Journal of the Neurological Sciences, 1977, 34 53-62 © Elsevier Scientific Pubhshmg Company, Amsterdam - Printed m The Netherlands
53
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 IN T H E G U I N E A PIG Failure to Suppress with Crude Human Chorlonic Gonadotrophln
E A
CASPARY and D HUGHES
MRC Demyehnatmg Diseases Unit, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne NE4 6BE (Great Brttaln)
(Received 1 April, 1977)
SUMMARY A single intraperltoneal dose of 3000 1U of crude human chorlonic gonadotrophin (HCG), of proven in vitro lmmunosuppresslve activity, gave only marginal effect on the clinical and hlstopathological course of experimental allergic encephalomyelitis (EAE) in guinea pigs, which did not reach statistical significance Treatment 7 and 11 days after immunization significantly reduced the Mantoux reaction concomitant with decreased perlvascular inflammation, but with unaltered or (when treated at onset) slightly enhanced clinical disease, with in vitro lymphocyte reactivity increased in accord Much earlier treatment gave the opposite effect of a marginal improvement H C G in vitro immunosuppresslve potency was dependent on its concentration, intensity of immune response and sequence of addition While the action of H C G on EAE does not point to an effective therapy for multiple sclerosis, as suggested by the influence of pregnancy on this disease, n o n - H C G fractions of urine during pregnancy may yet contain highly efficacious lmmunosuppressants
INTRODUCTION The finding that a factor associated with human chorionlc gonadotrophln (HCG) (Caldwell, Stltes and Fudenberg 1975, Morse, Stearns, Arden, Agosto and Canfield 1976, Muchmore and Blaese 1977) has an inhibitory effect on cell-mediated immunity (Han 1975) and its possible role in suppression of maternal lymphocyte reactivity (Adcock, Teasdale, August, Cox, Meschla, Battaglia and Naughton 1973) suggested that crude preparations of the hormone containing this factor as a contaminant may be effective in the treatment of experimental allergic disease or "autoallergic" diseases in man The observation that in multiple sclerosis (MS), where there is a strong indication of immune-mediated damage, relapses occur at a greater rate in
54 the puerperium than m the 9 month~ of pregnancy (Mfllar, Alh~on, Chee~eman ,tnd Merrett 1959, rewewed by Prmeas 1970) also lends support to the thesJ~ that depression of some immune responses m pregnancy may also suppress the d~sease process The present work examines this hypothesis using experimental allergic encephalomyeht~s (EAE) as a model and assesses the chmcal slgns and hJstopathology, Mantou× reaction and maogen- and antigen-reduced lymphoc~yte transformation after treatment with a commercial preparation of H C G used without additional pumficat~on Immunosuppressive potency of the same batch of H C G was assessed by suppression of lymphocyte blastogemc response to phytohaemagglutmm (Contractor and Dawes 1973 Han 1974, Caldwell et al 1975) MATERIALS AND METHODS
EAE This was induced in adult (350-450 g) random bred Hartley guinea pigs with a single lntradermal injection of 0 1 ml into the dorsum of one foot The encephahtogemc mixture was either 25 o/~o(w/v) homologous spinal cord in Freund's complete adjuvant (CFA) (Dlfco-H37Ra) containing 10 mg/ml M tuberculosis or 30/~g of myehn basic protein (MBP) in C F A without additional mycobacterla, Animals were weighed and examined daily and definite neurological signs were scored on a 5-point scale The hlstopathology ofhaematoxyhn and eosln sections of brain and spinal cord was assessed on an arbitrary 3-polnt scale for the brain and spinal cord at the end of the experimental period, the results were expressed as the sum of the maximum score for the brain and cord Seven areas of the brain and transverse sections taken at the level of each spinal root of the cervical, thoracic and lumbar cord were examined and the most severe score for pathological change m brain and cord was used to calculate the pathological index Mantoux tests were carried out by the conventional lntradermal inJection of 0 1 ml of tubercuhn (Weybmdge) at dilutions of 1/ 100 and 1/ 1000 15 days after lmmumzatlon and the maximum diameter of the skin reaction was measured Measurements were converted into grades from + to + + + according to whether they fell within the ranges 5-10, 11-15 or 16-20 m m respectively
Treatment Ammals were treated with a single mtrapemtoneal injection of 3000 I U of H C G either 3 days before, simultaneously with, 7, 10 or I I days after challenge or at the onset of disease One group received 3 dally doses of 1500 I U following the onset of disease The preparation used was chorionic gonadotrophIn BP (Pregnyl, Organon Laboratories L t d , Batch N o E1888/1030) dissolved in water for injection and given m a volume of 1 ml (added substances mono- and all-basic phosphate, mannltol and methyl-p-hydroxybenzoate) Lymphocyte transformatton EA E guinea ptgs The effect of in vitro H C G treatment on lymphocyte reactivity in vitro to PHA, PPD and MBP was tested Blood samples (2-4 ml) were taken 22-27
55 days after immunization by cardiac puncture into 50 IU hthlum heparln The packed cells were washed 3 times in TC 199, with centrlfugatlons at 500 × g (max) for l0 rain at 4 °C, and finally resuspended in RPMI 1640 with HEPES buffer (Glbco-Blocult) at l0 times the original blood volume Master cultures of 1 ml volume were made diluted whole blood equivalent to original undiluted blood at final concentration of 5 °o~, 15 ~ pooled human AB serum (heat-inactivated for 30 mln/56 °C), 1-100 PHA-M (Dlfco) or either PPD or MBP at 25 or 100 #g/ml with stimulant-free controls, made up to volume with RPMI-1640-Hepes Master solutions were transferred to 8 × 12 well mlcroplates (Cooke Mlcrotlter System M25 AR), 150 #1 to each of six 250 #l v-bottom wells and sealed with plastic sheet before humidified incubation at 37 °C After incubation (3 days for P H A , 6 days for PPD and MBP) the cultures were pulse labelled for 7 hr with [3H]thymldlne (5C1/mM TRA-61 - Radlochemlcal Centre, Amersham, Bucks, England), 75 #l of RPMI-1640 containing 6 #C1/ml was added per well giving a final concentration of 2 #Cl/ml (0 45/~C1 per well) After pulsing the cultures were harvested on to glass fibre sheets using a cell harvester (Skatron, Norway) w~th distilled water washing only The dried filter discs were transferred to scintillation vials containing 5 ml of toluene-based sclntlllant (5 0 g 2,5-dlphenyloxazole (PPO), 0 3 g 1,4-bls-2-(5-phenyloxazolyl)-benzlne (POPOP), made up to 1 1 with toluene) Radioactivity was counted in a Trl-Carb hquld scintillation spectrometer (Packard Instruments) with gate settings of A = 50, B = 1,000 and 50°/~, gain Results were expressed as counts/mm with background subtracted The effect of stimulation by PHA, PPD or MBP on thymldlne incorporation was expressed as the numerical dtfference between stimulated and unstlmulated counts in addition to the conventional ratio of these counts or stimulation index (SI) Normalgumea ptgs The direct in vitro effect of H C G on PHA stimulated normal lymphocytes was tested H C G was added 4 hr before (pretreatment), immediately after or 24 hr following addition of PHA at 3 concentrations (10, 100, 500 IU/ml) with HCG-free controls Two PHA concentrations (1/500 and 1/100) were used with PHA-free controls Master cultures were prepared with diluted whole blood equwalent to 15 5 %o original undiluted blood, 1 5 ~ heat inactivated human AB serum, PHA (l/100, 1/500 or none), H C G (500, 100, l0 IU/ml or none) for"pretreatment" and"immediately after" groups with volumes made up to 666 #1 with RPMI-1640 Hepes Six replicates of 100 #1 were set up for each P H A / H C G combination The " H C G 24 hr after P H A " cultures received additional RPMI 1640 Hepes in lieu of H C G and after 24 hr incubation 50 #l of H C G in RPMI 1640-Hepes was added to each well The final volume of 150 #1 per well consequently altered the serum concentration to 1 0 ~ and PHA concentrations to 1/150 and 1/750 Cultures were pulse-labelled as previously to give a final concentration of 2 #C1/ml (50 #l added to 100/~1 cultures and 75 #l to 150 #l cultures gwlng 0 3 and 0 45/zCl per well respectively) Cultures were harvested and counted as before Statistical significance of results was assessed by the Mann-Whltney U test
56 RESULTS I n Vll~O
A p r e h m m a r y experiment using a m m a l s sensltlsed with M B P gave the results shown m Table I There was n o s~gmficant change either m the chmcal seventy of EAE or the pathology of the i n f l a m m a t o r y CNS lesions, t h o u g h the results suggest that late t r e a t m e n t w~th H C G may mcrease chmcal seventy The results o b t a i n e d with a m m a l s challenged wLth h o m o l o g o u s spinal cord and treated with H C G at different times m relation to l m m u m s a t l o n are gwen m Table 2 T r e a t m e n t at day 11 a n d at the onset of the d~sease resulted m e q m v a l e n t chmcal seventy to the untreated g r o u p whde the groups treated 3 days before, simultaneously with a n d 7 days after challenge appeared to have marginally milder chnlcal s y m p t o m s However the &fferences between the &sease scores were not statistically slgmficant O n the other h a n d the pathological appearance of the m f l a m m a t o r y process seemed relatively reduced after t r e a t m e n t with H C G at 7 a n d 11 days m parallel with the M a n toux reaction, these changes also laded to achieve statlstlcal slgmficance The M a n t o u x reaction at 1/100 was reduced m a m m a l s gwen H C G at days 0, 7 a n d 11 tested at 15 days after l m m u n l s a t l o n TABLE 1 EFFECT OF HCG ON EAE 1N GUINEA PIGS CHALLENGED WITH MBP 1N FCA Treatment
Mean chnlcal disease score a
Mean h~stologlcal score a
NIL 28 3000 IU HCG~p at day 10 30 1500 IU HCGIp for 3 days starting at onset of symptoms 33
20 20 18
Derived from groups of 5 ammals - based on highest score recorded m each mdwldual ammal TABLE 2 EFFECT OF HCG ON EAE IN GUINEA PIGS INDUCED WITH HOMOLOGOUS SPINAL CORD IN CFA Treatment
Days after lmmumzat~on
No of guinea pigs
Mean chnlcal score
Mean h~stological score
Mantoux a
NIL 3000 IU 3000 IU 3000 IU 3000 IU 3000 IU
---3 0 7 11 at onset of chmcal signs
19 7 6 12 7
31 24 28 26 33
27 31 27 21 25
-4-++ -t- 6- + ÷ ~~ + -~ J-
11
31
29
NT
HCGIp HCGIp HCGIp HCGIp HCGJp
a Tested at 1/100, 15 days after immunization Mean chmcal and histological scores based on highest score recorded m each mdlv~dual ammal
57 TABLE 3 IN VITRO WHOLE BLOOD LYMPHOCYTE TRANSFORMATION RESPONSES OF HCGTREATED GUINEA PIGS W1TH EAE Group
Treatment a
Number of gumea pigs tested
lncrease in stimulated uptake over control (cpm) and Stimulation Index (SI) PHA 1 100
TubercuhnPPD 25 or 100/~g/ml
mean
range
mean
range
Control
no HCG
4
cpm SI
1394 (733-1989) 142 (8 0-21 4)
28 15
(--13-179) (063-34)
HCG treatment
--3 days
2
simultaneous
1 3
+ 11 days
4
2947 194 1515 61 2131 18 1 4871 103
27 14 4 1I 24 14 396 54
(--33-87) (054-23) --
-t 7 days
cpm S| corn SI cpm SI cpm SI
(1009-4885) (45 34 2) -(1766-2819) (13 6-22 3) (2355-12135) (7 3-13 2)
(12-43) (1 3-1 7) (24-895) (18-156)
T~mmg of treatment (3000 IU of HCG per ammal) m relation to immumzatton
In vitro
H C G - t r e a t e d E A E guinea pigs In vitro t r a n s f o r m a t i o n responses I n VlVO H C G t r e a t m e n t of E A E guinea pigs did n o t suppress the lymphocyte t r a n s f o r m a t i o n reactivity of washed blood lymphocytes either to P H A or P P D The results (Table 3) however show that H C G t r e a t e m e n t at 11 days after i m m u n i z a t i o n did give the opposite effect of a n Increased response to b o t h P H A (P = 0 03) a n d P P D (not significant) with respect to the n u m e r i c a l increase of stimulated over u n s t l m u l a t e d counts which was paralleled by a n Increase in SI to P P D The opposite effect of a reduction in SI to P H A in the presence of increased stimulated counts is due to a rise in u n s t l m u l a t e d counts of untreated animals from a m e a n of 128 cpm (range ~ 66-258) to a m e a n of 552 cpm (range 208-1397) in the 11-day t r e a t m e n t group Responses to M B P were all too low to give any m e a n i n g f u l i n f o r m a t i o n N o r m a l guinea pigs effect of H C G o n P H A induced t r a n s f o r m a t i o n I n contrast to the absence of any m VlVO i n h i b i t o r y action of H C G reported above, direct in vitro a p p h c a t l o n to lymphocytes gave a clear inhibitory effect However, this action vamed with the c o n c e n t r a t i o n of H C G and the P H A stimulus and also the sequence of a d d i t i o n relative to P H A Results in Table 4 have been presented in cpm a n d as a percentage to facilitate c o m p a r i s o n of the varying effects It can be seen that 100 a n d particularly 500 I U / m l of H C G produced I n h i b i t i o n u n d e r all circumstances b u t varied between 59 1-23 9 ~o at 500 1 U / m l a n d 83 8-47 9 % at 100 I U / m l In general, at these c o n c e n t r a t i o n s maxi m u m I n h i b i t i o n was seen where application was delayed There was a tendency for delayed application to be more active on the weaker P H A responses whereas the strong
58 TABLE 4 EFFECT OF HCG ON UNSTIMULATED AND PHA STIMULATED WHOLE BLOOD LYMPHOCYTE TRANSFORMATION Varmt~on w~th concentration and t~rne of apphcahon Timing of HCG
Conc NCG (IU/ml)
[3H]Thymldme uptake Unshmulated
PHA 1 500
PHA 1 100
cpm
°o
cpm
°o
cpm
°o
Nfl 10 100 500
419 353 351 234
100 84 2 83 8 55 8
10122 8945 7795 4715
100 88 4 77 0 46 6
15927 17663 13179 6580
100 110 9 82 7 41 3
Simultaneous Nfl 10 100 500 After 24 hr Nd 10 100 500
456 408 311 197 392 428 312 156
100 89 5 68 2 43 2 100 109 2 79 6 39 8
8527 8839 7041 5042 11258 8660 5399 2697
100 103 7 82 6 59 1 100 76 9 47 9 23 9
17646 17742 13943 5183 17370 13112 11950 6156
100 100 5 78 4 29 4 100 75 5 68 8 35 4
Pretreatment
response was reduced more by s i m u l t a n e o u s H C G t r e a t m e n t especially at 500 I U / m l T r e a t m e n t with only 10 I U / m l H C G gave even more variable results r a n g i n g between mild stimulation a n d todd mhlbltxon U n s t l m u l a t e d cells showed m d d l n h l b m o n ff pretreated, b u t stimulation if t r e a t m e n t was delayed, while the h~gher P H A stimulation gave the reverse situation with m t e r m e & a t e effects at the weaker P H A c o n c e n t r a t i o n I n general at th~s low c o n c e n t r a t i o n of H C G u n s t i m u l a t e d cells r e q m r e d p r e t r e a t m e n t to effect i n h i b i t i o n whereas strongly stimulated cells were affected more by a delayed treatment DISCUSSION O u r results have shown that while a crude h u m a n H C G p r e p a r a t i o n with dem o n s t r a b l e in vitro l m m u n o s u p p r e s s i v e actlwty reduces a delayed hypersensmvity ( M a n t o u x ) reaction the clinical a n d hlstopathologlcal course of E A E m guinea pigs was n o t significantly affected The influence of p r e g n a n c y o n the course of MS has received attention m the past (reviewed by Prmeas 1970) A l t h o u g h the evidence did n o t p o i n t to p r e g n a n c y having an adverse effect on the long term course of MS or its prognosis it appeared that p a r t u r m o n might precipitate a relapse when the disease was active (Mfllar 1961) While the aetlology a n d pathogenesls o f MS remains u n k n o w n it is p r o b a b l e that cellm e & a t e d a u t o - I m m u n e d a m a g e Is involved EAE, where the role o f a u t o - a g g r e s s w e lymphocytes is established, is accepted as a w o r k i n g model for MS a n d m a y provide a fairly close parallel for the m e c h a m s m o f i m m u n e C N S change C r u d e h u m a n H C G p r e p a r a t i o n s have been variously shown to inhibit m vitro
59 lymphocytic responses to P H A (Kaye and Jones 1971, Adcock et al 1973, Contractor and Davies 1973, H a n 1974), allogenelc cells (Bellng and Weksler 1974, Han 1974) and specific antlgen(Han 1974) Moreover H C G t r e a t m e n t ofgulneaplgslmmunlsedtoglve delayed hypersensitivity to tuberculin has shown reduced in vitro lymphocyte responses to mltogen, allogenelc cells and antigen in addition to moderate inhibition of in VlVO reactivity by direct skin testing (Han 1975) Our results on the in VlVO H C G treatment of EAE guinea pigs make it clear that the small influence on EAE does not warrant further study with a view to possible therapy of MS Although our dose of 3000 IU per animal was lower than the 4000 IU used by Han (1975) this was calculated to give a serum level of 170 IU/ml equivalent to the average maximum serum concentration in human pregnancy (Brody and Carlstrom 1965) and in excess of the 30 I U / m l found after the first trimester peak (Kaye and Jones 1971) As in H a n ' s study (1975), actual serum levels of H C G were not measured While our preparation of H C G contained phosphate, and Han (1974, 1975) has stressed the use of phosphate-free H C G , the reported toxicity attributed to phosphate was only "suggested" at the exceedingly high concentration of 10,000 IU/ml (Adcock et al 1973) far in excess of that used in the present study The effect of H C G treatment as seen by changes in the clinical signs of disease was too small to be of statistical significance While treatment given at onset of clinical signs either had no effect or produced a slight worsening, early treatment gave a marginal improvement A similar pattern had been observed in attempts to suppress EAE with oral llnoleic acid (Hughes, Keith, Mertln and Caspary 1977) In contrast small but definite reductions in the delayed skin reactions to PPD were apparent up to 15 days after H C G in all treatment groups except that treated before immunization In addition H C G treatment at 7 and I 1 days after immunization seemed to affect the intensity of the perlvascular Inflammatory reaction Treatment with MBP, either soon after immunization (Alvord, Shaw, Hornby and Kies 1965) or during clinical signs of EAE (Eylar, Jackson, Rothenberg and Brostoff 1972) is known to suppress the disease Consequently skin testing with MBP, although giving a direct measure to any H C G induced reduction of lymphocyte sensitization to the encephalltogenlc antigen, was not attempted In vitro lymphocyte reactivity to P H A and PPD in treated EAE guinea pigs did not suggest in contrast to the findings of Han (1975) an in VlVOinhibitory effect on cell-mediated immunity Thus the reductions in clinical severity, perlvascular cuffing and Mantoux reactions were not paralleled by reduced in vitro lymphocyte reactivity On the contrary, response to both P H A and PPD was increased in animals treated 1 l days after immunization This is not in accord with the small reduction in the inflammatory process and the reduced Mantoux reaction, though in line with the unaltered clinical severity Han 0975) showed reduced P H A and PPD lymphocyte reactivity when measured 1-7 days after H C G treatment However, in order not to influence the course of EAE by stressing the animals our blood sampling was delayed until 11-37 days after H C G administration, which would give a considerably reduced H C G concentration Moreover, whereas the blood cells were washed 3 times prior to culture in our tests Han (1975) simply diluted the blood with synthetic medium and consequently measured lymphocyte reactivity In the presence of circulating H C G As H C G has been
60 shown to exert a d~rect m wtro depressive effect on lymphocyte reactw~ty ~ts presence where autologous serum of a recently H C G reJected ammal is used as part of the culture medmm, could be directly responsible for any depresswe effects found Its presence m the circulation could also &rectly depress skin test reactiwty, where we are m agreement with Han (1975), and points to any lmmunomhlbltory effect of H C G requiring the actual presence of H C G m the test system In addition we have shown that low concentratlons of H C G (10 IU/ml) can gwe increased reactwlty to 1 100 PHA, which may provide an explanation for the increased stimulation at this m~togen concentration found m the present study Ad&tlonal m vitro tests showed that the H C G preparation used had inhibitory potency comparable to that previously reported and was similarly dose-related (Kaye and Jones 1971, Adcock et al 1973, Contractor and Davies 1973, Han 1974) However, the degree of inhibition depended not only on the concentration of HCG, but also on the intensity of the immune response and ad&t~onally upon whether the lymphocytes were exposed to H C G before P H A or vice versa H C G stimulated responses were reported by Adcock et al (1973) and Han (1974) after washing off htghly inhibitory concentrations, a sttuation effectively comparable to continuous low dose exposure and used in the present study At high dilution hormonal growth factors present in the crude H C G preparation might retain st~mulatory actw~ty beyond the active level of the inhibitory component(s) Han's (1975) evidence of H C G inhibition of in VlVOcell-medmted lmmumty hes in a reduction of about one grade (5 mm) m the mean s~ze of the skin test but th~s was not against the background of an ~mmunologlcal disease We achieved an equwalent reduction in skin test responses, but only minimal effects on the EAE &sease process An inhibitory effect sufficient to abohsh the delayed skin reaction would probably be needed to suppress EAE Such suppression of lymphocytic responses has been achieved m vitro using concentrations as high as 5000 IU/ml, near to the actual local concentration at the placenta during pregnancy However, the dose required to elevate circulating H C G to this concentration would be masswe w~th unacceptable side effects The immune response In EAE is enhanced by the use of Freund's complete adjuvant and consequently hkely to be resistant to inhibition by the "non-placental'" H C G concentration used in the present study Some recent investigations (Caldwell et al 1975, Morse et al 1976, Muchmore and Blaese 1977), into the speofic lmmunosuppresswe activity of pure and crude H C G has shown clearly that while most crude preparations have considerable inhibitory capability this was lost on purification However the demonstratmn (Morse et al 1976, Muchmore and Blaese 1977) that the actual lmmuno-suppresswe activity can be concentrated in a n o n - H C G fraction points to the existence of an unidentified lmmunosuppresslve agent recoverable from urine in high concentration during pregnancy Its purification could prowde a natural and physiologically acceptable lmmunosuppressant free from the undesirable side effects of crude H C G preparations H C G is not the only immunosuppresslve factor during pregnancy Progesterone has such activity (Mort, Kobayashi, Nlshlmura, Morn, Fujn and Inov 1975) and a whole complex of non-hormonal humoral lmmunosuppresswe factors (Cooperband, Nlmberg, Schmld
61 and Manntck 1976) may also be elevated during pregnancy (Kasakura 1971, Stlmson and Blackstock 1975) in connection with tolerance to the foetus with sudden depression at parturition Arnason and Richman (1969) have shown suppression of EAE with ethlnyl oestradlol and 3 different oral contraceptive preparations, though a group treated with one of the latter developed htstologlcal signs after a longer period Medroxy progesterone potentiated the disease The occurrence of any natural lmmunosuppressant, occurring at higher concentrations during pregnancy, able to hold back exacerbations of EAE, and by analogy the less active cell-mediated responses m MS also, would be worthy of further study ACKNOWLEDGEMENTS
We wish to thank Mr A B Kelth, F I A T , Mrs Joyce Woodburn and Miss Judith Home for thetr invaluable technical assistance
REFERENCES Adcock, E W , F Teasdale, C S August, S Cox, G Meschla, F C Battagha and M A Naughton (1973) H u m a n chonomc gonadotrophm - - Its possible role m maternal lymphocyte suppression, Scwnce, 811 845-847 Alvord, E C , C -M Shaw, S Hruby and M W Kles (1965) Encephahtogen-mduced inhibition of experimental allergxc encephalomyehtls - - Preventxon, suppression and therapy, Ann N Y Acad Sct , 122 333-345 Arnason, B G a n d D P Rlchman (1969) Effect of oral contraceptwes on experlmental demyehnatmg disease, Arch Neurol (Chtc ), 21 103-108 Behng, C G and M E Weksler (1974) Suppresszon of mixed lympocyte reactwlty by human chorlomc gonadotrophm, Chn exp hnmunol , 18 537-541 Brody, S and G Carlstrom (1965) In G E W Wolstenholme and J Knight (Eds), Gonadotrophms - - Physwal and Immunologtcal Propertws (Clba Foundataon Study Group, No 22), Churchill, London, p 70 Caldwell, J L , D P Stltes and H H Fudenberg (1975) Human chonomc gonadotrophm - - Effects of crude and purified preparations on lymphocyte responses to phytohaemagglutmm and allogenelc stimulation, J Immunol, 115 1249-1253 Contractor, S F and H Davies (1973) Effect of human chonomc somatomammotrophm and human chonomc gonadotrophm on phytohaemagglutmm induced lymphocyte transformation, Nature New Btol, 243 284-286 Cooperband, S R , R Nimberg, K Schmld and J A Manmck (1976)Humoral lmmunosuppresslve factors, Transplant Proe, 8 225-242 Eylar, E H , J Jackson, B Rothenberg and S W Brostoff(1972) Suppression of the immune response Reversal of the d~sease state with antlgen m allergic encephalomyehtls, Nature (Lond), 236 74-76 Han, T (i 974) Inhlb~tor~ effect of human chorlonlc gonadotrophm onlymphocyte blastogemcresponse to m~togen, antigen, and allogene~c cells, Chn exp lmmunol, 18 529-535 Han, T (1975) Human chonomc gonadotrophm - - Its mhlbltory effect on cell medmted lmmumty m wvo and m vitro, hnmunology, 29 509-515 Hughes, D , A B Ke~th, J Mertm and E A Caspary, In preparation Kasakura, S (1971) A factor m maternal plasma during pregnancy that suppresses the reactxwty of mixed leukocyte culture, J lmmunol, 107 1296-1301 Kaye, M D a n d W R J o n e s ( 1 9 7 1 ) E f f e c t s o f h u m a n c h o r l o m c g o n a d o t r o p h m o n m w t r o l y m p h o c y t e transformation, .4rner J Obstet Gynec, 109 1029-1031 Mdlar, J H D (1961)The influence of pregnancy on dlssemmatedsclerosls, Proe roy Soc M e d , 54 4-7 -
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62 Mfllar, J H D , R S Allison, E A Cheeseman and J D Merrett (1959I Pregnancy as ,i lactol i1~ fluencmg relapse in multiple sclerosis, Brain, 82 417-426 Morl, T , H Kobayashl, T Nlshlmura, T S MorJ, G Fujn and T Inov (1975) Inhibitory effect ol progesterone on the phytohaemagglutmln-mduced transformation of human peripheral [ynlphocytes, lmmunol Commun, 4 519-527 Morse, J H , G Stearns, J Alden, G M Agosto and R E Canfield (1976) The effects ot crude and purified human gonadotrophm on m vitro stimulated human lymphocyte cultures, CellulaJ bnmunologv, 25 178 188 Muchmore, A V and R M Blaese (1977) Immunoregulatory properties of fractions from human pregnancy urine - - Ewdence that human chor~onlc gonadotrophm ts not responsible, J Immunol, 118 881-886 Prmeas, J W (1970) The etiology and pathogenesls of multiple sclerosis In P J Fmken and G W Bruyn (Eds), Handboo~ of Chmeal Neurology, Vol 9 (Multiple Sclerost~ and Other Det~oehnatmg Diseases), North-Holland, Amsterdam, pp 107-160 Stlmson, W H and J C Blackstock (1975) The effects of h u m a n pregnancy serum on the binding of sheep erythrocytes to human lymphocytes, Behrmg lnst Mitt, No 57, pp 92-97
53
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 IN T H E G U I N E A PIG Failure to Suppress with Crude Human Chorlonic Gonadotrophln
E A
CASPARY and D HUGHES
MRC Demyehnatmg Diseases Unit, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne NE4 6BE (Great Brttaln)
(Received 1 April, 1977)
SUMMARY A single intraperltoneal dose of 3000 1U of crude human chorlonic gonadotrophin (HCG), of proven in vitro lmmunosuppresslve activity, gave only marginal effect on the clinical and hlstopathological course of experimental allergic encephalomyelitis (EAE) in guinea pigs, which did not reach statistical significance Treatment 7 and 11 days after immunization significantly reduced the Mantoux reaction concomitant with decreased perlvascular inflammation, but with unaltered or (when treated at onset) slightly enhanced clinical disease, with in vitro lymphocyte reactivity increased in accord Much earlier treatment gave the opposite effect of a marginal improvement H C G in vitro immunosuppresslve potency was dependent on its concentration, intensity of immune response and sequence of addition While the action of H C G on EAE does not point to an effective therapy for multiple sclerosis, as suggested by the influence of pregnancy on this disease, n o n - H C G fractions of urine during pregnancy may yet contain highly efficacious lmmunosuppressants
INTRODUCTION The finding that a factor associated with human chorionlc gonadotrophln (HCG) (Caldwell, Stltes and Fudenberg 1975, Morse, Stearns, Arden, Agosto and Canfield 1976, Muchmore and Blaese 1977) has an inhibitory effect on cell-mediated immunity (Han 1975) and its possible role in suppression of maternal lymphocyte reactivity (Adcock, Teasdale, August, Cox, Meschla, Battaglia and Naughton 1973) suggested that crude preparations of the hormone containing this factor as a contaminant may be effective in the treatment of experimental allergic disease or "autoallergic" diseases in man The observation that in multiple sclerosis (MS), where there is a strong indication of immune-mediated damage, relapses occur at a greater rate in
54 the puerperium than m the 9 month~ of pregnancy (Mfllar, Alh~on, Chee~eman ,tnd Merrett 1959, rewewed by Prmeas 1970) also lends support to the thesJ~ that depression of some immune responses m pregnancy may also suppress the d~sease process The present work examines this hypothesis using experimental allergic encephalomyeht~s (EAE) as a model and assesses the chmcal slgns and hJstopathology, Mantou× reaction and maogen- and antigen-reduced lymphoc~yte transformation after treatment with a commercial preparation of H C G used without additional pumficat~on Immunosuppressive potency of the same batch of H C G was assessed by suppression of lymphocyte blastogemc response to phytohaemagglutmm (Contractor and Dawes 1973 Han 1974, Caldwell et al 1975) MATERIALS AND METHODS
EAE This was induced in adult (350-450 g) random bred Hartley guinea pigs with a single lntradermal injection of 0 1 ml into the dorsum of one foot The encephahtogemc mixture was either 25 o/~o(w/v) homologous spinal cord in Freund's complete adjuvant (CFA) (Dlfco-H37Ra) containing 10 mg/ml M tuberculosis or 30/~g of myehn basic protein (MBP) in C F A without additional mycobacterla, Animals were weighed and examined daily and definite neurological signs were scored on a 5-point scale The hlstopathology ofhaematoxyhn and eosln sections of brain and spinal cord was assessed on an arbitrary 3-polnt scale for the brain and spinal cord at the end of the experimental period, the results were expressed as the sum of the maximum score for the brain and cord Seven areas of the brain and transverse sections taken at the level of each spinal root of the cervical, thoracic and lumbar cord were examined and the most severe score for pathological change m brain and cord was used to calculate the pathological index Mantoux tests were carried out by the conventional lntradermal inJection of 0 1 ml of tubercuhn (Weybmdge) at dilutions of 1/ 100 and 1/ 1000 15 days after lmmumzatlon and the maximum diameter of the skin reaction was measured Measurements were converted into grades from + to + + + according to whether they fell within the ranges 5-10, 11-15 or 16-20 m m respectively
Treatment Ammals were treated with a single mtrapemtoneal injection of 3000 I U of H C G either 3 days before, simultaneously with, 7, 10 or I I days after challenge or at the onset of disease One group received 3 dally doses of 1500 I U following the onset of disease The preparation used was chorionic gonadotrophIn BP (Pregnyl, Organon Laboratories L t d , Batch N o E1888/1030) dissolved in water for injection and given m a volume of 1 ml (added substances mono- and all-basic phosphate, mannltol and methyl-p-hydroxybenzoate) Lymphocyte transformatton EA E guinea ptgs The effect of in vitro H C G treatment on lymphocyte reactivity in vitro to PHA, PPD and MBP was tested Blood samples (2-4 ml) were taken 22-27
55 days after immunization by cardiac puncture into 50 IU hthlum heparln The packed cells were washed 3 times in TC 199, with centrlfugatlons at 500 × g (max) for l0 rain at 4 °C, and finally resuspended in RPMI 1640 with HEPES buffer (Glbco-Blocult) at l0 times the original blood volume Master cultures of 1 ml volume were made diluted whole blood equivalent to original undiluted blood at final concentration of 5 °o~, 15 ~ pooled human AB serum (heat-inactivated for 30 mln/56 °C), 1-100 PHA-M (Dlfco) or either PPD or MBP at 25 or 100 #g/ml with stimulant-free controls, made up to volume with RPMI-1640-Hepes Master solutions were transferred to 8 × 12 well mlcroplates (Cooke Mlcrotlter System M25 AR), 150 #1 to each of six 250 #l v-bottom wells and sealed with plastic sheet before humidified incubation at 37 °C After incubation (3 days for P H A , 6 days for PPD and MBP) the cultures were pulse labelled for 7 hr with [3H]thymldlne (5C1/mM TRA-61 - Radlochemlcal Centre, Amersham, Bucks, England), 75 #l of RPMI-1640 containing 6 #C1/ml was added per well giving a final concentration of 2 #Cl/ml (0 45/~C1 per well) After pulsing the cultures were harvested on to glass fibre sheets using a cell harvester (Skatron, Norway) w~th distilled water washing only The dried filter discs were transferred to scintillation vials containing 5 ml of toluene-based sclntlllant (5 0 g 2,5-dlphenyloxazole (PPO), 0 3 g 1,4-bls-2-(5-phenyloxazolyl)-benzlne (POPOP), made up to 1 1 with toluene) Radioactivity was counted in a Trl-Carb hquld scintillation spectrometer (Packard Instruments) with gate settings of A = 50, B = 1,000 and 50°/~, gain Results were expressed as counts/mm with background subtracted The effect of stimulation by PHA, PPD or MBP on thymldlne incorporation was expressed as the numerical dtfference between stimulated and unstlmulated counts in addition to the conventional ratio of these counts or stimulation index (SI) Normalgumea ptgs The direct in vitro effect of H C G on PHA stimulated normal lymphocytes was tested H C G was added 4 hr before (pretreatment), immediately after or 24 hr following addition of PHA at 3 concentrations (10, 100, 500 IU/ml) with HCG-free controls Two PHA concentrations (1/500 and 1/100) were used with PHA-free controls Master cultures were prepared with diluted whole blood equwalent to 15 5 %o original undiluted blood, 1 5 ~ heat inactivated human AB serum, PHA (l/100, 1/500 or none), H C G (500, 100, l0 IU/ml or none) for"pretreatment" and"immediately after" groups with volumes made up to 666 #1 with RPMI-1640 Hepes Six replicates of 100 #1 were set up for each P H A / H C G combination The " H C G 24 hr after P H A " cultures received additional RPMI 1640 Hepes in lieu of H C G and after 24 hr incubation 50 #l of H C G in RPMI 1640-Hepes was added to each well The final volume of 150 #1 per well consequently altered the serum concentration to 1 0 ~ and PHA concentrations to 1/150 and 1/750 Cultures were pulse-labelled as previously to give a final concentration of 2 #C1/ml (50 #l added to 100/~1 cultures and 75 #l to 150 #l cultures gwlng 0 3 and 0 45/zCl per well respectively) Cultures were harvested and counted as before Statistical significance of results was assessed by the Mann-Whltney U test
56 RESULTS I n Vll~O
A p r e h m m a r y experiment using a m m a l s sensltlsed with M B P gave the results shown m Table I There was n o s~gmficant change either m the chmcal seventy of EAE or the pathology of the i n f l a m m a t o r y CNS lesions, t h o u g h the results suggest that late t r e a t m e n t w~th H C G may mcrease chmcal seventy The results o b t a i n e d with a m m a l s challenged wLth h o m o l o g o u s spinal cord and treated with H C G at different times m relation to l m m u m s a t l o n are gwen m Table 2 T r e a t m e n t at day 11 a n d at the onset of the d~sease resulted m e q m v a l e n t chmcal seventy to the untreated g r o u p whde the groups treated 3 days before, simultaneously with a n d 7 days after challenge appeared to have marginally milder chnlcal s y m p t o m s However the &fferences between the &sease scores were not statistically slgmficant O n the other h a n d the pathological appearance of the m f l a m m a t o r y process seemed relatively reduced after t r e a t m e n t with H C G at 7 a n d 11 days m parallel with the M a n toux reaction, these changes also laded to achieve statlstlcal slgmficance The M a n t o u x reaction at 1/100 was reduced m a m m a l s gwen H C G at days 0, 7 a n d 11 tested at 15 days after l m m u n l s a t l o n TABLE 1 EFFECT OF HCG ON EAE 1N GUINEA PIGS CHALLENGED WITH MBP 1N FCA Treatment
Mean chnlcal disease score a
Mean h~stologlcal score a
NIL 28 3000 IU HCG~p at day 10 30 1500 IU HCGIp for 3 days starting at onset of symptoms 33
20 20 18
Derived from groups of 5 ammals - based on highest score recorded m each mdwldual ammal TABLE 2 EFFECT OF HCG ON EAE IN GUINEA PIGS INDUCED WITH HOMOLOGOUS SPINAL CORD IN CFA Treatment
Days after lmmumzat~on
No of guinea pigs
Mean chnlcal score
Mean h~stological score
Mantoux a
NIL 3000 IU 3000 IU 3000 IU 3000 IU 3000 IU
---3 0 7 11 at onset of chmcal signs
19 7 6 12 7
31 24 28 26 33
27 31 27 21 25
-4-++ -t- 6- + ÷ ~~ + -~ J-
11
31
29
NT
HCGIp HCGIp HCGIp HCGIp HCGJp
a Tested at 1/100, 15 days after immunization Mean chmcal and histological scores based on highest score recorded m each mdlv~dual ammal
57 TABLE 3 IN VITRO WHOLE BLOOD LYMPHOCYTE TRANSFORMATION RESPONSES OF HCGTREATED GUINEA PIGS W1TH EAE Group
Treatment a
Number of gumea pigs tested
lncrease in stimulated uptake over control (cpm) and Stimulation Index (SI) PHA 1 100
TubercuhnPPD 25 or 100/~g/ml
mean
range
mean
range
Control
no HCG
4
cpm SI
1394 (733-1989) 142 (8 0-21 4)
28 15
(--13-179) (063-34)
HCG treatment
--3 days
2
simultaneous
1 3
+ 11 days
4
2947 194 1515 61 2131 18 1 4871 103
27 14 4 1I 24 14 396 54
(--33-87) (054-23) --
-t 7 days
cpm S| corn SI cpm SI cpm SI
(1009-4885) (45 34 2) -(1766-2819) (13 6-22 3) (2355-12135) (7 3-13 2)
(12-43) (1 3-1 7) (24-895) (18-156)
T~mmg of treatment (3000 IU of HCG per ammal) m relation to immumzatton
In vitro
H C G - t r e a t e d E A E guinea pigs In vitro t r a n s f o r m a t i o n responses I n VlVO H C G t r e a t m e n t of E A E guinea pigs did n o t suppress the lymphocyte t r a n s f o r m a t i o n reactivity of washed blood lymphocytes either to P H A or P P D The results (Table 3) however show that H C G t r e a t e m e n t at 11 days after i m m u n i z a t i o n did give the opposite effect of a n Increased response to b o t h P H A (P = 0 03) a n d P P D (not significant) with respect to the n u m e r i c a l increase of stimulated over u n s t l m u l a t e d counts which was paralleled by a n Increase in SI to P P D The opposite effect of a reduction in SI to P H A in the presence of increased stimulated counts is due to a rise in u n s t l m u l a t e d counts of untreated animals from a m e a n of 128 cpm (range ~ 66-258) to a m e a n of 552 cpm (range 208-1397) in the 11-day t r e a t m e n t group Responses to M B P were all too low to give any m e a n i n g f u l i n f o r m a t i o n N o r m a l guinea pigs effect of H C G o n P H A induced t r a n s f o r m a t i o n I n contrast to the absence of any m VlVO i n h i b i t o r y action of H C G reported above, direct in vitro a p p h c a t l o n to lymphocytes gave a clear inhibitory effect However, this action vamed with the c o n c e n t r a t i o n of H C G and the P H A stimulus and also the sequence of a d d i t i o n relative to P H A Results in Table 4 have been presented in cpm a n d as a percentage to facilitate c o m p a r i s o n of the varying effects It can be seen that 100 a n d particularly 500 I U / m l of H C G produced I n h i b i t i o n u n d e r all circumstances b u t varied between 59 1-23 9 ~o at 500 1 U / m l a n d 83 8-47 9 % at 100 I U / m l In general, at these c o n c e n t r a t i o n s maxi m u m I n h i b i t i o n was seen where application was delayed There was a tendency for delayed application to be more active on the weaker P H A responses whereas the strong
58 TABLE 4 EFFECT OF HCG ON UNSTIMULATED AND PHA STIMULATED WHOLE BLOOD LYMPHOCYTE TRANSFORMATION Varmt~on w~th concentration and t~rne of apphcahon Timing of HCG
Conc NCG (IU/ml)
[3H]Thymldme uptake Unshmulated
PHA 1 500
PHA 1 100
cpm
°o
cpm
°o
cpm
°o
Nfl 10 100 500
419 353 351 234
100 84 2 83 8 55 8
10122 8945 7795 4715
100 88 4 77 0 46 6
15927 17663 13179 6580
100 110 9 82 7 41 3
Simultaneous Nfl 10 100 500 After 24 hr Nd 10 100 500
456 408 311 197 392 428 312 156
100 89 5 68 2 43 2 100 109 2 79 6 39 8
8527 8839 7041 5042 11258 8660 5399 2697
100 103 7 82 6 59 1 100 76 9 47 9 23 9
17646 17742 13943 5183 17370 13112 11950 6156
100 100 5 78 4 29 4 100 75 5 68 8 35 4
Pretreatment
response was reduced more by s i m u l t a n e o u s H C G t r e a t m e n t especially at 500 I U / m l T r e a t m e n t with only 10 I U / m l H C G gave even more variable results r a n g i n g between mild stimulation a n d todd mhlbltxon U n s t l m u l a t e d cells showed m d d l n h l b m o n ff pretreated, b u t stimulation if t r e a t m e n t was delayed, while the h~gher P H A stimulation gave the reverse situation with m t e r m e & a t e effects at the weaker P H A c o n c e n t r a t i o n I n general at th~s low c o n c e n t r a t i o n of H C G u n s t i m u l a t e d cells r e q m r e d p r e t r e a t m e n t to effect i n h i b i t i o n whereas strongly stimulated cells were affected more by a delayed treatment DISCUSSION O u r results have shown that while a crude h u m a n H C G p r e p a r a t i o n with dem o n s t r a b l e in vitro l m m u n o s u p p r e s s i v e actlwty reduces a delayed hypersensmvity ( M a n t o u x ) reaction the clinical a n d hlstopathologlcal course of E A E m guinea pigs was n o t significantly affected The influence of p r e g n a n c y o n the course of MS has received attention m the past (reviewed by Prmeas 1970) A l t h o u g h the evidence did n o t p o i n t to p r e g n a n c y having an adverse effect on the long term course of MS or its prognosis it appeared that p a r t u r m o n might precipitate a relapse when the disease was active (Mfllar 1961) While the aetlology a n d pathogenesls o f MS remains u n k n o w n it is p r o b a b l e that cellm e & a t e d a u t o - I m m u n e d a m a g e Is involved EAE, where the role o f a u t o - a g g r e s s w e lymphocytes is established, is accepted as a w o r k i n g model for MS a n d m a y provide a fairly close parallel for the m e c h a m s m o f i m m u n e C N S change C r u d e h u m a n H C G p r e p a r a t i o n s have been variously shown to inhibit m vitro
59 lymphocytic responses to P H A (Kaye and Jones 1971, Adcock et al 1973, Contractor and Davies 1973, H a n 1974), allogenelc cells (Bellng and Weksler 1974, Han 1974) and specific antlgen(Han 1974) Moreover H C G t r e a t m e n t ofgulneaplgslmmunlsedtoglve delayed hypersensitivity to tuberculin has shown reduced in vitro lymphocyte responses to mltogen, allogenelc cells and antigen in addition to moderate inhibition of in VlVO reactivity by direct skin testing (Han 1975) Our results on the in VlVO H C G treatment of EAE guinea pigs make it clear that the small influence on EAE does not warrant further study with a view to possible therapy of MS Although our dose of 3000 IU per animal was lower than the 4000 IU used by Han (1975) this was calculated to give a serum level of 170 IU/ml equivalent to the average maximum serum concentration in human pregnancy (Brody and Carlstrom 1965) and in excess of the 30 I U / m l found after the first trimester peak (Kaye and Jones 1971) As in H a n ' s study (1975), actual serum levels of H C G were not measured While our preparation of H C G contained phosphate, and Han (1974, 1975) has stressed the use of phosphate-free H C G , the reported toxicity attributed to phosphate was only "suggested" at the exceedingly high concentration of 10,000 IU/ml (Adcock et al 1973) far in excess of that used in the present study The effect of H C G treatment as seen by changes in the clinical signs of disease was too small to be of statistical significance While treatment given at onset of clinical signs either had no effect or produced a slight worsening, early treatment gave a marginal improvement A similar pattern had been observed in attempts to suppress EAE with oral llnoleic acid (Hughes, Keith, Mertln and Caspary 1977) In contrast small but definite reductions in the delayed skin reactions to PPD were apparent up to 15 days after H C G in all treatment groups except that treated before immunization In addition H C G treatment at 7 and I 1 days after immunization seemed to affect the intensity of the perlvascular Inflammatory reaction Treatment with MBP, either soon after immunization (Alvord, Shaw, Hornby and Kies 1965) or during clinical signs of EAE (Eylar, Jackson, Rothenberg and Brostoff 1972) is known to suppress the disease Consequently skin testing with MBP, although giving a direct measure to any H C G induced reduction of lymphocyte sensitization to the encephalltogenlc antigen, was not attempted In vitro lymphocyte reactivity to P H A and PPD in treated EAE guinea pigs did not suggest in contrast to the findings of Han (1975) an in VlVOinhibitory effect on cell-mediated immunity Thus the reductions in clinical severity, perlvascular cuffing and Mantoux reactions were not paralleled by reduced in vitro lymphocyte reactivity On the contrary, response to both P H A and PPD was increased in animals treated 1 l days after immunization This is not in accord with the small reduction in the inflammatory process and the reduced Mantoux reaction, though in line with the unaltered clinical severity Han 0975) showed reduced P H A and PPD lymphocyte reactivity when measured 1-7 days after H C G treatment However, in order not to influence the course of EAE by stressing the animals our blood sampling was delayed until 11-37 days after H C G administration, which would give a considerably reduced H C G concentration Moreover, whereas the blood cells were washed 3 times prior to culture in our tests Han (1975) simply diluted the blood with synthetic medium and consequently measured lymphocyte reactivity In the presence of circulating H C G As H C G has been
60 shown to exert a d~rect m wtro depressive effect on lymphocyte reactw~ty ~ts presence where autologous serum of a recently H C G reJected ammal is used as part of the culture medmm, could be directly responsible for any depresswe effects found Its presence m the circulation could also &rectly depress skin test reactiwty, where we are m agreement with Han (1975), and points to any lmmunomhlbltory effect of H C G requiring the actual presence of H C G m the test system In addition we have shown that low concentratlons of H C G (10 IU/ml) can gwe increased reactwlty to 1 100 PHA, which may provide an explanation for the increased stimulation at this m~togen concentration found m the present study Ad&tlonal m vitro tests showed that the H C G preparation used had inhibitory potency comparable to that previously reported and was similarly dose-related (Kaye and Jones 1971, Adcock et al 1973, Contractor and Davies 1973, Han 1974) However, the degree of inhibition depended not only on the concentration of HCG, but also on the intensity of the immune response and ad&t~onally upon whether the lymphocytes were exposed to H C G before P H A or vice versa H C G stimulated responses were reported by Adcock et al (1973) and Han (1974) after washing off htghly inhibitory concentrations, a sttuation effectively comparable to continuous low dose exposure and used in the present study At high dilution hormonal growth factors present in the crude H C G preparation might retain st~mulatory actw~ty beyond the active level of the inhibitory component(s) Han's (1975) evidence of H C G inhibition of in VlVOcell-medmted lmmumty hes in a reduction of about one grade (5 mm) m the mean s~ze of the skin test but th~s was not against the background of an ~mmunologlcal disease We achieved an equwalent reduction in skin test responses, but only minimal effects on the EAE &sease process An inhibitory effect sufficient to abohsh the delayed skin reaction would probably be needed to suppress EAE Such suppression of lymphocytic responses has been achieved m vitro using concentrations as high as 5000 IU/ml, near to the actual local concentration at the placenta during pregnancy However, the dose required to elevate circulating H C G to this concentration would be masswe w~th unacceptable side effects The immune response In EAE is enhanced by the use of Freund's complete adjuvant and consequently hkely to be resistant to inhibition by the "non-placental'" H C G concentration used in the present study Some recent investigations (Caldwell et al 1975, Morse et al 1976, Muchmore and Blaese 1977), into the speofic lmmunosuppresswe activity of pure and crude H C G has shown clearly that while most crude preparations have considerable inhibitory capability this was lost on purification However the demonstratmn (Morse et al 1976, Muchmore and Blaese 1977) that the actual lmmuno-suppresswe activity can be concentrated in a n o n - H C G fraction points to the existence of an unidentified lmmunosuppresslve agent recoverable from urine in high concentration during pregnancy Its purification could prowde a natural and physiologically acceptable lmmunosuppressant free from the undesirable side effects of crude H C G preparations H C G is not the only immunosuppresslve factor during pregnancy Progesterone has such activity (Mort, Kobayashi, Nlshlmura, Morn, Fujn and Inov 1975) and a whole complex of non-hormonal humoral lmmunosuppresswe factors (Cooperband, Nlmberg, Schmld
61 and Manntck 1976) may also be elevated during pregnancy (Kasakura 1971, Stlmson and Blackstock 1975) in connection with tolerance to the foetus with sudden depression at parturition Arnason and Richman (1969) have shown suppression of EAE with ethlnyl oestradlol and 3 different oral contraceptive preparations, though a group treated with one of the latter developed htstologlcal signs after a longer period Medroxy progesterone potentiated the disease The occurrence of any natural lmmunosuppressant, occurring at higher concentrations during pregnancy, able to hold back exacerbations of EAE, and by analogy the less active cell-mediated responses m MS also, would be worthy of further study ACKNOWLEDGEMENTS
We wish to thank Mr A B Kelth, F I A T , Mrs Joyce Woodburn and Miss Judith Home for thetr invaluable technical assistance
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