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Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Center, University of Louisville, Louisville, KY 40202
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The adoptive transfer of autoreactive T cells has a number of advantages over active immunization, including avoidance of the use of adjuvant, mycobacterium, or pertussis toxin. In addition, in several experimental models the autoreactive T cell subsets elicited by an autoantigen differ greatly in their disease-inducing ability and immunoregulatory activity (11, 12, 13). In the present study we have shown that the adoptive transfer of T cell subsets specific for residues 1177–1191 of IRBP (peptide R16) into naive Lewis rats induced a chronic relapsing uveitis, rather than the monophasic disease induced by immunization with R16 emulsified in CFA. The model of recurrent uveitis induced by the transfer of autoreactive T cell subsets was determined by both the number of autoreactive T cells injected and their activation state. This experimental model should help greatly in the study of the pathogenesis of recurrent uveitis in humans.
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Materials and Methods |
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Pathogen-free female Lewis rats (5–6 wk old) were purchased from Harlan Sprague Dawley (Indianapolis, IN) and were housed and maintained at the animal facilities of the University of Louisville.
All animal studies conformed to the Association of Research in Vision and Ophthalmology statement on the use of animals in Ophthalmic and Vision Research. Institutional approval was obtained, and institutional guidelines regarding animal experimentation were followed.
Animal model of EAU
The induction of uveitis in Lewis rats using peptide R16 has been previously described (13, 14). Briefly, the rats were immunized s.c. with 200 µl of an emulsion containing 30 µg of R16 and 500 µg of Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI) in CFA (Sigma-Aldrich, St. Louis, MO), distributed over six spots on the tail base and flank.
For induction of uveitis by adoptive transfer of R16-specific T cells (tEAU), naive Lewis rats were injected i.v. either with in vitro restimulated R16-specific T cells prepared from rats with uveitis (induced either using Ag immunization or cell transfer) or with R16-specific T cell lines in 0.5 ml of PBS and were examined daily for clinical signs of uveitis by slit-lamp biomicroscopy. The intensity of uveitis was scored blind on an arbitrary scale of 0–4 (13), with 0 as no disease, 1 as engorged blood vessels in the iris and an abnormal pupil configuration, 2 as a hazy anterior chamber, 3 as a moderately opaque anterior chamber with the pupil still visible, and 4 as an opaque anterior chamber, obscured pupil, and frequently proptosis.
Inflammation in the eye was confirmed by histopathology. Many inflammatory cells were seen in both anterior and posterior chambers together with a disorganized retinal architecture, retinal detachment, and photoreceptor cell damage.
R16-specific T cell lines
R16-specific T cell lines were isolated from R16-immunized Lewis rats, using the methods of generating Ag-specific T cells as previously described (15, 16, 17, 18). T cells were isolated 10 days postimmunization (p.i.) from lymph node cells or spleen cells by passage through a nylon wool column. The cells (1 x 107) were first stimulated with 20 µg/ml of R16 in 2 ml of medium in a six-well plate (Costar, Cambridge, MA) with 2 x 107 irradiated syngeneic spleen cells as APCs. After 2 days the activated lymphoblasts were isolated by gradient centrifugation in Lymphoprep (Robbins Scientific, Mountain View, CA) and cultured in RPMI 1640 medium supplemented with 15% IL-2-containing medium (supernatant from Con A-stimulated rat spleen cells). These T cell lines were maintained by periodic restimulation for 48 h (about once every 10 days) with Ag in the presence of irradiated syngeneic APCs. The lines were used after three or four stimulation/resting cycles.
Cell proliferation assay
R16-specific T cells (3 x 104 cells/well) in a total volume of 200 µl were cultured at 37°C for 48 h in 96-well microtiter plates with medium or R16 and irradiated syngeneic spleen APCs (2 x 105), and [3H]thymidine incorporation during the last 8 h was assessed using a microplate scintillation counter (Packard Instruments, Meriden, CT). The proliferative response was expressed as the mean counts per minute ± SD of triplicate determinations.
Isolation of cells from inflamed eyes
After perfusion of the anesthetized rat with PBS on the indicated day after immunization or injection of T cells, the eyes were collected, and a cell suspension was prepared by digestion for 10 min at 37°C with collagenase (1 mg/ml) and DNase (100 µg/ml) in RPMI 1640, followed by gradient centrifugation on 25% Percoll and subsequent Ficoll separation; in some experiments, cells separated only by Percoll were used, as indicated in the text. The cells were washed and resuspended in staining buffer (PBS containing 3% FCS and 0.1% sodium azide) for Ab staining. The cells obtained using the full separation procedure consisted mainly of inflammation-recruited immune cells as well as a minor population of intraocular resident macrophages and dendritic cells.
Immunofluorescence flow cytometry
Aliquots of 2 x 105 cells were double-stained with combinations of FITC- or PE-conjugated mAbs against rat 
TCR (R73), NK cells (NKR-P1a), macrophages (ED1), dendritic cells (OX-62), or bone-marrow-derived cells (CD45). All Abs were purchased from BD Bioscience (La Jolla, CA). Data collection and analysis were performed on a FACSCalibur flow cytometer using CellQuest software.
Pathological examination
For histology, whole eyes were collected, immersed for 1 h in 4% phosphate-buffered glutaraldehyde, then transferred to 10% phosphate-buffered formaldehyde until processed. The fixed and dehydrated tissue was embedded in methacrylate, and 5-µm sections were cut through the pupillary-optic nerve plane and stained with H&E. The presence or the absence of disease was evaluated blind by examining six sections cut at different levels for each eye. The severity of EAU was scored on a scale of 0 (no disease) to 4 (maximum disease) in half-point increments, as described previously (13).
Statistical analysis
The data are expressed as the mean ± SD. Each experiment was repeated at least three times. Student’s t test was used to analyze the results.
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Results |
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Lewis rats immunized with R16 developed acute and monophasic uveitis, showing overt clinical symptoms at 8–9 days p.i. Maximum disease was observed in animals immunized with 
30 µg of peptide/rat. The clinical signs observed by slit-lamp biomicroscopy included engorged iris blood vessels, abnormal pupil configuration, and hazy anterior chamber. The disease persisted for 7–10 days. No spontaneous recurrence was observed. Fig. 1 illustrates the histopathology of the inflamed eye in Lewis rats at the peak (12 days p.i.) of the monophasic disease. Inflammatory cell infiltrates were present in both anterior and posterior chambers (Fig. 1C), the vitreous, and the retina (Fig. 1D). Damage to the photoreceptor layer, retinal folding, and retinal vasculitis were also observed (Fig. 1D).
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In initial experiments uveitogenic T cells were prepared from the draining lymph nodes and spleens 10–14 days after active immunization with R16 and were stimulated for 3 days with R16 in the presence of syngeneic APC. The Ficoll-isolated T cell blasts (3 x 106/rat) were then injected i.v. into naive Lewis rats. Over the following 80 days the majority of the recipient rats developed acute uveitis with several recurrences. Each recurrent episode lasted 1–7 days and was followed by varying periods of remission. Fig. 2 shows a typical experiment in which six recipient rats received 3 x 106 R16-specific T cells and showed a similar, but not identical, pattern of relapsing EAU with neither the time nor the duration of the relapse predictable. Interestingly, the right and left eyes of a single recipient showed a different pattern of relapse (data not shown), analogous to that frequently observed in human disease.
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Recurrent uveitis after the adoptive transfer of uveitogenic T cells is dose dependent
To determine whether recurrent uveitis was dose dependent, we injected different numbers (0.5, 1, or 3 x 106) of a uveitogenic T cell line (IRBP-3) into groups of naive Lewis rats. The results showed that transfer of 1 or 3 x 106 T cells consistently induced acute uveitis in all recipient rats, whereas 0.5 x 106 T cells induced disease in only 50%. Furthermore, only rats receiving 3 x 106 autoreactive T cells consistently showed relapsing disease, whereas 0.5 x 106 T cells induced only monophasic disease. Those animals who received 1 x 106 cells developed either monophasic or recurrent disease (Table IIA).
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Based on our previous observations in EAE that autoreactive T cell lines are only encephalitogenic within 3 days after each in vitro restimulation, we tested whether the uveitogenic activity of R16-specific T cell lines was similarly restricted by the activation status of the T cells. An R16-specific T cell line (IRBP-3) was stimulated in vitro for 2 days with an optimal dose (10 µg/ml) of R16 and APCs. The activated T cell blasts were then harvested and cultured in IL-2-containing medium for an additional 0, 3, or 5 days before being collected and injected into naive recipient rats (3 x 106 cells/rat). As shown in Table 2B, when cells were used immediately after in vitro restimulation (day 0) all recipients developed uveitis, whereas T cells injected 3 or 5 days after restimulation induced EAU in only some or none, respectively. Interestingly, only T cells used immediately after restimulation induced recurrent EAU.
Cellular infiltration in monophasic and recurrent uveitis
We then studied whether the number and/or pattern of eye-infiltrating cells differed between the acute (day 6 p.i.) and the relapsing (second relapse, day 30 p.i.) phase of EAU induced by the transfer of 3 x 106 IRBP-3 T cells. Histological examination (Fig. 4) of the eyes did not show a significant difference between the two phases of the disease, except that the damage to the photoreceptor layer was more severe (Fig. 4D), and mild cellular infiltration never resolved in recurrent disease (not shown). Thus, in monophasic EAU there were no clinical signs or histologic evidence of inflammation upon resolution of the disease, whereas in recurrent EAU, although there were no clinical signs of inflammation upon resolution of disease, there was mild chronic inflammation on histologic examination.
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TCR), macrophages (CD11b/c, ED1), dendritic cells (OX-62), NK/NKT cells (NKR-P1a), and hemopoietic (nonparenchymal) cells (CD45+) and analyzed by flow cytometry, recurrent EAU (day 30) was found to be associated with increased infiltration of NKT cells and a decrease in macrophages/dendritic cells (Fig. 5, 2D-2F) compared with acute EAU (day 6, Fig. 5, 2A–2C). It was also noted that the NKR-P1a+TCR+ (NKT) cell profile became more heterogeneous when examined by FACS analysis (Fig. 5, 2D) during the relapsing phase of the disease.
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In parallel with the chronic cellular infiltration observed in recurrent EAU, R16-specific T cells were consistently detected in the spleens of recipient rats for 3–6 mo after the injection of a single dose of 3 x 106 cells. This contrasted sharply with rats actively immunized with R16, in which R16-specific T cells were undetectable following the acute phase of the disease (>30 days p.i.; Fig. 6A). Limiting dilution assays showed that the frequency of R16-specific T cells in the periphery ranged from 1/5,000 to 1/10,000 in animals at the peak of a relapse, whereas animals with monophasic disease after active immunization had many fewer autoreactive T cells (data not shown). Importantly, after in vitro restimulation, splenic T cells from rats that had received 3 x 106 R16-specific IRBP-3 T cell line cells 60 days previously still proliferated in response to R16 (Fig. 6A) and were uveitogenic when adoptively transferred into naive recipients (Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals were either actively immunized with R16 or adoptively transferred with R16-specific T cells and closely monitored for >80 consecutive days. Surprisingly, most of the animals with uveitis induced by the adoptive transfer of T cells experienced several recurrent episodes of EAU, whereas relapses were not seen in actively induced uveitis, i.e., the disease was monophasic.
To explain the chronic relapsing nature of EAU observed after adoptive transfer, we considered the possibility that adoptive transfer provided a larger number of highly activated autoreactive T cells than active immunization. This idea is supported by the previous observations that although BALB/c mice and Fischer 344 rats are resistant to the active induction of autoimmune encephalomyelitis (EAE), they are susceptible to adoptively transferred EAE (20, 21, 22). It is likely that these so-called resistant strains generate fewer autoreactive T cells on Ag immunization than autoimmune-prone strains and that the injection of sufficient autoreactive T cells renders recipients susceptible. Thus, we injected different numbers of R16-specific T cells into naive recipient Lewis rats. Interestingly, the recipient rats receiving 3 x 106 R16-specific T cells consistently developed chronic relapsing uveitis, which was associated with early disease onset, whereas rats receiving lower numbers (0.5 x 106) of IRBP-specific T cells showed only monophasic disease and later disease onset.
We also tested the hypothesis that a weaker autoimmune attack induced monophasic disease, whereas a stronger autoimmune attack resulted in relapsing disease, by injecting recipient rats with R16-specific T cells of varying activation status. The rationale for this experiment is that Ag-specific T cell lines require periodic in vitro restimulation and expansion in IL-2-containing medium, and cells harvested at different times after in vitro restimulation have different activation status. R16-specific T cell lines were restimulated in vitro with an optimal dose of Ag and APC and were then maintained in IL-2-containing medium for 0, 3, or 5 days before being used for disease induction. Only T cells used immediately after activation (0 days in IL-2 medium) induced chronic relapsing uveitis. After 3 days of maintenance, T cells remained uveitogenic, but only induced monophasic disease, whereas those maintained for 5 days lost uveitogenic activity. It is interesting to note that we have seen similar results in rat uveitis induced by T cells specific for myelin basic protein, which induced both uveitis and encephalomyelitis. In those experiments the encephalitogenic activity was strictly restricted to highly activated T cells (i.e., those maintained for 0 day poststimulation), whereas the same T cells were still uveitogenic 2 days later (D. Sun, S. L. Sun, H. J. Kaplan, and H. Shao, manuscript in preparation). Our results imply that the number of autoreactive T cells and their activation status will determine whether EAU is monophasic or relapsing.
Our study of a panel of R16-specific T cells showed that R16-specific T cell lines vary greatly in their ability to induce monophasic or relapsing uveitis. This observation matches our previous studies of autoreactive T cells induced by myelin proteins (12, 17, 18). It is likely that the pathogenic capability of autoreactive T cell subsets is determined by multiple factors, including the ability of autoreactive T cells to access Ag, enter their target organ, and interact with parenchymal cells of the tissue.
We have noted that the NKR-P1a+TCR+ (NKT) cell profile became more heterogeneous during the relapsing phase of the disease. In a previous report we have shown that NK/NKT cells are the major infiltrating cells of the inflamed eye actively induced by immunization with peptide R16; however, functional tests on eye-derived NK/NKT cells showed increased suppressive and decreased cytolytic activities (23). It remains to be determined whether the infiltration of an excessive number of NK/NKT cell subsets will be associated with increased tissue damage and chronic disease progression.
We have observed that parenchymal cells showed increased granularity and a higher side scatter profile in relapsing disease compared with active immunization (Fig. 5, 1B and 1C), suggesting that local parenchymal cells were activated in recurrent disease in contrast to the monophasic disease seen after active immunization. Such an observation agrees with previous reports that parenchymal cells of the autoimmune organ are not simply the victims of an autoimmune attack and that their functional alteration is closely linked to the course of the disease (24, 25, 26, 27, 28, 29, 30).
Most cases of human uveitis are of unknown etiology and thus are considered to be autoimmune in etiology. The clinical course of intraocular inflammation (i.e., uveitis) in these patients is frequently unpredictable, but is often characterized by recurrent episodes that affect first one eye and then the other. Additionally, although there may be minimal evidence of intraocular inflammation following resolution of an acute episode, the patient’s vision is frequently decreased as the result of cystoid macular edema, which suggests that a low grade, chronic subclinical inflammatory reaction is present, but undetectable. This EAU exhibits resolution of the acute episode with spontaneous recurrences and evidence of chronic, subclinical intraocular inflammation. It should provide important insights into the immunological mechanisms responsible for both the resolution of an acute episode and the subsequent relapse, similar to the human disease.
In summary, we have shown that R16-specific T cells can induce either monophasic or chronic recurrent EAU depending on the autoreactive load, i.e., the number of autoreactive T cells and/or their activation status. The creation of a relapsing model of autoimmune uveitis should allow studies to identify those factors that are important in disease recurrence. Because chronic, relapsing disease is the major cause of the visual disabilities associated with uveitis, this model of recurrent EAU promises to be very important in our understanding and treatment of this disease.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hui Shao, Kentucky Lions Eye Center, Department of Ophthalmology and Vision Sciences, University of Louisville, 301 E. Muhammad Ali Bouleveard, Louisville, KY 40202. E-mail address: hd0shaoun001{at}louisville.edu
3 Abbreviations used in this paper: EAU, experimental autoimmune uveitis; aEAU, experimental autoimmune uveitis induced by immunization of Ag; IRBP, interphotoreceptor retinoid-binding protein; MAA, melanin-associated Ag; p.i., postimmunization; R16, bovine IRBP peptide 1177–1191; tEAU, experimental autoimmune uveitis induced by adoptive transfer of T cells.
Received for publication May 27, 2003. Accepted for publication September 16, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R-Ig treatment blocks actively induced, but not adoptively transferred, uveitis in Lewis rats. Eur. J. Immunol. 33:1736.[Medline]
8 T cell receptors. Eur. J. Immunol. 22:591.[Medline]
cells activates autoreactive T cells in virus-mediated autoimmune diabetes. J Clin. Invest. 109:79.[Medline]
as a result of glia-T cell interaction correlates with the pathogenic activity of myelin basic protein-reactive T cells in experimental autoimmune encephalomyelitis. J. Neurosci. Res. 45:400.[Medline]This article has been cited by other articles:
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  Y. Ke, G. Jiang, D. Sun, H. J. Kaplan, and H. Shao Ocular Regulatory T Cells Distinguish Monophasic from Recurrent Autoimmune Uveitis Invest. Ophthalmol. Vis. Sci., September 1, 2008; 49(9): 3999 - 4007. [Abstract] [Full Text] [PDF]
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 M. Diedrichs-Mohring, C. Hoffmann, and G. Wildner Antigen-dependent monophasic or recurrent autoimmune uveitis in rats Int. Immunol., March 1, 2008; 20(3): 365 - 374. [Abstract] [Full Text] [PDF]
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 N. J. Singh, M. Cox, and R. H. Schwartz TLR Ligands Differentially Modulate T Cell Responses to Acute and Chronic Antigen Presentation J. Immunol., December 15, 2007; 179(12): 7999 - 8008. [Abstract] [Full Text] [PDF]
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Y. Peng, H. Shao, Y. Ke, P. Zhang, G. Han, H. J. Kaplan, and D. Sun Minimally Activated CD8 Autoreactive T Cells Specific for IRBP Express a High Level of Foxp3 and Are Functionally Suppressive Invest. Ophthalmol. Vis. Sci., May 1, 2007; 48(5): 2178 - 2184. [Abstract] [Full Text] [PDF]
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P. Zhang, D. Sun, Y. Ke, H. J. Kaplan, and H. Shao The Net Effect of Costimulatory Blockers Is Dependent on the Subset and Activation Status of the Autoreactive T Cells J. Immunol., January 1, 2007; 178(1): 474 - 479. [Abstract] [Full Text] [PDF]
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Y. Peng, H. Shao, Y. Ke, P. Zhang, J. Xiang, H. J. Kaplan, and D. Sun In Vitro Activation of CD8 Interphotoreceptor Retinoid-Binding Protein-Specific T Cells Requires not only Antigenic Stimulation but also Exogenous Growth Factors. J. Immunol., April 15, 2006; 176(8): 5006 - 5014. [Abstract] [Full Text] [PDF]
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T. Liao, Y. Ke, W.-H. Shao, B. Haribabu, H. J. Kaplan, D. Sun, and H. Shao Blockade of the interaction of leukotriene b4 with its receptor prevents development of autoimmune uveitis. Invest. Ophthalmol. Vis. Sci., April 1, 2006; 47(4): 1543 - 1549. [Abstract] [Full Text] [PDF]
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H. Shao, Y. Peng, T. Liao, M. Wang, M. Song, H. J. Kaplan, and D. Sun A Shared Epitope of the Interphotoreceptor Retinoid-Binding Protein Recognized by the CD4+ and CD8+ Autoreactive T Cells J. Immunol., August 1, 2005; 175(3): 1851 - 1857. [Abstract] [Full Text] [PDF]
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H. Shao, Y. Fu, T. Liao, Y. Peng, L. Chen, H. J. Kaplan, and D. Sun Anti-CD137 mAb Treatment Inhibits Experimental Autoimmune Uveitis by Limiting Expansion and Increasing Apoptotic Death of Uveitogenic T Cells Invest. Ophthalmol. Vis. Sci., February 1, 2005; 46(2): 596 - 603. [Abstract] [Full Text] [PDF]
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H. Shao, S. L. Sun, H. J. Kaplan, and D. Sun Characterization of Rat CD8+ Uveitogenic T Cells Specific for Interphotoreceptor Retinal-Binding Protein 1177-1191 J. Immunol., August 15, 2004; 173(4): 2849 - 2854. [Abstract] [Full Text] [PDF]
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