prevention of experimental autoimmune uveoretinitis by intrathymic s-antigen injection

Ocular Immunology and Inflammation Prevention of experimental autoimmune uveoretinitis by in trat hymic S-ant igen inject ion 0927-3948/97/US$ I 2.00

Ocular Immunology and Inflammation

0 Eolus Press Buren (The Netherlands) 1997

-I997, Val. 5, NO. 3, pp . 165-172

Accepted 18 March 1997

Steven B. Koevaryl Rachel R.Caspi

New England College of Optometry, Ocular Research Center, Boston, MA 021 15; *Section of Immunoregulation, Laboratory of Immunology, National Eye Institute, National Institutes of Health,

Bethesda, MD20892; USA

Abstract The objective of this paper was to determine whether intrathymic injection of retinal S-antigen (S-Ag) can prevent experimental autoimmune uveoretinitis (EAU) in Lewis rats. Lewis rats were injected intrathymically with 25-100 pg of S-Ag in IOO p1 split between thymic lobes. Controls received vehicle alone (PBS) or IOO pg of BSA. Animals were immunized two weeks later with IOO pg of S-Ag in CFA with or without pertussis toxin (0.5 pg/rat). Clinical ocular disease was confirmed by histopathology. Splenocytes and lymph node cells were assayed, in vitro, for their

Correspondence to: Steven B. Koevary, Ph.D., New England College of Optometry, Ocular Research Center, 424 Beacon Street, Boston, MA 02115, USA. Feel free to FAX correspondence to: 6 17-424- 9202. e-mail: KoevarysQne- optometry-edu.

ability to proliferate in response to various concentrations of S-Ag. Further- more, attempts were made to adoptively transfer protection to naive rats using spleen cells from intrathymically injected animals and to adoptively transfer EAU to protected rats using Con A activated cells from affected animals. Intrathymic injection of S-Ag reduced the incidence of EAU in animals subsequently immunized with S-Ag and pertussis, and prevented it en-

Acknowledgments: The authors wish to thank Dr. Chi-Chao Chan for grading the histopathology slides, and Robin Segal and Kerry Sullivan for their technical assistance.

tirely in rats immunized in the absence of pertussis. Splenic and lymph node cells from intrathymically injected animals showed reduced reactivity to S- Ag compared to controls, suggesting that intrathymic S-Ag injection may have rendered them tolerant to this antigen. We were unable to adoptively transfer protection to naive rats, nor were intrathymically injected rats protected from EAU induced by the adoptive transfer of primed lymph node cells. These data demonstrate that intrathymic S-Ag injection can be an effective method for protection from EAU, apparently through the induction of immunological tolerance and not active suppression. The tolerance was not absolute and could be overcome by increasing the intensity of the antigenic challenge.

Key words intrathymic; prevention; pertussis

Experimental autoimmune uveoretinitis (EAU); S-antigen;

Introduction Experimental autoimmune uveoretinitis (EAU) is an organ-specific, T cell-mediated disease primarily affecting the posterior seg- ment of the eye.',' It is induced in rats (as well as other species) by immunization with retinal antigens, such as S-antigen (S-Ag) and interphoto-

Intrathymic S-Ag injection prevents EAU 165

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receptor retinoid-binding protein (IRBP). EAU serves as a model for several human sight-threatening ocular inflammation^,^ including sympathetic oph- thalmia, birdshot retinochoroidopathy, Vogt-Koyanagi-Harada syndrome, and Behqet's disease. The disease can be transferred by immune lymph node cells and long-term CD4+, MHC class 11-restricted T cell lines4q5 and major histocompatibility (MHC) genes were found to play a role in susceptibility.2 Therapeutic strategies for the suppression of EAU have included the use of cyclosporin,6 FK506? oral tolerance? and ACAID.9

The thymus plays a major role in the development of self tolerance." Around 30 years ago, it was suggested that the thymus might also induce acquired tolerance to exogenous antigens.' ',Iz lnterest in this area was rekin- dled by the work of Posselt et al.I3 who induced donor-specific unresponsiveness to islet allografts by intrathymic islet transplantation. Many reports have followed confirming that intrathymic antigen administration induced a state of specific systemic unresponsiveness in transplantation models (see reference l4 for review). It has also been demonstrated that such a procedure was effective in preventing some autoimmune diseases in rodent models. 1 5 -

l 7 EAU shares many characteristics with these autoimmune models, the chief difference being the target organ. The objective of this study was to determine whether intrathymic injection of S- Ag can prevent the development of EAU in Lewis rats.

Materials and methods

ANIMALS Female Lewis rats, five to seven weeks of age, were supplied by Harlan Sprague Dawley (Indianapolis, IN) from colony 202B. All proce- dures utilizing these animals adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

INTRATHYMIC INJECTION OF S - A G Under ketamine/xylazine anesthesia (0.1 ~ V I O O g body weight), the thymus was exposed via a partial median sternotomy. Under direct visualization, various concentrations of S-Ag, ranging from 25 to roo pg, were injected intrathymically in a total volume of IOO ~ 1 , distributed equally between the two thymic lobes. The wound was sealed using surgical staples. Control rats were injected intrathymically with either IOO pg of BSA or PBS alone.

INDUCTION A N D EVALUATION OF EAU Two weeks after intrathymic injections, rats were immunized subcutaneously in the right thigh with IOO

pg of S-Ag emulsified in complete Freund's adjuvant (CFA; Sigma Chemi- cal Co., St. Louis, MO), supplemented with a total of 2.5 mg/ml of Myco- bacterium tuberculosis H37Ra (Difco Laboratories, Detroit, MI). In rats in which pertussis toxin (Sigma) was used, 0.5 pg/rat was injected i.p. Nega- tive controls included animals immunized with CFA 5 pertussis toxin. All animals were evaluated for clinical ocular changes beginning five days after immunization. Clinical EAU was graded on an arbitrary scale as previously described.2 Eyes were collected for histopathology approximately seven days following the onset of clinical disease, fixed in 4% glutaraldehyde for one hour, and postfixed in 10% formalin. Tissues were embedded in paraffin and 4 to 6 pm sections stained with hematoxylin and eosin. Histopathology was graded on a scale of o to 4 as previously described.2

166 S. B. Koevary & R. R. Caspi

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PROLIFERATION ASSAY Four rats intrathymically injected with S-Ag and four rats intrathymically injected with BSA were sacrificed approximately three weeks after immunization and their spleen and draining lymph nodes were removed and dissociated into single-cell suspensions and counted in a Coulter Z I cell counter. Spleen cells werc pre-treated with ammonium chlo- ride to remove red blood cells. Washed cells were adjusted to 106 celldm1 in RPMI media supplemented with 1.5% syngeneic rat serum, 5 mM gluta- mine, 5 x I o - ~ M p-mercaptoethanol, and penicillirdstreptomycin ( IOO U/ml and IOO pg/ml, respectively). Aliquots (100 pl) were added to microtiter wells of a 96-well, round-bottomed plate (Falcon), to which IOO pl of media containing either 40, 4, or 0.4 pg of S-Ag, or media alone, were added in quadruplicate. The plates were incubated for four days at 37°C in a humidi- fied 5% CO, atmosphere. Tritiated thymidine ( I pCi; New England Nuclear, Boston, MA) was added to the cultures eight hours prior to harvesting and samples were counted in a Packard 2500 TR scintillation counter. Back- ground counts were negligible and were subtracted from each group.

ADOPTIVE TRANSFER To determine whether protection can be transferred, spleen cells from intrathymically injected rats ( 10' cells), obtained between three and four weeks after immunization, were injected into naive Lewis rats one to two weeks prior to immunization with S-Ag in order to determine whether they can suppress the induction of EAU. Injection of untreated spleen cells from non-uveitic control rats served as controls. To determine whether intrathymically injected rats were protected from adoptively transferred EAU, draining lymph nodes from rats with EAU were cultured for 72 hours in RPMI containing 10% heat inactivated fetal calf serum, 5 mM glu- tamine, 5 x I O - ~ M P-mercaptoethanol, and penicillin and streptomycin ( IOO

U/ml and IOO pg/ml, respectively), and 5 pg/ml of Con A (Sigma). Cells were counted and 50 x 106 cells injected i.p. into rats which were intrathymically injected with S-Ag six weeks earlier and which were protected from developing EAU following immunization, and naive controls.

STATISTICS Comparisons of the incidence of EAU in the different groups were made by logistic regression modeling.'8 Significance was assessed by likelihood ratio tests. Results of the proliferation assay were analyzed using an analysis of variance for repeated measures employing an SPSS for Win- dows program.

Results

INTRATHYMIC S-AG INJECTION PROTECTS FROM E A U I N D U C T I O N In- jection of various concentrations of S-Ag into the thymus of Lewis rats prior to immunization with S-Ag reduced or prevented the development of EAU (see Table I ) . Sham operated rats developed a high incidence of EAU in both the pertussis and non-pertussis groups. Similarly, rats intrathymically injected with BSA also developed EAU following immunization. There was a dose-dependent decrease @=0.02) in EAU in rats intrathymically injected with doses between 25 and IOO pg of S-Ag in those immunized with S-Ag and pertussis. However, when immunizations were carried out in the absence of pertussis, all intrathymic doses of S-Ag completely prevented EAU (~<o.oooI). Injection of rats with CFA alone or with pertussis failed to in-

Intrathymic S-Ag injection prevents EAU 167

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TABLE I Effects of intrathymic S-Ag injection on induction of EAU

Intrathymic injection Immunization protocols

antigen+ CFA +pertussis antigen+CFA, no pertussis *Incidence of disease (positiveltotal)

controls sham operated 8/ro* 8/12 BSA (100 pg) 313 7/14

25 515 015 50 315 018

I00 318 0120

S-Ag (P.g)

TABLE 2 EAU histopathology in experimental and control rats

duce EAU (0/4 total). Clinical disease was confirmed histopathologically approximately one

week following onset and, in many of these animals, was quantitated using an arbitrary grading scale in a masked fashion. Nearly all cases of EAU that developed in control and intrathymically injected rats immunized with or without pertussis were severe (3.3 to 4.0; see Table 2 for histopathology results). Examples of retinal histology from an intrathymically injected and control rat are shown in Figures IA and IB. Fig. IA was taken from a control rat nearly four weeks after clinical disease onset, and shows post-acute disease in which inflammation has subsided. The inflammatory cells are ab- sent in most of the photoreceptor cell layer. The retina has re-attached to the choroid. Fig. IB shows a normal retina obtained from an intrathymically injected rat.

LYMPHOCYTE PROLIFERATIVE RESPONSE TO S-AG IS REDUCED I N PRO-

TECTED RATS The reactivity of spleen and lymph node cells to S-Ag in intrathymic S-Ag-treated and sham-injected rats was measured in a proliferation assay. Results are shown in Table 3 . Spleen and lymph node cells from rats intrathymically injected with S-Ag showed significantly reduced proliferation to all three in vitro S-Ag concentrations compared to controls.

PROTECTION IS NOT TRANSFERABLE WITH SPLEEN CELLS O F INTRA-

THYMICALLY S-AG INJECTED RATS Adoptive transfer of spleen cells from non-uveitic intrathymically injected rats failed to protect three of four naive rats from developing EAU 12.350.7 (meankSEM) days following S- Ag immunization. Three of five uninjected controls similarly developed disease.

Intrathyrnic injection 2 pertussis EAU score + SE (a )

IOO Pg S-Ag 50 Pg S-Ag 25 Pg S-Ag

*Score was calculated as the average of the number (n) of positive eyes examined in each group. Sham

Sham IOO pg BSA


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Fig. I . Retinal histology from a representative control (A) and intrathymically injected (B) rat. The retina from the control rat, sacrificed 26 days after onset, revealed a paucity of inflammatory cells. V = vitreous; C = choroid; S = sclera; PE =pigment epithelium. Most of the photoreceptor cell layer is gone, except for a few dark nuclei (arrowheads). Fig. rB from an intrathymically injected rat shows normal retinal histology.

INTRATHYMICALLY S-AG-INJECTED RATS ARE N O T PROTECTED FROM

UVEITOGENIC C H A L L E N G E BY ADOPTIVE TRANSFER Of the five rats previously intrathymically injected with S-Ag to protect them from EAU which received Con A-activated cells from uveitic rats, three developed EAU I ok0.4 (mean+SEM) days post injection. Histopathologically, the

Lymphocyte source [S-As] irz yg

40 4 0.4

Control spleen 57303* 3,3001 1.558-1: Experimental spleen 1,183 949 653 Control node 78,2475 70,865" 33293" Experimental node 8,654 9.643 5,211

Intrathymic S-Ag injection prevents EAU

TABLE 3 Proliferative response of experimental and control immune cells to S-Ag.

Results are CPM; *p<0.0004 compared to comparable experimental spleen; tp<o.o3 compared to comparable experimental spleen; $p<o.or compared to comparable experimental spleen; §p<o.oz compared to comparable experimental node; "p<0.009 compared to comparable experimental node; @<0.03 compared to comparable experimental node

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mean+SEM EAU score of positive animals was 2.1k0.7. Two of four naive controls similarly developed EAU.

Discussion This study showed that intrathymic S-Ag injection can protect Lewis rats from EAU. Intrathymic injection of increasing concentrations of S-Ag significantly reduced the incidence of EAU in the pertussis group while all tested concentrations of S-Ag completely prevented EAU in the group immunized in the absence of pertussis. It thus appeared that the tolerance to S-Ag was not absolute and could be overcome by increasing the intensity of the antigenic challenge.

Intrathymic injection of self antigens has been used successfully to prevent other autoimmune diseases in rodents such as experimental autoimmune encephalomyelitis (EAE) and Type I diabetes. In some of those cases, cellular antigens were intrathymically injected’ 5~19320 while others utilized soluble antigens such as myelin basic protein (EAE)17 or glutamic acid decarboxylase (diabetes).”

Prevention of EAU in this model appeared to be due to a reduction in the cell mediated response to S-Ag. Our results showed a significant reduction, in intrathymically injected rats, in spleen and draining lymph node cell proliferation to three S-Ag concentrations compared to controls. Others have shown that such a reduction in proliferation following intrathymic antigen injection was due to a suppression of ThI cells,22 which have also been im- plicated in EAU.23

Intrathymic self antigen injection probably heightens self tolerance by either deleting antigen-reactive cells or by inducing their functional inactiva- tion (anergy). Anergy has been shown to play a role in the prevention of EAU produced by the injection of antigen-coupled spleen cellsz4 and was reported to be one of several mechanisms which played a role in oral toler- a n ~ e . ’ ~ What did not appear to play a role in EAU protection following intrathymic S-Ag injection was active suppression. This was suggested by our results showing an inability to adoptively transfer disease protection into naive recipients and our ability to transfer EAU into rats previously injected intrathymically with S-Ag, supporting the interpretation that the mechanism of tolerance involves anergy or deletion of S-Ag-reactive cells, rather than the induction of regulatory cells. It is conceivable, but unlikely, that the cho- sen intervals for injection, harvesting, and transfer of cells may not have been ideal for revealing suppressor cells. Failure to adoptively transfer disease protection was similarly reported following intrathymic and myelin basic protein17 injection.

Tolerance induction following intrathymic antigen injection need not pre- suppose that the experimental antigen is normally expressed there, since alloantigens can induce intrathymic tolerance. In the case of alloantigens, the normal tolerizing potential of the thymus is merely usurped by the foreign antigen. Data are emerging, however, suggesting that peripheral, organ specific antigens, thought to be tolerized peripherally, may normally be expressed in the thymus. Thus, mRNA of the acetylcholine receptor26 and S- Ag27 were recently detected in the thymus. At least in the case of S-Ag, it is not yet clear that the protein is indeed expressed.

The intrathymic tolerance technique is valuable as a model system to study tolerance mechanisms which normally play a role in preventing autoimmune eye disease. Not only can soluble antigen be used but isolated


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photoreceptors can be implanted into the thymus to study the long term effects of their tolerization on development of disease and to investigate the role of the thymus in normal retinal tolerance induction. With further development and understanding of its mechanism of action, the intrathymic S-Ag approach could conceivably also be utilized in humans to prevent (or re- verse) disease, joining oral and intravenous tolerance schemes in this regard. It is possible that more than one of these approaches will be required to prevent disease in humans. Though the thymus undergoes involution after pu- berty, it remains a viable, T cell-producing organ throughout life. Attempts at intrathymic antigen injection in humans have begun28,2g and it thus far appears that the technique can be performed without undesired side effects.29

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172 S. B. Koevary & R. R. Cuspi

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prevention of experimental autoimmune uveoretinitis by intrathymic s-antigen injection

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