Suppression of chronic-relapsing experimental allergic encephalomyelitis in strain-13 guinea pigs by administration of liposome-associated myelin basic protein

Journal of Neurotmmunolog~', 7 (1984/85) 27-41

27

Elsevier JNI 00215

Suppression of Chronic-Relapsing Experimental Allergic Encephalomyelitis in Strain-13 Guinea Pigs by Administration of Liposome-Associated Myelin Basic Protein G.H. Strejan, J.J. Gilbert and J.St. Louis Department of Mtcroblologv and lmmunoloKv and Department of Pathologo', Umverstty of Western Ontarto, London, Ont. (Canada)

(Received 19 December, 1983) (Revised, received 14 February, 1984) (Accepted 29 February, 1984)

Summary Juvenile strain-13 guinea pigs were challenged with isologous spinal cord in C F A . A f t e r recovery from the first E A E e p i s o d e the animals were treated with guinea pig M B P inserted into liposomes, with c y t o c h r o m e - c - l i p o s o m e s , with M B P in saline or with M B P in I F A . G u i n e a pigs treated with M B P - l i p o s o m e s showed a striking r e d u c t i o n in clinical signs a n d in the n u m b e r a n d intensity of relapses. T h e y d i s p l a y e d virtually no d e m y e l i n a t i n g lesions, and had c o m p a r a t i v e l y little p a r e n c h y m a l i n f l a m m a t i o n in the spinal cord. Early T rosette levels showed an inverse c o r r e l a t i o n with the severity of histological lesions in the spinal c o r d but c o r r e l a t i o n with the clinical status at the time of rosette assay was less well defined. K e y words: D e m v e l i n a t i o n - E x p e r i m e n t a l allergic e n c e p h a l o m y e l i t i s - G u i n e a p i g M y e l i n basic p r o t e i n - R o s e t t e levels

This work was supported by a Multiple Sclerosis Society of Canada grant. Correspondence: Dr. G.H. Strejan, Department of Microbiology and Immunology, University of Western Ontario, Health Sciences Center, London, Ont., Canada. 0165-5728/84/$03.00 © 1984 Elsevier Science Publishers B.V.

28 Introduction

Experimental allergic encephalomyelitis (EAE), an autoimmune disease of the central nervous system (CNS) is regarded as the animal prototype for multiple sclerosis (MS) and for other human demyelinating diseases with suspected autoimmune etiology (Paterson 1976; McFarlin and Waksman 1982). The usual course of the disease is acute and monophasic. Chronic-relapsing forms of EAE (CR-EAE) were reported by several laboratories and have been shown to resemble far better than acute EAE, the clinico-pathological and immunological manifestations of MS (Stone and Lerner 1965; Raine et al. 1974; Panitch and Ciccone 1981; Fritz et al. 1983). In guinea pigs it is induced in juvenile strain 13 by the administration of whole CNS tissue or of myelin, emulsified in complete Freund's adjuvant (CFA). MBP is ineffective. After challenge, guinea pigs undergo the first clinical episode 14-21 days after sensitization, followed in many animals, by complete recovery. The chronic stage of the disease appears approximately 2 months after challenge and is characterized by a series of relapses and remissions or by a progressive worsening of the neurological deficits (Wisniewski and Keith 1977). Histologically there is a high incidence of demyelination in the brain and spinal cord, perivenous inflammation, oligodendrocyte loss and active gliosis. Immunologically, CR-EAE is characterized by the presence of sensitized lymphocytes, and of antibodies to MBP and to CNS glycolipids (Raine and Traugott 1982), IgG oligoclonal bands in the cerebrospinal fluid (CSF) (Mehta et al. 1981), and by significant decreases in the number of early (active) T cell rosettes in the peripheral blood of animals during clinical episodes (Traugott et al. 1979). Suppression of CR-EAE has been achieved recently by the administration of repeated doses of MBP in IFA or of a mixture of MBP and galactocerebrosides (GC) in IFA (Raine et al. 1978; Traugott et al. 1982a,b). We have reported previously that acute EAE can be effectively suppressed by the intracardiac (i.c.) administration of human MBP inserted into phosphatidylserine (PS) liposomes given in 1 or 2 doses before challenge with MBP in CFA (Strejan et al. 1981). As a logical extension of those findings, MBP-liposomes (MBP-L) were used in the present study, for the treatment of strain-13 guinea pigs undergoing CR-EAE. The results show that as little as 3 weekly injections of 50 ~tg guinea pig MBP-L given after recovery from the acute attack were sufficient to determine a remarkable reduction in both clinical and histological manifestations of the chronic phase of the disease.

Materials and Methods

Myelin basic protein Guinea pig brains and spinal cords were obtained from Pel-Freez Biologicals Rogers Ark. MBP was isolated and purified according to Deibler et al. (1972). The purity of the preparation was assessed by polyacrylamide gel electrophoresis. The MBP was lyophilized and stored in a desiccator at room temperature under reduced pressure.

29

Liposomes Myelin basic protein- and cytochrome-c (CYC)-liposomes were generated as previously described (Boggs and Moscarello 1978; Strejan et al. 1981). Briefly, phosphatidylserine (PS) (Serdary Research Laboratory, London, Canada) was dissolved in chloroform at a concentration of 5 m g / m l . Between 2.5 and 5 mg PS were dried in a conical glass tube and then mixed with an equal amount of MBP or CYC dissolved in 2 ml hypotonic E D T A - H E P E S - N a C 1 buffer pH 7.4. The liposomeprotein complexes formed after 5-10 min of Vortex mixing were washed by centrifugation in the same buffer. The amount of protein inserted into liposomes was determined by radioactivity (~25NaI) and the amount of phospholipid, by phosphorus analysis (Bartlett 1959). Since the hypotonic buffer interfered in the assay, the aliquot of liposomes to be assayed for phosphorus content was extensively washed and resuspended in isotonic NaCI solution.

Induction of chronic-relapsing EA E Strain-13/N guinea pigs were bred in our animal facility from a starting nucleus of 5 male/female pairs kindly supplied by the Veterinary Resources Branch, NIH. C R - E A E was induced in 200-220 g guinea pigs of both sexes essentially as described by Traugott et al. (1979). One gram wet isologous spinal cord was homogenized in 1 ml of isotonic saline and each ml of the 50% spinal cord homogenate was emulsified with an equal volume of CFA supplemented with 10 m g / m l dried M. tuberculosis H37RA (Difco Laboratories, Detroit, MI). Each guinea pig was injected intradermally (i.d.) at several sites in the nuchal area with 0.5 ml emulsion.

Treatment The animals were allowed to develop the acute attack (first episode) and those that recovered were divided into various treatment groups. The treatment started usually 2 - 4 weeks after clear indication of clinical remission. Each injection consisted of 50/~g MBP or CYC. The details of routes and schedules are presented at Results (Table 1). The animals were weighed at least once a week and were observed daily for clinical signs. These were scored on an arbitrary scale from 0 to 4: 0, no visible abnormality; 1, weight loss associated with poor righting reflex, ruffled fur, or tilted head; 2, hind leg weakness, urinary incontinence or retention, fecal impaction; 3, hind leg paresis or paralysis; 4, death or advanced paralysis justifying euthanasia. When weight loss was the only visible manifestation, it was scored as 0.5 since it was difficult to determine whether it was attributable to EAE or not.

Histopathology The animals were kept under observation for up to 70 weeks, and were killed by Na-pentobarbital overdose. At the time of killing the entire central nervous system of the guinea pig was dissected and immersed in 10% buffered formaldehyde. After fixation, the tissue was serially sectioned in 1-2 mm coronal cuts and the entire specimen was serially embedded in paraffin. Sections were stained with hematoxylin and eosin, Solochrome R for myelin and Bodian's stain for axis cylinders. The slides were viewed by

30 one observer (JJG) independently and without prior knowledge of the treatment or clinical condition of the animal at the time of killing. The pathologic changes were scored systematically from 0 to 4 for the cerebrum, brain stem and cerebellum, and separately for the spinal cord as follows: Inflammatory reaction in the meninges O, no changes; 1, perivascular a n d / o r meningeal infiltration by mononuclear cells, 1-3 vessels in any one section involved; 2, 4 - 6 vessels involved; 3, more than 6 vessels involved; 4, dense infiltration of meninges and nearly all or all blood vessels involved. Parenchymal perivascular infiltration: O, no changes; 1, 1-3 parenchymal vessels infiltrated in Virchow-Robin spaces in any one section 2, 4 - 6 vessels involved; 3, more than 6 vessels involved; 4, virtually all or nearly all vessels involved. Encephalitis (cerebritis): 0, no invasion of the neural parenchyma; microglial or inflammatory cells invading neural parenchyma; 1, a few scattered cells; 2, invasion by cells from several perivascular cuffs; 3, large areas of neural parenchyma involved; 4, virtually the entire section is infiltrated. Demyelination, remyelination and myelin debris: O, no demyelination; 1, a single small focus of subpial demyelination or of myelin debris; 2, several small foci of demyelination; 3, one large confluent area of demyelination on a single section; 4, several large confluent areas of demyelination in any one section. Since certain histological alterations were considered more likely than others to have clinical relevance they were weighted by the following factors: meningeal inflammation score times a factor of 1; parenchymal perivascular inflammation score times a factor of 2; encephalitis score times a factor of 3; and demyelination score times a factor of 4. Thus a maximum score for cerebrum, brain stem, and cerebellum was 40; and for the spinal cord was 40.

Cell-mediated immune functions Delayed hypersensitivity (DTH) reactions and lymphocyte transformation (LT) to MBP and to MBP-L were carried out as previously described (Strejan et al. 1981). The enumeration of early rosette-forming T lymphocytes was carried out essentially according to Traugott and Raine (1977). Briefly, mononuclear cells from blood obtained by cardiac puncture were isolated by F i c o l l / H y p a q u e gradient centrifugation. The cells recovered from the interface were washed 3 times in Hanks' balanced salt solution (BSS) diluted 1 : 2 with phosphate-buffered saline, pH 7.4. The number of phagocytic cells was determined after incubation with latex particles. These cells ranged between 7% and 15% of total mononuclear cells isolated. One million guinea pig lymphocytes in 0.5 ml BSS were mixed with 0.1 ml 1% suspension of rabbit erythrocytes in saline, and incubated for 15 min at 37°C. The suspension was centrifuged at 500 rpm for 5 min and the pellet was resuspended in 0.2 ml BSS. One drop of the cell suspension was mixed with one drop of 0.5% toluidine blue solution and 200 mononuclear cells were counted in each preparation. A rosette was defined as a lymphocyte with 3 or more erythrocytes attached to it. Variability between counts of the same preparation performed on replicate slides was 10% or less. Total (late) T rosette-forming cells were not determined.

31

Statistical analysis T h e clinical a n d histological scores were subjected to n o n p a r a m e t r i c tests. All groups were c o m p a r e d by the M a n n - W h i t n e y U - d i s t r i b u t i o n test with tied ranks, or whenever possible, by the K r u s k a l l - W a l l i s , single-factor analysis of variance (for the groups c o n t a i n i n g equal n u m b e r s of animals). The statistical significance between groups with respect to T rosette levels was d e t e r m i n e d by 95% confidence limits, after arcsine t r a n s f o r m a t i o n of the percentages. The correlation between T rosette levels a n d clinicopathological scores was analyzed by simple linear regression a n d the significance of the regression line, by analysis of variance (Zar 1974).

Results A total of 83 strain-13 guinea pigs were challenged with spinal cord h o m o g e n a t e in C F A a n d all b u t one (82) developed the disease. The average time of onset was 18 days a n d the first episode was characterized by drastic weight loss, poor righting reflexes, u r i n a r y i n c o n t i n e n c e , a n d m o d e r a t e to severe paraparesis. Twenty-six a n i m a l s died or had to be euthanized 4 - 6 days after onset of clinical signs, a n d 57 a n i m a l s recovered within 10 days after onset. These were divided into 6 groups a n d treated according to the schedule shown in T a b l e 1. The t r e a t m e n t was started 2 weeks after clear i n d i c a t i o n of clinical remission. The ratio of protein to lipid in the M B P - l i p o s o m e p r e p a r a t i o n s was d e t e r m i n e d o n 10 different batches. The M B P / P S weight ratio was 1.12 + 0.11, thus a s s u m i n g a molecular weight of 800 for PS a n d 18000 for M B P the m o l a r P S / M B P ratio was 21.

TABLE

I

TREATMENT

O F C R - E A E IN STRAIN-13 G U I N E A

PIGS

Group

Treatment schedule a

Control CYC-L

None Cytochrome-c-liposomeswere injected once a week, for 3 weeks MBP dissolved in saline was injected once a week, for 3 weeks MBP emulsified in IFA was injected once a week, for 3 weeks b MBP-liposomes injected once a week, for 3 weeks MBP-liposomes injected once a week for 3 weeks then once a month for 3 months.

MBP-SAL

MBP-IFA

MBP-L x3 MBP-L x6

a MBP and CYC were injected intracardially under ether anesthesia in a dose of 50 #g/injection. b MBP-IFA was injected subcutaneously.

32

Figure 1 shows the evolution of clinical signs of representative individual animals in all groups. The animals developed the first clinical signs between 2 and 3 weeks after challenge, with clinical manifestations ranging from moderate to severe. The chronic-relapsing phase that followed, proceeded along a strictly individual pattern in terms of intensity, duration and frequency of episodes. In several animals the disease had a progressive rather than a relapsing-remitting pattern and the animals died or had to be killed long before the planned termination of the experiments (50-70 weeks). The animals treated with MBP-L ×3, had clinical manifestations that were much milder than those of the animals in the other groups. They had very few and infrequent relapses, and the clinical manifestations of each relapse seldom exceeded a score of 1 (Panel F, lanes 5 and 6). The animals treated with MBP-L ×6 A CONTROL

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Fig. 1. Clinical evolution and active T rosettes of individual guinea pigs undergoing chronic-relapsing E A E . For details see Table 1. A: u n t r e a t e d ; B: treated with C Y C - L ; C: treated with M B P - S A L ; D: treated with M B P - I F A ; E: treated with M B P - L ×6; F: treated with M B P - L x3. Vertical bars indicate % active T rosettes.

33 TABLE 2 CLINICAL SCORES OF GUINEA PIGS BEFORE AND AFTER TREATMENT WITH MBP-LIPOSOMES Treatment"

1st Attack

Relapses

EAE/total

CS b

EAE/Rec c

CCS d

% Relapsing c

None CYC-L MBP-SAL MBP-IFA MBP-L x3 MBP-L x6

38/38 8/8 8/8 9/9 9/10 10/10

2.68 2.00 2.40 2.60 2.50 2.90

18/24 6/7 6/6 7/7 3/6 0/7

4.28 5.70 8.60 3.10 1.17 f 0

75 86 100 100 50 0

a See Table 1 for details. b Mean clinical score of group. ¢ Number of animals undergoing relapses out of total recovered from 1st episode. d Mean cumulative clinical score was calculated by adding the maximal clinical score of each relapse and dividing by the number of animals remaining in the group after the first attack. c Percentage of animals undergoing one or more relapses, out of total recovered from 1st episode. r Values in italics indicate statistical significance(P < 0.05) with respect to all other groups; determined by Mann-Whitney test.

had virtually no clinical manifestations. Weight loss was the only objective sign (Panel E). A s u m m a r y of the clinical assessment of guinea pigs by groups is shown in T a b l e 2. It can be seen that d u r i n g the first episode (before treatment), there were no significant differences between the m e a n clinical scores of the groups. After treatm e n t however, the m e a n c u m u l a t i v e clinical scores of animals treated with M B P - L were significantly lower than the m e a n scores of all other groups. The group treated with M B P - I F A also had reduced scores b u t these were significantly lower only when c o m p a r e d to the group treated with MBP-SAL. These results indicate that t r e a t m e n t with 3 or 6 injections of 50 p.g M B P - L reduces significantly the n u m b e r of relapsing animals, the clinical m a n i f e s t a t i o n s of each relapse as well as their frequency.

Histological manifestations Systematic e x a m i n a t i o n of the b r a i n a n d spinal cord sections revealed several distinct types of lesions. There was m e n i n g e a l and perivascular meningeal infiltration by m o n o n u c l e a r cells, i n c l u d i n g occasional macrophages. These tended to be most p r o m i n e n t a r o u n d venules, particularly in the anterior m e d i a n sulcus of the spinal cord. Similarly, in the p a r e n c h y m a there was perivascular infiltration by m o n o n u c l e a r cells. This varied in degree from few cells affecting only part of the vessel wall to a dense infiltrate m a n y layers thick. Again, the small blood vessels, venules, were the most affected. In some a n i m a l s killed after 50 weeks or more, some b l o o d vessels showed fibrosis of the vessel wall a n d few i n f l a m m a t o r y cells. The degree a n d extent of fibrosis was not scored separately. In moderately a n d severely affected animals, there were m o n o n u c l e a r cells, and microglial cells within the p a r e n c h y m a extending from one blood vessel to the next. This was seen both in gray and white matter b u t virtually only in the b r a i n stem a n d

34

spinal cord. The pattern resembled an encephalitis and was not seen in protected animals and in those in long remission. Demyelination took several forms but tended to occur in a subpial distribution as well as at root entry and exit zones. Small areas of subpial demyelination adjacent to

Fig. 2. Histopathoiogy. A: Guinea pig treated with MBP-SAL; thoracic spinal cord; large area of

demyelination. × 100, Solochrome R; B: Guinea pig untreated; lumbar spinal cord; large area of demyelination with mononuclear infiltrate, astrogliosis and fibrosis of small blood vessels. × 100, H&E.

35 TABLE 3 HISTOLOGICAL SCORES OF GUINEA PIGS UNDERGOING CR-EAE Treatment a None CYC-L MBP-SAL MBP-IFA MBP-L ×3 MBP-Lx6

No h 19 4 5 6 6 7

Mean score Brain

Spinal cord

7.7 6.0 7.2 7.8 3.5 7.4

8.1 7.3 15.8 ¢ 13.7 6.5 6.3

See Table 1 for details. b Number of animals examined. Values in italics are s~gmficantlylower than the scores of this group: P < 0.05 Mann-Whitney test).

a meningeal vessel showing perivascular infiltration were p r o m i n e n t . In areas of large c o n f l u e n t d e m y e l i n a t i o n the blood vessels showed fibrosis, a n d astrogliosis (Fig. 2A, B). R e m y e l i n a t i o n could not be accurately j u d g e d in paraffin preparations. All treatment groups showed the same type of lesions as did the controls. The variation was in frequency, severity a n d topography of the lesions. The findings were assessed a n d scored as shown in Table 3. The lesions were more severe in the spinal cord than in the brain. The lowest scores in the spinal cord were found in the groups treated with MBP-L. The a n i m a l s in these groups had the smallest n u m b e r of lesions a n d had no or very little d e m y e l i n a t i o n and p a r e n c h y m a l i n f l a m m a t i o n . W h e n subjected to statistical analysis significant differences were found only between these two groups and the groups treated with MBP-SAL. In the brain, the group treated with M B P - L × 3 had the lowest score.

Cell-mediated immunity In the guinea pig, the b i n d i n g of peripheral blood lymphocytes to rabbit erythrocytes is a sensitive assay for T-cell activity. Recent studies in both acute a n d chronic E A E have revealed a significant decrease in the levels of early (5 min) T rosettes, d u r i n g overt clinical episodes. N o r m a l or even increased levels were observed d u r i n g s p o n t a n e o u s remissions a n d following M B P - i n d u c e d i m m u n o s u p p r e s s i o n (Traugott et al. 1982a). At various times throughout the present experiments, the percentages a n d absolute n u m b e r s of early T rosettes were determined. I n d i v i d u a l percentages are shown in Fig. 1 (vertical bars) superimposed on the clinical diagram. It can be seen that two o p p o s i n g patterns were o b t a i n e d : on one h a n d were the expected decreases in rosette levels d u r i n g relapses (Panel C, lanes 1, 2 and 4; Panel D, lanes 2, 3, 4 and 5) a n d the n o r m a l levels d u r i n g remissions (Panel B, lanes 1 and 6; Panel F); on the other hand, on m a n y occasions there were high or n o r m a l rosette levels d u r i n g severe clinical episodes (Panel A, lane 6; Panel B, lanes 1 a n d 2; Panel C, lanes 3 and 4; a n d decreased levels d u r i n g extended remissions (Panel F, lane 6; Panel D, lane 6, Panel E, lanes 2, 4 a n d 7, etc.). In order to allow a m e a n i n g f u l evaluation of the data, the rosette percentages recorded on all guinea pigs were

36

40

i 30

i 2o 10

'~ = 40 - 1 51 I)<0 0 2

2

4

6

8

10

12

14

16

18

20

SPINAL CORD HISTOLOGICAL

SCORE

Fig. 3. Simple linear regression of histologic scores vs. early T rosette levels. Each point represents a pair of histologic scores and 70 early T rosette averages taken in relapse. In the case of the group treated with MBP-L ×6 the rosette values plotted are from remission. The equation Y = a+ bx defines the regression line.

divided into 2 separate categories depending on whether they were obtained during relapses, or d u r i n g remissions. Table 4 shows the early T rosette averages for each g r o u p . It c a n b e s e e n t h a t d u r i n g r e m i s s i o n s all g r o u p s h a d T r o s e t t e a v e r a g e s t h a t fell w i t h i n t h e n o r m a l r a n g e . W h e n t h e T r o s e t t e s w e r e d e t e r m i n e d d u r i n g r e l a p s e s , 2 o f t h e 6 g r o u p s h a d a v e r a g e s w h i c h w e r e s i g n i f i c a n t l y l o w e r t h a n n o r m a l . It is

TABLE 4 DISTRIBUTION OF EARLY T ROSETTES DURING REMISSIONS AND RELAPSES Treatment"

% Rosettes Remission

No. b

70 Rosettes Relapse

No.

None CYC-L MBP-SAL MBP-IFA MBP-Lx3 MBP-L ×6 Normal

25.0 (20-31) c 27.2 (17-39) 32.0 (22-42) 28.0 (20-36) 25.0 (23-28) 27.0 (22-32) 28.0 (25-31 )

8 5 5 10 32 26 28

27.5 (24-31) 27.5 (19-36) 1 6 . 0 d (9-25) 2 0 . 0 (13-29) 36.0 (11-66) -

6 7 9 8 3 0

" b c d

See Table 1 for details. Number of assays. In parantheses are 95% confidence limits. Values in italics are significantly lower than normal.

37 interesting to note that the decreased rosettes occurred in the groups displaying the most severe histological lesions in the spinal cord. Moreover, a simple regression analysis in which the T rosettes were plotted against the histological scores (Fig. 3) indicated that decreases in early T rosettes depended upon and correlated with the extent of the spinal cord lesions. The statistical significance of the regression line was tested by analysis of variance ( P < 0.02). Delayed hypersensitivity reactions to MBP were carried out at the beginning of the treatment and were all positive. Lymphocyte transformation assays on lymphnode lymphocytes draining the injection site, performed at the time of killing, in the presence of 1, 5 and 25/~g MBP or of MBP-L were all negative regardless of whether the animals were in remission or in relapse (results not shown).

Discussion

The present experiments demonstrated that chronic-relapsing EAE induced in juvenile strain-13 guinea pigs could be effectively treated after recovery from the first attack, by the intracardiac administration of MBP inserted into PS liposomes. The clinical course of CR-EAE in the guinea pig follows one of 2 distinct patterns. One is characterized by a delayed onset (8-12 weeks) and by progressive worsening of the neurological deficits. Relapses and remissions are few and unpredictable. This pattern has been described in animals challenged with isologous spinal cord at multiple intradermal sites in the nuchal area (Stone and Lerner 1965; Traugott et al. 1979). The second pattern is characterized by a first episode with early onset (14-21 days) followed by a series of relapses, and remissions of variable length and intensity. This pattern occurs after foot-pad inoculation of the CNS tissue (Wisniewski and Keith 1977; Keith and McDermott 1980). In our experiments, the guinea pigs were challenged in the nuchal area at multiple sites, nonetheless most displayed the second pattern. The cause of this discrepancy is not clear but may be attributed to small changes in the genetic background of the strain-13/N guinea pigs maintained in different animal facilities. The clinical manifestations of the chronic phase of the disease were significantly reduced after treatment with MBP-L x 3 and MBP-L x6. These groups showed also decreased histological modifications in the spinal cord; in addition, the group treated with MBP-L ×3 had the lowest brain score of all groups (3.5). In two groups (untreated and CYC-L) unexpectedly moderate histologic modifications were associated with severe and frequent clinical episodes. The cause is obscure. It may be due in part to the fact that the lesions were localized in areas of the CNS controlling vital functions. Lack of correlation between clinical and histological manifestations is well documented. There are many reports describing CNS lesions that occur in the absence of any clinical signs in both EAE and MS (Paterson 1982; Gilbert and Sadler 1983; Stone et al. 1983). On the other hand clinical episodes of EAE take place in the absence of morphologic abnormalities and there is the suggestion that the mononuclear cell infiltrates may not be the cause of clinical manifestations. This

38 is based on findings linking clinical EAE with extensive accumulation of fibrin in the CNS, in the absence of any evidence of perivascular mononuclear infiltrates (Paterson 1982). These modifications, which are best detected by immunofluorescence would have remained undetected in the present experiments. In any case, the differences in the histological scores between these control groups and groups with similar clinical manifestations (MBP-SAL, MBP-IFA) were not statistically significant and therefore do not detract from the clinical suppression achieved in the groups treated with MBP-L x3 and MBP-L ×6. Treatment of C R - E A E recently reported, consisted of the intramuscular administration of 1.4 mg or 2.8 mg bovine MBP in IFA divided into 10 doses over a period of 28 days, or 2.8 mg MBP mixed with 6.7 mg GC (Traugott et al. 1982b). The treatment with M B P - G C - I F A was more effective than with MBP-IFA alone. It is important to note that these doses were 9 and 18 times higher than the lowest effective dose of MBP-L used in the present study. It is generally accepted that the major antigen responsible for the induction of EAE is myelin basic protein (Hashim 1978). Chronic EAE, however, does not develop unless whole CNS tissue or myelin is present in the inoculum. It has been recently shown that Hartley guinea pigs which develop only an inflammatory CNS infiltrate when challenged with MBP, display large demyelinating plaques if injected with a mixture of MBP and bovine brain GC in CFA (Raine and Traugott 1982). Coupled with the observations that divergent clinical manifestations occur in whole CNS- and in MBP-induced forms of EAE (Hoffman et al. 1973), this observation implies that CNS antigens other than MBP are involved in the induction of C R - E A E and thus may be also required for effective suppression. Both GC and PS are components of the CNS and may contribute to suppression of CR-EAE. However, to our knowledge, results of suppressive treatment with GC alone have not been reported and the administration of PS-liposomes either alone or in combination with cytochrome-c failed to protect against either acute (Strejan et al. 1981), or chronic EAE. Recent investigations have shown that the administration of MBP-bacterial LPS complexes to Lewis rats prior to encephalitogenic challenge, inhibited the development of EAE and led to the appearance of T suppressor cells (Raziuddin et al. 1982). The treatments with MBP-GC, MBP-L and MBP-LPS have one element in common: they are all administered as MBP-lipid vesicle complexes. Taken collectively these observations suggest that the presentation of MBP in association with lipid may be essential in achieving lasting protection from EAE. Guinea pigs undergoing clinical episodes of EAE have decreased early (active) T cell rosettes during overt clinical signs in both acute and chronic EAE, and revert to normal or slightly increased levels in remission. Normal or increased rosette levels are also observed following treatment with MBP-IFA (Traugott et al. 1979) or with MBP-liposomes (Strejan et al. 1981). The early T rosette levels are thought to follow closely the clinical course of EAE, and their decrease in the peripheral blood reflects their mass migration to the CNS. In the present experiments the majority of the animals remaining after the first attack were sampled repeatedly for early T rosettes, as a cell-mediated immune correlate of the disease. Individual assays showed that both decreased and normal or slightly increased rosette levels were present during

39 clinical episodes. On the other hand in addition to normal values, animals in prolonged remission had decreased rosettes. It has been reported that too frequent sampling of blood can alter significantly the clinical course of both acute and C R - E A E (Traugott and Raine 1979). This may also influence the results of subsequent rosette assays. We believe however, that this was not the case here since the assays were not frequent enough to justify such an interpretation. When the rosette assays were grouped under the 2 clinical categories (remission or relapse), two of the 6 groups showed decreased rosettes during relapses and none had decreased rosettes during remissions. Unexpected were the normal rosette levels in 2 control groups (untreated and CYC-L ×3), despite frequent and severe relapses. It became immediately apparent that the T rosette levels reflected more closely the severity of the histological manifestations than of the clinical course. In addition to histologic modifications other immunological events occurring in the CNS may contribute to the migration patterns of T lymphocytes; these include systemic or intrathecal production of specific antibody to various CNS antigens. The isoelectric focusing pattern of the Ig and the quantitation of anti-CNS antibodies in both serum and CSF of these animals may provide important information in this regard. The premise leading to the use of MBP-inserted liposomes in the suppression of EAE (Strejan et al. 1981) derived from observations that the i.v. administration of hapten- or protein-coupled syngeneic cells, or cell membranes resulted in tolerance induction and in generation of T suppressor cells for T cell-mediated immune functions (Claman et al. 1980). Implicit in this premise was the assumption that the insertion of MBP into artificial model membranes mimics the coupling of protein antigens to cell membranes. This assumption remains largely untested because the exact topography of MBP in the liposome and of other extrinsic proteins in the cell membrane has not been elucidated. A recent electron-microscopic study on the insertion of MBP in PS vesicles (Naughton and Smith 1982) does not provide sufficient information to allow comparisons between the 2 systems. It is interesting to note that the i.v. administration of mouse MBP coupled to syngeneic spleen cells prevented the subsequent induction of EAE (Sriram et al. 1983). One obvious distinction between the liposomes and the cell membrane systems is the absence of M H C gene products from the liposomes. Using hapten-coupled solubilized lymphoid cell membranes, it has been shown that for the induction of T suppressor cells in contact sensitivity, the hapten must be presented in association with products of the H - 2 D locus (Miller et al. 1980). Since liposomes are known to fuse readily with cell membranes, the requirement for association with D locus products may be satisfied. It is also known that the generation of T suppressor cells is favored when antigen association with Ia-bearing macrophages is bypassed (Benacerraf 1980). It is conceivable that the fusion of the liposomes with an antigen-presenting cell occurs in a way that would prevent such an association.

Acknowledgements The authors wish to thank Dr. D.W.T. Watson for providing the strain-13/N guinea pig breeding nucleus, Dr. N.R. Sinclair for his advice with the statistical

40 a n a l y s e s , M r s . N . E s s a n i a n d M r . D. S u r l a n f o r t h e i r h e l p w i t h p o r t i o n s o f t h i s i n v e s t i g a t i o n , a n d M r s . D. J e l l i n e k f o r h e r e x c e l l e n t w o r k i n t y p i n g t h e m a n u s c r i p t .

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