in DC in vitro 30 37 and of their ability to acquire soluble Ag in vivo. 38 39 Uptake of dextran by DC and macrophages, possibly mediated through mannose receptors, 40 is widely considered to model uptake of bacterial polysaccharides. 41 Of interest, dextran has also been extensively used as a tracer to track short-term fluid dynamics in the eye. 42 43 44 Indeed, recent studies 24 44 of fluid movement in the anterior segment have reported homogeneous distribution of fluorescence in the iris, cornea, ciliary body, and sclera within a few hours of injection, and, although these researchers were not specifically investigating cellular uptake from an immunologic perspective, they noted that after 24 hours the fluorescence became punctate in a pattern similar to that observed in the present study. Preliminary in vivo and in vitro pilot experiments in our laboratory (data not shown) comparing dextran with protein Ag revealed an identical pattern of uptake of fluorescently labeled bovine serum albumin and ovalbumin (Ova) by cells in the iris. Similarly, in a recent study the uptake of intratracheal instilled FITC-dextran and FITC-Ova by respiratory tract DCs was also found to be identical with both of these Ags. 39  
Based on our previous immunophenotypic and functional studies of rat iris-ciliary body DC and macrophages we fully anticipated that intracamerally placed Ag would be captured by iris DCs. However the majority of the fluorescent Ag-positive cells in the iris express ED1, ED2, and ED3 but not MHC class II, indicative of cells of the monocyte-macrophage lineage. 45 The detection of ED2+ OX62+ double-positive cells in the present study was unexpected, because ED2 is considered to be a pan-specific marker for mature tissue macrophages and OX62 to be a DC marker, although other cell types have been noted to express the α E2-integrin recognized by this latter mAb. 46 47 We propose that the ED2+/OX62+ double-positive, Ag-bearing cells are macrophages and not DCs. This is consistent with the noticeable paucity of MHC class II+ Ag-bearing cells. 
Our results showing Ag-bearing cells in the conventional outflow pathways corresponds with the aqueous humor drainage pathways 48 and the well-described populations of macrophages 49 and DCs 11 in the iridocorneal angle. Furthermore, the phagocytic capacity of the trabecular meshwork cells is well known. 50 However, the presence of Ag-bearing cells in the episcleral tissues was less expected and suggests that Ag may leave the AC by nonconventional pathways or the uveoscleral route through the connective tissue in the iris root-ciliary body base and anterior suprachoroidal space. From there, it may either diffuse through the sclera 51 or pass through the loose connective tissue spaces around vessels piercing the sclera 52 to gain access to the episcleral tissues, from where it would have access to conjunctival lymphatic vessels. Our data revealed Ag-bearing cells at all points along these outflow routes. A recent study using identical molecular mass dextran injected into the mouse AC confirms the importance of uveoscleral outflow pathways in rodent eyes. 24 This potential pathway of Ag exit from the AC correlates with recent data showing intracameral Ag, 33 53 and passenger cells from murine corneal transplants 54 are indeed in communication with submandibular and cervical lymph nodes. The precise phenotype of the Ag-bearing cells in the peripheral cornea observed during our in vivo video microscopy and how they may relate to the newly described populations of DCs and macrophages in the central cornea 55 56 57 is an area for future investigations. Drainage of Ag from the eye through the conventional and nonconventional aqueous outflow pathways is consistent with the observation of McKenna et al. 58 that small amounts of Ova injected into the AC reach all peripheral lymphoid tissue through the venous circulation but that higher amounts drain to local lymph nodes through the afferent lymphatics. The possibility that leakage from the corneal cannulation site may have acted as a potential source of the Ag later identified in the episcleral tissues in the present study must be considered, although every attempt was made to minimize trauma and leakage from the cannulation site. 
The gradual dimunition in the number of Ag-positive cells in the iris after intracameral Ag injection is consistent with the idea that some Ag may subsequently leave the eye in a cell-associated form. However, it is possible that a large amount of Ag leaves the eye directly without being captured and processed by the anterior segment APCs and indeed experiments currently in progress in our laboratory suggest that this is most likely the case. 
The ocular sequestration of Ag has been postulated to be an important underlying factor influencing the unusual nature of immune responses that follow Ag injection into the AC. Evidence that the eye acts as an Ag depot was first provided by Wilbanks and Streilein, 8 who showed that after an intracameral injection of I125BSA 95% of the Ag leaves the eye through the blood circulation, whereas 5% of the Ag remains in the eye at 48 hours and 0.7% for up to 2 weeks. They also demonstrated that the release of Ag from the eye differed from an intravenous injection, where Ag was undetectable several days later at the venipuncture site. Such early studies were motivated by the suggestion that the unusual form of tolerance induced by Ag placement in the eye was in effect the equivalent of a low-dose intravenous Ag injection. A more recent study has also shown that after a subconjunctival injection of 70-kDa dextran, Ag is still present within the eye 72 hours after injection. 44 Consistent with these two studies we demonstrated that Ag retention within the anterior segment after experimental injections is predominantly associated with uveal tract macrophages and episcleral macrophages. It seems probable, although untested, that retention of Ag for long periods by such cells may make them likely candidates (on activation and expression of MHC class II and costimulatory molecules) to be APCs in secondary immune responses on subsequent Ag challenge. 
There are several possible explanations for the lack of Ag-bearing DCs in the anterior uvea after intracameral Ag injection. First, the quantity of fluorescent Ag trapped by MHC class II–positive DCs may be small and thus difficult to detect. Second, perhaps Ag-laden DCs migrate quickly from the eye to local lymphoid tissues and are thus difficult to detect after 24 hours. Studies are in progress to examine this possibility, but data from Becker et al. 36 support this suggestion. Furthermore, although the expression of MHC class II on uveal tract DCs is similar to other immature nonlymphoid DC populations such as Langerhans’ cells and respiratory tract DCs, the uveal tract DCs may be functionally downregulated by the suppressive microenvironment of the eye that may serve to limit their capability to respond to danger signals which in other tissues usually results in the internalization of Ag and migration to lymphoid tissues. It may be possible that ocular DCs have a limited capacity to perform the sentinel role characteristic of these cells in other tissues, a factor that may have a bearing on the immune-privileged status of the eye. 
 
Figure 1.
 
Confocal microscopic images of the distribution of FITC-Dx in anterior segment tissue wholemounts after 48 hours of organ culture in the presence of this fluorescent antigen. The images of the iris (A), ciliary body (B), anterior choroid (C) and limbus (D) are planar views. Note that extensive networks of cells in all these tissues have internalized the FITC-Dx from the culture medium. Magnification, ×40.
Figure 1.
 
Confocal microscopic images of the distribution of FITC-Dx in anterior segment tissue wholemounts after 48 hours of organ culture in the presence of this fluorescent antigen. The images of the iris (A), ciliary body (B), anterior choroid (C) and limbus (D) are planar views. Note that extensive networks of cells in all these tissues have internalized the FITC-Dx from the culture medium. Magnification, ×40.
Figure 2.
 
Detection of FITC-Dx–positive cells in the limbus and cornea. (A, B) Two frames of a time-lapse video obtained by in vivo video microscopy of the limbus and cornea of an experimental eye 24 hours after injection with FITC-Dx. Arrowheads: limbus; ( Image not available ) peripheral cornea. Note that the density of FITC-Dx–positive cells decrease from the limbus to the peripheral and central (not included in frame) cornea. (CF) Confocal images of frozen sections from CB-Dx–injected eye at day 7. (C) Labeled cells were located in the pars plicata of the ciliary body and iridocorneal angle in close proximity to Schlemm’s canal ( Image not available ) and on the inner and outer aspects of the sclera. (D) Ag-positive cells in the ciliary body stroma and in the episcleral tissue associated with vessels. (E) CB-Dx–bearing cells in the choroid, sclera, and episcleral tissues close to vessels ( Image not available ). Note the absence of Ag-positive cells in the retina. (F) CB-Dx–bearing cells were visible in the iris stroma close to vessels. The lens and central cornea were devoid of CB-Dx–bearing cells. (A, B, C) Magnification ×20; (D, E, F) ×40.
Figure 2.
 
Detection of FITC-Dx–positive cells in the limbus and cornea. (A, B) Two frames of a time-lapse video obtained by in vivo video microscopy of the limbus and cornea of an experimental eye 24 hours after injection with FITC-Dx. Arrowheads: limbus; ( Image not available ) peripheral cornea. Note that the density of FITC-Dx–positive cells decrease from the limbus to the peripheral and central (not included in frame) cornea. (CF) Confocal images of frozen sections from CB-Dx–injected eye at day 7. (C) Labeled cells were located in the pars plicata of the ciliary body and iridocorneal angle in close proximity to Schlemm’s canal ( Image not available ) and on the inner and outer aspects of the sclera. (D) Ag-positive cells in the ciliary body stroma and in the episcleral tissue associated with vessels. (E) CB-Dx–bearing cells in the choroid, sclera, and episcleral tissues close to vessels ( Image not available ). Note the absence of Ag-positive cells in the retina. (F) CB-Dx–bearing cells were visible in the iris stroma close to vessels. The lens and central cornea were devoid of CB-Dx–bearing cells. (A, B, C) Magnification ×20; (D, E, F) ×40.
Figure 3.
 
Uptake of Ag by cells in the anterior segment in vivo. Frames from time-lapse videos recorded by in vivo video fluorescence microscopy of the rat iris 1 day PI with sterile PBS (control) (A) or with CB-Dx (B). The video is available on the Internet at the address provided in the Methods section. Note the dendriform or pleomorphic morphology of the CB-Dx–positive cells and their extravascular distribution. Note the general lack of anterior segment inflammation or haze. (CF) Montages composed of video frames showing the distribution of Ag-positive cells in the iris of individual animals. The first animal was filmed at day 1 (C) and again at day 3 (D). Note the identical vessel topography in montages (C) and (D) and that the CB-Dx+ cells were reduced in number between the two time points, particularly in the more pupillary aspects. The second animal was filmed at days 5 (E) and 12 (F) after injection. Note the similar vascular pattern in (E) and (F). Magnification, ×20.
Figure 3.
 
Uptake of Ag by cells in the anterior segment in vivo. Frames from time-lapse videos recorded by in vivo video fluorescence microscopy of the rat iris 1 day PI with sterile PBS (control) (A) or with CB-Dx (B). The video is available on the Internet at the address provided in the Methods section. Note the dendriform or pleomorphic morphology of the CB-Dx–positive cells and their extravascular distribution. Note the general lack of anterior segment inflammation or haze. (CF) Montages composed of video frames showing the distribution of Ag-positive cells in the iris of individual animals. The first animal was filmed at day 1 (C) and again at day 3 (D). Note the identical vessel topography in montages (C) and (D) and that the CB-Dx+ cells were reduced in number between the two time points, particularly in the more pupillary aspects. The second animal was filmed at days 5 (E) and 12 (F) after injection. Note the similar vascular pattern in (E) and (F). Magnification, ×20.
Figure 4.
 
Kinetics of CB-Dx–positive cells in the rat iris. Mean number of iris CB-Dx–positive cells per square millimeter were counted on montages such as those shown in Figure 3 . The connected points indicate data gathered from the same animal at several time points.
Figure 4.
 
Kinetics of CB-Dx–positive cells in the rat iris. Mean number of iris CB-Dx–positive cells per square millimeter were counted on montages such as those shown in Figure 3 . The connected points indicate data gathered from the same animal at several time points.
Figure 5.
 
Confocal microscopic analysis of iris wholemounts 24 hours PI. (A) Control (PBS injected) eye showing the presence of OX62+ (red) and ED2+ (green) double-positive cells. (B) Iris wholemount from CB-Dx–injected animal (blue) showing the even distribution of internalized Ag. The tissue has been stained with a nonspecific antibody (negative control). Iris wholemounts of CB-Dx–injected eyes stained with ED1 (C), ED3 (D), and OX6 (E). In these images CB-Dx appears blue, the mAb green, and colocalization cyan. Most of the CB-Dx–bearing cells were ED1+ and ED3+ but OX6. (F) Experimental eye showing the presence of CB-Dx–bearing cells that are OX62+ (red) and ED2+ (green). Note that the cell morphology of these triple-labeled cells varied from round to bipolar to dendriform. Bars, 20 μm.
Figure 5.
 
Confocal microscopic analysis of iris wholemounts 24 hours PI. (A) Control (PBS injected) eye showing the presence of OX62+ (red) and ED2+ (green) double-positive cells. (B) Iris wholemount from CB-Dx–injected animal (blue) showing the even distribution of internalized Ag. The tissue has been stained with a nonspecific antibody (negative control). Iris wholemounts of CB-Dx–injected eyes stained with ED1 (C), ED3 (D), and OX6 (E). In these images CB-Dx appears blue, the mAb green, and colocalization cyan. Most of the CB-Dx–bearing cells were ED1+ and ED3+ but OX6. (F) Experimental eye showing the presence of CB-Dx–bearing cells that are OX62+ (red) and ED2+ (green). Note that the cell morphology of these triple-labeled cells varied from round to bipolar to dendriform. Bars, 20 μm.
The authors thank Paul Rigby for helpful advice and technical help with confocal microscopy and Guy Ben-Ary and the staff at the Image Acquisition and Analysis Facility for help in image processing and in vivo video fluorescence microscopy. 
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Figure 1.
 
Confocal microscopic images of the distribution of FITC-Dx in anterior segment tissue wholemounts after 48 hours of organ culture in the presence of this fluorescent antigen. The images of the iris (A), ciliary body (B), anterior choroid (C) and limbus (D) are planar views. Note that extensive networks of cells in all these tissues have internalized the FITC-Dx from the culture medium. Magnification, ×40.
Figure 1.
 
Confocal microscopic images of the distribution of FITC-Dx in anterior segment tissue wholemounts after 48 hours of organ culture in the presence of this fluorescent antigen. The images of the iris (A), ciliary body (B), anterior choroid (C) and limbus (D) are planar views. Note that extensive networks of cells in all these tissues have internalized the FITC-Dx from the culture medium. Magnification, ×40.
Figure 2.
 
Detection of FITC-Dx–positive cells in the limbus and cornea. (A, B) Two frames of a time-lapse video obtained by in vivo video microscopy of the limbus and cornea of an experimental eye 24 hours after injection with FITC-Dx. Arrowheads: limbus; ( Image not available ) peripheral cornea. Note that the density of FITC-Dx–positive cells decrease from the limbus to the peripheral and central (not included in frame) cornea. (CF) Confocal images of frozen sections from CB-Dx–injected eye at day 7. (C) Labeled cells were located in the pars plicata of the ciliary body and iridocorneal angle in close proximity to Schlemm’s canal ( Image not available ) and on the inner and outer aspects of the sclera. (D) Ag-positive cells in the ciliary body stroma and in the episcleral tissue associated with vessels. (E) CB-Dx–bearing cells in the choroid, sclera, and episcleral tissues close to vessels ( Image not available ). Note the absence of Ag-positive cells in the retina. (F) CB-Dx–bearing cells were visible in the iris stroma close to vessels. The lens and central cornea were devoid of CB-Dx–bearing cells. (A, B, C) Magnification ×20; (D, E, F) ×40.
Figure 2.
 
Detection of FITC-Dx–positive cells in the limbus and cornea. (A, B) Two frames of a time-lapse video obtained by in vivo video microscopy of the limbus and cornea of an experimental eye 24 hours after injection with FITC-Dx. Arrowheads: limbus; ( Image not available ) peripheral cornea. Note that the density of FITC-Dx–positive cells decrease from the limbus to the peripheral and central (not included in frame) cornea. (CF) Confocal images of frozen sections from CB-Dx–injected eye at day 7. (C) Labeled cells were located in the pars plicata of the ciliary body and iridocorneal angle in close proximity to Schlemm’s canal ( Image not available ) and on the inner and outer aspects of the sclera. (D) Ag-positive cells in the ciliary body stroma and in the episcleral tissue associated with vessels. (E) CB-Dx–bearing cells in the choroid, sclera, and episcleral tissues close to vessels ( Image not available ). Note the absence of Ag-positive cells in the retina. (F) CB-Dx–bearing cells were visible in the iris stroma close to vessels. The lens and central cornea were devoid of CB-Dx–bearing cells. (A, B, C) Magnification ×20; (D, E, F) ×40.
Figure 3.
 
Uptake of Ag by cells in the anterior segment in vivo. Frames from time-lapse videos recorded by in vivo video fluorescence microscopy of the rat iris 1 day PI with sterile PBS (control) (A) or with CB-Dx (B). The video is available on the Internet at the address provided in the Methods section. Note the dendriform or pleomorphic morphology of the CB-Dx–positive cells and their extravascular distribution. Note the general lack of anterior segment inflammation or haze. (CF) Montages composed of video frames showing the distribution of Ag-positive cells in the iris of individual animals. The first animal was filmed at day 1 (C) and again at day 3 (D). Note the identical vessel topography in montages (C) and (D) and that the CB-Dx+ cells were reduced in number between the two time points, particularly in the more pupillary aspects. The second animal was filmed at days 5 (E) and 12 (F) after injection. Note the similar vascular pattern in (E) and (F). Magnification, ×20.
Figure 3.
 
Uptake of Ag by cells in the anterior segment in vivo. Frames from time-lapse videos recorded by in vivo video fluorescence microscopy of the rat iris 1 day PI with sterile PBS (control) (A) or with CB-Dx (B). The video is available on the Internet at the address provided in the Methods section. Note the dendriform or pleomorphic morphology of the CB-Dx–positive cells and their extravascular distribution. Note the general lack of anterior segment inflammation or haze. (CF) Montages composed of video frames showing the distribution of Ag-positive cells in the iris of individual animals. The first animal was filmed at day 1 (C) and again at day 3 (D). Note the identical vessel topography in montages (C) and (D) and that the CB-Dx+ cells were reduced in number between the two time points, particularly in the more pupillary aspects. The second animal was filmed at days 5 (E) and 12 (F) after injection. Note the similar vascular pattern in (E) and (F). Magnification, ×20.
Figure 4.
 
Kinetics of CB-Dx–positive cells in the rat iris. Mean number of iris CB-Dx–positive cells per square millimeter were counted on montages such as those shown in Figure 3 . The connected points indicate data gathered from the same animal at several time points.
Figure 4.
 
Kinetics of CB-Dx–positive cells in the rat iris. Mean number of iris CB-Dx–positive cells per square millimeter were counted on montages such as those shown in Figure 3 . The connected points indicate data gathered from the same animal at several time points.
Figure 5.
 
Confocal microscopic analysis of iris wholemounts 24 hours PI. (A) Control (PBS injected) eye showing the presence of OX62+ (red) and ED2+ (green) double-positive cells. (B) Iris wholemount from CB-Dx–injected animal (blue) showing the even distribution of internalized Ag. The tissue has been stained with a nonspecific antibody (negative control). Iris wholemounts of CB-Dx–injected eyes stained with ED1 (C), ED3 (D), and OX6 (E). In these images CB-Dx appears blue, the mAb green, and colocalization cyan. Most of the CB-Dx–bearing cells were ED1+ and ED3+ but OX6. (F) Experimental eye showing the presence of CB-Dx–bearing cells that are OX62+ (red) and ED2+ (green). Note that the cell morphology of these triple-labeled cells varied from round to bipolar to dendriform. Bars, 20 μm.
Figure 5.
 
Confocal microscopic analysis of iris wholemounts 24 hours PI. (A) Control (PBS injected) eye showing the presence of OX62+ (red) and ED2+ (green) double-positive cells. (B) Iris wholemount from CB-Dx–injected animal (blue) showing the even distribution of internalized Ag. The tissue has been stained with a nonspecific antibody (negative control). Iris wholemounts of CB-Dx–injected eyes stained with ED1 (C), ED3 (D), and OX6 (E). In these images CB-Dx appears blue, the mAb green, and colocalization cyan. Most of the CB-Dx–bearing cells were ED1+ and ED3+ but OX6. (F) Experimental eye showing the presence of CB-Dx–bearing cells that are OX62+ (red) and ED2+ (green). Note that the cell morphology of these triple-labeled cells varied from round to bipolar to dendriform. Bars, 20 μm.
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