Original Paper
Ophthalmic Res 1992:24:92-98
Doheny Eye Institute and Department of Ophthalmology, University of Southern California. Los Angeles, Calif.; Department of Ophthalmology. Indiana University, Indianapolis, Ind.: Department of Medicine, UCLA/Cedars Sinai Medical Center and Department of Neurology and Pediatrics, Los Angeles; Stanford University School of Medicine, Stanford, Calif.. USA
KeyWords
Autoimmune uveitis Anti-I-A Suppressor T cell Adoptive transfer Retinal S-antigen
Antigen-Specific Suppressor Cells in Experimental Autoimmune Uveitis
Abstract
Anti-I-A antibodies, administered in vivo at the time of Santigen injection, suppress development of experimental au toimmune uveitis (EAU) in Lewis rats. While the effects of anti-I-A are profound, the exact mechanism for this suppres sion is unknown. We attempted adoptive transfer of this form of suppression by injecting lymphocytes from anti-I-A-trcatcd animals into syngeneic recipients which were later injected with S-antigen. Histologically, globes of 75% of the anti-I-Atreated animals showed no inflammation while 25% of these animals developed mild uveitis. In the group of animals which were injected with S-antigen and also received spleen cells from anti-I-A-treated rats, only 1 showed mild uveitis while the remaining 7 had no inflammation. The animals undergo ing adoptive transfer of spleen cells and which were primed with an irrelevant antigen, readily developed uveitis. Suppres sion of S-antigcn-induced EAU was abrogated by pretreat ment of donor animals with cyclophosphamide. In vitro stud ies revealed that spleen cells of S-antigen-primed, anti-I-Atreated donors specifically suppressed lymphocyte responses to S-antigen. These in vivo and in vitro results suggest that generation of antigen-specific suppressor cells play a role in the anti-I-A immunotherapy of EAU.
Introduction
Uveoretinitis induced by retinal S-antigen has been shown to be a useful animal model for uveitis. This experimental intraocular in
Received: August 15, 1991 Accepted: October 4. 1991
flammation can be produced in various labo ratory animals, including rats. Lewis rats are highly susceptible and readily develop uveo retinitis when injected with S-antigen [1], In these animals, the development of uveitis has
Narsing A. Rao. MD Doheny Eye Institute 1355 San Pablo St. Los Angeles. CA 90033 (USA)
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:09:11 PM
NarsingA. Raoa Lily Atal/a3 Shao-ling Fongb Fen Chena M. Linker-Israelic Lawrence Steinm and
Materials and Methods The studies described herein conformed to the ARVO Resolution on the Use of Animals in Research. Twenty-four Lewis rats were initially injected with 50 pg of S-antigen synthetic peptide (HSA 320) [7] in complete Freund’s adjuvant in the foot pad followed by 1 pg pertussis toxin intraperitoneally. The immu nized rats were divided into two groups: group 1 was treated with anit-I-A (OX 6), 1 mg/injection on days - 1 , + 1 and +2; group 2 was injected with RPMI 1640 medium according to the same time schedule. All ani mals were killed on day 15 after antigen administra tion. The eyes were enucleated for histologic study, and individual spleens from 8 animals in each of the two groups and from naive animals were used for isola tion of spleen mononuclear cells that were then trans ferred to three additional groups of syngeneic recipi ents. Donor spleen cells were separated on Ficollpaque gradients and resuspended in saline prior to intraperitoneal injection, 5 X 106 in each recipient. A group of S animals (group 3) received cells from antiI-A-treated donors (group 1). Another group of 6 ani mals (group 4) received spleen cells from RPMItreated donors (group 2), and another 6 animals (group 5) received cells from naive animals. On the day of spleen cell transfer, all recipients (groups 3-5) were injected with 50 pg of S-antigen synthetic peptide (HSA 320) in complete Freund’s adjuvant and pertus sis toxin as described above. The recipients were killed on day 15 after antigen administration. The eyes were enucleated from all animals (group 3 through 5) and processed for histopathologic examination. An additional group (group 6) was injected with cyclophosphamide, 20 mg/kg 2 days prior to 50 pg of S-antigen synthetic peptide administration in com plete Freund’s adjuvant. These animals were treated with anti-l-A antibody, 1 mg/injection, on days - 1 , +1 and +2 after the antigen injection. On day 15, after the administration of antigen, these animals were killed and the eyes were enucleated and processed for histo pathologic examination. Six other animals (group 7) were injected with 50 pg of synthetic peptide of IRBP [8] (HIRBP 715) in complete Freund’s adjuvant in the right hind foot pad. On the same day, these animals received first 1 pgof pertussis toxin, injected intraperi toneally, and later on the same day an intraperitoneal injection of 50 X 106 cells from group 1 animals. These animals were sacrificed on day 15 and the enu cleated eyes were fixed in formalin, embedded in par affin and stained with HE.
93
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been shown to be mediated by CD4 + major histocompatibility complex (MHC) class 11 restricted helper/inducer T cells [2], We have demonstrated that development of uveoretinitis in Lewis rats can be prevented by admin istration of anti-I-A antibody at the time of sensitization with S-antigen [3]. This inhibi tion was found to be dose-dependent, and when treated with 750 pg of anti-I-A, about 90% of animals showed amelioration of clini cal signs and absence of histopathologic evi dence of the disease. Even though the successful inhibition of uveitis could be achieved with an appropriate dose of anti-I-A antibody, the precise mechanism(s) underlying such inhibition was not clear [3], As the development of S-antigeninduced uveitis consists of a complex interac tion of various immunoreactive cells and their products, disturbance of any aspect of these interactions could lead to repercussions involving some other cellular response. One such repercussion could be the production of antigen-specific suppressor cells. Several pos sible mechanisms have been proposed for the anti-I-A-mediated suppression, including: (a) blocking or impairment of antigen presen tation in context with class II MHC results in a lack of activation of the antigen-specific CD4 + cells that mediate development of dis ease; (b) the anti-I-A antibodies may impair the antigen-presenting functions of macro phages and dendritic cells and induce sup pressor cells [4-6]; (c) compensatory antigenspecific activation of suppressor cells that rec ognize antigen in context with class I MHC when the class II MHC-restricted activation of helper/inducer cells is blocked [4], The studies reported here support the idea that at least 1 of the mechanisms of uveitis inhibition by anti-I-A antibodies is by generation of anti gen-specific suppressor cells.
Fig. 1. Animal treated with anti-I-A monoclonal antibody shows normal retina, ciliary body, iris and choroid. HE. X 150.
Fig. 2. Control animal treated with RPMI 1640 shows retinitis and iridocyclitis. HE. X 150.
94
incubated at 37 °C in 5% CO2 for 5 days. 1 pCi of 3Hthymidine was used to pulse the cultures similar to the method described earlier [9]. Results are expressed as stimulation index.
Rao/Atalla/Fong/Chen/Linker-Israeli/ Steinman
Suppressor Cells in EAU
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Lymphocyte Stimulation Studies In vitro lymphocyte stimulation was performed on 2 X 105 spleen cells of animals injected with S-antigen synthetic peptide (group 2) in the presence or absence of 5 X 104 spleen cells of animals treated with anti-I-A (similar to group 1). Retinal S-antigen synthetic pep tide, 4 |ig per well, and concanavalin A (ConA), 2 pg per well, were used for stimulation. Cultures were
Fig. 3. Animal pretreated with cyclophosphamide and injected with anti-I-A antibody demon strates development of iridocycli tis. HE. X 150.
Table 1. Suppression of uveitis by transfer of spleen cells
Experimental group and treatment
Number of animals
Animals with uveitis n
%
Treatment with anti-I-A Treatment with RPMI Recipient of spleen cells from group 1 Recipients of spleen cells from group 2 Recipient o f spleen cells from naive animals Pretreated with cyclophosphamide and injected with anti-I-A Recipients of spleen cells of group 1, and injected with IRBP
12 12 8 6 6
3/12 12/12 1/8 6/6 6/6
25 100 12 100 100
mild, focal severe, diffuse mild, focal severe, diffuse severe, diffuse
6
6/6
100
severe, diffuse
6
6/6
100
severe, diffuse
Histologically, globes of 9 of the 12 (75%) rats treated with anti-I-A showed no inflam mation (fig. 1), while 3 animals had mild uve itis. This inflammation was focal, involving the choroid and the retina. All animals treated with RPMI 1640 developed severe uveitis and retinits. The inflammation was diffuse
and there was destruction of various retinal layers (fig. 2). Of the spleen cell recipient ani mals in group 3, only 1 showed mild uveitis, and the remaining 7 had no inflammation. All animals in groups 4 and 5 developed severe retinitis and uveitis (table 1). All animals in group 6 and 7 showed severe choroiditis, iri docyclitis (fig. 3) and focal retinitis with de struction of retinal tissue.
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Results
Severity of uveitis
Table 2. Antigen-spccific suppression of stimulation index in the presence of spleen cells of animals treated with anti-I-A
Experiment
ConA
In the presence of cells from controlled nonimmunized animals In the presence of cells from anti-I-A-treated rats injected with S-antigen
S-antigen
count/min
SI
count/min
SI
2,533.0±48.1
8.37 ± 1.42
1.345.5 ±55.8
4.29 ± 1.16
2.213.0 ± 32.5 (8% suppression)
7.69± 3.25
260.0 ±57.9 1.20 ±0.53 (72% suppression)
Spleen cells from anti-I-A-treated donors had antigen-specific suppressive activity. There was a 72% suppression of the lympho cyte stimulation index when spleen cells of anti-I-A-treated animals were added to the Santigen synthetic-peptide-containing cultures and minimal (8%) suppression when these cells were added to the ConA-containing cul tures (table 2).
Discussion
The results of this study clearly show the effectiveness of immunotherapy in the pre vention of S-antigen-induced uveitis and in the generation of suppressor cells. In vivo administration of anti-I-A antibody has pre viously been shown to induce suppressor T cells that abrogate a delayed-type hypersensi tivity response to tumor antigens [6]. Anti-I-A treatment was also found to induce suppres sor T cells that attenuate the autoimmune response to myelin basic protein [10], As in these earlier experimental studies, the present study also demonstrated generation of sup pressor cells. These suppressor cells were found to be antigen-specific, as the spleen cell recipient animals in the adoptive transfer
96
study, animals which were also primed with IRBP. readily developed uveitis and retinitis, whereas such animals failed to show any clini cal or histopathologic evidence of uveitis when injected with S-antigen. Therefore, at tenuation of the disease in some animals and suppressed development of uveitis in others may be attributed to the generation of sup pressor function (table 1). The generation of suppressor cells was cyclophosphamide-sensi tive, as the protective effect could be nullified by pretreatment of donor animals with a lowdose of cyclophosphamide. These results sug gest that antigen-specific immune suppres sion of uveitis is feasible. Several mechanisms could account for the generation of suppressor cells following treat ment with anti-I-A monoclonal antibodies. The I-A-specific antibodies may affect the dis tribution of S-antigen within the plasma membrane of the antigen-presenting macro phages, thereby blocking the interaction of antigen with I-A, which may prompt the anti gen to shift to other determinants of the la, such as I-E or I-J [ 11. 12], Thus. S-antigen pre sentation, where I-A is blocked, may provide an activation signal for suppressor cell precur sors. Other mechanisms for activation of sup pressor T cells include direct stimulation of
Rao/Atalla/Fong/Chen/Linker-Israeli/ Steinman
Suppressor Cells in EAU
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SI = Stimulation index.
hans cells of the dermis. Anti-I-A antibodies could nullify the antigen-presenting function of splenic cells, but not that of Langerhans cells [20], Similarly, the inhibition noted with anti-I-A treatment could be from the effect of anti-I-A antibody on the peripheral lymphoid and splenic I-A-positive cells rather than on the la-positive cells, such as retinal vascular endothelial cells and Müller cells. As reported earlier, maximum effect of anti-I-A on the inhibition of uveitis was seen when the anti body was administered early, within a week after administration of S-antigen [3], If the antibody administration were delayed, the prevention of uveitis was minimal or nonsig nificant, which also suggests that the anti-I-A antibody effect was extraocular. In vivo adoptive transfer experiments, as well as in vitro lymphocyte proliferation (ta bles 1,2) in the presence of spleen cells from naive versus anti-I-A antibody-treated ani mals, show antigen-specific suppression and intact T-cell response to nonspecific mito gens, and a specific response to IRBP. These studies suggest that anti-I-A therapy may not lead to the global immune suppression that occurs with various cytotoxic agents currently used in the treatment of severe recalcitrant uveitis unresponsive to corticosteroids. Even though ciclosporin is known to selectively in hibit T-cell helper function, this drug is not antigen-specific, as is anti-I-A therapy. More over, ciclosporin and other cytotoxic agents are associated with severe side effects. Even though prelim inary studies with anti-I-A ther apy showed no untoward effects in the form of glomerulonephritis or serum sickness in the experimental animals [3], further indepth studies in nonhuman primates having experi mental autoimmune uveitis should be carried out prior to any consideration of anti-I-A treatment in humans with severe uveitis.
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these suppressor cells by anti-I-A antibody. Such a mechanism appears unlikely, however, as various antigen-specific suppressor cells have been shown to express I-J-encodcd de terminants and consistently lack an I-A deter minant. A 3rd possible mechanism may result from a high concentration of circulating Santigcn due to diminished antigen ingestion by antigen-presenting cells, including macro phages. However, there is no evidence to sup port the idea that anti-I-A antibodies prevent ingestion of antigen, which would lead to high concentrations of circulating soluble antigens [13], However, further experimental studies are required to determine which, if any, of these putative mechanisms is operative. Thus far. anti-I-A antibody administration has reversed conditions triggered by immuni zation with self antigens, including experi mental allergic encephalomyelitis after im munization with myelin basic protein [14], experimental autoimmune myasthenia gravis following acetylcholine receptor inoculation [15], experimental autoimmune thyroiditis after thyroglobulin immunization [16], colla gen-induced arthritis due to type II collagen injection and experimental autoimmune uveitis induced by S-antigen [3], Anti-I-A an tibodies have been used successfully to treat 2 spontaneously occurring autoimmune condi tions: the lupus-like disease in NZB/W mice and the diabetes-like condition in BB rats [ 17, 18], Such immunotherapy was also found beneficial in the reversal of chronic paralysis in mice infected with Theiler’s virus [19], All of these experimental studies demonstrate the utility of anti-I-A antibodies for treatment of autoimmune conditions triggered by organspecific antigens, and where the disease sus ceptibility is linked to class II MHC genes. It has been demonstrated that in vivo ad ministration of anti-I-A antibody can differ entially affect la antigen expression by var ious cells including spleen cells and Langer
Acknowledgements These studies were supported in part by National Eye Institute grant EY09088, and by the Research to Prevent Blindness, Inc., NY., and Core Grant EY03040. The animals used in this study were main
tained in animal care facilities fully accredited by the American Association of Laboratory Animal Science. The authors express their appreciation to Ms. Irene Cumplido for preparation of the manuscript and to Ms. Ann Dawson for editorial assistance.
References
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9 Rao NA, Robin J, Hartmann D. Sweeney JA, Marak GE Jr: The role of the penetrating wound in sympa thetic ophthalmia: Experimental observations. Arch Ophthalmol 1983:101:102-103. 10 Steinman L. Schwartz G, Waldor M, O’Hcarn M, Lim M, Sriram S: Gene specific and antigen specific strate gics for the induction of suppressor T cells to myelin basic protein; in Alvord EC Jr. Kies MW, Suckling AJ (eds): Experimental Allergic En cephalomyelitis: A Useful Model for Multiple Sclerosis. New York. Alan Liss, 1984, pp 393-397. 11 Zembala M, Asherson GL: T cell suppression of contact sensitivity in the mouse. II. The role of soluble suppressor factor and its interaction with macrophages. Eur J Immunol 1974;4:799-803. 12 Ptak W. Zembala M. Gershon RK: Intermediary' role of macrophages in the passage of suppressor signals be tween T-cell subsets. J Exp Med 1978:148:424-433. 13 Ziegler K, Unanue ER: Identifica tion of a macrophage antigen-pro cessing event required for I-regionrestricted antigen presentation to T lymphocytes. J Immunol 1981; 127: 1869-1875. 14 Steinman L, Rosenbaum JT, Sriram S. McDevitt HO: In vivo effects of antibodies to immune response gene products. Prevention of experimen tal allergic encephalitis. Proc Natl Acad Sci USA 1981:78:7111-7114.
15 Waldor MK. Sriram S. McDevitt HOV, Steinman L: In vivo therapy with monoclonal anti-l-A antibody suppresses immune responses to acetylcholine receptor. Proc Natl Acad Sci USA 1983;80:2713-2717. 16 Vladutiu AO. Steinman L: Inhibi tion of experimental autoimmune thyroiditis (EAT) induction in mice by monoclonal anti-I-A. Fed Proc 1984;43:1991-1997. 17 Adelman NE. Walling DL, McDe vitt HO: Treatment of NZB x NZW)F|, disease with anti-I-A monoclonal antibodies. J Exp Med 1983;158:1350-1354. 18 Boitard C, Michie S. Serrurier P. Butcher GW. Larkins A, McDevitt HO: In vivo prevention of thyroid and pancreatic autoimmunity in the BB rat by antibody to class II major histocompatibility complex gene products. Proc Natl Acad Sci USA 1985;82:6627-6631. 19 Friedman AN, Frankel G, Lorch Y, Steinman L: Monoclonal anti-I-A antibody reverses chronic paralysis and demyelination in Theiler’s vi rus-infected mice: Critical impor tance of timing of treatment. J Virol 1987:61:898-903.
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Suppressor Cells in EAU
20 Aberer W. Kruisbeek AM. Shimada
S, Katz SI: in vivo treatment with anti-I-A antibodies: Differential ef fects on la antigens and antigen-pre senting cell function of spleen cells and epidermal Langerhans cells. J Immunol 1986:136:830-836.
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1 Marak GE Jr. Rao NA: Retinal Santigen disease in rats. Ophthalmic Res 1982;14:29-39. 2 Mochizuki M. Kuwabara T. McAl lister C, Nussenblatt RB. Gcry 1: Adoptive transfer of experimental autoimmune uveoretinitis in rats: Immunopathogenic mechanisms and histologic features. Invest Oph thalmol Vis Sci 1985;26:1-9. 3 Rao NA, Atalla L. Linkcr-Isracli M, Chen FY, George FW IV, Martin WJ, Steinman L: Suppression of ex perimental uveitis in rats byanti-I-A antibodies. Invest Ophthalmol Vis Sci 1989;30:2348-2355. 4 Carrol A, Perry LL. Lowy A. Green MI: Regulation of immune response by in vivo administration of mono clonal anti-I-A antibodies; in Brochier J. Clot J. Sany J (eds): Anti-la Antibodies in the Treatment of Au toimmune Disease. New York, Aca demic Press. 1986. pp 59-70. 5 Steinberg AD, Santoro TJ; Anti-la antibodies: A revolutionary therapy for immune-mediated diseases. ImmunolToday 1984;5:13-14. 6 Perry LL. Greene MI: Conversion of immunity to suppression by in vivo administration of I-A subregionspecific antibodies. J Exp Med 1982;156:480-488. 7 Donoso LA. Yamaki K, Merryman CF, Shinohara T, Yuc S, Scry TW: Human S-antigen: Characterization of uveitopathogenic sites. Curr Eye Res 1988;7:1077-1085. 8 Donoso LA. Merryman CF, Sery TW, Vrabec T, Arbizo V. Fong SL: Human IRBP: Characterization of uveitopathogenesis site. Curr Eye Res 1988;7:1087-1095.
Ophthalmic Res 1992:24:92-98
Doheny Eye Institute and Department of Ophthalmology, University of Southern California. Los Angeles, Calif.; Department of Ophthalmology. Indiana University, Indianapolis, Ind.: Department of Medicine, UCLA/Cedars Sinai Medical Center and Department of Neurology and Pediatrics, Los Angeles; Stanford University School of Medicine, Stanford, Calif.. USA
KeyWords
Autoimmune uveitis Anti-I-A Suppressor T cell Adoptive transfer Retinal S-antigen
Antigen-Specific Suppressor Cells in Experimental Autoimmune Uveitis
Abstract
Anti-I-A antibodies, administered in vivo at the time of Santigen injection, suppress development of experimental au toimmune uveitis (EAU) in Lewis rats. While the effects of anti-I-A are profound, the exact mechanism for this suppres sion is unknown. We attempted adoptive transfer of this form of suppression by injecting lymphocytes from anti-I-A-trcatcd animals into syngeneic recipients which were later injected with S-antigen. Histologically, globes of 75% of the anti-I-Atreated animals showed no inflammation while 25% of these animals developed mild uveitis. In the group of animals which were injected with S-antigen and also received spleen cells from anti-I-A-treated rats, only 1 showed mild uveitis while the remaining 7 had no inflammation. The animals undergo ing adoptive transfer of spleen cells and which were primed with an irrelevant antigen, readily developed uveitis. Suppres sion of S-antigcn-induced EAU was abrogated by pretreat ment of donor animals with cyclophosphamide. In vitro stud ies revealed that spleen cells of S-antigen-primed, anti-I-Atreated donors specifically suppressed lymphocyte responses to S-antigen. These in vivo and in vitro results suggest that generation of antigen-specific suppressor cells play a role in the anti-I-A immunotherapy of EAU.
Introduction
Uveoretinitis induced by retinal S-antigen has been shown to be a useful animal model for uveitis. This experimental intraocular in
Received: August 15, 1991 Accepted: October 4. 1991
flammation can be produced in various labo ratory animals, including rats. Lewis rats are highly susceptible and readily develop uveo retinitis when injected with S-antigen [1], In these animals, the development of uveitis has
Narsing A. Rao. MD Doheny Eye Institute 1355 San Pablo St. Los Angeles. CA 90033 (USA)
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:09:11 PM
NarsingA. Raoa Lily Atal/a3 Shao-ling Fongb Fen Chena M. Linker-Israelic Lawrence Steinm and
Materials and Methods The studies described herein conformed to the ARVO Resolution on the Use of Animals in Research. Twenty-four Lewis rats were initially injected with 50 pg of S-antigen synthetic peptide (HSA 320) [7] in complete Freund’s adjuvant in the foot pad followed by 1 pg pertussis toxin intraperitoneally. The immu nized rats were divided into two groups: group 1 was treated with anit-I-A (OX 6), 1 mg/injection on days - 1 , + 1 and +2; group 2 was injected with RPMI 1640 medium according to the same time schedule. All ani mals were killed on day 15 after antigen administra tion. The eyes were enucleated for histologic study, and individual spleens from 8 animals in each of the two groups and from naive animals were used for isola tion of spleen mononuclear cells that were then trans ferred to three additional groups of syngeneic recipi ents. Donor spleen cells were separated on Ficollpaque gradients and resuspended in saline prior to intraperitoneal injection, 5 X 106 in each recipient. A group of S animals (group 3) received cells from antiI-A-treated donors (group 1). Another group of 6 ani mals (group 4) received spleen cells from RPMItreated donors (group 2), and another 6 animals (group 5) received cells from naive animals. On the day of spleen cell transfer, all recipients (groups 3-5) were injected with 50 pg of S-antigen synthetic peptide (HSA 320) in complete Freund’s adjuvant and pertus sis toxin as described above. The recipients were killed on day 15 after antigen administration. The eyes were enucleated from all animals (group 3 through 5) and processed for histopathologic examination. An additional group (group 6) was injected with cyclophosphamide, 20 mg/kg 2 days prior to 50 pg of S-antigen synthetic peptide administration in com plete Freund’s adjuvant. These animals were treated with anti-l-A antibody, 1 mg/injection, on days - 1 , +1 and +2 after the antigen injection. On day 15, after the administration of antigen, these animals were killed and the eyes were enucleated and processed for histo pathologic examination. Six other animals (group 7) were injected with 50 pg of synthetic peptide of IRBP [8] (HIRBP 715) in complete Freund’s adjuvant in the right hind foot pad. On the same day, these animals received first 1 pgof pertussis toxin, injected intraperi toneally, and later on the same day an intraperitoneal injection of 50 X 106 cells from group 1 animals. These animals were sacrificed on day 15 and the enu cleated eyes were fixed in formalin, embedded in par affin and stained with HE.
93
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been shown to be mediated by CD4 + major histocompatibility complex (MHC) class 11 restricted helper/inducer T cells [2], We have demonstrated that development of uveoretinitis in Lewis rats can be prevented by admin istration of anti-I-A antibody at the time of sensitization with S-antigen [3]. This inhibi tion was found to be dose-dependent, and when treated with 750 pg of anti-I-A, about 90% of animals showed amelioration of clini cal signs and absence of histopathologic evi dence of the disease. Even though the successful inhibition of uveitis could be achieved with an appropriate dose of anti-I-A antibody, the precise mechanism(s) underlying such inhibition was not clear [3], As the development of S-antigeninduced uveitis consists of a complex interac tion of various immunoreactive cells and their products, disturbance of any aspect of these interactions could lead to repercussions involving some other cellular response. One such repercussion could be the production of antigen-specific suppressor cells. Several pos sible mechanisms have been proposed for the anti-I-A-mediated suppression, including: (a) blocking or impairment of antigen presen tation in context with class II MHC results in a lack of activation of the antigen-specific CD4 + cells that mediate development of dis ease; (b) the anti-I-A antibodies may impair the antigen-presenting functions of macro phages and dendritic cells and induce sup pressor cells [4-6]; (c) compensatory antigenspecific activation of suppressor cells that rec ognize antigen in context with class I MHC when the class II MHC-restricted activation of helper/inducer cells is blocked [4], The studies reported here support the idea that at least 1 of the mechanisms of uveitis inhibition by anti-I-A antibodies is by generation of anti gen-specific suppressor cells.
Fig. 1. Animal treated with anti-I-A monoclonal antibody shows normal retina, ciliary body, iris and choroid. HE. X 150.
Fig. 2. Control animal treated with RPMI 1640 shows retinitis and iridocyclitis. HE. X 150.
94
incubated at 37 °C in 5% CO2 for 5 days. 1 pCi of 3Hthymidine was used to pulse the cultures similar to the method described earlier [9]. Results are expressed as stimulation index.
Rao/Atalla/Fong/Chen/Linker-Israeli/ Steinman
Suppressor Cells in EAU
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Lymphocyte Stimulation Studies In vitro lymphocyte stimulation was performed on 2 X 105 spleen cells of animals injected with S-antigen synthetic peptide (group 2) in the presence or absence of 5 X 104 spleen cells of animals treated with anti-I-A (similar to group 1). Retinal S-antigen synthetic pep tide, 4 |ig per well, and concanavalin A (ConA), 2 pg per well, were used for stimulation. Cultures were
Fig. 3. Animal pretreated with cyclophosphamide and injected with anti-I-A antibody demon strates development of iridocycli tis. HE. X 150.
Table 1. Suppression of uveitis by transfer of spleen cells
Experimental group and treatment
Number of animals
Animals with uveitis n
%
Treatment with anti-I-A Treatment with RPMI Recipient of spleen cells from group 1 Recipients of spleen cells from group 2 Recipient o f spleen cells from naive animals Pretreated with cyclophosphamide and injected with anti-I-A Recipients of spleen cells of group 1, and injected with IRBP
12 12 8 6 6
3/12 12/12 1/8 6/6 6/6
25 100 12 100 100
mild, focal severe, diffuse mild, focal severe, diffuse severe, diffuse
6
6/6
100
severe, diffuse
6
6/6
100
severe, diffuse
Histologically, globes of 9 of the 12 (75%) rats treated with anti-I-A showed no inflam mation (fig. 1), while 3 animals had mild uve itis. This inflammation was focal, involving the choroid and the retina. All animals treated with RPMI 1640 developed severe uveitis and retinits. The inflammation was diffuse
and there was destruction of various retinal layers (fig. 2). Of the spleen cell recipient ani mals in group 3, only 1 showed mild uveitis, and the remaining 7 had no inflammation. All animals in groups 4 and 5 developed severe retinitis and uveitis (table 1). All animals in group 6 and 7 showed severe choroiditis, iri docyclitis (fig. 3) and focal retinitis with de struction of retinal tissue.
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Results
Severity of uveitis
Table 2. Antigen-spccific suppression of stimulation index in the presence of spleen cells of animals treated with anti-I-A
Experiment
ConA
In the presence of cells from controlled nonimmunized animals In the presence of cells from anti-I-A-treated rats injected with S-antigen
S-antigen
count/min
SI
count/min
SI
2,533.0±48.1
8.37 ± 1.42
1.345.5 ±55.8
4.29 ± 1.16
2.213.0 ± 32.5 (8% suppression)
7.69± 3.25
260.0 ±57.9 1.20 ±0.53 (72% suppression)
Spleen cells from anti-I-A-treated donors had antigen-specific suppressive activity. There was a 72% suppression of the lympho cyte stimulation index when spleen cells of anti-I-A-treated animals were added to the Santigen synthetic-peptide-containing cultures and minimal (8%) suppression when these cells were added to the ConA-containing cul tures (table 2).
Discussion
The results of this study clearly show the effectiveness of immunotherapy in the pre vention of S-antigen-induced uveitis and in the generation of suppressor cells. In vivo administration of anti-I-A antibody has pre viously been shown to induce suppressor T cells that abrogate a delayed-type hypersensi tivity response to tumor antigens [6]. Anti-I-A treatment was also found to induce suppres sor T cells that attenuate the autoimmune response to myelin basic protein [10], As in these earlier experimental studies, the present study also demonstrated generation of sup pressor cells. These suppressor cells were found to be antigen-specific, as the spleen cell recipient animals in the adoptive transfer
96
study, animals which were also primed with IRBP. readily developed uveitis and retinitis, whereas such animals failed to show any clini cal or histopathologic evidence of uveitis when injected with S-antigen. Therefore, at tenuation of the disease in some animals and suppressed development of uveitis in others may be attributed to the generation of sup pressor function (table 1). The generation of suppressor cells was cyclophosphamide-sensi tive, as the protective effect could be nullified by pretreatment of donor animals with a lowdose of cyclophosphamide. These results sug gest that antigen-specific immune suppres sion of uveitis is feasible. Several mechanisms could account for the generation of suppressor cells following treat ment with anti-I-A monoclonal antibodies. The I-A-specific antibodies may affect the dis tribution of S-antigen within the plasma membrane of the antigen-presenting macro phages, thereby blocking the interaction of antigen with I-A, which may prompt the anti gen to shift to other determinants of the la, such as I-E or I-J [ 11. 12], Thus. S-antigen pre sentation, where I-A is blocked, may provide an activation signal for suppressor cell precur sors. Other mechanisms for activation of sup pressor T cells include direct stimulation of
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SI = Stimulation index.
hans cells of the dermis. Anti-I-A antibodies could nullify the antigen-presenting function of splenic cells, but not that of Langerhans cells [20], Similarly, the inhibition noted with anti-I-A treatment could be from the effect of anti-I-A antibody on the peripheral lymphoid and splenic I-A-positive cells rather than on the la-positive cells, such as retinal vascular endothelial cells and Müller cells. As reported earlier, maximum effect of anti-I-A on the inhibition of uveitis was seen when the anti body was administered early, within a week after administration of S-antigen [3], If the antibody administration were delayed, the prevention of uveitis was minimal or nonsig nificant, which also suggests that the anti-I-A antibody effect was extraocular. In vivo adoptive transfer experiments, as well as in vitro lymphocyte proliferation (ta bles 1,2) in the presence of spleen cells from naive versus anti-I-A antibody-treated ani mals, show antigen-specific suppression and intact T-cell response to nonspecific mito gens, and a specific response to IRBP. These studies suggest that anti-I-A therapy may not lead to the global immune suppression that occurs with various cytotoxic agents currently used in the treatment of severe recalcitrant uveitis unresponsive to corticosteroids. Even though ciclosporin is known to selectively in hibit T-cell helper function, this drug is not antigen-specific, as is anti-I-A therapy. More over, ciclosporin and other cytotoxic agents are associated with severe side effects. Even though prelim inary studies with anti-I-A ther apy showed no untoward effects in the form of glomerulonephritis or serum sickness in the experimental animals [3], further indepth studies in nonhuman primates having experi mental autoimmune uveitis should be carried out prior to any consideration of anti-I-A treatment in humans with severe uveitis.
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these suppressor cells by anti-I-A antibody. Such a mechanism appears unlikely, however, as various antigen-specific suppressor cells have been shown to express I-J-encodcd de terminants and consistently lack an I-A deter minant. A 3rd possible mechanism may result from a high concentration of circulating Santigcn due to diminished antigen ingestion by antigen-presenting cells, including macro phages. However, there is no evidence to sup port the idea that anti-I-A antibodies prevent ingestion of antigen, which would lead to high concentrations of circulating soluble antigens [13], However, further experimental studies are required to determine which, if any, of these putative mechanisms is operative. Thus far. anti-I-A antibody administration has reversed conditions triggered by immuni zation with self antigens, including experi mental allergic encephalomyelitis after im munization with myelin basic protein [14], experimental autoimmune myasthenia gravis following acetylcholine receptor inoculation [15], experimental autoimmune thyroiditis after thyroglobulin immunization [16], colla gen-induced arthritis due to type II collagen injection and experimental autoimmune uveitis induced by S-antigen [3], Anti-I-A an tibodies have been used successfully to treat 2 spontaneously occurring autoimmune condi tions: the lupus-like disease in NZB/W mice and the diabetes-like condition in BB rats [ 17, 18], Such immunotherapy was also found beneficial in the reversal of chronic paralysis in mice infected with Theiler’s virus [19], All of these experimental studies demonstrate the utility of anti-I-A antibodies for treatment of autoimmune conditions triggered by organspecific antigens, and where the disease sus ceptibility is linked to class II MHC genes. It has been demonstrated that in vivo ad ministration of anti-I-A antibody can differ entially affect la antigen expression by var ious cells including spleen cells and Langer
Acknowledgements These studies were supported in part by National Eye Institute grant EY09088, and by the Research to Prevent Blindness, Inc., NY., and Core Grant EY03040. The animals used in this study were main
tained in animal care facilities fully accredited by the American Association of Laboratory Animal Science. The authors express their appreciation to Ms. Irene Cumplido for preparation of the manuscript and to Ms. Ann Dawson for editorial assistance.
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