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Thymus-dependent immune functions in chickens bursectomized with colchicine applied to the anal lips
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 7, pp. 525-534, 0145-305X/83 $3.00 +.00 Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.
1983.
T H Y M U S - D E P E N D E N T IMMUNE F U N C T I O N S IN C H I C K E N S B U R S E C T O M I Z E D WITH C O L C H I C I N E APPLIED TO THE A N A L LIPS
T . Romppanen, J. Eskola, O. Lassila, M . K . Viljanen and T . E . S o r v a r i D e p a r t m e n t of P a t h o l o g y , U n i v e r s i t y of Kuopio, Kuopio, and D e p a r t m e n t of Medical M i c r o b i o l o g y , U n i v e r s i t y of T u r k u , T u r k u , Finland
ABSTRACT
Thymus-dependent immune f u n c t i o n s were i n v e s t i g a t e d in c h i c k e n s b u r s e c t o m i z e d n e o n a t a l l y w i t h colchicine solution g i v e n p e r anum. A n t i b o d y responses to t h y m u s - d e p e n d e n t a n t i g e n s sheep red blood cells ( S R B C ) and human gammaglobulin ( H G G ) were d e l a y e d , r e a c h i n g the normal level a f t e r the t h i r d antigen stimulation. Also the mitogenic responses of p e r i p h e r a l blood l y m p h o c y t e s were p r e s e r v e d , and no changes in the t h y m i c m o r p h o l o g y were f o u n d . In contrast, a n t i b o d y responses to b u r s a - d e p e n d e n t a n t i g e n B r u c e l l a a b o r t u s were low and the switch of immunoglobulin i s o t y p e s from IgM to IgA and IgG was d i s t u r b e d . It can be c o n c l u d e d t h a t neonatal b u r s e c t o m y with cloacal a d m i n i s t r a tion of colchicine does not s i g n i f i c a n t l y a f f e c t T cell f u n c t i o n s , whereas B cell f u n c t i o n s are p a r t i a l l y d e f i c i e n t .
INTRODUCTION R e c e n t l y , a new model of chemical b u r s e c t o m y f o r neonatal c h i c k e n s was described, where colchicine solution applied p e r anum d e s t r o y e d both lymphoid cells and cells of t h e f o l l i c u l a r e p i t h e l i u m and f o l l i c l e - a s s o c i a t e d e p i t h e l i u m in t h e b u r s a ( 1 ) . T h e i n t e r f o l l i c u l a r s u r f a c e e p i t h e l i u m and t h e b u r s a l stroma remained r e l a t i v e l y i n t a c t . A few weeks a f t e r colchicine t r e a t m e n t some b u r s a l epithelial buds were r e g e n e r a t e d and r e p o p u l a t e d w i t h lymphoid cells (1, 2). T h e humoral immunity was p a r t i a l l y impaired as measured b y total IgG and IgM c o n c e n t r a t i o n s in t h e plasma and b y t h e a n t i b o d y r e s p o n s e to B r u c e l l a a b o r t u s a f t e r i n t r a p e r i t o n e a l or p e r anum immunization ( 3 ) . T h e a n t i b o d y r e s p o n s e to a t h y m u s - d e p e n d e n t a n t i g e n , sheep red blood cells ( S R B C ) , h o w e v e r , was o n l y s l i g h t l y d i m i n i s h e d . F u r t h e r m o r e , c o l c h i c i n e t r e a t m e n t y i e l d e d no a p p a r e n t d i r e c t e f f e c t s on the t h y m u s and spleen ( 1 ) . T h e s e f i n d i n g s s u g g e s t t h a t o n l y B cell lineage is impared, w h e r e a s t h e T cell f u n c t i o n s remain i n t a c t . T h e p u r p o s e of t h e p r e s e n t w o r k was to c h a r a c t e r i z e t h y m u s - d e p e n d e n t immune f u n c t i o n s in c h i c k e n s b u r s e c t o m i z e d w i t h colchicine b y s t u d y i n g 525
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t h y m i c m o r p h o l o g y and T c e l l - d e p e n d e n t a n t i b o d y responses as well as mitogenic responses of p e r i p h e r a l blood T l y m p h o c y t e s . In a d d i t i o n , t h i s b u r s e c t o m y model was compared to that induced by cyclophosphamide.
MATERIALS AND METHODS E x p e r i m e n t a l Animals I n b r e d c h i c k e n s of line P ( D e p a r t m e n t of Medical M i c r o b i o l o g y , U n i v e r s i t y of T u r k u , T u r k u , F i n l a n d ) were used. The chickens are homozygous for the major h i s t o c o m p a t i b i l i t y locus, g e n o t y p e B2B 2 . The care of the chickens has been d e s c r i b e d e a r l i e r ( 4 ) .
Colchicine A p p l i c a t i o n T h i r t y MI of colchicine in saline (2 m g / m l , i . e . 60 IJg per c h i c k ) was applied on the anal lips of c h i c k e n s on f o u r c o n s e c u t i v e days s t a r t i n g on the day of h a t c h i n g as d e s c r i b e d e a r l i e r ( 1 ) .
Cyclophosphamide Treatment F r e s h l y p r e p a r e d aqueous solution of cyclophosphamide ( S y k l o f o s f a m i d , L ~ k e O y , T u r k u , F i n l a n d ) was injected i n t r a p e r i t o n e a l l y on f o u r consecu t i v e days s t a r t i n g on the day of h a t c h i n g . The d a i l y dose was 3 mg in 0.1 ml per c h i c k .
Anticjenic S t i m u l a t i o n s and A n t i b o d y D e t e r m i n a t i o n s Sheep e r y t h r o c y t e s ( S R B C ) and f o r m a l i n - k i l l e d B r u c e l l a a b o r t u s organisms were p r e p a r e d as d e s c r i b e d e a r l i e r ( 4 ) . Human gammaglobulin (HGG) was p u r c h a s e d from Finnish Red Cross ( H e l s i n k i , F i n l a n d ) . At 3, 4 and 5 weeks of age the chickens were immunized i n t r a p e r i t o n e a l l y with 1.0 ml of a a n t i g e n m i x t u r e c o n t a i n i n g 1 mg of HGG, 10 ? B r u c e l l a organisms and 5 SRBC. H e p a r i n i z e d blood samples were collected j u s t before the a n t i g e n s t i m u lations and 6 days a f t e r the last s t i m u l a t i o n . A g g l u t i n i n s to B r u c e l l a and SRBC were measured by m i c r o t i t r a t i o n as d e s c r i b e d e a r l i e r ( 4 ) . The t i t r e s are e x p r e s s e d as a g g l u t i n i n end point d i l u t i o n s in log 2. E n z y m e - l i n k e d immunosorbent assay was used f o r q u a n t i t a t i o n of IgM, IgG and IgA a n t i bodies a g a i n s t B r u c e l l a ( 5 ) and HGG ( 6 ) . The c o n c e n t r a t i o n s of a n t i b o d i e s in the samples are e x p r e s s e d as ELISA u n i t s where one u n i t c o r r e s p o n d s to 1/100 of the c o n c e n t r a t i o n of the c o r r e s p o n d i n g a n t i b o d y in the r e f e r ence plasma.
Lymphocyte Transformation L y m p h o c y t e responses to p h y t o h a e m a g g l u t i n i n ( P H A ) and concanavalin A (Con A) were measured at the age of 3 and 6 weeks with a whole blood micromethod as d e s c r i b e d e a r l i e r ( 7 ) . The r e s u l t s are e x p r e s s e d as cpm v a l u e s of i n c o r p o r a t e d [ 1 2 S l ] l U d R .
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A u t o p s y and Microscopical Examination A f t e r collection of the last serum sample ( a t age of 6 w e e k s ) , the chickens were k i l l e d w i t h an o v e r d o s e of e t h y l e t h e r , weighed and a u t o p s i e d . The b u r s a , spleen and all t h y m i c lobes were removed and weighed. The o r g a n s were f i x e d in 10 per cent n e u t r a l b u f f e r e d f o r m a l i n and embedded in paraffin. Histological sections, 5 tJm t h i c k were stained w i t h the van G i e s o n - h e m a t o x y l i n method. From the b u r s a , one cross section p e r p e n d i c u l a r to the b u r s a l s u r f a c e e p i t h e l i u m was examined in o r d e r to e n s u r e the e f f i c i e n c y of the b u r s e c t o m i e s . T h r e e t h y m i c sections, 0 . 5 - 1 . 0 mm a p a r t from each o t h e r , were analyzed m o r p h o m e t r i c a l l y using the p o i n t counting method (8-10). The relative volume densities of the c o r t e x and medulla were calculated a f t e r c o u n t i n g 300-400 p o i n t s per thymus.
RESULTS Body and Organ Weights The mean body w e i g h t of c h i c k e n s t r e a t e d with colchicine was s i g n i f i c a n t l y lower than t h a t of the c o n t r o l s . It was, h o w e v e r , d i s t i n c t l y h i g h e r than t h a t of c y c l o p h o s p h a m i d e - t r e a t e d chickens ( T a b l e 1). The b u r s a l w e i g h t s in both b u r s e c t o m y g r o u p s were o n l y 21-24 % of those in u n t r e a t e d c o n t r o l c h i c k e n s . No s i g n i f i c a n t d i f f e r e n c e s were found in the mean r e l a t i v e w e i g h t of the spleen and t h y m u s between d i f f e r e n t g r o u p s .
TABLE 1 Body and organ w e i g h t s of c h i c k e n s t r e a t e d w i t h colchicine or cyclophosphamide a
Untreated chickens (n : 4)
Colchicinet r e a t e d chickens (n = 13)
Cyclophosphamidetreated chickens (n = 8)
Body w e i g h t ( g )
465.0 ± 4 2 . 2
*403.4 ± 3 6 . 1 " * *
312.6 ± 5 1 . 0 " * *
Bursa weight b
362.5 ± 51.4
* * * 8 6 . 3 ± 79.1
Spleen w e i g h t b
234.8 ± 66.4
244.1 ± 60.2
216.3 ± 72.7
Thymus weight b
671.0 + 159.8
567.0 ± 111.4
555.1 ± 122.9
a
74.9 ± 12.2 **~
Mean _+ SD are g i v e n . mg/100 g of body w e i g h t . The a s t e r i s k s at the l e f t side of the values of colchicine g r o u p show the s t a t i s t i c a l s i g n i f i c a n c e in a comparison to c o n t r o l g r o u p and the a s t e r i s k s at the r i g h t side in a comparison to c y c l o p h o s p h a m i d e g r o u p . The a s t e r i s k s at the r i g h t side of the values of c y c l o p h o s p h a m i d e g r o u p show the s t a t i s t i c a l s i g n i f i c a n c e in a comparison to control g r o u p . *P < 0.05, **P < 0.01, * * * P < 0.001 b y StudentSs t w o - t a i l e d t - t e s t . b
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TABLE Volumetric
Cortex
7, No. 3
2
p r o p o r t i o n s o f t h e t h y m i c c o r t e x a n d m e d. u l laa in c h i c k e n s treated with colchicine or cyclophosphamlde
(-~)
M e d u l l a (-~)
a
Vol.
Untreated chickens ( n = 4)
Colchicinetreated chickens ( n = 13)
Cyclophosphamidetreated chickens ( n = 8)
7 2 . 8 4- 7.1
74.1 _+ 5.1
7 3 . 6 4- 3 . 4
2 7 . 2 +_ 7.1
2 5 . 9 4- 5.1
2 6 . 4 _+ 3 . 4
Mean 4- SD a r e g i v e n .
TABLE Agglutinin
r e s p o n s e s a g a i n s t B r u c e l l a a n d S R B C . ina c h i c k e n s with colchicine or cyclophosphamlde
Untreated chickens ( n = 4)
Agglutinins
3
Colchicinetreated chickens ( n = 13)
treated
Cyclophosphamidetreated chickens ( n = 8)
to B r u c e l l a
After
1st s t i m u l a t i o n
3 . 5 4. 0 . 6
**0.5
4. 1.7
0"**
After
2nd stimulation
1 0 . 8 4. 1 . 0
***2.2
+ 3.8
0"**
After
3rd
1 2 . 0 _+ 0 . 0
**4.6
Agglutinins
stimulation
0 . 4 4- 0 . 7 * * *
to S R B C
After
1st s t i m u l a t i o n
1 . 5 4. 0 . 6
After
2nd s t i m u l a t i o n
6 . 8 _+ 1.3
3rd stimulation
7 . 8 _+ 2 . 4
After
4- 3 . 7 * *
Mean + SD o f log 2 a r e g i v e n . For a s t e r i s k s , see T a b l e 1.
0 . 4 + 1.3 **2.2
+ 2.5*
8 . 0 +_ 3 . 3 * * *
0.1 + 0 . 4 * * * 0.3 + 0.7*** 1.1 + 3 . 2 * * *
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Histology of the Bursa The histological findings of the bursae of chickens treated with colchicine o r c y c l o p h o s p h a m i d e w e r e e s s e n t i a l l y t h e same as d e s c r i b e d e a r l i e r ( 1 - 3 , 11-13). The bursae of colchicine-treated c h i c k e n s c o n s i s t e d o f a sac c o v e r e d b y f o l l i c u l a r s u r f a c e e p i t h e l i u m w i t h a f e w l y m p h o i d c e l l s in t h e f i b r o u s s t r o m a a n d a f e w l y m p h o i d f o l l i c l e s (mean 56 ± SD 64 p e r a c r o s s s e c t i o n ) as w e l l as e p i t h e l i a l b u d s (46 ± 6 1 ) , w h e r e a s in t h e b u r s a e o f t h e c o n t r o l g r o u p n u m e r o u s l y m p h o i d f o l l i c l e s (456 ± 77) w e r e p r e s e n t . The lymphoid follicles and the epithelial buds were covered with follicle-associated epithelium. The bursae of cyclophosphamide-treated chickens were d e v o i d o f l y m p h o i d f o l l i c l e s , w h e r e a s n u m e r o u s e p i t h e l i a l b u d s w e r e seen (305 ± 1 0 6 ) .
TABLE IgM,
IgA and
4
IgG a n t i - B r u c e l l a a n t i b o d i e s in • c hai c k e n s colchicine or cyclophosphamlde
Untreated chickens ( n = 4)
Antibodies
Colchicinetreated chickens ( n = 13)
treated
with
Cyclophosphamidetreated chickens ( n = 8)
IgM a n t i - B r u c e l l a Before stimulations
0.78 + 0.05
0.61 ± 1.90
0.78 ± 0.25
After
1st s t i m u l a t i o n
1.40 ± 0.53
1.53 ± 1 . 6 4
1.25 ± 0 . 7 6
After
2nd stimulation
18.9 ± 13.8
After
3rd stimulation
7 . 4 0 _+ 4 . 6 0
8.60 ± 22.5
0.46 ± 0.32***
stimulations
1.25 ± 0 . 3 3
*0.72 ± 0.40**
0.26 ± 0.07***
After
1st s t i m u l a t i o n
1.60 ± 0.24
1.38 ± 0 . 8 2 * *
0.36 ± 0.17"**
After
2rid s t i m u l a t i o n
13.5 ± 3.50 **'1.65
± 1.15"*
0.46 ± 0.36***
After
3rd
60.3 ± 26.6 **'7.01
± 18.7
0.36 ± 0.63***
**2.70
± 3.80
0.65 ± 0.36**
IgA anti-Brucella Before
stimulation
IgG a n t i - B r u c e l l a Before stimulations
0.33 ± 0.13
0.25 ± 0.13
0.21 + 0 . 1 2
After
1st s t i m u l a t i o n
0 . 3 5 _+ 0 . 1 7
0.37 ± 0.13
0.28 + 0.12
After
2nd stimulation
2 . 4 0 _+ 0 . 9 0 * * * 0 . 4 4
± 0.41
0.18 ± 0.16"**
After
3rd stimulation
6 8 . 4 _+ 4 1 . 6 * * ' 1 . 8 0
± 4.90
0.08 + 0.09"**
Mean _+ SD a r e g i v e n . T h e r e s u l t s For a s t e r i s k s , see T a b l e 1.
are expressed
as E L I S A u n i t s .
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H i s t o l o g y of t h e T h y m u s T h e l y m p h o i d and e p i t h e l i a l cell c o m p o s i t i o n of t h e t h y m u s was i d e n t i c a l in all c h i c k e n g r o u p s . A l s o , no s i g n i f i c a n t d i f f e r e n c e s w e r e f o u n d in the v o l u m e t r i c p r o p o r t i o n s of t h e c o r t e x and medulla when t h e t h r e e g r o u p s w e r e compared w i t h each o t h e r ( T a b l e 2 ) . Antibody
Responses to T h y m u s - i n d e p e n d e n t
Antigen
(Brucella abortus)
In c o l c h i c i n e - t r e a t e d c h i c k e n s , low a g g l u t i n i n r e s p o n s e s a g a i n s t B r u c e l l a w e r e f o u n d a f t e r each a n t i g e n s t i m u l a t i o n and t h e r e s p o n s e s w e r e s i g n i f i c a n t l y l o w e r t h a n in u n t r e a t e d b i r d s ( T a b l e 3 ) . On t h e o t h e r h a n d , chickens treated w i t h c y c l o p h o s p h a m i d e had p r a c t i c a l l y no a g g l u t i n i n s against Brucella (Table 3). Colchicine-treated c h i c k e n s had v e r y low c o n c e n t r a t i o n s of I g A , IgM and IgG a n t i - B r u c e l l a a n t i b o d i e s ( T a b l e 4 ) .
TABLE IgM,
IgA and
5
gG a n t i - H G G a n t i b o d i e s in c h i c k e n s t r e a t e d w i t h colchicine or c y c l o p h o s p h a m i d e a
Antibodies
Untreated chickens (n = 4)
Colchicinetreated chickens (n = 13)
Cyclophosphamidetreated chickens (n : 8)
IgM a n t i - H G G Before stimulations
0.03 _+ 0.05
0.06 ,+ 0 . 0 8 *
0
After
1st s t i m u l a t i o n
10.0 _+ 9.60
3.00 ,+ 6.00
0.13 ,+ 0 . 0 7 *
After
2nd s t i m u l a t i o n
44.1 _+ 10.6
After
3rd stimulation
19.8 _+ 4.22
± 12.0"
0.11 + 0 . 1 0 " * *
29.9 ,+ 2 9 . 9 *
0.33 + 0 . 9 2 " * *
**'11.0
IgA anti-HGG Before stimulations
0
After
1st s t i m u l a t i o n
4.00 _+ 3.40
After
2nd s t i m u l a t i o n
16.4 ,+ 9.10
After
3rd stimulation
48.4 _+ 17.6
0
0
* 0 . 8 3 _+ 2.00 ***3.40
+ 4.00"
22.0 _+ 35.0
0"* 0"** 0.18 _+ 0.51"**
IgG a n t i - H G G Before stimulations
0
After
1st s t i m u l a t i o n
7.50 + 7.20
After
2rid s t i m u l a t i o n
25.7 + 17.5
After
3rd stimulation
5.15 +_ 3.46
0 " 1 . 2 8 _+ 2.90 ***4.20
+ 5.40*
21.4 _+ 29.2
0 O* 0.01 _+ 0 . 0 3 " * * 0.18 + 0 . 5 1 " *
a Mean + SD a r e g i v e n . T h e r e s u l t s a r e e x p r e s s e d as ELISA u n i t s . For a s t e r i s k s , see T a b l e 1.
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A n t i b o d y Responses to T h y m u s - d e p e n d e n t A n t i g e n s (SRBC and HGG) A g g l u t i n i n s to SRBC of c o l c h i c i n e - t r e a t e d c h i c k e n s were low a f t e r the f i r s t and second s t i m u l a t i o n s , b u t reached the mean t i t e r of c o n t r o l chickens a f t e r t h e t h i r d s t i m u l a t i o n ( T a b l e 3 ) . The chickens t r e a t e d w i t h c y c l o p h o s phamide had p r a c t i c a l l y no a g g l u t i n i n s a g a i n s t SRBC, even a f t e r the t h i r d antigen stimulation (Table 3). IgM, IgA and IgG class a n t i b o d y responses against another thymus-dependent antigen, HGG, of c o l c h i c i n e - t r e a t e d c h i c k e n s were lower than those of u n t r e a t e d chickens a f t e r the f i r s t and second s t i m u l a t i o n s , b u t a f t e r the t h i r d s t i m u l a t i o n t h e y reached the a n t i b o d y l e v e l s of u n t r e a t e d c h i c k e n s ( T a b l e 5). Cyclophosphamide-treated c h i c k e n s had p r a c t i c a l l y no a n t i b o d i e s a g a i n s t HGG.
Mitocjen Responses of P e r i p h e r a l Blood L y m p h o c y t e s The responses of p e r i p h e r a l blood l y m p h o c y t e s to PHA and Con A in c o l c h i c i n e - t r e a t e d c h i c k e n s were s i m i l a r to those in u n t r e a t e d c o n t r o l b i r d s (Table 6). The mitogen responses of c y c l o p h o s p h a m i d e - t r e a t e d chickens were o n l y s l i g h t l y lower than those in c o n t r o l s and c o l c h i c i n e - t r e a t e d chickens both at t h r e e and s i x weeks of age.
TABLE 6 Mitogen
Age of chickens (wks)
•
responses in c h i c k e n s t r e a t e d w i t h colchicine or c y c l o p h o s p h a m l d e
Mitogen
Untreated chickens (n = 4)
Colchicinet r e a t e d chickens (n = 13)
a
Cyclophosphamidet r e a t e d chickens (n = 8)
-
179 ± 86
220 ± 158
186 ± 145
PHA
17216 ± 9999
9305 ± 6410
5666 ± 3956*
Con A
6814 ± 5573
5673 ± 7305
1963 ± 3024
-
325 ± 214
268 ± 315
137 ± 62
PHA
24826 ± 20230
25019 ± 20909
10092 ± 11678
Con A
5531 ± 7042
4272 ± 5949
1688 ± 846
Mean + SD of cpm are g i v e n . For a s t e r i s k s , see Table 1.
DISCUSSION Colchicine g i v e n p e r anum a f t e r h a t c h i n g induced a d e f i c i e n c y of humoral i m m u n i t y , which c o n f i r m s the r e s u l t s of a p r e v i o u s s t u d y ( 3 ) . The a n t i b o d y response to a t h y m u s - i n d e p e n d e n t a n t i g e n B r u c e l l a a b o r t u s of these
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chickens was v e r y low a f t e r the f i r s t and second s t i m u l a t i o n s , b u t a f t e r the t h i r d s t i m u l a t i o n the a g g l u t i n i n t i t e r s were about 40 96 of those of control chickens. T h i s f i n d i n g w i t h the r e s t o r a t i o n of SRBC responses i n d i c a t e s t h a t the i m m u n o d e f i c i e n c y induced by a p p l i c a t i o n of colchicine on the anal lips is followed by a p a r t i a l spontaneous r e c o n s t i t u t i o n of humoral i m m u n i t y . R e l a t i v e l y tow B r u c e l l a responses i n d i c a t e t h a t t h i s a n t i g e n may r a t h e r be r e g a r d e d as a b u r s a - d e p e n d e n t a n t i g e n than as a t h y m u s - independent antigen. On the c o n t r a r y , the t r e a t m e n t of c h i c k e n s with c y c l o p h o s p h a m i d e in the n e w l y hatched p e r i o d leads to a s e v e r e r d e f i c i e n c y in humoral i m m u n i t y ( 1 1 - 1 4 ) , which a p p a r e n t l y is due to local d e s t r u c t i o n of b u r s a cells as well as d e s t r u c t i o n of B cell p r e c u r s o r s and mature B cells in e x t r a b u r s a l s i t e s . It is l i k e l y t h a t in c o l c h i c i n e - t r e a t e d b i r d s e x t r a b u r s a l B cell p r e c u r s o r s l a t e r colonize the b u r s a r e s u l t i n g in a p a r t i a l r e g e n e r a t i o n of the b u r s a l s t r u c t u r e and p a r t i a l r e s t o r a t i o n of humoral immune f u n c t i o n s . It is also e v i d e n t t h a t the b u r s a of c o l c h i c i n e - t r e a t e d c h i c k e n s r e t a i n s its l y m p h o t a c t i c and l y m p h o c y t e - d i f f e r e n t i a t i n g c a p a c i t y and t h u s these c h i c k e n s can be used as r e c i p i e n t s in cell t r a n s f e r e x p e r i ments like c y c l o p h o s p h a m i d e - t r e a t e d b i r d s ( 4 ) . In c o n t r a s t , it has been d e m o n s t r a t e d t h a t t e s t o s t e r o n e t r e a t m e n t a f f e c t s the b u r s a l stroma and d e s t r o y s its c a p a c i t y to s e r v e as a m a t u r a t i o n site f o r B cell p r e c u r s o r s (15). In c o l c h i c i n e - t r e a t e d c h i c k e n s , the a n t i b o d y responses to the t h y m u s d e p e n d e n t a n t i g e n s SRBC and HGG were d i s t u r b e d a f t e r the f i r s t and second a n t i g e n s t i m u l a t i o n s . H o w e v e r , the responses were p r a c t i c a l l y the same as in u n t r e a t e d c h i c k e n s a f t e r the t h i r d s t i m u l a t i o n . This indicates t h a t the responses are o n l y d e l a y e d , p r o b a b l y due to a slow r e s t o r a t i o n of the b u r s a l s t r u c t u r e . Also the l y m p h o c y t e responses to PHA and Con A were p r e s e r v e d . In a d d i t i o n , no changes in the t h y m i c m o r p h o l o g y were observed. T h u s , it appears t h a t the c e l l - m e d i a t e d i m m u n i t y in chickens t r e a t e d w i t h colchicine p e r anum is not s i g n i f i c a n t l y a f f e c t e d . This finding is a n t i c i p a t e d because colchicine causes a s e l e c t i v e local i n i u r y in the b u r s a , whereas no d i r e c t e f f e c t s in the spleen, t h y m u s or caecal t o n s i l s have been o b s e r v e d ( 1 ) . On the o t h e r hand, c y c l o p h o s p h a m i d e , g i v e n i.p., causes a s e v e r e i n j u r y in the whole lymphoid s y s t e m , i n c l u d i n g the t h y m u s , a l t h o u g h d u r i n g a few weeks a f t e r the i n i t i a l i n j u r y the t h y m i c m o r p h o l o g y r e g e n e r a t e s (12, 16). In p r e v i o u s r e p o r t s v a r i a b l e defects of c e l l - m e d i a t e d i m m u n i t y have been r e p o r t e d a f t e r c y c l o p h o s p h a m i d e t r e a t m e n t (12, 14, 16-18). The d i f f e r e n c e s are p r o b a b l y due m a i n l y to the v a r i a t i o n of d r u g doses as well as t i m i n g of the t r e a t m e n t , g e n e t i c b a c k g r o u n d of the c h i c k e n s and the e x p e r i m e n t a l s e t - u p used. Of p a r t i c u l a r i n t e r e s t are the f i n d i n g s of IgA and IgG class a n t i b o d i e s against Brucella. IgA and IgG a n t i b o d i e s in h o r m o n a l l y bursectomized c h i c k e n s are p r a c t i c a l l y absent and in s u r g i c a l l y bursectomized c h i c k e n s o n l y s l i g h t l y d i m i n i s h e d (19, 20). The p r e s e n t s t u d y i n d i c a t e s t h a t in c h i c k e n s t r e a t e d w i t h colchicine the switch from IgM to both IgA and IgG may be e i t h e r delayed or d i s t u r b e d . The l a t t e r p o s s i b i l i t y seems more p r o b a b l e since in these c h i c k e n s c l a s s - s p e c i f i c a n t i - H G G a n t i b o d i e s a f t e r the t h i r d a n t i g e n s t i m u l a t i o n were on the same level as in normal c o n t r o l s , while c l a s s - s p e c i f i c a n t i - B r u c e l l a a n t i b o d i e s were s i g n i f i c a n t l y lower than those of control c h i c k e n s even a f t e r the t h i r d a n t i g e n s t i m u l a t i o n . It is possible t h a t the absence of IgA and IgG a n t i b o d i e s a g a i n s t B r u c e l l a is due to a local d e s t r u c t i o n of T cells r e g u l a t i n g the switch of immunoglobulin i s o t y p e s in the b u r s a . Whether t h i s d i s t u r b a n c e also applies to o t h e r t h y m u s - i n d e p e n d e n t or b u r s a - d e p e n d e n t a n t i g e n s remains to be shown.
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In c o n c l u s i o n , o u r r e s u l t s d e m o n s t r a t e t h a t t h y m u s - d e p e n d e n t immune f u n c t i o n s of c h i c k e n s t r e a t e d n e o n a t a l l y p e r anum w i t h c o l c h i c i n e remain p r a c t i c a l l y normal. Due to o n l y local d e s t r u c t i o n of B l y m p h o c y t e s in t h e b u r s a this t y p e of b u r s e c t o m y p r o v i d e s a new model f o r s t u d i e s of s p o n t a neous r e g e n e r a t i o n of t h e humoral immune system and e x t r a b u r s a l r e s e r v e s of B l y m p h o c y t e p r o g e n i t o r s .
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Romppanen T , S o r v a r i TE. Late e f f e c t s of colchicine on the b u r s a of F a b r i c i u s a f t e r neonatal application on the anal lips of c h i c k e n s . Acta Pathol Microbiol Scand ( A ) . 1980; 88:407-413.
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Romppanen T , V i l j a n e n MK, S o r v a r i TE. Humoral immunodeficiency in c h i c k e n s i n d u c e d by chemical b u r s a c t o m y w i t h colchicine applied on the anal lips. Immunology. 1981; 42:391-399.
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T o i v a n e n P, T o i v a n e n A. B u r s a l and p o s t b u r s a l stem cells in c h i c k e n . Functional c h a r a c t e r i s t i c s . Eur J Immunol. 1973; 3:585-595.
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Soppi E, Lassila O, Viljanen MK, Lehtonen O-P, Eskola J. In v i v o e f f e c t of levamisole on c e l l u l a r and humoral immunity in normal c h i c k e n s . Clin Exp Immunol. 1979; 38:609-614.
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Hirota Y, M a r t i n M - T , Viljanen M, T o i v a n e n P, F r a n k l i n RM. Immun o p a t h o l o g y of c h i c k e n s i n f e c t e d in ovo and at h a t c h i n g w i t h the avian o s t e o p e t r o s i s v i r u s M A V . 2 - 0 . Eur J Immunol. 1980; 10:929-936.
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Lassila O, Eskola J, T o i v a n e n P. A micromethod c h i c k e n l y m p h o c y t e s in v i t r o using whole blood. 1976; 26:641-646.
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Glick B. Morphological changes and humoral immunity in p h a m i d e - t r e a t e d c h i c k s . T r a n s p l a n t a t i o n . 1971; 11:433-439.
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Linna T J, Frommel D, Good RA. Effects of e a r l y c y c l o p h o s p h a m i d e t r e a t m e n t on t h e d e v e l o p m e n t of lymphoid o r g a n s and immunological f u n c t i o n s in t h e c h i c k e n . Int A r c h A l l e r g y . 1972; 42:20-39.
13.
S o r v a r i T , T o i v a n e n A, T o i v a n e n P. T r a n s p l a n t a t i o n of b u r s a l stem cells into c y c l o p h o s p h a m i d e - t r e a t e d chicks. Transplantation. 1974; 17 : 584-592.
cyclophos-
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14.
Lerman SP, Weidanz WP. The e f f e c t of cyclophosphamide on the o n t o g e n y of the humoral immune response in c h i c k e n s . J Immunol. 1970; 105: 614-619.
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Eskola J, Ruuskanen O, Fr~ki J, Viljanen MK, T o i v a n e n A. A l k a l i n e phosphatase in the d e v e l o p i n g b u r s a of F a b r i c i u s . A c o m p a r a t i v e s t u d y of the c y c l o p h o s p h a m i d e - and t e s t o s t e r o n e - i n d u c e d immunod e f i c i e n c i e s in the c h i c k e m b r y o . Scand 3 Im.munol. 1977; 6:185-194.
16.
Rouse B T , Szenberg A. Functional and morphological o b s e r v a t i o n s on the e f f e c t of c y c l o p h o s p h a m i d e on the immune response of the c h i c k e n . A u s t J Exp Biol Med Sci. 1974; 52:873-885.
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Eskola J, T o i v a n e n P. Effect of phamide on lymphoid system in 459-471.
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Sharma JM, Lee LF. S u p p r e s s i v e e f f e c t of c y c l o p h o s p h a m i d e T - c e l l system in c h i c k e n s . I n f e c t Immun. 1977; 17:227-230.
19.
Perey DYE, B i e n e n s t o c k J. Effects of b u r s e c t o m y and t h y m e c t o m y on the o n t o g e n y of fowl I g A , IgG and IgM. J Immunot. 1973; 111:633-637.
20.
Kowalski WJ, Malkinson M, Leslie GA, Small PA. The s e c r e t o r y immunological system of the fowl. V I . The e f f e c t of chemical b u r s e c t o m y on immunoglobulin c o n c e n t r a t i o n in t e a r s . Immunology. 1978; 34:663-667.
Received Accepted
: September, 1982 : March, 1983
in ovo t r e a t e m e n t w i t h chicken. Cell Immunol.
cyclophos1974; 13:
on the
3
1983.
T H Y M U S - D E P E N D E N T IMMUNE F U N C T I O N S IN C H I C K E N S B U R S E C T O M I Z E D WITH C O L C H I C I N E APPLIED TO THE A N A L LIPS
T . Romppanen, J. Eskola, O. Lassila, M . K . Viljanen and T . E . S o r v a r i D e p a r t m e n t of P a t h o l o g y , U n i v e r s i t y of Kuopio, Kuopio, and D e p a r t m e n t of Medical M i c r o b i o l o g y , U n i v e r s i t y of T u r k u , T u r k u , Finland
ABSTRACT
Thymus-dependent immune f u n c t i o n s were i n v e s t i g a t e d in c h i c k e n s b u r s e c t o m i z e d n e o n a t a l l y w i t h colchicine solution g i v e n p e r anum. A n t i b o d y responses to t h y m u s - d e p e n d e n t a n t i g e n s sheep red blood cells ( S R B C ) and human gammaglobulin ( H G G ) were d e l a y e d , r e a c h i n g the normal level a f t e r the t h i r d antigen stimulation. Also the mitogenic responses of p e r i p h e r a l blood l y m p h o c y t e s were p r e s e r v e d , and no changes in the t h y m i c m o r p h o l o g y were f o u n d . In contrast, a n t i b o d y responses to b u r s a - d e p e n d e n t a n t i g e n B r u c e l l a a b o r t u s were low and the switch of immunoglobulin i s o t y p e s from IgM to IgA and IgG was d i s t u r b e d . It can be c o n c l u d e d t h a t neonatal b u r s e c t o m y with cloacal a d m i n i s t r a tion of colchicine does not s i g n i f i c a n t l y a f f e c t T cell f u n c t i o n s , whereas B cell f u n c t i o n s are p a r t i a l l y d e f i c i e n t .
INTRODUCTION R e c e n t l y , a new model of chemical b u r s e c t o m y f o r neonatal c h i c k e n s was described, where colchicine solution applied p e r anum d e s t r o y e d both lymphoid cells and cells of t h e f o l l i c u l a r e p i t h e l i u m and f o l l i c l e - a s s o c i a t e d e p i t h e l i u m in t h e b u r s a ( 1 ) . T h e i n t e r f o l l i c u l a r s u r f a c e e p i t h e l i u m and t h e b u r s a l stroma remained r e l a t i v e l y i n t a c t . A few weeks a f t e r colchicine t r e a t m e n t some b u r s a l epithelial buds were r e g e n e r a t e d and r e p o p u l a t e d w i t h lymphoid cells (1, 2). T h e humoral immunity was p a r t i a l l y impaired as measured b y total IgG and IgM c o n c e n t r a t i o n s in t h e plasma and b y t h e a n t i b o d y r e s p o n s e to B r u c e l l a a b o r t u s a f t e r i n t r a p e r i t o n e a l or p e r anum immunization ( 3 ) . T h e a n t i b o d y r e s p o n s e to a t h y m u s - d e p e n d e n t a n t i g e n , sheep red blood cells ( S R B C ) , h o w e v e r , was o n l y s l i g h t l y d i m i n i s h e d . F u r t h e r m o r e , c o l c h i c i n e t r e a t m e n t y i e l d e d no a p p a r e n t d i r e c t e f f e c t s on the t h y m u s and spleen ( 1 ) . T h e s e f i n d i n g s s u g g e s t t h a t o n l y B cell lineage is impared, w h e r e a s t h e T cell f u n c t i o n s remain i n t a c t . T h e p u r p o s e of t h e p r e s e n t w o r k was to c h a r a c t e r i z e t h y m u s - d e p e n d e n t immune f u n c t i o n s in c h i c k e n s b u r s e c t o m i z e d w i t h colchicine b y s t u d y i n g 525
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t h y m i c m o r p h o l o g y and T c e l l - d e p e n d e n t a n t i b o d y responses as well as mitogenic responses of p e r i p h e r a l blood T l y m p h o c y t e s . In a d d i t i o n , t h i s b u r s e c t o m y model was compared to that induced by cyclophosphamide.
MATERIALS AND METHODS E x p e r i m e n t a l Animals I n b r e d c h i c k e n s of line P ( D e p a r t m e n t of Medical M i c r o b i o l o g y , U n i v e r s i t y of T u r k u , T u r k u , F i n l a n d ) were used. The chickens are homozygous for the major h i s t o c o m p a t i b i l i t y locus, g e n o t y p e B2B 2 . The care of the chickens has been d e s c r i b e d e a r l i e r ( 4 ) .
Colchicine A p p l i c a t i o n T h i r t y MI of colchicine in saline (2 m g / m l , i . e . 60 IJg per c h i c k ) was applied on the anal lips of c h i c k e n s on f o u r c o n s e c u t i v e days s t a r t i n g on the day of h a t c h i n g as d e s c r i b e d e a r l i e r ( 1 ) .
Cyclophosphamide Treatment F r e s h l y p r e p a r e d aqueous solution of cyclophosphamide ( S y k l o f o s f a m i d , L ~ k e O y , T u r k u , F i n l a n d ) was injected i n t r a p e r i t o n e a l l y on f o u r consecu t i v e days s t a r t i n g on the day of h a t c h i n g . The d a i l y dose was 3 mg in 0.1 ml per c h i c k .
Anticjenic S t i m u l a t i o n s and A n t i b o d y D e t e r m i n a t i o n s Sheep e r y t h r o c y t e s ( S R B C ) and f o r m a l i n - k i l l e d B r u c e l l a a b o r t u s organisms were p r e p a r e d as d e s c r i b e d e a r l i e r ( 4 ) . Human gammaglobulin (HGG) was p u r c h a s e d from Finnish Red Cross ( H e l s i n k i , F i n l a n d ) . At 3, 4 and 5 weeks of age the chickens were immunized i n t r a p e r i t o n e a l l y with 1.0 ml of a a n t i g e n m i x t u r e c o n t a i n i n g 1 mg of HGG, 10 ? B r u c e l l a organisms and 5 SRBC. H e p a r i n i z e d blood samples were collected j u s t before the a n t i g e n s t i m u lations and 6 days a f t e r the last s t i m u l a t i o n . A g g l u t i n i n s to B r u c e l l a and SRBC were measured by m i c r o t i t r a t i o n as d e s c r i b e d e a r l i e r ( 4 ) . The t i t r e s are e x p r e s s e d as a g g l u t i n i n end point d i l u t i o n s in log 2. E n z y m e - l i n k e d immunosorbent assay was used f o r q u a n t i t a t i o n of IgM, IgG and IgA a n t i bodies a g a i n s t B r u c e l l a ( 5 ) and HGG ( 6 ) . The c o n c e n t r a t i o n s of a n t i b o d i e s in the samples are e x p r e s s e d as ELISA u n i t s where one u n i t c o r r e s p o n d s to 1/100 of the c o n c e n t r a t i o n of the c o r r e s p o n d i n g a n t i b o d y in the r e f e r ence plasma.
Lymphocyte Transformation L y m p h o c y t e responses to p h y t o h a e m a g g l u t i n i n ( P H A ) and concanavalin A (Con A) were measured at the age of 3 and 6 weeks with a whole blood micromethod as d e s c r i b e d e a r l i e r ( 7 ) . The r e s u l t s are e x p r e s s e d as cpm v a l u e s of i n c o r p o r a t e d [ 1 2 S l ] l U d R .
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A u t o p s y and Microscopical Examination A f t e r collection of the last serum sample ( a t age of 6 w e e k s ) , the chickens were k i l l e d w i t h an o v e r d o s e of e t h y l e t h e r , weighed and a u t o p s i e d . The b u r s a , spleen and all t h y m i c lobes were removed and weighed. The o r g a n s were f i x e d in 10 per cent n e u t r a l b u f f e r e d f o r m a l i n and embedded in paraffin. Histological sections, 5 tJm t h i c k were stained w i t h the van G i e s o n - h e m a t o x y l i n method. From the b u r s a , one cross section p e r p e n d i c u l a r to the b u r s a l s u r f a c e e p i t h e l i u m was examined in o r d e r to e n s u r e the e f f i c i e n c y of the b u r s e c t o m i e s . T h r e e t h y m i c sections, 0 . 5 - 1 . 0 mm a p a r t from each o t h e r , were analyzed m o r p h o m e t r i c a l l y using the p o i n t counting method (8-10). The relative volume densities of the c o r t e x and medulla were calculated a f t e r c o u n t i n g 300-400 p o i n t s per thymus.
RESULTS Body and Organ Weights The mean body w e i g h t of c h i c k e n s t r e a t e d with colchicine was s i g n i f i c a n t l y lower than t h a t of the c o n t r o l s . It was, h o w e v e r , d i s t i n c t l y h i g h e r than t h a t of c y c l o p h o s p h a m i d e - t r e a t e d chickens ( T a b l e 1). The b u r s a l w e i g h t s in both b u r s e c t o m y g r o u p s were o n l y 21-24 % of those in u n t r e a t e d c o n t r o l c h i c k e n s . No s i g n i f i c a n t d i f f e r e n c e s were found in the mean r e l a t i v e w e i g h t of the spleen and t h y m u s between d i f f e r e n t g r o u p s .
TABLE 1 Body and organ w e i g h t s of c h i c k e n s t r e a t e d w i t h colchicine or cyclophosphamide a
Untreated chickens (n : 4)
Colchicinet r e a t e d chickens (n = 13)
Cyclophosphamidetreated chickens (n = 8)
Body w e i g h t ( g )
465.0 ± 4 2 . 2
*403.4 ± 3 6 . 1 " * *
312.6 ± 5 1 . 0 " * *
Bursa weight b
362.5 ± 51.4
* * * 8 6 . 3 ± 79.1
Spleen w e i g h t b
234.8 ± 66.4
244.1 ± 60.2
216.3 ± 72.7
Thymus weight b
671.0 + 159.8
567.0 ± 111.4
555.1 ± 122.9
a
74.9 ± 12.2 **~
Mean _+ SD are g i v e n . mg/100 g of body w e i g h t . The a s t e r i s k s at the l e f t side of the values of colchicine g r o u p show the s t a t i s t i c a l s i g n i f i c a n c e in a comparison to c o n t r o l g r o u p and the a s t e r i s k s at the r i g h t side in a comparison to c y c l o p h o s p h a m i d e g r o u p . The a s t e r i s k s at the r i g h t side of the values of c y c l o p h o s p h a m i d e g r o u p show the s t a t i s t i c a l s i g n i f i c a n c e in a comparison to control g r o u p . *P < 0.05, **P < 0.01, * * * P < 0.001 b y StudentSs t w o - t a i l e d t - t e s t . b
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BURSECTOMY BY COLCHICINE
TABLE Volumetric
Cortex
7, No. 3
2
p r o p o r t i o n s o f t h e t h y m i c c o r t e x a n d m e d. u l laa in c h i c k e n s treated with colchicine or cyclophosphamlde
(-~)
M e d u l l a (-~)
a
Vol.
Untreated chickens ( n = 4)
Colchicinetreated chickens ( n = 13)
Cyclophosphamidetreated chickens ( n = 8)
7 2 . 8 4- 7.1
74.1 _+ 5.1
7 3 . 6 4- 3 . 4
2 7 . 2 +_ 7.1
2 5 . 9 4- 5.1
2 6 . 4 _+ 3 . 4
Mean 4- SD a r e g i v e n .
TABLE Agglutinin
r e s p o n s e s a g a i n s t B r u c e l l a a n d S R B C . ina c h i c k e n s with colchicine or cyclophosphamlde
Untreated chickens ( n = 4)
Agglutinins
3
Colchicinetreated chickens ( n = 13)
treated
Cyclophosphamidetreated chickens ( n = 8)
to B r u c e l l a
After
1st s t i m u l a t i o n
3 . 5 4. 0 . 6
**0.5
4. 1.7
0"**
After
2nd stimulation
1 0 . 8 4. 1 . 0
***2.2
+ 3.8
0"**
After
3rd
1 2 . 0 _+ 0 . 0
**4.6
Agglutinins
stimulation
0 . 4 4- 0 . 7 * * *
to S R B C
After
1st s t i m u l a t i o n
1 . 5 4. 0 . 6
After
2nd s t i m u l a t i o n
6 . 8 _+ 1.3
3rd stimulation
7 . 8 _+ 2 . 4
After
4- 3 . 7 * *
Mean + SD o f log 2 a r e g i v e n . For a s t e r i s k s , see T a b l e 1.
0 . 4 + 1.3 **2.2
+ 2.5*
8 . 0 +_ 3 . 3 * * *
0.1 + 0 . 4 * * * 0.3 + 0.7*** 1.1 + 3 . 2 * * *
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529
Histology of the Bursa The histological findings of the bursae of chickens treated with colchicine o r c y c l o p h o s p h a m i d e w e r e e s s e n t i a l l y t h e same as d e s c r i b e d e a r l i e r ( 1 - 3 , 11-13). The bursae of colchicine-treated c h i c k e n s c o n s i s t e d o f a sac c o v e r e d b y f o l l i c u l a r s u r f a c e e p i t h e l i u m w i t h a f e w l y m p h o i d c e l l s in t h e f i b r o u s s t r o m a a n d a f e w l y m p h o i d f o l l i c l e s (mean 56 ± SD 64 p e r a c r o s s s e c t i o n ) as w e l l as e p i t h e l i a l b u d s (46 ± 6 1 ) , w h e r e a s in t h e b u r s a e o f t h e c o n t r o l g r o u p n u m e r o u s l y m p h o i d f o l l i c l e s (456 ± 77) w e r e p r e s e n t . The lymphoid follicles and the epithelial buds were covered with follicle-associated epithelium. The bursae of cyclophosphamide-treated chickens were d e v o i d o f l y m p h o i d f o l l i c l e s , w h e r e a s n u m e r o u s e p i t h e l i a l b u d s w e r e seen (305 ± 1 0 6 ) .
TABLE IgM,
IgA and
4
IgG a n t i - B r u c e l l a a n t i b o d i e s in • c hai c k e n s colchicine or cyclophosphamlde
Untreated chickens ( n = 4)
Antibodies
Colchicinetreated chickens ( n = 13)
treated
with
Cyclophosphamidetreated chickens ( n = 8)
IgM a n t i - B r u c e l l a Before stimulations
0.78 + 0.05
0.61 ± 1.90
0.78 ± 0.25
After
1st s t i m u l a t i o n
1.40 ± 0.53
1.53 ± 1 . 6 4
1.25 ± 0 . 7 6
After
2nd stimulation
18.9 ± 13.8
After
3rd stimulation
7 . 4 0 _+ 4 . 6 0
8.60 ± 22.5
0.46 ± 0.32***
stimulations
1.25 ± 0 . 3 3
*0.72 ± 0.40**
0.26 ± 0.07***
After
1st s t i m u l a t i o n
1.60 ± 0.24
1.38 ± 0 . 8 2 * *
0.36 ± 0.17"**
After
2rid s t i m u l a t i o n
13.5 ± 3.50 **'1.65
± 1.15"*
0.46 ± 0.36***
After
3rd
60.3 ± 26.6 **'7.01
± 18.7
0.36 ± 0.63***
**2.70
± 3.80
0.65 ± 0.36**
IgA anti-Brucella Before
stimulation
IgG a n t i - B r u c e l l a Before stimulations
0.33 ± 0.13
0.25 ± 0.13
0.21 + 0 . 1 2
After
1st s t i m u l a t i o n
0 . 3 5 _+ 0 . 1 7
0.37 ± 0.13
0.28 + 0.12
After
2nd stimulation
2 . 4 0 _+ 0 . 9 0 * * * 0 . 4 4
± 0.41
0.18 ± 0.16"**
After
3rd stimulation
6 8 . 4 _+ 4 1 . 6 * * ' 1 . 8 0
± 4.90
0.08 + 0.09"**
Mean _+ SD a r e g i v e n . T h e r e s u l t s For a s t e r i s k s , see T a b l e 1.
are expressed
as E L I S A u n i t s .
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H i s t o l o g y of t h e T h y m u s T h e l y m p h o i d and e p i t h e l i a l cell c o m p o s i t i o n of t h e t h y m u s was i d e n t i c a l in all c h i c k e n g r o u p s . A l s o , no s i g n i f i c a n t d i f f e r e n c e s w e r e f o u n d in the v o l u m e t r i c p r o p o r t i o n s of t h e c o r t e x and medulla when t h e t h r e e g r o u p s w e r e compared w i t h each o t h e r ( T a b l e 2 ) . Antibody
Responses to T h y m u s - i n d e p e n d e n t
Antigen
(Brucella abortus)
In c o l c h i c i n e - t r e a t e d c h i c k e n s , low a g g l u t i n i n r e s p o n s e s a g a i n s t B r u c e l l a w e r e f o u n d a f t e r each a n t i g e n s t i m u l a t i o n and t h e r e s p o n s e s w e r e s i g n i f i c a n t l y l o w e r t h a n in u n t r e a t e d b i r d s ( T a b l e 3 ) . On t h e o t h e r h a n d , chickens treated w i t h c y c l o p h o s p h a m i d e had p r a c t i c a l l y no a g g l u t i n i n s against Brucella (Table 3). Colchicine-treated c h i c k e n s had v e r y low c o n c e n t r a t i o n s of I g A , IgM and IgG a n t i - B r u c e l l a a n t i b o d i e s ( T a b l e 4 ) .
TABLE IgM,
IgA and
5
gG a n t i - H G G a n t i b o d i e s in c h i c k e n s t r e a t e d w i t h colchicine or c y c l o p h o s p h a m i d e a
Antibodies
Untreated chickens (n = 4)
Colchicinetreated chickens (n = 13)
Cyclophosphamidetreated chickens (n : 8)
IgM a n t i - H G G Before stimulations
0.03 _+ 0.05
0.06 ,+ 0 . 0 8 *
0
After
1st s t i m u l a t i o n
10.0 _+ 9.60
3.00 ,+ 6.00
0.13 ,+ 0 . 0 7 *
After
2nd s t i m u l a t i o n
44.1 _+ 10.6
After
3rd stimulation
19.8 _+ 4.22
± 12.0"
0.11 + 0 . 1 0 " * *
29.9 ,+ 2 9 . 9 *
0.33 + 0 . 9 2 " * *
**'11.0
IgA anti-HGG Before stimulations
0
After
1st s t i m u l a t i o n
4.00 _+ 3.40
After
2nd s t i m u l a t i o n
16.4 ,+ 9.10
After
3rd stimulation
48.4 _+ 17.6
0
0
* 0 . 8 3 _+ 2.00 ***3.40
+ 4.00"
22.0 _+ 35.0
0"* 0"** 0.18 _+ 0.51"**
IgG a n t i - H G G Before stimulations
0
After
1st s t i m u l a t i o n
7.50 + 7.20
After
2rid s t i m u l a t i o n
25.7 + 17.5
After
3rd stimulation
5.15 +_ 3.46
0 " 1 . 2 8 _+ 2.90 ***4.20
+ 5.40*
21.4 _+ 29.2
0 O* 0.01 _+ 0 . 0 3 " * * 0.18 + 0 . 5 1 " *
a Mean + SD a r e g i v e n . T h e r e s u l t s a r e e x p r e s s e d as ELISA u n i t s . For a s t e r i s k s , see T a b l e 1.
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BY COLCHICINE
531
A n t i b o d y Responses to T h y m u s - d e p e n d e n t A n t i g e n s (SRBC and HGG) A g g l u t i n i n s to SRBC of c o l c h i c i n e - t r e a t e d c h i c k e n s were low a f t e r the f i r s t and second s t i m u l a t i o n s , b u t reached the mean t i t e r of c o n t r o l chickens a f t e r t h e t h i r d s t i m u l a t i o n ( T a b l e 3 ) . The chickens t r e a t e d w i t h c y c l o p h o s phamide had p r a c t i c a l l y no a g g l u t i n i n s a g a i n s t SRBC, even a f t e r the t h i r d antigen stimulation (Table 3). IgM, IgA and IgG class a n t i b o d y responses against another thymus-dependent antigen, HGG, of c o l c h i c i n e - t r e a t e d c h i c k e n s were lower than those of u n t r e a t e d chickens a f t e r the f i r s t and second s t i m u l a t i o n s , b u t a f t e r the t h i r d s t i m u l a t i o n t h e y reached the a n t i b o d y l e v e l s of u n t r e a t e d c h i c k e n s ( T a b l e 5). Cyclophosphamide-treated c h i c k e n s had p r a c t i c a l l y no a n t i b o d i e s a g a i n s t HGG.
Mitocjen Responses of P e r i p h e r a l Blood L y m p h o c y t e s The responses of p e r i p h e r a l blood l y m p h o c y t e s to PHA and Con A in c o l c h i c i n e - t r e a t e d c h i c k e n s were s i m i l a r to those in u n t r e a t e d c o n t r o l b i r d s (Table 6). The mitogen responses of c y c l o p h o s p h a m i d e - t r e a t e d chickens were o n l y s l i g h t l y lower than those in c o n t r o l s and c o l c h i c i n e - t r e a t e d chickens both at t h r e e and s i x weeks of age.
TABLE 6 Mitogen
Age of chickens (wks)
•
responses in c h i c k e n s t r e a t e d w i t h colchicine or c y c l o p h o s p h a m l d e
Mitogen
Untreated chickens (n = 4)
Colchicinet r e a t e d chickens (n = 13)
a
Cyclophosphamidet r e a t e d chickens (n = 8)
-
179 ± 86
220 ± 158
186 ± 145
PHA
17216 ± 9999
9305 ± 6410
5666 ± 3956*
Con A
6814 ± 5573
5673 ± 7305
1963 ± 3024
-
325 ± 214
268 ± 315
137 ± 62
PHA
24826 ± 20230
25019 ± 20909
10092 ± 11678
Con A
5531 ± 7042
4272 ± 5949
1688 ± 846
Mean + SD of cpm are g i v e n . For a s t e r i s k s , see Table 1.
DISCUSSION Colchicine g i v e n p e r anum a f t e r h a t c h i n g induced a d e f i c i e n c y of humoral i m m u n i t y , which c o n f i r m s the r e s u l t s of a p r e v i o u s s t u d y ( 3 ) . The a n t i b o d y response to a t h y m u s - i n d e p e n d e n t a n t i g e n B r u c e l l a a b o r t u s of these
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BURSECTOMY BY COLCHICINE
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chickens was v e r y low a f t e r the f i r s t and second s t i m u l a t i o n s , b u t a f t e r the t h i r d s t i m u l a t i o n the a g g l u t i n i n t i t e r s were about 40 96 of those of control chickens. T h i s f i n d i n g w i t h the r e s t o r a t i o n of SRBC responses i n d i c a t e s t h a t the i m m u n o d e f i c i e n c y induced by a p p l i c a t i o n of colchicine on the anal lips is followed by a p a r t i a l spontaneous r e c o n s t i t u t i o n of humoral i m m u n i t y . R e l a t i v e l y tow B r u c e l l a responses i n d i c a t e t h a t t h i s a n t i g e n may r a t h e r be r e g a r d e d as a b u r s a - d e p e n d e n t a n t i g e n than as a t h y m u s - independent antigen. On the c o n t r a r y , the t r e a t m e n t of c h i c k e n s with c y c l o p h o s p h a m i d e in the n e w l y hatched p e r i o d leads to a s e v e r e r d e f i c i e n c y in humoral i m m u n i t y ( 1 1 - 1 4 ) , which a p p a r e n t l y is due to local d e s t r u c t i o n of b u r s a cells as well as d e s t r u c t i o n of B cell p r e c u r s o r s and mature B cells in e x t r a b u r s a l s i t e s . It is l i k e l y t h a t in c o l c h i c i n e - t r e a t e d b i r d s e x t r a b u r s a l B cell p r e c u r s o r s l a t e r colonize the b u r s a r e s u l t i n g in a p a r t i a l r e g e n e r a t i o n of the b u r s a l s t r u c t u r e and p a r t i a l r e s t o r a t i o n of humoral immune f u n c t i o n s . It is also e v i d e n t t h a t the b u r s a of c o l c h i c i n e - t r e a t e d c h i c k e n s r e t a i n s its l y m p h o t a c t i c and l y m p h o c y t e - d i f f e r e n t i a t i n g c a p a c i t y and t h u s these c h i c k e n s can be used as r e c i p i e n t s in cell t r a n s f e r e x p e r i ments like c y c l o p h o s p h a m i d e - t r e a t e d b i r d s ( 4 ) . In c o n t r a s t , it has been d e m o n s t r a t e d t h a t t e s t o s t e r o n e t r e a t m e n t a f f e c t s the b u r s a l stroma and d e s t r o y s its c a p a c i t y to s e r v e as a m a t u r a t i o n site f o r B cell p r e c u r s o r s (15). In c o l c h i c i n e - t r e a t e d c h i c k e n s , the a n t i b o d y responses to the t h y m u s d e p e n d e n t a n t i g e n s SRBC and HGG were d i s t u r b e d a f t e r the f i r s t and second a n t i g e n s t i m u l a t i o n s . H o w e v e r , the responses were p r a c t i c a l l y the same as in u n t r e a t e d c h i c k e n s a f t e r the t h i r d s t i m u l a t i o n . This indicates t h a t the responses are o n l y d e l a y e d , p r o b a b l y due to a slow r e s t o r a t i o n of the b u r s a l s t r u c t u r e . Also the l y m p h o c y t e responses to PHA and Con A were p r e s e r v e d . In a d d i t i o n , no changes in the t h y m i c m o r p h o l o g y were observed. T h u s , it appears t h a t the c e l l - m e d i a t e d i m m u n i t y in chickens t r e a t e d w i t h colchicine p e r anum is not s i g n i f i c a n t l y a f f e c t e d . This finding is a n t i c i p a t e d because colchicine causes a s e l e c t i v e local i n i u r y in the b u r s a , whereas no d i r e c t e f f e c t s in the spleen, t h y m u s or caecal t o n s i l s have been o b s e r v e d ( 1 ) . On the o t h e r hand, c y c l o p h o s p h a m i d e , g i v e n i.p., causes a s e v e r e i n j u r y in the whole lymphoid s y s t e m , i n c l u d i n g the t h y m u s , a l t h o u g h d u r i n g a few weeks a f t e r the i n i t i a l i n j u r y the t h y m i c m o r p h o l o g y r e g e n e r a t e s (12, 16). In p r e v i o u s r e p o r t s v a r i a b l e defects of c e l l - m e d i a t e d i m m u n i t y have been r e p o r t e d a f t e r c y c l o p h o s p h a m i d e t r e a t m e n t (12, 14, 16-18). The d i f f e r e n c e s are p r o b a b l y due m a i n l y to the v a r i a t i o n of d r u g doses as well as t i m i n g of the t r e a t m e n t , g e n e t i c b a c k g r o u n d of the c h i c k e n s and the e x p e r i m e n t a l s e t - u p used. Of p a r t i c u l a r i n t e r e s t are the f i n d i n g s of IgA and IgG class a n t i b o d i e s against Brucella. IgA and IgG a n t i b o d i e s in h o r m o n a l l y bursectomized c h i c k e n s are p r a c t i c a l l y absent and in s u r g i c a l l y bursectomized c h i c k e n s o n l y s l i g h t l y d i m i n i s h e d (19, 20). The p r e s e n t s t u d y i n d i c a t e s t h a t in c h i c k e n s t r e a t e d w i t h colchicine the switch from IgM to both IgA and IgG may be e i t h e r delayed or d i s t u r b e d . The l a t t e r p o s s i b i l i t y seems more p r o b a b l e since in these c h i c k e n s c l a s s - s p e c i f i c a n t i - H G G a n t i b o d i e s a f t e r the t h i r d a n t i g e n s t i m u l a t i o n were on the same level as in normal c o n t r o l s , while c l a s s - s p e c i f i c a n t i - B r u c e l l a a n t i b o d i e s were s i g n i f i c a n t l y lower than those of control c h i c k e n s even a f t e r the t h i r d a n t i g e n s t i m u l a t i o n . It is possible t h a t the absence of IgA and IgG a n t i b o d i e s a g a i n s t B r u c e l l a is due to a local d e s t r u c t i o n of T cells r e g u l a t i n g the switch of immunoglobulin i s o t y p e s in the b u r s a . Whether t h i s d i s t u r b a n c e also applies to o t h e r t h y m u s - i n d e p e n d e n t or b u r s a - d e p e n d e n t a n t i g e n s remains to be shown.
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In c o n c l u s i o n , o u r r e s u l t s d e m o n s t r a t e t h a t t h y m u s - d e p e n d e n t immune f u n c t i o n s of c h i c k e n s t r e a t e d n e o n a t a l l y p e r anum w i t h c o l c h i c i n e remain p r a c t i c a l l y normal. Due to o n l y local d e s t r u c t i o n of B l y m p h o c y t e s in t h e b u r s a this t y p e of b u r s e c t o m y p r o v i d e s a new model f o r s t u d i e s of s p o n t a neous r e g e n e r a t i o n of t h e humoral immune system and e x t r a b u r s a l r e s e r v e s of B l y m p h o c y t e p r o g e n i t o r s .
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Romppanen T , S o r v a r i TE. Late e f f e c t s of colchicine on the b u r s a of F a b r i c i u s a f t e r neonatal application on the anal lips of c h i c k e n s . Acta Pathol Microbiol Scand ( A ) . 1980; 88:407-413.
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Hirota Y, M a r t i n M - T , Viljanen M, T o i v a n e n P, F r a n k l i n RM. Immun o p a t h o l o g y of c h i c k e n s i n f e c t e d in ovo and at h a t c h i n g w i t h the avian o s t e o p e t r o s i s v i r u s M A V . 2 - 0 . Eur J Immunol. 1980; 10:929-936.
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11.
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Received Accepted
: September, 1982 : March, 1983
in ovo t r e a t e m e n t w i t h chicken. Cell Immunol.
cyclophos1974; 13:
on the
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