Journal of the Neurological Sciences, 1978, 35:291-308 © Elsevier/North-Holland Biomedical Press

291

R E D U C T I O N BY L I N O L E I C A C I D OF THE SEVERITY OF E X P E R I M E N T A L A L L E R G I C E N C E P H A L O M Y E L I T I S I N T H E G U I N E A PIG

C_ J. MEADE, J. MERTIN, JINAN SHEENA and RUTH HUNT Transplantation Biology Section, Surgical Sciences Division, Clinical Research Centre, Watford Road, Harrow HA1 3UJ, Middlesex (Great Britain)

(Received 20 July, 1977) (Accepted 19 October, 1977)

SUMMARY This paper reports the effects of supplementation of the diet with linoleic acid on the severity of experimental allergic encephalomyelitis (EAE) in guinea pigs. Clinical signs of disease (e.g. paresis, paraplegia, urinary incontinence), weight loss, frequency of perivascular lesions in the central nervous system and ability of isolated lymph node cells to respond to myelin basic protein in vitro were all reduced by linoleic acid supplementation. Linoleic acid was effective when fed at a dose of 0.5 ml/day from 7 to 21 days after sensitization of the animals with basic protein, i.e., before and during the time in which clinical signs normally appeared. The same daily dose fed from 7 days before to 7 days after sensitization, i.e., ceasing about 7 days before the normal time of appearance of clinical signs, produced no significant effect. Feeding linoleic acid to normal guinea pigs significantly altered the fatty acid composition of their serum and lymph nodes, but not of their brain. Of several possible explanations for the protective effect of linoleic acid in EAE, we considered action by this essential fatty acid on the immune system most likely.

INTRODUCTION Multiple sclerosis (MS) patients have been claimed to have lowered levels of linoleic acid (C 18 : 2") in plasma, brain tissue and lymphocytes (Thompson 1975; Tsang, * The number preceding the colon indicates number of atoms in the carbon chain; the number following indicates the number of double bonds. Thus linoleic acid (C18:2) has 18 carbon atoms and 2 double bonds, whilst oleic acid (C18:1) has one double bond less. Other fatty acids mentioned in this paper are palmitic acid (C16:0), stearic acid (C18:0), linolenic acid (C18:3) and arachidomc at,id (C20:4). As our gas chromatographic technique could not distinguish different isomers with identical numbers of carbon atoms and double bonds, fatty acids identified only by gas chromatography are identified first by an abbreviation, then by the trivial name of the predominant isomer in mammalian tissues possessing the indicated number of carbon atoms and double bonds.

292 Belin, Monro, Smith, Thompson and Zilkha 1976). Supplementing patients' diet with sunflower seed oil, rich in linoleic acid, has been reported to produce a slight beneficial effect on the clinical course of MS (Millar, Zilkha, Langman, Payling Wright, Smith, Belin and Thompson 1975). In MS some evidence implicates autoimmunity in the disease process. There is no ideal animal model for MS, but a cell-mediated autoimmune disease of the central nervous system, experimental allergic encephalomyelitis (EAE), can readily be induced in susceptible animals by immunisation with myelin basic protein in complete Freund's adjuvant (CFA) (Paterson 1960). This work uses EAE as a model to examine effects of dietary linoleic acid on an autoimmune brain disease. Two previous studies investigated the effect of dietary fatty acids on EAE. Clausen and M~ller (1969) showed that a diet deficient in essential fatty acids (including linoleic acid) potentiated the ability of injected brain homogenates to produce EAE in rats. Selivonchick and Johnston (1975), confirming this, also showed a protective effect of supplementary oral ethyl linoleate in rats fed a fat-deficient diet. Both groups gave experimental diets of different fatty acid composition from birth (i.e., to both parents and offspring). Detailed analyses of brain lipids were made, and results interpreted in terms of altered susceptibility of the central nervous system to attack. The study reported here was designed on a different basis. Altered dietary linoleate levels were provided only after weaning, not from birth. This situation parallels more closely that of MS patients receiving dietary linoleate supplementation, such patients being generally young or middle-aged adults. Immunological parameters as well as brain composition were examined. METHODS Animals Young female Dunkin-Hartley guinea pigs, weighing 250-350 g, were obtained from the National Institute for Medical Research, Mill Hill. Diet All guinea pigs were fed Spratt's guinea pig diet and water ad lib. The manufacturers report 18.7~ protein, 12.2~ fibre, and 2.8 ~ ether extractable material. Principal fatty acids were C18:2 (linoleic acid) 41 ~ ; C l6:0 (palmitic acid) 17 ~o; C18 : 3 (a- and 7-1inolenic acids) 14 ~o and C18 : 1 (oleic acid) 15 ~o. Total tocopherols (as a-tocopherol) were 81 #g/g. Linoleic acid supplementation 0.5 ml 99 % pure (Sigma) linoleic acid (cis-9-cis-12-octadecadienoic acid) was fed daily from a small syringe. Where stated, control animals were fed 0.5 ml of water, liquid paraffin or oleic acid in the same way. Vitamin injection Where stated, guinea pigs (both fat supplemented animals and unsupplememed

293 controls) received an intraperitoneal injection of 0.05 ml of a mixture of fat-soluble vitamins on the first and eighth day after commencing supplementary feeding. This injection contained 100 IU vitamin A (Crooke's), 300 U of antirachitic activity vitamin D (Evans Medical), 0.1 mg vitamin E (Sigma) and 0.1 mg vitamin K1 (Roche), all in a polyethylene glycol ester dispersing agent. Myelin basic protein (BP) was supplied by Dr. E. A. Caspary from a batch prepared from human brain (Caspary and Field 1965). Only 2 bands (identified as BP and its polymer) were observed after agar gel electrophoresis (Bignami and Eng 1973). Solutions of BP were prepared immediately before use from a lyophilised stock.

Induction of EAE Guinea pigs were injected with 10 #g BP emulsified in CFA (Difco Bacto adjuvant, containing Mycobacterium tuberculosis). The ratio of aqueous to oily phase was 1:2 and total volume injected was 0.1 ml, divided between the 2 hind foot pads. The dose of BP was selected following a preliminary experiment in which 10 #g was found to be sufficient to produce clinical signs of disease in most, but not all animals (i.e., 14/20 compared with 6/20 when the immunising dose was 2 #g, and 20/20 when the dose was 50 #g). We aimed to produce a moderate disease, as we considered the incidence of such disease would be more susceptible to modification by a weak agent. Clinical scorhlg Each guinea pig, individually identified, was weighed and assessed for clinical signs of disease daily. (Prior to the seventh day after injection of BP, weighing and assessment was only at 2-4 daily intervals.) The clinical scale used was: Grade O. No disease. Grade 1. Mild ataxia, e.g., an insecure gait. Mild paresis, particularly of hind limbs. Hypotonia (particularly noticeable in hind limbs and abdominal muscles). Grade 2. As 1, but ataxia, paresis or hypotonia marked. Animal still able to move hind limbs and walk. Grade 3. Slight movement of the hind limbs possible after strong stimuli, but the animal normally moves on its forepaws whilst dragging the hind limbs. Grade 4. No movement of hind limbs, even following a strong stimulus. Alternatively, paralysis as in 3 accompanied by urinary incontinence and/or bowel disturbances. The latter (rarer than bladder disturbances) could take the form of faecal retention. Rarely, urinary incontinence is not accompanied by dragging of the hind limbs, in which case the animals are scored 3. Grade 5. A moribund condition of complete paralysis of hind limbs and involvement of forelimbs. The animal is unable to get about or raise its head on its forelimbs to reach the food bowl. Respiration is frequently impaired. Grade 6. Death. Severely paralysed guinea pigs were moved to new cages in which food was on the ground, so the animals could still reach food despite their paralysis.

294

Histological examination Brains and spinal cords were removed as rapidly as possible after death and fixed in formol-acetic acid. Longitudinal sections were made in a standard manner through the posterior part of the brain (cerebellum and brain stem) and through the spinal cord at 2 standard places. Sections were stained with haematoxylin and eosin. The severity and distribution of perivascular infiltrations were assessed by an observer unaware of the origin of the material he was reviewing, and graded on an arbitrary scale 1~,. Gold uptake assay This assay measures the ability of lymphocytes to produce soluble factors able to 'activate' macrophages, an 'activated' macrophage taking up more 19aAu than an unactivated macrophage. Lymph node cell supernatants were assayed on monolayers of exudate cells (95 % macrophages) exactly as described by Meade, Lachmann and Lowe (1976). Test supernatants were prepared by incubating, with rotation, 1 × 107/ml isolated popliteal lymph node cells for 40 hr at 37 °C with 20 #g/ml BP in TC 199 ÷ I00 U/ml penicillin ~- 100 #g/ml streptomycin q- 0.22% sodium bicarbonate q- 5 ~ normal guinea pig serum. Control supernatants were prepared by incubating lymph node cells in the same medium but without BP, 20/zg/ml BP being added at the end of the culture. 'Test' and 'control' supernatants from each animal were stored at --70 °C and assayed, diluted I :4 in supplemented TC 199 ÷ 15 ~ normal guinea pig serum, on quadruplicate monolayers. The difference between the geometric mean values of gold uptake by monolayers in contact with 'test' and 'control' supernatants was taken as a measure of antigen-stimulated lymphokine production, and is henceforth abbreviated A gold uptake. Fatty acid analysis Extraction of lipids from tissue samples and diets. Appropriate amounts were weighed accurately and extracted with chloroform-methanol (2: 1, v/v) to which had been added: 2 mg a-tocopheryl acetate, as antioxidant, and 1 mg/100 mg tissue of heptadecanoic acid (C 17:0) as internal standard. Extraction was aided by homogenizing in presence of solvent. Extracts, if necessary filtered through glass wool, were washed with an equal volume of 1% sodium chloride solution. The lower, lipid containing phase was dried over magnesium sulphate and stored at --20 °C in the dark. Fatty acid methyl esters were prepared by refluxing this lipid extract with methanoltoluene-HeSO4 (10:20:1, v/v/v) for 30 min. Water was added and methyl esters then extracted into ether. Extracts were dried over magnesium sulphate, then evaporated under nitrogen, dissolved in 0.4 ml cyclohexane, and stored at --20 °C until analysed by gas liquid chromatography. Extraction oflipidsfrom sera. An appropriate volume (0.5 or 1.0 ml) of serum was refluxed with 2 ml 10 % potassium hydroxide in methanol for 10 min. a-Tocopheryl acetate (2 mg) and heptadecanoic acid, 1 mg/ml serum, were included in the reflux mixture. The sample was then acidified with 4.5 ml 1 N hydrochloric acid, and the partially esterified fatty acids were extracted with ether.

295 Extracts dried over magnesium sulphate were methylated with excess diazomethane (Schlenk and Gellerman 1960), then evaporated to dryness and taken up in 0.2 ml cyclohexane for analysis. All reagents used were of 'Analar' purity except cyclohexane which was 'spectrograde' (British Drug Houses). Chromatography. Fatty acid methyl esters were analysed on a Pye Unicam series 104 gas chromatograph. The stationary phase was 10 ~ diethylene glycol succinate on an 100-120 mesh, acid washed, diatomite 'C' support. Carrier gas (argon) flowed at 50 ml/min. Operating temperature was 200 °C. Output was analysed by a chromatographic data processor (Digital Equipment Corporation) or a DP88 computing integrator (Pye-Unicam). Identification of fatty acids was carried out with reference subs tances. Statistical analysis. The number of animals showing signs of disease was analysed by the X2 test. Clinical scores are arbitrary figures and have no quantitative meaning except to indicate a rank order of disease intensity. They were therefore handled with a nonparamctric test (Wilcoxon sum of ranks test). All animals, including those scored '0', were ranked. The Wilcoxon test was also used to compare 'day of onset of clinical signs'. A gold uptake was handled by parametric statistics. Preliminary experiments showed that, over the supernatant range under study, there was a good linear relationship between log supernatant concentration and log A gold uptake, so A gold uptake was treated in logarithmic transformation. A gold uptake was influenced not only by the nature of the test supernatant, but also by the monolayer used for assay. This 'monolayer associated' variance was separated from the 'treatment associated' variance by an appropriate multifactorial analysis of variance using the general linear interactive model computer programme (Royal Statistical Society). To test for a link between clinical score and either weight loss or A gold uptake, an analysis of variance was performed to test for differences between 3 groups: animals with clinical score 0, those with score I or 2 and those with score 3, 4, 5 or 6. Fisher's test was used to examine for significance between group variance in this and all other analyses of variance. RESULTS

Effect of feeding linoleic acid, oleic acid, paraffin oil or water on serum fatty acids Fatty acid levels in each animal of groups of 4 normal animals were measured in sera taken 5 hr after feeding a single dose of 0.5 ml of each supplement. Following a single feed oflinoleic acid, both the amount and proportion of Cl8:2 (linoleic acid) in the serum increased (Table 1). Whilst the proportion of other fatty acids was necessarily decreased, the absolute levels (not shown in t a b l e ) o f C18:1 (oleic acid), C18:3 (linolenic acid) and C20:4 (arachidonic acid) were not significantly affected. A single feed of oleic acid did not significantly affect the absolute level of C l 8 : 2 (linoleic acid) but the concentration of C18:1 (oleic acid) was increased, from

296 TABLE 1 S E R U M FATTY ACID LEVELS IN N O R M A L ANIMALS 5 hr A F T E R A SINGLE 0.5 ml FEED Each value is the mean of 4 values :t: 1 SE_ The ratio CI 8:2 (linoleic acid)/C16:0 (palmitic acid) -i C18:0 (stearie acid) + C18:1 (oleic acid) was significantly different in unsupplemented and linoleic fed animals (0.025 > P > 0.01). No other dietary supplement produced a significant alteration in this ratio. Dietary supplement

Concentration C18 : 2 (linoleic acid) in serum (mg/ml)

No supplement Water Liquid paraffin Oleic acid Linoleic acid

0.13 0.15 0.17 0.17 0.24

Ratio CI 8 : 2 (linoleic acid) C16:0 (palmitic acid) + C18 : 0 (stearic acid) + C18:1 (oleic acid)

+ 0.02 + 0.01 4- 0.02 4- 0.02 I- 0.05

0.63 1.07 0.78 0.51 1.83

a mean value of 0.07 mg/ml in the serum of control animals to a mean value of 0.20 mg/ml in the serum of animals given a single feed of oleic acid and killed at the same time as the controls. This difference was significant (P < 0.001). Feeding liquid paraffin or water was without effect on fatty acid levels. The elevated ratio of C18:2 (linoleic acid) to the major non-essential fatty acids of serum (C16:0 (palmitic acid), C18:0 (stearic acid) and C18:1 (oleic acid)) was at

1~

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W ItJ'lOIJt]li~elc acid { expe~mer~.al amrnals i~'eviou sly fed l i ~ acicl 0.5 ml ~ clay for 14 clays )

Fig. 1. Effect of feeding linoleic acid for different lengths of time o n the ratio of C18 : 2 (linolelc acid) to the major non-essential fatty acids of serum. Shaded bars: animals fed linoleic acid. Open bars: controls killed at the same time. Each bar represents the mean of measurements on 4 animals (with standard error).

297 its height 5 hr after a single feed. Repeated daily dosing with linoleic acid did not raise the level more than a single feed (Fig. 1). Effects of feeding linoleic acid, oleic acid, paraffin oil or water on E A E Animals fed linoleic acid, or oleic acid, in comparison with animals fed no supplement, water or liquid paraffin, received modest but significant protection from the encephalitogenic action of injected BP. Three experiments were performed. In the first, 0.5 ml of each supplement was fed to groups of 12 guinea pigs daily between 7 and 21 days after immunisation with 10 #g BP in CFA. A fifth group of guinea pigs received no supplement. The experiment was continued for 30 days to permit the animals to recover, The second experiment repeated the first except that guinea pigs were killed for histological examination 21 days after BP injection. Serum was obtained for fatty acid analysis at the same time. In the third experiment, the animals were allowed to recover, but only 2 feeding supplements were used: linoleic acid (49 guinea pigs) and liquid paraffin (40 guinea pigs). Weight loss was used as an indicator of disease. Since many other factors influence weight gain apart from EAE, in the first 2 experiments, groups of 6 animals injected with CFA alone were fed the same supplements and kept in similar cages to those injected with CFA q- BP. The difference between the mean weight gain of the animals injected with CFA q- BP and those injected with CFA alone was considered as 'weight loss due to EAE'. Linoleic acid and oleic acid both significantly (0.05 > P > 0.025) reduced the number of animals showing clinical signs of disease (i.e., scoring grade '1' or above) whilst water or liquid paraffin had no effect (Fig. 2). The incidence of clinical signs up to 21 days after injection of BP was, in the first and second experiments together: fed linoleic acid 4/20 (20%); fed no supplement 12/24 (50%); fed liquid paraffin 11/24 100--

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Fig. 2. Disease incidence compared in groups of guinea pigs fed linoleic acid (---), with no supplement ( ), with water ( . . . . ), with liquid paraffin (---), or with oleic acid ( ), daily between 7 and 21 days after injection of BP + CFA. Vitamins were injected on day 8 and 15.

298 TABLE 2 CLINICAL SIGNS OF EAE - - MAXIMUM SEVERITY DURING THE PERIOD OF DIETARY SUPPLEMENTATION Figures show maximum severity of disease up to the end of the period of dietary supplementation (i.e. the 21st day after injection of BP). Experiments I and 1I have been pooled, but experiment lIl (which used a different batch of guinea pigs) is shown separately. The asterisked numbers (*) include one animal in each group killed by us for humane reasons when quite paralysed and on the point of death. Exclusions of such animals reduces the number of deaths by 4 ~ in each case. The double asterisk (**) indicates that the cause of death in 2 cases in this group was probably accidental inhalation of fatty acid rather than EAE. However, as both animals showed mild (grade I) EAE the previous day, this is not certain, so both cases have been included in the number of deaths due to EAE. If these animals are excluded, the percentage of deaths in the linoleic acid fed group is reduced to 17 ~. Number of animals examined

of animals with different clinical scores Score Score Score Score Score Score 1 or

2 or

3 or

4 or

5 or

6

above above above above above (dead) Experiments I and 1I No supplement Water Liquid paraffin Linoleic acid Oleic acid Experiment 111

24 23 24 20 22

50 52 46 20 14

38 35 29 10 9

29 26 21 5 5

17 17 8 5 0

13 17 8 5 0

13 17" 8" 5 0

Liquid paraffin 40 Linoleic acid 47

63 49

50 38

40 23

30 21

30 21

30 21 **

(46~o); fed w a t e r 12/23 ( 5 2 ~ ) ; fed oleic acid 3/22 ( 1 4 ~ ) . I n a d d i t i o n , in the first e x p e r i m e n t one oleic acid fed a n d one u n s u p p l e m e n t e d a n i m a l d e v e l o p e d signs o f disease after s u p p l e m e n t a t i o n was d i s c o n t i n u e d on d a y 21. In the t h i r d experiment, 23/47 (49 ~ ) linoleic acid fed guinea pigs a n d 25/40 (63 ~ ) liquid paraffin fed guinea pigs showed clinical disease. The overall differences in disease incidence between linoleic acid fed a n d liquid paraffin fed animals were j u s t outside the n o r m a l l y accepted limits for statistical significance (for the c o m p a r i s o n 27/67 vs. 36/64, 0.1 > P ~ 0.05). H o w e v e r , when n o t only presence o f disease b u t also clinical severity were included in the statistical analysis, r a n k i n g a p p r o p r i a t e p o o l e d values for m a x i m u m disease severity d u r i n g the p e r i o d o f d i e t a r y s u p p l e m e n t a t i o n , linoleic acid a n d oleic acid b o t h p r o d u c e d significant (0.05 > P ) p r o t e c t i o n in c o m p a r i s o n with no s u p p l e m e n t a t i o n , feeding w a t e r o r feeding liquid paraffin. Table 2 shows the effect o f o u r different feeding regimens on disease severity in all 3 experiments. ' W e i g h t loss due to disease' was similar in linoleic acid o r oleic acid fed animals, in b o t h cases being less t h a n in u n s u p p l e m e n t e d , water fed o r liquid paraffin fed guinea pigs (Fig. 3). The same t r e n d s ( p r o t e c t i o n by linoleic acid or oleic acid, b u t n o t by liquid paraffin o r water) were seen in histological scores with a reduced size a n d frequency o f perivascular lesions in the central nervous system, p a r t i c u l a r l y the spinal c o r d (Table

3).

299 v. untreeted

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P), whilst linoleic acid from day - - 7 to + 6 had no detectable effect at all. In the second experiment, there was significant monolayer associated variance, so results cannot be readily pictorially represented, but again feeding linoleic acid from day 7 significantly reduced gold uptake (0.05 --~ P > 0.01) whilst feeding linoleic acid up to day 6 had no significant effect. Viability of the lymph node cells used for preparing supernatants, where examined, was similar, no matter what dietary treatment the guinea pigs supplying the cells received.

Effect of feeding linoleic acid on fatty acid composition of lymph nodes and brain F a t t y acid composition of lymph nodes from linoleic acid fed guinea pigs differed markedly from that of lymph nodes from untreated animals. Differences occurred in both CFA stimulated and unstimulated nodes, but were more pronounced in the for-

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Fig. 6. Total C18:2 (linolcic acid) content/g wet weight of popliteal lymph nodes from guinea pigs fed linoicic acid, olcic acid or no supplement for 14 days, and injected with C F A 21 days prior to sacrifice. Shaded part of bars: C l 8 : 2 (linotvic acid); open part: other fatty acids of chain length C > 14. Means of measurement on 4 animals (lino|¢ic acid fed 3 only), with smndaxd errors shown as vertical bars.

303 25.

20

15 !

10

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I 12:.0 14;0 16:0

16:1 18:0 18:1 18:2 18:3 20:0 20:1 20:4 22:0 22:1 22:4 22:6 24:0 F a t t y actd ?

Fig. 7. Percentage of different fatty acids in guinea pig whole brain_ Shaded bars: animals fed linoleic acid for 14 days prior to sacrifice. Open bars: animals fed no supplement and killed at the same time. Vertical lines : standard error. Each value is the mean of determinations made on 4 animals_ See footnote, p. 291, for explanation of fatty acid abbreviations.

mer; 14 days feeding linoleic acid increased the total yield of long-chain fatty acids per gram of lymph node tissue. This increase consisted of a large and statistically significant (0.001 :> P) increase in tissue linoleate, along with smaller increases in several other fatty acids (Fig. 6). By contrast, supplementary dietary linoleic acid had no significant effect on either the proportion (anyway small) or amount of linoleic acid in the brain. Nor was there any significant effect on levels of the other major fatty acids. Although there were some differences in the minor fatty acids, these are not meaningful in view of the difficulty in assaying fatty acids present only in small proportions (Fig. 7). Correlation between different parameters used to assess disease and between disease and immunological parameters Histological scores, particularly those of spinal cord sections, correlated well with clinical scores. Of the 53 animals examined, each of the 19 which at some time manifested clinical signs of disease showed perivascular cuffs, and in 18 out of 19 cases cuffs were present in both brain and spinal cord. By contrast, only 18/34 (53 %) of clinically unaffected animals showed lesions, and with one exception the lesion score was the lowest on our scale. Spinal cord lesions were seen in only 3 out of 34 clinically unaffected animals. Animals with spinal cord lesions always had lesions in the brain, but the converse was not necessarily true.

304 A gold uptake did not correlate either with weight loss in the last 72 hr of life o~ clinical score when the guinea pigs were killed on day 14 or 15 (P - 0.05). The same group of animals gave a significant correlation between 72-hr weight loss and clinical score (differences in weight loss between group scoring '0', °1 or 2', or '3 to 6' all significant, 0.05 > P).

Side effects of treatment Provided they received vitamin supplementation, animals fed supplementary linoleic acid and injected with CFA but not BP (so they would not get EAE) appeared healthy and their weight gain was not significantly different from controls. There was occasional yellowish discolouration of the fur and fur loss around the mouth if linoleic acid was spilled during feeding (the mouth region was therefore carefully wiped after each feed). A small percentage (15/185 ~-- 8 ~ ) of linoleic acid fed, CFA + BP injected guinea pigs died during the experiments without previously showing any signs of EA E. We think accidental inhalation of fatty acid, with consequent lung damage, caused the majority of these deaths. Such deaths also occurred in linoleic acid fed animals injected with CFA alone (13/60) and in oleic acid ted animals (4/36), but the proportion of water, paraffin fed or unsupplemented animals dying from causes other than EAE was much lower (1/36, 1/76 and 3/219 respectively). With greater experience, the proportion of animals dying as a result of accidents in feeding declined, yet suppression of EAE was similar. Where death was clearly due to causes other than EAE (e.g. where death occurred before day 10, the earliest day even mild signs of EAE were ever noted, and when there was post-mortem evidence for lung damage), such death has been excluded from the statistical analyses. This is one of the reasons why figures set out in the results sometimes total less than the number of animals used in each experiment. In some experiments not otherwise described, linoleic acid was injected subcutaneously (s.c.) rather than fed. This injection was highly effective in suppressing EAE. In one experiment, clinical signs appeared in 5/20 (25 ~ ) of guinea pigs injected w~th 0.1 ml linoleic acid s.c. daily from 7 days before till 7 days after 10 #g BP + CFA, whilst 14/20 (70 ~ ) BP + CFA injected animals not given linoleic acid showed signs of EAE (difference significant 0.005 > P > 0.001). These experiments were not pursued because granulomata formed at the site of injection, and in a few cases ulceration occurred. DISCUSSION This work supports the report of Selivonchick and Johnston (1975)that feeding linoleate can suppress EAE, and further extends their results by showing that this protective effect can occur in animals fed a normal basic diet. Theeffect was small but significant, and could be observed using all 3 of the parameters used to assess the disease - - clinical score, weight loss and histological changes in the central nervous system (CNS). Selivonchick and Johnston killed the guinea pigs used for their study at day 14. By continuing some of our experiments until all animals had either complete-

305 ly recovered or died, we were convinced that the effect was a true reduction in the severity of EAE and not just a delay in the onset of clinical signs. A protective effect was only achieved if raised linoleic acid serum levels were maintained during the time of clinical disease. This suggested that linoleic acid effects are short-lived. Feeding linoleic acid for 14 days (the longest period of dietary supplementation used by us to suppress EAE) did not significantly alter brain fatty acid composition, although the composition of stimulated lymph nodes could be altered. This makes it unlikely that in our experiments linoleic acid acted through gross overall changes in brain lipids. Resistance of the CNS to the effects of differing dietary linoleate intakes has also been reported in calves by Erwin and Sterner (1963). Paoletti and Galli (1972) demonstrated that a diet deficient in linoleic acid and other essential fatty acids given to rats from the age of 30 days onwards only slightly altered the fatty acid composition of the brain unlike other tissues. Changes in the fatty acid composition of the brain could only be achieved when the rats were fed the diet from birth. Effects on immune function Linoleic acid suppressed the immune response to BP as seen in the decreased gold uptake by macrophages, resulting from a reduced production of macrophage activating factor by lymph node cells. Soluble products of lymphocytes have been implicated in demyelination in studies by Stoner, Brosnan, Wi~niewski and Bloom (1977), in which mononuclear cell cuffing and demyelination in the optic nerve followed injection of such materials into the eyes of rabbits primed with spinal cord in CFA. In our study only the regimen which was effective in suppressing EAE brought about suppression in the in vitro immune response to BP. However, in individual animals within each group we failed to correlate in vitro lymphocyte reactivity to BP with the extent of clinical signs. Failures to correlate disease intensity and in vitro measures of delayed hypersensitivity have been reported by other workers (Dau and Peterson 1969; Warnatz, Scheiffarth and Kuntz 1970). Most agents that suppress immune function suppress EAE. Linoleic acid has elsewhere been shown to have a suppressive effect on lymphocyte function in vitro (Mertin, Hughes, Shenton and Dickinson 1974; Offner and Clausen 1974; Mertin and Hughes 1975; Mihas, Gibson and Hirschowitz 1975; Weyman, Belin, Smith and Thompson 1975), and in vivo findings of weak effects of oral linoleic acid on graft rejection have also been interpreted in immunological terms (Mertin and Hunt 1976). The ease with which linoleic acid can cross the blood-brain barrier may contribute to its beneficial effect in EAE. Speculations about the ways in which oral linoleic acid might influence the immune system in vivo include: (1) Linoleic acid is a prostaglandin precursor. Increased levels of such precursors can stimulate prostaglandin biosynthesis (Hong, Polsky-Cynkin and Levine 1976). Prostaglandins, especially those of the E series, are immunosuppressive both in vivo and in vitro (Pelus and Strausser 1976) and also inhibit the release of lysosomal enzymes (Ignarro, Oronsky and Perper 1973), i.e., substances possibly involved in demyelination. (2) Effects of dietary linoleate on fl-lipoprotein levels (Nichaman, Sweeley and

306 Olson 1967) could possibly extend to the immunoregulatory subspecies of this group of molecules, described by Curtiss, De Heer and Edgington (1977). (3) Changes in membrane fluidity brought about by an increase in the level of arachidonic acid in the membranes may have a role in lymphocyte activation (Ferber and Resch 1976). Abnormally high levels of linoleic acid, an arachidonic acid precursor, might modify this process. Immune phenomena, including those in EAE, can also be suppressed by nonspecific stress (Levine, Strebel, Wenk and Harman 1962). It is therefore necessary, when evaluating an agent claimed to suppress an immune function, also to evaluate the possible stress caused by the treatment. Selivonchick and Johnston (1975) reported that feeding ethyl linoleate alone depressed weight gain. This we also found in preliminary experiments. However, weight gain was not significantly different in either linoleic acid fed animals or unsupplemented controls if a supplement of fat-soluble vitamins was also injected. Stress, by causing lipolysis, can affect fatty acid concentrations in the serum (Engel and White 1960). The failure to demonstrate an absolute increase in serum C18:2 (linoleic acid) concentration in linoleic acid fed animals after the onset of the disease (on day 21 after sensitization) is thus not surprising. The stress caused by EAE itself might be expected to affect total fatty acid levels. However, the proportion of linoleic acid in the serum was still raised by linoleic acid feeding even when a high proportion of animals showed signs of the disease.

Effects of oleie acid (C18:1) on EAE The effect of oleic acid must also be considered in any hypothesis to explain the protective effect of linoleic acid. Oleic acid, unlike linoleic acid, can be synthesized by mammals, and is thus not essential in the diet. This substance was included as a control in a trial of a high linoleate diet in multiple sclerosis (Millar et al. 1975). However, in this trial the amount of oleate (olive oil) was considerably less than the amount of linoleate (in sunflower seed oil) provided to the patients, and no significant change in levels of C18 : 1 (oleate) in the serum of the control patients was produced. By contrast, in the present study the quantities of oleic acid and linoleic acid used were equivalent, and C18:1 (oleate) levels in the serum were significantly raised by the feeding regimen. Oleic acid is not a control in the sense of being an inert substance. It competitively affects linoleic acid metabolism (Dhopeshwarkar and Mead 1961 ; Lowry and Tinsley 1966). Whilst mammals themselves cannot convert oleic to linoleic acid, certain gut micro-organisms can (Sklan, Volcani and Budowski 1971). We found in our experiments no evidence for significant conversion of oteic to linoleic acid, but the possibility of altered linoleic acid metabolism must be seriously considered in view of the large alteration in serum oleate levels. Mertin and Hughes (I975) and Weyman et al. (1975) found oleic acid also suppressed in vitro transformation. In structure activity studies on the effect of different fatty acids (Meade, unpublished), oleic and linoleic acid were equally effective in depressing thymus weight if given in high doses, but at low dosage linoleic acid was more effective.

307 Implications o f these results f o r multiple sclerosis research The effects of linoleic acid o n E A E f o u n d in this study were reproducible b u t modest. Agents such as steroids a n d nucleotide analogues appear from the literature to be more effective. A s s u m i n g that E A E m a y - - at least in part - - be a worthwhile model for MS, our experiments s u p p o r t c o n t i n u e d studies o n linoleate rich diets in this disease. The a p p a r e n t l y low toxicity of even large doses of oils such as sunflower seed oil renders such therapy attractive i n c o m p a r i s o n with other agents, also of uncertain value in MS treatment, b u t involving greater risks. If further clinical trials are held, we suggest, on the basis of results o b t a i n e d in this study that (1) it m a y be worthwhile to m o n i t o r i m m u n o l o g i c a l parameters; (2) careful consideration should be given to whether oleic acid rich supplements are a good c o n t r o l ; (3) deficiency in fat-soluble vitamins, c o n s e q u e n t u p o n ingestion of increased q u a n t i t y of oil, should be guarded against. ACKNOWLEDGEMENTS

We thank Dr. E. A. Caspary for providing the basic protein, and Mr. C. Sowter and colleagues of the Histology Section (Clinical Research Centre) for preparing material for microscopic examination. We are grateful to Sir Peter Medawar and D1. A. J. Edwards for reading and criticising the manuscript. The brain lipid extracts were gas-chromatographed by M. P. Robertson and M. I. Gurr.

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