Modulation of experimental autoimmune neuritis in Lewis rats by oral application of myelin antigens

Journal of Neuroimmunology 79 Ž1997. 129–137

Modulation of experimental autoimmune neuritis in Lewis rats by oral application of myelin antigens Stefanie Gaupp, Hans-Peter Hartung, Klaus Toyka, Stefan Jung

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Department of Neurology, Neuroimmunology Branch and MS Clinical Research Group, Julius-Maximilians UniÕersitat Wurzburg, Germany ¨ Wurzburg, ¨ ¨ Received 10 December 1996; revised 23 April 1997; accepted 16 May 1997

Abstract Experimental autoimmune neuritis ŽEAN. in Lewis rats is a T cell-mediated disease and serves as an animal model of human inflammatory demyelinating neuropathies. EAN can be induced by immunization with complete bovine peripheral nerve myelin ŽBPM., the myelin protein P2 or its neuritogenic peptide, each emulsified in complete Freund’s adjuvant ŽCFA.. The present study evaluates the effect of oral tolerization with BPM or P2 protein on the development of actively induced EAN. Oral administration of BPM strongly suppressed clinical and histological signs of EAN subsequently induced by BPMrCFA, but feeding of P2 protein alone did not affect its course. In contrast, feeding of BPM did not mitigate the course of EAN subsequently induced by immunization with neuritogenic P2 peptiderCFA. Oral therapy with BPM after onset of myelin-induced EAN only slightly ameliorated the further course of disease, but significantly reduced lethality of this severe form of disease. The findings suggest that immunogenicity of the antigens fed determine strength of tolerance, that downregulation of EAN occurs at the site of immunization and not in the nerve, and that active suppression rather than specific anergization is operative in mediating resistance to EAN. However, only partial tolerance to myelin-induced EAN was achieved in naive animals by transfer of spleenrLN cells from rats orally tolerized with BPM. Although methodic factors may have limited the effect of the cells, the result is suggestive of some contribution of anergy to oral tolerance in the present model. Cholera toxin and LPS were identified as oral adjuvants for BPM and prolonged the state of tolerance. However, LPS exhibited proinflammatory properties if EAN was induced early after BPMrLPS-feeding. Thus, oral application of a mixture of myelin components in combination with cholera toxin may be a useful treatment for chronic inflammatory neuropathies considered autoimmune in nature. q 1997 Elsevier Science B.V. Keywords: Oral tolerance; GBS; Immunomodulation; Adjuvant; Antigen therapy

1. Introduction Oral application of an individual protein results in an antigen-specific non-responsiveness of the immune system which is called oral tolerance ŽMowat, 1987; Weiner et al., 1994.. Induction and maintenance of oral tolerance relies

Abbreviations: BPM, bovine peripheral nerve myelin; CIDP, chronic inflammatory demyelinating neuropathy; CFA, complete Freund’s adjuvant; CT, cholera toxin; EAE, experimental autoimmune encephalomyelitis; EAN, experimental autoimmune neuritis; EAU, experimental autoimmune uveoretinitis; GBS, Guillain–Barre´ syndrome; LN, lymph node; LNC, lymph node cells; MBP, myelin basic protein; MLN, mesenteric lymph node; MLNC, mesenteric lymph node cells; p.i., postimmunization; PNS, peripheral nervous system ) Corresponding author. Neurologische Universitatsklinik, Josef¨ Schneider-Str. 11, D-97080 Wurzburg, Germany. Tel.: q49-931¨ 2012262; fax: q49-931-2012697. 0165-5728r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 7 . 0 0 1 1 5 - X

on two immunological mechanisms, anergization ŽWhitacre et al., 1991., with subsequent elimination of antigen-reactive T-lymphocytes ŽChen et al., 1995., and generation of antigen-specific suppressive T cells ŽLider et al., 1989; Miller et al., 1993.. Application of high Ag-doses may favor the first ŽGregerson et al., 1993; Friedman and Weiner, 1994., while repeated feeding of low Ag-doses may support the latter mechanism ŽGregerson et al., 1993; Friedman and Weiner, 1994.. Active suppression is thought to be mediated by classical Th2- ŽChen et al., 1994. andror so-called Th3-T cells which preferentially secrete TGFb ŽMiller et al., 1993; Fukaura et al., 1996.. Oral tolerization has been reported to prevent or suppress a number of experimental autoimmune diseases such as experimental autoimmune encephalomyelitis ŽEAE. ŽBitar and Whitacre, 1988; Weiner et al., 1994., uveitis ŽEAU. ŽSingh et al., 1992., myasthenia gravis ŽEAMG. ŽOkumura et al., 1994; Wang et al., 1994., and various

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forms of experimental arthritis ŽWeiner et al., 1994.. Results of a pilot trial in patients suffering from rheumatoid arthritis suggest clinical benefit of this treatment ŽTrentham et al., 1993.. We undertook the present study to evaluate and optimize the immunomodulatory potency of oral tolerization in experimental autoimmune neuritis ŽEAN. of Lewis rats, which can be induced by active immunization with bovine peripheral nerve myelin ŽBPM. ŽKadlubowski and Hughes, 1979., P2 protein ŽSmith et al., 1979., or its neuritogenic peptide ŽOlee et al., 1988., each emulsified in CFA. The monophasic disease is characterized by ascending paresis of the tail and limbs due to inflammatory demyelination in spinal nerve roots and peripheral nerves. Thus, EAN replicates salient clinical and histological features of the Guillain–Barre´ syndrome ŽGBS. and of relapses that occur during the course of chronic inflammatory demyelinating neuropathies, and hence serves as their animal model ŽHartung et al., 1993..

2. Material and methods 2.1. Animals Female and male Lewis rats obtained from Charles River ŽSulzfeld. or from our own breeding facility, were 6–8 weeks old and weighed 130–240 g. All experiments were approved by the Bavarian State authorities and were carried out in accordance with their regulations.

emulsion of equal volumes of PBS and Freund’s incomplete adjuvant oil ŽGibco. containing 3.8 or 4.6 mg of bovine peripheral nerve myelin and 25 m g Mycobacterium tuberculosis H37Ra ŽDifco, Augsburg.. P2 peptide-induced EAN was provoked by immunization in the same manner, using 50 m l of an PBSrCFA-emulsion containing 100 m g of P2 peptide Žaa53–78. and 25 m g of Mycobacterium. 2.5. AdoptiÕe transfer of tolerance Spleen cells and mesenteric lymph node cells ŽMLNC. were taken from donor rats, that had been fed with BPMrCT as described above, 2 days after the last oral treatment. 152 = 10 6 spleen cells together with 102 = 10 6 MLNC were transferred i.p. to each of 4 naive rats. 2 h after injection of the cells recipients were immunized with BPMrCFA for induction of EAN. Naive rats which were immunized only served as controls. 2.6. Scoring of disease Rats were weighed daily and inspected for disease severity. Clinical scores were given according to the following scale: 0 s normal; 1 s reduced tone of the tail, hanging tail tip; 2 s limp tail, impaired righting; 3 s absent righting; 4 s gait ataxia, abnormal positioning; 5 s mild paraparesis of the hind limbs; 6 s moderate paraparesis; 7 s severe paraparesis or paraplegia of the hind limbs; 8 s tetraparesis; 9 s moribund; 10 s death.

2.2. Antigens and reagents 2.7. Histology Bovine peripheral nerve myelin ŽBPM. was prepared from intra- and extradural spinal nerve roots according to Norton and Poduslo Ž1973.. P2 protein was isolated from BPM as described by Kitamura et al. Ž1976.. The neuritogenic peptide P2 53–78 representing the corresponding amino acids of bovine P2 was purchased from Biotrend ŽKoln ¨ .. LPS, and cholera toxin ŽCT. were obtained from Sigma ŽDeisenhofen.. 2.3. Induction of oral tolerance Lewis rats received 50 mg of BPM or 1 mg of P2-protein in 0.5 ml saline per feeding by oropharyngeal gavaging. Rats were fed five times on alternate days. Where indicated, 1 mg LPS or 10 m g cholera toxin were added per antigen dose as adjuvant. Animals were immunized 2 or 8 d after the last oral treatment. Animals that received saline, or saline with adjuvant only, served as controls. 2.4. Induction of EAN For active induction of myelin-EAN rats were immunized into the right hind footpad with 50 or 60 m l of an

Rats were anesthetized with Narcoren w ŽIffa Merieux, Laupheim. and perfused through the left cardiac ventricle with Ringer solution ŽFresenius, Bad Homburg. containing 20,000 Url heparin, followed by 4% paraformaldehyde ŽMerck. in a 0.1 M phosphate buffer, pH 7.4. Spinal cords with adjacent roots and right sciatic nerves were dissected and fixed for an additional 14 h. After postfixation in 2% osmium tetroxide for at least 4 h material was embedded in Araldit ŽServa, Heidelberg.. Semithin sections from the cauda equina and sciatic nerves were stained with toluidine blue. Areas surrounding intraneural vessels were examined by a blinded investigator. All perivascular areas present in cross sections were evaluated, and the degree of pathological alterations was graded semiquantitatively on the following scale ŽHartung et al., 1988.: 0 was a normal perivascular area; 1 was mild cellular infiltrate adjacent to the vessel; 2 was cellular infiltration plus demyelination in immediate proximity to the vessel; 3 was cellular infiltration and demyelination throughout the section. The mean histological score for individual animals was calculated dividing the summed scores by the number of perivascular areas examined.

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2.8. Statistical eÕaluation Clinical scores on individual days were compared by the Wilcoxon rank sum test and during the indicated observation period by ANOVA for two variables. Student’s t-test was used for comparison of mean weights on individual days, of days of disease onset, and histological findings. The t-test could be applied to histological scores because the high number of samples Žperivascular areas. permitted assumption of a normal distribution according to the central limit theorem ŽHartung et al., 1988.. The incidence of the EAN-associated death rate was compared by the x 2 analysis.

3. Results 3.1. Influence of adjuÕants on strength and duration of myelin-induced tolerance The tolerogenic effect of myelin alone or in combination with LPS or CT as adjuvants was examined by feeding Lewis rats 5 times within 10 d. Two days later EAN was actively induced by immunization with BPM in CFA. Beginning on day 11r12 PBS-fed controls rapidly developed severe paraparesis within 4 d ŽFig. 1A. and recovered slowly. Animals fed with myelin alone or in combination with CT were all protected from severe EAN and did not suffer from any deterioration of gait Žsignificantly lower daily disease scores from day 13 on.. Addition of CT during feeding of myelin did not further enhance tolerance induction, but rather resulted in a slightly earlier appearance of attenuated disease ŽFig. 1A.. Rats fed with BPM and LPS showed a much earlier onset of the disease than control animals but recovered rapidly within 6 d ŽFig. 1A.. Therefore, the overall severity of EAN in the BPMrLPS-fed rats was significantly suppressed Ž p - 0.0001.. Body weight closely correlated with clinical signs and BPM- as well as BPMrCT-fed rats did not exhibit any weight loss ŽFig. 1B.. Histological examination of sciatic nerves and spinal nerve roots dissected from animals on day 25r26 p.i. confirmed the clinical observations ŽFig. 2.. Sections from rats pretreated with BPM and CT revealed mild to moderate histological changes with a higher incidence of demyelination restricted to the perivascular area Žscore 2., but lacking widespread, diffuse demyelination Žscore 3.. Therefore, mean histological scores of the two groups were lower than in the control group Ž p - 0.05.. Although the shift to lower histopathological scores was less conspicuous in the BPMrLPS-fed group it resulted in a relevant reduction of the corresponding mean score Ž p - 0.05. compared to control animals. We next assessed persistence of orally induced tolerance according to the above protocol with an extended

Fig. 1. Induction of resistance to myelin-induced EAN by oral application of myelin. Six Lewis rats per group were gavaged every other day with 50 mg BPM per animal without or with 10 m g CT or 1 mg LPS as adjuvants or were sham-treated with 0.5 ml saline on days y10, y8, y6, y4, y2 before immunization. On day 0 all rats were immunized with BPM Ž3.5 mg.rCFA Žcontaining 25 m g M. tuberculosis. as described in Section 2. Animals were scored for clinical signs of disease ŽA. as specified and weighed ŽB. every morning. Bars indicate SD.

period between the last feeding and induction of EAN Ž8 versus 2 d.. As shown in Fig. 3, animals fed with BPMrCT still were nearly resistant to EAN, while the suppression of clinical signs in the group fed with BPM alone was almost abrogated. Interestingly, in this experimental setting rats fed with BPMrLPS were protected from EAN and developed only mild to moderate signs of disease Žmean clinical score 2.. As in Fig. 1 body weight was linked to the course of disease Žnot shown.. The weights of the BPMrCT- or BPMrLPS-treated group only slightly decreased and were significantly higher than in controls from day 14 or 15 p.i. on, respectively Ž p - 0.05.. In the BPM-fed group the difference to the controls was significant Ž p - 0.05. on day 14 and day 16 only. A control experiment Žnot shown. demonstrated an identical course of myelin-induced EAN in rats that had been fed with saline or CT alone and excluded the possi-

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Fig. 4. Tolerance to myelin-EAN is not induced by feeding of P2 protein. 1 mg of the P2 protein was given orally 5 times to 6 out of 12 Lewis rats which were immunized with BPMrCFA 8 d after the last feeding. Mean clinical scores q or ySD are given.

bility that the observed effects of BPMrCT-feeding as depicted in Figs. 1 and 3 relied on CT alone. 3.2. Effect of the oral application of P2 protein on the deÕelopment of myelin-induced EAN Five Lewis rats were fed 5 times either with saline or 1 mg of P2 within 10 d, followed by the induction of EAN with BPMrCFA eight days after the last feeding. Fig. 4 illustrates that oral administration of P2 did not affect the severity of EAN at all. Accordingly, P2-fed rats lost weight to the same extent as control animals Žnot shown.. Fig. 2. Prevention of widespread nerve damage in BPM-induced EAN by oral administration of BPM. Sciatic nerves ŽA. and lumbar spinal nerve roots ŽB. were removed 25r26 d p.i. and histopathology around intraneural blood vessels was scored by a blinded investigator in semithin sections as specified in Section 2. Columns ŽqSD. represent the mean percentage of perivascular areas with the defined alteration in each group Ž ns 3.. Mean histological scores of each group Žgiven in the symbol boxes. were calculated from the mean histological scores of the corresponding individual animals. ) ) ) p- 0.01; ) ) p- 0.02; ) p- 0.05.

Fig. 3. Adjuvants enhance duration of oral tolerance to myelin-induced EAN. Five Lewis rats per group were fed either with BPM without or with CT or LPS and immunized with BPMrCFA 8 d after the last feeding. Mean clinical scores"SD are shown.

3.3. Feeding BPM r CT protects from myelin- but not from peptide-induced EAN In previous studies EAN provoked by immunization with the neuritogenic peptide Žaa53–78. was more suscep-

Fig. 5. Feeding myelin prevents BPM- but not peptide-induced EAN. Each group of 4 animals was fed either with BPMrCT or salinerCT 5 times within 9 d, followed by immunization with BPM Ž3.5 mg.rCFA or neuritogenic peptide Ž100 m g.rCFA 2 d after the last feeding. Mean clinical scores q or ySD are given.

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tible to immunosuppression than myelin-induced disease. We therefore examined the tolerogenicity of BPM in this form of disease. Eight animals were sham-fed five times with salinerCT, 8 rats received BPMrCT. Two days later, 4 rats of each group were immunized with BPMrCFA, the others with P2 53–78rCFA. Again, rats treated with BPMrCT and immunized with BPM were nearly completely protected from EAN ŽFig. 5.. However, oral administration of BPMrCT did not even ameliorate the clinical severity of peptide-induced EAN or disease-associated weight loss. The observation that animals recover much more rapidly from peptide- than from myelin-induced EAN has also been made in previous experiments ŽJung et al., 1996.. 3.4. AdoptiÕe transfer of orally induced tolerance The latter experiment suggested the contribution of active suppression in oral tolerance to EAN. To corroborate this we attempted to demonstrate its transfer to naive rats by cells.

Fig. 7. Oral administration of myelin after onset of EAN slightly downregulates the further course of myelin-induced disease. Lewis rats were immunized with BPMrCFA and after the appearance of the first clinical signs Žday 0. each group of 6 animals was fed with either BPM, BPMrCT, or saline on day 0, 1, 3, 5. Hatched line indicates mean clinical scores of surviving animals. Mean clinical scores q or ySD are given.

Splenocytes together with MLNC from animals fed five times with BPMrCT adoptively were transferred i.p. to naive rats, which were immunized with BPMrCFA on the same day. Untreated rats served as controls and were immunized without precedent cell transfer. In a separate experiment transfer of splenocytes and MLNC from CT-fed animals did not alter the course of EAN compared to that in naive rats Žnot shown.. In recipients of orally sensitized splenocytes and MLNC onset of EAN was significantly delayed by around 2 d Ž p - 0.01; Fig. 6A.. Furthermore, progression of this severe form of EAN was slightly mitigated in this animal group resulting in reduced lethality from EAN Ž2r6. compared to that of control animals Ž4r6.. However, disease severity in surviving animals did not differ between the groups Žhatched lines in Fig. 6A.. In line with these findings, weight loss in rats which had received BPM-sensitized lymphocytes was significantly lower than in controls ŽFig. 6B.. 3.5. Therapeutical feeding of BPM during ongoing EAN

Fig. 6. Partial transfer of resistance to EAN by lymphocytes from BPMrCT-fed animals. Lewis rats were fed with BPMrCT five times within 9 d. Two days after the last feeding, spleens and mesenteric lymph nodes of the fed animals were removed. 105=10 6 MLN cells together with 152=10 6 spleen cells were transferred i.p. to naive animals Ž ns 4.. Naive animals served as controls. Hatched lines indicate mean clinical scores of surviving animals. Mean clinical scores ŽA. and body weights ŽB. q or ySD are shown. ) ) ) p- 0.01; ) ) p- 0.02; ) p- 0.05.

In this experiment, rats were first immunized with BPMrCFA and when individual animals showed the first clinical signs of EAN Žin Fig. 7 normalized to day 0. BPMrCT, BPM, or saline was administered orally on days 0, 1, 3, and 5. Although the therapeutic antigen feeding did not alter mean clinical scores significantly, it is of note that both the feeding of BPM and BPMrCT protected all 12 myelin-fed animals from death, while 2 out of 6 control rats died due to severity of EAN Ž p - 0.05.. Again, a beneficial effect Ž p - 0.0001. of treatment with BPMrCT could be demonstrated by ANOVA when all disease scores during the observation period were con-

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sidered. Likewise analysis of body weights in this manner disclosed differences in favor of both groups of antigen-fed rats compared to the controls Ž p - 0.05..

4. Discussion The experiments of this study were designed to evaluate the hitherto unknown influence of orally induced antigenspecific tolerance on autoreactive inflammation in the PNS. With respect to possible therapeutic strategies applicable to GBS or CIDP, they also aimed at designing the most efficient treatment schedule for suppression of EAN, the animal model of GBS. 4.1. The role of adjuÕants in myelin-mediated tolerance to myelin-induced EAN Clinical and histological signs of myelin-induced EAN could be prevented most efficiently by prior feeding with myelin. Duration of orally-induced protection from disease by myelin feeding turned out to be enhanced by addition of CT or LPS as oral adjuvants in the experiment in which we induced disease not before 8 d after the last feeding. However, immunization early after tolerization did not reveal superiority of BPMrCT over BPM alone and prior application of BPMrLPS even accelerated development of EAN while disease severity was slightly ameliorated. CT is known to be a potent oral adjuvant ŽElson and Ealding, 1984; Vajdy and Lycke, 1992; Xu-Amano et al., 1993; Weiner, 1994. but has so far not been successfully used for oral induction of antigen-specific tolerance. In fact there is reported evidence for abrogation of antigenspecific oral tolerance on the humoral and cellular level of the immune response by unbound CT ŽElson and Ealding, 1984; Sun et al., 1994.. In our experiments CT exhibited a significant and, in contrast to LPS, also safe adjuvant effect. The induction of persistent tolerance to EAN by BPMrCT in contrast to BPM alone is in line with the induction of long-term mucosal immunological memory by the adjuvant CT ŽLycke and Holmgren, 1986; Vajdy and Lycke, 1992.. Our observation that CT promotes oral induction of tolerance to experimental autoimmune disease which is thought to be mediated at least in part by Th2 cytokines and may be explained by the ability of the CT subunit A to increase intracellular levels of cAMP ŽSpangler, 1992.. Direct or indirect elevation of cAMP levels are known to skew the immune response to a Th2-dominated pattern ŽMunoz et al., 1990. and this property has been demonstrated for CT ŽMunoz et al., 1990; Xu-Amano et al., 1993.. On the other hand, abrogation of oral tolerance by CT ŽElson and Ealding, 1984; Sun et al., 1994. may be attributed to its stimulatory effect on the secretion of the proinflammatory cytokine IL-6 by intestinal epithelial line cells ŽMcGee et al., 1993.. Since we, unlike others who

utilized unbound CT ŽPierre et al., 1992; Sun et al., 1994. did not observe a negative influence of CT on the development of myelin-induced tolerance the possibility exists that our antigen-suspension did not contain free CT. Thus, CT is known to bind to GM1-gangliosides via its pentameric ring of B subunits ŽSpangler, 1992., which also mediates binding of CT to eukaryotic cell surfaces. Since preparations of peripheral nerve myelin contain very high amounts of GM1 it is conceivable that a relevant number of CT molecules are trapped by the antigen emulsion. This could reduce the concentration of free CT to a tolerogenic one. It may on the other hand potentiate the tolerogenicity of myelin because conjugation of CT–B to MBP or GM1coated red blood cells has been shown to greatly augment the capacity of the tolerogen to prevent EAE or to induce systemic unresponsiveness ŽSun et al., 1994; Sun et al., 1996.. As CT may in our experiments have been bound to the BPM fed, we currently investigate whether the adjuvant effect of oral CT differs between myelin and other proteins lacking GM1. The finding that in certain instances addition of LPS can accelerate appearance of subsequently induced disease is in contrast to results of Khoury et al. These authors describe beneficial adjuvant effects of orally administered LPS although animals were also challenged early Ž2–3 d. after the last feeding with antigenrLPS ŽKhoury et al., 1990, 1992.. However, subcutaneous administration of LPS abrogated oral induction of tolerance and oral treatment with LPS prevented humoral tolerance ŽKhoury et al., 1990.. These results raise the possibility that the oral adjuvant LPS may systemically act in an immunostimulatory fashion. This may in certain experimental settings overcome the development of cellular tolerance. 4.2. Mechanism of orally induced tolerance to EAN and its site of action Since myelin-feeding conferred a marked resistance to myelin-induced EAN it was surprising that myelin-fed rats developed full blown peptide-induced disease. This was the case although peptide-induced EAN in our hands always takes a milder clinical course than myelin-provoked disease and was in previous studies consistently easier to suppress ŽJung et al., 1992, 1996.. Because two main mechanisms, induction of antigenspecific anergy and active suppression, are thought to be operative in oral tolerance Žreviewed in Weiner, 1994., the result obtained here may be interpreted in two ways: first, with respect to anergy, which is assumed to be induced by high doses of antigen ŽWhitacre et al., 1991; Gregerson et al., 1993., the myelin inoculum may not have contained a sufficient amount of neuritogenic P2 epitopes to silence the corresponding T cells. However, a further experiment Žnot shown. suggests that feeding comparably high amounts of the peptide with CT does not render rats resistant to peptide-induced EAN. The second possibility which we

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would consider as decisive refers to active suppression. Peripheral nerve myelin contains a multitude of protein antigens and sugar-like epitopes and in additional experiments turned out to be highly immunogenic, even immunostimulatory. Thus, naive lymphocytes proliferated in vitro in the presence of BPM, and in draining lymph nodes from BPM-immunized rats we always noted a higher ex vivo lymphocyte proliferation than from animals challenged with proteinrCFA or CFA only ŽJung and Gaupp, unpublished results.. We therefore assume that only feeding of myelin and its presence at the site of immunization ensures optimal recruitment to and activation of downregulatory myelin-specific lymphocytes at the locus of generation of neuritogenic T cells. This notion is supported by our finding that feeding of one myelin component alone, the P2 protein, failed to mitigate the clinical course of myelin-induced EAN. Similarly, EAN actively induced by P2 peptiderCFA was not mitigated by precedent feeding of the peptiderCT in our hands Žnot shown. and only slightly ameliorated in a preliminary study of others ŽRostami and Cronkhite, 1992.. However, a study in spinal cord-induced EAE comparable to the present one lined out that oral application of whole myelin and purified MBP equally ameliorated relapses of the disease ŽBrod et al., 1991.. Our observation of myelin-mediated protection from myelin- but not peptide-induced EAN implies that suppression of EAN mainly takes place in lymphatic organs and not within the nerve. Otherwise, myelin-fed animals would have been resistant to myelin- as well as to peptide-induced EAN since intraneural antigens are identical in these groups of rats. Also in EAU orally-induced antigen-specific immunoregulation was shown to take place at the site of immunization ŽWildner and Thurau, 1995.. Furthermore, our conclusion is concordant with results that demonstrated identical suppression of EAE by oral MBP as well as by orally induced OVA-specific bystander suppression at the site of immunization ŽMiller et al., 1991.. On the other hand, in EAE much evidence indirectly ŽKhoury et al., 1992. or directly points to orally-induced active suppression in the target organ of inflammation ŽAl-Sabbagh et al., 1994; Chen et al., 1994.. While the latter downregulation is thought to be mediated by TGFb 1-secreting cells ŽAl-Sabbagh et al., 1994; Chen et al., 1994., neuroantigen-specific Th2 cells did not display suppressive activity of clinical relevance in the brain ŽKhoruts et al., 1995.. Therefore, it is possible that CT-biased oral tolerization generated a predominantly Th2-mediated downregulation of EAN operating in lymph nodes but not in the target organ. Since our findings in EAN and those in EAU ŽGregerson et al., 1993; Wildner and Thurau, 1995. differ from observations made in EAE, one may speculate that individual experimental autoimmune diseases vary in their main site of immunoregulation. Although the experiments discussed so far clearly indicated active suppression at the site of immunization as

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decisive, the results of the transfer experiment seemed not to unequivocally corroborate this notion. Transfer of spleen and MLN cells from myelinrCT-fed animals did not inhibit myelin-evoked EAN in recipients as impressively as direct application of BPM alone. However, it is possible that the number of spleen cells transferred Žcorresponding to only half of the organ. was too low or that further immunoregulatory lymphocytes which may reside in Peyer’s patches, thymus, LN, and liver were not transmitted. Furthermore, transferred protection seems to be only optimally detectable if recipients are irradiated ŽLider et al., 1989; Miller et al., 1991.. Although these facts may have limited the effect of the cells transferred in our experiment, the latter finding is suggestive of some contribution of anergy to oral tolerance in the model tested. We obtained no evidence that the cells need to be reactivated before transfer ŽLider et al., 1989; Miller et al., 1991., because this manipulation rather reduced protective capacity of the cells in our experiments Žnot shown.. 4.3. Therapeutic application of myelin during ongoing EAN Oral administration of myelin or myelinrCT after onset of clinical signs of myelin-induced EAN did not significantly ameliorate mean severity in surviving animals but it was of note that myelin feeding prevented rats from disease-associated death. Thus, oral application of putative autoantigens may even be useful for the treatment of GBS if it is diagnosed at an early stage of the disease. But so far, only a few studies ŽZhang et al., 1990; Brod et al., 1991; Meyer et al., 1996. provided evidence for some benefit of therapeutic oral antigen application on the further clinical course of disease, while a further investigation failed ŽYoshino, 1995.. In conclusion we have shown that autoimmunity directed to the PNS can be markedly downregulated by oral application of peripheral nerve myelin. Oral tolerization by myelinrCT or a mixture of myelin proteins may be a useful and well tolerated add-on therapy for the treatment of CIDP and possibly also GBS.

Acknowledgements This work was supported by the Gemeinnutzige Hertie¨ Stiftung ŽGHS 307r94. and by funds of the Julius-Maximilian University. We wish to thank Gabriele Kollner for ¨ expert technical assistance.

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