J. OF IMMUNOPHARMACOLOGY, 2 ( 2 ) , 245-256 (1980)

ASSESSMENT OF TOLERANCE TO IMMUNOSUPPRESSIVE ACTIVITY OF

A’-TETRAHYDROCANNABINOL I N RATS

Yugal K. Lu thra , Henry J . Esber*, Diane M. L a r i v i e r e and H a r r i s Rosenkrantz

Department o f Biochemical Pharmacology and Labora tory o f Immunobiology and Exper imental Oncology

EG&G Mason Research I n s t i t u t e 57 Union S t r e e t

Worcester, Massachusetts 01 608

ABSTRACT

Immunosuppression evoked by Aq- te t rahydrocannab ino l (A9-THC) has been a c o n s i s t e n t f i n d i n g i n r a t s b u t t h e development o f t o le rance t o t h i s phenomenon has n o t been exp lo red . There fore , F i sche r r a t s o f bo th sexes were o r a l l y g i ven A9-THC a t 6 o r 12 mg/kg o r sesame o i l as v e h i c l e c o n t r o l f o r 5-26 days be fo re and a f t e r I . P . a n t i g e n i c s t imu- l a t i o n w i t h sheep r e d b lood c e l l s (SRBC). A9-THC doses were r e l e v a n t t o those o f man and produced m i l d CNS- inh ib i t i on f o l l o w e d by CNS- s t i m u l a t i o n , t o le rance deve lop ing t o bo th behav io ra l phases. The p r imary immune response was eva lua ted by de termin ing s p l e n i c an t ibody- fo rming c e l l s (AFC), hemagg lu t in in (HT) and/or hemolysin ( H S ) t i t e r s . Simultaneous a d m i n i s t r a t i o n o f A9-THC and SE induced dose- re la ted s p l e n i c a t rophy and reduced AFC p r o l i f e r a t i o n as w e l l as HT and HS responses. These changes were n o t e l i c i t e d by sesame o i l . To le rance d i d n o t develop t o imnunosuppression d u r i n g 26 days o f cannabinoid t rea tment . A9-THC g i v e n 3 days p o s t SEBC i n o c u l a t i o n induced immuno- suppression a t 12 b u t n o t 6 mg/kg. Immunosuppression was d i r e c t l y r e l a t e d t o A9-THC r a t h e r than t o non -spec i f i c d e b i l i t a t i n g f a c t o r s s ince body we igh ts a re s t a b l e . The i n d u c t i v e phase o f t h e p r imary immune response was most s e n s i t i v e t o impairment a l t hough t h e rep ro - d u c t i v e phase was a l s o a f f e c t e d a t t h e h i g h dose l e v e l .

245

Copyright 0 1980 by Marcel Dekker, Inc.

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246 LUTHRA ET AL.

INTRODUCTION

Impairment o f both the c e l l mediated and humoral immune pathways

has been observed i n rodents t r e a t e d w i t h A9-tetrahydrocannabinol

(n9-THC) and marihuana smoke (1,2). o f 1-10 mg/kg g i ven p . 0 . o r 0.7-4 mg/kg by the i n h a l a t i o n r o u t e were r e l e v a n t t o heavy, ch ron ic marihuana use by humans as demonstrated by p h y s i o l o g i c responses and

cannabinoid b lood l e v e l s (3-5) . Suppression o f bo th t h e i n d u c t i v e and

p roduc t i ve phases o f t h e pr imary immune response was ev iden t as de te r -

mined by t h e r e d u c t i o n i n t h e number o f v i a b l e s p l e n i c ant ibody forming

c e l l s (AFC), serum ant ibody t i t e r s and changes i n s p l e n i c weights ( 2 ) .

i . p . o r p . 0 has a l s o been demonstrated (6-10). According t o recen t

s tud ies , marihuana and o t h e r cannabinoids have r e p o r t e d l y caused de fec ts

i n e a r l y r o s e t t e fo rma t ion by human T - c e l l s (11-13). Conversely, ab- sence o f o r t r a n s i e n t changes i n the imnune response system have a l s o

been demonstrated (14-17).

The d iscrepancy i n human f i n d i n g s may, i n p a r t , be r e l a t e d t o t h e

development o f t o le rance and t h e p o s s i b l e l o s s o f t o le rance d u r i n g pro-

longed marihuana usage. For example, sub jec ts a b s t a i n i n g f o r one month

(16) may be i n a t o l e r a n t s t a t e .

s i n g l e t reatment r e s u l t s i n r e t e n t i o n o f cannabinoids i n t i s s u e s and

f a t depots f o r a few weeks (18-20) .

t i o n o f t reatment a l s o requ i res severa l weeks t o occur (21-22).

case of c h r o n i c marihuana users (23 ) , excess i ve l y e leva ted cannabinoid

t i s s u e l e v e l s may have a l ready induced a s t a t e o f immunosuppression, t o

which to le rance had as y e t n o t occurred o r had been overwhelmed (24) . The development of t o le rance and i t s l o s s a r e d i r e c t l y r e l a t e d t o can-

nab ino id dosages (5,25-26), a f a c t o r n o t e a s i l y v a l i d a t e d i n vo lun tee r marihuana subjects .

Al though development o f t o le rance t o cannabinoid- induced behav io ra l ,

p h y s i o l o g i c a l and neurochemical parameters has been w e l l es tab l i shed i n

mammals i n c l u d i n g man (15,24-26), no evidence f o r t o l e r a n c e t o t h e i m - munosuppressive a c t i v i t y o f marihuana o r i t s psychoact ive cons t i t uen t , a9-THC, has been demonstrated. Therefore, t h i s comnunicat ion presents r e s u l t s on t h e assessment o f t o le rance t o imnunosuppression induced by prolonged t reatment w i t h A9-THC p r i o r t o a n t i g e n i c chal lenge as w e l l as

t h e e f f e c t o f A9-THC on both t h e i n d u c t i v e and p roduc t i ve phases o f t h e

pr imary immune response.

Doses

Suppression o f AFC by A9-THC and o t h e r cannabinoids i n mice t r e a t e d

It has been demonstrated t h a t even a

The l o s s o f t o l e r a n c e a f t e r cessa-

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TOLERANCE TO IMMUNOSUPPRESSION BY Ag-THC 2 4 .

MATERIAL AND METHOD

Animals :

Inb red F i sche r 344 r a t s (Char les R i v e r Breeding Labora to r ies ,

Wi lmington, Massachusetts) o f bo th sexes, weighing 100-140 g, were

housed 5 per sex i n a po lycarbonate cage o f dimensions 36x31~17 cm. The cages were i n a f a c i l i t y w i t h a 12-hour l i g h t / d a r k c y c l e and am-

b i e n t temperature o f 23°+20C. a v a i l a b l e ad l i b i t u m .

be fo re t rea tment .

Commercial r a t chow and water were

Animals were a l lowed t o a c c l i m a t i z e f o r 5 days

Druq f o r m u l a t i o n :

on Drug Abuse, R o c k v i l l e , Maryland. The compound was s t o r e d a t 5 O C as

a s tock s o l u t i o n o f 300 mg/ml i n sesame o i l and f u r t h e r d i l u t e d w i t h

t h e same v e h i c l e t o o b t a i n 1.8 mg/ml f o r t h e 6 mg/kg dosage and 3.6 mg/ml

f o r t h e 10 o r 12 mg/kg dosage. needle and sy r inge .

Syn the t i c A9-THC (96% pure) was p rov ided by t h e Na t iona l I n s t i t u t e

A9-THC was o r a l l y admin is te red by gavage

Exper imental design:

s t i m u l a t i o n w i t h a 50% suspension of t h r i c e washed sheep r e d b lood (SRBC)

i n i s o t o n i c s a l i n e as l i s t e d i n Tab le 1.

Ora l t rea tment was v a r i e d i n r e l a t i o n s h i p t o t ime o f a n t i g e n i c

Immunological Parameters:

method us ing 0.2% t r y p a n b lue , were used i n t h e l o c a l i z e d hemolysis

i n g e l p laque fo rmat ion assay f o r de te rm in ing AFC number (28-30). Con t ro l s u s i n g SRBC and/or sp leen c e l l s w i t h complement or a combina- t i o n o f SRBC and spleen c e l l s w i t h o u t complement, were determined d a i l y f o r e s t a b l i s h i n g background i n t e r f e r i n g substances. Values ob- t a i n e d f o r t h e c o n t r o l p l a t e s were sub t rac ted f rom t h e exper imenta l

values .

as p r e v i o u s l y descr ibed (30 ) . Mercaptoethanol (ME) s e n s i t i v i t y was a l s o determined by m i x i n g an equal volume o f serum and 0.2M ME and i n c u b a t i n g a t 37OC f o r 30 minutes. t h e i r HT and HS a c t i v i t i e s .

V iab le s p l e n i c lymphocytes, as determined by t h e dye exc lus ion

Hemolysin (HS) and hemagg lu t ina t ion t i t e r (HT) were determined

Treated sera were then t e s t e d f o r

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248 LUTHRA ET AL.

TABLE I EXPERIMENTAL PROTOCOL^

A'-THC Time of

in study in study Monitored (days) Group mg/kg Treatment days SRBCb day Phase' Sacr i f ice

1 2 3

4 5 6

7 8 9

10 11 12

13

14

Vehicle 6

1 2

Vehicle 6 12

Vehicle 6 12

Vehicle 6 12

Vehicle 10

1-5 1-5 1-5

3- 5 3-5 3-5

1-7 1-7 1-7

1-12 1-12 1-12

1-26 1-26

1 1 1

1 1 1

8 8

8

8

8 8

21 21

Inductive

Productive

Inductive

Inductive and

Productive

Inductive and

Productive

5 5 5

5 5 5

13 13 13

13 13 13

26 26

aGroups of animals consis ted o f 8-11 rats/sex/dose.

A 9 - T H C was administered p . 0 . and SRBC i .p .

Phase o f the primary immune response.

Biological Parameters:

f o r assessing gross organ malfunction. da i ly behavioral observations were made. paid t o manifestations of CNS-inhibition, a t a x i a , p ros t ra t ion , hyper- a c t i v i t y , tremors, convulsions o r the "popcorn" response. These be-

A t time o f s a c r i f i c e the spleen, thymus and adrenals were weighed During the experimental period

Par t icu lar a t ten t ion was

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TOLERANCE TO IMMUNOSUPPRESSION BY A'-THC 249

h a v i o r a l parameters were mon i to red so as t o be aware o f t h e e x t e n t of

n e u r o t o x i c i t y and/or development o f t o le rance .

Resu l ts :

A9-THC t rea tment d u r i n g t h e i n d u c t i v e phase o f t h e p r imary immune

response e l i c i t e d sex -spec i f i c changes i n organ we igh ts (Tab le 2 ) . There

was a 25% inc rease f o r spleen i n bo th groups o f t r e a t e d males and a 91%

r i s e i n thymus a t 6 mg/kg male group.

s p l e n i c we igh ts occur red a t t h e h i g h dose and a 23-52% dose- re la ted drop

occur red i n thymus we igh t .

females b u t n o t males.

I n females, a 23% d e c l i n e i n

Adrenal we igh t inc reased 37% among high-dosed

A s i g n i f i c a n t and dose - re la ted decrease i n s p l e n i c AFC was seen i n

bo th males (64-735) and females (49-65%). HT and HS t i t e r s d e c l i n e d

48-60% i n males a t bo th doses and i n high-dose female groups by 42%.

TABLE 2

EFFECT OF A'-THC ON THE INDUCTIVE PHASE OF THE PRIMARY IMMUNE RESPONSE I N FISCHER RATSa

Parameters

(Mean 2 S E )

Organ Wts (mg/l 009 FBW)

Spleen Thymus Adrenal s

Sp len i c AFC/ l o 6 v i a b l e c e l l s

HT

HS

A9-THC, mg/kg/day on days 1-5

b n a l es

( 9 ) ( 9 ) ( 8 ) Cont ro l 6 12

234i16 294~8 ' 293i2OC 109k17 209t15' 23-tl3

23c2 2 4 ~ 0 24+1

214i16 77513' 57i14'

267217 140i25c 108t15'

889+128 5205112 302t49

b females Cont ro l 6 12

(9 ) (9 ) (10)

296+5 295t9 229i9' 221 +6 171i-7 105&

30cl 33+1 41 51

170k13 87+6' 6021 1'

234+17 164212 136_+16'

84021 34 335t50 240550

C C

C

a.

b. F igures i n parentheses a r e number o f animals/dose c . P<O.01 (S tudent t t e s t ) .

Animals were t r e a t e d o r a l l y w i t h A9-THC. on day one and animals s a c r i f i c e d on day f i v e .

SRBC an t i gen was g i ven i . p .

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250 LUTHRA ET AL.

The e f f e c t of a9-THC on the productive phase of the primary im- mune response was i n i t i a t i o n o f a non-dose-related 18-34% increase i n splenic weights and a 21-26% increase i n adrenal weights i n the males of t rea ted groups (Table 3 ) . Female sp len ic weights were increased by about 14% a t e i t h e r dose; thymus weight was u p by 23% a t the low dose and adrenal weights rose by 13-20% a t both dose l e v e l s . high dose, sp len ic AFC decreased by 66% and HT and HS by 50% in males. Corresponding values f o r females were 50% and 36% respect ively.

Antigenic treatment a f t e r cessat ion of treatment with a9-THC in- duced a decrease i n sp len ic weights (11-13%) and an increase i n adrenal weights (29-36%) among t rea ted males (Table 4 ) . Female thymus weights f e l l by 20% and adrenal weights increased by 11% a t the high dose.

A t the

T A B L E 3 EFFECT OF A 9 - T H C ON THE PRODUCTIVE PHASE OF THE

PRIMARY IMMUNE RESPONSE OF FISCHER RATSa

Parameters (Mean t SE)

Organ Wts (mg/100g FBW

Spleen Thymus Adrenal s Splenic AFC/106 Viable Cel ls HT HS

A 9 - T H C , mg/ kg/da, malesb

_______ ( 9 ) ( 9 ) (10) Iontrol 6 12

255i19 343221' 302+8d 153211 18229 143+10

d 1921 23-c 1 24 +1

1861-18 181222 6627'

267217 252k24 13328' 1031+128 5605131 400255

on days 3-5 b females

Control 6 1 2 (10) (10) ( 9 )

257kC 29524' 2G7igd 175210 21627C 77325

3021 34t1 36t lC

172'13 169213 86+6'

267515 259214 1711-17' 1056k117 1029i105 5692103

a.

b. Figures i n parentheses a r e number of animals/dose. c . Pc0.01 (Student t t e s t ) . d . Pc0.05 (Student t t e s t ) .

Animals were t rea ted o r a l l y with A9-THC. on day one and animals sacr i f iced on day f i v e .

SRBC antigen was given i . p . Imm

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TOLERANCE TO IMMUNOSUPPRESSION BY .i9-THC 251

Parameters

(Mean f SE)

Organ Wts (mg/lOOg FBW

Spleen

Thymus

Adrenal s

Splen ic A F C / ~ O ~ V iab le C e l l s

HT

HS

TABLE 4

OF A9-THC TREATMENT OF FISCHER RATSa ASSESSMENT OF THE IMMUNE RESPONSE AFTER CESSATION

A9-THC, mg/kg/daj b males

28729 256+8d 249+5'

137+10 137t7d 135t6

1421 1 9 i l 1850 C

195t28 11 2 ~ 9 ' 11 0214'

656+144 3 2 0 ~ 5 8 340271

i n days 1-7 b females

Contro l 6 12 (10) (10) ( 9 )

29027 26627 2 6 0 ~ 6

194t9 1 9 4 ~ 5 15624'

27+1 2811 30?1

1 4 5 ~ 1 4 91t2' 8 1 ~ 6 '

208t22 144t3' 113?8'

768+119 416+48 302k49

a.

b . c. P<O.O1 (Student t t e s t ) . d. P<0.05 (Student t t e s t ) .

Animals were t r e a t e d o r a l l y w i t h A9-THC. i ,p. on day e i g h t and animals s a c r i f i c e d on day t h i r t e e n . F igures i n parentheses a re number o f animals/dose.

SRBC an t i gen was g i ven

Splenic AFC showed a dose-re la ted decrease o f 54-61%, and HT as w e l l as HS dec l i ned by t h e same e x t e n t (43%) among t r e a t e d males. i n g changes f o r females were 31-36?; f o r t h e va r ious immunologic para- meters.

Correspond-

Tolerance t o t h e immunosuppressive a c t i v i t y o f A9-THC was assessed

by pro longing the t ime o f a d m i n i s t r a t i o n o f A9-THC p r i o r t o a n t i g e n i c s t i m u l a t i o n . Organ weight changes were apparent i n females on ly : 15%

decrease i n s p l e n i c weight a t t h e 6 and 12 mg/kg doses, a 30% drop i n thymus weight a t t h e h igh dose and a non-dose-related increase o f 21-34?; i n adrenals (Table 5 ) . Splen ic AFC f e l l 34% and 63% a t 6 and 12 mg/kg respec t i ve l y , i n males. 46-66% i n males and 48-55% i n females a t both t h e doses tested.

HT and HS decreased i n a dose-re la ted manner

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252 LUTHRA ET AL.

TABLE 5

ASSESSMENT OF TOLERANCE DEVELOPMENT TO THE IMMUNE SUPPRESSIVE ACTIVITY OF A9-THC I N FISCHER RATSa

a9-THC, mg/kg/day on days 1-12 Parameters

(Mean i SE) males"

( 9 ) ( 8 ) ( 9 ) Contro l 6 12

Organ wts. (rng/100g FBW

Spleen

T hymu s

Adrenal s

Splen ic AFCjl O6 Viab le C e l l s

HT

HS

263+19 238k8 260i13

139+14 12817 136+9 1621 2oi 7 231 2

170-+16 113-+2lC 63i1OC

224f23 144+19' 77i1OC

10672107 447k121 260t60

femal es b

Contro l 6 1 2 ( 9 ) (11) (10)

d 293+16 250+!jd 243+9

195+6 177i5 130+13' d 291 1 3 9 + l C 35+2

146+9 E 2 i 8 6218'

192119 100111' 86+9'

978+121 462256 352k52

a.

b. F igures i n parentheses a r e number o f animals/dose. c. P<O.O l (Student t t e s t ) . d. P<0.05 (Student t t e s t ) .

Animals were t r e a t e d o r a l l y w i t h A9-THC. i ,p. on day e i g h t and animals s a c r i f i c e d on day t h i r t e e n .

SRBC an t i gen was g i ven

Pro longa t ion o f t h e A9-THC t reatment f o r 21 days p r i o r t o SRBC

i n j e c t i o n d i d n o t induce a s t a t e o f t o le rance t o the innnunosuppressive

a c t i v i t y o f t h i s compound (Tab le 6).

t h i s p e r i o d revealed t h a t synthes is of t h e 19s ant ibody was predorn-

i n a n t l y a f f e c t e d .

The e f f e c t o f A9-THC t reatment of t he 19s and 7s responses d u r i n g

DISCUSSION This s tudy was the f i r s t probe i n t o p o t e n t i a l t o le rance develop-

ment t o t h e immunosuppressive a c t i o n o f cannabinoids. The imnunosup- p ress i ve a c t i v i t y o f A9-THC on t h e pr imary imnune response o f r a t s t o a chal lenge w i t h sheep e ry th rocy tes was moni tored us ing ant ibody form-

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9 TOLERANCE TO IMMUNOSWPRESSION BY h -THC 253

TABLE 6

SUPPRESSIVE ACTIVITY OF A9-THC I N RATS ASSESSMENT OF TOLERANCE DEVELOPMENT TO THE IgMUNO-

~~

Parameter Contro 1 b Ag-THCb (Mean 2 S.E.) 10 mg/kglday

i n days 1-26

d I Sp len i c we igh t

' Sp len ic kFC/106

(mg/lOOg FBW) 296f11.9 24921 1 .2

v i a b l e c e l l s 189Q7 49+15'

HT 185f21 66+7 HS 980i41 280i18c

a. Resu l ts a re expressed as the average i S.E. on 10 female r a t s pe r group.

b. SRBC were admin is te red i . p . on day 21 and t h e animals were s a c r i f i c e d on day 26.

c . P<O.O1 (Student t t e s t ) . d. PcO.05 (S tudent t t e s t ) .

C

i n g c e l l s and serum hemagg lu t in in and hemolysin t i t e r s as i n d i c a t o r

systems.

an t ibody- fo rming c e l l m u l t i p l i c a t i o n which, i n t u r n , d imin ished hemag-

g l u t i n i n and hemolysin responses.

That A ~ - T H C a d m i n i s t r a t i o n can induce marked immunosuppression was apparent and t h e t i m i n g o f A9-THC a d m i n i s t r a t i o n i n re fe rence t o

a n t i g e n i c s t i m u l a t i o n determined i t s d i r e c t e f f e c t on t h e i n d u c t i v e and p roduc t i ve phases o f t h e humoral response. The imnunosuppression was r e l a t e d t o A9-THC and n o t t o nonspec i f i c d e b i l i t a t i n g f a c t o r s .

D e b i l i t a t i n g pharmacotoxic s igns and body we igh t losses were absent. No to le rance t o t h e d e l e t e r i o u s e f f e c t on t h e imnune response

was developed as a r e s u l t o f p ro long ing t h e a d m i n i s t r a t i o n o f a9-THC

p r i o r t o a n t i g e n i c s t i m u l a t i o n . However, t h e p e r i o d o f t rea tment may have been too sho r t . The i n t e r v a l o f t ime f o r t h e development o f t o le rance i s known t o vary f rom a few days ( a t a x i a ) t o a few weeks ( e x p l o r a t o r y a c t i v i t y , f i g h t i n g aggression) (24,27). I t remains t o

d e f i n e t h e pe r iod o f imnunosuppression assoc ia ted w i t h acu te and

A9-THC induced a non-dose-related s p l e n i c a t rophy and reduced

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254 LUTHRA ET AL.

ch ron ic a d m i n i s t r a t i o n o f A9-THC and t o de termine t h e t ime r e q u i r e d

t o f u l l y recove r f rom immune impairment.

ACKNOWLEDGEMENT

we a r e indebted t o D r . Monique C . Braude o f t h e Na t iona l I n s t i t u t e on Drug Abuse f o r her encouragement.

Th is i n v e s t i g a t i o n was supported by Con t rac t HSM 42-71-79, and

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