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Immune response to different bacterial and synthetic antigens during ontogeny
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 7, pp. 743-744, 1983. 0145-305X/83 $3.00 + .00 Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.
IMMUNE RESPONSE TO DIFFERENT BACTERIAL AND SYNTHETIC ANTIGENS DURING ONTOGENY
H.Tlaskalov&-Hogenov&, O.KopeEek I, M. Pospi~il, B.~lhov&, M.Nov&k 2, K. Ulbrich I, O. Hofman, V.V~tviEka Institute of Microbiology, Czech.Acad.Sci,, CS-142 20 Prague 4; llnstltute of Macromolecular Chemistry, Czech.Acad,Sci., CS162 06 Prague 6; 21nstltute of Virology, Slovak Acad.Scl,, CS-800 000 Bratlslava, Czechoslovakia
For i n d u c t i o n of a n t i b o d y response to d i f f e r e n t b a c t e r i a l and synthetic antigens the cells from fetal liver or spleen of C3H/DiScSN mice of various ages were cultivated in H-MEM supplemented with i0 ~ fetal calf serum, 2 mercaptoethanol and antibiotics in microtest plates (1). On day 4 of cultivation the number of plaque-formlng cells (PFC) was estimated using haptenated sheep red blood cells: TNP-SRBC, FITC-SRBC, ARS-SRBC, The proliferative response was studied after 3 days of cultivation of cells isolated from mice at various ages by adding 3H-thymldine 8-12 h before the end of cultivation. After activation of cells with lipopolyssccharlde (LPS) the degree of polyclonal response was demonstrated by simultaneous appearance of TNP, FITC and ARS-PFC in embryonal liver cells beginning with the 18th day. Using haptenated polymerized flagellins (FLA) specific antibody response against all the haptens was first found in lO-day-old mice (Table I). To study their specificity, PFC were inhibited by appropriate hapten conjugates. In our system we did not find any sequential maturation of responsiveness to the haptens used. When comparing the antibody response of young, adult and old mice we found that fetal liver cells and spleen cells of lO-dayo l d mice r e q u i r e d l O 0 - f o l d h i g h e r doses of p a r t i c u l a t e a n t i g e n s ( s u s p e n s i o n of heat i n a c t i v a t e d E . c o l i c o n j u g a t e d w i t h DNP) than the spleen c e l l s from a d u l t mice. On the o t h e r hand, the optimum dose of s o l u b l e b a c t e r i a l (LPS, D N P - f l e g e l l i n ) or s y n t h e t i c a n t i g e n s evoking a p o l y c l o n a l or s p e c i f i c response i n v i t r o was comparable i n young and a d u l t mice ( T a b l e 1 ) . I n a l l groups of mice i n c o r p o r a t i o n of 3 H - t h y m i d i n e was the highest after in vitro stimulation with particulate bacterial antigens. In aged mice both the antibody and proliferative response of cells to different antigens was decreased. To study the role of antigen structure in the development of 743
744
ANTIBODY RESPONSES DURING ONTOGENY
Vol. 7, No. 4
a n t i b o d y response, B c e l l s were s t i m u l a t e d w l t h a n t l g e n a a y n t h e t l z e d f o r the purpose, i . e . s o l u b l e methacrylamtde polymers (P-Acap-Leu-HHDA-DNP) of d i f f e r e n t m o l e c u l a r w e i g h t ( 2 ) . A n t i body response to these polymers i n nude mlce i n d i c a t e d the T independence of these a n t i g e n s . DNP-polymers (DNP-POL) of high m o l . w t , evoked a peak response at lower doses than d i d low mol e c u l a r p o l y m e r s . High m o l e c u l a r polymers s t i m u l a t e d the a n t i body response e a r l l e r i n ontogeny than low m o l e c u l a r ones. Our r e s u l t a l n d t c a t e t h a t the a c t i v a t i n g p r o p e r t i e s of the antlgen (carrier) are h i g h l y I n f l u e n c e d by i t s p h y s l c a l s t a t e a n d / o r s t r u c t u r e whlch p l a y s a d e c l s l v e r o l e i n the onset of a n t l b o d y f o r m a t i o n and the h e i g h t of the response. Table 1. In v i t r o Response to D i f f e r e n t T Independent A n t l g e n s d u r l n g Ontogeny. Hean Number of TNP-PFC/106 c u l t i v a t e d c e l l s . Age 18th day fetal liver
A n t t g e n ii
_i ~
=ii
/
0.1 1
)
(/ug/ml
10
DNP-FLA (rig)
IL
43.2
0 22.2 18.4 0
103 105 107
0 0 7.8
63.1 123.4 83.3
109
12.9
0 0 21.2 36.4
12.2
26.3 61.5 54.3 24.2
i0
0
0
75.3
n,d.
100
0 0
5.2 4.8
98.7
0
0
i0
0
0
i00 i000 10000
0 0 0
0
0
0
•
ii
-
i
25.4 40.8 36.4 20.7
20.1 22.5 26.4 40.8 l
,
12.3
91.2 80.3
0 0 ,
i.
i
11.3 16.8
0 0 0 0
m.w.
i
14.8
i
1 10 100 1000
1000 143 x 103 10000
I
3.5 5.6
i
0
(ng) m.w.
-
_
33.5 55.6 97.6 24.5 45.3 81.8 56.5 47.1
0 11.2
0
DNP-POL
~
0
100
DNP-COLI microbes
25 x 103
donors 4 months 18 months spleen spleen
ii
LPS
IL
of cell 10 days spleen
~
....
•
_ _ _
7.6
,,,
9.2 _
TLASKALOVA-HOGENOVA,H. , ~IHOVA,B., HOFMAN,D., NOVAK,M. , Z I KAN,D. S p e c i f i c i t y and a f f i n i t y of antibodies produced a f t e r in v i t r o and in vivo a c t i v a t i o n of B c e l l s with l l p o p o l y e a c c h e r i d e , DNP-1ipopoZyeacchartde, f l a g e l l i n and D N P - f l a g e l l t n . F o l i a b t o l j , 2 5 , 393, 1979. 2. KOPE~EKoO. R e a c t i v e copoZymera of N - ( 2 - h y d r o x y p r o p y l ) - m e t h a c r y l a m i d e w i t h N - m e t h a c r y l o y l a t e d d e r i v a t i v e s of L - l e u c t n e and L - p h e n y l a l a n i n e . I . P r e p a r a t i o n , c h a r a c t e r i z a t i o n and r e a c t i o n w i t h d i a m i n e s . Macromol.Chem. 178, 2169, 1977.
IMMUNE RESPONSE TO DIFFERENT BACTERIAL AND SYNTHETIC ANTIGENS DURING ONTOGENY
H.Tlaskalov&-Hogenov&, O.KopeEek I, M. Pospi~il, B.~lhov&, M.Nov&k 2, K. Ulbrich I, O. Hofman, V.V~tviEka Institute of Microbiology, Czech.Acad.Sci,, CS-142 20 Prague 4; llnstltute of Macromolecular Chemistry, Czech.Acad,Sci., CS162 06 Prague 6; 21nstltute of Virology, Slovak Acad.Scl,, CS-800 000 Bratlslava, Czechoslovakia
For i n d u c t i o n of a n t i b o d y response to d i f f e r e n t b a c t e r i a l and synthetic antigens the cells from fetal liver or spleen of C3H/DiScSN mice of various ages were cultivated in H-MEM supplemented with i0 ~ fetal calf serum, 2 mercaptoethanol and antibiotics in microtest plates (1). On day 4 of cultivation the number of plaque-formlng cells (PFC) was estimated using haptenated sheep red blood cells: TNP-SRBC, FITC-SRBC, ARS-SRBC, The proliferative response was studied after 3 days of cultivation of cells isolated from mice at various ages by adding 3H-thymldine 8-12 h before the end of cultivation. After activation of cells with lipopolyssccharlde (LPS) the degree of polyclonal response was demonstrated by simultaneous appearance of TNP, FITC and ARS-PFC in embryonal liver cells beginning with the 18th day. Using haptenated polymerized flagellins (FLA) specific antibody response against all the haptens was first found in lO-day-old mice (Table I). To study their specificity, PFC were inhibited by appropriate hapten conjugates. In our system we did not find any sequential maturation of responsiveness to the haptens used. When comparing the antibody response of young, adult and old mice we found that fetal liver cells and spleen cells of lO-dayo l d mice r e q u i r e d l O 0 - f o l d h i g h e r doses of p a r t i c u l a t e a n t i g e n s ( s u s p e n s i o n of heat i n a c t i v a t e d E . c o l i c o n j u g a t e d w i t h DNP) than the spleen c e l l s from a d u l t mice. On the o t h e r hand, the optimum dose of s o l u b l e b a c t e r i a l (LPS, D N P - f l e g e l l i n ) or s y n t h e t i c a n t i g e n s evoking a p o l y c l o n a l or s p e c i f i c response i n v i t r o was comparable i n young and a d u l t mice ( T a b l e 1 ) . I n a l l groups of mice i n c o r p o r a t i o n of 3 H - t h y m i d i n e was the highest after in vitro stimulation with particulate bacterial antigens. In aged mice both the antibody and proliferative response of cells to different antigens was decreased. To study the role of antigen structure in the development of 743
744
ANTIBODY RESPONSES DURING ONTOGENY
Vol. 7, No. 4
a n t i b o d y response, B c e l l s were s t i m u l a t e d w l t h a n t l g e n a a y n t h e t l z e d f o r the purpose, i . e . s o l u b l e methacrylamtde polymers (P-Acap-Leu-HHDA-DNP) of d i f f e r e n t m o l e c u l a r w e i g h t ( 2 ) . A n t i body response to these polymers i n nude mlce i n d i c a t e d the T independence of these a n t i g e n s . DNP-polymers (DNP-POL) of high m o l . w t , evoked a peak response at lower doses than d i d low mol e c u l a r p o l y m e r s . High m o l e c u l a r polymers s t i m u l a t e d the a n t i body response e a r l l e r i n ontogeny than low m o l e c u l a r ones. Our r e s u l t a l n d t c a t e t h a t the a c t i v a t i n g p r o p e r t i e s of the antlgen (carrier) are h i g h l y I n f l u e n c e d by i t s p h y s l c a l s t a t e a n d / o r s t r u c t u r e whlch p l a y s a d e c l s l v e r o l e i n the onset of a n t l b o d y f o r m a t i o n and the h e i g h t of the response. Table 1. In v i t r o Response to D i f f e r e n t T Independent A n t l g e n s d u r l n g Ontogeny. Hean Number of TNP-PFC/106 c u l t i v a t e d c e l l s . Age 18th day fetal liver
A n t t g e n ii
_i ~
=ii
/
0.1 1
)
(/ug/ml
10
DNP-FLA (rig)
IL
43.2
0 22.2 18.4 0
103 105 107
0 0 7.8
63.1 123.4 83.3
109
12.9
0 0 21.2 36.4
12.2
26.3 61.5 54.3 24.2
i0
0
0
75.3
n,d.
100
0 0
5.2 4.8
98.7
0
0
i0
0
0
i00 i000 10000
0 0 0
0
0
0
•
ii
-
i
25.4 40.8 36.4 20.7
20.1 22.5 26.4 40.8 l
,
12.3
91.2 80.3
0 0 ,
i.
i
11.3 16.8
0 0 0 0
m.w.
i
14.8
i
1 10 100 1000
1000 143 x 103 10000
I
3.5 5.6
i
0
(ng) m.w.
-
_
33.5 55.6 97.6 24.5 45.3 81.8 56.5 47.1
0 11.2
0
DNP-POL
~
0
100
DNP-COLI microbes
25 x 103
donors 4 months 18 months spleen spleen
ii
LPS
IL
of cell 10 days spleen
~
....
•
_ _ _
7.6
,,,
9.2 _
TLASKALOVA-HOGENOVA,H. , ~IHOVA,B., HOFMAN,D., NOVAK,M. , Z I KAN,D. S p e c i f i c i t y and a f f i n i t y of antibodies produced a f t e r in v i t r o and in vivo a c t i v a t i o n of B c e l l s with l l p o p o l y e a c c h e r i d e , DNP-1ipopoZyeacchartde, f l a g e l l i n and D N P - f l a g e l l t n . F o l i a b t o l j , 2 5 , 393, 1979. 2. KOPE~EKoO. R e a c t i v e copoZymera of N - ( 2 - h y d r o x y p r o p y l ) - m e t h a c r y l a m i d e w i t h N - m e t h a c r y l o y l a t e d d e r i v a t i v e s of L - l e u c t n e and L - p h e n y l a l a n i n e . I . P r e p a r a t i o n , c h a r a c t e r i z a t i o n and r e a c t i o n w i t h d i a m i n e s . Macromol.Chem. 178, 2169, 1977.