IMMUNOLOGICAL INVESTIGATIONS, 21(1),
1-10 (1992)
STUDIES ON THE IMMUNOSUPPRESSIVE ROLE OF STEROID HORMONES
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
DURING PREGNANCY
M.A.Saleem. , P . J h a . , K . Bucksheel and A.Farooq* Departments of Reproductive Biology and O b s t e t r i c s and Gynecology1 A l l I n d i a I n s t i t u t e o f Medical S c i e n c e s , New Delhi-110029, I N D I A . ABSTRACT I t h a s been r e c o g n i s e d t h a t s t e r o i d s c a n e x e r t a profound i n f l u e n c e o v e r immunological r e a c t i v i t y . The p r e s e n t s t u d y a n a l y z e s t h e r o l e of s t e r o i d hormones e s t r o g e n , p r o g e s t e r o n e and c o r t i s o l - and t h e i r i n v o l v e ment i n immunoregulation d u r i n g pregnancy. A s i s known, t h e endogenous l e v e l s of a l l t h e t h r e e hormones i n c r e a s e d u r i n g pregnancy. When t h e s t e r o i d l e v e l s i n pregnancy serum w e r e c o r r e l a t e d w i t h t h e lymphocyte r e s p o n s e t o mitogen, no c o r r e l a t i o n w a s o b s e r v e d . The s u p p r e s s i v e e f f e c t o f pregnancy serum w a s found t o have no c o r r e l a t i o n with i t s s t e r o i d content. I n general, s t e r o i d s did not seem t o a f f e c t t h e m a t e r n a l immune system a s e v i d e n c e d by t h e p r e s e n t s t u d y . INTRODUCTION
The mechanism by which t h e f e t u s , which i s e s s e n t i a l l y an a l l o g r a f t , i s a b l e t o s u r v i v e a g a i n s t t h e immunologic d e f e n c e s o f t h e mother i s one of the less u n d e r s t o o d phenomena I t h a s been r e p o r t e d t h a t t h e r e i s i n p r e s e n t day immunology.
a b l o c k i n g of t h e m a t e r n a l immune system by t h e r e g u l a t o r y f a c t o r s p r e s e n t i n t h e b l o o d , p a r t i c u l a r l y s t e r o i d s , which i n d u c e changes i n t h e immunological a c t i v i t y i n t h e m a t e r n a l h o s t d u r i n g pregnancy. I n g e n e r a l , s t e r o i d hormones-progesterone, e s t r o g e n i n h i b i t normal T - c e l l p r o l i f e r a t i o n by and c o r t i c o s t e r o i d s v a r i o u s a g e n t s such a s PHA, Con A o r PPD (1,2). S i g n i f i c a n t
-
*To whom a l l c o r r e s p o n d e n c e should be a d d r e s s e d .
Copyright 0 1992 by Marcel Dekker, Inc.
2
SALEEM ET AL.
s u p p r e s s i o n by p r o g e s t e r o n e w a s o b s e r v e d on many o c c a s i o n s ( 3 , 4 ) . But i n h i b i t i o n of mitogen, a n t i g e n and a l l o g e n i c c e l l a c t i v a t i o n by e s t r o g e n w a s found o n l y a t h i g h p h y s i o l o g i c a l l e v e l s ( 5 , 6 ) . Marked r e d u c t i o n i n PHA t r a n s f o r m a t i o n o f lymphocytes was shown by Tomoda e t a l . ,
(7) when c o r t i c o s t e r o i d s were added t o t h e
cultures.
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
On t h e c o n t r a r y , it h a s a l s o been r e p o r t e d t h a t serum and p l a c e n t a l l e v e l s o f s t e r o i d s a r e too low t o have s i g n i f i c a n t e f f e c t on lymphocytes (5,7). P o s k i t t e t a l . , ( 8 ) found t h a t p r o g e s t e r o n e and e s t r o g e n s have l i t t l e e f f e c t on t h e r e s p o n s e of lymphocytes t o PHA and PWM u n l e s s p r e s e n t i n h i g h c o n c e n t r a t i o n s n o t u s u a l l y p r e s e n t i n pregnancy blood.
Although profound
q u a l i t a t i v e and q u a n t i t a t i v e changes o c c u r d u r i n g pregnancy, t h e e f f e c t of t h e s e changes on t h e m a t e r n a l immune system i s n o t
w e l l u n d e r s t o o d and r e q u i r e s f u r t h e r i n v e s t i g a t i o n . MATERIALS AND METHODS Blood samples: Blood samples from p r e g n a n t women were c o l l e c t e d from t h e c l i n i c s of t h e Department o f O b s t e t r i c s and Gynecology, A.I.I.M.S.,
New D e l h i .
Blood from v o l u n t a r y d o n o r s s e r v e d a s
c o n t r o l . The blood samples w e r e c o l l e c t e d i n screw-capped t u b e s w i t h h e p a r i n (Centron Laboratories, Bombay, I n d i a ) f o r lymphocyte i s o l a t i o n and w i t h o u t h e p a r i n f o r serum s e p a r a t i o n . Lymphocyte c u l t u r e s : Lymphocytes w e r e i s o l a t e d by t h e F i c o l l Hypaque d e n s i t y g r a d i e n t method. The c u l t u r e s were done i n 96 w e l l m i c r o t i t r e p l a t e s (Nunc, Denmark). C e l l s u s p e n s i o n (50 u l ) c o n t a i n i n g 0.2 t o 0.3 m i l l i o n c e l l s w a s t a k e n f o r c u l t u r e . A s t a n d a r d i s e d optimum c o n c e n t r a t i o n of PHA and Con A was added t o each w e l l and t h e t o t a l volume o f t h e c u l t u r e s w a s a d j u s t e d t o 0.25 m l w i t h RPMI-1640 medium (Gibco L a b o r a t o r i e s , USA).
Wells w i t h o u t mitogen s e r v e d as c o n t r o l s . The c u l t u r e s w e r e i n c u b a t e d a t 37OC w i t h 5% C 0 2 . A f t e r 48 h o u r s 0.5 uCi of t r i t i a t e d thymidine (Bhabha A t o m i c Research C e n t r e , Bombay, I n d i a ) was added t o e a c h w e l l and t h e c u l t u r e s w e r e t e r m i n a t e d 2 4 h o u r s a f t e r thymidine i n c o r p o r a t i o n . The c e l l s w e r e h a r v e s t e d on a Whatman GF/C f i l t e r p a p e r u s i n g a c e l l h a r v e s t e r (Nunc, D e n m a r k ) . The f i l t e r p a p e r w a s t r a n s f e r r e d t o a s c i n t i l l a t i o n v i a l and 8 m l o f s i m p l e s c i n t i l l a t i o n m i x t u r e (POPOP 100 mg and PPO 4 9 p e r l i t r e s u l f u r - f r e e t o l u e n e ) was
STEROID HORMONES DURING PREGNANCY
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
added t o e a c h v i a l . The r a d i o a c t i v i t y p r e s e n t i n t h e v i a l s w a s counted i n a LKB Wallac Minibeta l i q u i d s c i n t i l l a t i o n counter. The p r o l i f e r a t i v e c a p a c i t y of t h e lymphocytes w a s analyzed as follows: Counts i n cpm o f c e l l s w i t h mitogen ( T ) Transformation index= Counts i n cpm of c e l l s w i t h o u t mitogen ( C ) The p e r c e n t a g e t r a n s f o r m a t i o n of lymphocytes from p r e g n a n t p a t i e n t s w a s c a l c u l a t e d by t a k i n g t h e t r a n s f o r m a t i o n i n d e x of non-pregnant c o n t r o l s as 1 0 0 % . E f f e c t o f pregnancy serum on normal lymphocyte c u l t u r e s : _
~
_
Normal lymphocyte c u l t u r e s were done as d e s c r i b e d e a r l i e r
and 1 0 % pregnancy serum w a s added t o each w e l l . C u l t u r e s w i t h o u t pregnancy serum s e r v e d as controls. By t a k i n g t h e p e r c e n t t r a n s f o r m a t i o n i n c u l t u r e s w i t h o u t pregnancy serum a s 0% s u p p r e s s i o n , t h e p e r c e n t s u p p r e s s i o n i n c u l t u r e s w i t h
pregnancy serum w a s c a l c u l a t e d . Radioimmunoassay o f hormones ( R I A ) : E s t i m a t i o n o f s t e r o i d hormones 1 7 - f i e s t r a d i o l , p r o g e s t e r o n e and c o r t i s o l w a s c a r r i e d o u t a c c o r d i n g t o t h e WHO method manual ( 9 ) . Within and between a s s a y c o e f f i c i e n t s of v a r i a t i o n f o r e s t r a d i o l , p r o g e s t e r o n e and c o r t i s o l were found t o be 7 . 8 % and 10.2%, 9 . 3 % and 1 0 . 9 % and 9 . 2 % and 10.8%, respectively. A n a l y s i s of t h e s u p p r e s s i v e e f f e c t o f serum a f t e r t h e e x t r a c t i o n of hormones: A f t e r t h e e x t r a c t i o n of hormones f o r R I A from t h e serum, t h e r e s i d u e c o n t a i n i n g serum p r o t e i n s w a s once a g a i n a n a l y z e d f o r i t s s u p p r e s s i v e e f f e c t on lymphocyte c u l t u r e s . The r e s i d u e w a s f i r s t d i s s o l v e d i n RPMI-1640 medium and added t o normal lymphocyte c u l t u r e s t o s t u d y i t s e f f e c t . RESULTS
S t e r o i d hormone p r o f i l e d u r i n g pregnancy: Endogenous l e v e l s of e s t r o g e n , p r o g e s t e r o n e and c o r t i s o l were e s t i m a t e d by R I A i n pregnancy sera from d i f f e r e n t trimesters of pregnancy and t h e r e s u l t s a r e summarized i n Table I .
3
SALEEM ET AL.
4
TABLE I
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
S t e r o i d Hormone L e v e l s (mean
ESTRADIOL
3154
(pmol/l)
t
1183
5 SD)
6558
(25)
PROGESTERONE 20.84
+
CORTISOL
411.4
33.42
+
1768
11003
+
146.44
+
616
5
3886
(22)
7.39
48.87
( i 81
( 2 01
(nmOl/l)
+
(19)
5.72
( i 61
(nmol/l)
During Normal Pregnancy
189.51
769
+
4.25
(is) + 266.52 (301
(i7)
F i g u r e s i n p a r e n t h e s e s r e p r e s e n t t h e number of samples s t u d i e d .
120 100
-
80 - ' *
.
.
60 40
-
..
20 n " 0
1
2
3
4
5
6
ESTRADIOL LEVELS ng/ml FIGURE 1 C o r r e l a t i o n b e t w e e n E s t r a d i o l l e v e l a n d PHA i n d u c e d lymphocyte t r a n s f o r m a t i o n .
STEROID HORMONES DURING PREGNANCY
5
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
120
100
-
80
-
80
-
. . ..
. '
t ,
0'
0
20
10
30
40
50
PROGESTERONE LEVEL (nmol\l) FIGURE 2
Correlation between Progesterone level and PHA induced lymphocyte transformation.
100
-
..
m
..
.
80 60
-
40
-
2o 0
t 0
I
I
100
200
, 300
400
CORTISOL LEVEL (nmol\l) FIGURE 3
Correlation between Cortisol level and PHA induced lymphocyte transformation.
500
6
SALEEM ET AL.
C o r r e l a t i o n of s t e r o i d l e v e l s w i t h lymphocyte t r a n s f o r m a t i o n : The l e v e l s o f s t e r o i d hormones, e s t r a d i o l , p r o g e s t e r o n e and c o r t i s o l , were compared w i t h the p r o l i f e r a t i v e c a p a c i t y of lymphocytes from t h e same s u b j e c t t o f i n d whether t h e r e w a s any c o r r e l a t i o n between t h e hormone l e v e l s and t h e lymphocyte t r a n s f o r m a t i o n . F i g u r e s . 1 , 2 and 3 show t h e r e l a t i o n between
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
t h e hormone l e v e l s and t h e PHA i n d u c e d t r a n s f o r m a t i o n of lymphocytes from p r e g n a n t women.
N o c o r r e l a t i o n w a s found
between t h e l e v e l s o f s t e r o i d hormones and t h e p a t t e r n o f lymphocyte t r a n s f o r m a t i o n .
The same o b s e r v a t i o n w a s made i n
Con A induced c u l t u r e s . S u p p r e s s i v e e f f e c t o f pregnancy serum i n r e l a t i o n t o s t e r o i d levels: A f t e r e s t i m a t i n g t h e hormone l e v e l s i n pregnancy sera t h e samples w e r e a n a l y z e d f o r t h e i r e f f e c t o n normal lymphoc y t e c u l t u r e s . The i n h i b i t o r y e f f e c t of t h e s e pregnancy
sera was once a g a i n compared w i t h t h e serum l e v e l s of s t e r o i d hormones. The r e s u l t s i n d i c a t e a l a c k of c o r r e l a t i o n between s t e r o i d l e v e l s and i n h i b i t i o n o f PHA i n d u c e d normal lymphocyte b l a s t t r a n s f o r m a t i o n by t h e pregnancy serum ( F i g 4,5 and 6 ) . The same o b s e r v a t i o n s w e r e a l s o made i n Con A i n d u c e d c u l t u r e s . E f f e c t of p r e c i p i t a t e d serum p r o t e i n s a f t e r hormone e x t r a c t i o n on normal lymphocyte c u l t u r e : A f t e r t h e e x t r a c t i o n of hormones f o r R I A ,
the p e l l e t
c o n t a i n i n g t h e r e s i d u a l serum p r o t e i n s w a s a n a l y z e d f o r i t s e f f e c t on lymphocyte c u l t u r e s t o a s c e r t a i n whether t h e serum, d e p l e t e d of s t e r o i d hormones, s t i l l h a s any i n h i b i t o r y a c t i v i t y on normal lymphocyte c u l t u r e .
No s i g n i f i c a n t r e d u c t i o n i n t h e s u p p r e s s i v e a c t i v i t y of t h e s t e r o i d d e p l e t e d serum w a s observed. DISCUSSION
The immunosuppressive r o l e o f s t e r o i d hormones, p a r t i c u l a r l y e s t r o g e n , p r o g e s t e r o n e and c o r t i s o l , i n g e n e r a l
a s w e l l a s d u r i n g pregnancy has been r e p o r t e d t o be c o n f l i c t ing. The serum l e v e l s o f p r o g e s t e r o n e , e s t r o g e n and c o r t i s o l i n c r e a s e c o n s i d e r a b l y d u r i n g pregnancy. It was interesting t o f i n d o u t w h e t h e r i n c r e a s e d l e v e l s of s t e r o i d s had any r e l a t i o n w i t h lymphocyte t r a n s f o r m a t i o n r a t e or t h e s u p p r e s s -
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
S T E R O I D HORMONES DURING PREGNANCY
80
-
20
I
FIGURE 4 Correlation between Estradiol level and % suppression of PHA induced normal lymphocyte transformation.
100
80
-
60
-
40 -
20 -
I
0
FIGURE 5 Correlation between Progesterone level and % suppression of PHA induced normal lymphocyte transformation.
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
8
SALEEM ET AL.
40
-
20
-
0-
I
1
FIGURE 6
C o r r e l a t i o n between C o r t i s o l l e v e l and % s u p p r e s s i o n of PHA induced normal lymphocyte t r a n s f o r m a t i o n .
i v e e f f e c t on pregnancy serum. The p r e s e n t s t u d y f a i l e d t o o b s e r v e any c o r r e l a t i o n between s t e r o i d c o n c e n t r a t i o n i n pregnancy serum w i t h e i t h e r lymphocyte r e s p o n s i v e n e s s or immunosuppressive e f f e c t of serum d u r i n g pregnancy. This i s i n agreement w i t h t h e e a r l i e r r e p o r t of Yoshida e t a l . ,
(10)
who have shown t h a t t h e amount of s t e r o i d s p r e s e n t i n pregnancy serum d i d n o t i n h i b i t T - c e l l p r o l i f e r a t i o n .
Poskitt et al.,
( 8 ) found t h a t p r o g e s t e r o n e and e s t r o g e n s have l i t t l e e f f e c t
on t h e r e s p o n s e o f lymphocytes t o PHA o r PWM u n l e s s p r e s e n t i n h i g h c o n c e n t r a t i o n s , n o t u s u a l l y p r e s e n t d u r i n g pregnancy. The s u p p r e s s i v e e f f e c t of e s t r o g e n on immune c e l l f u n c t i o n was found o n l y a t h i g h p h y s i o l o g i c a l c o n c e n t r a t i o n s (5,6) w h i l e no s u p p r e s s i o n was o b s e r v e d by S z e k e r e s e t a l . ,
(4).
The work of
Kasakura (11) a l s o showed a l a c k of c o r r e l a t i o n between t h e c o r t i s o l l e v e l s i n pregnancy plasma and t h e i n h i b i t i o n of MLC d u r i n g pregnancy. Another a p p r o a c h w a s f o l l o w e d t o s t u d y t h e s u p p r e s s i v e e f f e c t of s t e r o i d hormones. The p e l l e t , which remained a f t e r t h e e x t r a c t i o n of s t e r o i d s from t h e serum, w a s a n a l y z e d f o r
STEROID HORMONES DURING PREGNANCY
9
i t s i n h i b i t o r y a c t i v i t y . The r e s u l t s d i d n o t show any d i f f e r e n c e i n t h e i n h i b i t o r y a c t i v i t y a f t e r t h e e x t r a c t i o n of t h e s t e r o i d s . T h i s r e a f f i r m s t h a t serum s t e r o i d hormones are n o t r e s p o n s i b l e f o r t h e o b s e r v e d i n h i b i t o r y a c t i v i t y of pregnancy serum. The p r e s e n t s t u d y s t r o n g l y s u g g e s t s t h a t t h e s t e r o i d
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
hormones
-
e s t r a d i o l , p r o g e s t e r o n e and c o r t i s o l
an immunosuppressive role.
-
do n o t p l a y
Although a number of s t u d i e s have
i n d i c a t e d t h a t s t e r o i d s are immunosuppressive i n v i t r o (5,6), t h e s e s t u d i e s u t i l i z e d much h i g h e r c o n c e n t r a t i o n o f s t e r o i d s t h a n t h e normal p h y s i o l o g i c a l l e v e l s . T h e r e f o r e , it h a s been s u g g e s t e d t h a t t h e hormones might b e r e s p o n s i b l e f o r l o c a l immunosuppression around t h e p l a c e n t a which c o n t a i n s enormous amount o f these s t e r o i d s .
Even k h i s p o s s i b i l i t y d o e s n o t
s e e m t o be t r u e a s Yoshida e t a l . ,
(10) have found t h a t t h e amounts of hormones p r e s e n t i n t h e k e t r o p l a c e n t a l serum d i d However, Stimson and not i n h i b i t T-cell proliferation. Hunter ( 1 2 ) proposed t h a t s t e r o i d s may a f f e c t t h e immune r e s p o n s e by i n d u c i n g t h e thymus t o produce immunoregulatory f a c t o r s o r immunoregulatory c e l l s . The e v i d e n c e p r e s e n t e d i n d i c a t e s t h a t t h e i n c r e a s i n g amount o f s t e r o i d hormones d u r i n g pregnancy p l a y a p h y s i o l o g i c a l s u p p o r t i v e r o l e r a t h e r t h a n an immunological r o l e . ACKNOWLEDGMENTS M.A.
Saleem w a s s u p p o r t e d by a f e l l o w s h i p of t h e
Directorate G e n e r a l o f H e a l t h S e r v i c e s . The a u t h o r s a r e
g r a t e f u l t o t h e World H e a l t h O r g a n i s a t i o n f o r p r o v i d i n g R I A r e a g e n t s and wish t o t h a n k D r . J . K .
D u t t a and D r . S. W i s e 1
f o r t h e i r help i n e s t r a d i o l assay. REFERENCES
2.
Wyle and J . R . Kent, C l i n . Exp. Immunol., 27, 407-415 (1977). J. Mendelsohn, M.M. M u l t e r and J . L . B e r n h e h , C l i n . Exp.
3.
P.K.
1.
F.A.
Immunol., 27, 127-134 (1977). S i i t e r i , F . F e b r e s , L . E . Clemens, R . J .
B. Gondos and D. S t i t e s , Ann. N . Y . 397 (1977).
Chang,
Acad. S c i . ,
286,
384-
SALEEM ET AL.
10 4.
J. S z e k e r e s , V.
C s e r n u s , S. P e j t s i k , L. Emody a n d A . S .
P a s c a s , J . Reprod.
Imunol.,
3,
5.
R.I. S c h i f f , D. Mercier and R.H. 20, 69-80 (1975).
6.
L.E.
Clemens, P.K.
333-339 (1981). Buckly, C e l l .
S i i t e r i and D.P.
Immunol.,
S t i t e s , J . Immunol.,
122, -
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
7. 8.
1978-1985 (1979). Y. Tomoda, M . Fuma, T. M i w a , N. S a i k i a n d N . I s h i z u k a , Gynecol. I n v e s t . , L, 280-282 (1976). P.K.F.
Poskitt, E.A. S b a r r a a n d G.W.
A.J.
K u r t , B.B.
Paul, R.J.
Selvaraj,
M i t c h e l l J r . , O b s t e t . Gynecol,
5 0 , 319-321 (1977). 9.
WHO programme f o r t h e p r o v i s i o n of m a t c h e d a s s a y r e a g e n t s
f o r t h e radioimmunoassay of hormones i n R e p r o d u c t i v e P h y s i o l o g y Method Manual, T h i r t e e n t h e d i t i o n ,
10.
N. Y o s h i d a ,
A.
Uchida,
(1989).
H. K a n z a k i , M. T o k u s h i g e , S . S a t o , M . K a r i y a , S . Kasakura a n d T. Mori, Am.
J . Reprod.
19, 141-145 (1989). 11.
S. K a s a k u r a , N a t u r e ,
12.
W.H.
S t i m s o n a n d I.C.
4, 27-31 (1980).
246,
496-497 (1973).
H u n t e r , J . C l i n . Lab. Med.,
Immunol
1-10 (1992)
STUDIES ON THE IMMUNOSUPPRESSIVE ROLE OF STEROID HORMONES
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
DURING PREGNANCY
M.A.Saleem. , P . J h a . , K . Bucksheel and A.Farooq* Departments of Reproductive Biology and O b s t e t r i c s and Gynecology1 A l l I n d i a I n s t i t u t e o f Medical S c i e n c e s , New Delhi-110029, I N D I A . ABSTRACT I t h a s been r e c o g n i s e d t h a t s t e r o i d s c a n e x e r t a profound i n f l u e n c e o v e r immunological r e a c t i v i t y . The p r e s e n t s t u d y a n a l y z e s t h e r o l e of s t e r o i d hormones e s t r o g e n , p r o g e s t e r o n e and c o r t i s o l - and t h e i r i n v o l v e ment i n immunoregulation d u r i n g pregnancy. A s i s known, t h e endogenous l e v e l s of a l l t h e t h r e e hormones i n c r e a s e d u r i n g pregnancy. When t h e s t e r o i d l e v e l s i n pregnancy serum w e r e c o r r e l a t e d w i t h t h e lymphocyte r e s p o n s e t o mitogen, no c o r r e l a t i o n w a s o b s e r v e d . The s u p p r e s s i v e e f f e c t o f pregnancy serum w a s found t o have no c o r r e l a t i o n with i t s s t e r o i d content. I n general, s t e r o i d s did not seem t o a f f e c t t h e m a t e r n a l immune system a s e v i d e n c e d by t h e p r e s e n t s t u d y . INTRODUCTION
The mechanism by which t h e f e t u s , which i s e s s e n t i a l l y an a l l o g r a f t , i s a b l e t o s u r v i v e a g a i n s t t h e immunologic d e f e n c e s o f t h e mother i s one of the less u n d e r s t o o d phenomena I t h a s been r e p o r t e d t h a t t h e r e i s i n p r e s e n t day immunology.
a b l o c k i n g of t h e m a t e r n a l immune system by t h e r e g u l a t o r y f a c t o r s p r e s e n t i n t h e b l o o d , p a r t i c u l a r l y s t e r o i d s , which i n d u c e changes i n t h e immunological a c t i v i t y i n t h e m a t e r n a l h o s t d u r i n g pregnancy. I n g e n e r a l , s t e r o i d hormones-progesterone, e s t r o g e n i n h i b i t normal T - c e l l p r o l i f e r a t i o n by and c o r t i c o s t e r o i d s v a r i o u s a g e n t s such a s PHA, Con A o r PPD (1,2). S i g n i f i c a n t
-
*To whom a l l c o r r e s p o n d e n c e should be a d d r e s s e d .
Copyright 0 1992 by Marcel Dekker, Inc.
2
SALEEM ET AL.
s u p p r e s s i o n by p r o g e s t e r o n e w a s o b s e r v e d on many o c c a s i o n s ( 3 , 4 ) . But i n h i b i t i o n of mitogen, a n t i g e n and a l l o g e n i c c e l l a c t i v a t i o n by e s t r o g e n w a s found o n l y a t h i g h p h y s i o l o g i c a l l e v e l s ( 5 , 6 ) . Marked r e d u c t i o n i n PHA t r a n s f o r m a t i o n o f lymphocytes was shown by Tomoda e t a l . ,
(7) when c o r t i c o s t e r o i d s were added t o t h e
cultures.
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
On t h e c o n t r a r y , it h a s a l s o been r e p o r t e d t h a t serum and p l a c e n t a l l e v e l s o f s t e r o i d s a r e too low t o have s i g n i f i c a n t e f f e c t on lymphocytes (5,7). P o s k i t t e t a l . , ( 8 ) found t h a t p r o g e s t e r o n e and e s t r o g e n s have l i t t l e e f f e c t on t h e r e s p o n s e of lymphocytes t o PHA and PWM u n l e s s p r e s e n t i n h i g h c o n c e n t r a t i o n s n o t u s u a l l y p r e s e n t i n pregnancy blood.
Although profound
q u a l i t a t i v e and q u a n t i t a t i v e changes o c c u r d u r i n g pregnancy, t h e e f f e c t of t h e s e changes on t h e m a t e r n a l immune system i s n o t
w e l l u n d e r s t o o d and r e q u i r e s f u r t h e r i n v e s t i g a t i o n . MATERIALS AND METHODS Blood samples: Blood samples from p r e g n a n t women were c o l l e c t e d from t h e c l i n i c s of t h e Department o f O b s t e t r i c s and Gynecology, A.I.I.M.S.,
New D e l h i .
Blood from v o l u n t a r y d o n o r s s e r v e d a s
c o n t r o l . The blood samples w e r e c o l l e c t e d i n screw-capped t u b e s w i t h h e p a r i n (Centron Laboratories, Bombay, I n d i a ) f o r lymphocyte i s o l a t i o n and w i t h o u t h e p a r i n f o r serum s e p a r a t i o n . Lymphocyte c u l t u r e s : Lymphocytes w e r e i s o l a t e d by t h e F i c o l l Hypaque d e n s i t y g r a d i e n t method. The c u l t u r e s were done i n 96 w e l l m i c r o t i t r e p l a t e s (Nunc, Denmark). C e l l s u s p e n s i o n (50 u l ) c o n t a i n i n g 0.2 t o 0.3 m i l l i o n c e l l s w a s t a k e n f o r c u l t u r e . A s t a n d a r d i s e d optimum c o n c e n t r a t i o n of PHA and Con A was added t o each w e l l and t h e t o t a l volume o f t h e c u l t u r e s w a s a d j u s t e d t o 0.25 m l w i t h RPMI-1640 medium (Gibco L a b o r a t o r i e s , USA).
Wells w i t h o u t mitogen s e r v e d as c o n t r o l s . The c u l t u r e s w e r e i n c u b a t e d a t 37OC w i t h 5% C 0 2 . A f t e r 48 h o u r s 0.5 uCi of t r i t i a t e d thymidine (Bhabha A t o m i c Research C e n t r e , Bombay, I n d i a ) was added t o e a c h w e l l and t h e c u l t u r e s w e r e t e r m i n a t e d 2 4 h o u r s a f t e r thymidine i n c o r p o r a t i o n . The c e l l s w e r e h a r v e s t e d on a Whatman GF/C f i l t e r p a p e r u s i n g a c e l l h a r v e s t e r (Nunc, D e n m a r k ) . The f i l t e r p a p e r w a s t r a n s f e r r e d t o a s c i n t i l l a t i o n v i a l and 8 m l o f s i m p l e s c i n t i l l a t i o n m i x t u r e (POPOP 100 mg and PPO 4 9 p e r l i t r e s u l f u r - f r e e t o l u e n e ) was
STEROID HORMONES DURING PREGNANCY
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
added t o e a c h v i a l . The r a d i o a c t i v i t y p r e s e n t i n t h e v i a l s w a s counted i n a LKB Wallac Minibeta l i q u i d s c i n t i l l a t i o n counter. The p r o l i f e r a t i v e c a p a c i t y of t h e lymphocytes w a s analyzed as follows: Counts i n cpm o f c e l l s w i t h mitogen ( T ) Transformation index= Counts i n cpm of c e l l s w i t h o u t mitogen ( C ) The p e r c e n t a g e t r a n s f o r m a t i o n of lymphocytes from p r e g n a n t p a t i e n t s w a s c a l c u l a t e d by t a k i n g t h e t r a n s f o r m a t i o n i n d e x of non-pregnant c o n t r o l s as 1 0 0 % . E f f e c t o f pregnancy serum on normal lymphocyte c u l t u r e s : _
~
_
Normal lymphocyte c u l t u r e s were done as d e s c r i b e d e a r l i e r
and 1 0 % pregnancy serum w a s added t o each w e l l . C u l t u r e s w i t h o u t pregnancy serum s e r v e d as controls. By t a k i n g t h e p e r c e n t t r a n s f o r m a t i o n i n c u l t u r e s w i t h o u t pregnancy serum a s 0% s u p p r e s s i o n , t h e p e r c e n t s u p p r e s s i o n i n c u l t u r e s w i t h
pregnancy serum w a s c a l c u l a t e d . Radioimmunoassay o f hormones ( R I A ) : E s t i m a t i o n o f s t e r o i d hormones 1 7 - f i e s t r a d i o l , p r o g e s t e r o n e and c o r t i s o l w a s c a r r i e d o u t a c c o r d i n g t o t h e WHO method manual ( 9 ) . Within and between a s s a y c o e f f i c i e n t s of v a r i a t i o n f o r e s t r a d i o l , p r o g e s t e r o n e and c o r t i s o l were found t o be 7 . 8 % and 10.2%, 9 . 3 % and 1 0 . 9 % and 9 . 2 % and 10.8%, respectively. A n a l y s i s of t h e s u p p r e s s i v e e f f e c t o f serum a f t e r t h e e x t r a c t i o n of hormones: A f t e r t h e e x t r a c t i o n of hormones f o r R I A from t h e serum, t h e r e s i d u e c o n t a i n i n g serum p r o t e i n s w a s once a g a i n a n a l y z e d f o r i t s s u p p r e s s i v e e f f e c t on lymphocyte c u l t u r e s . The r e s i d u e w a s f i r s t d i s s o l v e d i n RPMI-1640 medium and added t o normal lymphocyte c u l t u r e s t o s t u d y i t s e f f e c t . RESULTS
S t e r o i d hormone p r o f i l e d u r i n g pregnancy: Endogenous l e v e l s of e s t r o g e n , p r o g e s t e r o n e and c o r t i s o l were e s t i m a t e d by R I A i n pregnancy sera from d i f f e r e n t trimesters of pregnancy and t h e r e s u l t s a r e summarized i n Table I .
3
SALEEM ET AL.
4
TABLE I
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
S t e r o i d Hormone L e v e l s (mean
ESTRADIOL
3154
(pmol/l)
t
1183
5 SD)
6558
(25)
PROGESTERONE 20.84
+
CORTISOL
411.4
33.42
+
1768
11003
+
146.44
+
616
5
3886
(22)
7.39
48.87
( i 81
( 2 01
(nmOl/l)
+
(19)
5.72
( i 61
(nmol/l)
During Normal Pregnancy
189.51
769
+
4.25
(is) + 266.52 (301
(i7)
F i g u r e s i n p a r e n t h e s e s r e p r e s e n t t h e number of samples s t u d i e d .
120 100
-
80 - ' *
.
.
60 40
-
..
20 n " 0
1
2
3
4
5
6
ESTRADIOL LEVELS ng/ml FIGURE 1 C o r r e l a t i o n b e t w e e n E s t r a d i o l l e v e l a n d PHA i n d u c e d lymphocyte t r a n s f o r m a t i o n .
STEROID HORMONES DURING PREGNANCY
5
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
120
100
-
80
-
80
-
. . ..
. '
t ,
0'
0
20
10
30
40
50
PROGESTERONE LEVEL (nmol\l) FIGURE 2
Correlation between Progesterone level and PHA induced lymphocyte transformation.
100
-
..
m
..
.
80 60
-
40
-
2o 0
t 0
I
I
100
200
, 300
400
CORTISOL LEVEL (nmol\l) FIGURE 3
Correlation between Cortisol level and PHA induced lymphocyte transformation.
500
6
SALEEM ET AL.
C o r r e l a t i o n of s t e r o i d l e v e l s w i t h lymphocyte t r a n s f o r m a t i o n : The l e v e l s o f s t e r o i d hormones, e s t r a d i o l , p r o g e s t e r o n e and c o r t i s o l , were compared w i t h the p r o l i f e r a t i v e c a p a c i t y of lymphocytes from t h e same s u b j e c t t o f i n d whether t h e r e w a s any c o r r e l a t i o n between t h e hormone l e v e l s and t h e lymphocyte t r a n s f o r m a t i o n . F i g u r e s . 1 , 2 and 3 show t h e r e l a t i o n between
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
t h e hormone l e v e l s and t h e PHA i n d u c e d t r a n s f o r m a t i o n of lymphocytes from p r e g n a n t women.
N o c o r r e l a t i o n w a s found
between t h e l e v e l s o f s t e r o i d hormones and t h e p a t t e r n o f lymphocyte t r a n s f o r m a t i o n .
The same o b s e r v a t i o n w a s made i n
Con A induced c u l t u r e s . S u p p r e s s i v e e f f e c t o f pregnancy serum i n r e l a t i o n t o s t e r o i d levels: A f t e r e s t i m a t i n g t h e hormone l e v e l s i n pregnancy sera t h e samples w e r e a n a l y z e d f o r t h e i r e f f e c t o n normal lymphoc y t e c u l t u r e s . The i n h i b i t o r y e f f e c t of t h e s e pregnancy
sera was once a g a i n compared w i t h t h e serum l e v e l s of s t e r o i d hormones. The r e s u l t s i n d i c a t e a l a c k of c o r r e l a t i o n between s t e r o i d l e v e l s and i n h i b i t i o n o f PHA i n d u c e d normal lymphocyte b l a s t t r a n s f o r m a t i o n by t h e pregnancy serum ( F i g 4,5 and 6 ) . The same o b s e r v a t i o n s w e r e a l s o made i n Con A i n d u c e d c u l t u r e s . E f f e c t of p r e c i p i t a t e d serum p r o t e i n s a f t e r hormone e x t r a c t i o n on normal lymphocyte c u l t u r e : A f t e r t h e e x t r a c t i o n of hormones f o r R I A ,
the p e l l e t
c o n t a i n i n g t h e r e s i d u a l serum p r o t e i n s w a s a n a l y z e d f o r i t s e f f e c t on lymphocyte c u l t u r e s t o a s c e r t a i n whether t h e serum, d e p l e t e d of s t e r o i d hormones, s t i l l h a s any i n h i b i t o r y a c t i v i t y on normal lymphocyte c u l t u r e .
No s i g n i f i c a n t r e d u c t i o n i n t h e s u p p r e s s i v e a c t i v i t y of t h e s t e r o i d d e p l e t e d serum w a s observed. DISCUSSION
The immunosuppressive r o l e o f s t e r o i d hormones, p a r t i c u l a r l y e s t r o g e n , p r o g e s t e r o n e and c o r t i s o l , i n g e n e r a l
a s w e l l a s d u r i n g pregnancy has been r e p o r t e d t o be c o n f l i c t ing. The serum l e v e l s o f p r o g e s t e r o n e , e s t r o g e n and c o r t i s o l i n c r e a s e c o n s i d e r a b l y d u r i n g pregnancy. It was interesting t o f i n d o u t w h e t h e r i n c r e a s e d l e v e l s of s t e r o i d s had any r e l a t i o n w i t h lymphocyte t r a n s f o r m a t i o n r a t e or t h e s u p p r e s s -
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
S T E R O I D HORMONES DURING PREGNANCY
80
-
20
I
FIGURE 4 Correlation between Estradiol level and % suppression of PHA induced normal lymphocyte transformation.
100
80
-
60
-
40 -
20 -
I
0
FIGURE 5 Correlation between Progesterone level and % suppression of PHA induced normal lymphocyte transformation.
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
8
SALEEM ET AL.
40
-
20
-
0-
I
1
FIGURE 6
C o r r e l a t i o n between C o r t i s o l l e v e l and % s u p p r e s s i o n of PHA induced normal lymphocyte t r a n s f o r m a t i o n .
i v e e f f e c t on pregnancy serum. The p r e s e n t s t u d y f a i l e d t o o b s e r v e any c o r r e l a t i o n between s t e r o i d c o n c e n t r a t i o n i n pregnancy serum w i t h e i t h e r lymphocyte r e s p o n s i v e n e s s or immunosuppressive e f f e c t of serum d u r i n g pregnancy. This i s i n agreement w i t h t h e e a r l i e r r e p o r t of Yoshida e t a l . ,
(10)
who have shown t h a t t h e amount of s t e r o i d s p r e s e n t i n pregnancy serum d i d n o t i n h i b i t T - c e l l p r o l i f e r a t i o n .
Poskitt et al.,
( 8 ) found t h a t p r o g e s t e r o n e and e s t r o g e n s have l i t t l e e f f e c t
on t h e r e s p o n s e o f lymphocytes t o PHA o r PWM u n l e s s p r e s e n t i n h i g h c o n c e n t r a t i o n s , n o t u s u a l l y p r e s e n t d u r i n g pregnancy. The s u p p r e s s i v e e f f e c t of e s t r o g e n on immune c e l l f u n c t i o n was found o n l y a t h i g h p h y s i o l o g i c a l c o n c e n t r a t i o n s (5,6) w h i l e no s u p p r e s s i o n was o b s e r v e d by S z e k e r e s e t a l . ,
(4).
The work of
Kasakura (11) a l s o showed a l a c k of c o r r e l a t i o n between t h e c o r t i s o l l e v e l s i n pregnancy plasma and t h e i n h i b i t i o n of MLC d u r i n g pregnancy. Another a p p r o a c h w a s f o l l o w e d t o s t u d y t h e s u p p r e s s i v e e f f e c t of s t e r o i d hormones. The p e l l e t , which remained a f t e r t h e e x t r a c t i o n of s t e r o i d s from t h e serum, w a s a n a l y z e d f o r
STEROID HORMONES DURING PREGNANCY
9
i t s i n h i b i t o r y a c t i v i t y . The r e s u l t s d i d n o t show any d i f f e r e n c e i n t h e i n h i b i t o r y a c t i v i t y a f t e r t h e e x t r a c t i o n of t h e s t e r o i d s . T h i s r e a f f i r m s t h a t serum s t e r o i d hormones are n o t r e s p o n s i b l e f o r t h e o b s e r v e d i n h i b i t o r y a c t i v i t y of pregnancy serum. The p r e s e n t s t u d y s t r o n g l y s u g g e s t s t h a t t h e s t e r o i d
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
hormones
-
e s t r a d i o l , p r o g e s t e r o n e and c o r t i s o l
an immunosuppressive role.
-
do n o t p l a y
Although a number of s t u d i e s have
i n d i c a t e d t h a t s t e r o i d s are immunosuppressive i n v i t r o (5,6), t h e s e s t u d i e s u t i l i z e d much h i g h e r c o n c e n t r a t i o n o f s t e r o i d s t h a n t h e normal p h y s i o l o g i c a l l e v e l s . T h e r e f o r e , it h a s been s u g g e s t e d t h a t t h e hormones might b e r e s p o n s i b l e f o r l o c a l immunosuppression around t h e p l a c e n t a which c o n t a i n s enormous amount o f these s t e r o i d s .
Even k h i s p o s s i b i l i t y d o e s n o t
s e e m t o be t r u e a s Yoshida e t a l . ,
(10) have found t h a t t h e amounts of hormones p r e s e n t i n t h e k e t r o p l a c e n t a l serum d i d However, Stimson and not i n h i b i t T-cell proliferation. Hunter ( 1 2 ) proposed t h a t s t e r o i d s may a f f e c t t h e immune r e s p o n s e by i n d u c i n g t h e thymus t o produce immunoregulatory f a c t o r s o r immunoregulatory c e l l s . The e v i d e n c e p r e s e n t e d i n d i c a t e s t h a t t h e i n c r e a s i n g amount o f s t e r o i d hormones d u r i n g pregnancy p l a y a p h y s i o l o g i c a l s u p p o r t i v e r o l e r a t h e r t h a n an immunological r o l e . ACKNOWLEDGMENTS M.A.
Saleem w a s s u p p o r t e d by a f e l l o w s h i p of t h e
Directorate G e n e r a l o f H e a l t h S e r v i c e s . The a u t h o r s a r e
g r a t e f u l t o t h e World H e a l t h O r g a n i s a t i o n f o r p r o v i d i n g R I A r e a g e n t s and wish t o t h a n k D r . J . K .
D u t t a and D r . S. W i s e 1
f o r t h e i r help i n e s t r a d i o l assay. REFERENCES
2.
Wyle and J . R . Kent, C l i n . Exp. Immunol., 27, 407-415 (1977). J. Mendelsohn, M.M. M u l t e r and J . L . B e r n h e h , C l i n . Exp.
3.
P.K.
1.
F.A.
Immunol., 27, 127-134 (1977). S i i t e r i , F . F e b r e s , L . E . Clemens, R . J .
B. Gondos and D. S t i t e s , Ann. N . Y . 397 (1977).
Chang,
Acad. S c i . ,
286,
384-
SALEEM ET AL.
10 4.
J. S z e k e r e s , V.
C s e r n u s , S. P e j t s i k , L. Emody a n d A . S .
P a s c a s , J . Reprod.
Imunol.,
3,
5.
R.I. S c h i f f , D. Mercier and R.H. 20, 69-80 (1975).
6.
L.E.
Clemens, P.K.
333-339 (1981). Buckly, C e l l .
S i i t e r i and D.P.
Immunol.,
S t i t e s , J . Immunol.,
122, -
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
7. 8.
1978-1985 (1979). Y. Tomoda, M . Fuma, T. M i w a , N. S a i k i a n d N . I s h i z u k a , Gynecol. I n v e s t . , L, 280-282 (1976). P.K.F.
Poskitt, E.A. S b a r r a a n d G.W.
A.J.
K u r t , B.B.
Paul, R.J.
Selvaraj,
M i t c h e l l J r . , O b s t e t . Gynecol,
5 0 , 319-321 (1977). 9.
WHO programme f o r t h e p r o v i s i o n of m a t c h e d a s s a y r e a g e n t s
f o r t h e radioimmunoassay of hormones i n R e p r o d u c t i v e P h y s i o l o g y Method Manual, T h i r t e e n t h e d i t i o n ,
10.
N. Y o s h i d a ,
A.
Uchida,
(1989).
H. K a n z a k i , M. T o k u s h i g e , S . S a t o , M . K a r i y a , S . Kasakura a n d T. Mori, Am.
J . Reprod.
19, 141-145 (1989). 11.
S. K a s a k u r a , N a t u r e ,
12.
W.H.
S t i m s o n a n d I.C.
4, 27-31 (1980).
246,
496-497 (1973).
H u n t e r , J . C l i n . Lab. Med.,
Immunol
