Unresponsiveness in Balb/c mice following repeated doses of sheep erythrocytes
by Philip Depoyster Thomson
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE in Microbiology
Montana State University
© Copyright by Philip Depoyster Thomson (1972)
Abstract:
Balb/c mice were rendered unresponsive to sheep red blood cells by injections of 5 x 10 8 sheep red
blood cells every other day from 7 days of age to adulthood. It was shown that unresponsiveness was
specific by injection of burro red blood cells which produced normal plaque-forming cell responses in
SRBC-unresponsive mice. Neither normal thymus nor bone marrow alone or in combination, nor
normal peritoneal exudate cells could reconstitute unresponsive mice. However, SRBC-primed
peritoneal exudate cells (80-90%,macrophage cells) did reconstitute the plaque-forming response of
unresponsive mice. Normal Balb/c mice were rendered unresponsive by injection of 32 hemagglutinin,
units of serum from unresponsive mice. Serum from unresponsive mice was absorbed with anti-IgG2.
Serum absorbed in this manner failed to render mice unresponsive when administered passively. It was
concluded that unresponsiveness to sheep red blood cells involves IgG2 which interacts with the
macrophage and, in turn, inhibits antigen-macrophage interaction. Thus this system represents a
feedback mechanism which prevents the presentation of antigen to the lymphocyte. In p r e s e n tin g t h i s t h e s i s in p a r t i a l f u lf illm e n t o f th e r e q u ir e ­
m e n ts f o r a n .a d v a n c e d d e g r e e a t M ontana S t a t e U n i v e r s i t y , I a g r e e t h a t
t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r i n s p e c t i o n .
I fu rth e r
a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y in g o f t h i s t h e s i s f o r
, s c h o l a r l y p u r p o s e s may b e g r a n t e d by my m a jo r p r o f e s s o r , o r , i n h i s
a b se n c e , by th e D ire c to r o f L i b r a r ie s .
I t i s u n d e r s to o d t h a t any
c o p y in g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a in s h a l l n o t
b e a llo w e d w i t h o u t my w r i t t e n p e r m i s s i o n .
Z
UNRESPONSIVENESS IN BALE/C MICE FOLLOWING REPEATED
DOSES OF SHEEP ERYTHROCYTES
by
PH ILIP DEPOYSTER THOMSON
A t h e s i s s u b m itte d to t h e G ra d u a te F a c u l t y i n p a r t i a l
f u l f i l l m e n t o f t h e r e q u i r e m e n ts f o r t h e d e g re e
of
MASTER OF SCIENCE
in
M ic r o b io lo g y
A p p ro v ed :
'C h a irm a n , E x a m in in g C om m ittee
MONTANA STATE UNIVERSITY
Bozem an, M ontana
M arch , 1972
iii
ACKNOWLEDGEMENTS
I th a n k D r. Jo h n W. J u t i l a f o r h i s a s s i s t a n c e d u r in g t h e c o u rs e
o f th is rese a rc h .
I a l s o th a n k D r. W illia m D. H i l l , D r. D av id G.
S t u a r t , D r. Norman D. Reed and D r. Dean D, M anning f o r t h e i r h e lp i n
p r e p a r in g t h i s m a n u s c rip t.
T h is w ork was s u p p o r te d i n p a r t by U .S .
P u b l i c H e a l th S e r v i c e T r a i n i n g G ra n t A I0 0 1 3 1 -1 1 and by U .S . P u b l i c
H e a l th S e r v i c e G ra n t A I0 6 5 5 2 -0 7 .
iv
TABLE OF CONTENTS
P age
V I T A .................................................
ii
ACKNOWLEDGEMENTS............................................................................
iii
TABLE OF CONTENTS..........................................................................................................
LIST OF TABLES .
. .....................................................................................................
iv
vi
LIST OF FIGURES....................................................................................................................v i i
ABSTRACT . . ......................................................................................................................v i i i
' INTRODUCTION ............................................................................................ . . . . .
I
MATERIALS AND METHODS.......................................'.
8
M ice . . . . . . . . . . .
. ... ...........................................’
............................................
8
Im m u n o lo g ic U n r e s p o n s i v e n e s s .................................. ....
.
8
I r r a d i a t i o n . ................................................ , . . . • ......................................
9
R e c o n s t i t u t i o n w i t h Bone M arrow - T h y m u s .......................................
9
R e c o n s t i t u t i o n w i t h P e r i t o n e a l E x u d a te C e l l ..................................
10 .
Im m u n o g lo b u lin Q u a n t i t a t i o n .
11
. . . . . . .
. . . y .
..................................
P a s s i v e " T u r n o f f " ..............................................................
Im m u n o g lo b u lin C l a s s o f T u r n o f f A n tib o d y
12
.
.
.
.
.
.
12
RESULTS..................................................................................................................................
14
U n r e s p o n s iv e n e s s ................................................................................................
14
R e c o n s t i t u t i o n w i t h Bone M arrow - T h y m u s .......................................
18
R e c o n s t i t u t i o n w i t h P e r i t o n e a l E x u d a te C e l l ..................................
18
V
V
P age
Im m u n o g lo b u lin Q u a n t i t a t i o n ............................
21
P a s s i v e T u r n o f f ..............................................................
21
Im m u n o g lo b u lin C l a s s o f T u r n o f f A n tib o d y ..................................
.
21
D ISCU SSIO N ...................................................................
26
SUMMARY................................................................................................
29
LITERATURE C li1E D .......................................................................................................... ' 1
30
vi
LIST OF TABLES
P age
T a b le
I
T a b le I I
S am p les o f e x p e r i m e n t a l m o d els o f
t o l e r a n c e 1962 -1 9 6 6 . . . »
............................................
4
. P l a q u e - f o r m i n g c e l l (PFC) r e s p o n s e o f
S R B C ru n re sp o n siv e B a l b / c m ic e to b u r r o
e r y t h r o c y t e s ..................................................................................
16
v ii
LIST OF FIGURES
P age
F ig u re I .
F ig u re 2 .
F ig u re 3.
F ig u re 4 .
F ig u re 5 .
F ig u re 6 .
F i g u r e 7.
P l a q u e - f o r m i n g c e l l (PFC) r e s p o n s e
i n B a l b / c m ic e t r e a t e d w i t h r e p e a t e d
d o s e s o f s h e e p e r y t h r o c y t e s ............................................
15
H e m a g g lu tin in (HA) and h e m o ly s in (HL)
a n ti b o d y t i t e r i n B a l b /c m ic e t r e a t e d
w i t h r e p e a t e d d o s e s o f sh e e p e r y t h r o c y t e s . . .
17
P la q u e - f o r m in g c e l l (PFC) r e s p o n s e o f
n o rm a l B a l b /c m ic e r e c o n s t i t u t e d w ith
v a r i o u s c o m b in a tio n s o f b o n e m arrow and
thym us fro m n o rm a l and u n r e s p o n s i v e m ic e . . . .
19
P l a q u e - f o r m i n g c e l l (PFC) r e s p o n s e o f
- u n r e s p o n s i v e B a l b /c m ic e r e c o n s t i t u t e d
w i t h p rim e d p e r i t o n e a l e x u d a te c e l l s (P F C ). . .
20
Im m u n o g lo b u lin l e v e l s i n serum from
n o rm a l and u n r e s p o n s i v e m ic e ............................................
22
P l a q u e - f o r m in g c e l l (PFC) r e s p o n s e o f
n o rm a l B a l b / c m ic e t r e a t e d w ith serum
from u n r e s p o n s i v e m ic e . .....................................................
23
P la q u e - f o r m in g c e l l (PFC) r e s p o n s e o f
n o rm a l B a l b /c m ic e t r e a t e d w i t h a n ti- I g G g
a b s o rb e d " t u r n o f f " a n ti b o d y p l u s sh e ep
e r y t h r o c y t e s ........................ .........................................................
25
v iii
ABSTRACT
B a l b / c m ic e w e re r e n d e r e d u n r e s p o n s i v e to sh e e p r e d b lo o d c e l l s
by i n j e c t i o n s o f 5 x 10° s h e e p r e d b lo o d c e l l s e v e ry o t h e r d ay from
7 d a y s o f a g e t o a d u lt h o o d .
I t was shown t h a t u n r e s p o n s i v e n e s s was
s p e c i f i c by i n j e c t i o n o f b u r r o r e d b lo o d c e l l s w h ich p ro d u c e d n o rm a l
p l a q u e - f o r m i n g c e l l r e s p o n s e s i n S R B C -u n re sp o n siv e m ic e . N e i t h e r
n o rm a l thym us n o r b o n e m arrow a lo n e o r i n c o m b in a tio n , n o r n o rm a l
p e r i t o n e a l e x u d a te c e l l s c o u ld r e c o n s t i t u t e u n r e s p o n s i v e m ic e . How­
e v e r , SR BC-prim ed p e r i t o n e a l e x u d a te c e l l s (80-907=,m ac ro p h ag e c e l l s )
d i d r e c o n s t i t u t e t h e p l a q u e - f o r m i n g r e s p o n s e o f u n r e s p o n s i v e m ic e .
N orm al B a l b / c m ic e w ere r e n d e r e d u n r e s p o n s i v e by i n j e c t i o n o f 32 .
h e m a g g lu tin in , u n i t s o f serum fro m u n r e s p o n s i v e m ic e . Serum, from
u n r e s p o n s i v e m ic e was a b s o rb e d w i t h a h t i - I g G g . Serum a b s o rb e d i n t h i s
m an n er f a i l e d t o r e n d e r m ic e u n r e s p o n s i v e when a d m i n is t e r e d p a s s i v e l y .
. I t w as c o n c lu d e d t h a t u n r e s p o n s i v e n e s s t o sh e e p r e d b lo o d c e l l s
i n v o l v e s IgGg w h ic h i n t e r a c t s w ith t h e m ac ro p h ag e a n d , I n t u r n ,
i n h i b i t s a n tig e n - m a c r o p h a g e i n t e r a c t i o n .
Thus, t h i s s y s te m r e p r e s e n t s
a f e e d b a c k m echanism w h ic h p r e v e n t s t h e p r e s e n t a t i o n o f a n t i g e n to
t h e ly m p h o c y te .
INTRODUCTION
B u r n e t and F e n n e r (1 9 4 9 ) p r e d i c t e d t h a t e m b ry o n ic e x p o s u re to an
a n t i g e n i c s t i m u l u s w ould l e a d to s p e c i f i c u n r e s p o n s i v e n e s s o r t o l e r a n c e
to t h a t a n tig e n in l a t e r l i f e .
T h is c o n c e p t g e n e r a te d i n t e n s i v e
r e s e a r c h i n t o t h e n a t u r e o f t h e u n r e s p o n s i v e s t a t e and t h e c o n d i t i o n s
r e q u i r e d to in d u c e i t .
The te rm t o l e r a n c e was o r i g i n a l l y a p p l i e d to
th e u n re s p o n s iv e s t a t e fo llo w in g th e i n j e c t i o n o f l iv i n g a n tig e n , i . e . ,
t h e i n j e c t i o n o f a l l o g e n i c s p le e n c e l l s i n t o new born m ice-.
"Immuno­
l o g i c u n r e s p o n s i v e n e s s " , on t h e o t h e r h a n d , i s a te rm o r i g i n a l l y
s u g g e s te d by C hase and u s e d b y D ixon t o i d e n t i f y t h e g ro u p o f
phenom ena w h e re b y s p e c i f i c r e p r e s s i o n o f t h e v e r t e b r a t e immune r e s p o n s e
f o ll o w s e x p o s u re to n o n l i v i n g a n t i g e n s ( i n Sm ith., 1 9 6 1 ) .
N o n liv in g
a n t i g e n s a s d e s c r i b e d b y S m ith (1 9 6 1 ) i n c l u d e c h e m ic a ls , p r o t e i n s ,
c a r b o h y d r a t e s , r e d b lo o d c e l l s , b a c t e r i a l a n t i g e n s , and s i m i l a r
com plex m i x t u r e s .
I t h a s g e n e r a l l y b e e n a c c e p te d by m o st t h a t t h e te r m s " t o l e r a n c e " ,
" p a r a l y s i s " , " p r o t e i n o v e r l o a d i n g " , and " u n r e s p o n s iv e n e s s " may repre*s e n t a s p e c t s o f t h e same b a s i c p r o c e s s ( L e s k o w itz , 1 9 6 7 ).
E a r ly s t u d i e s i l l u s t r a t i n g t h e u n r e s p o n s i v e c o n d i t i o n i n c l u d e t h e
f e e d i n g o f cows m i l k , o% b l o o d , and egg w h ite to g u in e a p i g s (W e lls
and O s b o rn e , 1 9 1 1 ) , t h e i n j e c t i o n '- o f m ic e w i t h p n e u m o c o c c a l p o l y ­
s a c c h a r i d e ( F e l t o n and O t t i n g e r , 1 9 4 2 ) , t h e v a s c u l a r a n a s to m o s is o f
b o v in e tw in s b y Owen ( 1 9 4 5 ) , and t h e - i n h i b i t i o n o f e x p e r i m e n t a l d ru g
2
a l l e r g y by p r i o r f e e d i n g o f t h e s e n s i t i z i n g a g e n t by C h ase ( 1 9 4 6 ) .
Mope d e f i n i t i v e s t u d i e s w ere p e rf o r m e d by D ixon and M a u re r (1 9 5 5 )
w ho, u s i n g r a b b i t s g iv e n r e p e a t e d i n j e c t i o n s o f human p la s m a o r
b o v in e serum a lb u m in (BSA) from s h o r t l y a f t e r b i r t h u n t i l a p p r o x i ­
m a te ly 100 d a y s o f l i f e ,
d e t e c t e d no BSA a n ti b o d y , and t h e y c o u ld
d e t e c t no a n ti b o d y f o r a s lo n g a s 11 m o n th s a f t e r c e s s a t i o n o f a n t i g e n
in fu s io n .
The w ork o f S m ith ert a J . ( 1 9 5 7 ) and S m ith and B r id g e s
(1 9 5 8 ) sh e d m ore l i g h t on t h e m echanism o f t o l e r a n c e to p r o t e i n
a n tig e n s .
T h e se e x p e r im e n ts showed t h a t t h e r e was a r e l a t i o n b e tw e e n
a n t i g e n d o s e and d u r a t i o n o f t o l e r a n c e , t h a t p e r s i s t a n c e o f t h e u n ­
r e s p o n s i v e s t a t e was d e p e n d e n t upon a d d i t i o n a l in c r e m e n ts o f a n ti g e n
g iv e n w h ile t h e a n im a l was s t i l l t o l e r a n t , t h a t t o l e r a n c e i s h i g h ly
s p e c i f i c , t h a t a n t i g e n i c a l l y p rim e d lym ph n o d e c e l l s and s p le e n c e l l s
h a d no m ore r e c o n s t i t u t i v e e f f e c t t h a n d i d n o rm a l lym ph n o d e c e l l s
and s p le e n c e l l s when a d m i n is t e r e d to t o l e r a n t a n im a ls , and t h a t
n e i t h e r lym ph n o d e c e l l s n o r s p le e n c e l l s fro m t o l e r a n t a n im a ls ,
when g iv e n t o n o rm a l r e c i p i e n t s ,
c o u ld e l i c i t a n tib o d y p r o d u c t i o n .
S m ith (1 9 6 1 ) p r o p o s e d t h a t i n m o st c ir c u m s ta n c e s n o n e o f th e
l a t t e r , p r o d u c t i v e p h a s e s o f t h e immune r e s p o n s e o c c u r i n t h e
t o l e r a n t a n im a l.
S m ith , h o w e v e r, a g r e e s t h a t t h e r e a r e o t h e r i n t e r ­
p r e t a t i o n s , e . g . , t h a t a n tib o d y i s b e in g p ro d u c e d c o n s t a n t l y b u t
bound to a n t i g e n im m e d ia te ly o r t h a t t h e a n tib o d y p r o d u c in g c e l l i s
x
3
p o i s e d , b u t e n v ir o n m e n ta l c o n d i t i o n s a r e n o t f a v o r a b le f o r a n tib o d y
p rp d u c tio n .
S in c e 1 9 6 1 , much w ork h a s b e e n done on i n d u c t i o n o f t h e u n ­
r e s p o n s i v e s t a t e and on t h e c e l l u l a r m ech an ism s i n v o lv e d .
T a b le I
shows some o f t h e w ork done and i s p r e s e n t e d to show t h e d i v e r s i t y i n
e x p e r i m e n t a l m o d e ls .
From t h e s e m o d e ls , D r e s s e r and M itc h is o n (1 9 6 8 )
sum m arize t h e m ech an ism s in v o lv e d i n u n r e s p o n s i v e n e s s .
They s t a t e ,
"Im m u n o lo g ic p a r a l y s i s i s h e r e d e f i n e d a s a c e n t r a l f a i l u r e o f
r e s p o n s i v e n e s s , b r o u g h t a b o u t by e x p o s u re t o a n t i g e n , i n w h ic h immunol o g i c a l l y c o m p e te n t c e l l s (IC C ) becom e u n a b le to i n i t i a t e
o f a r e s t r i c t e d ran g e o f a n tib o d ie s ."
s y n th e s is
F o r many a n t i g e n s y s te m s , t h e
u n r e s p o n s i v e c o n d i t i o n f o llo w s am e n c o u n te r o f ICC w i t h u n p r o c e s s e d
a n t i g e n o r m a s s iv e d o s e s o f a n t i g e n w h ic h r e s t r i c t t h e a c t i v i t y o f
p o t e n t i a l a n ti b o d y - p r o d u c in g c e l l s .
I n some i n s t a n c e s , p a r a l y s i s o r
u n r e s p o n s i v e n e s s was th o u g h t to b e b r o u g h t a b o u t b y a n ti b o d y b l o c k i n g
a c c e s s o f a n t i g e n t o t h e r e c e p t o r s o f t h e ICC.
R e g a r d le s s o f th e
m echanism i n v o lv e d , u n r e s p o n s i v e n e s s seem s to d epend on t h e r e c r u i t ­
m en t o f new c o m p e te n t c e l l s by a th y m u s -d e p e n d e n t m ec h a n ism .
H ence,
i n a g e d o r i n th y m e c to m iz e d a n im a ls , r e c r u i t m e n t may o c c u r s lo w ly o r
not at a ll,
and t h e p r e c u r s o r a l a n tib o d y - f o r m in g c e l l t h a t was
a f f e c t e d i n t h i s m ech an ism was c o n c lu d e d t o b e a ly m p h o c y te .
4
T a b le I .
The d i v e r s i t y i n e x p e r i m e n t a l m o d els o f t o l e r a n c e ,
1 9 6 2 -1 9 6 6 .
R e fe re n c e
B a t t i s t o and M i l l e r 1962
M cG regor and Gowans 1963
B a th s o n , e t a l . 1963
D i e t r i c h and W e ig le 1963
Clam an and T alm age 1963
S c h w a rtz and Dam eshek 1963
S e r c a r z and Coons 1963
Gorman and C h a n d le r 1964
D o rn e r and U hr 1964
F rie d m a n 1964
M itc h is o n 1964
B ro o k e 1965
Row ley and F i t c h 1965
L i n s c o t t and W e ig le 1965
N o s s a l e t a l . 1965
S i s k i n d and Howard 1966
A u s t in and N o s s a l 1966
A z a r 1966
C ru c h a u d 1966
S t a p l e s , ^ t al_. 1966
W e ig le 1966
Thompson et: a l . 1966
M i t c h e l l and N o s s a l 1966
A n t i g e n ; A n im a l; A ssa y s y s te m
BCG; g u in e a p i g ; d e la y e d s k i n r e a c t i o n
BSA; m ic e ; m ac ro p h ag e i n d u c t i o n
K l e b s i e l l a p o l y s a c c h a r i d e ; m ic e ; a n tib o d y
tite r ,
SA and GG; m ic e ; I .
e lim in a tio n
BGG; m ic e ; p r o l o n g a t i o n o f t o l e r a n c e by
thym ectom y
BSA; r a b b i t ; ' a g g l u t i n a t i o n , p r e c i p i t a t i o n
■BSA; m ic e ; F a r r t e c h n i q u e , a g g l u t i n a t i o n
h y p o t h e s i s o f t h e in c o m p e te n t ly m p h o c y te
BSA; r a b b i t ; l ! 3 1 e l i m i n a t i o n , a g g l u t i n ­
a tio n
S h i g e l l a p o l y s a c c h a r i d e ; m ic e , c e l l
tra n s fe r
BSA; m ic e ; F a r r t e c h n i q u e , p r e c i p i t a t i o n
P neum ococcus p o l y s a c c h a r i d e ; m ic e ;
a g g lu tin a tio n
SRBC; r a t s ; p la q u e - f o r m in g c e l l s
BSA; r a b b i t ; F a r r t e c h n i q u e , p r e c i p i t a t i o n
S a lm o n e lla f l a g e l l i n ; r a t ; a n tib o d y
t i t e r , 2ME
P seudom onas p o l y s a c c h a r i d e ; m ic e ;
s u rv iv o rs
S a lm o n e lla f l a g e l l i n ; r a t ; a n tib o d y t i t e r
HGG; m ic e ; immune e l i m i n a t i o n , IEP
BSA; r a t ; a n tib o d y t i t e r , s k i n g r a f t
BGG; r a t ; i r r a d i a t i o n o f thym us and s p le e n
BSA; m ic e ; a g g l u t i n a t i o n , immune
e lim in a tio n
1 __
A l l o g e n i c RBC; m ic e ; I
e lim in a tio n
S a lm o n e lla f l a g e l l i n , ; r a t ; m echanism o f
to le ra n c e
5
C h i l l e r e t a!L. (1 9 7 1 ) h a v e shown e v id e n c e t h a t b o t h thym us and
b o n e m arrow c e l l s o f a d u l t m ic e c a n b e made s p e c i f i c a l l y u n r e s p o n s iv e
to human g l o b u l i n s , b u t t h a t e a c h c e l l p o p u l a t i o n h a s i t s own s p e c i a l
k in e tic s .
Human IgG (HGG), a s an a n t i g e n , shows s lo w e r i n d u c t i o n o f
u n r e s p o n s i v e n e s s i n b o n e m arrow th a n i n thym us c e l l s and i s a ls o
n e e d e d i n g r e a t e r q u a n t i t y i n b o n e m arro w .
T h is w ork a l s o show s, by
m eans o f r e c o n s t i t u t i o n e x p e r i m e n t s , t h a t o n l y o n e c e l l ty p e n e e d b e
t o l e r a n t to r e n d e r t h e a n im a l u n r e s p o n s i v e .
N o s s a l (1 9 5 8 ) h a s shown t h a t when s u c k l i n g r a t s a r e i n j e c t e d
r e p e a t e d l y w i t h s h e e p r e d b lo o d c e l l s
(SRBC) f o r a t l e a s t 7 w e e k s,
th e y becom e s p e c i f i c a l l y t o l e r a n t t o SRBC.
T h is phenom enon was
t h o u g h t to b e due t o a n t i g e n i c m a t e r i a l i n t h e c i r c u l a t i o n w h ich was
p re su m e d t o r e a c t w i t h and n e u t r a l i z e a n ti b o d y m o le c u le s .
shown n o t b e b e t h e c a s e .
T h is was
N o s s a l r e p e a t e d t h i s work u s i n g m ouse
e r y t h r o c y t e s i n r a t s and d i s c o v e r e d t h a t t o l e r a n c e p r o d u c e d was m ore
n e a r l y c o m p le te and\ o f l o n g e r d u r a t i o n , l a s t i n g up t o 9 m o n th s .
Row ley and F i t c h (1 9 6 4 ) p r o p o s e d a m echanism o f t o l e f 'a n c e i n
w h ic h r a t s made t o l e r a n t to SRBC w e re com pared t o r a t s made t o l e r a n t
by p a s s i v e im m u n iz a tio n w i t h hom ologous anti-S R B C se ru m .
The p la q u e ­
fo rm in g r e s p o n s e was r e d u c e d b o t h i n t o l e r a n t and p a s s i v e l y im m unized
ra ts .
T h u s , t h e m echanism p r o p o s e d was o n e o f " f e e d b a c k i n h i b i t i o n " ,
i n w h ic h an ti-S R B C i n h i b i t e d p o t e n t i a l a n tib o d y - f o r m in g c e l l s from
6
s y n t h e s i z i n g a n ti b o d y o r p r e v e n t e d a n t i g e n from r e a c h i n g and t r i g g e r ­
in g t h e a n ti b o d y p r o d u c in g c e l l ( a f f e r e n t b r a n c h ) .
M cC u llag h ( 1 9 7 0 ) , u s i n g r a t s made t o l e r a n t to SRBC, showed t h a t
t h e h e m o ly s in r e s p o n s e was e n h a n c e d i n t h o s e r a t s w h ic h r e c e i v e d
t h o r a c i c d u c t ly m p h o c y te s in c u b a t e d i n v i t r o w ith p rim e d p e r i t o n e a l
e x u d a te c e l l s .
He e n v is i o n e d some f a c t o r i n t h e t o l e r a n t r a t w hich
i s i n i m i c a l to t h e i n i t i a t i o n o f an immune r e s p o n s e i n t r a n s f u s e d ,
n o rm a l ly m p h o c y te s .
M i l l e r and M i t c h e l l ( 1 9 7 0 ) , w o rk in g w i t h a d u l t CBA m ic e r e n d e r e d
t o l e r a n t to SRBC and m a i n ta i n e d i n t h e t o l e r a n t s t a t e w i t h c y c lo ­
p h o s p h a m id e , h a v e d e m o n s tr a te d t h a t t o l e r a n c e i s n o t a c h ie v e d a t th e
n o n - th y m u s - d e r iv e d ly m p h o c y te l e v e l .
T hym ocytes a l s o d i d n o t e x h i b i t .
t o l e r a n c e b u t , i n c o n t r a s t , th y m u s - d e r iv e d c e l l s o f t h e t h o r a c i c d u c t
d id e x h i b i t t o l e r a n c e .
F eldm an and D ie n e r (1 9 7 0 ) h a v e shown s u p p r e s s io n to SRBC to b e
m e d ia te d b y p r e i n c u b a t i o n o f ly m p h o id c e l l s w ith a n tib o d y JLn v i t r o ,
and h a v e s t a t e d t h a t t h i s s u p p r e s s i o n i s a n a g o lo u s t o t o l e r a n c e on
■
t h e b a s i s o f a n t i g e n s p e c i f i c i t y and a c e n t r a l u n r e s p o n s i v e n e s s .
The a v a i l a b l e e v id e n c e d o e s n o t c o n c l u s i v e l y d e te r m in e t h e p r e c i s e
n a tu r e o f th e u n re s p o n s iv e s t a t e to e r y th r o c y te a n tig e n s n o r does i t
d e f i n i t e l y e s t a b l i s h t h e r o l e o f v a r i o u s c e l l u l a r e le m e n ts i n th e
in d u c tio n o f u n re s p o n s iv e n e s s .
T h u s , t h e p u r p o s e o f t h i s s tu d y was to
7
ex am in e t h e s t a t e o f s p e c i f i c u n r e s p o n s i v e n e s s o f m ic e t o r e p e a t e d
d o s e s o f s h e e p e r y t h r o c y t e s and to d e te r m in e t h e c e l l t y p e and
m echanism in v o lv e d
MATERIALS AND METHODS
M ice
I n b r e d B a l b /c m ic e w ere u s e d t h r o u g h o u t t h i s s t u d y .
The m ice
w e re o b t a i n e d from t h e N a t i o n a l I n s t i t u t e s o f H e a l th ( B e th e s d a ,
M a ry la n d ) i n 1966 and w ere i n t r o d u c e d i n t o t h e c o n v e n t i o n a l l y ^ r e a r e d
s to c k in 1967.
The m ic e a r e c u r r e n t l y m a i n ta i n e d by b r o t h e r - s i s t e r
m a tin g s and f e d a u to c l a v e d P u r i n a 5010C and- c h l o r i n a t e d , a c i d i f i e d
w a t e r , ad l i b i t u m .
Im m u n o lo g ic U n r e s p o n s iv e n e s s
M ice w ere r e n d e r e d immunol o g i c a l l y u n r e s p o n s i v e by i n t r a p e r i t o n e a l
( l.P .)
i n j e c t i o n s p f 5 x IO^ t h r i c e - w a s h e d SRBC i n a volum e o f 0 .1 ml
g iv e n e v e r y o t h e r d ay o f l i f e b e g in n i n g a t 7 d a y s o f a g e .
C o n tr o l
m ic e w ere g iv e n 0 .1 m l o f s t e r i l e p h y s i o l o g i c a l s a l i n e i n t h e same
r e g im e n .
The m ic e w e re a s s a y e d f o r r e s p o n s i v e n e s s to t h e re g im e n o f
a n t i g e n a t i o n a t d ay 1 0 , a t d ay 3 0 , and a t 30' day i n t e r v a l s .
was rem oved b y r e t r o - o r b i t a l b l e e d i n g and was p o o le d .
B lood
,Serum was
c o l l e c t e d fro m c l o t s , w h ic h s to o d a t room t e m p e r a t u r e f o r I h o u r ,
c e n t r i f u g e d a t 4 - 1 0 C , and a s s a y e d f o r b o t h h e m a g g lu tin in (HA) and
h e m o ly s in (HL) t i t e r
( J u t i l a , 1 9 6 9 ).
M ice w ere t h e n s a c r i f i c e d and
s p le e n s rem oved f o r a s s a y i n th e s l i d e m o d i f i c a t i o n o f t h e J e m e
p la q u e a s s a y ( M i s h e ll and D u tto n , 1 9 6 7 ) .
9
■ Irra d ia tio n
(
M ice w e re g iv e n .900 r a d w h o le b o d y gamma i r r a d i a t i o n u s i n g a ^ C o
s o u r c e , d e l i v e r i n g 128 r a d /m in o n to a p l e x i g l a s s c a g e (H and H
p l a s t i c s , L in c o ln , N e b ra sk a ).
T h is c a g e was p l a c e d i n a 19 x 19 cm
f i e l d on to p o f a t u r n t a b l e ( S ieck m an C o n s t r u c t i o n C o ., L in c o ln ,
N e b r a s k a ) , w h ic h was h e l d 60 cm b e lo w t h e s o u r c e .
12 m ic e ^ e r e i r r a d i a t e d a t p n e t im e .
I n t h i s m a n n e r,
U n ifo rm i r r a d i a t i o n was a c h ie v e d
b y r o t a t i n g t h e c a g e w i t h m ic e a t 2 rpm d u r in g i r r a d i a t i o n .
Maximum
'
b a c k s c a t t e r c o n d i t i o n s w e re p r o d u c e d by p l a c i n g a f i v e - i n c h m a s o n ite
b lo c k u n d e r th e c a g e .
R e c o n s t i t u t i o n w i t h Bone M arrow - Thymus
Bone m arrow c e l l s w e re o b t a i n e d fro m t h e fem ur and t i b i a l b o n e s
o f u n r e s p o n s i v e and n o rm a l m ic e b y c u t t i n g o f f t h e e p ip h y s e s o f b o n e s
f r e e o f a l l e x t e r n a l t i s s u e , and f o r c i n g t h e m arrow from t h e m arrow
c a v i t y by i n j e c t i n g 0.83% s a l i n e w i t h a 26 gu ag e n e e d le a f f i x e d to a
5 ml s y rin g e .
A s i n g l e c e l l s u s p e n s io n was made by g e n t l y p i p e t t i n g
w ith a P a s te u r p i p e t t e .
The c e l l s w e re w ashed i n s a l i n e ., l a r g e r
p a r t i c l e s a llo w e d t o s e t t l e o u t , and t h e c e l l s u s p e n s io n c o u n te d
u s i n g a h e m a c y to m e te r.
Bone m arrow c e l l s w ere u s e d i n d o s e s o f 5 x 10
fo r re c o n s titu tio n .
Thymus g la n d s w e re rem oved from u n r e s p o n s i v e and n o rm a l m ic e ,
and c e l l s w e re p r e p a r e d b y g e n t l y s c r e e n i n g th r o u g h #80 m esh s t a i n l e s s
10
s t e e l s c r e e n s i n t o 0.837» s a l i n e .
and a llo w e d t o s e t t l e
d o s e o f 3 .5 x 10
8
The c e l l s w ere w ashed o n c e i n s a l i n e
f o r 10 m i n u t e s .
The c e l l s w ere c o u n te d and a
thym us c e l l s w ere u s e d f o r r e c o n s t i t u t i o n .
A ll
m a n i p u l a t i o n s i n v o l v i n g c e l l s w ere c a r r i e d o u t a t 4 - 1 0 C.
M ice w e re r e c o n s t i t u t e d w i t h v a r i o u s c o m b in a tio n s o f n o rm a l and
u n r e s p o n s i v e thym us and b o n e m arrow c e l l s n o t m ore t h a n '5 h o u r s
f o ll o w i n g i r r a d i a t i o n .
Thymus and b o n e m arrow c e l l s w e re m ix e d w ith
I x IO8 SRBC and 0.837» s a l i n e was u s e d to b r i n g th e r e c o n s t i t u t i n g
d o s e to a volum e o f 0 .5 m l.
C e l l s u s p e n s i o n s w ere a d m i n i s t e r e d i n t r a ­
v e n o u s ly ( l . V . ) v i a t h e t a i l v e i n .
A f t e r 8 d a y s , t h e s p le e n s w ere
rem oved fro m r e c i p i e n t s and c e l l a s s a y s w e re p e rfo rm e d b y a s l i d e
m o d i f i c a t i o n o f t h e J e r n e p la q u e a s s a y ( M i s h e ll and D u t t o n , 1 9 6 7 ).
R e c o n s t i t u t i o n w i t h P e r i t o n e a l E x u d a te C e l l
Nonimmune B a l b /c m ic e w ere g iv e n 3 ml o f f l u i d t h i o g l y c o l l a t e
( D i f c o , D e t r o i t , M ic h ig a n ) b y t h e I . P . r o u t e .
t h i o g l y c o l l a t e , p e r i t o n e a l e x u d a te c e l l s
F iv e d a y s f o llo w in g
(PEC) w ere c o l l e c t e d by
w a s h in g t h e p e r i t o n e a l c a v i t y w i t h 5 m l o f H anks m in im a l e s s e n t i a l
medium (MEM) ( M i c r o b i o l o g i c a l A s s o c i a t e s , B e t h e s d a , M a ry la n d ) p l u s 57»
f e t a l c a l f se ru m .
I n a n o t h e r e x p e r im e n t, SRBC w ere g iv e n i n t r a -
8
p e r i t o n e a l l y i n a d o s e o f 5 x 10
to h a r v e s t .
c o n ta i n e d i n 0 .2 5 ml two h o u r s p r i o r
The p e r i t o n e a l C e l l s w e re w ashed o n c e i n MEM, c o u n te d
and i n j e c t e d i n t o u n r e s p o n s i v e m ic e .
PECs (5 x 1 0 ^ ). w e re a d m i n is t e r e d
11
i n t r a p e r i t o n e a l l y i n I ml d o s e s .
C o n t r o l m ic e r e c e i v e d 5 x 10
u n p rim e d p e r i t o n e a l c e l l s o r r e c e i v e d 5 x IO^ SRBC a l o n e .
A fte r 5
d a y s ,, s p l e e n s from a l l m ic e w ere a s s a y e d by t h e m o d if ie d p la q u e a s s a y .
Im m u n o g lo b u lin Q u a n t i t a t i o n
,
U n r e s p o n s iv e m ic e w ere s e l e c t e d a t d a y 210 f o r im m u n o g lo b u lin
(Ig ) q u a n tita tio n .
M ice w ere b l e d by r e t r o - o r b i t a l p u n c tu r e and
serum was c o l l e c t e d f o r a s s a y i n a m o d i f i c a t i o n o f t h e s i n g l e r a d i a l
immune d i f f u s i o n t e c h n i q u e o f M a s s e y e ff and Z i s s w i l l e r ( 1 9 6 9 ) .
D is c s ,
10 mm i n d i a m e t e r , w e re c u t from 1.0% a g a r o s e (S igm a C h e m ic a l C o .,
S t." L o u i s , M i s s o u r i ) p r e p a r e d i n 0 .2 M b a r b i t o l b u f f e r , pH 8 . 6 .
was p o u r e d i n t o l e v e l ,
in d e p th .
A gar
s h a llo w t r a y s r e n d e r i n g d i s c s u n i f o r m i l y 1 .5 mm
D is c s w e re p l a c e d a t room te m p e r a t u r e i n an o p e n , m o is t
cham ber f o r I h o u r .
A n t i s e r a (M e lp a r , F a l l s C h u rc h , V i r g i n i a )
s p e c i f i c f o r o n e o f t h e I g c l a s s e s w e re d i l u t e d 1 :2 0 i n b a r b i t o l b u f f e r
and 0 .0 5 ml was a p p l i e d to e a c h d i s c .
The d i s c s w ere t h e n p l a c e d i n ,
a m o i s t , c o v e re d cham ber a t room t e m p e r a t u r e f o r 1 2 -1 8 h o u r s .
S e r a t o b e q u a n t i t a t e d 'f o r im m u n o g lo b u lin s w ere p i p e t t e d i n t o a
2 mm w e l l c u t i n t o t h e c e n t e r o f e a c h d i s c w ith a b l u n t e d 18 guage
h y p o d e rm ic n e e d l e .
W e lls w ere t h e n f i l l e d w i t h 5
o f a n tig e n
(s e ru m ) p r e p a r e d i n tw o ^ f o ld d i l u t i o n s t e p s from 1 :1 2 8 .
The p r e c i p i t a t i o n r e a c t i o n was a llo w e d to p r o g r e s s f o r 48 h o u rs
a t room t e m p e r a t u r e a t w h ic h tim e e x c e s s p r o t e i n was rem oved by
12
w a s h in g t h r i c e w i t h 0.83% s a l i n e .
D is c s w ere th e n s t a i n e d w ith A cid
F u c h s in f o r 30 m in u te s and d e s t a i n e d w i t h a m ix tu r e o f w a t e r ,
m e th a n o l, and a c e t i c a c i d ( 4 : 5 : 1 ) .
The d i a m e te r s o f t h e p r e c i p i t a t i n g r i n g s w ere m e a s u re d w ith th e
a i d o f a m e a s u r in g m ic r o s c o p e .
P a s s iv e "T u rn o ff"
Serum was c o l l e c t e d from im m u n e lo g ic a lly u n r e s p o n s i v e m ic e by
c e n t r i f u g i n g b lo o d t a k e n b y r e t r o - o r b i t a l b l e e d i n g .
The seru m was
t i t e r e d and d i l u t i o n s made w i t h 0.83% s a l i n e to g iv e an a n tis e r u m
c o n t a i n i n g 32 HA u n i t s i n e a c h 0 .1 m l.
u n i t s o f se ru m , f o llo w e d b y I x 10
i n j e c t i o n s w e re a d m i n i s t e r e d I . P .
8
T e s t m ic e w ere g iv e n 32 HA
SRBC o n e h o u r l a t e r ; b o t h
C o n tr o l m ic e w ere g iv e n I x 10
SRBC a l o n e , o r e x p e r i e n c e d no a n t i g e n a t i o n .
5 days l a t e r ,
8
A l l m ic e w e re s a c r i f i c e d
and t h e i r s p le e n c e l l s w e re a s s a y e d f o r p la q u e - f o r m in g
c e lls .
Immunog l o b u l i n C la s s o f T u r n o f f A n tib o d y
Serum was c o l l e c t e d fro m u n r e s p o n s i v e m ic e and d i v i d e d i n t o 3
a liq u o ts .
One a l i q u o t was a b s o rb e d w i t h r a b b i t a n ti- m o u s e IgG^
(M e lp a r , F a l l s C h u rc h , V i r g i n i a ) , o n e a l i q u o t was a b s o rb e d w ith
r a b b i t a n ti- m o u s e IgG ( p r e p a r e d b y D. M an n in g , M ontana S t a t e U n i v e r s i t y ,
B ozem an, M o n ta n a ), a n d o n e a l i q u o t was n o t a b s o r b e d .
A ll a b s o rp tio n
13
was done a t 37 C.
N orm al m ic e r e c e i v e d 32 HA u n i t's o f o n e serum
a l i q u o t i n 0 .1 m l I . P .
f o llo w e d i n I h o u r w i t h I x IO^ SRBC I . P .
C o n tr o l m ic e r e c e i v e d I x IO^ SRBC a l o n e .
F iv e d a y s l a t e r , s p le e n
c e l l s w ere h a r v e s t e d and a s s a y e d f o r p l a q u e - f o r m in g c e l l s .
RESULTS
U n r e s p o n s iv e n e s s
The s p l e e n s o f H O m ic e r e c e i v i n g SRBC e v e ry o t h e r d ay fro m day
7 a f t e r b i r t h w e re a s s a y e d f o r t h e i r p la q u e - f o r m in g c e l l (PFC)
r e s p o n s e i n g ro u p s o f 10 m ic e e a c h b e g in n i n g a t day 1 0 , a g a in a t day
3 0 , and t h e n a t t h i r t y d ay i n t e r v a l s t h e r e a f t e r .
th e s e a s s a y s ap p e ar in F ig u re I .
The r e s u l t s o f
A v ig o r o u s d i r e c t and i n d i r e c t
p la q u e r e s p o n s e d e v e lo p e d i n t r e a t e d m ic e b y day 30 and p e a k e d a t
two m o n th s o f a g e .
T h e r e a f t e r , t h e r e s p o n s e r a p i d l y d e c l i n e d to l e s s
t h a n 100 PFCs p e r 10^ c e l l s .
- U n r e s p o n s iv e n e s s t o d i r e c t p la q u e a s s a y
was r e a c h e d e a r l i e r (b e tw e e n 60 and 90 d a y s ) w h ile u n r e s p o n s i v e n e s s
t o i n d i r e c t p l a q u e a s s a y was r e a c h e d som ew hat l a t e r (b e tw e e n 120 and
150 d a y s ) .
The u n r e s p o n s i v e s t a t e c o n tin u e d to p e r s i s t a t day 300
and t h e immune r e s p o n s e showed no s i g n s o f r e c o v e r y 45 d a y s ,a f t e r
c e s s a t i o n o f t r e a t m e n t w i t h SRBC.
T h a t t h e u n r e s p o n s iv e s t a t e i s
s p e c i f i c was shown when b u r r o c e l l s g iv e n t o u n r e s p o n s i v e m ic e (150
d a y s a f t e r i n i t i a t i o n o f t r e a t m e n t ) o r t o n o rm a l m ic e , p ro d u c e d a
n o rm a l r e s p o n s e ( T a b le I I ) .
The r e s u l t s o f HA and HL t i t e r s
a p p e a r i n F ig u r e 2 .
The k i n e t i c s
o f a n tib o d y s y n t h e s i s i n t h e i n t e r v a l d a y 10 t o day 120 seem to m im ic
t h e PFC r e s p o n s e .
H ow ever, m o d e r a te ly h i g h t i t e r s o f a n t i b o d y ,
p re s u m a b ly o f t h e IgG c l a s s , w ere m a i n ta i n e d i n t h e u n r e s p o n s i v e
a n im a l f o r some tim e a f t e r t h e p l a q u e - f o r m i n g r e s p o n s e w aned.
15
12001
IOOO-
INDIRECT
DIRECT
RFC
RFC
-
BACKGROUND RFC *
IO
30
60
90
12 0
ISO
ISO 2 IO 2 4 0
DAY
Figure I.
Plaque -forming cell (RFC) response in Balb / c mice
treated with repeated doses of sheep erythrocytes.
16
Table II.
Plaque forming cell (PFC) response of SRBCunresponsive Balb/c mice to burro erythrocytes.
Treatment
PFC/IO6
Burro cells
Sheep cells
direct
indirect
indirect
direct
Unresponsive mice0
given 5XIO8 burro
erythrocytes.
317
68
9
22
Normal mice given
5X108 burro erythro­
cytes.
541
82
2
O
0
2
I
Untreated mice
a
I
Mice assayed at day 150
17
2560-
HL
TITER
ha
TITER
Zj 1280
10
30
60
90
120 150 180 210
240
DAY
Figure 2. Hemagglutinin (HA) and hemolysin (HL) antibody titer in
Balb/c mice trea ted with repeated doses of sheep
erythrocytes.
18
R e c o n s t i t u t i o n w i t h Bone M arrow - Thymus
I r r a d i a t i o n f o llo w e d b y b o n e m arrow - thym us r e c o n s t i t u t i o n was
done i n h o p e s o f i d e n t i f y i n g t h e o r i g i n o f t h e c e l l i n v o lv e d i n u n ­
re s p o n s iv e n e s s .
g ro u p s.
Rec o n s t i t u t i o n was p e rf o r m e d u s in g 36 m ic e i n 4
The f o u r g ro u p s in c l u d e d r e c i p i e n t s o f n o rm a l b o n e m arrow -
n o rm a l thym us (N B-N T), n o rm a l b o n e m arrow = u n r e s p o n s i v e thym us (NBUT) , u n r e s p o n s i v e b o n e m arrow - n o rm a l thym us (UB-NT)3 and u n r e s p o n s iv e
b o n e m arrow - u n r e s p o n s i v e thym us (UB-UT).
A l l b o n e m arrow c e l l s
w ere a d m i n i s t e r e d i n a d o s e o f 5 x IO ^ , w h ile a l l thym us c e l l s w ere
8
g iv e n i n a d o s e o f 3 .5 x 10 .
in F ig u re 3 .
The r e s u l t s o f t h i s e x p e r im e n t a p p e a r
E x c e p t f o r NB-NT3 t h e d i r e c t PFC r e s p o n s e was d e p r e s s e d
Thfereas t h e i n d i r e c t PFC r e s p o n s e was e l e v a t e d i n a l l c o m b in a tio n s
o f c e l l s e x a m in e d , p a r t i c u l a r l y t h e UB-UT c o m b in a tio n .
R e c o n s t i t u t i o n w i t h P e r i t o n e a l E x u d a te C e l l
A d o s e o f 3 .3 x IO^ u n p rim e d p e r i t o n e a l e x u d a te c e l l s
(PEC)
g iv e n IoP« t o 8 u n r e s p o n s i v e m ic e f a i l e d to r e c o n s t i t u t e t h e PFC
resp o n se.
I n a n o t h e r e x p e r i m e n t , a d o s e o f 5 x IO^ p rim e d ( w ith I
h o u r e x p o s u re t o SRBC _in v i v o ) PECs c o n t a i n i n g 80-907, m ac ro p h ag e c e l l s
wa$ u s e d to r e c o n s t i t u t e 8 u n r e s p o n s i v e m ic e .
a s s a y s 5 days l a t e r a p p e a r in F ig u re 4 .
The r e s u l t s o f PFC
A n im als r e c e i v i n g p rim e d
PECs r e s p o n d e d v i g o r o u s l y i n c o m p a ris o n t o u n r e c o n s t i t u t e d , u n ­
r e s p o n s i v e a n im a ls o r t o n o rm a l c o n t r o l a n im a ls .
S i g n i f i c a n t l y , th e
19
140
120^
DIRECT
IQO
INDIRECT
RFC
RFC
CD
O
U
£
60
irnmrn
NB-NT
Figure 3.
NB-UT
UB-NT
UB-UT
P la q u e for min g cell (RFC) r e s p o n s e of no rm a l B a l b / c mice
r e c o n s t i t u t e d with v a ri ous combin at ion s of bone m a rr o w
a n d t h y m u s cells fr om normal and unresponsive m i c e .
20
DIRECT
□
F i g u r e 4.
INDIRECT
RFC
RFC
P l a q u e fo r m in g cell (RFC) r e s p o n s e of unresponsive
B alb/c
mice r e c o n s t i t u t e d with primed peritoneal
exudate cells ( PEC).
21
i n d i r e c t PFC r e s p o n s e , i n c o n t r a s t t o t h e d i r e c t PFC r e s p o n s e , was
e le v a te d .
Im m u n o g lo b u lin Q u a n t i t a t i o n
Im m u n o g lo b u lin ( I g ) q u a n t i t a t i o n was c a r r i e d o u t i n 9 u n r e s p o n s iv e
a n im a ls (2 1 0 d a y s a f t e r i n i t i a t i o n o f t r e a t m e n t ) and 9 c o n t r o l a n im a ls .
The r a d i a l im m u n o d iffu s io n l e v e l s o f seru m I g C lgG ^, Ig G ^ , IgM and
IgA ) a r e p r e s e n t e d i n F i g u r e 5 .
A l l I g s a p p e a r to b e p r e s e n t i n
s i m i l a r q u a n t i t i e s e x c e p t IgG^ w hich, shows a tw o - f o l d i n c r e a s e i n
u n r e s p o n s i v e a n im a ls .
P a s s iv e T u rn o ff
Three groups of 8 mice each received either serum from unrespon­
sive mice plus SRBC, or SRBC alone, or were untreated, normal,
control mice.
Figure 6.
The PFC response of these three groups is shown in
A singular finding was that passively administered serum
from unresponsive mice reduced the PFC response to SRBC to background
levels.
Im m u n o g lo b u lin C la s s o f T u r n o f f A n tib o d y
T w elve m ic e w e re d i v i d e d i n t o 5 g ro u p s t o i n c l u d e 2 m ic e
r e c e i v i n g u n r e s p o n s i v e serum p l u s S R B C ,.3 m ic e r e c e i v i n g u n r e s p o n s iv e
serum a b s o rb e d w i t h a n t i - I g G ^ , IgGg p l u s SRBC, 2 m ic e r e c e i v i n g u n ­
r e s p o n s i v e serum a b s o rb e d w i t h a n ti- I g G g p l u s SRBC, 2 m ic e r e c e i v i n g
22
UNRESPONSIVE LEVELS
□
IgG,
Figure 5.
IgG2
NORMAL LE VEL S
IgM
Immunoglobulin levels in s e r u m from normal and
unresponsive m ic e .
IgA
23
UNRESPONSIVE
170-
□
SRBC
SERUM + SRBC
ALONE
BACKGROUND
150130-
IIOPFC/IO
<o
90J
70
5030-
io |
5
432-
I-
DIRECT RFC
Figure 6.
INDIRECT RFC
P la q u e for ming cell (PFC) r e s p o n s e of n o r m a l B al b/c
mice trea ted with s e r u m from unre s p o n si v e m ic e.
24
SRBC a l o n e , and 2 m ic e s e r v e d t o e s t a b l i s h b a c k g ro u n d l e v e l s .
r e s u l t s o f PFC a s s a y s a p p e a r i n F i g u r e 7 .
The
Serum w h ich was a b s o rb e d
w i t h b o t h a n t i - l g G ^ and a n ti - I g G ^ f a i l e d to " tu r n - o f f " "the PEC
r e s p o n s e t o SRBC i n t h o s e a n im a ls w h ic h r e c e i v e d 32 HA u n i t s o f t h a t
se ru m .
A s i n g l u l a r f i n d i n g was t h a t serum a b s o rb e d w i t h a n ti- I g G ^
a lo n e l o s t i t s
h o s t.
a b i l i t y t o s u p p r e s s t h e immune r e s p o n s e i n a n e u t r a l
H e n c e , t h e im m u n o g lo b u lin s p e c i e s o f t h e " t u r n o f f " a n tib o d y
a p p e a r s t o b e o f t h e IgGg c l a s s .
25
2 9 0
250210
D
170-
O
\ 130
^
•
a. 905010%
3■
I-
■
SRBC
alone
Figure
7.
SRBC
+
serum
abs.
a n ti IgGl-G2
SRBC
+
ser um
abs.
antiIgG2
SRBC
+
whole
s er um
BACK­
GROUND
Plaque -forming cell (PFC) response of n o r m a l Balb/c
mice treated with anti - I g G 2 a b s o r b e d turnoff antibody
plus s h e e p e r y th r o c y te s .
DISCUSSION
B a l b / c m ic e t r e a t e d w i t h m u l t i p l e d o s e s o f SRBC fro m t h e
n e o n a t a l p e r i o d i n t o a d u lth o o d becam e s p e c i f i c a l l y u n r e s p o n s i v e to
SRBC a t 90 to 120 d a y s o f a g e .
A r a p i d d e c l i n e i n t h e PFC r e s p o n s e
and a n ti b o d y t i t e r a f t e r an i n i t i a l v ig o r o u s r e s p o n s e s u g g e s te d t h a t
t h e r e yras <a d e f e c t i n o n e o f t h e t h r e e c e l l t y p e s (T alm ag e j s t a l . ,
1 9 7 0 ) in v o lv e d i n t h e immune r e s p o n s e .
R e c o n s titu tio n w ith bone
m arrow and thym us showed t h a t t h e s e c e l l s w e re p rim e d b y p r i o r
e x p o s u re t o a n t i g e n and when g iv e n t o i r r a d i a t e d , n e u t r a l h o s t s gave
r i s e to a se c o n d a ry , i n d i r e c t p la q u e -fo rm in g re s p o n s e .
A lth o u g h
t h e r e i s e v id e n c e f o r a d i r e c t (1 9 S ) memory c e l l (C u n n in g h am , 1969)
n o n e was shown i n t h i s s y s te m .
The i n d i r e c t p l a q u e - f o r m in g
r e s p o n s e s u g g e s t s t h a t t h e r e i s some f a c t o r ( s ) p r e s e n t i n t h e o r i g i n a l
h o s t t h a t p r e v e n t e d a n ti b o d y p r o d u c t i o n .
S in c e b o n e m arrow and thym us
c e l l s fro m u n r e s p o n s i v e m ic e w ere a b l e t o r e c o n s t i t u t e t h e i n d i r e c t
(7 S ) p la q u e r e s p o n s e i n i r r a d i a t e d r e c i p i e n t s , t h e m a c ro p h a g e becam e
s u s p e c t a s t h e c e l l i n v o lv e d i n u n r e s p o n s i v e n e s s .
I t was e x p e c te d
t h a t p e r i t o n e a l e x u d a te c e l l s (80-90% m ac ro p h ag e c e l l s ) w ould r e s t o r e
r e s p o n s i v e n e s s t o SRBC, b u t t h i s was n o t t h e c a s e .
f a c t t h a t a n ti b o d y t i t e r s
H ow ever, th e
(p re s u m a b ly o f t h e IgG c l a s s ) re m a in e d
r e l a t i v e l y h i g h a f t e r t h e PFC r e s p o n s e w an ed , s u g g e s te d t h a t a n t i ­
b o d y p l a y e d some p a r t i n t h i s u n r e s p o n s i v e s t a t e .
In t h i s re g a rd ,
i t h a s b e e n shown t h a t a n tib o d y may r e n d e r m a c ro p h a g e s i n a c t i v e ,
27
m ak in g f u r t h e r a n t i g e n p r o c e s s i n g im p o s s ib l e ( P i e r c e , 1 9 6 9 ) and
(R y d e r and S c h w a r tz , 1 9 6 9 ) .
The p r o c e s s i s b e l i e v e d to be a f e e d ­
b a c k i n h i b i t i o n i n w h ic h a p ti b o d y p r e s e n t i n t e r a c t s w i t h t h e m ac ro ­
p h a g e and r e n d e r s i t i n c a p a b l e o f r e a c t i o n w i t h s p e c i f i c a n t i g e n .
T h u s , i n t h e s e s t u d i e s , when SRBC-prim ed p e r i t o n e a l e x u d a te c e l l s
w e re g iv e n to u n r e s p o n s i v e m ic e , t h e PFC r e s p o n s e was r e c o n s t i t u t e d .
T h is i s e v id e n c e t h a t u n r e s p o n s i v e n e s s i s a p p a r e n t l y a s s o c i a t e d w ith
t h e a f f e r e n t lim b o f t h e immune r e s p o n s e , and t h a t o n c e t h e m ac ro ­
p h a g e i s a llo w e d to i n g e s t and p r o c e s s t h e a n t i g e n , t h e PFC r e s p o n s e
w ill o c c u r.
The f a c t t h a t m ic e , when p a s s i v e l y g iv e n seru m from
u n r e s p o n s i v e m ic e , f a i l e d to r e s p o n d to SRBC le n d s g r e a t e r c re d e n c e
to t h e s e a rg u m e n ts .
T h e s e f i n d i n g s a r e u n l i k e t h o s e o f F eldm an and D ie n e r (1 9 7 0 )
who show ed t h a t ly m p h o id c e l l s , i n c u b a t e d i n v i t r o w i t h a n ti b o d y , w ere
m e d i a to r s o f SRBC s u p p r e s s i o n , and a r e n o t u n l i k e t h o s e o f Row ley
and F i t c h (1 9 6 4 ) who, u s i n g r a t s made t o l e r a n t to SRBC, showed t h a t
a n tib o d y was in v o lv e d i n t h e t o l e r a n t s t a t e .
H ow ever, Row ley and
F i t c h s p e c u l a t e d t h a t t h e s i t e o f a c t i o n o f a n tib o d y was t h e lym pho­
c y te .
M i l l e r and M i t c h e l l ( 1 9 7 0 ) , w o rk in g w i t h CBA m ic e t o l e r a n t to
SRBC and m a i n ta i n e d i n t h e t o l e r a n t s t a t e w i t h c y c lo p h o s p h a m id e ,
d e m o n s tr a te d t h a t th y m u s - d e r iv e d c e l l s o f t h e t h o r a c i c d u c t w ere
in v o lv e d i n t o l e r a n c e .
M cC u llag h ( 1 9 7 0 ) , u s i n g r a t s made t o l e r a n t to
28
r e p e a t e d d o s e s o f SKBC, a l s o b e l i e v e s t h a t t h e c e l l in v o lv e d i n h i s
s y s te m i s t h e t h o r a c i c d u c t ly m p h o c y te w h ic h i s im p a ir e d b y some
" fa c to r" .
T h is f a c t o r u n d o u b te d ly i s a n ti b o d y , b u t we b e l i e v e th e
c e l l i n v o lv e d i s t h e m a c ro p h a g e .
Row ley and F i t c h (1 9 6 4 ) s t a t e d t h a t i n h i b i t i o n o f r e s p o n s e to
a n t i g e n was a p p a r e n t l y due t o 19S o r 7S a n ti b o d y .
T h u s, a search
was made t o d e te r m in e t h e c l a s s o f a n tib o d y in v o lv e d i n fe e d b a c k
i n h i b i t i o n o f t h e PFC r e s p o n s e .
W hole t u r n o f f serum was a b s o rb e d w i t h a n ti - I g G ^ a n d a n ti - I g G ^ o r
a n ti-Ig G
2
a lo n e .
i
The f a c t t h a t t h e e l i m i n a t i o n o f IgGh fro m u n 6
r e s p o n s i v e serum p ro d u c e d a s u b s t a n t i a l r e c o v e r y o f t h e PFC r e s p o n s e
i n m ic e , p r o v id e d e v id e n c e t h a t IgGg i s t h e t u r n o f f a n ti b o d y in v o lv e d
i n t h i s s y s te m .
J u tila
T h e se f i n d i n g s a r e s u p p o r te d by t h e o b s e r v a t i o n s o f
(1 9 6 8 ) i n a n o th e r s y s te m .
I t i s th u s b e lie v e d t h a t in un­
r e s p o n s i v e n e s s t o r e p e a t e d d o s e s o f s h e e p e r y t h r o c y t e s i n B a l b /c m ic e
Ig G g , b y a t t a c h i n g to t h e m a c ro p h a g e , p r e v e n t s t h e i n t e r a c t i o n o f
a n t i g e n w i t h t h e c e l l s u r f a c e i n some f a s h i o n .
T h is p r o d u c e s a f e e d ­
b a c k i n h i b i t i o n o f t h e PFC r e s p o n s e , b u t l e a v e s p o t e n t i a l i n d i r e c t
p l a q u e - f o r m i n g c e l l s o f b o n e m arrow and thym us o r i g i n i n t h e u n re s p o n
s i v e m o u se.
SUMMARY
B a l b / c m ic e w e re r e n d e r e d u n r e s p o n s i v e t o sh e e p r e d b lo o d c e l l s
b y i n j e c t i o n s o f 5 x IQ^ s h e e p r e d b lo o d c e l l s e v e ry o t h e r d ay from
7 d a y s o f a g e to a d u lt h o o d .
I t was shown t h a t u n r e s p o n s i v e n e s s was
s p e c i f i c by i n j e c t i o n o f b u r r o r e d b lo o d c e l l s yrhich p ro d u c e d n o rm a l
p l a q u e - f o r m i n g c e l l r e s p o n s e s i n S R B C -u n re sp o n siv e m ic e .
N e ith e r
n o rm a l thym us n o r b o n e m arrow a lo n e o r i n c o m b in a tio n , n o r n o rm a l
p e r i t o n e a l e x u d a te c e l l s r e c o n s t i t u t e d u n r e s p o n s iv e m ic e .
SKBC-prim ed p e r i t o n e a l e x u d a te c e l l s
H ow ever,
(80-907» m acro p h ag e c e l l s ) d id
r e c o n s t i t u t e t h e p l a q u e - f o r m i n g r e s p o n s e o f u n r e s p o n s i v e m ic e .
N orm al B a l b / c m ic e w e re r e n d e r e d u n r e s p o n s i v e by i n j e c t i o n o f 32
h e m a g g l u ti n in u n i t s o f serum from u n r e s p o n s i v e m ic e .
u n r e s p o n s i v e m ic e was a b s o rb e d w ith Ig G ^ .
Serum from
Serum a b s o rb e d i n t h i s .
m an n er f a i l e d t o r e n d e r m ic e u n r e s p o n s i v e when a d m i n i s t e r e d p a s s i v e l y .
I t was c o n c lu d e d t h a t u n r e s p o n s i v e n e s s t o sh e e p r e d b lo o d c e l l s
i n v o l v e s IgG^ w h ic h i n t e r a c t s w i t h t h e m ac ro p h ag e a n d , i n t u r n ,
i n h i b i t s a n tig e n - m a c r o p h a g e i n t e r a c t i o n .
Thus t h i s s y s te m r e p r e s e n t s
a f e e d b a c k m echanism w h ic h p r e v e n t s t h e p r e s e n t a t i o n o f a n t i g e n to
t h e ly m p h o c y te .
LITERATURE CITED
j
A u s t i n , C.M. and N o s s a l , G .J .V .
1 9 6 6 . M echanism o f i n d u c t i o n o f
im m u n o lo g ic a l t o l e r a n c e . A u s t. J . E xp. B i o l . Med. S c i . 4 4 : 34 1 .
A z a r , M.M. 1 9 6 6 . The e f f e c t o f a c t i v e and i n a c t i v e r h e u m a to id f a c t o r s
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