Lundeberg 91 Neuroimmunomodulatory Effects of Acupuncture in Mice

8/11/2019 Lundeberg 91 Neuroimmunomodulatory Effects of Acupuncture in Mice

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Neuroscience Letters, 128 (1991) 161-164 1991 Elsevier Scientific Publ ishers Irel and Ltd. 0304-3940/91/ 03.50A DON1S 0304394091003498

NSL 07864

161

euro im m uno m odu latory effects of acupuncture in m ice

T. L u n d e b e rg 1 S . V.E r i k s s o n 2 a n d E . T h e o d o r s s o n 31Department of Physiology 11, Karolinska lnstitutet, Stockholm Sweden), Z Department of Medicine, Danderyds Hospital, Danderyd Sweden) and

3Department of Clinical Chemistry, Karolinska Hospital, Stockholm Sweden)

(Received 2 Novem ber 1990; Revised version received 10 April 1991; Accept ed 12 April 199 I)

Key words: Acupuncture; Immune response

The purpose of this study was to assess the effect of acupuncture on the immunological response. The induction of anti-sheep red blood cells(SRBC) plaque-forming cells (PFC) was used as a measurement o f the immune response to treatment. In normal no n-immunized mice, enhancem entof PFC was seen after a single acupuncture treatment when spleen cells from sti mulated mice were cultured with SRBC in vitro. Aft er 3 acupuncture

treatments, spleen cells from mice did not show PFC enhancement after treatment with anti-Thy-1.2 antibody an d complement, nor aft er the removalof non-adh erent cells. Serum obtained from mice I h after acupuncture stimulation enhanced the PFC of normal spleen cells in vitro, but the enhance-ment was abolished by the addition of propranolol. These results suggest that acupuncture, by activation of the autonomic nervous system,modulates the immune response.

Recen t r epor t s have sugges ted tha t the immune reac -t ion is regulated by the centra l nervous system [4, 8] .This is in teres t ing to note , as exper imental s tudies havea l so shown tha t acupun c tu re s t imula t ion a ffec ts the ac -t ivi ty of several systems within the brain and induces there lease o f endo genou s op io ids [11].

O p i a t e s a n d e n d o g e n o u s o p i o i d s h av e b e e n s h o w n t ohave immuno-compromising effects [2 , 3 , 7] . Fur ther,o the r s tud ies have shown tha t e ry th rocy tes , monocy tes ,g ranu locy tes and lymphocy tes a l l have spec i f i c op ia tebinding s i tes [1 , 14] . These f indings, and the fact th atacupu nc tu re h as been t r ied in the t rea tment o f pa ti en t swi th imm uno-def ic iency d i seases , induced us to s tudy thee ffec ts o f acupunc tu re on the imm une responses [17].

Acupunc tu re t r ea tment was ca r r i ed ou t fo r pe r iods o f

30 min . S ix po in t s were chosen ( J ingmen G25; Zhaoha iK6; Dazhu Bl l , b i l a t e ra l ly ) . Th i r ty male mice , 6 -10weeks o ld , were ass igned to 3 g roups wi th 10 mice ineach, for each of the t reatm ent categories .

G rou p 1 (acupunc tu re ): the need les were inse rt edbe tween 0 .3 and 0 .9 cm an d ro ta ted . Th i s caused a loca lmusc le con t rac t ion , r ecogn ized as de Qi , and wasrepeated f or 10 sec every 5 min.

Group 2 ( super f i c ia l acupunc tu re ) : the need les were

Correspondence: T. Lundeberg, Department of Physiology II, Karo-linska Instit utet, S- 104 01 Stockholm, Sweden.

inser ted int radermal ly and lef t for 30 min without e l ic i t -ing any response.

Gr ou p 3 (con t ro ls ) : t en mice , un t rea ted , se rved as con-t rois . After the t r ia ls the mice were ki l led by decapi-ta t ion.

Spleens were rem ove d asept ical ly, s l iced in t issue cul-tu re media (RPMI 1640 , S igma) so lu t ion , and passedth rough a wi re mesh . The ce l ls ob ta ined w ere washed 3t imes in phospha te -buffe red sa l ine (pH 7 .4 ) and thenhemolyzed wi th 0 .75 Tr i s -amm onium ch lo ride so lu t ion(pH 7.65) . The cel ls were washed again 3 t imes and thensuspended in t i s sue cu l tu re media (RPMI 1640 , S igma)in a conc en trati on of I x 107 cells/ml [9].

Measurement o f an t i - sheep red b lood ce l l s (SRBC)IgM p laque- fo rming ce l l s (PFC) in v ivo was pe r fo rmed

b y d e te r m i n in g t h e n u m b e r o f a n t i -S R B C P F C s p le e ncells on the 5th d ay af ter sensi t izat ion w ith 5 x 107 SR BC[6].

The f l - adrenerg ic b lock ing d rug p roprano lo l ( Indera l ) ,the ~ -adrenergic b lock ing agen t phen to lamine (Reg i t ine )and hexam ethon ium (Meth obrom ine) were d i s so lved inphysiological sa l ine and adminis tered oral ly to the mice.The local anaesthet ic l idocaine (Xylocain) was injectedsubcu taneous ly. Al l d rugs were g iven in amo unts o f 0 .1ml /10 g bod y we igh t .

Regulatory cel ls : spleen or thymic cel ls f rom CBF1

mice were t reated with mitomycin C (25 pg/ml , 37C, 30min) , washed 3 times wi th RP M I 1640 , and cu l tu red a t


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TA B L E I

E F F EC T S O F S U P E R F I C IA L A C U P U N C T U R E O R A C U P U N C -T U R E O N A N T I - S H E E P R E D B L O O D C E L L S (S R BC ) Ig MP L A Q U E - F O R M I N G C E L L S ( PF C) P R O D U C T I O N I N I M M U -

N I Z E D M I C E

Tr e a t m e n t w a s g i v e n o n c e a d a y f r o m d a y 1 t o d a y 3 a f te r i m m u n i s a -t i o n w it h S R B C . I n t e r a c t i o n o f p h e n t o l a m i n e , p r o p r a n o l o l , h e x a m e t h -o n i u m , a n d l i d o c a in e w i t h t h e d i f fe r e n t m o d e s o f t r e a t m e n t . A l l d r u g sw e r e a d m i n i s t e r e d 9 0 m i n b e f o r e t r e a t m e n t . S a l i n e w a s a d m i n i s t e r e d

s .c . d a i l y b e fo r e e a c h m o d e o f t r e a t m e n t . Va l u e s ( a n t i - S R B C P F C ,P F C / 1 0 6 , s p l e e n c el l s) a r e g i v e n a s m e a n + S . D .

M o d e o f t r e a t m e n t

N o t r e a t m e n t S u p e r fi c ia l A c u p u n c t u r e

( c o n t r o l ) a c u p u n c t u r e

S a li n e 2 0 8 3 3 6 8 2 1 6 8 + 3 2 8 4 1 6 5 5 5 0P h e n t o l a m i n e 3 3 0 4 + 5 0 8 3 3 4 5 4 3 6 5 7 3 9 + 7 0 5 *Pro pra no l o l 2668 339 2656__+ 337 2619 425

H e x a m e t h o n i u m 2 0 7 6 3 6 2 2 1 0 5 2 9 6 2 5 3 3 +__347Lid oca ine 2003 270 2081 223 2926__+ 590

* P < 0 . 0 5 c o m p a r i n g n o t r e a t m e n t ( c o n tr o l s ) w it h t h e d i f f er e n t m o d e s

o f a c u p u n c t u r e .

5 106 ce l l s /wel l w i th 5 106 no rm al sp leen ce l l s and5 x 1 06 SRBC (9 ) .

R P M I 1 640 w a s s u p p le m e n t e d w i th 2 5 m M H E P E S ,300 /~g /ml o f L -g lu t amine , 100 U/m l o f pen ic il l in , 100 / tg /m l o f s t r e p t o m y c i n , 5 10 - 5 M 2 - m e r c a p t o e t h a n o l , a n d10 f e t a l bov in e se rum fo r t he cu l tu re o f mice sp l eencells [16].

Sp leen ce ll s , ad jus t ed wi th R PM I 1640, were p l aced inPe t r i d i shes t ha t had been t r ea t ed wi th 10 f e t a l bov inese rum, a nd in cub a ted fo r I h a t 37C [9] . Th e f r ac t ion o fc e l l s n o n - a d h e r e n t t o t h e d i s h e s w a s r e m o v e d . T h e p r o -cedure was r epea t ed 3 t imes .

A n t i - T h y - l . 2 a n t i b o d y w a s d i l u t e d t o 1 / 5 0 0 0 w i t hR P M I 1 64 0 a n d w a s u s e d t o a d j u st t h e c o n c e n t r a t i o n o fspleen ce l l s to 1 107 ce l l s /ml . Th e s usp ens ion was lef ta t 4 C fo r 30 min . The ce l l s were t hen r e suspended in

R P M I 1 6 4 0 w h i c h c o n t a i n e d g u i n e a p i g d r i e d c o m p l e -men t , i ncuba ted a t 37C fo r a fu r the r 30 min , t henwash ed 3 t imes wi th RP M I 1640 [9] . Co n t ro l ce ll s weret r e a t e d w i th c o m p l e m e n t o n l y.

Th e values pre sen ted rep rese nt mea n ___ S.D. S ignif i -c a n c e s o f d i f fe r e n c e s b e t w e e n g r o u p s w e r e t e s t e d b y a n a -ly si s o f va r i ance . In t e rg r oup d i f f e r ences o f ca t ego r i ca lva r i ab l e s were t e s t ed fo r s ign i f icance wi th F i she r ' s exac tt e s t. S t a t is t i ca l s ign if i cance was de f ined a s P < 0 .05.

A c u p u n c t u r e s t i m u l a t i o n r e s u l t e d i n a n i n c r e a se i n t heP F C p r o d u c t i o n . P r e - a d m i n i s t r a ti o n ( 9 0 m i n b e f o r e st i-m u l a t i o n ) o f p h e n t o l a m i n e 5 m g / k g p . o . f u r t h e re n h a n c e d t h e in c r e a s e o f P F C p r o d u c t i o n . H o w e v e r, th e

TA B L E I I

T H E E F F E C T O F S U P E R F I C I A L A C U P U N C T U R E O R A C U -P U N C T U R E O N A N T I - SR B C P F C I N D U C T I O N O F S PL E E NC E L L S F R O M N O R M A L A N D I M M U N I Z E D M I C E IN V I T R O

T h e g r o u p i m m u n i z e d w i th S R B C w a s u s e d d a y 5 af t e r im m u n i z a t i o n .T h e m i c e w e r e g i v e n a c u p u n c t u r e o n c e a d a y f o r 3 d a y s . Va l u e s a r e

g i v e n a s m e a n S . D .

S p l ee n c e ll s f r o m m i c e P F C / C u l t u r e

N o t r e a t m e n t ( c o n tr o l )I m m u n i z e d w i t h S R B C

S u p e r fi c ia l a c u p u n c t u r eA c u p u n c t u r e

I m m u n i z e d w i t h S R B C a n d g i v e n s u p e rf i c ia la c u p u n c t u r e

I m m u n i z e d w i t h S R B C a n d g i v e n a c u p u n c t u r e

2 5 3 4 + 2 3 34 7 4 0 4 3 44758 3296 0 3 2 3 0 9 *

4 7 4 0 + 4 1 5

7372 1123

* P < 0 . 0 5 c o m p a r i n g n o t r e a t m e n t ( c o n t r o ls ) w i t h t h e d i ff e r e n t m o d e so f a c u p u n c t u r e .

i n c r e a s e i n d u c e d b y a c u p u n c t u r e w a s b l o c k e d b y p r e -t r e a t m e n t w i t h p r o p r a n o l o l 5 m g / k g p . o ., h e x a m e t h o -n ium 5 mg/kg p .o . and l i doca ine 5 mg /kg s . c . (Tab le I ) .M i c e , n o r m a l o r i m m u n i z e d w i t h S R B C , w e r e t r e a t e dwi th acu pun c tu re fo r 3 days . Sp leen ce l ls f rom these micew e r e t h e n c u l t u r e d w i t h S R B C i n v i t r o . A s s h o w n i nTab le I I , t he sp l een ce l l s f rom bo th t r ea t ed g roupss h o w e d a n i n c r e a s e i n P F C d u e t o a c u p u n c t u r e s t i m u l a -t io n . T h i s i n c r e a s e a p p e a r e d 3 h a f t e r a n a c u p u n c t u r et r ea tm en t a nd l a s t ed fo r a t l e a s t 24 h .

T h e e f fe c t o f a n t i - T h y - l . 2 a n t i b o d y o n t h e h e l p e r a c -t iv i ty w a s e n h a n c e d b y a c u p u n c t u r e a n d t h e e n h a n c e -m e n t w a s r e d u c e d b y t h e a d d i t io n o f a n t i - T h y - l . 2 a n t i -b o d y a n d c o m p l e m e n t , b u t n o t b y c o m p l e m e n t a l o n e(Tab le I I I ) . The he lpe r ac t iv i ty was seen in sp l een ce l l st h a t w e r e n o n - a d h e r e n t t o t h e d is h e s, b u t n o t i n a d h e r e n tc e ll s o r t h y m i c c el ls . T h e n u m b e r o f a n t i - S R B C P F C w a si n c r ea s e d b y t r e a t m e n t w i th s e r u m t a k e n f r o m m i c e 1 ha f t e r a c u p u n c t u r e s t i m u l a t i o n ( Ta b l e I V ) . T h i s s e r u m -in f luenced ac t iv i ty was abo l i shed by the ad d i t i on o f p ro -

p r a n o l o l 1 0 - 7 g / m l , b u t n o t b y p h e n t o l a m i n e 1 0 - 7 g / m l(Tab le V) .

T h e p r e s e n t s t u d y s h o w s t h a t a c u p u n c t u r e t r e a t m e n tm a y r e s u lt in e n h a n c e m e n t o f t h e P F C a c t iv i ty. T h r e eh o u r s a f t e r t r e a t m e n t , t h e r e w a s e n h a n c e d P F C a c t iv i ty.T h i s e n h a n c e d P F C a c t i v it y w a s n o t b l o c k e d b y in v i t rot r e a t m e n t w i t h m i t o m y c i n C , b u t w a s a b o l is h e d b y t h ea d d i t i o n o f a n ti - T h y - 1 . 2 a n t i b o d y a n d c o m p l e m e n t , a n db y t h e r e m o v a l o f n o n - a d h e r e n t c el ls . S e r u m o b t a i n e df r o m m i c e l h a f t e r a c u p u n c t u r e t r e a t m e n t s h o w e denh anc ed P FC in no rm a l m ice sp l een ce l ls , bu t t h i s e f fec twas r eve r sed by the add i t i on o f t he f l - ad rene rg i cb l o c k i n g a g e n t p r o p r a n o l o l .


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TABLE I I I

C H A R A C T E R I Z AT I O N O F H E L P E R A C T I V IT Y O N P F CE N H A N C E D B Y S U P E R F I C I A L A C U P U N C T U R E O R A C U -P U N C T U R E

Spleen and thymic cells were used 3 h a fter treatment. These cells weretreated with mitomycin and were used as regu latory cells of in vitro

anti-SRBC PF C p roduc tion. No rma l spleen cells were used as effectorcells. Values (PFC/culture) are given as mean + S.D.

No treatme nt Superficial Acupuncture(controls) acupuncture

3 2 0 _ + 4 7 4 2 4 1 4 _ + 3 8 6 4 79 7 3 56 *

2337415 4707594*

Spleen cellsTreated with

complementTreated with anti-

Thy- l .2 andcomplement

Adh erent cellsNon-adherent cells

Thymic cells 2377435

2280_+403 28772612320474 2230_+3382377435 4152_+437

2520360 2427427

*P < 0.05 compa ring no treatm ent (controls) with the different modesof acupuncture or within the treatm ent groups.

T h e p r e s e n t a n d p r e v i o u s r e s u l t s i n d i c a t e t h a t t h e

e n h a n c e m e n t o f P F C b y a c u p u n c t u r e m a y b e d u e to t he

a c t i v a t i o n o f n o n - s p e c if i c h e l p e r T l y m p h o c y t e s b y f l- a c-

t i o n o f e n d o g e n o u s e p i n e p h r i n e , r e l e a s e d t h r o u g h s t i m u -

l a t i o n o f t h e s y m p a t h e t i c n e r v o u s s y s t e m [ 9] . T h i s r e s u l t

i s s u p p o r t e d b y t h e f a c t t h a t a n i . p . i n j e c t i o n o f p a i n f u l

s u b s t a n c e r a is e s t h e le v e l o f e n d o g e n o u s e p i n e p h r i n e i n

t h e s p le e n [1 0] . F u r t h e r m o r e , t h e a u t o n o m i c n e r v o u s s y s-

t e m m a y b e a c t i v a t e d b y t h e l o c a l s t i m u l u s o f e x t e r n a l ly

a d m i n i s t e r e d c a t e c h o l a m i n e s [ 9, 10 ], a n d i n c r e a s e d a n t i -

b o d y p r o d u c t i o n i s c a u s e d v i a t h e fl - a c ti o n o f e p i n e p h -

TABLE IV

E F F E C T O F S E R U M F R O M M I C E G I V E N S U P E R F I C IA L A C U -P U N C T U R E O R A C U P U N C T U R E O N A N T I -S R B C P F C O FS P L E E N C EL L S F R O M N O R M A L M I C E I N V I T R O

Sera were added at 0.3 ml to culture plate before beginning culture.Values (PFC/culture) are given as mean S.D. NS between groups.

Time No treatm ent Superficial Acupuncture(min) acupuncture

0 1077_+8210 1070 4- 91 1041_+ 7430 1095_+ 97 1175_+ 11060 1142__+ 87 1761_+ 97

120 1145 _+ 135 1401 + 183

240 1083 _+ 106 1094 + 194

163

TA B L E V

E F F E C T S O F P R O P R A N O L O L A N D P H E N T O L A M I N E I NV I TR O O N T H E E N H A N C E M E N T O F P F C B Y S E R U M F R O MM I C E T R E AT E D W I T H S U P E R F I C I A L A C U P U N C T U R E O RA C U P U N C T U R E

Sera were obtained from mice 60 min after superficial acupuncture or

acupuncture treatment. P hentolamine and pro pran olol were used atdose 10 -7 g/ml. Values (PFC/culture) are given as mean + S.D. NSbetween groups.

No treatme nt Superficial Acu punctu reacupuncture

No drug 1750+477 1851 +47 0 2506__+381Prop ranolo l 1830+307 2010+ 383 1279+183Phentholam ine 17 28 __428 1793 193 2408 332

r i n e f r o m t h e s y m p a t h e t i c n e r v o u s s y s t e m [ 9 ]. It is l i k e l y

t h a t p a r t o f t h e a c ti v a t i o n o f th e a u t o n o m i c n e r v o u s s y s -

t e m i s m e d i a t e d t h r o u g h s e n s o r y n e u r o p e p t i d e s r e l ea s e dd u r i n g a c u p u n c t u r e [ 13 ]. T h i s i s i n t e r e s t i n g t o n o t e a s

l y m p h o c y t e s p o s s e s s r e c e p t o r s f o r t h e se m e d i a t o r s a n d

a r e a f fe c t e d b y s u ch m e d i a t o r s n o t o n l y i n p r o l i fe r a t i o n

[ 1 5 ] b u t a l s o i n r o s e t t e f o r m a t i o n [ 1 2 ] , a n t i b o d y f o r m a -

t i o n [ 5] , c y t o t o x i c i t y [ 1 8] a n d t h e i n t r a c e l l u l a r l e v e l o f

c y c l i c n u c l e o t i d e s [ 1 9 ].

I n s u m m a r y , i t h a s b e e n r e p o r t e d t h a t t h e p e r i p h e r a l

a n d c e n t r a l n e r v o u s s y s t e m m a y e x e r t a n e f f ec t o n t h e

i m m u n o l o g i c a l r e s p o n s e [2 , 3]. A c u p u n c t u r e t r e a t m e n t

r e s u l t s in c h a n g e s i n t h e a c t i v i t y o f th e c e n t r a l a n d p e r i -

p h e r a l n e r v o u s s y s t e m [ 11 , 1 3]. T h e r e s u l t s o f th e p r e s e n t

s t u d y i n d ic a t e t h a t a c u p u n c t u r e e n h a n c e s t h e i m m u n e

r e a c t i o n t h r o u g h t h e a c t i v a t i o n o f n o n -s p e c i fi c T l y m -

p h o c y t e s t h r o u g h t h e a u t o n o m i c n e r v o u s s y s t em .

T h e a s s i s t a n c e o f M s . U l l a L i n d g r e n i n p r e p a r i n g t h e

m a n u s c r i p t i s g r e a t l y a c k n o w l e d g e d . A i d i n a n a l y s i s w a s

k i n d l y p r o v i d e d b y E n r a f - N o n i u s , t h e N e t h e r l a n d s . T h i s

w o r k w a s s u p p o r t e d b y T o r e o c h R a g n a r S 6 d e r b e r g s

S t i f te l s e r , K o n u n g G u s t a v V: s 8 0 - ~ r s f o n d , S t i f te l s e nP r o f . N a n n a S v a r t z ' F o n d , S t if t el s en L a r s H i e r t a s

M i n n e , T o r e N i l so n s F o n d f 6 r M e d i c in s k F o r s k n i n g ,

R i k s f 6 r b u n d e t m o t R e u m a t i s m .

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Lundeberg 91 Neuroimmunomodulatory Effects of Acupuncture in Mice