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Resident and Infiltrating Immune Cells in the Uveal Tract in the Early and Late Stages of Experimental Autoimmune Uveoretinitis T. L. Butler and P. G. McMenamin Purpose. To investigate the dynamics of resident and infiltrating immune cells in the choroid and iris during the early and late stages of experimental autoimmune uveitis (EAU) in Lewis rats. Methods. Uveoretinitis was induced by footpad injection of crude retinal extract and complete Freund's adjuvant with concurrent intraperitoneal injection of Bordetella pertussis. Five experimental (EAU) and five control animals (adjuvant alone) were studied at days 5, 7, 9, 11 (prodromal stage) and 42 (late stage) after immunization. Five normal animals and five animals injected with B. pertussis alone served as further controls. Immunohistochemical localization of resident macrophages, major histocompatibility complex class II (la)+ dendritic cells (DC), infiltrating mononuclear cells, and T cells was performed on wholemounts of isolated choroidal and iris tissue. Results. Double immunolabeling confirmed the presence of distinct networks of macrophages (591 ± 52 cells/mm2) and DC (746 ± 38 cells/mm2) in the rat choroid. No marked qualitative and quantitative changes were observed in the density or morphologic appearance of ED2+ resident tissue macrophages in the choroid and iris before clinical onset of ocular disease. On day 11, infiltration of ED1+ monocytes had occurred in the iris but not in the choroid; however, marked infiltration of T cells was evident in both choroid (286 ± 161 cells/mm2) and iris (196 ± 72 cells/mm2). The total density of Ia+ cells was significantly elevated in the choroid (1152 ± 192 cells/mm2) at day 11, and small, round Ia+ cells were two to three times more frequent than normal at both sites. The density of T cells and Ia+ cells remained significantly elevated in the choroid and iris in the late stages of EAU. Conclusions. These data suggest resident uveal tract macrophages undergo no significant alteration in density in the early stages of EAU and that the earliest site of mononuclear cellular infiltrate in EAU occurs in the iris. The increased total density of Ia+ cells in the choroid on day 11 and the presence of significantly increased numbers of small, round Ia+ cells in the iris and choroid may represent increased trafficking of DC in the eye during uveoretinitis. Furthermore, the raised numbers of Ia+ cells, concurrent with the influx of T cells, suggests Ia+ DC and macrophages may act as local antigen-presenting cells in the induction of uveoretinitis. Invest Ophthalmol Vis Sci. 1996;37:2195-2210. Hixperimental autoimmune uveitis (EAU) is recognized as a useful model of human posterior uveitis.1'2 The target of the autoimmune response in EAU and human posterior uveitis is the retinal photoreceptors. The immunopathologic processes in EAU, namely vasFrom the Department of Anatomy and Human Biology, The University of Western Australia, Nedlands, Perth, Western Australia. Supported l/y National Health and Medical Research Council. Submitted for publication December 1, 1995; reviled March 26, 1996; accepted June 4, 1996. Profnietary interest category: N. liefnint mjuests: P. G. McMenamin, Department of Anatomy and Human Biology, The University of Western Australia, Nedlands, Western Australia, 6907 Australia. culitis and focal retinochoroidal infiltrates, closely mimic the signs observed in various forms of human posterior uveitis (see reviews2"5). Experimental autoimmune uveitis is a CD4 T cell-mediated immune response5'6 that can be induced in susceptible species and strains by injection of a variety of retinal antigens3'7 in association with appropriate adjuvants8 at sites distant from the eye. More recently, modified uveal and retinal pigment epithelial melanin has been used to induce a predominantly anterior segment disease, known as experimental melanin-protein-induced uveitis. Investigative Ophthalmology & Visual Science, October 1996, Vol. 37, No. 11 Copyright © Association for Research in Vision and Ophthalmology 2195 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933414/ on 05/10/2017 2196 Investigative Ophthalmology & Visual Science, October 1996, Vol. 37, No. 11 The initiation of primary cell-mediated immune responses requires presentation of antigen within the groove of the major histocompatibility complex (MHC) class II molecules on the membrane of antigen-presenting cells (APC), such as dendritic cells (DC). This peptide-MHC class II complex interacts with the T-cell receptor14 and is aided by accessory cell adhesion and co-stimulatory molecules.1516 Although it is well known that uveitis is T cell-mediated, the cell(s) within the eye responsible for presentation of retinal autoantigens and induction of human endogenous uveitis is unknown and has been the subject of much recent research. Although ocular tissues were long thought to lack a distinct population of APCs and represent an "immunologically privileged site,"17 recent immunomorphologic studies have revealed extensive networks of macrophages and MHC class II + DC in the anterior18"21 and posterior portions22 of the uveal tract (see review23). Recent studies in our laboratory have shown that purified isolated iris DC function as potent APCs on exposure to cytokine-mediated maturation signals, such as granulocyte-macrophage colony-stimulating factors.24 Studies of isolated choroidal DC have shown they have similar functional capacity to act as APCs.25 The retina, by comparison, appears to lack MHC class II+ cells almost completely in most experimental animals,26"28 although there are some reports that human retinal microglia express MHC class II.2930 However, the appropriate turnover, functional, and double-immunophenotypic studies have yet to be performed to support the suggestion that cells of the DC lineage exist within the neural retina. Histologic, immunopathologic, ultrastructural, and depletion studies have highlighted the significance of macrophages as effector cells in target organ damage in EAU (see review4) and experimental autoimmune (allergic) encephalomyelitis.31 Macrophages also may play an important role in perpetuating local secondary immune responses by presenting antigens to T cells within ocular tissues. Macrophages, however, function poorly as APCs in primary immune responses when compared to DC because they lack the appropriate secondary messenger signals or co-stimulatory molecules.14'32 This general phenomenon has been confirmed recently for rat iris macrophages.24 Although it is well established that the tissues of the uveal tract are involved in EAU, it is unclear whether this involvement is primary or secondary. There is evidence to support the suggestion that the uveal tract, not the retina, is the initial site of inflammatory cell infiltration into the eye (see reviews4'33). Furthermore, the presence of a rich network of MHC class II (Ia) + DC in the uveal tract and their likely absence from the neural retina suggests an active role by these cells in disease induction. The general aim of this study was to perform a time course study of immune cells in the uveal tract during the early and late phases of EAU using tissue wholemounts. It was hoped this technique would provide novel en face perspectives of changes in resident immune cells and infiltrating inflammatory cells occurring throughout large areas of the choroid and iris, not achievable by conventional histologic approaches. The first aim of this study was to extend our previous data of the morphology, immunophenotype, density, and distribution of various resident immune cell types in the normal rat choroid with improved wholemount and double immunohistochemical techniques. Second, we aimed to determine the response of these cells and the dynamics of infiltrating inflammatory cells in the uveal tract during the prodromal stage of EAU. Finally, we sought to determine the long-term effects of the disease process on the networks of immune cells in the rat choroid and iris. MATERIALS AND METHODS Animals Sixty-three female Lewis rats (6 to 8 weeks of age, specific pathogen free), obtained from the Animal Resources Centre (Murdoch, Australia), were used in this study. Throughout the study, all procedures conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Preparation of Crude Retinal Extract and Immunization Bovine crude retinal extract (CRE; 2 mg/ml) was prepared as described in our previous studies.34 Experimental animals received a 100 /xl injection of an emulsion, consisting of equal proportions of CRE and complete Freund's adjuvant (Bacto Adjuvant Complete H37 Ra; Difco, Detroit, MI), subcutaneously into the right hind footpad. Animals received a concurrent 100 //I intraperitoneal injection of Bordetella pertussis (109 organisms; C.S.L. Parkeville, Victoria, Australia). The footpad injection corresponded to a dose of 100 fxg CRE, which in the current and previous studies has produced a mild to moderate form of EAU (Fig. 1). Control animals were treated in an identical manner except that phosphate-buffered saline (PBS) was substituted for CRE in the inoculum. Five control and five experimental animals were killed at days 5, 7, 9, 11 (prodromal stage) and 42 after immunization (late stage). The early time points were chosen carefully after pilot studies revealed that staining reproducibility and quality were severely compromised and quantitation of individual immunolabeled cells was impractical during the peak of the disease (days, 12 to 28) Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933414/ on 05/10/2017 2197 Uveal Tract Immune Cells in Experimental Uveoretinitis and irides were incubated in EDTA (20 mM) for 30 minutes (37°C). Each choroid was dissected from the sclera as an intact sheet and was divided by radial incisions into six pie-shaped pieces. Immunohistochemistry Day FIGURE l. The clinical score of ocular disease in experimental autoimmune uveoretinitis as a function of time. Values represent means (± SEM), and there were a minimum of five observations per time point. because the choroid, ciliary body, and iris were swollen by massive inflammatory cell infiltrates. Two groups acted as additional controls to provide baseline data: normal animals (n = 5) and a group that received only an intraperitoneal injection of B. pertussis (n = 5). Clinical Grading of Eye Disease All eyes were graded clinically on a scale of 0 to 4 widi the aid of an operating microscope 35 at days 5, 7, 9, and 11 after immunization until they were killed. Animals studied long term were graded clinically on these days and on every alternate day thereafter until day 42. Grade 1 (mild) disease was evident as discernible hyperemia with occasional cells adhering to die lens and pupil margin. Grade 2 (mild to moderate) disease was characterized by iris vessel dilation, flare, and iridocyclitis producing large numbers of inflammatory cells in the anterior chamber; however, the pupil remained visible. Grade 3 (moderate) disease manifested as dense inflammatory cell infiltrates within the anterior chamber sufficient to obscure the pupil, hypopyon uveitis, and corneal haze. Grade 4 (acute to severe) disease was similar to grade 3 disease but included intraocular hemorrhage. Choroidal and Iris Wholemount Preparation and Immunohistochemistry Animals were anesthetized (sodium pentobarbitone, 100 mg/kg) before whole body perfusion through the left ventricle with cold heparinized PBS followed by cold 2% paraformaldehyde. Eyes were posdixed in 2% paraformaldehyde after enucleation. Each eye was divided into anterior and posterior segments, and die lens and capsule were removed carefully. The irides were dien separated from the cornea-sclera and divided by radial incisions into six equal portions. The retina was dissected from the posterior segment of the globe, and the remaining choroid-sclera complex Samples of choroid and iris tissue from each eye were incubated with a panel of primary monoclonal antibodies (mAbs). Negative controls included either PBS alone or an inappropriate mAb (e.g., anti-human macrophage marker). The specificities of primary mAbs to rat macrophages, DC, and other leukocytes are listed in Table 1. Single and double immunohistochemical studies were performed as previously described. 2137 For single immunostaining, portions of tissue were blocked with PBS-bovine serum albuminTween and then were incubated widi the primary mAb followed by biotinylated sheep antimouse and streptavidin-horseradish peroxidase (Amersham Laboratories, Buckinghamshire, UK). Primary antibody incubations were performed at 4°C overnight. Other steps were performed at room temperature widi incubation times of 30 to 45 minutes. The horseradish peroxidase was visualized by 3-amino-9-ethyl carbazole (0.2 mg/ml in acetate buffer pH 5; plus 0.35 /xl/ml H 2 O 2 [30% wt/vol]). Very few cells displaying endogenous activity were noted in negative controls. A double-chromogen-immunostaining procedure diat relies on biotinylation of one primary mAb (ED2) 38 was used to investigate die extent of co-expression of leukocyte markers on macrophage and DC populations in normal choroid wholemounts. Choroidal wholemounts were double stained with die following combinations: OX6-ED2, ED3-ED2, and E D 1 ED2. Specimens were incubated widi a nonbiotinylated primary mAb, such as OX6, followed by direcdy conjugated secondary antibody (sheep anti-mouseTABLE l. Anti-Rat Monoclonal Antibodies* Monoclonal Specificity OX-6 MHC class II (la) antigen Glycosylated lysosomal antigen (putative CD68) in most rat monocytes, macrophages, and 90% of DC Membrane differentiation marker: recognizes glycoprotein on resident/ mature connective tissue macrophages and small subpopulation in lymphoid tissues Macrophage subset in lymphoid organs: a 175-185 kDa sialoadhesin-like glycoprotein CD5/a/3 TCR, pan T<ell EDI ED2 ED3 OX-19/R73 * For full description and individual references, see Dijkstra et ;t " Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933414/ on 05/10/2017 2198 Investigative Ophthalmology & Visual Science, October 1996, Vol. 37, No. 11 horseradish peroxidase; Amersham). The tissues were washed three times in PBS and then incubated with the biotinylated primary mAb (ED2-biotin) localized by streptavidin-alkaline phosphatase. The ED2-alkaline phosphatase-labeled cells were visualized with the substrate naphthol AS-MX (0.12 mg/ml) and Fast blue BB (0.25 mg/ml) in Tris buffer (pH 8.5). Levamisole (0.25 mg/ml) was added to the substrate to block endogenous alkaline phosphatase activity. Immunolabeled cells were visualized as a blue reaction product. Cells labeled with the other primary monoclonal (e.g., OX6) were visualized using 3-amino-9-ethyl carbazole (vide supra), which produces a red product. Double staining or co-localization of both phenotypic markers resulted in purple cells or occasionally cells that contained discrete blue- and red-stained regions. All visualization agents and chemicals were purchased from Sigma (St. Louis, MO). Stained choroid and iris-ciliary body wholemounts were mounted in aqueous medium on glass slides and were coverslipped. Quantitative Analysis Three animals from each group were subjected to quantitative analysis. These were chosen on the basis of disease onset closest to day 11 in that group and for clarity and reproducibility of immunoperoxidase staining. Immunopositive cells within choroid and iris wholemounts were counted using a calibrated eyepiece graticule and a X40 objective lens. The method of counting immunostained cells in iris has been described.21 Six counts were performed on each piece of immunostained choroidal tissue—two random fields from the posterior choroid, two from the mid-choroid, and two from the peripheral choroid. Immunostained cells were counted using the forbidden line method.39 Cells were classified into three morphologic categories2137 as dendriform, pleomorphic, and round-ovoid. The mean area of iris and choroid tissue sampled for each mAb was 0.504 mm2. Data from six counts were averaged to give a mean cell density for each morphologic type and each mAb per individual animal. Group means represent the combined means of the animals within each experimental group. All cell counts were performed on coded slides by a blinded observer (TB). Cell density data were analyzed using one-way analysis of variance. Differences between individual groups were subjected to Rest for invariate small samples. Significance values indicated differences between experimental and both normal and control groups unless otherwise stated. RESULTS Clinical Evaluation The onset of clinical disease, as determined by macroscopic clinical examination of the anterior segment, usually occurred on day 11 after immunization (Fig. 1). Ocular disease was maximal at day 17; by day 32, manifestations of active disease were minimal or absent. Control animals did not exhibit ocular disease. Density, Distribution, and Phenotype of Resident Tissue Macrophages and Dendritic Cells in the Normal Rat Choroid In a previous study,22 we described the morphology, location, distribution, and density of macrophages and MHC class II (la) + cells in the normal rat choroid largely on the basis of appearance in sections with only limited use of tissue wholemounts. Recent advances in the fixation, preparation, and immunostaining of choroidal wholemounts allows us to upgrade the description of normal choroidal macrophages and Ia+ DC and, moreover, has revealed important supplementary data on these cells. The mAb ED2 is a pan-specific resident tissue macrophage marker.40 Resident macrophages were distributed uniformly within the choroid and exhibited a predominandy perivascular location (Fig. 2). Large numbers of ED2+ macrophages were associated with the long posterior ciliary arteries and vortex veins (Fig. 2B). They were predominandy bipolar-pleomorphic in shape (Fig. 3A). Although dendriform ED2+ cells were noted, they did not display the extremely branched dendritic cell processes or ruffled appearance of Ia+ cells (vide infra). Rounded ED2+ cells constituted less than 1 % of the population (Table 2). The mean cell density of 591 ± 52 cells/mm 2 observed in the current study was higher than previously reported (200 ± 13 cells/mm2).22 The discrepancy most likely was caused by major improvements in the fixation, wholemount preparation, and immunostaining protocols. For example, in our previous study, choroid and sclera were stained as one, whereas we now routinely remove the choroid from the sclera, which markedly improves antibody penetration and staining. The mAb EDI recognizes a cytoplasmic antigen associated with phagolysosomes present in monocytes, most tissue macrophages, and some DC subpopulations.40"42 Immunopositive cells thus tend to have a "beaded" appearance. Previous studies have shown that 100% of ED2+ iris tissue macrophages are ED1 + . 37 In the current study, pleomorphic and dendriform ED1 + cells were present in equal numbers in the choroid, and rounded cells constituted only 4% of the total (Table 2). The distribution and predominantly perivascular location of EDI + cells in the normal choroid was identical to ED2+ macrophages. Their mean density was estimated as 723 ± 47 cells/mm2 (Table 2). The mAb ED3 is considered a marker of activated macrophages and reacts strongly with macrophages in Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933414/ on 05/10/2017 Uveal Tract Immune Cells in Experimental Uveoretinitis 2199 2. Choroidal wholemount from a normal Lewis rat stained with the resident tissue macrophage marker ED2. (A) Low-power view of the entire choroid. Note the long posterior ciliary arteries orientated in the horizontal plane and the vortex veins peripherally. (B) Medium-power view to illustrate the dense network of ED2+ macrophages that appear particularly dense around the long posterior ciliary artery (A). V = vortex vein. Magnifications: (A) X20; (B) X45. FIGURE lymphoid tissues and at sites of inflammation.40 There have been few reports that resident tissue macrophages in nonlymphoid tissues are immunoreactive to ED3; however, ED3+ macrophages were found to be more widely distributed in the normal rat choroid (Fig. 3C) than was previously described.22 The ED3+ macrophages displayed a similar density (580 ± 26 cells/mm2) and morphology to ED2+ resident tissue macrophages (Table 2, Fig. 3C). The OX6 mAb is specific for the rat MHC class II molecule, constitutively expressed on the surface of DC and some macrophages.43 In die absence of a panspecific anti-rat DC marker, conventional classification of DC relies on die demonstration of dendriform or pleomorphic morphology, constitutive MHC class II (la) expression, and lack of coexpression of pan-macrophage markers.14 In the current study, choroidal wholemounts stained with the OX6 mAb revealed an extensive network of dendriform, pleomorphic, and bipolar Ia+ cells (Fig. 3D). Many appeared close to die focal plane of the retinal pigment epithelium. Only a small proportion (5%) of Ia+ cells exhibited a rounded morphology in normal eyes. The density of Ia+ cells (746 ± 37 cells/mm2) in the normal rat choroid was considerably higher than that obtained in a previous study (373 cells/mm2).22 As stated above, this probably was caused by improved fixation, wholemount preparation, and immunostaining procedures. Double immunostaining with the combination of mAb OX6-ED2 confirmed our earlier preliminary studies22 that Ia+ DC and ED2+ resident tissue macrophages are distinct populations (Figs. 3E, 3F). Macrophages that express la appear purple in the doublestained preparations (Fig. 3F) and form only a minor subpopulation. The ED1-ED2 double-stained choroidal wholemounts revealed that the majority of ED2+ resident tissue macrophages also contained ED1+ phagolysosomes (Fig 3G); however, there are ED2~, ED1 + cells that are most likely DC. This is in accordance with our previous studies, which revealed diat approximately 35% of EDI+ cells in the iris were Ia+. Furthermore, ED3-ED2 double-stained preparations revealed that most ED2+ tissue macrophages were also ED3+ (Fig. 3H). Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933414/ on 05/10/2017 2200 Investigative Ophthalmology & Visual Science, October 1996, Vol. 37, No. 11 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933414/ on 05/10/2017 Uveal Tract Immune Cells in Experimental Uveoretinitis 2201 FIGURE 3. (A) High-power view of normal rat choroidal wholemount showing the network of resident ED2+ tissue macrophages beneath the retinal pigment epithelium, whose regular hexagonal cell borders can be identified clearly. (B) Medium-power view of EDl-stained wholemount. (C) ED3+ macrophages in the choroid. (D) Major histocompatibility complex class II (Ia)+ dendritic cells in choroidal wholemount. Note the irregular distribution, which contrasts with the regular perivascular location of macrophages. (E,F). Low- and high-power views of double-color immunohistochemical-stained normal rat choroidal wholemount. The mAbs ED2 and anti-la (OX6) have been used to co-localize resident tissue macrophages (blue) and dendritic cells (red). Note that both networks are distinct, and only a few cells (arrorvheads) coexpress both markers. (G) High-power view of choroidal cells double stained with ED2 (blue) and EDI (red). Note that almost all cells are double positive (purple). (H) Choroid double stained with the mAbs ED2 and ED3. Almost all choroidal macrophages are double positive (purple). Magnifications: (A) X300; (B) X200; (C) X200; (D) X200; (E) X80; (F) X310; (G) X310; (H) X200. Morphology and Density of Macrophages and increase in T-cell density throughout the choroid on MHC class II Dendritic Cells in the Choroid the day of disease onset, day 11, in EAU animals (Fig. During the Prodromal Stage of Experimental 4C, Table 2). A very small subpopulation of unusual Autoimmune Uveoretinitis dendriform T cells was observed in day 11 animals + (not illustrated). The density and regular distribution of ED2 resident tissue macrophages in the choroid was largely unaffected Choroidal Immune Cells in the Late Phase of during the prodromal stage of EAU. There was a marginal Experimental Autoimmune Uveoretinitis (Day 42) shift from a dendriform to a pleomorphic morphology The density of macrophages (ED2, EDI, and ED3+) evident on days 5, 7, and 9 (Table 2). There was no + was not conspicuously altered in day 42 EAU animals. observed increase in the number of ED1 round cells The total density of Ia+ cells was significantly elevated (monocytes) on day 11 as would have been expected if (1233 ±146 cells/mm2) in long-term animals (Table the mononuclear cell infiltrate characteristic of EAU had + 2), primarily because of increases in dendriform and begun. By contrast, a conspicuous ED1 mononuclear pleomorphic cells. T-cell density in the choroid was cell infiltrate began in the iris by day 11 (vide infra). The significandy elevated in day 42 EAU animals (Table 2). only detectable difference in EDI staining in the choroid was a generalized adjuvant effect (not observed in pertussis controls) on days 5, 7, and 9 in which there was a slight Morphology, Distribution, and Density of Immune Cells in the Iris During the Prodromal shift from a predominantly dendriform to pleomorphic Stage of Experimental Autoimmune morphology (Table 2). The pattern of ED3 staining Uveoretinitis changed little during EAU apart from a slightly increased density in experimental and control animals on days 5, The morphology, distribution, density, and phenotype 7, and 9 (Table 2), which indicated an adjuvant effect of resident tissue macrophages and Ia+ DC in the nor+ mal rat iris corresponded with our previous studThe density of the la (OX6) cell network in the choroid increased marginally at days 5, 7, 9, and 11. ies.20'2137 Iris macrophages were more weakly stained The majority of these cells were dendriform or pleowith ED3 than those in the choroid. morphic (Table 2). The total density of Ia+ cells (1152 The network of ED2+ resident tissue macrophages ±192 cells/mm2) in experimental animals at the day in the rat iris was largely unaffected during the prodroof clinical onset, day 11 (Fig 4A)—although greater mal phase of EAU. Infiltrates of round ED1+ monothan normal—did not reach statistical significance cytic cells were evident in the iris on day 11 in most (Table 2). Part of this increase was the result of a eyes (Table 3). Interestingly, clinically detectable dismarked rise in the number of round Ia+ cells, which ease was evident in only one of these animals in whose was particularly evident in the peripheral choroid eye the ED1+ monocytic infiltrate was so severe that (Fig. 4B, Table 2). it formed a complete cellular "sheet" over the anterior iris surface (Fig. 5A). Such dense inflammatory T Cells in the Choroid During the Prodromal infiltrates precluded counting individual cells and preStage of Experimental Autoimmune vented optimal immunostaining of the underlying iris. Uveoretinitis Occasionally, the mononuclear cell infiltrate was less well developed and involved only focal patches of ciliThe density of T cells in the normal choroid was ex2 ary body and iris vasculature and stroma (Fig. 5B). tremely low (16 ± 7 cells/mm ). There was a marked Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933414/ on 05/10/2017 Choroid Cell Density Day 5 Day 7 Normal Pertussis EAU c 591 (52) 122 (34) 465 (50) 4(1) 562 (37) 133 (26) 423 (48) 6(1) 659 (33) 40(3) 614 (22) 5(2) 521 65 451 5 c 723 350 342 31 647 268 339 40 807 (83) 140 (9) 630 (79) 37(6) c 580 (26) 59 (12) 515 (18) 6(2) 505 (18) 56 (8) 442 (24) 7(2) 922 (45) 35 (18) 861 (20) 26(9) c 746 136 571 39 674 157 456 61 c (47) (66)* (40) (7) (38) (15) (32) (6) 16(7) 0 0 16(7) (38) (26)* (56) (8) (28) (35) (36) (13) 71 (4) 0 15(1) 56(3) 1040 84 895 61 (83) (24) (115) (15) 64 (18) 0 6(3) 58 (15) Control Control EAU Day Control EAU Day 42 Control 670 (10) 48 (19) 618 (13) 4(2) 580 (69) 67 (13) 509 (55) 4(2) 625 48 574 3 (21) (10) (26) (2) 573 (27) 117 (7) 450 (18) 6(3) 595 (35) 65 (29) 520 (24) 10(2) 565 126 436 3 (52) (21) (63) (1) 744 (61) 147 (32) 578 (73) 19(7) 724 143 558 23 (31) (28) (30) (10) 791 163 599 29 (152) (5) (123) (4) 737 199 518 20 (204) (32) (130) (4) 748 178 547 23 (51) (21) (47) (2) 531 137 379 15 620 166 424 30 672 (14) 22 (11) 643 (13) 7(4) 798 32 746 20 (68) (12) (83) (3) 864 53 801 10 (80) (12) (73) (5) 893 34 838 21 (81) (11) (96) (5) 777 57 710 10 (21) (20) (29) (3) 625 (72) 28(5) 593 (76) 4 (2) 895 89 753 43 (48) (19) (50) (12) 952 190 722 40 (128) (36) (87) (5) 916 130 730 56 (42) (24) (64) (4) 1035 126 841 68 (26) (42) (66) (4) 1111 161 915 35 (11) (19) (14) (1) EAU Day 9 (94) (37) (92) (3) 57 (16) 0 6(2) 51 (15) 61 (18) 0 3(2) 58 (18) 59 (24) 0 7 (4) 52 (21) 50 (4) 0 7 (6) 43(7) 76 (10) 0 8(6) 68 (5) mental autoimmune uveitis. an cell density (±SEM). sus normal. sus normal. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933414/ on 05/10/2017 1152 101 941 110 (106) (39) (46) (2) (192) (43) (139) (53) 286 (161) 0 14(9) 272 (152) EAU (21) (34) (34) (1) 624 56 565 3 (86) (9) (82) (8) 667 (71) 137 (22) 515 (69) 15 (6) 595 122 461 12 694 52 632 10 (35) (20) (55) (2) 664 (25) 44 (18) 616 (25) 4(1) 630 33 589 8 916 165 700 51 (91) (15) (84) (21) 1233 (146)f 158 (29) 1038(144) 37 (8) 823 120 671 32 187 (54) t 0 16(3) 171 (56) 66 0 9 57 57 (3) 0 8(3) 49 (2) 535 75 457 3 C Uveal Tract Immune Cells in Experimental Uveoretinitis -at. 2203 marked ED1+ mononuclear cell infiltration of the iris at day 11 also exhibited marked focal infiltrates of small, round Ia+ cells throughout the iris stroma (Fig. 5C, Table 3). In addition, the sheet of mononuclear cells that covered the anterior iris surface in one animal contained numerous round Ia+ cells. These are most likely a mixture of Ia+ monocytes-exudate macrophages, activated T cells, and DC. T cells are extremely rare in the normal iris (16 ± 6 cells/mm^) (Fig. 5E). In the early stages of EAU before disease onset (days 5, 7, and 9), there was no quantitative change in T-cell numbers. However, by day 11, disease onset was characterized by an influx of small, round OX19-R73+ T cells throughout the iris (Table 3, Fig. 5F). The density (196 ± 72 cells/ mm2) was evident as focal aggregates and as isolated individual cells. Immune Cells in the Iris in Late-Phase Experimental Autoimmune Uveoretinitis (Day 42) The total number of Ia+ cells in the iris of die day 42 animals was approximately four times greater than normal values (Table 3). Of special significance, however, was the presence of a distinct layer of extremely dendriform Ia+ cells that appeared to be situated on or close to the anterior iris surface (Fig. 5D). T cells remained significandy elevated in the iris of day 42 animals (Table 3). DISCUSSION FIGURE4. Ia(OX6)+ cells in the central (A) and peripheral (B) choroid in a day 11 experimental autoimmune uveoretinitis (EAU) Lewis rat. Note the increased number of predominantly round and pleomorphic cells close to vessel walls. (C) Day 11 EAU choroid stained with pan-T-cell marker (Tc). Note the exclusively small round cells evenly distributed in the choroid. Magnifications: (A) X200; (B) X80; (C) X200. There was a slight decrease in the total density of ED3 + iris macrophages in day 11 EAU animals (Table 3). The network of Ia + DC in the iris was not markedly altered in die early prodromal stages of EAU (days 5, 7, and 9) (Table 3). Those EAU animals that showed Networks of DC are distributed ubiquitously in most tissues and are particularly well developed and characterized in epithelial sites close to the external environment, such as skin, gut, and respiratory tract, where they function as sentinels to trap and sample large numbers of exogenous antigens.14 On migration to Tcell rich zones of draining lymphoid tissues, they mature into potent APCs and may present antigen to naive T cells, thus initiating primary immune responses. In addition, DC are significantly more efficient APCs than B cells and macrophages in the induction of secondary immune responses.14 Dendritic cell populations in the interstitium of nonrymphoid organs4445 are also likely to encounter altered self-antigens as a result of tissue injury, regeneration, and repair.46 Many forms of endogenous intraocular inflammation are diought to have an autoimmune etiology, and several autoantigens have been identified that can induce symptoms similar to posterior uveitis. In these models, immunization at distant sites with a potential uveitogen leads to antigen-specific T-cell proliferation in local lymph nodes. On entering the circulation, T Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933414/ on 05/10/2017 ris Cell Density Normal 634 (41) c 361 (25) hic 264 (29) 9 (2) c c c ic Day 7 Day 5 603 399 187 17 (42) (41) (16) (5) Pertussis EAU 533 (24) 203 (18) 299 (17) 31 (4) 552 (38) 113 (11) 419 (36) 20 (6) 468 131 317 19 (22) (11) (18) (3) 496 107 371 18 (34) (24) (50) (4) 514 125 365 24 (47) (16) (38) (3) 406 106 288 12 (22) (16) (7) (2) 360 153 165 43 356 110 229 17 383 72 269 42 (103) (18) (94) (21) 331 89 208 35 (55) (31) (16) (11) 260 83 162 15 (58) (23) (38) (4) 392 136 233 23 (86) (42) (52) (10) (32) (13) (25) (6) (59) (18) (48) (5) Control EAU Day 9 Control EAU Day 11 Control Day 42 EAU Control EAU 416 (72) 133 (30) 263 (47) 20 (1) 423 (132) 94 (51) 301 (87) 28 (10) 491 (56) 130 (35) 336 (30) 25 (4) 317 (77) 56 (4) 248 (72) 13 (2) 35 4 28 410 154 233 23 369 48 179 141 340 88 215 37 540 110 380 50 31 7 21 3 (42) (19) (40) (13) (112) (26) (68) (60) (54) (18) (45) (4) (101) (45) (75) (10) C 385 (29) 210 (8) 264 (35) 254 (44) 281 (66) 316 (85) 336 (62) 336 (115) 173 (89) 270 (67) 356 (52) 108 (17) 29 (3) 20 (4) 9 (6) 2 3 (12) 14 (5) 27 (4) 31 (18) 14 (10) 30 (15) 20 (7) 1 262 (24) 16 (3) 136 (9) 45 (1) 228 (37) 16 (9) 224 (45) 21 (5) 231 (45) 27 (13) 278 (84) 24 (3) 271 (75) 39 (10) 270 (79) 35 (18) 146 (75) 13 (6) 214 (60) 26 (6) 291 (55) 44 (9) 19 1 436 298 124 14 395 159 215 21 607 194 393 20 459 121 319 19 483 161 297 25 436 139 280 18 544 188 330 27 (129) (15) (110) (6) 606 207 378 21 (31) (27) (30) (7) 495 93 325 78 (162) (51) (156) (46) 465 135 306 25 60 2 11 46 (23) (2) (9) (12) 35 (11) 0 1 (1) 34 (11) 196 1 9 186 (72) (1) (6) (68) (43) (28) (19) (4) 16 (6) 0 0 16 (27) (18) (16) (4) 12 (5) 0 1 (1) 11 (5) 25 1 1 23 (106) (30) (94) (10) (9) (1) (1) (8) (48) (24) (24) (1) 44 (13) 0 1 (1) 43 (13) (104) (33) (5) (16) 27 (13) 0 1 (1) 25 (14) (63) (23) (34) (8) 12 (3) 0 .1 (1) 10 (2) mental autoimmune uveitis. an cell density (±SEM). sus normal. rsus normal. ersus normal. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933414/ on 05/10/2017 (24) (19) (17) (6) 41 (10) 0 2 (1) 40 (10) 1691 454 1191 46 (108)* (56) (78) (9)f 152 (24)} 0 23 (7) 129 (29) 23 66 18 46 1 2 2 Uveal Tract Immune Cells in Experimental Uveoretinitis Tc FIGURES. Light micrographs of immunostained rat iris wholemounts in experimental autoimmune uveoretinitis (EAU). (A) Low-power view of dense aggregates of EDI+ mononuclear cells forming a cellular "sheet" that covered the anterior surface of the iris in a day 11 EAU animal. (B) Focal aggregate of ED1+ cells in the iris in a day 11 EAU animal. (C) Highpower view of la stained iris from a day 11 EAU animal, illustrating the focal aggregation of Ia+ cells and the predominantly rounded and pleomorphic shape of the cells. (D) Ia+ cells in the rat iris in the late phase of EAU. Note the increased density of the DC network and, in particular, the highly dendriform cells in focus lying close to the iris surface. The remainder of the DC population is out of the focal plane. (E) Normal rat eye stained with the pan-T-cell (Tc) marker combination of mAbs OX19-R73. (F) T cells in day 11 EAU animal. Magnifications: (A,B) X78; (C to F) X195. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933414/ on 05/10/2017 2205 2206 Investigative Ophthalmology 8c Visual Science, October 1996, Vol. 37, No. 11 cells may adhere to postcapillary venules in the retina or uveal tract before extravasating into the surrounding tissue, where it is postulated they interact with autoantigen in the context of MHC class II+ APC. The precise nature of the APC in the eye in the EAU model is subject to speculation. The current study confirms previous evidence that rich networks of DC exist in the iris, ciliary body,18"21 and choroid22 at comparable densities to other nonocular tissues.47 Recent evidence has demonstrated that the functional capacity of DC isolated from anterior24 and posterior portions of the uveal tract25 are comparable to other DC, such as epidermal Langerhans cells. This makes them likely APCs in the sensitizing and inductive phases of endogenous uveitis. Furthermore, because of their anatomic location, immediately on either the internal or external interface of the bloodocular barrier (iris stroma and basal aspect of retinal pigment epithelium, respectively), uveal tract DC are probably exposed to retinal antigens. The mechanisms that modulate immune responses within the normal eye by prevention or downregulation of unwanted antigen presentation of ocular-derived antigen by the rich networks of intraocular DC are now under critical investigation in many laboratories (see reviews23'48). In light of the potential importance of uveal tract DC to the pathogenesis of posterior uveitis, one of the specific aims of the current study was to obtain data on the dynamics of DC in the early and late phase of uveitis using the EAU model. Although several studies have reported MHC class II upregulation on retinal pigment epithelium and retinal vascular endothelium in EAU models,4'1"52 sympathetic ophthalmia, and uveitis,53 there have been only limited reports of MHC class II expression on individual cells in the choroid or retina. However, a recent semiquantitative histologic study of adhesion molecules and MHC class II expression in melanin-associated, antigen-induced uveitis described an increase in MHC class II+ cells in the iris and choroid during the onset and peak of the disease.54 Our study, with the advantage of wholemount staining, has demonstrated obvious increases in the density of MHC class II+ cells in the iris and choroid in the early stages of EAU. Although some of the increase may be accounted for by MHC class II+ activated T cells or macrophages, it probably represents enhanced recruitment of DC precursors from the circulation or an upregulation of la on previously Ia~ DC. There is evidence that a population of DC precursors (OX62'Ia) exists within the choroid22 and iris (data not shown). These may provide a rapidly recruitable source of DC in the event of local inflammatory episodes. The concurrent increase in density of Ia+ DC and T-cell infiltration in the uveal tract would provide the appropriate environment for local activation of uveitogenic T cells. The crucial importance of the interaction between the T-cell receptor and peptidebearing MHC class II molecule in induction of autoimmune disease has underpinned the rationale of experimental attempts to disrupt the complex using anti-la antibody therapy and, therefore, to ameliorate diseases such as EAU51'55 and experimental autoimmune (allergic) encephalomyelitis (EAE) .r>(1 The partial success of such studies supports the concept that MHC class II expression is involved in the induction and perpetuation of EAU. There is evidence that EAE may be transferred to naive animals by sensitized DC57; however, similar studies have yet to be performed with ocular autoimmune models. Macrophages are long lived, functionally heterogeneous cells whose phenotype is determined by their developmental stage, state of activity, and location. This heterogeneity and associated variable expression of surface and cytoplasmic markers is commonly used for the characterization of various macrophage subpopulations.3640 The current study has revealed that uveal tract macrophages are a heterogeneous population. For example, normal iris macrophages are weakly ED3+, but choroidal macrophages are strongly ED3+. This mAb recognizes sialoadhesins and is weakly expressed on other resident tissue macrophages but is strongly expressed on "activated" lymphoid macrophages, where it is postulated these cells play an immunosuppressive role.40'58 The significance of the differential expression of ED3 between the iris and choroidal macrophages is unclear. Uveal tract macrophages differ radically in immunophenotype from the resident tissue macrophage population within the retina (microglia), which are ED2", ED3~, and Ia~ but weakly ED1+.20 Similar observations have been made in the brain and spinal cord, in which meningeal macrophages differ widely in phenotype and function from microglia within the neural parenchyma.28'59-W) Data on the phenotypic and morphologic changes in retinal microglia during EAU would have complemented the current study. This was not possible, however, because of difficulties in obtaining immunostaining throughout the entire thickness of retinal wholemounts that was sufficiendy reliable and consistent for quantitative analysis. The fixation and staining protocols used to preserve and visualize macrophages in the uveal tract do not reveal retinal microglia. Therefore, an investigation of these cells in EAU would have to be undertaken as a distinct exercise. Our data suggest that immediately before fullscale inflammation, there is surprisingly little alteration in the resident iris and choroidal macrophages, although the slighdy more pleomorphic shape may Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933414/ on 05/10/2017 Uveal Tract Immune Cells in Experimental Uveoretinitis 2207 indicate evidence of motility and activation. Similarly, the resident macrophages showed little alteration in form during the late phase of EAU. Our data suggest the majority of macrophages in choroidal lesions in EAU are newly extravasated ED1 + monocytes-macrophages. This concurs with similar histologic observations in IRBP-induced EAU61 and in the experimental melanin-protein-induced uveitis model. 10 The role of infiltrating macrophages in the immunopathology of EAE, both in directly mediating damage to the central nervous system (CNS) and in attracting other cells to lesions, is well accepted62'63 and is supported by depletion studies in which marked suppression of the clinical signs of disease have been obtained. 31 To date, there are no comparable published reports of prevention of EAU using macrophage depletion. 8 days after onset65; in another nonquantitative study,66 T-helper cells were noted in 4 of 8 eyes examined between days 21 and 31 after immunization. Evidence from EAE studies suggest few T cells remained in the CNS after recovery, although the meninges were not specifically studied.71 These authors proposed that the decline in T cells and macrophages in the CNS in the recovery phase of EAE was caused by apoptosis in situ.72 On the basis of newly emerging evidence, it appears that similar mechanisms may operate in the normal eye, thus reducing the likelihood of unwanted tissue damage. 73 The situation in the recovery phase of ocular inflammatory disease remains to be elucidated. The consistent identification of small numbers of T cells (approximately 10 to 20 cells/mm 2 ) in the normal iris and choroid adds support to the recently advanced concept that the CNS is normally patrolled by a small number of T cells.64 Characterization of Tcell subsets was not performed in the current investigation because of the number of mAbs investigated and the limited amount of tissue from each eye. In previous immunohistochemical studies of T-cell subsets in EAU6506 the ciliary body, followed closely by the iris, was identified as the earliest site of cellular infiltration.65 Initially, our data appeared to suggest that the choroid contained T-cell numbers similar to those in the iris on day 11; however, in light of the fact that Tcell numbers were marginally elevated in the iris on day 9 and monocytic infiltrate began earlier in the iris than in the choroid, we support the contention that inflammatory cell infiltration begins in the anterior uvea before the posterior uveal tract. Increased T-cell density in the choroid and iris in EAU was caused by either continuous extravasation from the circulation, local in situ proliferation, or both. Studies of BrdU incorporation in EAE lesions suggests that T-cell proliferation in the CNS is minimal,67 leading to the suggestion that T cells enter an unresponsive state in the CNS microenvironment. The iris and choroid, because of their anatomic homology, are more likely to behave similarly to the leptomeninges and subarachnoid space, which are reported to act as important sites of T-cell precursor proliferation and effector cell selection in EAE.68'69 Recent studies have suggested antigen nonspecific T cells have an important role in amplification of the disease process in EAU.70 The demonstration of persistently raised T-cell numbers in the uveal tract 42 days after immunization (approximately 30 days after disease onset) and after subsidence of the active inflammatory phase of EAU has not been described in previous studies. Other studies have described T cells returning to near normal levels autoimmune, choroid, dendritic cell, iris, macrophages, T cell, uveitis Key Words Acknowledgments The authors thank Julie Crewe for performing the doublecolor immunolabeling. References 1. Forrester JV, Liversidge J, Towler H, McMenamin PG. Comparison of clinical and experimental uveitis. 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