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Published online before print June 14, 2004
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Department of Ophthalmology, University of Minnesota, Minneapolis
1Correspondence: Department of Ophthalmology, University of Minnesota, Lions Research Building, Rm. 314, 2001 6th Street, S.E., Minneapolis, MN 55455. E-mail: grege001{at}tc.umn.edu
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ABSTRACT |
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Key Words: autoimmunity • antigen presentation • arrestin • uveoretinitis • T cells
Several experimental autoimmune disease models, including experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune uveoretinitis (EAU), have been developed to study inflammatory diseases of the nervous system. Pathogenesis in these models is mediated by CD4 T cells directed against self-antigens (Ags) expressed in brain and retina, respectively. The identities of the cells responsible for local Ag presentation in these models are of interest, not only for their role in the pathogenesis of autoimmune disease but also for their participation in maintaining peripheral tolerance to tissue-specific Ags. Ag-presenting cell (APC) candidates include bone marrow (BM)-derived cells and several types of non-BM-derived cells with inducible major histocompatibility complex (MHC) class II expression [1 2 3 4 ]. Hickey and Kimura [5 ] and Hinrichs et al. [6 ] used rat radiation/BM chimeras to show that EAE expression in T cell adoptive transfer experiments was dependent on a BM-derived population, postulated to be perivascular microglia (PVMG) [5 ]. More recently, the radiation/BM chimera strategy was used to show that a BM-derived population was also required to support the pathogenesis of EAU by transfer of activated T cells specific for a retinal Ag [7 ]. Results like these formed the basis for the widely accepted concept that local, BM-derived APC, probably PVMG, are responsible for the Ag presentation required for expression of EAE and EAU. Although some retinal CD45+ cells express low levels of MHC class II and CD80, functional assays show that they have little in vitro ability to present Ag to naive or Ag-experienced T cells, unlike the CD45+ cells from brain [8 ]. In this report, we re-examine the origin of the APC responsible for adoptive-transfer EAU and ask if BM-derived APC in BM chimeras are required.
EAU is well-known to be mediated by MHC class II-restricted CD4 T cells specific for arrestin (a/k/a S-Ag), a 48,000 molecular weight photoreceptor cell protein, and can be induced in Lewis (LEW) rats by transfer of activated, arrestin-specific CD4 T cells [9
]. The Ag specificity and pathogenicity of the arrestin-specific LEW T cell lines have been described previously [10
]. For adoptive transfer of EAU, the T cell lines were Ficoll gradient-purified after activation for 1–2 days with 5 µg/ml concanavalin A in the presence of irradiated APC and transferred intravenously (i.v.) into recipients. Radiation/BM chimeras were constructed using conventional techniques [5
]. Recipient rats were irradiated (10 Gy) and reconstituted on the same day with BM taken from tibia, femur, and humerus. Recipients received 5–10 x 107 viable cells in 1.0 ml saline intraperitoneally (i.p.). Animals were used 8–11 weeks after BM reconstitution. Some animals were evaluated after 2 months for the presence of chimerism. Donors and recipients were chosen to allow detection of the RT7.1 and RT7.2 alleles of CD45 or LEW-specific MHC class II (OX3) or a common epitope (OX6). Chimerism was confirmed by staining spleen cells for flow cytometry. In contrast to prior studies showing that Lewis x Brown Norway F1 (LBNF1) 
Brown Norway (BN) radiation/BM chimeras were highly susceptible to adoptive transfer EAU [7
] or EAE [5
, 6
] by pathogenic LEW T cells, we found that LBNF1 BN radiation/BM chimeras were substantially less susceptible to EAU induced by T cell transfer, in incidence and severity, than were F1 LEW rats, which were as susceptible as unmanipulated LEW control rats (Table 1
). In several separate experiments, only the occasional LBNF1 BN recipient developed significant, local retinal lesions, in which parts of the photoreceptor layer were destroyed (Fig. 1D
). A control experiment uisng LBNF1 LBNF1-grafted rats also showed substantially reduced susceptibility. In a separate experiment, only four of 12 ungrafted LBNF1 rats developed EAU with an average severity of 0.32, indicating that the LBNF1 host was poorly supportive of EAU development, despite the presence of compatible LEW MHC class II. Disease induction in normal LEW control rats confirmed the pathogenicity of the LEW T cells.
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BUF developed severe EAU that destroyed the entire photoreceptor cell layer (Fig. 1C)
. LBuF1 BUF chimeras were also found to be more susceptible than ungrafted LBuF1 controls. The relative resistance of the ungrafted LBuF1 was confirmed in a separate experiment, where only one of six recipients developed EAU using a different purified LEW T cell line, which was fully (six of six) pathogenic in LEW recipients (data not shown).
The resistance of F1 F1, F1 BN, and F1 BUF radiation/BM chimeras to EAU induced by the T cell line was unexpected, given the previous reports showing that F1 BN chimeras were as susceptible as LEW controls to EAE and also highly susceptible to EAU, although the severity was less than found in LEW recipients [7
]. There is no question of inadequate "reconstitution" using the LBNF1 LBNF1 animals or unmanipulated LBNF1 recipients in our experiments, but they remained resistant. Chimeras were also made using fetal liver to reconstitute lethally irradiated recipients. These chimeras were no more susceptible than those made by grafting BM (data not shown). In summary, none of our stem cell-grafting strategies produced a consistently high level of susceptibility to EAU in allogeneic recipients and suggested that results obtained for the EAE system did not fully generalize to EAU.
The experiments in Table 1
were done with T cells purified from cocultures with irradiated splenocytes that were used to activate the T cells. This is unlike prior studies of EAE using radiation/BM chimeras, where the T cells were not purified or consisted of unseparated spleen or lymph node (LN) cell cultures. As very large numbers of APC would contaminate T cell transfers using these crude cultures, experiments were done to determine if purification of the T cells was a factor that contributed to the minimal pathogenesis found in our radiation/stem cell chimeras. Density gradient purification of the activated LEW T cells prior to adoptive transfer to semiallogeneic LBNF1 BN chimeric recipients resulted in a loss of pathogenicity, relative to the crude, unseparated inoculum, but had no effect on the severity of EAU induced by transfer to the syngeneic LEW recipient (Table 2
). The results show that the T cells are still fully active in syngeneic LEW recipients but that pathogenicity was lost in allogeneic recipients if the T cells were purified.
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In preliminary experiments, irradiation of semiallogeneic recipients with 7.5 Gy immediately before adoptive transfer of activated LEW T cells increased EAU susceptibility. In one experiment, eight of eight irradiated LBuF1 rats developed EAU with an average severity of 3.3, and zero of 12 nonirradiated LBuF1 rats developed EAU. As a result, all recipient rats were irradiated in subsequent experiments, except for LEW-positive controls used to demonstrate pathogenicity of the T cells. Using this strategy, transfer of 10 Gy-irradiated LEW splenocytes into fully allogeneic BUF recipients, together with pathogenic LEW T cells, provided an environment in which mild EAU was elicited in a few recipients (Table 3 , Exp. 1). Transfer of irradiated LEW splenocytes or nonirradiated LBuF1 splenocytes also supported mild EAU induction in BUF recipients (Table 3 , Exps. 2 and 3). Overall, no BUF rats (zero of 21) developed EAU when transferred with purified LEW T cells, but several (10 of 36) developed EAU if also given LEW or LBuF1 spleen cells (P=0.009; two-tailed Fisher’s exact test). The APC transfer results show that resident retinal APC or BM donor-derived PVMG are not absolutely required for induction of EAU. Instead, APC could be recruited from the transferred spleen cells. The results suggest that sufficient numbers of donor APC, even if irradiated, can contaminate T cell line transfers and affect EAU susceptibility. Fully allogeneic BN rats (zero of 10) were not susceptible to the transferred APC strategy, indicating that additional, unidentified factors contribute. Regardless, the ability of LEW T cells to induce EAU in BUF recipients makes the point that recruited APC can support the pathogenesis of EAU.
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The potential contribution of APC present in T cell inocula for adoptive transfers was not considered in previous reports. Myers et al. [18 ], in asserting that parenchymal cells had sufficient APC activity to support EAE, transferred nonirradiated LN cells as the source of pathogenic T cells. Hinrichs et al. [6 ] used immune spleen cells, which contain many cells in addition to T cells. Hickey and Kimura [5 ] and Ishimoto et al. [7 ] used 15 x 106 T cells from lines supported by irradiated splenocytes without specifying their preparation prior to transfer.
The possibility that whole body irradiation of the recipient rats altered the BRB has been considered. Although systematic studies of the effect of low-dose radiation on the BRB have not been done, several studies of the BBB are applicable. Doses of 2 and 8 Gy did not induce leaks in the BBB [19
, 20
]. A 2-Gy dose had no acute or long-term effects on intercellular adhesion molecule-1 (ICAM-1) or tumor necrosis factor 
mRNA levels, and 10 Gy induced a twofold increase in ICAM-1 at 48 h [21
]. Another study of ICAM-1 mRNA levels found that 5 Gy had no effect but that 15 Gy gave significant induction [22
]. As leukocyte adhesion is a factor in BBB breakdown, a few reports surgically placed glass windows over the brains of rats to allow direct visualization of the adherence of fluorescent leukocytes to pial blood vessels and fluorescein isothiocyanate–dextran leakage. A dose of 5 Gy had no effect. Using anti-ICAM-1-coated fluorescent beads or in vivo-labeled leukocytes, a dose of 10 Gy was found to increase adherence to the endothelium [23
24
25
]. The use of cranial glass windows is an elegant strategy but raises questions regarding the degree to which surgery and window placement sensitize the vessels to react more strongly to further insult. Our study used 7.5 Gy, which is at the lower limit of detectable effects on the BBB. Hu et al. [15
] provide the important precedent that activated T cells trafficked into retina and brought monocyte-like cells with them in nonirradiated recipients. We conclude that 7.5 Gy is unlikely to be responsible for our observations.
Our results do not rule out contributory or regulatory roles for resident retinal cells, BM-derived and non-BM-derived. Although efficient costimulation is often a property of BM-derived, "professional" APC, non-BM-derived cells with inducible class II such as glia or vascular endothelium also express CD80, CD86, B7RP-1, CD137L, and CD134L under certain conditions and could alter responses locally. Assessing the contributions of resident cells in retina to the pathogenesis of EAU may reveal fundamental differences relative to brain, especially given the disparity in the presence of abundant, meningeal DC in brain [26 ] but not retina, which lacks the equivalent tissue.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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Received April 22, 2004; revised May 21, 2004; accepted May 24, 2004.
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REFERENCES |
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TOPABSTRACTREFERENCES |
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2-like genes J. Immunol. 146,75-80[Abstract]
or anti-CD40 Invest. Ophthalmol. Vis. Sci. 44,3083-3093RIII(+)) subset of human monocytes preferentially becomes migratory dendritic cells in a model tissue setting J. Exp. Med. 196,517-527 expression between large single fraction and fractionated irradiation in mouse brain Int. J. Radiat. Biol. 79,359-366[CrossRef][Medline]
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