specific T cells were present in the retina within a few hours after cell transfer. Because S-Ag is ... characterized by Savion et al.,6 for adoptive transfer of EAU.
T Cell Traffic and the Inflammatory Response in Experimental Autoimmune Uveoretinitis Robert A. Prendergast,1 Charles E. Iliff,1 Nezih M. Coskuncan,1 Rachel R. Caspi,2 Gil Sartani,2 Teresa K. Tarrant,2 Gerard A. Lutty,1 and D. Scott McLeod1 quantify S-antigen-specific (S-Ag) T cells in the retina after adoptive transfer, and to evaluate their role in the initiation and progress of destructive ocular inflammation in experimental autoimmune uveoretinitis (EAU).
PURPOSE. TO
Lewis rats were administered 10 X 106 S-Ag-specific T cells from the SP35 cell line or 10 X 10 concanavalin A-stimulated syngeneic spleen cell lymphoblasts labeled with lipophilic PKH26 fluorescent dye immediately before intravenous inoculation. Labeled cells in each retina were counted at various times from 4 to 120 hours after cell transfer by fluorescence microscopic analysis of each dissociated retina. Recipient eyes were examined within the same period by light and confocal microscope. METHODS.
6
RESULTS. SP35 T cells showed a biphasic distribution in the retina. Thefirstpeak of 160 cells/retina was noted at 24 hours. A steady decline of labeled cells at 48 and 72 hours was followed by a rapid increase at 96 and 120 hours. Concanavalin A-stimulated, control-labeled cell populations showed an identical peak at 24 hours but a persistent decline thereafter; only two or three T cells were present in each retina at 120 hours. Concurrent inoculation of SP35 cells and nonspecific T cell blasts did not produce more SP35 cells than control cells in the retina at any time. Microscopic analysis showed mononuclear cell infiltration of the iris, ciliary body, and aqueous humor at 48 hours, which intensified rapidly and persisted through 120 hours. Retinal inflammation did not begin until 80 hours. Mononuclear cell adherence to vascular endothelium and perivascular macrophage infiltration of the innermost layers progressed to edema, and profound destructive inflammation and loss of retinal stratification were observed at 120 hours.
There is no evidence of a blood- ocular or blood-retinal barrier to activated T cell blasts. Autologous S-Ag does not provoke a more rapid entry of specific T cells at that site. The data confirm that anterior segment inflammation precedes retinal inflammation, even though S-Agspecific T cells were present in the retina within a few hours after cell transfer. Because S-Ag is clearly present in the retina, delay in antigen presentation at that site may account for the temporal difference between retinal and anterior segment inflammation. (Invest Ophthalmol Vis Sci. 1998; 39:754-762)
CONCLUSIONS.
E
xperimental autoimmune uveoretinitis (EAU) is a wellcharacterized, lymphocyte-mediated autoimmune disease model that can be induced by active immunization of susceptible species with whole S-antigen (S-Ag) protein (arrestin) or by immunization with certain immunopathogenic peptides derived from this molecule.1"3 It can be induced in naive syngeneic recipients by transfer of activated CD4+ major
From 'The Johns Hopkins University, Baltimore; and the 2National Eye Institute, National Institutes of Health, Bethesda, Maryland. Supported in part by Research to Prevent Blindness and Fight For Sight Research Division of Prevent Blindness America (CEI), New York, New York; National Institutes of Health National Research Service Award Institutional Training grant EY07047 (NMC); Research to Prevent Blindness Foreign Fellowship (NMC); and National Institutes of Health Core Center grant EY01765 (Wilmer Institute). GAL is an American Heart Association Established Investigator. Submitted for publication July 14, 1997; revised November 26, 1997; accepted December 9, 1997. Proprietary interest category: N. Reprint requests: Robert A. Prendergast, The Wilmer Eye Institute, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287-9142.
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histocompatibility complex-restricted T cells specific for retinal S-Ag.4"6 The presence of S-Ag-specific T cells within the globe is requisite for the initiation and rapid development of retinal and uveal inflammation.7'8 Because the retina is a major target organ, this exquisitely defined and readily isolated tissue presents a unique structure for the study of the traffic and specificity of T cells within the target tissue at sequential times after immunization. Antigen-specific migration or retention of immune effector T cells at sites of cell-mediated immunity, including delayed hypersensitivity, allograft rejection reactions, and autoimmune T cell-driven lesions, has been studied for many years.9"1' In general, tritiated thymidine-labeled lymphocytes, transferred from immunized donors to syngeneic recipients for quantitative analysis of the number of transferred cells at antigenspecific or appropriate nonspecific challenge sites, have been used in these studies. In the case of skin allograft rejection in the rabbit, cells from a specific draining lymph node reacting to one allograft were labeled in vivo, and the presence of these cells calibrated at test transplant rejection sites of identical or allogeneic skin grafts in the same animal.10 In none of these instances has a clear preponderance of labeled sensitized lymInvestigative Ophthalmology & Visual Science, April 1998, Vol. 39, No. 5 Copyright © Association for Research in Vision and Ophthalmology
IOVS, April 1998, Vol. 39, No. 5 phocytes been demonstrated at the specific immune reactive site in excess of the number of these cells at appropriate control sites. Several objections could be raised to these experiments, however. The specific donor lymphocytes from animals initially responding to antigen had, in general, been accumulated from disrupted lymphoid tissue in which antigen-responding cells were actively engaged in clonal expansion and were harvested at various stages of maturity. The specific cells within the donor population were present in low concentrations in a highly random, immunologically naive group. Donor cells were usually examined autoradiographically at reactive sites in minute samples taken from inflammatory lesions, or the number of donor cells at the reactive inflammatory site was inferred by analysis of total radioactivity of that area when the donor population was labeled ex vivo with a radioactive tag before transfer.12 In the latter instance, it is unclear precisely how radiation comes to this site, whether from the original labeled lymphocyte population, through generation of these cells in the host animal, or by leakage of label and possible uptake by nonspecific inflammatory cells. The precise mechanism of T cell-mediated inflammatory disease is not fully understood. Furthermore, in EAU, the complete temporal sequence of ocular inflammation, including chemotaxis and activation of mononuclear cells, is also unclear. In the experiments described in the present report, two experimental techniques were used to examine T cell traffic in the EAU model of ocular autoimmune disease. First, the precise number of T cells reactive to retinal antigen were calibrated by using a uniformly labeled S-Ag-specific T cell line, initially characterized by Savion et al.,6 for adoptive transfer of EAU. The second technique used was cytologic examination of the entire end organ—that is, the retina, for the presence of effector cells. Thus, it was possible to quantify all labeled T cells involved at the end organ-reactive site and so to obviate the sampling error inherent in previous studies. These data demonstrate that activated T cells of S-Ag or random specificity invade retinal tissue early after transfer, and that the total number of these T cells can be directly measured during early phases before and after development of destructive disease. The sequence of pathologic changes induced by these T cells will also be shown throughout the entire sequence from the time of T cell transfer through complete destruction of the retina.
MATERIALS AND METHODS Animals Young male Lewis rats, 175 to 225 g, and adult female BALB/c mice were obtained from Charles River (Wilmington, MA) and were maintained in the animal facilities at the Wilmer Institute. The maintenance, care, and all experimental use of these animals were in strict compliance with guidelines issued by the National Institutes of Health and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Cells SP35, the T cell line derived from reactive lymph nodes of Lewis rats immunized with bovine S-Ag plus complete Freund's adjuvant, was used throughout this study. SP35 is a class II-restricted T cell line specific to the P35 peptide composed
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of amino acids 337 to 356 of human S-Ag. This line was maintained by restimulation with S-Ag peptide P35 antigen, then by interleukin-2 (IL-2)- driven expansion, according to the method of Savion et al.6 After culture, all cells were frozen while in the active blast state and were maintained in liquid nitrogen for 3 to 6 months before their use in these experiments. Aliquots from tissue culture harvests of these effector T cells were tested for their pathogenicity by adoptive cell transfer to naive Lewis rats, in which 0.2 X 106 cells injected intraperitoneally consistently induced EAU. Fluorescent Labeling of T Cells and Induction of Experimental Autoimmune Uveoretinitis Vials of frozen SP35 and y-irradiated splenic feeder cells were rapidly thawed under 56°C tap water and were allowed to recover at 5 million SP35 cells/ml for 4 hours in RPMI medium containing 10% activated spleen cell supernatant and antibiotics. Cells were harvested, washed in Hanks' balanced salt solution (HBSS), and placed over Ficoll (Sigma, St. Louis, MO) to separate viable SP35 lymphoblasts from irradiated rat feeder cells used during proliferative expansion. After centrifugation at 400g for 22 minutes, SP35 blast cells were harvested, washed three times in HBSS, and labeled, using the red fluorescein dye cell linker kit (PKH26; Sigma), precisely as directed by the manufacturer. PKH26 is a patented fluorescent cell linker technology that incorporates fluoresceinated lipophilic molecules into the cell membrane by selective partitioning. The precise nature and chemical composition of all of the components of the cell linker kit are unavailable from Sigma or from the original manufacturer.13>l4 Briefly, a suspension of 20 to 25 X 106 HBSS-washed SP35 cells was exposed to 2 X 10" 6 M PKH26 dye in PKH26 buffer for 4 minutes in polypropylene tubes. The reaction was stopped by addition of an equal volume of fetal bovine serum for 1 minute, and the cell solution was diluted 1:1 with complete medium and washed three times before injection. The cells were brilliantly red with a smooth, uniformly fluorescent membrane label, visible under epifluorescent microscope equipped with a rhodamine detection filter system. Labeled SP35 cells were concentrated to 10 X 106 cells in 0.3 ml HBSS and injected into the penile vein of normal Lewis rats anesthetized by xylazine hydrochloride (Rompun; Bayer Animal Health, Leverkusen, Germany) and ketamine hydrochloride (Ketaset; Bristol-Myers Squibb, New York, NY). Injection was simple to perform; the nits awakened approximately 15 to 30 minutes later and displayed no evidence of injury. A control population of normal Lewis rat spleen cells, stimulated by concanavalin A (Con A; Sigma) at 35 /Jig/ml for 60 hours, was used after PKH26 labeling at the same concentration and was injected intravenously in an identical fashion. Enumeration of S-Antigen T Cell Population in Retinal Tissue Labeled SP35 or control cell recipient rats were killed using an overdose of sodium pentobarbital delivered intraperitoneally and the vascular system was flushed by left ventricular injection of 200 ml HBSS. The eyes were carefully dissected, the anterior segment removed, and the retinas separated from the choroid within 30 minutes after death. All ocular tissues were fixed for 2 days in 4% paraformaldehyde in phosphate-buffered saline containing 6% sucrose (pH 7.2). After washing, fixed
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retinas were digested with elastase, according to the method described by Laver et al.15 After removal of intact retinal vessels, the total number of disaggregated retinal cells isolated by this technique from normal Lewis retinas was 12 X 106 ± 8% cells/retina, measured by Coulter counter. Cells from one retina were concentrated to a volume of 150 A HBSS. Labeled cells were counted, using a standard hemocytometer and a Zeiss (Carl Zeiss, Thornwood, NY) epifluorescent microscope with a dry X16 planapochromatic objective. Labeled cells in the peripheral blood were counted in a similar fashion after Ficoll separation of mononuclear cells from 2 ml heparinized peripheral blood.
Histopathology The sequence of pathologic changes was studied at various times after injection of labeled SP35 or labeled Lewis rat Con A-stimulated splenic lymphocytes. At the time of death, the eyes were removed, stabbed with a number 11 blade at four sites in the pars plana, and fixed in 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.2) for 20 hours. The retinas were removed, washed in cacodylate buffer, hemisectioned, and embedded in glycol methacrylate. Two-micrometer sections were stained with toluidine blue and basic fuchsin (Polysciences, Warrenton, PA). Anterior segments and posterior sclera with overlying choroid and retinal pigment cells were embedded in paraffin, sectioned at 5 jLim, and stained by hematoxylin-eosin. Confocal microscopic study of phosphatebuffered saline-washed, paraformaldehyde-fixed retinal whole mounts was performed, using a Zeiss laser scanning confocal microscope, courtesy of Marshall Montrose, Department of Medicine, Johns Hopkins University. Lectin-Induced Proliferation and Cytokine Production PKH26-labeled and unlabeled rat and mouse spleen cells were cultured at 2 X 106 cell/ml in 5-ml quantities in 6-well plates (Costar, Cambridge, MA). Con A was added to these cultures at concentrations of 3-5 /xg/ml for rat and 2 ju-g/ml for mouse spleen cultures. Control and Con A culture supernatants were tested for IL-2, IL-4, and interferon-y, by using enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN). Tritiated thymidine incorporation in control and lectin-stimulated spleen cells was determined for PKH26-labeled and unlabeled cells. A mean of 5 wells was used for each measurement in a standard microculture technique.'6 RESULTS
T Cell Traffic These studies were carried out in Lewis rats, using S-Agspecific T cell line SP35, generated from S-Ag-immune Lewis rats and stimulated in vitro with the human S-Ag peptide P35 and lectin-stimulated lymphoblasts. The presence of fluoresceinated SP35 cells in the retina was examined in rats injected intravenously with 10 X 106 PKH26-labeled cells. The animals were killed from 12 to 120 hours thereafter. Removal of the intact retinal vasculature after elastase treatment permitted epifluorescent microscopic analysis of labeled cells in the entire retinal cell population. As shown in Figure 1, S-Ag-specific cells demonstrated a biphasic population density within the retina as time elapsed. An earlyfirstpeak occurred at 24 hours.
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Hours After Cell Transfer FIGURE 1. Measurements of extravascular PKH26-labeled SP35 T cells in the retina at various times after intravenous transfer of 10 X 106 cells. Each bar represents the mean number of labeled cells in four to six individual enzyme-dissociated retinas examined.
There was a steady decrease during the next 2 days, then a rapid increase in labeled cells at 96 and 120 hours after SP35 T cell transfer. Retinal inflammation was not detected during the initial peak or during its decline, as will be shown. Several features of this portion of the experiment should be noted. The number of labeled cells in each retina from a single animal was very similar. In addition, comparison of the labeled cell number in retinas examined at the same time after adoptive transfer also showed close adherence to the mean. The first peak in this biphasic time course experiment at 24 hours showed approximately 160 labeled SP35 cells within each retina. This peak was followed by a steady decline, to the extent that at 72 hours, fewer than 40 cells were present within the retinal tissue. The rapid increase at 96 and 120 hours shown in Figure 1 marks only the specifically labeled T cells. The pathologic condition of the retina at those times demonstrates that this represents only a small portion of infiltrating mononuclear cells (see later discussion). Normal Lewis rat spleen cells activated by Con A stimulation were labeled with PKH26 and were used as a control population. Injection of 10 X 106 labeled T cell blasts induced a peak of activity very similar to that seen with S-Ag-specific T cells during the first phase of the response 24 hours after injection (Fig. 2). Subsequently, however, the cells decreased to much lower numbers at 96 and 120 hours, the time of the second peak after SP35 labeled cell transfer (Fig. 1). No inflammation was present at any time after injection of normal Con A-activated T cell blasts. To study the specificity of cells in the reactive retinal target, an experiment was performed in which inoculation of SP35 S-Ag-specific T cells and Con A Lewis rat T cell blasts was used. Only one of these two cell populations was labeled with PKH26 dye. Thus, 10 X 106 labeled S-Agspecific cells were injected with 10 X 106 unlabeled Con A-activated spleen cells. The converse experiment was performed in which the label was placed on the Con A-stimulated control T cell blasts. Experimental autoimmune uveoretinitis was induced in all animals. No recognizable preponderance of specific or nonspecific lectin-activated T cells at 24 hours or later times could be demonstrated in this experiment in which 4 to 8 eyes were examined for each time point for each labeled population (Fig. 3). The differences in
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