Antibody regulation in birds by thyroid hormones

DEVELOPMENTAL AND COMLPARATIVE IM~CUNOLOGY, Vol. 4, pp. 323-330, 1980. 0145-305X/80/020323-08502.00/0 Printed in the USA. Copyright (c) 1980 Pergamon Press Ltd. All rights reserved.

ANTIBODY

REGULATION

BY T H Y R O I D

D. Keast Department University Queen

HORMONES

and D.J.

Ayre

of M i c r o b i o l o g y

of W e s t e r n

Elizabeth

NEDLANDS,

IN BIRDS

Australia

II M e d i c a l

Western

Centre

Australia,

6009

ABSTRACT It has b e e n p r e v i o u s l y shown that the t h y r o i d g l a n d and its a s s o c i a t e d h o r m o n e s are able to i n f l u e n c e m a m m a l i a n immune responses. The p u r p o s e of this study was to d e t e r m i n e w h e t h e r h u m o r a l immune r e s p o n s e s are under similar c o n t r o l in avian systems. We r e p o r t that d u r i n g the p r i m a r y r e s p o n s e to sheep e r y t h r o c y t e s (SRBC), s e r u m a n t i b o d y titres and d e p r e s s i o n of serum T3 levels are s i g n i f i c a n t l y correlated. We also show that c h e m i c a l m o d i f i c a t i o n of t h y r o i d f u n c t i o n inhibits the p r o d u c t i o n of serum a n t i b o d y titres, f o l l o w i n g b o t h p r i m a r y and s e c o n d a r y c h a l l e n g e w i t h SRBC. However, s e r u m a n t i b o d y titres w e r e found to rise d r a m a t i c a l l y w i t h i n 48 h of c e s s a t i o n of treatment. These results i n d i c a t e that T3 may play an i m p o r t a n t role as a r e g u l a t o r of avian h u m o r a l immune responses.

INTRODUCTION

It has b e e n k n o w n for m a n y years that the d e v e l o p m e n t of m a m m a l i a n immune systems is d e p e n d e n t upon the t h y r o i d gland (i). M o r e recently, the role of the t h y r o i d hormones,

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ANTIBODY REGULATION IN BIRDS

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t h y r o x i n e (T4) and t r i o d o t h y r o n i n e (T3), as r e g u l a t o r s of immune r e s p o n s e s has been f r u i t f u l l y investigated. Fabris (2) d e m o n s t r a t e d that t h y r o i d e c t o m y of rats p r o d u c e d i m m u n o - d e p r e s s i o n , w h i l s t i n j e c t e d doses of thyroxine p r o d u c e d immune enhancement. Panousopoulos et aZ, (3) c l a i m further that the n u m b e r of a n t i b o d y p r o d u c i n g cells and serum a n t i b o d y titres induced by sheep e r y t h r o c y t e (SRBC) inoculation, vary i n d e p e n d e n t l y in response to changes of thyroid status. In the avian s y s t e m (4), there is also e v i d e n c e to suggest that thyroid a c t i v i t y is r e f l e c t e d in v a r i a t i o n in the l y m p h o i d system. S e a s o n a l v a r i a t i o n of thyroid hormone levels, a s s o c i a t e d with moulting, are well c o r r e l a t e d w i t h changes of thymus w e i g h t in w i l d p h e a s a n t s and a d m i n i s t r a t i o n of large doses of t h y r o x i n e induces both thymic e n l a r g e m e n t and m o u l t i n g in the d o m e s t i c fowl (4). In o r d e r to test the h y p o t h e s i s that a l t e r e d T3 a n d / o r T4 levels were a s s o c i a t e d w i t h changes in the immune r e s p o n s i v e n e s s and a n t i b o d y p r o d u c t i o n in birds, we m o n i t o r e d s e r u m T3 and T4 and h a e m a g g l u t i n a t i n g a n t i b o d y titres in 'thyroid inhibited' and 'normal' b i r d s f o l l o w i n g primary i n o c u l a t i o n with SRBC. In a s e c o n d series of e x p e r i m e n t s the a b i l i t y of 'thyroid inhibited' birds to m o u n t a s e c o n d a r y immune r e s p o n s e to SRBC was investigated.

M A T E R I A L S AND M E T H O D S Birds A d u l t b u d g e r y g a h s (Melopsittacus undulatus) and b a r b a r y doves (Streptopelia risona) of both sexes w e r e m a i n t a i n e d in large f r e e - f l y i n g aviaries. SRBC SRBC w e r e c o l l e c t e d in A l s e v e r s solution, s t o r e d at 4°C and used w i t h i n 6 days of collection. Prior to inoculation, SRBC were w a s h e d 3 times in p h o s p h a t e b u f f e r e d saline. I n o c u l a t i o n and s a m p l i n g p r o t o c o l s Three groups of 12 b u d g e r y g a h s w e r e c h o s e n at r a n d o m from a b r e e d i n g colony. G r o u p i, a c o n t r o l group, r e c e i v e d a single i n t r a - p e r i t o n e a l (i/p) i n j e c t i o n of 0.i ml saline at day 0. G r o u p s 2 and 3 r e c e i v e d single a n t i g e n i c doses of SRBC (0.i mls 10% SRBC) by i/p i n j e c t i o n at day 0. In addition, group 3 birds r e c e i v e d daily i / p

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ANTIBODY REGULATION IN BIRDS

i n o c u l a t i o n s of t h i o u r e a (7 ~g/0.1 mls 89% EtOH) from day -i to day 4. Three birds from each group w e r e e x a n g u i n a t e d at days i, 2, 4 and 6 and sera stored at -20°C. S a m p l i n g was always c o n d u c t e d b e t w e e n 10.30 and 11.30 a.m. Seven b a r b a r y doves were h y p e r i m m u n i z e d by i/p i n o c u l a t i o n (0.1 ml 10% SRBC) at days 0, l0 and 20. On day 37 the birds were given t h i o u r e a i/p (15 ug/100 gm body wt.). This t r e a t m e n t was c o n t i n u e d over a 7 day period. On day 38 the birds were given a fourth i/p i n o c u l a t i o n of SRBC. B l o o d samples were c o l l e c t e d from the w i n g vein as i n d i c a t e d in Table i. All b l o o d samples w e r e a s s a y e d for h a e m a g g l u t i n a t i n g a n t i b o d y levels u s i n g 2% SRBC and a s t a n d a r d technique. Ts, T4 d e t e r m i n a t i o n s T3 and T4 levels w e r e d e t e r m i n e d for all b u d g e r y g a h s e r u m samples. H o r m o n e levels w e r e determined, single for T3 and in d u p l i c a t e for T4, using an 1 2 5 I - r a d i o i m m u n o assay t e c h n i q u e (Abbott's D i a g n o s t i c Kits 2635 and 5927).

RESULTS B u d g e r y g a h s g i v e n a single i n o c u l a t i o n of SRBC (Group 2) e x h i b i t e d a d e p r e s s i o n of serum T3 levels w h i c h r e a c h e d a low of 0.4 ng/ml 48 h after i n i t i a t i o n of the p r i m a r y immune response. This c o i n c i d e d with the o n s e t of d e t e c t a b l e a n t i b o d y p r o d u c t i o n (Fig. i). There w e r e no s i g n i f i c a n t d e t e c t a b l e changes in T4 levels over the course of the p r i m a r y immune r e s p o n s e in these birds. A s i g n i f i c a n t d e p r e s s i o n of both serum T3 and T4 levels was produced, and m a i n t a i n e d in the t h i o u r e a treated birds. D u r i n g this time little or no d e t e c t a b l e a n t i b o d y r e s p o n s e was m a d e to SRBC. Once the t h i o u r e a t r e a t m e n t was w i t h d r a w n T4 levels r e t u r n e d to normal but T3 levels r e m a i n e d reduced. This c o n t i n u e d r e d u c t i o n c o i n c i d e d with the rapid p r o d u c t i o n of a n t i b o d y to levels greater than those e x h i b i t e d at the peak of the control (Group 2) p r i m a r y response. All results w e r e s t a n d a r d i s e d a g a i n s t the s a l i n e - r e c e i v i n g control group (Group i) w h i c h w o u l d n e g a t e any i n f l u e n c e s to h o r m o n e levels of h a n d l i n g procedures. G r o u p 1 T3 and T4 values showed little variation, the m e a n values ± s t a n d a r d errors w e r e 1.73 ± 0.07 ng/ml and 0.93 ± 0.06 ~g/100 ml respectively.

325

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ANTIBODY REGULATION IN BIRDS

Vol. 4, No. 2

3 14 -,-4

2 0

1 ~4 O O

-4

-0.2 -0.4

4

-0.6

..0

-0.2 O O

~4

-0.4

v

-0.6 -0.8

A v

1

2

3 DAYS

Fig.

4 AFTER

A

•

5

6

INOCULATION

i.

V a r i a t i o n s of s e r u m a n t i - S R B C h a e m a g g l u t i n a t i n g a n t i b o d y t i t r e s of the p r i m a r y i m m u n e r e s p o n s e of 'normal' and t h i o u r e a t r e a t e d b u d g e r y g a h s , and c h a n g e s in s e r u m T3 and T4 l e v e l s up to 6 d a y s a f t e r i/p i n o c u l a t i o n . A n t i b o d y t i t r e s as s h o w n r e p r e s e n t the log2 m e a n ± s t a n d a r d e r r o r for g r o u p s of 3 b i r d s (shaded = 'normal'). / ~ T 3 a n d / ~ T 4 r e p r e s e n t the d i f f e r e n c e s b e t w e e n the s e r u m T3 a n d T4 l e v e l s of n o r m a l (group 2 - O) and t h i o u r e a t r e a t e d (group 3 - 0) b i r d s and a c o n t r o l at e a c h s a m p l e time.

mean

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ANTIBODY REGULATION IN BIRDS

327

It is clear (Fig. 2) that the m a g n i t u d e of the antibody titre produced is significantly c o r r e l a t e d with the extent to which T3 levels are depressed, b e l o w control values.

4

•o

•a

2

0 1

,

0,

a

0.2

0

•

-0.2

. . . . . -0.4

AT3 Fig.

-0.6

, -0.8

ng/100

-i.0

ml

2.

Comparison antibody against group,

of log2 SRBC serum h a e m a g g l u t i n a t i n g

titres

and

AT3

the day means Group

i).

birds,

after cessation

The plotted

for the saline

Results

the onset of antibody regression

2 values

P <0.05.

( •

of t h i o u r e a represents P <0.01).

the r e l a t i o n s h i p

calculated

receiving

are for G r o u p

production

(n = 12, Y = 3.85X-0.12, Group

(T3 depression,

control

2 birds,

from

), and Group

treatment all points

( •

3 ).

shown

For the 9

is Y = 3.62X-0.18,

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ANTIBODY REGULATION IN BIRDS

Vol. 4, No. 2

Barbary doves already hyperimmunised with SRBC but with low levels of circulating antibodies failed to m o u n t a f u r t h e r h u m o r a l r e s p o n s e w h e n c h a l l e n g e d during a period of thiourea treatment ( T a b l e i). H o w e v e r , o n c e s s a t i o n of t h e t h i o u r e a t r e a t m e n t a n t i b o d y synthesis commenced and levels two days later were the highest recorded. TABLE

1

Haemagglutination titres following intraperitoneal inoculation with SRBC t h i o u r e a of b a r b a r y d o v e s (Streptopelia

Days of

0

5

i0

14

18

20

22

24

SRBC i/p

+

-

+

-

-

+

-

-

Thiourea

.

.

Log 2 HA tit_re

+

days

÷

i/p

HA

ND

of

3.8

i/p

.

.

2.6

. 3.2

. 3.4

inoculation

inoculation

with

haemagglutination birds per sample vein.

15

.

32

.

3.4

37

t 3.4

with ~g

and

risona)

3.4

2.6

ND

38

44

46

daily

+

-

2

2

4

2 x 10 8 S R B C

thiourea

t i t r e as m e a n o f f r o m 2 to 7 blood was obtained from wing

DISCUSSION

The r e s u l t s o v e r a l l i n d i c a t e t h a t b o t h the p r i m a r y and s e c o n d a r y h u m o r a l r e s p o n s e , w i t h r e s p e c t to its a m p l i f i c a t i o n to d e t e c t a b l e levels, m a y be u n d e r the c o n t r o l s of T3. W h e t h e r this is as a d i r e c t m e t a b o l i c m e c h a n i s m of t h r o u g h i n t e r a c t i o n w i t h o t h e r h o r m o n a l p a t h w a y s (5, 6, 7) has n o t b e e n d e t e r m i n e d . T h i o u r e a , an e f f e c t i v e i n h i b i t o r of t h y r o i d f u n c t i o n in m a m m a l s (8), was f o u n d to p r o d u c e m a r k e d l o w e r i n g of T3 and T4 l e v e l s in b u d g e r y g a h s . Elevated serum antibody t i t r e s w e r e n o t d e t e c t e d in the t h i o u r e a - t r e a t e d b i r d s , g i v e n p r i m a r y and s e c o n d a r y a n t i g e n i c s t i m u l a t i o n , u n t i l thiourea treatment was discounted. In a d d i t i o n d e p r e s s i o n of s e r u m T3 l e v e l s of u n t r e a t e d b i r d s , m a k i n g a

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ANTIBODY REGULATION IN BIRDS

primary immune response, was found to be c o r r e l a t e d s i g n i f i c a n t l y with serum a n t i b o d y titre. We i n t e r p r e t these results to indicate that a n t i b o d y p r o d u c t i o n and p o s s i b l y l y m p h o c y t e p r o l i f e r a t i o n in h u m o r a l immune r e s p o n s e s is d e p e n d e n t upon the p r e s e n c e and/or a v a i l a b i l i t y of s u f f i c i e n t l y large amounts of free T3 (or T4 for conversion) (7). If the above is true it is also a p p a r e n t that d u r i n g the i n d u c t i o n of both the primary and s e c o n d a r y h u m o r a l immune response, s u f f i c i e n t i m m u n o g e n is r e t a i n e d in the t h i o u r e a - t r e a t e d birds to e n s u r e a h i g h l y s i g n i f i c a n t level of humoral a n t i b o d y once the T3/T4 c o n t r o l s are r e l e a s e d (Fig. i, Table i). This r e t e n t i o n time for i m m u n o g e n w o u l d be at least five days. An a l t e r n a t i v e e x p l a n a t i o n of the o b s e r v e d effects of t h i o u r e a treatment, on serum a n t i b o d y titres, is that such t r e a t m e n t increases c a t a b o l i s m of i m m u n o g l o b u l i n s u f f i c i e n t l y to p r e v e n t d e t e c t i o n of a n t i b o d y at the low titres c h a r a c t e r i s t i c of avian antiSRBC r e s p o n s e s (9). It c a n n o t be d e d u c e d from our e x p e r i m e n t s at w h a t stage in the progress of an immune response T3 first becomes c r i t i c a l l y important. H u l b e r t (i0) has r e v i e w e d the role of this hormone and suggests that it may be able to r e g u l a t e gene t r a n s l a t i o n and t r a n s c r i p t i o n at the r i b o s o m a l level. It is k n o w n (ii) that h u m a n l e u k o c y t e s possess n u c l e a r r e c e p t o r sites, h a v i n g specific a f f i n i t y for T3 and T4. P o t e n t i a l l y then T3 may play a d e c i s i v e role as a r e g u l a t o r and a m p l i f i e r of l y m p h o c y t e p r o l i f e r a t i o n a n d / o r a n t i b o d y p r o d u c t i o n in both m a m m a l i a n and avian systems.

ACKNOWLEDGEMENTS We thank Dr. S.J.J.F. Davies, D i r e c t o r of W.A. D i v i s i o n of W i l d l i f e Research, C.S.I.R.O., H e l e n a Valley, W e s t e r n Australia, for p r o v i s i o n of aviaries and facilities.

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i.

DOUGHERTY, T.F. E f f e c t of h o r m o n e s on lymphatic tissue. Physiol. R e v . 32, 379, 1952.

2.

FABRIS, N. I m m u n o d e p r e s s i o n in t h y r o i d - d e p r i v e d animals. C l i n . Exp. I m m u n o l . 15, 69, 1973.

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ANTIBODY REGULATION IN BIRDS

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3.

PANOUSSOPOULOS, D.G., HUMPHREY, P.A., HUMPHREY, and MEEK, J. Effect of various thyroid states on immunity. Surg. Forum. XXVII, 138, 1976.

4.

ANDERSON, W.L. Seasonal changes in thymus weights in ring-necked pheasants. The C o n d o r , 7_22, 205, 1970.

5.

DOUGHERTY, T.F., BERLINER, M.L. and BERLINER, D.L. Hormonal control of lymphocyte production and destruction. P r o g . S e m a t . 3, 155, 1962.

6.

AHLQUIST, J. Endocrine influences on lymphatic organs, immune responses, inflammation and autoimmunity. A c t a . E n d o c . 83, Suppl. 206, 1976.

7.

BESEDOVSKY, H., SORKIN, E., KELLER, M. and MOLLER, J. Changes in blood hormone levels during the immune response. P r o c . S o c . Exp. B i o l . M e d . 150, 466, 1975.

8.

CAMPBELL, D., LANDGREBE. F.W. and MORGAN, T.N. Pharmacology of Thiourea. The L a n c e t , i, 630, 1944.

9.

KEILY, D. and ABRAMOFF, P. The Chicken Immune Response III. Cellular and Humoral Antibody Production in the Splenectomised Chicken. J. I m m u n o l . 102, 1958, 1969.

i0.

HULBERT, A.J. The Thyroid Hormones : A thesis concerning their action. J. T h e o r . B i o l . 73, 81, 1978.

ii.

LIEWENDAHL, K., ROSENGARD, S. and LAMBERG, B.S. Nuclear binding of the tri-iodothyronine and thyroxine in lymphocytes from subjects with hyperthyroidism, hypothyroidism and resistance to thyroid hormones. C l i n . C h i m . A c t a . 8_~3, 41, 1978.

J.L.