163Immunosuppression and BMT in ocular autoimmunity

Correspondence and reprintrequests to:Dr. Shoshana SavionDept. of Embryology and TeratologySackler School of MedicineTel Aviv UniversityRamat Aviv, Tel Aviv 69978IsraelTel: 972-3-640-9626Fax: 972-3-640-6149e-mail: shoshans@plato.tau.ac.il

Research reports

Acute immunosuppression and syngeneicbone marrow transplantation in ocularautoimmunity abort disease, but do not

result in induction of long-term protection

Shoshana SavionPhyllis B. SilverChi-Chao ChanRachel R. Caspi

Laboratory of Immunology, National Eye Institute, NationalInstitutes of Health, Bethesda, MD, USA

Ocular Immunology and Inflammation0927-3948/98/US$ 12.00

Ocular Immunology and Inflammation –1998, Vol. 6, No. 3, pp. 163-172© Æolus PressBuren (The Netherlands) 1998

Accepted 23 April 1998

Abstract Acute immunosuppression induced by total body irradiation(TBI) or cyclophosphamide (Cy) treatment, followed by syngeneic bonemarrow transplantation (SBMT), was reported to be effective in inducinglong-term tolerance in some autoimmune disease models. We examinedthe efficacy of this approach in the mouse model of experimental auto-immune uveoretinitis (EAU). Development of EAU induced by theinterphotoreceptor retinoid binding protein (IRBP) was abolished almostcompletely by either TBI or Cy treatment, followed by SBMT, institutedone week after priming. In parallel, IRBP-specific delayed-type hyper-sensitivity (DTH) responses and lymph node cell proliferation were stronglysuppressed or abolished. However, when these IRBP-immunized, lympho-ablated and BM reconstituted mice were rechallenged with the immunizingantigen seven weeks after the primary immunization, they were notprotected from developing disease, despite the fact that DTH and lymphnode cell proliferation to the antigen were suppressed relative to controls.TBI treatment appeared somewhat more effective than Cy treatment asjudged by its more profound effect on immunological responses. Theseresults demonstrate the ability of acute immunosuppression followed byreconstitution of the immune system to inhibit the development of EAU,although long-term protection from disease was not achieved.

Key words Experimental autoimmune uveoretinitis; acute immunosup-pression; total body irradiation; cyclophosphamide; syngeneic bone marrowtransplantation

Introduction EAU is a T cell-mediated autoimmune disease thatserves as a model for several human sight-threatening ocular inflammatoryconditions that affect mainly the posterior segment of the eye and areimmune-mediated or autoimmune by nature.1-4 These include sympatheticophthalmia, birdshot retinochoroidopathy, Vogt-Koyanagi-Harada syn-

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S. Savion et al.164

drome, and Behcet’s disease. In animals, EAU can be induced by immu-nization with several different retinal proteins and can be adoptivelytransferred to syngeneic hosts with immune T cells or T cell lines.2,5-8

The most widely studied and characterized uveitogens are the 48-kDsoluble antigen (S-Ag) and the 140-kD interphotoreceptor retinoid bindingprotein (IRBP). Both are major components of the photoreceptor celllayer, which is the primary target of the autoimmune attack in EAU.2,9-11

In addition to providing a model for the study of basic immunologicalmechanisms in ocular inflammation, EAU also provides a means of testingthe efficacy of various treatment modalities. In recent years, attentionhas centered on various immunomodulatory agents as a means forcontrolling immune-mediated inflammatory processes in the eye. Theseinclude the immunosuppressive drugs cyclosporin A (CsA) and FK506,monoclonal antibodies against S-Ag, interleukin-2 receptor and MHCClass II molecules, as well as oral administration of S-Ag.12-15

Karussis et al.16 treated experimental autoimmune encephalomyelitis(EAE), which is also a model for T cell-mediated immunity, by meansof immunosuppression with cyclophosphamide (Cy) or total bodyirradiation (TBI), followed by syngeneic bone marrow transplantation(SBMT). Both treatments have also been utilized by other investigatorsto treat EAE; however, these reports were somewhat controversial.17-21

Since EAU and EAE share essential immunological mechanisms andfrequently respond to similar immunotherapeutic approaches,15 we thoughtit relevant to examine the possible therapeutic effect of lymphoablationin combination with SBMT on EAU development. We present data showingthat acute immunosuppression followed by reconstitution of the immunesystem abort the development of EAU, but do not result in induction oflasting tolerance to the immunizing antigen that would manifest itself asprotection from disease.

Materials and methods

mice Six- to seven-week-old female B10.A mice were purchased fromthe Jackson Laboratory (Bar Harbor, ME, USA). Animals were housedunder specific pathogen-free conditions and kept on a regular laboratorydiet. Care and use of the animals conformed to ARVO and NIH guidelines.

antigens IRBP was prepared from bovine retinas by ConA-Sepharoseaffinity chromatography and fast performance liquid chromatography asreported previously.18 Purified protein derivative of tuberculin (PPD)was obtained from Connaught Laboratories (Swiftwater, PA, USA); keyholelimpet hemocyanin (KLH) and phytohemagglutinin (PHA) were purchasedfrom Sigma (St. Louis, MO, USA). IRBP for proliferation and DTHassays was dialyzed against PBS and sterile-filtered.

induction of eau For EAU induction by either primary challenge(day 1) or secondary challenge (day 50), mice were immunized S.C.with 50 ªg IRBP in a 1:1 (v/v) emulsion with CFA containing 2.5 mg/ml Mycobacterium tuberculosis H37RA in a total volume of 0.2 ml dividedbetween base of tail (0.1 ml) and both thighs (0.05 ml/each). Bordetellapertussis toxin (PTX), 0.5 mg in a volume of 0.2 ml, was injected I.P. at

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165Immunosuppression and BMT in ocular autoimmunity

the same time. In some experiments, mice were given the secondarychallenge as a mixure of IRBP and KLH in CFA at 50 ªg/ml each.KLH, an irrelevant antigen, was included as an additional measure ofthe effectiveness of the reconstitution.

tbi Seven days after the primary IRBP immunization, mice were placedin irradiation chambers in a GammaCell irradiator utilizing a 137Cs source,and exposed to a single dose of 1100 rad.

cy treatment Seven days after the primary IRBP immunization,mice received a single I.V. injection of Cy (Sigma, St. Louis, MO, USA)at 300 mg/kg.

reconstitution by sbmt Syngeneic bone marrow cells were obtainedby flushing freshly dissected femora and humora with RPMI-1640 througha 25-gauge needle. The cells were teased into a single cell suspensionand viability was determined by trypan blue exclusion. Twenty millioncells in 0.5 ml RPMI supplemented with 1.5% mouse serum were injectedI.V. into irradiated or Cy-treated mice, eight days after IRBP immunization(=24h after lymphoablation treatment).

clinical and histopathological evaluation of eau Animalswere examined weekly by fundoscopy starting two weeks afterimmunization. At the end of the experiments (10 weeks following IRBP-primary immunization), eyes were collected for histopathologicalevaluation. Freshly enucleated eyes were fixed for one hour in 4%phosphate-buffered glutaraldehyde and transferred into 10% phosphate-buffered formalin until processing. Fixed and dehydrated tissue wasembedded in methacrylate and 4-6 ªm sections were stained withhematoxylin and eosin for examination by light microscopy. Slides werescored in a masked fashion by one of us, an ophthalmic pathologist(CCC). At least six sections were examined per eye. Clinical or histologicalseverity of the disease as recorded by fundoscopy or by examination ofthe sections, respectively, was graded on a scale of 0-4 according to thetype and extent of the lesions, using the criteria described previously.22

proliferative responses of lymph node cells Draining lymphnodes were collected at the end of the experiments (10 weeks followingIRBP-primary immunization), pooled within each group, and teased intosingle cell suspensions. The cells were suspended in RPMI-1640supplemented with 2 mM L-glutamine, 1 mM nonessential amino acids,1 mM sodium pyruvate, 5×10-5 M 2-mercaptoethanol, 50 ªg/mlgentamycin, 1% normal mouse serum and alpha methyl mannopyranoside(20 mg/ml), which was included in the medium to neutralize traces ofConA used for IRBP purification. It had previously been determinedthat this concentration of ConA has no effect on cell proliferation. Triplicatecultures (4×105 cells/well) were plated in 96-well flat-bottom multiwellplates in the presence of 30 ªg/ml IRBP or KLH, 20 ªg/ml PPD, 1 ªg/ml PHA or in medium only for 72 hours. The plates were pulsed with 1ªCi/well 3H-thymidine (New England Nuclear, Boston, MA, USA) duringthe last 18 hours of culture and harvested and 3H-thymidine uptake wasdetermined by standard liquid scintillation techniques.

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delayed hypersensitivity measurement Twenty-four hoursbefore termination of the experiments, mice were anesthetized and injectedintradermally into the ear pinna with IRBP or KLH at 1 mg/ml in 10 ªlPBS/ear using a 30-gauge needle. Each antigen was injected in a differentear. Untreated naive mice, which were injected in the same way, servedas controls. At the end of the experiments (10 weeks following IRBP-primary immunization), ear thickness was measured with a spring-loadedmicrometer (3 measurements/ear) and the specific response was calculated.

Results Mice were immunized for EAU induction with IRBP, weretreated with TBI or Cy followed by SBMT as described in Materials andmethods, and were challenged with a second uveitogenic immunizationseven weeks after the primary immunization. In some cases, KLH (as anirrelevant antigen) was included in the challenge as an additional measureof the effectiveness of the reconstitution. The experiments were terminatedthree weeks later, a total of ten weeks after the primary immunization.The timeframe of the experiments is shown in Figure 1.

The effect of TBI or Cy treatment followed by SBMT on the clinicalcourse of IRBP-induced EAU, as evaluated by fundoscopy, is shown inFigure 2. Mice were followed by fundoscopy starting two weeks afterthe primary immunization, when full-blown EAU had already developedin nonlymphoablated animals. As can be seen, the irradiation as well asthe Cy treatment were very effective and resulted in a complete abrogationof the disease, as compared to the nonlymphoablated control group.However, when challenged with the immunizing antigen seven weeksafter the primary challenge, all mice developed comparable disease scores,although its onset in the treated mice was somewhat delayed.

The effect of TBI or Cy in combination with SBMT on the histopathologyof EAU is shown in Table 1. Histopathology was always evaluated threeweeks after the uveitogenic challenge (primary or secondary). Groups 1-3 were not rechallenged and were evaluated for EAU induction threeweeks after the primary immunization. The disease scores of the firstgroup show that the primary uveitogenic challenge was effective in inducingEAU in the expected three weeks timeframe. In contrast, none of thelymphoablated and SBMT-treated animals developed disease for as long

Fig. 1. Timescale of the experiments.

Fig. 2. The effect of TBI or Cytreatment in combination with SBMTon the clinical course of EAU. Sevendays after IRBP immunization, themice were either irradiated (}) orinjected with Cy (i), followed bySBMT the next day. IRBP-immunizedmice only (]) served as controls. Onday 50 of the experiment, all threegroups of mice were challenged withIRBP and KLH and the experimentwas terminated three weeks later, asdescribed in Materials and methods.Each point represents the meanclinical score of six mice. Shown isone out of three separate experiments.

1 2

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167Immunosuppression and BMT in ocular autoimmunity

as three weeks (groups 2 and 3) or seven weeks (groups 5 and 6) afterthe primary immunization, when they were rechallenged (groups 5 and6). Upon IRBP rechallenge, the lymphoablated and reconstituted animalsdeveloped EAU whose severity was only marginally lower than in theuntreated/rechallenged group (group 4). To test the efficiency of thereconstitution of the immune system after SBMT, previously unimmunizedmice were treated with TBI or Cy followed by SBMT and were challengedwith IRBP six weeks later. These animals (groups 7 and 8) indeeddeveloped EAU, although of a reduced severity than that developed byunmanipulated mice (group 1), indicating that although the reconstitutionwas sufficient to support the development of EAU, it was not complete.

The histopathology of EAU that developed after lymphoablationtreatment followed by SBMT is presented in Figure 3. Examination ofsections collected from eyes of IRBP-immunized mice demonstratedthe typical pathology of EAU, mainly an extensive destruction of thephotoreceptor cell layer (Fig. 3A). In contrast, eyes of IRBP-immunizedmice that were treated with TBI followed by SBMT and not rechallengedstill showed no evidence of EAU pathology as long as seven weeksafter the primary immunization (Fig. 3B).

Table 2 shows the effect of lymphoablation and reconstitution on DTHresponses of mice immunized for EAU induction. IRBP-immunized anduntreated mice (group 1) developed a strong DTH reaction towards theimmunizing antigen, but had no preexisting background responses toKLH. Treatment with TBI and SBMT completely abrogated the DTHreaction, while after Cy + SBMT treatment it was reduced but detectable.When the animals were challenged with IRBP and KLH, they developeda strong DTH response, which was significantly decreased in mice afterTBI or Cy treatment followed by SBMT (groups 5 and 6). Mice thatwere not immunized before lymphoablation developed reduced DTHresponses in comparison to similarly immunized, unmanipulated mice,confirming that although reconstitution was effective, it was not complete.

Group # Treatment Incidenceb Severityc

1 IRBP 4/4 2.192 IRBP+TBI+SBMT 0/4 03 IRBP+Cy+SBMT 0/4 0

IRBP Challenge4 IRBP 4/4 3.885 IRBP+TBI+SBMT 3/4 2.066 IRBP+Cy+SBMT 4/4 2.447 TBI+SBMT 3/3 0.798 Cy+SBMT 2/2 1.75

aSeven days after IRBP immunization, the mice were either irradiated or Cy-treated,followed by SBMT the next day. On day 50 of the experiments, mice were challengedwith IRBP and the pathology of EAU was evaluated three weeks later.bIncidence is shown as positive out of total animals.cSeverity represents mean scores of only those animals that developed EAU.

table 1. The effect of TBI or Cy incombination with SBMT on thepathology of EAU.a

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Fig. 3. The effect of TBI incombination with SBMT on thehistological manifestation of EAU.(A) Eyes of IRBP-immunized micedemonstrate an extensive destructionof the photoreceptor cell layer. (B)Treatment of IRBP-immunized micewith TBI and SBMT results in anintact retinal architecture (×160).

Group # Treatment DTH (x10-2 mm)b

IRBP KLH

1 IRBP 14.95 -0.82 IRBP+TBI+SBMT -1.7 2.73 IRBP+Cy+SBMT 11.5 3.95

IRBP+KLH Challenge4 IRBP 15.45 19.455 IRBP+TBI+SBMT -0.8 8.456 IRBP+Cy+SBMT 5.2 15.77 TBI+SBMT 4.95 11.98 Cy+SBMT 8.3 18.1

aTime scale of the experiments is similar to that described in Table 1.bDTH challenge was given 24 hours before termination of the experiments and earthickness was measured the next day as described in Materials and methods.

table 2. DTH in EAU-induced micetreated with TBI or Cy in combinationwith SBMT.a

All KLH-challenged animals responded very well to KLH, confirmingthe specificity of the cellular responses.

Lymph node cell proliferation in mice immunized for EAU inductionafter the various treatments is shown in Table 3. IRBP-immunized miceshowed a strong proliferative response to the immunizing antigens, whichwas absent in mice that received treatment with either TBI or Cy followedby SBMT. Cy treatment was found somewhat less ablative than TBI forthe DTH and EAU responses. Immunized, lymphoablated, reconstituted,and rechallenged mice (groups 5 and 6) had lower proliferative responsesto IRBP and to PPD than IRBP-rechallenged mice that had not beenimmunosuppressed and reconstituted (group 4). As for other responses,mice that had not been immunized before immunosuppression, recon-stitution, and rechallenge (groups 7 and 8) demonstrated a depressedproliferative response, especially after TBI treatment, indicating thatreconstitution was not complete. However, it should be pointed out thatlymph node cells of groups that underwent the various treatments respondedvery well to KLH as well as to PHA (data not shown), suggesting thatalthough not completely reconstituted, the immune system had thecapability of supporting diverse immune responses.

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169Immunosuppression and BMT in ocular autoimmunity

Discussion Allogeneic bone marrow transplantation has been usedsuccessfully for the treatment of several animal models of autoimmunediseases including EAE, lupus-like syndrome in MRL, NZB, and BXSBmice, and insulin-dependent diabetes in NOD mice.23-27 These studiesemphasize the potential value of such combined procedures for treatmentof intractable autoimmune diseases. Nevertheless, there is substantialrisk involved in an allogeneic procedure in which the host’s immunecells are replaced by a normal donor-type immune system. Syngeneic(or autologous) transplantation is a safer procedure, and has been reportedas successful in inducing long-term tolerance in the EAE model.16 In thepresent study, we addressed the therapeutic potential of a combinedtreatment of acute immunosuppression and syngeneic bone marrowtransplantation to abort EAU and to induce long-lasting tolerance.

Our results showed that acute immunosuppression induced by Cy orTBI followed by reconstitution through SBMT aborted the developmentof EAU and suppressed immunological responses to IRBP. TBI wasmore effective than Cy as an immunosuppressive regimen, as demonstratedby its more drastic effect on various disease-associated immunologicalresponses. The antigen-adjuvant emulsion persists and remains immuno-genic for many weeks. Nevertheless, the treated mice remained disease-free for six weeks after the procedure, when they were rechallenged.Prevention of EAU development during this time period was not causedby a lack of reconstitution of the immune system, since non-immunizedmice treated with TBI or Cy followed by SBMT, that were challengedwith IRBP for the first time after reconstitution, did develop EAU (albeitwith reduced scores) and demonstrated immunological responses to IRBP,PPD, and KLH. Thus, lymphoablation and reconstitution preventeddevelopment of disease in the face of an immune system apparentlycapable of supporting its pathogenesis. While this might suggest thatsome level of tolerance had been induced by the process, it was clearlyinsufficient to result in long-term protection (see ahead).

Karussis et al.16 demonstrated a long-lasting protective effect of

Group # Treatment Proliferative response (CPM)b to:

IRBP PPD Culture medium

1 IRBP 31,690 41,197 1,2642 IRBP+TBI+SBMT 132 N.D. 1213 IRBP+Cy+SBMT 7,423 8,366 530

IRBP Challenge4 IRBP 77,780 92,555 1,9355 IRBP+TBI+SBMT 2,059 33,061 3846 IRBP+Cy+SBMT 22,315 25,790 1,2487 TBI+SBMT 9,003 9,668 2958 Cy+SBMT 21,696 41,449 2,132

aTime scale of the experiments is similar to that described in Table 1.bDraining lymph node cells were collected at the end of the experiments and theirproliferative response measured after 72 hours in culture as described in Materialsand methods.

table 3. Lymph node cellproliferation in EAU-induced miceafter combined treatment with TBI orCy and SBMT.a

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lymphoablation and reconstitution on the progression of EAE, andsuggested development of long-lasting tolerance with an element of activeimmunoregulation. Our results are in partial agreement with these data,as far as initial reversal of EAU development by the procedure. However,our results are at variance with these investigators in that mice in whichEAU development was initially abrogated were not protected from diseaseinduced by a rechallenge with a uveitogenic regimen of IRBP. Thisindicated that, unlike in the study of Karussis et al.,16 long-lasting protectionfrom disease was not induced. Thus, although the initial disease-freeperiod until rechallenge and the relatively low immunological responsesin the treated and reconstituted mice might have indicated some level oftolerance, this was not translated to protection from disease after a seconduveitogenic immunization.

The difference between the present data and the report of Karussis etal.16 might be due in part to incomplete immunosuppression, leavingsome mature effector cells capable of responding to IRBP. The presenceof radiochemoresistant T cells has already been documented in the bloodof leukemia patients undergoing ABMT28 and in a primate model inwhich animals were conditioned exactly as leukemia patients.29 Anotherpossibility is the mature donor-derived T cells present in bone marrowthat might become activated after encountering the immunizing antigen,as well as are new thymic emigrants emerging after lymphoablation andreconstitution. Some of these issues might be addressed by higher dosesof TBI or Cy or by the elimination of the donor bone marrow-derived Tcells by use of pan-T antibodies, both of which have already been usedfor the enhancement of allograft acceptance.30-32 Irrespective of the actualmechanism, the lack of protection from disesase after rechallenge doesnot seem to support the notion of development of active regulation as aresult of the procedure.

The difference in degrees of suppression of DTH and disease are puz-zling. In part, the answer may lie in the fact that the T cells that mediateEAU and DTH in this system may not have the same antigenic specificity.The antigen used for immunization is bovine IRBP and contains non-self epitopes. Disease expression is, by definition, a manifestation of therecognition of autologous epitopes. The DTH response, however, maybe largely due to recognition of nonconserved bovine-specific epitopes.It is unclear why response to putative nonself epitopes would be moreeasily ablated than response to self epitopes under the present conditions.

In conclusion, inhibition of EAU by means of acute immunosuppressionfollowed by SBMT might provide a model for studying the basicimmunological mechanisms involved in ocular inflammation, leading tosolutions for controlling immune-mediated inflammatory processes inthe eye. Such drastic measures should, however, be considered only as atreatment for intractable cases of uveitis, or other autoimmune diseasesin combination with uveitis, when other treatments have failed.

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1986;5:75-109.3 Nussenblatt RB, Kuwabara T, de

Monasterio F, Wacker WB. S-Antigenuveitis in primates: a new model forhuman disease. Arch Ophthalmol

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