Neuroscience Letters, 128 (1991) 161-164

© 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50
A DON1S 0304394091003498
NSL 07864
161
Neuroimmunomodulatory effects of acupuncture in mice
T. Lundeberg 1, S.V. Er i ksson 2 and E. The odor s s on 3
1Department 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 November 1990; Revised version received 10 April 1991; Accepted 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 of the immune response to treatment. In normal non-immunized mice, enhancement
of PFC was seen after a single acupuncture treatment when spleen cells from stimulated mice were cultured with SRBC in vitro. After 3 acupuncture
treatments, spleen cells from mice did not show PFC enhancement after treatment with anti-Thy-1.2 antibody and complement, nor after the removal
of non-adherent 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.
Recent reports have suggested that the immune reac-
tion is regulated by the central nervous system [4, 8].
This is interesting to note, as experimental studies have
also shown that acupuncture stimulation affects the ac-
tivity of several systems within the brain and induces the
release of endogenous opioids [11].
Opiates and endogenous opioids have been shown to
have immuno-compromising effects [2, 3, 7]. Further,
other studies have shown that erythrocytes, monocytes,
granulocytes and lymphocytes all have specific opiate
binding sites [1, 14]. These findings, and the fact that
acupuncture has been tried in the treatment of patients
with immuno-deficiency diseases, induced us to study the
effects of acupuncture on the immune responses [17].
Acupuncture treatment was carried out for periods of
30 min. Six points were chosen (Jingmen G25; Zhaohai
K6; Dazhu Bl l , bilaterally). Thirty male mice, 6-10
weeks old, were assigned to 3 groups with 10 mice in
each, for each of the treatment categories.
Gr oup 1 (acupuncture): the needles were inserted
between 0.3 and 0.9 cm and rotated. This caused a local
muscle contraction, recognized as de Qi, and was
repeated for 10 sec every 5 min.
Gr oup 2 (superficial acupuncture): the needles were
Correspondence: T. Lundeberg, Department of Physiology II, Karo-
linska Institutet, S- 104 01 Stockholm, Sweden.
inserted intradermally and left for 30 min without elicit-
ing any response.
Gr oup 3 (controls): ten mice, untreated, served as con-
trois. After the trials the mice were killed by decapi-
tation.
Spleens were removed aseptically, sliced in tissue cul-
ture media ( RPMI 1640, Sigma) solution, and passed
through a wire mesh. The cells obtained were washed 3
times in phosphate-buffered saline (pH 7.4) and then
hemolyzed with 0.75 % Tris-ammonium chloride solution
(pH 7.65). The cells were washed again 3 times and then
suspended in tissue culture media ( RPMI 1640, Sigma)
in a concentration of I x 107 cells/ml [9].
Measurement of anti-sheep red blood cells (SRBC)
IgM plaque-forming cells (PFC) in vivo was performed
by determining the number of anti-SRBC PFC spleen
cells on the 5th day after sensitization with 5 x 107 SRBC
[6].
The fl-adrenergic blocking drug propranolol (Inderal),
the ~-adrenergic blocking agent phentolamine (Regitine)
and hexamethonium (Methobromine) were dissolved in
physiological saline and administered orally to the mice.
The local anaesthetic lidocaine (Xylocain) was injected
subcutaneously. All drugs were given in amounts of 0.1
ml/10 g body weight.
Regulatory cells: spleen or thymic cells from CBF1
mice were treated with mitomycin C (25 pg/ml, 37°C, 30
min), washed 3 times with RPMI 1640, and cultured at
162
TABLE I
EFFECTS OF S UP E RF I CI AL AC UP UNC T UR E OR AC UP UNC -
T UR E ON ANTI - S HEEP RED BLOOD CELLS ( SRBC) I gM
P L AQUE - F OR MI NG CELLS ( PFC) P R ODUC T I ON I N I MMU-
NI ZED MI CE
Tr e a t me nt was gi ven once a day f r om day 1 to day 3 af t er i mmuni s a -
t i on wi t h SRBC. I nt er act i on of phe nt ol a mi ne , pr opr anol ol , he xa me t h-
oni um, and l i docai ne wi t h t he di fferent mode s of t r eat ment . Al l dr ugs
were admi ni s t er ed 90 mi n bef or e t r eat ment . Sal i ne was admi ni s t er ed
s.c. dai l y before each mode of t r eat ment . Val ues ( ant i - SRBC PFC,
PFC/ 106, spl een cells) ar e gi ven as me a n + S. D.
Mode of t r e a t me nt
No t r e a t me nt Superfi ci al Ac upunc t ur e
(cont rol ) a c upunc t ur e
Sal i ne 2 0 8 3 ± 3 6 8 2168+328 4 1 6 5 ± 5 5 0 "
Phe nt ol a mi ne 3304 + 508 3345 ± 436 5739 + 705*
Pr opr anol ol 2668 ± 339 2656__+ 337 2619 ± 425
He x a me t h o n i u m 2076 ± 362 2105 ± 296 2533 +__347
Li docai ne 2003 ± 270 2081 ± 223 2926__+ 590
* P < 0 . 0 5 c ompa r i ng no t r e a t me nt ( cont r ol s) wi t h t he di fferent mode s
o f acupunct ur e.
5 × 106 cells/well with 5 × 106 nor mal spleen cells and
5 x 106 SRBC (9).
RPMI 1640 was suppl ement ed with 25 mM HEPES,
300/~g/ml of L-glutamine, 100 U/ ml of penicillin, 100/tg/
ml of st rept omyci n, 5 × 10 -5 M 2- mer capt oet hanol , and
10% fetal bovi ne serum f or t he cul t ure of mice spleen
cells [16].
Spleen cells, adj ust ed with RPMI 1640, were placed in
Petri dishes t hat had been t reat ed with 10 % fetal bovi ne
serum, and i ncubat ed for I h at 37°C [9]. The fract i on of
cells non- adher ent to the dishes was removed. The pro-
cedure was repeat ed 3 times.
Ant i - Thy- l . 2 ant i body was diluted to 1/5000 with
RPMI 1640 and was used t o adjust t he concent r at i on of
spleen cells to 1 × 107 cells/ml. The suspension was left
at 4°C f or 30 min. The cells were t hen resuspended in
RPMI 1640 which cont ai ned guinea pig dri ed compl e-
ment , i ncubat ed at 37°C for a f ur t her 30 min, t hen
washed 3 times with RPMI 1640 [9]. Cont r ol cells were
t reat ed with compl ement only.
The values present ed represent mean ___ S.D. Signifi-
cances of differences bet ween groups were tested by ana-
lysis of variance. I nt er gr oup differences of cat egori cal
variables were tested for significance with Fi sher' s exact
test. Statistical significance was defined as P < 0.05.
Acupunct ur e st i mul at i on resulted in an increase in the
PFC pr oduct i on. Pre-admi ni st rat i on (90 min before sti-
mul at i on) of phent ol ami ne 5 mg/ kg p.o. f ur t her
enhanced the increase of PFC pr oduct i on. However, the
TABLE II
THE E F F E CT OF S UP E RF I CI AL AC UP UNC T UR E OR ACU-
P UNC T UR E ON ANTI - S RBC PFC I NDUC T I ON OF SPLEEN
CELLS F R OM NOR MAL AND I MMUNI Z E D MI CE IN VI TRO
The gr oup i mmuni z e d wi t h SRBC was used day 5 af t er i mmuni z a t i on.
The mi ce were gi ven a c upunc t ur e once a day for 3 days. Val ues ar e
gi ven as me a n ± S. D.
Spleen cells f r om mi ce PFC/ Cul t ur e
No t r e a t me nt (cont rol )
I mmuni z e d wi t h SRBC
Superfi ci al a c upunc t ur e
Ac upunc t ur e
I mmuni z e d wi t h SRBC and gi ven superfi ci al
a c upunc t ur e
I mmuni z e d wi t h SRBC and gi ven a c upunc t ur e
2534+233
4 7 4 0 ± 434
4758 ± 329
6032 ± 309*
4 7 4 0 +4 1 5
7372 ± 1123"
* P < 0.05 c ompa r i ng no t r e a t me nt (cont rol s) wi t h t he di fferent mode s
o f acupunct ur e.
increase i nduced by acupunct ur e was bl ocked by pre-
t reat ment with pr opr anol ol 5 mg/ kg p. o. , hexamet ho-
nium 5 mg/ kg p.o. and lidocaine 5 mg/ kg s.c. (Tabl e I).
Mice, nor mal or i mmuni zed with SRBC, were t reat ed
with acupunct ur e for 3 days. Spleen cells from these mice
were t hen cul t ured with SRBC in vitro. As shown in
Tabl e II, the spleen cells f r om bot h t reat ed groups
showed an increase in PFC due to acupunct ur e stimula-
tion. This increase appear ed 3 h aft er an acupunct ur e
t r eat ment and lasted f or at least 24 h.
The effect of ant i - Thy- l . 2 ant i body on the helper ac-
tivity was enhanced by acupunct ur e and the enhance-
ment was reduced by the addi t i on of ant i - Thy- l . 2 anti-
body and compl ement , but not by compl ement al one
(Tabl e III). The helper activity was seen in spleen cells
t hat were non- adher ent to t he dishes, but not in adher ent
cells or t hymi c cells. The number of ant i -SRBC PFC was
increased by t r eat ment with serum t aken f r om mice 1 h
aft er acupunct ur e st i mul at i on (Tabl e IV). This serum-
influenced activity was abol i shed by the addi t i on of pro-
pranol ol 10 -7 g/ml, but not by phent ol ami ne 10 -7 g/ml
(Tabl e V).
The present st udy shows t hat acupunct ur e t r eat ment
may result in enhancement of the PFC activity. Thr ee
hours aft er t reat ment , t here was enhanced PFC activity.
This enhanced PFC activity was not bl ocked by in vi t ro
t r eat ment with mi t omyci n C, but was abol i shed by the
addi t i on of ant i -Thy-1. 2 ant i body and compl ement , and
by the removal of non- adher ent cells. Serum obt ai ned
f r om mice l h aft er acupunct ur e t r eat ment showed
enhanced PFC in nor mal mice spleen cells, but this effect
was reversed by the addi t i on of the fl-adrenergic
bl ocki ng agent pr opr anol ol .
TABLE III
CHARACTERIZATION OF HELPER ACTIVITY ON PFC
ENHANCED BY SUPERFICIAL ACUPUNCTURE OR ACU-
PUNCTURE
Spleen and thymic cells were used 3 h after treatment. These cells were
treated with mitomycin and were used as regulatory cells of in vitro
anti-SRBC PFC production. Normal spleen cells were used as effector
cells. Values (PFC/culture) are given as mean + S.D.
No treatment Superficial Acupuncture
(controls) acupuncture
320_+474 2414_+386 4797±356*
2337±415 4707±594*
Spleen cells
Treated with
complement
Treated with anti-
Thy-l.2 and
complement
Adherent cells
Non-adherent cells
Thymic cells 2377±435
2280_+403 2877±261
2320±474 2230_+338
2377±435 4152_+437"
2520±360 2427±427
*P < 0.05 comparing no treatment (controls) with the different modes
of acupuncture or within the treatment groups.
The p r e s e n t a n d p r e v i o u s r es ul t s i ndi c a t e t h a t t he
e n h a n c e me n t o f P F C by a c u p u n c t u r e ma y be due t o t he
a c t i v a t i o n o f non- s pe c i f i c h e l p e r T l y mp h o c y t e s by f l - ac-
t i on 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 mu-
l a t i o n o f t he s y mp a t h e t i c n e r v o u s s ys t e m [9]. Thi s r es ul t
is s u p p o r t e d b y t he f act t h a t a n i . p. i nj e c t i on o f p a i n f u l
s u b s t a n c e r ai s es t he l evel 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 he s pl een [10]. F u r t h e r mo r e , t he a u t o n o mi c n e r v o u s sys-
t e m ma y be a c t i v a t e d b y t he l oc a l s t i mu l u s o f e xt e r na l l y
a d mi n i s t e r e d c a t e c h o l a mi n e s [9, 10], a n d i n c r e a s e d a nt i -
b o d y p r o d u c t i o n i s c a u s e d vi a t he f l - a c t i on o f e p i n e p h -
TABLE IV
EFFECT OF SERUM FROM MICE GIVEN SUPERFICIAL ACU-
PUNCTURE OR ACUPUNCTURE ON ANTI-SRBC PFC OF
SPLEEN CELLS FROM NORMAL MICE IN VITRO
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 treatment Superficial Acupuncture
(min) acupuncture
0 1077_+82
10 10704- 91 1041_+ 74
30 1095_+ 97 1175_+ 110
60 1142__+ 87 1761_+ 97
120 1145 _+ 135 1401 + 183
240 1083 _+ 106 1094 + 194
163
TABLE V
EFFECTS OF PROPRANOLOL AND PHENTOLAMINE IN
VITRO ON THE ENHANCEMENT OF PFC BY SERUM FROM
MICE TREATED WITH SUPERFICIAL ACUPUNCTURE OR
ACUPUNCTURE
Sera were obtained from mice 60 min after superficial acupuncture or
acupuncture treatment. Phentolamine and propranolol were used at
dose 10 -7 g/ml. Values (PFC/culture) are given as mean + S.D. NS
between groups.
No treatment Superficial Acupuncture
acupuncture
No drug 1750+477 1851 +470 2506__+381
Propranolol 1830+307 2010+ 383 1279+183
Phentholamine 1728 ___428 1793 ± 193 2408 ± 332
r i ne f r o m t he s y mp a t h e t i c n e r v o u s s ys t em [9]. I t i s l i kel y
t h a t p a r t o f t he a c t i v a t i o n o f t he a u t o n o mi c n e r v o u s sys-
t e m is me d i a t e d t h r o u g h s e ns or y n e u r o p e p t i d e s r e l e a s e d
d u r i n g a c u p u n c t u r e [13]. Thi s i s i nt e r e s t i ng t o n o t e as
l y mp h o c y t e s pos s e s s r e c e p t o r s f or t hes e me d i a t o r s a n d
a r e af f ect ed by such me d i a t o r s n o t onl y i n p r o l i f e r a t i o n
[15] b u t al s o i n r os e t t e f o r ma t i o n [12], a n t i b o d y f o r ma -
t i on [5], c y t o t o x i c i t y [18] a n d t he i n t r a c e l l u l a r l evel o f
cycl i c nuc l e ot i de s [19].
I n s u mma r y , i t ha s be e n r e p o r t e d t h a t t he p e r i p h e r a l
a n d c e nt r a l n e r v o u s s ys t e m ma y e xe r t a n ef f ect on t he
i mmu n o l o g i c a l r e s p o n s e [2, 3]. Ac u p u n c t u r e t r e a t me n t
r es ul t s i n c ha nge s i n t he a c t i vi t y o f t he c e n t r a l a n d per i -
p h e r a l n e r v o u s s ys t e m [11, 13]. Th e r es ul t s o f t he p r e s e n t
s t udy i ndi c a t e t h a t a c u p u n c t u r e e nha nc e s t he i mmu n e
r e a c t i o n t h r o u g h t he a c t i v a t i o n o f non- s pe c i f i c T l ym-
p h o c y t e s t h r o u g h t he a u t o n o mi c n e r v o u s s ys t em.
The a s s i s t a nc e o f Ms . Ul l a Li n d g r e n i n p r e p a r i n g t he
ma n u s c r i p t is g r e a t l y a c k n o wl e d g e d . Ai d i n a na l ys i s wa s
k i n d l y p r o v i d e d by En r a f - No n i u s , t he Ne t h e r l a n d s . Thi s
wo r k wa s s u p p o r t e d by To r e oc h Ra g n a r S6 d e r b e r g s
St i f t el s er , Ko n u n g Gu s t a v V: s 80- ~r s f ond, St i f t el s en
Pr of . Na n n a Sv a r t z ' F o n d , St i f t el s en La r s Hi e r t a s
Mi n n e , To r e Ni l s o n s F o n d f 6r Me d i c i n s k F o r s k n i n g ,
Ri k s f 6 r b u n d e t mo t Re u ma t i s m.
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