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The Journal of Immunology, 2004, 172: 5388-5395.
Copyright © 2004 by The American Association of Immunologists

The Distribution of Antigen in Lymphoid Tissues following Its Injection into the Anterior Chamber of the Rat Eye1

Serge Camelo, Adam Shanley, Angel S. P. Voon and Paul G. McMenamin2

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Injection of Ag into the anterior chamber (AC) of the eye induces deviant immune responses. It has been proposed that Ag internalized by ocular APCs is presented in a tolerogenic fashion in the spleen. However, the nature and distribution of the Ag-bearing cells in the lymphoid organs remain unclear. Fluorescent-labeled Ag (dextran, BSA) injected into the AC of Lewis rats was detected in the subcapsular sinus of the right submandibular lymph nodes (LNs) and cervical LNs, the marginal zone of the spleen, and the medulla of the mesenteric LNs. In the spleen, Ag-bearing cells were CD1+, CD11b+, ED1+, ED2low, ED3+, CD86low, OX6+, CD11c–, ED5– and in the LNs were CD4+, CD8+, CD80+, and OX41+ suggesting these were lymphoid organ resident macrophages. These Ag-bearing macrophages were located adjacent to CD4+ cells, CD8+ cells, and NK cells in the LNs and spleen and to marginal zone B cells in the spleen. No interaction with {gamma}{delta} T cells was observed. The data demonstrates that Ag derived from the AC of the eye is mainly internalized by resident macrophages in the LNs and spleen which are ideally placed to interact with cells involved in the induction of deviant ocular immune responses. The extensive distribution of Ag in LNs draining the upper airway and gastrointestinal tracts, together with the phenotype of Ag-bearing cells in the lymphoid organs, suggests that Ag leaves the eye predominantly in a soluble form and implies other mechanisms of tolerance may contribute to ocular-specific immune responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Murine models attempting to explain specific ocular immune responses are based on the postulate that Ag injected into the anterior chamber (AC)3 is presented in a tolerogenic fashion in the spleen by iris/ciliary body-derived APCs (1, 2) whose specific identity remains in debate. It has been shown that regulatory CD4+ and CD8+ T cells are produced in the spleen in responses to ocular-derived Ag (3, 4). This has partly underpinned the concept of the "camerosplenic" axis (5). Similarly, a number of pieces of evidence suggest that submandibular lymph nodes (SMLN) and cervical LNs (CLN) are also important in ocular immune tolerance. For example, activation and anergy of peptide-specific T cells in the SMLNs occur after peptide immunization in the eye (6, 7, 8). Furthermore, melanoma cells placed in the AC are found in the ipsilateral, and to a lesser extent in the contralateral, CLN 4 days following placement into the AC (9). Similarly, in a transgenic murine model of corneal transplantation GFP+CD45+CD11c+I-Ab+ DCs are found in the recipient CLN after transplantation of GFP+ donor corneas (10). Therefore, the accumulated data suggest that Ag injected into the AC reaches both the spleen and the draining LN, in association with ocular APCs, where a state of Ag-specific systemic tolerance is induced. However, the presence of Ag-bearing ocular-derived APCs in the blood or in the different lymphoid organs has never been directly demonstrated.

In a recent study (11), we observed that fluorescent Ag injected in the AC is predominantly internalized by macrophages in the anterior segment tissues and that the number of these Ag+ macrophages decreases with time, suggesting that they may leave the eye. The aim of the present study was to determine the destiny of Ag placed in AC and phenotypically characterize Ag+ cells in a variety of lymphoid organs.

In this report, we demonstrate that fluorescent-labeled Ag placed in the AC of the eye is internalized in the marginal zone of the spleen and subcapsular sinus of the ipsilateral SMLN, CLN, and facial LN (FLN). Surprisingly however, we also detected Ag-bearing cells in the subcapsular sinus and medulla of the mesenteric LN (MLN). Phenotypic analysis of Ag-bearing cells in these organs reveals that they are macrophages rather than DCs. We observed that Ag-bearing cells are located in close proximity to a large array of cells previously implicated in the induction of the deviant ocular immune responses. This report constitutes the first direct observation of the nature and distribution of Ag-bearing cells in the lymphoid organs after an intracameral injection. Our data suggest the systemic immune deviant responses elicited by AC injection may result from a unique combination of drainage of soluble Ag to the local LNs, the spleen, and the MLN. This wide distribution suggests that Ag experimentally placed in the AC may have access to the nasal and oral mucosa. We believe these findings have major implications for investigators using AC injections and for those studying ocular immune responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Female Lewis rats, 8- to 11-wk-old, were obtained from the Animal Resources Center (Murdoch, Western Australia) and were kept under pathogen-free conditions in chaff-lined cages and housed in 12-h day/night cycles. Food (Stockfeeders RM2 autoclaved rat and mouse diet; Animal Resources Center, Murdoch Western Australia) and water were supplied ad libitum. All procedures conform to the Association for Research in Vision and Ophthalmology statement for the use of animals in research and local animal welfare regulations.

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 alcaine 0.5% (ALCON; New South Wales, Australia) was applied to the right eye. Following removal of 6–8 µl of aqueous humor with a fine glass microcannula (diameter of ~70 µm), a second glass micropipette was inserted obliquely through the same corneal wound and 2 µl (10–20 µg) of Alexa Fluor 488-conjugated BSA (A488-BSA; 67 kDa), lysine-fixable Cascade Blue-dextran (CB-Dx; 70 kDa), or in control experiments, sterile PBS (PBS, 0.015M, pH 7.4; Life Technologies) were injected. Lysine-fixable dextrans have covalently bound lysine residues, which bind to immediately surrounding tissue upon aldehyde fixation thereby preventing their further movement during tissue preparation. They have been widely used to study the endocytic capacity of DC (12) and Ag transport in lymph (13). Measures to avoid leakage of Ag from the eye included introducing a small air bubble into the AC and sealing the corneal wound with a small droplet of superglue. Spillage was kept to absolute minimum and any animal that displayed leakage at the time of the procedure was excluded from the study.

Reagents and Abs

Chemicals reagents and Abs were purchased from the following companies. BSA and paraformaldehyde (PFA) (BDH Laboratories Supplies, Poole, U.K.). Sodium pentobarbitone (Rhone Merieux, Queensland, Australia). OCT compound (ProSciTech, Queensland, Australia). Immunomount mounting medium (Lipshaw, Pittsburgh, PA). Primary mAbs used in the study are listed in Table I. Biotinylated sheep anti-mouse Ab and FITC-conjugated-streptavidin (emission 525 nm) were obtained from Amersham Pharmacia (Uppsala, Sweden). Biotinylated rabbit anti-goat IgG was obtained from Vector Laboratories (Burlingame, CA). Alexa Fluor 546-conjugated goat anti-mouse IgG (emission 572 nm); A488-BSA, 67 kDa, emission 520 nm; and CB-Dx, 70 kDa, emission 420 nm were purchased from Molecular Probes (Eugene, OR).


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  		Table I. mAbs used in the studya

 
Tissue collection and processing of frozen sections

On days 1, 3, 5, 7, and 12 after intracameral injection, animals were deeply anesthetized by i.p. injection of sodium pentobarbitone (100 mg/kg body weight) diluted in cold PBS. Animals that received a CB-Dx injection were then perfused via gravity flow with PBS plus heparin (1000 U/L) followed by perfusion with 4% PFA. Following perfusion, both eyes were enucleated and postfixed in 4% PFA. The spleen, thymus, MLN, right and left SMLN, superficial and deep CLN, FLN (located anteromedial to the parotid gland close to the origin of external jugular vein), brachial LN, and inguinal LN were collected, embedded in OCT, and stored at –20°C. Frozen sections (8-µm thick) were cut using a cryostat (Leica CM 3050; Deerfield, IL).

Immunohistochemical staining

The methods used to perform single immunohistochemistry have been described in detail elsewhere (14). Sections were incubated in a range of purified anti-rat primary mAbs followed by secondary goat anti-mouse IgG directly coupled to Alexa Fluor 546 or alternatively the secondary biotinylated sheep anti-mouse IgG and streptavidin-FITC. For double staining, the first mouse anti-rat primary mAb (mAb 1) was revealed by incubation with a secondary anti-mouse Ab directly coupled to Alexa Fluor 546. Tissues were then incubated in the second biotinylated mouse anti-rat primary mAb (mAb 2) followed by incubation with streptavidin-FITC. The first mAb was seen as red and the second mAb was seen as green in conventional epifluorescent microscopy. In the case of CD1 (goat polyclonal anti-murine CD1 Ab), a biotinylated rabbit anti-goat served as a secondary. Negative controls (no primary mAbs or incubation with purified anti-dinitrophenyl control mAb) were performed on some tissue sections in each experiment. Lymphoid tissues from uninjected animals acted as positive control and distribution of staining conformed to previous descriptions.

Confocal microscopy and image analysis

Ocular tissue whole mounts and tissue sections were examined for the presence of immunofluorescently labeled and fluorescent Ag-positive cells by confocal microscopy using a Bio-Rad MRC-1000/1024 UV laser scanning confocal microscope (Hercules, CA) equipped with argon lasers giving 351 (UV) and 488 nm (blue) laser lines, and a helium/neon laser giving 543 nm (green). Separate images of the pattern of staining with the three fluorochromes as observed with either a Nikon Fluor x20 NA 0.75 objective or a Nikon Fluor x40 NA 1.3 oil immersion objective (Nikon, Melville, NY) were collected sequentially. The images were merged using Confocal Assistant (version 4.02; Bio-Rad Microscience, Hemel-Hemestead, U.K.) to produce a composite multicolor image. Final image processing was performed using Adobe Photoshop (Mountain View, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distribution of fluorescent Ag in rats 24 h after an intracameral injection

Following a single injection of 2 µl of CB-Dx, the fluorescent Ag was observed consistently in the anterior segment of the right eye, the SMLN (right and left), superficial CLN (right and left), FLN (right and left), deep CLN (right), and the spleen. Surprisingly, Ag+ cells were also located in the MLN. Although the general distribution of Ag was similar in the right and left LNs of the neck, the overall amount of Ag was consistently greater in the LNs ipsilateral to the injection. The thymus, liver, and brachial LN were devoid of any CB-Dx-bearing cells or extracellular CB-Dx (Table II). No fluorescence could be detected in the eyes or lymphoid organs in animals injected with PBS or in uninjected animals.


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  		Table II. Distribution of CB-Dx 24 h following injection into the right AC

 
In the LNs of the neck, cells containing ocular-derived fluorescent Ag were located in the subcapsular sinus (Fig. 1). In the MLN, Ag+ cells were mainly situated in the medulla (Fig. 2) whereas in the spleen, Ag+ cells were located in the marginal zone (Fig. 3). Distribution and phenotype of Ag+ cells in the right SMLN, CLN, and spleen were equivalent at 1, 3, 5, 7, or 12 days postinjection (Table III). In the MLN, CB-Dx-bearing cells could be observed up to 7 days postinjection.



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  		FIGURE 1. Confocal microscopic images of the rat right SMLN 24 h postintracameral injection with either A488-BSA (A) or CB-Dx (B–F). A, Frozen section of right SMLN showing the presence of Ag+ cells (green) immunopositive for the mAb ED3 (red) in the subcapsular sinus. Colocalized fluorochromes result in a yellow appearance. B, Similar images were obtained on a frozen section of right SMLN of a CB-Dx (blue) injected animal stained with ED3 (red), showing the distribution of internalized Ag in the subcapsular sinus and that many of the Ag+ cells are ED3+ (purple). C, Right SMLN of CB-Dx injected animal stained with CD86 (C), OX6 (D), CD4 (E), and CD8 (F). In these images, Ag appears blue, the mAb red and colocalization is indicated by a purple color. The majority of Ag-bearing cells do not express OX6 but express CD86, CD4, and CD8. Note the presence of small rounded CD4+ and CD8+ cells (red) in contact with Ag+ cells. Bars represent 20 µm in each image.

 


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  		FIGURE 2. Confocal microscopic images of MLN from animals 24 h after intracameral injection with either A488-BSA (A) or CB-Dx (B–F). A, Frozen section of MLN showing the presence of A488-BSA (green) and ED3+ (red) cells in the medulla. Ag-bearing ED3+ cells appear yellow. Note also the presence of free Ag (green). B–E, Frozen section of MLN (from animal that has received an intracameral injection of CB-Dx) double stained with OX6 and either CD11b (B), ED1 (C), ED3 (D), and CD4 (E). Note that OX6+ cells appear green, all the others mAbs appear in red. The Ag derived from the eye is blue. The majority of Ag+ cells are CD11b+ (B). The ED1+ Ag-bearing cells (purple) do not express OX6 (green). Note also the presence of Ag– ED1+OX6+ cells (yellow) and single OX6+ cells in close proximity (C). The majority of ED3+Ag+ cells also coexpress OX6 and thus appear yellow and white (D). A few Ag+ cells also express CD4 (small areas of purple)(E). Note the close proximity of OX6+ to these large CD4+ Ag-bearing cells. In the MLN, Ag+ cells also express CD1 (purple/blue cells) and are associated with CD8+ cells (red) (F). Bars represent 20 µm in each image.

 


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  		FIGURE 3. Confocal microscopic images of the rat spleen 24 h after intracameral injection of Ag (CB-Dx). Frozen sections of spleen were single or double immunostained with mAbs specific for ED1, ED3, RLN-9D3 (B cell marker), CD1, CD4, CD8, and NKR-P1A. Ag appears in blue in all figures. ED1+ and CD1+ cells appear in green and all others mAbs appear red. Ag-bearing cells are located in the marginal zone and express ED1 (cyan cells in A), ED3 (purple cells in B). Ag+ CD1+ cells are closely associated with B cells (red) in the marginal zone (C). In D–F, CD4+ cells, CD8+ cells, and NK cells, respectively, appear in red. In these panels, CD1 appears in green and Ag in blue. Bars represent 20 µm.

 

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  		Table III. Kinetics of CB-Dx dissemination after injection into the right AC

 
We have previously reported that Ag injected into the AC accumulates in iris macrophages rather than in DC (11). In the present investigation, the intracameral Ag appeared to have concentrated in cells in the LNs of the neck and the spleen with Ag also surprisingly present in the MLN. Establishing the phenotype of Ag+ cells in the spleen and LNs after intracameral Ag injection was the next objective of the study.

Phenotypic characterization of Ag+ cells and of cells adjacent to Ag+ cells in the LNs of the neck, the MLN, and the spleen following an intracameral injection

To characterize the nature of the Ag+ cells, sections of right SMLN, right CLN, MLN, and spleen removed from rats 24 h or 7 days postintracameral injection were subjected to single immunofluorescent staining with a range of mAbs specific for phenotypic markers of T cells, B cells, macrophages, DC, Ag presentation, and costimulation. In addition, tissue sections were subjected to double immunofluorescent staining to determine whether Ag+ cells express CD1 and were situated adjacent to T cells, B cells, NK cells, and/or {gamma}{delta} T cells that are crucial in the induction of a specific ocular immune response (15, 16, 17, 18, 19, 20, 21, 22).

In the LNs of the neck (SMLN, FLN, and CLN), Ag+ cells consistently expressed the macrophage markers ED1 (data not shown) and ED3 (Fig. 1, A and B) with less frequent expression of ED2 and CD11b (data not shown). Many of these cells also consistently expressed CD86 (Fig. 1C). Some Ag+ cells were also positive for MHC class II (Fig. 1D), CD4 (Fig. 1E), and CD8 (Fig. 1F). Low numbers of Ag+ cells expressing the DC markers OX62 and OX41 were also observed (data not shown). By contrast, Ag+ cells did not express CD80, CD11c, and ED5 (data not shown). A fraction of Ag+, ED3+ macrophages expressed CD1 (data not shown) and a large proportion of these Ag-bearing cells were in close association with CD4+ T cells (Fig. 1E), CD8+ T cells (Fig. 1F), and NK cells (data not shown) but were absent from B cell follicles (data not shown).

The phenotype of Ag+ cells in the medulla of the MLN was equivalent to Ag+ cells in the LNs of the neck, namely most were ED3+ (Fig. 2A), CD11b+ (Fig. 2B), and ED1+ (Fig. 2C). By contrast, only few were ED2+ (data not shown). A few of the ED1+Ag+ cells and more of the ED3+ Ag-bearing cells coexpressed MHC class II (OX6+) (Fig. 2, C and D). Most of the Ag+ cells in the MLN were CD86+, CD11c– and only a few cells expressed CD80 (data not depicted) and OX6 (white cells in Fig. 2, B and D). The phenotypic profile described suggests that the majority of Ag reaching the LNs is taken up by cells of a macrophage phenotype. Although fewer Ag+ cells in the MLN were CD4+ (Fig. 2E), they failed to express CD8 (not shown) in contrast to the observations made in the LNs of the neck. Finally, in the MLN we observed cell contact between CD1+Ag+ macrophages CD4+ T cells (Fig. 2E), and CD8+ T cells (Fig. 2F) but not with NK cells or {gamma}{delta} T cells (data not shown).

In the marginal zone of the spleen, Ag+ cells were consistently positive for all the macrophage markers studied namely ED1 (Fig. 3A), ED3 (Fig. 3B), ED2, CD11b, and CD86 (data not shown) but did not express any DC markers (Table IV). B cells in the spleen were devoid of any Ag, but Ag-bearing macrophages were in close association with marginal zone B (MZB) cells (Fig. 3C and Table IV). Moreover Ag+ cells were closely approximated to CD4+ T cells (Fig. 3D), CD8+ T cells (Fig. 3E), and NK cells (Fig. 3F) but not with {gamma}{delta} T cells (data not shown).


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  		Table IV. Characterization of Ag+ cells in the lymphoid organsa

 
A summary of the pattern of marker expression by Ag+ cells in the different lymphoid organs is presented in Table IV and the phenotype of cells interacting with Ag+ macrophages is presented in Table V.


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  		Table V. Phenotypic characterization of cells adjacent to Ag+ cells

 
In conclusion, immunostaining data indicate that within 24 h of intracameral injection, fluorescent-labeled Ag (CB-Dx and A488-BSA) was present in the subcapsular sinus macrophages of the LNs of the neck, in subcapsular and medullary macrophages of the MLN and in the marginal zone macrophages of the spleen. We did not detect Ag in the typical T cell zones of the lymphoid organs and Ag-bearing cells only rarely expressed the DC marker OX62. Double immunostaining revealed that some ED3+Ag+ macrophages coexpressed MHC class II in all the lymphoid organs tested. Immunophenotypic analysis 7 days postinjection revealed that Ag+ cells in SMLN, CLN, and spleen were identical to those observed in day 1 experiments. However, in the spleen, after 7 days, Ag-bearing macrophages appeared to have migrated toward the red pulp (Fig. 3B). These observations strongly suggest that the great majority of the soluble fluorescent Ag injected into the AC leaves the eye in a soluble form and reaches the local draining LNs, the spleen, and MLN where it is taken up by resident ED3+ macrophages. Moreover, these macrophages express CD1 and are ideally located to present Ag to a large array of cells reported to be important in the induction of the deviant ocular immune responses.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This report constitutes the first direct description of the fate of soluble Ag injected into the AC of the eye and its subsequent distribution to lymphoid organs. In this confocal microscopic study, we demonstrate the presence of Ag+ cells in the subcapsular sinus of the ipsilateral and contralateral SMLN, FLN, and superficial CLN; the deep CLN on the ipsilateral side and the marginal zone of the spleen as early as 24 h postinjection and for a period of up to 12 days. Cells with low amounts of Ag were more rarely observed in the contralateral LNs of the neck. This general pattern of drainage partly accords with previous reports on Ag drainage from the AC of the eye. Surprisingly, however, Ag-bearing cells were also present in the subcapsular sinus and medulla of the MLN, a pattern not previously reported.

The phenotype of Ag-bearing cells in the different lymphoid organs, particularly their consistent expression of ED1 and ED3 (summarized in Table IV), identifies them as splenic marginal zone macrophages, subcapsular sinus macrophages in the LNs, and medullary macrophages in the MLN (23, 24, 25, 26). Our phenotypic analysis confirmed that some of these cells also express CD80, CD86, CD4, and OX41 (27, 28, 29). The nature and function of these macrophages may have important bearings on deviant ocular immune responses. Indeed, in contrast to DCs, splenic marginal zone macrophages are unable to prime specific T cell responses (30, 31), however, they may produce cytokines and/or chemokines such as IL-4, TGF-{beta}, or macrophage inflammatory protein-2 implicated in the generation of regulatory T cells (32, 33).

We also report that macrophages bearing Ag in the lymphoid organs are in close proximity to T cells, CD4+ cells, CD8+ cells, MZB cells, and cells expressing the NK cell marker NKR-P1A. Although the exact nature of the cells interacting with Ag+ cells cannot be determined with precision from our morphological and immunohistochemical data, it is likely that they are CD4+ T cells, CD8+ T cells, NK cells, and/or CD4+ NKT cells. These observations are in concordance with previous studies implicating CD4+ T (4) or NKT cells (16, 34), CD8 T cells (35), and MZB cells (17, 18, 19) in the suppression of delayed type hypersensitivity responses after injection of Ag into the AC of the eye. By contrast, although it has been proposed that {gamma}{delta} T cells play a role in ocular deviant immune responses (20, 21, 22), close interaction of Ag+ cells with {gamma}{delta} T cells was not observed.

Deviation of the immune response after injection of Ag into the AC of the eye also depends on the expression of CD1, an MHC class I homologue implicated in the presentation of nonpeptide Ags to CD8 T cells, NKT, and {gamma}{delta} T cells in several models of tolerance induction (36, 37, 38). In mice, rats, and humans, CD1 expression has been reported in the liver, spleen, thymus, lung, heart, kidney, small intestine, and skin (36, 37, 38, 39). In the mouse, CD1 is expressed on MHC class II+ macrophages, dendritic cells (DCs), and MZB cells in the spleen (36, 40). However, to our knowledge the precise nature of the cells expressing CD1 in the lymphoid organs of the rat has been less well studied. In this study, we report that Ag+ subcapsular sinus macrophages in LNs and marginal zone macrophages of the spleen express CD1. By contrast we did not detect CD1 expression on the MZB cells suggesting that CD1 expression might differ between mouse and rat. Furthermore, internalization of Ag by MZB cells was not observed suggesting that they do not transport or present Ag derived from the AC. However, this observation in itself does not preclude the possibility that they play a role in ocular immune responses as has previously been suggested (17, 18, 19).

On the basis of 1) the postulated mechanisms of anterior chamber associated deviation (1, 41, 42), 2) evidence that macrophages (11) and DC (43) in the uveal tract and aqueous humor outflow pathways of the eye (44, 45, 46, 47, 48, 49, 50, 51) can trap Ag in the AC, and 3) the known function and migratory capacity of DC (52), we postulated at the commencement of this study that intracamerally placed Ag would be trapped by uveal tract DC and transported from the eye to the secondary lymphoid organs. However, the presence of Ag in resident macrophages in the marginal zone of the spleen and subcapsular sinus macrophages in the SMLN and CLN strongly suggests that the majority of Ag leaves the eye and travels in a soluble form rather than associating with emigrating ocular APCs. This is in accordance with a previous report showing that 95% of Ag injected into the AC leaves the eye through the blood directly without being captured and processed by anterior segment APCs (53). However, we cannot completely exclude the possibility that a small number of Ag-loaded DCs may have migrated from the eye because small numbers of Ag+OX62+ cells were detected in the LNs. Additional experiments to determine whether Ag present in subcapsular sinus macrophages and marginal zone macrophages actually left the eye in a soluble form are currently in progress.

Advantages of using dextran or polysaccharide molecules in studies of Ag uptake or movement include the ability to use lysine fixable forms which are stabilized following fixation in paraformaldehyde thereby preventing their further movement during tissue preparation. Moreover, after uptake, dextran becomes localized primarily in compartments lacking endosomal and pan lysosomal markers (54) and thus avoids degradation. However, it could be argued that the unexpected localization and nature of CB-Dx+ cells we observe in this study is more reflective of the nature of the Ag and not of the site of injection. Indeed, uptake of dextran is possibly mediated in the mouse via mannose receptors (55) and via SIGN-R1 expressed on marginal zone macrophages in the spleen and subcapsular and medullary macrophages in LNs (54, 56) Moreover, dextran has recently been used successfully to investigate Ag and lymph flow through LNs where it was reported to remain in the subcapsular sinus (13). As several previous experimental studies of the deviant ocular immune responses had relied on a protein Ag, we decided to confirm that the phenotype and the distribution of Ag+ cells in the eye and in lymphoid organs following intracameral injection of fluorescent labeled BSA was equivalent to those of CB-Dx+ cells. This was indeed the case (Figs. 1A and 2A and data not shown). Similarly, in a recent study, the uptake of intratracheal instilled FITC-dextran and FITC-OVA by respiratory tract DC was also found to be identical with both of these Ags (57).

The demonstration that intracameral-injected Ag enters the spleen confirms previous studies showing that the presence of this organ is crucial to the induction of anterior chamber-associated immune responses (1, 41, 42). Faunce et al. (33) recently demonstrated clusters comprising F4/80+ APCs, T cells, and NKT cells in the marginal zone of the spleen of C57BL/6 mice injected with OVA into the AC, suggesting the presence of Ag. Unfortunately, the Ag was not labeled in this study. The present investigation goes further by providing direct demonstration of the presence of Ag within cells in the marginal zone of the spleen after AC injection. The marginal zone is important in the production of Abs against T-independent, blood-borne Ags (23, 58, 59) and pathogens including viruses (60), bacteria (61), and parasites (62). Whether the location of Ag in the marginal zone is sufficient to explain the specificity of the ocular deviant immune responses to viral, bacterial, or parasitic infections within the intraocular compartments of the eye is unclear.

The immunologic role of the LNs of the head and neck in the induction of ocular immune tolerance is controversial. A tolerogenic role for ipsilateral SMLNs (6, 7, 8, 63) has been suggested after peptide immunization in the eye caused activation and anergy of peptide-specific T cells in these nodes. Although in our study Ag was detected in some of the contralateral LNs in the neck, the quantity of Ag was not equivalent to the ipsilateral side. Activation of T cells in contralateral LNs has been shown to occur when the Ag is placed in the subconjunctival space (6). Therefore, it is possible that following intracameral injection, the drainage pattern of Ag from the AC into subconjunctival tissue via the uveoscleral route (vide infra) may partially mimic this later situation. A further issue is the confusion as to the precise identity of the nodes being studied due to anatomical variations between rats and mice and the choice of terminology in previous studies. For example, some authors fail to distinguish between SMLN, FLN, superficial CLN, or deep CLN. However, the correct identification of these various nodes would appear critical as some may drain oral and nasal mucosa from which we suspect Ag may originate following experimental injection into the AC of the eye. For the purposes of the present investigation, we relied partly on the terminology of Hebel and Stromberg (64).

Removal of SMLN but not superficial CLN or deep CLN (sometimes referred as "internal jugular LNs" in mice) prevented corneal graft rejection (65). The production of IFN-{gamma} by T cells in these same SMLNs was also induced following allogeneic corneal transplantation implicating SMLN in corneal rejection (66). However, in a separate study, corneal graft survival was greatly enhanced by the excision of superficial CLN and FLN but not SMLN (67, 68). Increased quantities of myelin Ags in the CLN of monkeys and marmosets with experimental autoimmune encephalomyelitis and humans suffering from MS (69) further supports the notion that CLN do indeed play a role in immune activation. In this context, implication of the superficial CLN which drain the nasal cavity, in the induction of nasal tolerance (70), further adds to the complexity of defining which LNs of the neck are involved in tolerance or immunity.

A rather unexpected finding in the present study was the detection of Ag-bearing macrophages in the subcapsular sinus and medulla of the MLN. This is the first report demonstrating such a link between the eye and lymphoid tissues associated with the gastrointestinal tract. Exposure of Ag to the mucosal surface of the gut, also a recognized route of mucosal tolerance induction, especially to soluble Ags (71, 72, 73, 74) suggests that immune deviation following AC injection of Ag may be partly explained by a systemic, rather than an ocular, mechanism of tolerance induction. However, further experimentations are required to verify this hypothesis.

The exact route by which Ag placed into the AC of the eye gains access to the spleen, LNs of the neck, and the MLN remains unclear. However, it is probable that soluble Ag travels from the eye to these lymphoid organs via different routes. The similarity between the distribution of Ag in the spleen reported here and that following an i.v. injection of dextran (54, 56) suggests that the majority of the Ag reaching the spleen travels via the vascular route. The presence of Ag+ cells in the subcapsular sinus of the draining LNs (SMLN, FLN, and CLN), in contrast, indicate that at least a portion of the Ag placed in the AC somehow gains access to lymph vessels in the head and neck. Although it is generally accepted that the internal compartments and uveal tract of the eye are devoid of lymphatic drainage, recent evidence suggests that a connection between the eye and local LNs exists (8, 10, 65, 66). Melanoma cells placed in the AC (9) gain access to LNs in the neck suggesting that a similar connection exists for nonsoluble cell-associated Ags. In addition, recent studies using a transgenic murine model of corneal transplantation have revealed that donor GFP+CD45+CD11c+I-Ab+ DCs were identified in the recipient CLN after transplantation of GFP+ donor corneas (10) suggesting that DCs from the cornea at least are capable of migrating to draining LNs. The precise route by which intraocular-derived Ag accesses these LNs is unclear but the demonstration by ourselves (11) and others (75) of Ag-bearing cells in the episcleral tissues following an intracameral injection suggests that Ag from this area has access to lymphatic vessels in the conjunctiva and episclera. It has been clearly demonstrated that a proportion of the aqueous humor leaving the eye passes via the "nonconventional" outflow route, namely posteriorly in the extracellular space of the ciliary body to the suprachoroidal space to enter the episcleral tissues by passing through the sclera (11, 75, 76, 77). Data on the relative importance of "conventional" aqueous outflow pathways vs nonconventional in rodents is scanty but recent studies by Hoffman et al. (66) using intracamerally placed radiolabeled colloidal albumin estimates that uveoscleral pathways represent ~16% of outflow in the mouse eye. Subconjunctival injection of this tracer was found to result in increased drainage to local nodes (up to 97%). Taken together with data presented here, we conclude that at least a portion of Ag placed in the AC of the eye reaches the SMLN, FLN, and CLN via lymphatic drainage from the loose connective tissue surrounding the eye which it accesses via the uveoscleral pathway.

It is also feasible that despite rigorous efforts to avoid it, some leakage of Ag may have occurred via the corneal cannulation wound site thereby allowing Ag to reach the tear film and drain via the nasolacrimal duct to the nasopharynx. Ag exposure to the mucosal system of the nasal cavity and upper airways would explain the presence of Ag in the CLN which are known to play a role in nasal tolerance (78). Dick et al. (79) have shown that intranasally administered Ag is detectable in the superficial CLN in the mouse as early as 30 min after application. The potential for intracameral Ags to access nasal mucosa may be important as it is recognized that the dose of Ag required to induce a state of tolerance via the nasal route is considerably less than that required via the oral route (78). In addition, passage of Ag from the nasopharynx to the oropharynx and gut would also explain the presence of Ag in the deep CLN and MLN. We believe that although the presence of intracamerally placed Ag in the MLN has not previously been reported, it likely represents a constant phenomenon occurring during and/or after intracameral injection. This pathway of Ag exposure may have important consequences on our understanding of the mechanisms of deviant ocular immune responses.

In conclusion, this study strongly suggests that Ag placed into the AC of the eye travels in a soluble form and is internalized by resident macrophages in the spleen, LNs of the head and neck, and, surprisingly, in the MLN draining the gastrointestinal tract. We propose that the various routes by which Ag reaches these lymphoid organs and the nature of the cells that internalize the Ag have major impacts on the induction of the deviant ocular immune responses.


   Acknowledgments
 
We thank Wendy Colangelo and Stephanie Cooper for their excellent technical assistance; Dr. Paul Rigby for his helpful advice and technical help with confocal microscopy; and Guy Ben-Ary for his help in image processing.


   Footnotes
 
1 This work was supported by Australian National Health and Medical Research Council Project Grant 37382000. S.C. is the recipient of an Eric Cyril Lawrence Medical Research Fellowship. The Biomedical Confocal Microscopy Research Centre (Department of Medicine and Pharmacology, University of Western Australia) and the Image Acquisition and Analysis Facility are supported by the Lotteries Commission of Western Australia. Back

2 Address correspondence and reprint requests to Prof. Paul G. McMenamin, School of Anatomy and Human Biology, University of Western Australia, Crawley, Perth, 6009 Western Australia. E-mail address: mcmenamin{at}anhb.uwa.edu.au Back

3 Abbreviations used in this paper: AC, anterior chamber; LN, lymph node; SMLN, submandibular LN; CLN, cervical LN; FLN, facial LN; MLN, mesenteric LN; CB-Dx, Cascade Blue-dextran; A488-BSA, Alexa Fluor 488-conjugated BSA; MZB, marginal zone B cell; DC, dendritic cell. Back

Received for publication August 11, 2003. Accepted for publication January 29, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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O. M. Z. Howard, H. F. Dong, S. B. Su, R. R. Caspi, X. Chen, P. Plotz, and J. J. Oppenheim
Autoantigens signal through chemokine receptors: uveitis antigens induce CXCR3- and CXCR5-expressing lymphocytes and immature dendritic cells to migrate
Blood, June 1, 2005; 105(11): 4207 - 4214.
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P. A. Dullforce, K. L. Garman, G. W. Seitz, R. J. Fleischmann, S. M. Crespo, S. R. Planck, D. C. Parker, and J. T. Rosenbaum
APCs in the Anterior Uveal Tract Do Not Migrate to Draining Lymph Nodes
J. Immunol., June 1, 2004; 172(11): 6701 - 6708.
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