The fate of autoreactive, GFP+ T cells in rat models of uveitis analyzed by intravital fluorescence microscopy and FACS
Stephan R. Thurau1,
Thorsten R. Mempel2,4,
Alexander Flügel3,
Maria Diedrichs-Möhring1,
Fritz Krombach2,
Naoto Kawakami3 and
Gerhild Wildner1
1 Section of Immunobiology, Department of Ophthalmology and 2 Institute for Surgical Research, Ludwig-Maximilians-University, Munich and 3 Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, Martinsried, Germany
4 Present address: Center for Blood Research and Department of Pathology, Harvard Medical School, Boston, USA
Correspondence to: S. R. Thurau; E-mail: Stephan.Thurau{at}med.uni-muenchen.de
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Abstract
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Experimental autoimmune uveitis (EAU) is an inflammatory disease of the immune privileged inner eye, mediated by CD4+ Th1 cells specific for retinal autoantigens. To elucidate the fate of the T cells in the eye we adoptively transferred green fluorescent protein-positive (GFP+) T cells with specificity for R14, a peptide from interphotoreceptor retinoid-binding protein (IRBP) or OVA as foreign control antigen to naive Lewis rats. We also used the model of immunogenic uveitis, an inflammatory eye disease induced by intraocular injection of soluble OVA 1 day post transfer of OVA-specific GFP+ cells. We investigated the timing of ocular T cell infiltration and their immunological activation state by intravital fluorescence microscopy (IVFM) of the iris until onset of intraocular inflammation. Within 30 min of injection, GFP+ cells invaded the iris tissue, irrespective of their antigen specificity, whereas intraocular inflammation was only observed 3 days later, if cells recognized their respective antigen (R14-specific cells in EAU, OVA-specific cells in immunogenic uveitis). Using FACS analysis we found that activation markers were upregulated only on cells from uveitic eyes, but not from other sources, suggesting that intraocularly presented specific antigen is a prerequisite for T cell reactivation and subsequent recruitment of inflammatory cells.
Keywords: activation marker, experimental autoimmune uveitis, immunogenic uveitis, interphotoreceptor retinoid binding protein, T cell migration
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Introduction
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Uveitis is an inflammatory autoimmune disorder of the eye, which is one of the major causes of blindness in industrialized countries. Animal models of uveitis share many features with the human disease and therefore have helped us to understand the pathophysiology of uveitis and to introduce new therapeutic regimens (1).
Experimental autoimmune uveitis in Lewis rats is mediated by CD4+ T cells with specificity for retinal antigens, secreting cytokines and chemokines to attract other leukocytes to the eye, which represent the inflammatory infiltrates in uveitis (2,3).
Through the transparent cornea the ocular tissues can easily be observed in vivo without the requirement of any surgical intervention (4,5). This offers the opportunity of directly following the infiltration of autoreactive T cells and the influx of inflammatory lymphocytes, whereas in other models of autoimmune diseases like EAE or arthritis, typical read out parameters like palsy or joint swelling are more indirect and depend on secondary mechanisms of inflammation. Here we investigated the ocular infiltration and the fate of adoptively transferred T cells with and without specificity for retinal autoantigen in Lewis rats. For this purpose, T lymphocytes with specificity for retinal IRBP peptide R14 and OVA as foreign control antigen were genetically engineered for stable expression of green fluorescent protein. This enabled us to easily follow them for extended periods of time even after several cycles of cell division, without losing the dye (6). After termination of the experiments these cells can either be retrieved and analyzed by flow cytometry (FACS) for the expression of surface and activation markers or visualized by conventional fluorescence microscopy in vitro. To investigate the importance of local antigen for reactivation of uveitogenic T cells and the subsequent recruitment of inflammatory cells, we used immunogenic uveitis, another model for uveitis (7), in which T cells specific for foreign proteins can induce inflammation of the eye if their respective antigen is injected intraocularly. Here we used OVA-specific GFP+ T cells and detected uveitis only in those eyes that had received injections of OVA into the anterior chamber. Our data show that activated T cells could easily pass the bloodretina barrier, irrespective of their antigen specificity, but only T cells that recognized their specific antigen in the eye were reactivated and enabled to induce uveitis. Recognition of the specific antigen is thus sufficient to induce uveitis and to overcome the immunosuppressive features of the ocular immune privilege.
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Methods
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Generation of GFP+ antigen-specific T cell lines
Lymph node cells from rats immunized with peptide R14 (aa 11691191) from bovine interphotoreceptor retinoid-binding protein or chicken ovalbumin (OVA, Sigma, Deisenhofen, Germany) for establishing T cells lines were obtained as described elsewhere (8). Transduction of T cells for GFP expression with a retroviral vector construct was performed according to Flügel et al. (6), and GFP+ T cells were selected with G418 (Life Technologies, Karlsruhe, Germany) and propagated as described (9). Antigen specificity of the T cell lines was tested by proliferation measured as [3H]thymidine incorporation. Cells were immediately transferred into rats 23 days after the fourth restimulation with antigen in vitro, or stored frozen and thawed immediately before adoptive transfer. Activated transduced T cells strongly express GFP, which is partially down modulated if cells are not activated (6).
Animals
Male and female Lewis rats were purchased from Janvier (Le-Genest-St Isle, France), Charles River (Sulzfeld, Germany) or bred in our own colony. They were housed in the animal facilities of the Department of Surgery or Institute for Surgical Research, University of Munich, and had unlimited access to standard rat chow and water. Rats were used for experiments at the age of 624 weeks. The experiments were approved by the Review Board of the Government (Regierung von Oberbayern) and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research.
Animal procedures
Anesthesia
Rats were anesthetized using 0.5 mg/kg Medetomidine (Domitor®) with or without 0.6 ml/kg Ketamine® 10% (both Pfizer, Karlsruhe, Germany) i.m. Additional topical anesthesia with 0.4% Oxybuprocaine eye drops (Dr Mann Pharma, Berlin, Germany) was applied. Sedation was antagonized with an i.m. injection of 2.5 mg/kg Atipamezol (Antisedan®, Pfizer).
Adoptive transfer of experimental autoimmune uveitis (EAU)
2.3x106 antigen-specific GFP+ cells were injected intravenously in naive Lewis rats. Cell lines were specific for IRBP peptide R14 or OVA. Experiments were terminated 5 days later. Organs and heparinized peripheral blood were immediately processed and stained for FACS analysis.
Induction of immunogenic uveitis
Immunogenic uveitis was induced by intraperitoneal injection of 2.3x106 GFP+ T cells specific for OVA. One day later, an injection of 6 µg OVA in 10 µl PBS (phosphate-buffered saline, pyrogen free) through the peripheral cornea into the anterior chamber of the right eye followed. The left eye received 6 µg/10 µl of bovine serum albumin (BSA, Sigma) and served as an internal control. Experiments were terminated on day 4 and organs and heparinized blood processed for FACS analysis.
Intravital fluorescence microscopy of iris
For excitation, a 75 W xenon lamp and a galvanometric scanner (450 nm, Polychrome II, Till Photonics, Germany) were attached to an Olympus BX50 upright microscope equipped with 10x and 20x water immersion lenses. Images were recorded by an analog video recorder and a black and white analog CCD camera (COHU 4920).
To prevent mydriasis, 0.5 % pilocarpine eye drops were applied. Vidisic gel (both Dr Mann Pharma, Berlin, Germany) was used to optically couple the objective front lens to the cornea of the rat eye. A masked observer scanned for GFP+ cells in a standardized way, screening the iris in a meandering fashion and recording short movie sequences of each field of view (631 x 474 µm at 10x objective magnification). The extent of infiltration by GFP+ cells was scored using the system summarized in Table 1. Similar to clinical and histological grading systems in humans and animal models, the scoring described here is non-linear. Representative areas of the iris were videotaped. Experiments were terminated after 5 days and organs collected and processed either for histology or for FACS analysis.
FACS analysis
T cell lines prior to adoptive transfer as well as single cell suspensions of minced spleens, inguinal and popliteal lymph nodes, uveas and peripheral blood lymphocytes, pooled from all animals of respective experimental groups were incubated on ice for 30 min with antibodies specific for various surface markers (anti TCR
/ß: R73; anti CD4: W3/25; anti MHC class II RT1.B: Ox6; CD25: anti IL-2R
, OX-39; and anti CD134: OX40; all BD, Heidelberg, Germany) all diluted 1:100 in PBS/1% FCS. After washing, the cells were subsequently stained for 45 min with a secondary goat anti-mouse IgG antibody coupled to RPE-Cy5 diluted 1:50 (6). Cells were fixed with 1% paraformaldehyde in PBS and analyzed with a Becton Dickinson FACScalibur using CellQuest software.
Histology
Tissue collected for histology was fixed in 4% paraformaldehyde in PBS and stored overnight at 4°C in the dark. After 24 h, tissue was transferred to 15% sucrose in PBS for another 24 h. Then specimens were embedded in TissueTec OCT compound (Sakura, Zoeterwoude, Netherlands) and snap frozen at 70°C. Cryosections were directly embedded with Immuno Fluore mounting medium (ICN, Eschwege, Germany) and viewed using phase contrast. Some sections were fixed for 15 min in ice-cold acetone, and nuclei counterstained with hematoxilin.
EAU grading
Severity of uveitis was clinically graded using an ophthalmoscope for illumination as described elsewhere (10). Briefly, score 0.5 indicates dilated iris vessels, partial inflammatory infiltrates of iris rim and hazy anterior chamber; score 1: complete circular infiltration of iris rim; score 2: pupil area completely filled with cells and fibrin; score 3: formation of hypopyon; score 4: anterior chamber completely filled with cells, fibrin and blood.
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Results
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Characterization of GFP+ T cell lines in vitro
GFP-transduced T cells were analyzed by FACS for expression of various surface markers prior to injection. More than 97% of GFP+ cells expressed TCR
/ß and CD4 (Fig. 1A and B), while class II (RT1.B, Ox6; Fig. 1C) was only marginally detectable. Activation markers CD25 (IL-2R
; Fig. 1D) and CD134 (OX40; Fig. 1E) were expressed on >99% of cells, indicating a state of high activation. After in vitro cultivation with medium containing IL-2, all cells upregulated CD25. Due to previous antigen-specific activation in vivo by immunization and in vitro by multiple cycles of antigen-specific stimulation and IL-2 mediated expansion these cells are not regulatory T cells, which is also demonstrated by their capability to induce disease after adoptive transfer. Both R14- and OVA-specific T cell lines had similar patterns of surface marker expression. The cell lines were highly specific for their respective antigens as determined by [3H]thymidine incorporation. Stimulation index (SI) of R14-specific line: 13.0 for R14; 1.01.8 for medium, OVA, the retinal peptide PDSAg (8) and four other unrelated control antigens; SI of the OVA-specific line: 5.8 for OVA, 0.81.2 for medium, R14, the retinal peptide PDSAg and four other unrelated antigens.

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Fig. 1. Representative FACS analysis of surface marker expression of GFP+ T cells prior to adoptive transfer. Gates were set to include GFP+ cells only. Repeated antigen stimulation in vitro resulted in >95% of cells expressing TCR /ß (96.9%) (A), CD4 (97.5%) (B). While only 18.4% of cells were positive for MHC class II (RT1.B) (C), the activation markers CD25 (99.0%) (D) and CD134 (99.1%) (E) were strongly upregulated on all cells. (F) GFP expression of in vitro stimulated T cell line.
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EAU
Clinical and IVFM score
Adoptive transfer of GFP+ T cells specific for IRBP-peptide R14 resulted in uveitis after 72130 h (35 days, average: 3.7 days) in all eyes. After 74 h, the average clinical score was 0.33, ranging from 0.5 to 1. Five days (120 h) post cell transfer, all animals had developed clinical disease. Control animals injected with OVA-specific GFP+ T cells remained negative (Fig. 2A). While the clinical score considers inflammatory cells that have left the iris tissue and infiltrated the anterior chamber, the IVFM score is only related to the number of GFP+ cells infiltrating the iris (Fig. 2B). Eyes were examined by IVFM at multiple time points starting at 30 min until 5 days after i.v. injection of antigen-specific GFP+ T cells. We screened iris tissue and anterior chamber, which is the part of the eye that is also considered for conventional clinical grading of rat EAU. As early as 30 min after injection, the first GFP+ cells were detected in the iris tissue (Fig. 3). Both R14- and OVA-specific T cells showed identical patterns, invading the iris tissue. These cells were not traveling in the vascular circulation or rolling along the vessel walls, but were extravascularly located in the tissue. Over the following 52 h (day 2) we observed a slight increase in cellular infiltrates, irrespective of the antigen specificity of injected GFP+ T cells using IVFM, although none of the animals developed any clinical signs of uveitis (Figs 2A and B, and 3).

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Fig. 2. EAU. (A) Time course of clinical score of adoptively transferred uveitis after i.v. injection of GFP+ R14-specific T cells (uveitogenic; dotted line, open symbols) and OVA (control; black line, closed symbols). Onset of uveitis was seen after 3 days (72 h) in eyes of animals injected with R14-specific T cells. (B) Time course of infiltration of GFP+ cells in the iris tissue after i.v. injection of GFP+ transduced T cells specific for R14 (dotted line, open symbols) and OVA (black line, closed symbols). Asterisks indicate significant differences between the groups [P < 0.005, MannWhitney test, n = 24 eyes (A), or 46 eyes per group (B)].
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Fig. 3. EAU. Intravital fluorescence microscopy at different time points following i.v. injection of GFP+ T cells specific for OVA (control) or R14 (uveitogenic). White dots represent fluorescent cells; blood vessels are depicted in black. Seventy-four hours: onset of uveitis in eyes of rats injected with R14-specific T cells.
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Despite the onset of clinical signs of disease observed on the third day (74 h) in the group receiving R14-specific T cells (three of five eyes had clinical sores of 1.0), the IVFM score was indistinguishable from that of the healthy control group (Fig. 2B). While the average clinical score markedly increased from 0.33 to 1.0 (99 h) and 1.4 (120 h) in the group injected with R14-specific cells, the IVFM score slightly raised from 3.5 (74 h) to 4.5 (99 h) and 4.3 (120 h), respectively (Fig. 2B), indicating an accumulation of GFP+ cells in uveitic eyes, predominantly in the iris, the aqueous humor of the anterior chamber and the anterior surface of the lens. In contrast, the IVFM score of eyes from rats injected with OVA-specific T cells drastically decreased from 3.25 (74 h) to 1.25 (99 h) and 1.75 (120 h), while the eyes of these rats were clinically negative at any time point.
Histology
In the EAU model, experiments were terminated 5 days (120 h) after adoptive transfer of cells, when all eyes of animals injected with R14-specific T cells had turned positive as determined by clinical examination. Eyes were processed for histology after they had developed clinical signs of uveitis. GFP-labeled cells could easily be detected due to their bright fluorescence (Fig. 4). Inflammatory cells have infiltrated the anterior chamber and adhered to iris and anterior lens surface as well as to the corneal endothelium. Only a minority of these cells were GFP-positive. The majority of infiltrating cells in rat eyes with EAU are macrophages as previously shown by others (11) and us (M. Diedrichs-Möhring et al., submitted for publication). GFP-transduced cells were not only found in the anterior segment, but also clustered within the ciliary body and dispersed within all layers of the retina and the vitreous. These findings in anterior as well as posterior segments are typical pathologic signs of uveitis in animal models and humans.

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Fig. 4. EAU. Fluorescence microscopy of anterior eye segment (A) and retina (B) from a rat adoptively transferred with R14-specific GFP+ T cells (clinical score of anterior chamber disease: 1). (A) Many inflammatory cells have invaded the iris and anterior chamber, but only few were GFP+ (arrowheads). (B) Retinal destruction and inflammatory infiltrate of the posterior eye segment.
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FACS analysis of surface markers on GFP+ cells
Eyes, spleens, lymph nodes and blood were collected, transferred to ice-cold PBS and immediately processed at 4°C for FACS analysis. In the eyes of animals adoptively transferred with R14-specific GFP transduced T cells we found significantly more GFP+ cells than in eyes from animals that had received OVA-specific GFP+ T cells (Fig. 5A). The other samples (spleen, lymph nodes and peripheral blood) revealed no differences between animals injected with the uveitogenic (R14) or the control cell line (OVA) (Fig. 5A). The GFP+ cells in spleen, lymph nodes or blood, irrespective of their antigen specificity, expressed TCR
/ß and CD4 (Fig. 5BD), whereas TCR
/ß expression was downregulated compared to CD4 in most compartments. The expression of activation markers CD25, MHC class II and CD134 was increased on GFP+ cells in uveitic eyes compared to cells from eyes of rats injected with OVA-specific cells (Fig. 5E). Interestingly, we observed stronger expression of CD25 and CD134 in the spleen (Fig. 5C) and also to a lesser extent in peripheral blood (Fig. 5B) as compared to non-uveitic eyes, but in these compartments there was no statistically significant difference between animals receiving R14- or OVA-specific T cells. However, the only significant difference of activation marker expression on R14- and OVA-specific GFP+ cells was observed in uveitic vs unaffected eyes (Fig. 5E). Figure 5 shows representative data from independently repeated experiments.
Immunogenic uveitis
Clinical score
Simultaneously, 1 or 2 days after intraperitoneal injection of GFP+ OVA-specific T cell lines we injected OVA into the anterior chamber of the right and BSA into the left eyes. One day later intraocular inflammation developed only in those eyes that were injected with OVA (Fig. 6). None of the animals receiving OVA-specific T cells and intraocular OVA at the same time developed uveitis. The eyes injected with BSA as unrelated control antigen remained negative. Experiments were terminated 45 days after adoptive transfer of OVA-specific cells.

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Fig. 6. Immunogenic uveitis: time course of average clinical score from nine rats per group. 2.6 x 106 OVA-specific GFP+ cells were injected i.p.; OVA and BSA were inoculated into the anterior chamber of the right and left eye 24 h later. Disease developed in OVA-injected (dotted line and open symbols) but not in BSA-injected eyes (filled symbols, control antigen). Asterisk indicates statistical significance (P < 0.05, MannWhitney test).
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Histology
Inflammation with infiltration of mononuclear cells in iris, ciliary body and anterior chamber was only observed in eyes that were injected with OVA into the anterior chamber (Fig. 7B). GFP+ cells were among the infiltrates (Fig. 7A). In addition we found infiltration of inflammatory cells and GFP+ cells in the retina, focal destruction of photoreceptors, vasculitis and vitritis (Fig. 7C and D), although antigen was injected only into the anterior chamber. The injection site at the corneal limbus was recognized by histology as a mild local inflammation with GFP+ infiltrates, but only in those eyes that had also developed uveitis (not shown). Control eyes injected with BSA showed no signs of inflammation (data not shown).

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Fig. 7. Immunogenic uveitis. Histology and fluorescence micrographs of uveitic and control eyes. (A and B) Infiltrates of the anterior segment [fluorescence microscopy (A) and phase contrast (B) of the same section]. The white line marks the iris. (A) White dots represent GFP+ cells. Some GFP+ cells have infiltrated the iris tissue. Most inflammatory cells in the anterior chamber were negative for GFP. (C and D) Posterior pole of the same eye (serial sections) was also involved in inflammation. Only few inflammatory cells were GFP+ and have infiltrated the inner retinal layers (C). Vasculitis and inflammatory infiltrates in the vitreous (D).
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FACS analysis
Experiments on immunogenic uveitis were terminated 4 days after injection of GFP+ OVA-specific T cells and 3 days post antigen injection into the anterior chamber. Many GFP+ cells had infiltrated the eyes that were injected with OVA, while from eyes receiving the control antigen BSA only very few, barely detectable, numbers of GFP+ cells were recovered (Fig. 8A) indicating that the surgical procedure of intraocular injection does not severely alter the bloodretina barrier. The percentage of GFP+ cells among total lymphocytes in control eyes was even lower than in other investigated organs (spleens, inguinal lymph nodes and blood). TCR
/ß and CD4 were expressed on the majority of GFP+ cells in all organs investigated. In peripheral blood and spleen, activation markers such as MHC class II, CD25 and CD134 were down regulated on most GFP+ cells (Fig. 8B and C). In cells recovered from lymph nodes (Fig. 8D) >50% expressed MHC class II and CD25 and >30% were positive for CD134. In contrast, all tested activation markers were detected on >60% of GFP+ cells recovered from OVA-injected eyes (Fig. 8E), whereas in BSA-injected eyes the number of activated OVA-specific GFP+ cells was significantly reduced (P < 0.05, paired t-test). The data displayed in Fig. 8 represent single measurements of three independent reproducible experiments.

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Fig. 8. Immunogenic uveitis. FACS analysis of pooled blood and organs from six rats per group 4 days post injection of OVA-specific GFP+ T cells. (A) Percentage of GFP+ cells of lymphocytes (data extracted from five FACS scans for GFP/surface marker double positive cells in panels BE). (BE) Surface marker expression on GFP+ cells from different organs. Expression of activation markers MHC class II (RT1.B), CD25 and CD134 on GFP+ cells was significantly increased in uveitic right eyes (light columns) compared to control-injected left eyes (E, black columns). P < 0.05, paired t-test.
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Discussion
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Here we investigate the migration of activated T cells in the induction of uveitis in two different experimental models. Instead of conventional biochemical staining of cells, we used antigen-specific T cells recombinantly expressing GFP, which allows tracking of injected T cells for an extended period of time without alteration of function or loss of the fluorescent dye due to proliferation. The expression of GFP does not impair the capacity of T cells to induce disease, which has extensively been investigated in the model of experimental autoimmune encephalitis in rats (6). In our system we did not see any differences in the pathogenicity of GFP transfected versus non-transfected cells (unpublished observation). GFP+ cells in the anterior segment of the eye can be observed by intravital fluorescence microscopy without the need of surgically creating a window and disturbing the tissue's microenvironment, due to the clarity of the optical structures of the eye (cornea, anterior chamber fluid). We found that activated T cells of any given antigen specificity (here: peptide R14 or OVA) have the capability to immigrate into ocular tissue within 30 min after injection into the tail vein. Prendergast et al. have made similar observations in retinal whole mounts using the membrane bound lipophilic fluorescent dye PKH26 to detect the cells (12).
Others have investigated the retina during EAU development using different techniques and differently activated T cells. Hossain et al. have demonstrated by laser scanning ophthalmoscopy that ConA-activated T cell blasts labeled with 6-carboxyfluorescein diacetate have become stationary in the retina and choroid within 4 s of injection into the carotid artery (13). This is much earlier than the increase in T cell numbers observed by Prendergast et al. who used a histological approach and found a biphasic infiltration with a primary extravasation of cells in the retina after 12 h, a peak after 24 h, a steady decline until 72 h and a secondary influx at the onset of clinical uveitis.
In our model of adoptively transferred EAU, the typical clinical signs of disease developed in the eyes of animals 74 h post injection of a uveitogenic R14-specific T cell line. This is in accordance with previously published data (12,14) and our own observations in many experiments (unpublished). From this time point the number of GFP+ cells increased only in those eyes (iris and anterior chamber) that developed uveitis, while in eyes of animals that had received a non-uveitogenic control line, the cell numbers decreased steadily until day 5 (120 h), which was the longest observation time in these experiments. Since the numbers of GFP+ cells increased only slightly prior to the development of uveitis but dramatically with the clinical onset of uveitis, it seems that low numbers of T cells are sufficient as primary inducers of uveitis and that they later comigrate with the unspecific inflammatory infiltrate (15).
The time course seen in anterior and posterior disease in adoptively transferred EAU is different in the presumed sister model of experimental autoimmune encephalomyelitis (6,16). While uveitogenic T cells specific for S-antigen peptide (12) or IRBP peptide as well as non-pathogenic OVA-specific T cells infiltrated iris and retina very early within the first hour, it seems that the CNS is infiltrated by encephalitogenic MBP-specific T cells only 6080 h post transfer, although very few cells have been detected at earlier time points (17,18). This CNS infiltrate precedes the clinical signs of demyelination and is not seen in the CNS of rats injected with non-pathogenic OVA-specific T cells (16). The difference in the two organs may be explained by variations in the blood organ barriers.
Upregulation of activation markers CD25 and CD134 was only observed on GFP+ cells that caused inflammation. We could show here that activated T cells have unrestricted access to the eye, easily cross the bloodeye barriers and cause inflammation after reactivation by local antigen recognition. Reactivation of retina autoantigen-specific T cells within the eye points to an important role of local antigen. To prove that the presence of antigen is a necessary prerequisite we used the model of immunogenic uveitis, in which the respective antigen has to be injected into the anterior chamber through the cornea, after activated, non-self antigen-specific GFP+ T cells have been transferred intraperitoneally. We found an activation pattern comparable to EAU on OVA-specific GFP+ cells recovered from uveitic eyes that were injected with OVA. BSA-injected control eyes, which did not develop uveitis and thus were not infiltrated with inflammatory cells, included very few, but activated GFP+ T cells, which argues against an alteration of the bloodretina barrier. These cells probably represent those activated T lymphocytes that normally transmigrate all tissues (19). The presence and local presentation of a soluble foreign antigen was sufficient to reactivate infiltrating T cells, which then initiated intraocular inflammation presenting as uveitis. Of interest was also the observation of retinal involvement in immunogenic uveitis induced by anterior chamber injection. In histological sections the proportion of GFP+ and other inflammatory cells in the retina of OVA-induced immunogenic uveitis is very similar to that seen in EAU. This leads to the conclusion that antigen from the anterior chamber might diffuse or is transported by antigen-presenting cells from the iris to the posterior pole, where it induces local inflammation. This is the opposite situation to EAU, which is induced with antigens from the photoreceptor layer, such as S-Ag or IRBP, which both induce anterior as well as posterior uveitis in rats. Whereas mRNA for S-Ag was detected in iris tissue (20), no expression of IRBP in the iris has been described so far. Nevertheless, it is a consistent observation that photoreceptor-derived antigens induce inflammation in the anterior chamber (8,21).
Our FACS analysis has shown that in eyes with uveitis, the number of GFP+ cells is highly increased compared to healthy eyes or lymphoid organs. Several mechanisms contribute to this finding. In response to antigen recognition, T cells are restimulated not only to secrete cytokines but also to proliferate. Since GFP is constitutively expressed in these cells its concentration will not diminish with division and therefore proliferation cannot be determined by reduced fluorescence intensity. In addition to an increase in their number by proliferation, T cells will largely be protected from apoptosis upon restimulation, both leading to accumulation of GFP+ cells in the eye. Another explanation for increased numbers of GFP+ cells could be the specific retention of locally reactivated cells in the tissue.
In both models of transferred disease, EAU and immunogenic uveitis, clinical disease with the typical signs of infiltrates in the iris, hyperemia and cellular extravasations to the iris rim, pupil and into the anterior chamber, became visible only 3 days after adoptive cell transfer, although potentially pathogenic GFP+ cells have invaded the iris tissue already a few minutes following injection. We can only speculate about the delay of clinical disease. At least the presence of antigen seems to be required. Day 3 after adoptive transfer of T cells seems to be the time point at which their fate becomes obvious: they either cause clinical signs of inflammation and increase in numbers or gradually disappear and do not cause inflammation. The immunogenic uveitis model as well as the adoptive transfer of T cells specific for a control antigen supports this notion, since eyes injected with control antigen did not become uveitic. During development of clinical disease at day 3 the numbers of GFP+ antigen-specific T cells increased slightly but not proportionally to the influx of host-derived inflammatory mononuclear cells. Histology revealed that only a small percentage of the cellular infiltrates were GFP+. We propose that these inflammatory cells are not specific for retinal autoantigens, for it is almost impossible to activate so many naive lymphocytes to ocular autoantigen within 3 days after adoptive transfer of immunogenic T cells. During these days the bloodeye barrier is still grossly intact and thus prevents entry of non-activated cells. This is in concordance with previously reported findings (12,22). With the breakdown of the bloodeye barriers, macrophages and other leucocytes can easily enter the ocular tissue. This might be achieved by the secretion of multiple cytokines, chemokines and upregulation of adhesion molecules, which promote the invasion of T cells and other inflammatory cells as an amplification mechanism ultimately leading to the clinical diagnosis of uveitis.
Our observation described here may have consequences for the therapy of uveitis. The treatment of acute uveitis requires an immediately acting, unspecific anti-inflammatory remedy such as corticosteroids, which are used due to their broad inhibitory activity on many different cells involved in inflammation. However, immunoregulatory therapies have no direct effect on the bloodeye barrier or inflammatory cells and will not control the acute episode, but might have an important impact on the prevention of further relapses, which in turn are initiated by antigen-specific T cells.
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Acknowledgements
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We thank Mrs Isabella Rädler-Angeli for outstanding technical assistance, Dr Hans-Peter Scheuber for access to the animal facilities and Professors Anselm Kampik and Hartmut Wekerle for their support. Deutsche Forschungsgemeinschaft (DFG) funded this work through SFB 571 and SFB 455 (A.F.).
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Abbreviations
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EAU | experimental autoimmune uveitis |
GFP | green fluorescent protein |
IRBP | interphotoreceptor retinoid binding protein |
IVFM | intravital fluorescence microscopy |
OVA | ovalbumin |
RT1.B | rat MHC class II antigen |
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Notes
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Transmitting editor: T. Hünig
Received 2 January 2004,
accepted 6 August 2004.
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