Journal ofNeuroimmunology, 39 (1992) 243-250 243 © 1992 Elsevier Science Publishers B.V. AII rights reserved 0165-5728/92/$05.00

JNI 02217

Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein. V. Hierarchy of suppression by myelin basic protein from different species

Ariel Miller, O f e r Lider , A h m a d AI-Sabbagh and H o w a r d L. W e i n e r

Center for Neurologic Diseases, Division of Neurology, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA

(Received 2 January 1992) (Revised, received 20 March 1992)

(Accepted 20 March 1992)

Key words: Myelin basic protein; Experimental autoimmune encephalomyelitis; Oral tolerance

Summary

We have been investigating the suppression of experimental autoimmune encephalomyelitis (EAE) by oral tolerization to autoantigens. In the present study the tolerizing effect of orally administered myelin basic protein (MBP) from different species was examined in the Lewis rat, Hartley guinea pig, and S J L / J mouse model of EAE. Animals were fed guinea pig, rat, bovine, human or mouse-MBP and then immunized with the homologous species of MBP or myelin: Lewis rats were immunized with rat MBP, Hartley guinea pigs with guinea pig-MBP, and S J L / J mice with mouse myelin. Clinical expression of EAE and delayed-type hypersensitivity (DTH) responses to MBP were assessed. In each species, suppression of disease and DTH responses were most pronounced by tolerization with the homologous species of MBP. In addition, cross-species tolerization was observed in each species and in general was less suppressive than homologous MBP although in some instances MBP from a heterologous species was as effective as tolerization with the homologous species. We also studied guinea pig-MBP induced EAE in the Lewis rat because it is a widely studied model of EAE and found that oral tolerization with guinea pig MBP was as suppressive as rat MBP. Of note is that oral tolerization with mouse MBP suppressed myelin-induced EAE in the SJL mouse in which autoimmunity to proteolipid protein appears to play a primary role, suggesting that antigen-driven bystander suppression following oral tolerization with autoantigens (Miller et al., 1991b) may be an important contributing mechanism for suppression of EAE following oral tolerization with MBP in this model. Taken together, these results suggest that epitopes of MBP distinct from the encephalitogenic region participate in triggering suppression following oral tolerization and have important implications for treating human diseases by oral tolerization to autoantigens.

Correspondence to: H.L. Weiner, Center for Neurologic Dis- eases, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA.

Introduction

Myelin basic protein (MBP) emulsified in Freund's adjuvant induces experimental autoim-

244

mune encephalomyelitis (EAE), the primary ani- mal model for multiple sclerosis (MS). The por- tions of the molecule responsible for the induc- tion of EAE have been determined for a number of species and strains within a species (reviewed in Zamvil and Steinman, 1990). MBP given in soluble form suppresses EAE and a variety of routes of administration have been employed to suppress EAE using such antigen-driven toler- ance, including administration of antigen intra- venously, in incomplete Freund's adjuvant (IFA), in liposomes, and coupled to autologous spleno- cytes (reviewed in Miller et aI., 1991a).

We have been investigating the oral adminis- tration of autoantigens as a method to downregu- late experimental autoimmune diseases (Higgins and Weiner, 1988; Nussenblatt et al., 1990; Zhang et al., 1990, 1991; Brod et al., 1991) and have initiated pilot clinical trials using this approach (Marx, 1991). We have previously demonstrated that feeding MBP generates MBP-specific CD8 + T cells that adoptively transfer protection against EAE (Lider et al., 1989; Miller et al., 1991b) and that feeding non-encephalitogenic MBP frag- ments also suppresses EAE in the Lewis rat (Higgins et al., 1988).

The studies reported herein investigate the species specificity of oral tolerance to MBP in suppressing EAE. We have found that cross- species suppression exists in a manner similar to previously demonstrated cross-species disease in- duction (Hashim, 1978; Happ and Heber-Katz, 1988) and that there is a hierarchy of suppression following oral administration by MBP from dif- ferent species, with the most profound suppres- sion observed following oral tolerization with ho- mologous MBP.

Materials and methods

Animals Female Lewis rats (6-8 weeks old) were pur-

chased from Harlan Sprague-Dawley Inc. (Indi- anapolis, IN). Outbred juvenile Hartley guinea pigs (18-21 days old) were purchased from Asso- ciated Rabbit Industries (East Bridgewater, MA). S J L / J mice, 7-10 weeks old, were purchased from Jackson Laboratories (Bar Harbor, ME).

Animals were housed in Harvard Medical School animal care facilities and maintained on standard laboratory chow and water ad libitum.

Antigens Myelin basic protein (MBP) was purified from

brain tissue by the method of Diebler et al. (1972) and purity checked by gel electrophoresis. No contamination with PLP was evident. Mouse myelin was prepared by the method of Norton and Poduslo (1973).

Induction of oral tolerance Rats were fed 1 mg of human, rat, bovine, or

guinea pig MBP, dissolved in PBS (1 mg/ml) , or PBS alone, five times, at intervals of 2-3 days using a 20-gauge animal feeding needle. Guinea pigs were fed 5 mg of human, rat, bovine, or guinea pig MBP, dissolved in PBS, using an iden- tical regimen. Mice were fed 0.5 mg of human, rat, bovine, guinea pig or mouse MBP, dissolved in PBS (0.5 mg/0.1 ml) given seven times at intervals of 2-3 days.

Induction of EAE Two days after the last feeding rats were im-

munized subcutaneously in the hind footpad with 0.1 ml of an emulsion containing 25 p.g of guinea pig MBP or rat MBP in PBS emulsified with an equal volume of complete Freund's adjuvant (CFA) containing 4 m g /m l of Mycobacterium tuberculosis H37R (Difco, Detroit, MI). Guinea pigs were injected with 0.2 ml of a similar emul- sion of guinea pig MBP at multiple sites at the nucal area. Mice were immunized with mouse myelin in CFA according to a modified method of Brown and McFarlin (1981) and Kennedy et al. (1988) 2 days after the fifth feeding. Each mouse received a subcutaneous injection in the shaved left flank of a 0.3-ml emulsion of 1.0 mg of myelin in 0.15 ml of Dulbecco's PBS and 0.03 mg of H37R in 0.15 ml of IFA (Difco, Detroit, MI). 7 days after the first injection, each mouse received a second injection of myelin into the shaved right flank.

Clinical evaluation Rats were monitored for clinical disease and

scored as follows: 0, no disease; + 1, limp tail;

+ 2, ataxia, mild hind limb paralysis; + 3, severe hind limb paralysis, limbs splayed apart; + 4, te- traplegy; and +5, death. Clinical severity for guinea pigs was graded as follows: 0, no disease; + 1, weakness, limpness, or reduced activity; + 2, mild paraparesis of hind legs; + 3, paraplegia of hind legs; +4, paresis of front legs; and +5, moribund. Mice were scored as follows: 0, no abnormality; + 1, limp tail; + 2, mild hind limb paraparesis; +3, moderate hind limb paralysis; + 4, complete hind limb paralysis; and + 5, death. All clinical evaluations were performed in a blinded fashion.

Delayed-type hypersensitivity (DTH) DTH responses in rats immunized with GP-

MBP or rat-MBP was tested by injecting 25/xg of GP- or rat-MBP in 50/xl of PBS subcutaneously in the ear 20 days after induction of disease. DTH in guinea pigs was measured by subcuta- neous injection of 100 ~g /0 .1 ml of GP-MBP subcutaneously in the ears. DTH in mice was measured by injection of 25/zg of mouse-MBP in 25 ~1 of PBS subcutaneously in the footpad. Ear or foot pad thickness were measured in a blinded fashion before and 48 h after challenge, using micrometer calipers (Mitutoyo, Utsinoia, Japan). Change in thickness pre and 48 h post challenge

245

were recorded for each animal and results ex- pressed as the mean for each experimental group _+ SEM.

Statistical analysis Clinical scores were analysed using a two-tailed

Wilcoxon rank sum test for score samples, chi square analysis was used in comparing the inci- dence of disease between groups, and the statisti- cal analysis of DTH responses between experi- mental groups was analysed using a one-tailed analysis of variance.

Results

Suppression of EAE in the Lewis rat The effect of oral tolerization with MBP from

different species on the subsequent induction of EAE was studied using rat or GP-MBP emulsi- fied in CFA (Fig. 1). In rats orally tolerized with various species of MBP and subsequently immu- nized with rat MBP plus CFA, significant sup- pression was observed with all species fed, though a hierarchy of suppressive effects was observed with GP- and rat-MBP being most suppressive, whereas intermediate suppression was observed with human- and bovine-MBP (Fig. 1A). Specifi-

5

4

3

2

1

0

r~ 4

"r,

Control H u m a n Borine Rat Guinea Pig Control Human Rat Bovine Guinea Pig

M B P Species Fed M B P Spec i e s F e d

Fig. 1. Suppression of EAE in Lewis rats by oral administration of MBP from various species. Lewis rats were fed 1 mg of human, bovine, rat, or guinea pig MBP and challenged with r a t - M B P / C F A (A) or G P - M B P / C F A (B) 2 days after the last feeding. Controls were fed PBS alone. For A, GP- and rat-MBP vs. control, P < 0.01; bovine and human MBP vs. control, P < 0.05; GP-MBP vs. human or bovine MBP, P < 0.05. For B, GP-MBP vs. control, P < 0.01; bovine, rat, and human MBP vs. control,

P < 0.05; GP-MBP vs. other species, P < 0.05.

246

4

"~ 2 e~

~a

Bovine Rat Human Guinea Pig

M B P Spec ies I n d u c i n g E A E

Fig. 2. Induction of EAE in Lewis rats by immunization with MBP of various species. Lewis rats were immunized with 25 /zg of MBP from different species emulsified in CFA and

scored for clinical disease.

cally, mean maximal disease severity for GP- and rat-MBP was 0.5 _+ 0.3 and 0.7 _+ 0.3 vs. 2.7 _+ 0.5 for control, P <0.01; bovine and human-MBP was 1.2 _+ 0.3 and 1.5 _+ 0.4 (P < 0.05 vs. control). GP-MBP suppressed significantly more than either human or bovine MBP, P < 0.(15. In rats immunized with GP-MBP (Fig. 1B), a hierarchy of suppression was observed with the most promi- nent suppression obtained with GP-MBP and in- termediate suppression with the other species tested. Specifically, mean maximal disease sever- ity for GP-MBP vs. control = 0.6 _+ 0.5 vs. 3.6 _+ 0.5 (P < 0.01). Bovine (1.7 _+ 0.3), rat (2.2 _+ 0.5) and human (2.3 + 0.4) were also significantly sup- pressive vs. control ( P < 0.05) though, GP-MBP was significantly more suppressive than the other species tested ( P < 0.05; vs. bovine, rat or human MBP). The ability of various species of MBP to induce EAE in the Lewis rat is shown in Fig. 2. Autologous (rat)-MBP resulted in less severe dis- ease than that induced by heterologous GP-MBP (mean maximal disease severity 2.4 _+ 0.3 vs. 3.7 _+ 0.5; P < 0.05) as has been previously reported (Martenson et al., 1972; McFarlin et al., 1973; Kibler et al., 1977; Happ and Heber-Katz, 1988).

Bovine-MBP was least enccphalitogenic (mean maximal score 1.6 ± 0.2, whereas human-MBP was similar to rat).

Suppression of EAE in the ttartley guinea pig The effect of oral tolerization of Hartley guinea

pigs immunized with G P - M B P / C F A with MBP of different species is shown in Fig. 3. GP-MBP was the most suppressive; mean maximal disease severity vs. control = 0.6 _+ 0.1 vs. 2.6 _+ 0.4, P < 0.01. Human (1.5 ± 0.6) and bovine (1.7_+ 0.2) MBP were also significantly suppressive vs. con- trol (P < 0.05) though GP-MBP was significantly more suppressive than bovine or human MBP ( P < 0.05). Feeding rat-MBP had no significant effect on EAE in the guinea pig. Of note is that batch preparat ion of rat MBP yields primarily the large isoform of MBP which lacks the encephali- togenie epitope for strain 13 guinea pigs and possibly Hartley guinea pigs used in these experi- ments.

Suppression of EAE in the SJL / J mouse A hierarchy of suppression was also observed

following oral administration of MBP from vari- ous species to SJL mice immunized with mouse myelin in CFA (Fig. 4). All species suppressed

4

.2 3

k~

2

Control Rat Bovine Human

M B P S p e c i e s F e d

Guinea Pig

Fig. 3. Suppression of EAE in Hartley guinea pigs by oral administration of MBP from various species. Guinea pigs were fed 5 mg MBP from different species five times over a 2-week period and 2 days after the last feeding EAE was induced with GP-MBP/CFA. Controls were fed PBS. GP- MBP vs. control, P < 0.01; human and bovine MBP vs. con- trol, P < 0.05; GP-MBP vs. human and bovine MBP, P < 0.05.

247

4

3

"9 2

Contro l Guinea Pig Rat Human

M B P Species Fed

Bovine Mouse

Fig. 4. Suppression of EAE in SJL / J mice by oral administra- tion of MBP from various species. SJL mice were fed 0.25 mg of MBP from different species seven times and induced for EAE with mouse myelin in CFA as described in Materials and methods. Mouse and bovine MBP vs. control, P < 0.01, and vs. rat and guinea pig MBP, P < 0.05; human, rat and

guinea pig MBP vs. control, P < 0.05.

EAE, though the most prominent suppression was observed with mouse and bovine MBP: mean maximal disease severity vs. control = 1.0 + 0.2 and 1.3 _+ 0.2 vs. 3.9 _+ 0.2 (P < 0.01). Human (1.5 +_ 0.3), rat (2.0 + 0.1) and guinea pig (2.6 + 0.3) MBP were also significantly suppressive vs. con- trol ( P < 0.05); mouse and bovine MBP were more suppressive than rat or guinea pig MBP (P < 0.05).

Delayed-type hypersensitivity (DTH) responses as- sociated with in vivo suppression

As shown in Table 1, in animals immunized with the homologous species of MBP or myelin, the most prominent suppression of DTH re- sponses was observed when the homologous species of MBP was fed. Of note is that in Lewis rats immunized with GP-MBP, feeding GP-MBP was more suppressive than feeding rat MBP. As was observed with clinical disease, cross-species suppression of DTH responses was also observed.

Discussion

The major encephalitogenic sites of the MBP molecule for various species have been identified and have been found to be distinct for a number of animal species, with a hierarchy of cross-species pathogenicity (Kibler and Shapira, 1968; Shapira et al., 1971; McFarlin et al., 1973; Happ and Heber-Katz, 1988). Other studies have suggested that structurally distinct T cell determinants of MBP regulate functionally diverse encephalito- genic and proliferative activities of MBP-specific T cell subsets (Mannie et al., 1989), anti-MBP antibody production (Whitaker et al., 1975) or DTH responses (Herzog et al., 1990). Dissocia- tion between proliferative responses and cytokine

TABLE 1

DTH RESPONSES FOLLOWING ORAL TOLERIZATION TO MBP FROM DIFFERENT SPECIES

Animals were fed over a period of 10-14 days. Rats were immunized with GP-MBP/CFA or rat-MBP/CFA, guinea pigs with GP-MBP/CFA, and mice with mouse-myelin/CFA. DTH responses to the sensitizing antigen or in the SJL mouse to mouse MBP were determined on day 20 after sensitization. Results are expresed as mean (inches x 10 -2) _+ SEM. The homologous species fed is underlined. Naive animals were not immunized nor fed, only tested for DTH. ND, not done; PBS, phosphate-buffered saline

Species fed Immunization

Lewis rat Hartley GP SJL mouse

Rat-MBP GP-MBP GP-MBP Mouse-myelin

Naive animals 0.01 _+ 0.05 0.03 _+ 0.05 0.2 _+ 0.1 0.2 _+ 0.0 PBS 1.50 _+ 0.12 2.20 + 0.15 1.6 _+ 0.2 1.4 _+ 0.1 Guinea pig 0.70 + 0.5 b 0.60 _+ 0.07 a 0.8 _+ 0.1 a 1.0 + 0.1 b

Rat 0.50 _+ 0.5 a 1.60 + 0.10 b 1.1 _+ 0.0 a 0.9 _+ 0.0 b

Human 1.30 _+ 0.7 2.10 _+ 0.11 0.9 +_ 0.0 a 0.8 _+ 0.0 a Bovine 1.10 _+ 0.6 2.00 _+ 0.10 1.0 _+ 0.0 a 0.8 +_ 0.1 a Mouse ND ND ND 0.5 + 0.0 a

a p < 0.01 vs. unfed b p < 0.05 vs. unfed

24~

production has also been demonstrated in other systems (Hecht et al., 1983).

The primary questions to be addressed in rela- tionship to tolerance induction by MBP whether administered by the oral or by other routes are the degree to which the epitopes responsible for encephalitogenicity are identical to those that induce tolerance, whether multiple tolerogenic epitopes exist in the MBP molecule, and whether the mechanism of tolerance induction by MBP differs depending on which epitopc (encephalito- genic or non-encephalitogenic) triggers suppres- sion. Depending on the species being studied and the tolerization protocol, investigators have re- ported that the epitopes responsible for antigen- driven suppression may or may not be the same as the encephalitogenic epitope (Driscoll et al., 1976; Chou et al., 1980; Swanborg, 1975). Raine et al. (1978) reported that bovine MBP sup- pressed EAE induced by isologous spinal cord in strain 13 guinea pigs bet ter than GP-MBP when given in lEA. Cross-species suppression using MBP as tolerogen following MBP/ IFA- induced tolerization was also previously demonstrated in the Lewis rat (Swanborg, 1973). Distinct reper- toire of suppression and helper T cells was demonstrated in the H E L system (Sercarz et al., 1978).

In previous studies, we have found that orally administered non-encephalitogenic fragments of MBP can suppress EAE (Higgins and Weiner, 1988) and that suppression of EAE following oral administration of MBP is related to the genera- tion of antigen-specific CD8 + T-cells that can adoptively transfer protection against EAE (Lider et al., 1989). We have also recently shown that suppression of F, AE induced by oral tolerization to MBP is driven in an antigen-specific way and mediated, both in vitro and in vivo, by the release of TGF/3 (Miller et al., 1992a). We have termed this phenomenon "antigen-driven bystander sup- pression" (Miller et al., 1991b).

In the present report we have demonstrated that the most effective suppression of EAE via oral tolerization in three different species tested is when the homologous species of MBP is fed. Nonetheless, in all instances, cross-species toler- ization existed and a hierarchy of suppression was found. In some instances oral administration of

heterologous MBP was as tolerogenic as homolo- gous MBP. The hierarchy of suppression we ob- served via oral tolerance by different species of MBP in the Lewis rat differs from the hierarchy of encephalitogenicity of these MBP species, sug- gesting that epitopes other than the encephalito- genic epitope in the Lewis rat are involved in suppression. We have recently found that epi- topes of MBP that trigger TGF/~ release follow- ing oral tolerization to MBP are different from the encephalitogenie epitope (Miller et al., 1992b).

Bitar and Whitacre (1989) studied oral toler- ization with GP, rat and human MBP in rats immunized with these species. In contrast to our results, in rats immunized with rat MBP, no sup- pression of clinical disease was observed with any of the species fed, including rat. In animals im- munized with GP-MBP, suppression of clinical disease was only observed in animals fed GP- MBP. Animals immunized with human MBP had very mild EAE (clinical score = 0.8) with suppres- sion of clinical disease by human BP and of histological disease by all species fed. The admin- istration of a much larger amount of MBP (20 mg per animal) in conjunction with soybean trypsin inhibitors and bicarbonate buffer make compari- son of these results with ours difficult. These investigators suggest that clonal anergy may be the primary mechanism of suppression in their model (Whitacre et al., 1991). However. anergy of autoreactive cells would be expected to be elicited most effectively by feeding homologous antigen. Thus, rat MBP would be expected to anergize and downregulate EAE in rats immunized with rat MBP, at least as effective as MBP from other species.

Of particular note in our results is the suppres- sion of mouse myelin-induced EAE in the SJL mouse by various species of MBP. Kennedy et al. (1990) have shown that in this model of EAE, proteolipid protein is the primary autoantigen although EAE can be induced in SJL mice with MBP alone with the use of pertussis (Zamvil et al., 1990). Our ability to suppress myelin-induced EAE in the SJL mouse by MBP most probably relates to the mechanism of antigen-driven by- stander suppression which we recently described in the Lewis rat model (Miller et al., 1991b). In ongoing studies, we have been able to suppress

PLP-induced EAE in the SJL mouse by oral tolerization with MBP (AI-Sabbagh et al., 1992).

The basis for the hierarchy of either suppres- sion or encephalitogenicity by MBP remains to be explained but could relate to the relative affinity of MBP-epitopes for MHC-encoded class I or class II molecules in the gut associated or periph- eral lymphoid tissue (Ria et al., 1990). Identifica- tion of suppressor epitopes of MBP responsible for downregulation of the autoimmune process either by oral tolerization or by other routes of administration will provide important insights into the mechanism(s) by which suppression is medi- ated. Of note is that intravenously administered MBP appears to act by a different mechanism than orally administered MBP (Miller et al., 1992c). If suppression is triggered only by en- cephalitogenic epitopes, mechanisms could in- volve either clonal anergy, clonal deletion or ac- tive suppression. If suppression is observed with non-encephalitogenic epitopes, mechanisms in- volving active suppression by cells distinct from encephalitogenic cells would be expected. Sup- pression by MBP from homologous species could involve either mechanism. The delineation of the mechanisms involved in suppression by MBP will require further investigations utilizing defined peptides both for immunization and tolerization.

Acknowledgements

Ariel Miller is the recipient of the Public Health Service Fogarty International Research Fellowship (1FO5TW04418, 1CP). This work was supported by NIH Grant NS 29352, a grant from Autolmmune Inc. and the MacArthur Founda- tion. We thank Amy Hostetter for help in prepa- ration of the manuscript.

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