Regulatory effects of the thymus on the IgE response of mice
by Rhet Lucy Schneller
A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE
in Microbiology
Montana State University
© Copyright by Rhet Lucy Schneller (1974)
Abstract:
The regulatory effects of the thymus gland on the production of reaginic immunoglobulin E (IgE) were
studied using congenitally athymic (nude) mice, phenotypically normal littermates (NLM) of nude
mice and Balb/c mice. Antigen specific IgE was quantitated in CFW mice by the passive cutaneous
anaphylaxis (PCA)assay. Alum, complete Freunds adjuvant and saline extract of Bordetella pertussis
were used as adjuvants. Nude mice failed to produce specific IgE following both primary and
secondary immunization with hen egg albumin (EA), crude ascaris extract and crude ascaris extract
coupled to dinitrophenyl groups. In contrast, NLM and Balb/c mice made strong IgE responses.
Nude mice implanted with thymus glands from neonatal Balb/c or NLM donors made IgE responses
equal to the responses of NLM mice. These data indicate that there is an absolute requirement for
thymus-derived cells in the formation by mice of specific IgE.
Nude mice were passively sensitized intradermally with an IgE-positive serum; when challenged
intravenously with specific antigen, these nude mice demonstrated strong PCA reactions. It is clear,
therefore, that although nude mice cannot make specific IgE, they do have the mechanism necessary to
elicit PCA reactions.
Attempts were made to show a negative regulatory effect of the thymus on IgE production by using
splenectomized, 60Co-irradiated or antilymphocyte serum-treated mice. No enhancement of IgE
production was observed in these experiments. In contrast to results obtained by others using rats,
infection of immunized mice with the nematode Nippostrongylus brasiliensis did not potentiate specific
IgE production. STATEMENT OF PERMISSION TO COPY
In p re s e n tin g
th is
th e sis
in p a r t i a l
f u l f i l l m e n t o f th e r e q u ir e r
m e n ts f o r an a d v a n c e d d e g r e e a t M o n tan a S t a t e U n i v e r s i t y ,
t h e L i b r a r y s h a l l m ake i t
I agree th a t
fre e ly a v a ila b le fo r in sp e c tio n .
a g re e t h a t p e r m is s io n f o r e x te n s iv e co p y in g o f t h i s
th e sis
I fu rth e r
fo 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 b y my m a j o r p r o f e s s o r ,
or,
a b s e n c e , by th e D i r e c t o r o f L i b r a r i e s .
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
th e sis
It
i s u n d e rsto o d
in h is
f o r f i n a n c i a l g a in s h a l l n o t be
a l l o 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 .
S ig n a tu r
D a te
l/ ✓
REGULATORY EFFECTS' OF THE THYMUS ON THE: I g E !RESPONSE.OF MICE
by
RHET' LUCY' SCHNELLER
A t h e s i s • s u b m i t t e d in- 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 t s f o r t h e d e g r e e
of
.MASTER OF SCIENCE
in
■M i c r o b i o l o g y
H e a d , . M ajo r D e p a rtm e n t
G rad u ate"D ean
MONTANA STATE UNIVERSITY
B o zem a n , M o n ta n a
A u gu st, . 1974'
iii
ACKNOWLEDGMENTS
I w o u ld l i k e
R eed f o r h i s
out th is
t o e x p r e s s my s i n c e r e a p p r e c i a t i o n t o D r . Norman D.
a ssista n c e ,
stu d y .
I w o u ld a l s o
.
lik e
p re p a ra tio n of th is
Thanks,
a lso ,
c o n tin u e d g u id a n c e and e n c o u ra g e m e n t th ro u g h ­
t o t h a n k D r . D ean M a n n in g f o r h i s h e l p
in
th e sis.
t o D r . J o h n Munoz f o r h i s h e l p w i t h t h e p a s s i v e
c u t a n e o u s a n a p h y l a x i s a s s a y a n d t o D r . H e r b e r t M o rs e f o r s u p p l y i n g
th e
D N P -a sc a ris.
I a l s o w i s h t o t h a n k D r . W i l l i a m G. W a l t e r f o r h i s
aid in o b ta in ­
i n g some o f t h e f i n a n c i a l s u p p o r t f o r t h i s w o r k .
I am e s p e c i a l l y g r a t e f u l t o t h e N a t i o n a l I n s t i t u t e
and I n f e c t i o u s D i s e a s e s
A l-1 0 3 8 4 ,
f o r t h e i r s u p p o rt th ro u g h R e se a rc h G ran t
and to t h e N a t i o n a l C a n c e r I n s t i t u t e
R e s e a r c h G r a n t C A -1 5 3 2 2 .
o f A llerg y
f o r s u p p o r t th ro u g h
iv
TABLE OF CONTENTS
VITA .
. ............................................................................... ..... .
ACKNOWLEDGMENTS
. . . . . . . . .
. . .
. . . .
.
. Page
■ ii
.
........................................
TABLE OF CONTENTS
iii
^ ......................................... .....
iy
LIS T OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . .
vi
ABSTRACT .............................................................
v ii.
INTRODUCTION ............................................................................. ............................................. :
I
MATERIALS AND M E T H O D S ...................................................
9
A n i m a l s .............................................................
. A n tig en s
A d ju v an ts
9.
...........................................................................................
...............................................................................................
10
Im m u n o su p p re ssiv e A g e n ts
. . . . . . . . . . . . . . . .
11
P a ra site s
.
11
.
.
.
.....................
. .
...
.
Thymus G r a f t s .......................... ..... . . . . .
. Thymus C e l l I n j e c t i o n s
Im m u n izatio n P ro c e d u re s
.
...
. .
• . . . .
..............................................
12
. . . . . . . . . . . . . . . . .
12.
...................................
.13.
P a s s i v e C u tan eo u s A n a p h y la x is R e a c tio n s
RESULTS ........................................................... ..... .
Ig E P r o d u c t i o n ■i n t h e M ouse .
'
9
.
.
.
.
.
. . .
.
. . . . . . . . .
. .
. . . . .
.
. . .
.
.
.
13
.
.
. ,1 5
.
.
15
’ ■ .
.
P r e l i m i n a r y S t u d i e s o n t h e A b i l i t y o f ■D i f f e r e n t . A n t i g e n s t o •
E l i c i t a n Ig E R e s p o n s e i n M i c e . '. '. . . . . .
• • i..
■■
.
15
V
Page
Thymus D e p e n d e n c y o f t h e Ig E R e s p o n s e ..................... .........................
17
Ig E P r o d u c t i o n i n Nude M ic e R e c o n s t i t u t e d w i t h Thymus
C e l l s ...................................................
20
Ig E P r o d u c t i o n i n Nude M ic e R e c o n s t i t u t e d w i t h Thymus
G l a n d s ......................................................................................................
21
Ig E R e s p o n s e o f Nude M ic e t o a Thymus I n d e p e n d e n t A n t i g e n
21
T he Thymus a s t h e O r i g i n o f t h e C e l l s R e s p o n s i b l e f o r PCA
R e a c t i o n s ........................................................................................
22
E f f e c t s o f i m m u n o s u p p r e s s i v e A g e n t s o n t h e Ig E R e s p o n s e i n
M i c e ............................................................................................................ . . .
22
E f f e c t o f I n f e c tio n w ith N ip p o stro n g y lu s b r a s i l i e n s i s
t h e Ig E R e s p o n s e i n M i c e ........................................
26
DISCUSSION .
on
. ■..............................
29
SUMMARY........................................................................................................................................•
34
BIBLIOGRAPHY
36
vi
L IS T .O F TABLES
Page
TABLE
TABLE
TABLE
TABLEIV
TABLE
I
II
III
E f f e c t s o f K e a tin g a hom ocyt o t r o p i c
a n tib o d y
p o s i t i v e s e r u m f r o m B a l b / c m i c e , 30 m i n u t e s
a t 56°C a s d e m o n s t r a t e d b y p a s s i v e c u t a n e o u s
a n a p h y l a x i s r e a c t i o n s .............................................. .....
16
E ffe c tiv e n e ss o f d if f e r e n t an tig e n -a d ju v a n t
co m b in atio n s i n j e c t e d i n t r a p e r i t o n e a l l y in
e l i c i t i n g Ig E p r o d u c t i o n i n B a l b / c m i c e . . . .
18
Thymus d e p e n d e n c y o f t h e Ig E r e s p o n s e
'19
. . . .
. I g E r e s p o n s e o f m i c e t o a th y m u s i n d e p e n d e n t
a n t i g e n , V i .................................................................................
V
TABLE
VI
TABLE
V II
.
23
P a s s iv e cu tan e o u s a n a p h y la x is re sp o n se o f nude
a n d CFW m i c e t o h o m o c y t o t r o p i c a n t i b o d y p o s i t i v e
serum . . . . . . . . . .
....................................................
24
Thymus r e g u l a t i o n o f t h e Ig E r e s p o n s e
25
. . . . .
E f f e c t s o n Ig E p r o d u c t i o n o f i n f e c t i o n w i t h
N ip p o stro n g y lu s b r a s i l i e n s i s te n days a f t e r
i n i t i a l i m m u n i z a t i o n w i t h 10 jug e g g a l b u m i n
a n d 10 mg a lu m
.........................................................
27
v ii
. ABSTRACT
T he r e g u l a t o r y e f f e c t s o f t h e th y m u s g l a n d on t h e p r o d u c t i o n o f
r e a g i i i i c im m u n o g lo b u lin E (I g E ) w ere s t u d i e d u s in g c o n g e n i t a l l y
a t h y m i c ( n u d e ) m i c e , p h e n o t y p i c a l I y n o r m a l l i t t e r m a t e s (NLM) o f n u d e
m ic e and B a l b / c m ic e .
A n t i g e n s p e c i f i c Ig E w as q u a n t i t a t e d i n CFW m i c e
by th e p a s s i v e c u ta n e o u s a n a p h y la x is (P C A )assay .
A lum , c o m p l e t e
F re u n d s a d j u v a n t and s a l i n e e x t r a c t o f B o r d e t e l l a p e r t u s s i s w ere u sed
as a d ju v a n ts.
Nude m i c e f a i l e d t o p r o d u c e s p e c i f i c I g E f o l l o w i n g b o t h
p r i m a r y a n d s e c o n d a r y i m m u n i z a t i o n w i t h h e n e g g a l b u m i n (E A ) , c r u d e
a s c a r i s e x t r a c t and c r u d e a s c a r i s e x t r a c t c o u p le d to d i n i t r o p h e n y l
groups.
I n c o n t r a s t , NLM a n d B a l b / c m i c e m ade s t r o n g I g E r e s p o n s e s .
Nude m i c e i m p l a n t e d w i t h th y m u s g l a n d s f r o m n e o n a t a l B a l b / c o r NLM
d o n o r s m ade I g E r e s p o n s e s e q u a l t o t h e r e s p o n s e s o f NLM m i c e .
These
d a t a i n d i c a t e t h a t t h e r e i s an a b s o l u t e r e q u ir e m e n t f o r th y m u s-d e riv e d
c e l l s i n th e fo r m a tio n by m ice o f s p e c i f i c Ig E .
Nude m i c e w e r e p a s s i v e l y s e n s i t i z e d i n t r a d e r m a l l y w i t h a n I g E p o s i t i v e s e r u m ; w hen c h a l l e n g e d I n t r a v e n o u s l y w i t h s p e c i f i c a n t i g e n ,
t h e s e n u d e m i c e d e m o n s t r a t e d s t r o n g PCA r e a c t i o n s .
I t is c lea r,
t h e r e f o r e , t h a t a l t h o u g h n u d e m i c e c a n n o t m ake s p e c i f i c I g E , t h e y do
h a v e t h e m e c h a n i s m n e c e s s a r y t o e l i c i t PCA r e a c t i o n s .
A t t e m p t s w e r e m ade t o show a n e g a t i v e r e g u l a t o r y e f f e c t . o f t h e
th y m u s o n I g E p r o d u c t i o n b y u s i n g s p l e n e c t o m i z e d , ^ ^ C o - i r r a d i a t e d o r
a n tily m p h o c y te s e r u m - tr e a te d m ice.
No e n h a n c e m e n t o f I g E p r o d u c t i o n
w as o b s e r v e d i n t h e s e e x p e r i m e n t s .
I n c o n t r a s t to r e s u l t s o b t a i n e d by
o t h e r s u s i n g r a t s , i n f e c t i o n o f im m u n iz e d m i c e w i t h t h e n e m a t o d e
N ip p o s t r o n g y l u s b r a s i l i e n s i s d i d n o t p o t e n t i a t e s p e c i f i c IgE p r o d u c t i o n
INTRODUCTION
P r o p e r t i e s o f H o m o cy to tro p ic A n t i b o d i e s .
In hum ans,
a n t i b o d i e s , w h i c h a r e r e s p o n s i b l e f o r some a l l e r g i c
Ig E i s . a
re a g in ic
re a c tio n s,
are in
th e im m u n o g lo b u lin c l a s s
IgE .
w e i g h t o f 2 0 0 ,0 0 0 a n d i s
s y n t h e s i z e d by p lasm a c e l l s p r e d o m in a n tly in
th e r e s p i r a t o r y and g a s t r o i n t e s t i n a l
tin a tin g
fix in g
g ly c o p ro te in w ith a m o lecu lar
tra c ts.
I t i s c a p a b le o f ag g lu ­
r e d b l o o d c e l l s c o a t e d w i t h a n t i g e n b u t p o s s e s s n o co m p lem en t­
a c tiv ity
in th a t i t
(I).
I t d iffe rs
is n o n -p re c ip ita tin g .
from o t h e r c l a s s e s o f im m u n o g lo b u lin
I t does n o t have th e a b i l i t y
t h e p l a c e n t a a n d i s n o t f o u n d i n hum an c o l o s t r u m
(2 ).
to c r o s s
Zone e l e c t r o ­
p h o r e s i s h a s shown t h a t Ig E m o v es i n t h e " f a s t l f 1 r e g i o n w h i l e z o n e
c e n trifu g a tio n
i n b u f f e r e d s u c r o s e . g r a d i e n t h a s shown t h a t i t
m e n t s i n t h e 7S r e g i o n . ( 2 ) .
Human Ig E c a n s e n s i t i z e
hum ans, b u t n o t t h e s k i n o f m o st o t h e r s p e c i e s ,
se d i­
th e s k in o f
f o r im m e d ia te -ty p e
h y p e rse n sitiv ity re a c tio n s.
,
A s i m i l a r t y p e o f a n t i b o d y , p o s s e s s i n g p r o p e r t i e s o f t h e human
r e a g in ic an tib o d y ,
I g E , h a s b e e n r e p o r t e d i n mammals o t h e r t h a n man b y
■s e v e r a l i n v e s t i g a t o r s .
b o d ie s
T hus, re a g in ic
I g E - I i k e ,' h o m o cy tro t r o p i c a n t i -
(HTA) h a v e b e e n r e p o r t e d i n s e v e r a l a n i m a l s p e c i e s ; i n c l u d i n g t h e
g u in ea p ig
(3),
fat
(4),
a n d m o u se ( 5 ) .
a n tib o d ie s are h e a t- la b ile ;
h e a tin g
L i k e t h e hum an r e a g i n ,
th e u n d ilu te d
s e r u m a t 56°C f o r
30 m i n u t e s d e s t r o y s . t h e i r s k i n - s e n s i t i z i n g a b i l i t y .
in c lu d in g
th e m ouse,
th e se
. Many s p e c i e s ,
a l s o p r o d u c e a n o t h e r hdm ocyt o t r o p i c a n t i b o d y
-■
;
2
w h ich b e lo n g s t o
p h ilic a lly
t h e IgG ^ s u b c l a s s
(5 ).
IgG ^ d o e s n o t p e r s i s t c y t o -
i n th e s k i n f o r lo n g p e r i o d s o f tim e ( h o u r s ,
a s com pared t o
weeks f o r Ig E ) b u t d o e s p e r s i s t i n t h e serum l o n g e r th a n t h e r e a g i n i c ty p e a n tib o d y .
IgG ^ i s h e a t - s t a b l e ,
does n o t d e s tro y i t s
a c tiv ity
(5 ).
d i s t i n g u i s h b etw e en th e a c t i v i t i e s
c u tan e o u s a n a p h y la x is
s o h e a t i n g f o r 30 m i n u t e s a t 5 6 ° c
It
is,
t h e r e f o r e , p o s s i b l e to
o f I g G ^ , a n d Ig E HTA i n t h e p a s s i v e
(PCA) a s s a y b y h e a t i n g
serum s o r v a r y in g th e tim e
of a n tig e n c h a lle n g e a f t e r s k in - s e n s itiz a tio n .
Ig G ^ -m ed iated r e a c t i o n s
a r e t y p i c a l l y d e v e lo p e d 2 h r a f t e r p a s s i v e s e n s i t i z a t i o n ,
m ed iated r e a c t i o n s
It
is
a r e d e v e l o p e d 72 h r a f t e r p a s s i v e s e n s i t i z a t i o n . .
th o u g h t t h a t IgE a t t a c h e s
to m ast c e l l s
r e l e a s e o f c h e m i c a l m e d i a t o r s o f t h e PCA r e a c t i o n
re p o rte d
w h e reas IgE -
and c a u s e s
(I).
th e
M o ta ( 6 ) a l s o
t h a t r a t m a s t c e l l s c a n b e s e n s i t i z e d p a s s i v e l y w i t h serum
c o n ta in in g w hat he c a ll e d
"m ast c e l l
se n sitiz in g
a n t i b o d y , " w h ich i s
c o m p a r a b l e t o hum an I g E .
He f o u n d t h a t s e n s i t i z e d m a s t c e l l s
re le a se
h i s t a m i n e and 5- h y d r o x y t r y p t a m i n e a f t e r . t h e a d d i t i o n o f s p e c i f i c
an tig en .
It
is
th o u g h t t h a t s e r o ti n i n
(5 -h y d ro x y t r y p tam in e) i s
th e
m a in c h e m i c a l m e d i a t o r o f a n a p h y l a x i s i n t h e r a t and t h e m ouse ( 6 ) .
In m ice,
Ig E p r o d u c t i o n d e p e n d s o n t h e a n t i g e n - a d j u v a n t s y s t e m a s
w e l l a s on t h e s t r a i n
re p o rte d
■
o f a n im a l u s e d . . R e v o l t e l l a and O vary (7 )
t h a t t h r e e o f s i x s t r a i n s o f m i c e im m u n iz e d w i t h DNP-haemo-
c y a n i n a n d AL(OH ) 3 w e r e a b l e t o p r o d u c e I g E .
SW R/j, C 5 7 B L /6 J a n d
A /H eJ m ade I g E , w h e r e a s C B A /J, DBA/J a n d SW m ade h i g h l e v e l s o f IgG^
b u t n o Ig E ( 7 ) .
C lau sen e t a l
( 8 ) fo u n d t h a t th e i n b r e d S t r a i n s o f
m i c e NBL/n^ C 5 7 B 1 / 6 J a n d C 5 7 B L /IO J w e r e f a r s u p e r i o r t o
th e s t r a i n s
AKR/n, STR /n a n d RML i n p r o d u c t i o n o f I g E when c h a l l e n g e d w i t h h e n e g g
a l b u m i n (EA) a n d . s a l i n e e x t r a c t s
from B b r d e t e l l a p e r t u s s i s
(P E ).
S e v e r a l d i f f e r e n t a n t i g e n s and a d j u v a n t s h av e b e e n u s e d to e l i c i t
Ig E p r o d u c t i o n .
U s i n g EA a s a n t i g e n ,
C lau sen ,
et a l.,
w ere s u c c e s s f u l
i n s t i m u l a t i n g I g E w i t h SE a s a d j u v a n t , w h e r e a s i n c o m p l e t e F r e u n d ' s
a d j u v a n t w as s u p e r i o r i n s t i m u l a t i n g
2 h r PCA a n t i b o d y ,
IgG ^ ( 9 ) .
Tumor a n t i g e n s a n d a l l b a n t i g e n s h a v e a l s o b e e n u s e d t o i n d u c e Ig E p r o ­
d u c tio n in m ice.
v ia b le
T h u s C57BL a n d B a l b / c m i c e im m u n iz e d w i t h I x IO^
tu m o r c e l l s
f r o m t h e B16 m e la n o m a e s t a b l i s h e d
r e s p o n d e d w i t h p r o d u c t i o n o f Ig E a n t i b o d y ( 1 0 ) .
i n C57B1 m i c e ,
K in d a n d M acedo-
S o b r i n h o w e r e a b l e t o g e t Ig E p r o d u c t i o n i n m i c e i n r e s p o n s e t o r a b b i t
a n ti- m o u s e th y m o c y te serum ( 11) .
S m a l l d o s e s o f a n t i g e n s u c h . a s p o l l e n , w h i c h c a u s e hum an a l l e r - .
' .
.-
.
■
■
,
x
g i e s , s e e m . t o b e b e s t f o r p r o d u c t i o n o f Ig E ( 1 2 ) .
;
■
-
-
/
■
.:
'•
•’
In g u in ea p ig s ,
P e r i n i a n d M o ta ( 1 3 ) d e m o n s t r a t e d t h a t lo w d o s e s o f EA t o g e t h e r w i t h
lo w d o s e s o f l i p o p o l y s a c c h a r i d e
IgE p r o d u c t i o n ,
(L P S ) w e r e . e f f e c t i v e i n s t i m u l a t i o n o f
and h i g h d o se s o f a n t i g e n and a d j u v a n t r e s u l t e d
in h ig h
p r o d u c t i o n o f IgG]/.
I t h a s a l s o been R ep o rted t h a t a s t a t e o f t o l e r a n c e can be in d u ced
4
t o t h e Ig E a n t i b o d y c l a s s .
K a t z a n d c o - w o r k e r s ( 1 4 ) sh o w ed t h a t two
p r e t r e a t m e n t s o f m i c e w i t h 500 jug o f t h e c o p o l y m e r DNP-D-g l u t a m i c a c i d
and D - I y s in e s i g n i f i c a n t l y re d u c e d th e a n i m a l s '
D N P -c ru d e a s c a r i s e x t r a c t s
a b ility
to resp o n d to
(DNP-CAE) w i t h p r o d u c t i o n o f I g E a n t i b o d y .
T h is to le r a n c e co u ld be t r a n s f e r r e d
r e c ip ie n ts w ith sp le e n c e l l s
to s y n g e n e ic , u n p rim ed ,
irra d ia te d
f r o m m i c e p r i m e d w i t h DNP-CAE a n d a lu m a n d
g i v e n two 500 jug i n j e c t i o n s o f D N P - D - g l u ta m ic a c i d a n d D - I y s i n e .25 d a y s
la te r.
S a v e r b ro n n M ala,
u g EA s i g n i f i c a n t l y
e t a l,
show ed t h a t t h r e e i m m u n i z a t i o n s o f 100
r e d u c e d Ig E p r o d u c t i o n o f DBA/I J m i c e w hen
im m u n iz e d s u b s e q u e n t l y w i t h 0 . 1 jig BA.
Thymus D e p e n d e n c y o f I g E .
T h y m u s-d e riv e d c e l l s
(T c e l l s )
are
n e c e s s a r y f o r p r o d u c t i o n o f IgM o r IgG t o t h e s o - c a l l e d "T h y m u sd ep e n d en t" a n t i g e n s .
A lth o u g h m ost a n t i g e n s ,
p r o t e i n s and h e t e r o l o g o u s e r y t h r o c y t e s ,
some " t h y m u s - i n d e p e n d e n t " a n t i g e n s ,
p o ly sa c c h a rid e ,
such as h e te ro lo g o u s
a r e thym us d e p e n d e n t ,
th e re are
such as e n d o to x in , pneum ococcal
a n d Vi. a n t i g e n .
U n til re c e n tly ,
o n l y s p e c u l a t i o n s h a v e b e e n m ade o n t h e T - c e l I .
c o lla b o r a tio n w ith B c e l l s
in re la tio n s h ip
t o t h e Ig E r e s p o n s e .
For
e x a m p l e , O kum ura a n d T a d a ( 1 6 ) d e m o n s t r a t e d t h a t n e o n a t a l th y m e c to m y o f
r a t s c a u s e d a r e d u c t i o n i n t h e p r o d u c t i o n o f Ig E i n r e s p o n s e t o
d i n i t r o p h e n o I - c r u d e A s c a r i s e x t r a c t (DNP-CAE ) 0
T he m o s t m e a n i n g f u l d a t a s u p p o r t i n g T - c e l l d e p e n d e n c y o f t h e Ig E
5
resp o n se i s
s e e n i n th e w ork o f M ic h a e l and B e r n s t e i n
t h a t c o n g e n i t a l l y ath y m ic
(n u d e) m ice f a i l e d
d u c t i o n t o BA, b o v i n e s e r u m a l b u m i n ,
(17).
T h ey f o u n d
t o r e s p o n d w i t h Ig E p r o ­
a n d hum an gamma g l o b u l i n , w h e r e a s
t h e i r norm al th y m u s-b e a rin g l i t t e r m a t e s d id resp o n d to th e s e a n t i g e n s .
i
•
H o w e v e r, n u d e m i c e - t h a t h a d r e c e i v e d
th y m u s c e l l s
'
from n o rm a l B a lb /c
m i c e w e r e a b l e t o m ake a n I g E r e s p o n s e t o t h e s e a n t i g e n s .
Some u n p u b l i s h e d w o r k . o f R eed a n d Munoz f u r t h e r i n d i c a t e s
r e s p o n s e s may b e th y m u s d e p e n d e n t .
t h a t Ig E
They fo u n d t h a t n o rm a l l i t t e r m a t e s
t o n u d e s c o u l d m ake a n Ig E r e s p o n s e t o EA a n d SE, b o v i n e g a m m a - g l o b u l i n
a n d SE a n d K e y h o l e L i m p e t h e m o c y a n i n , w h e r e a s n u d e m i c e d i d n o t r e s p o n d
to th e s e a n tig e n s .
T h e y a l s o m e a s u r e d t h e Ig E r e s p o n s e t o two a n t i g e n s
known t o b e th y m u s i n d e p e n d e n t f o r a n IgM r e s p o n s e , n a m e l y ,
en d o to x in .
S I I I and
N e i t h e r n u d e s n o r t h e i r n o r m a l l i t t e r m a t e s m ade a n y Ig E
re s p o n se to th e s e a n tig e n s .
Thymus R e g u l a t i o n o f t h e Ig E R e s p o n s e .
Ig E p r o d u c t i o n may be
th y m u s d e p e n d e n t b u t may a l s o b e r e g u l a t e d b y t h e t h y m u s .
w brkers
(1 6 ),
of ra ts
d e c r e a s e s t h e Ig E r e s p o n s e ,
response.
f o r e x a m p le ,
T ada and c o -
d e m o n s t r a t e d t h a t w h i l e n e o n a t a l th y m e c to m y
a d u l t th y m e c to m y i n c r e a s e s
th is
T h e y show ed a s i m i l a r e n h a n c e m e n t o f Ig E p r o d u c t i o n w i t h 400
r a d X -i r r a d i a t i o n
(1 8 ),
sp len ec to m y ( 1 6 ) ,
(1 9 ) and a n t i t h y m o c y t e t r e a t m e n t ( 2 0 ) .
enhanced e f f e c t i s
some i m m u n o s u p p r e s s i v e d r u g s
One e x p l a n a t i o n f o r s u c h a n
th e " tu r n in g o f f " o f s u p p re s s o r T - c e l l s o r th e
6
d e s t r u c ti o n o f T - c e lls ,. allo w in g B - c e lls
t o p r o d u c e Ig E i n a n u n r e g u l a ­
te d m anner.
T a d a a n d O kum ura ( 2 1 ) d e m o n s t r a t e d t h a t HTA f o r m a t i o n c a n b e
s u p p r e s s e d , by p a s s i v e a d m i n i s t r a t i o n o f hom o lo g o u s a n t i b o d y a g a i n s t t h e
sam e a n t i g e n .
T h is s u p p re s s io n ap p eared s e l e c t i v e
r e s p o n s e w as a f f e c t e d ,
i n t h a t t h e HTA
w h e r e a s t h e IgM r e s p o n s e w as n o t .
They s u g g e s te d
t h a t b l o c k i n g a n t i b o d y i n t h e p a s s i v e l y a d m i n i s t e r e d s e r u m a c t s by
s u p p r e s s i n g t h e s y n t h e s i s o f r e a g i n r a t h e r , t h a n b y p r e v e n t i n g t h e com- .
b i n a t i o n o f a l l e r g e n and r e a g i n
w ere:
(2 1 ).
T h e ir re a so n s fo r t h i s c o n c lu sio n
t h e f a c t t h a t t h e a m o u n t s o f p a s s i v e a n t i b o d y u s e d w e r e much l e s s
th a n th o s e needed to c o v e r a l l o f th e a n t i g e n i c d e te r m in a n ts ;
and a l s o
t h e f a c t t h a t Ig E w a s s u p p r e s s e d w h i l e h e m a g g l u t i n a t i n g a n t i b o d i e s w e r e
n o t.
O kum ura a n d T a d a ( 2 2 ) h a v e . u s e d a h a p t e n - c a r r i e r m o l e c u l e t o
in d u c e r e a g in f o r m a tio n .
By t r a n s f e r r i n g
th y m o c y te s from r a t s h y p e r - .
im m u n iz e d w i t h c r u d e . A s c a r i s e x t r a c t (CAE) o r DNP-CAE i n t o r e c i p i e n t s ' ,
t h a t w ere p ro d u c in g h ig h t i t e r s
o f HTA,. t h e y a c h i e v e d . a d r a s t i c r e d u c ­
t i o n o f HTA w i t h i n two d a y s o f t r a n s f e r .
B ecause t h i s e f f e c t o n ly to o k
p l a c e w hen t h e c a r r i e r w a s t h e sam e i n d o n o r a n d . r e c i p i e n t ,
clu d ed t h a t c a r r i e r - s p e c i f i c ,
re g u la te
th e y con­
th y m u s -d e riv e d ly m p h o cy tes ca n n e g a t i v e l y
t h e HTA f o r m a t i o n w h i c h i s know n t o b e h a p t e n - s p e c i f i c
■They f u r t h e r i n v e s t i g a t e d
(2 3 ).
t h i s n e g a tiv e c o n t r o l to f in d w hat th e y
7
■ c a l l e d a n " a n t i g e n - s p e c i f I c T - c e l l f a c t o r " w h i c h t h e y f e l t was
r e s p o n s i b l e f o r t h e s u p p r e s s i o n o f IgE p r o d u c t i o n .
th y m o c y te e x t r a c t s
They t r e a t e d
th e
fro m h y p e rim m u n iz e d d o n o rs w i t h d i f f e r e n t n u c l e a s e s
a n d p r o t e i n a s e s a n d f o u n d . t h a t t h e T - c e l l f a c t o r w as n o t d i g e s t i b l e b y
RNase b u t i s
clu d e d ,
d e s t r o y e d w i t h t r y p s i n and p r o n a s e ,
t h e T - c e l l f a c t o r w as o f p r o t e i n n a t u r e .
th e re fo re ,
Its
th ey con­
a c tiv ity
se d i­
m e n t e d s l o w e r t h a n IgG h e m a g g l u t i n i n s u c r o s e d e n s i t y g r a d i e n t u l t r a ­
c e n t r i f u g a t i o n a n d w as e l e c t r o p h o r e t i c a l I y l o c a t e d i n r e g i o n s c o r r e ­
s p o n d in g t o a l p h a and b e t a g l o b u l i n s .
They s u g g e s t ,
th e re fo re ,
th a t
t h e T - c e l l f a c t o r may b e a n u n i q u e r e c e p t o r o f t h e T - c e l l a n d n o t a n
u n u s u a l im m u n o g lo b u lin ( 2 4 ) .
In w o rk in g w ith r a t s ,
istra tio n
W h i t e a n d Holm ( 2 5 ) . d e m o n s t r a t e d t h a t a d m in ­
o f e q u i n e a n t i l y m p h o c y t e s e r u m b e f o r e i m m u n i z a t i o n w i t h KLH
y ie ld e d an enhancem ent o f e a r l y s y s t h e s i s o f r e a g i n i c a n tib o d y b u t
g i v i n g e q u i n e a n t i l y m p h o c y t e s e r u m a f t e r a d m i n i s t r a t i o n o f KLH r e s u l t e d
in l a t e
s y n th e s is enhancem ent (2 5 ).
A n tily m p h o c y te serum p r e t r e a t m e n t
w as s u p p r e s s i v e f o r KLH a g g l u t i n i n p r o d u c t i o n .
K in d a n d M a c e d o - S o b r i n h o ( 1 1 ) h a v e r e p o r t e d a s i m i l a r r e g u l a t o r y ,
c o n t r o l o f t h e th y m u s o n I g E f o r m a t i o n i n m i c e .
irra d ia tio n
(450 r a d ) r e s u l t e d
in su sta in e d
T h e y f o u n d t h a t X-
l e v e l o f IgE p r o d u c t i o n i n
m ice t r e a t e d w i t h r a b b i t a n ti-m o u s e th y m o cy te serum .
th y m e c tp m y d i d n o t r e s u l t i n i n c r e a s e d I g E p r o d u c t i o n .
S p le n e c to m y and
E f f e c ts o f N ip p o stro n g y lu s b r a s i l i e n s i s
o n Ig E P r o d u c t i o n .
S e v e ra l i n v e s t i g a t o r s have re p o rte d a p o te n tia te d re a g in re sp o n se f o l ­
lo w in g n e m ato d e i n f e c t i o n .
fa ts
Thus, B lo ck ,
et al
(2 6 ) d e m o n s tra te d t h a t
in f e c te d w ith N ip p o stro n g y lu s b r a s i l i e n s i s
la r v a e sec o n d ary to
i m m u n i z a t i o n w i t h EA o r KLH p l u s a l u m E x h i b i t e d i n c r e a s e d r e a g i n p r o ­
d u ctio n o f s h o rt d u ra tio n .
Ig E p r o d u c t i o n a n d n o t i n
et al
(2 7 ),
in th e r a t ,
T he p o t e n t i a t e d r e s p o n s e w as , o n l y f o u n d i n
th e o th e r c l a s s e s o f a n tib o d y .
.
show ed t h a t b o t h a n t i g e n a n d a d j u v a n t m u s t p r e
c e e d i n f e c t i o n w i t h N. b r a s i l i e n s i s
co u ld r i s e
Ja rre tt,
a s m uch a s 1 0 0 - f o l d .
f o r a p o t e n t i a t e d r e s p o n s e , w h ich
T h e y sh o w ed t h a t n o p o t e n t i a t e d
r e s p o n s e o c c u r r e d i f BE w as s u b s t i t u t e d
fo r th e p a r a s i t e ,
l e a d i n g th em
t o c o n c l u d e t h a t t h e N. b r a s i l i e n s i s d i d n o t a c t a s a n a d j u v a n t .
T he Thymus a s t h e O r i g i n o f t h e M a s t C e l l .
s i z e d by C sab a e t a I (2 8 ) and a l s o by G in s b e rg
th e o r i g i n o f th e m ast c e l l .
ev er,
stu d ie d
P ritc h a rd ;
th e c o n n e c tiv e t i s s u e . o f
I t h as been h y p o th e­
(29)
t h a t t h e thy m u s i s
V i k l i c k y a n d S im a ( 3 0 ) , how­
t h e s k i n a n d t h e lym ph n o d e s o f
n u d e m ice and fo u n d w h a t th e y p ro p o s e d t o be m a s t c e l l s .
n u d e m ouse i s c o n g e n i t a l l y a th y m ic ,
th e y co n c lu d e d t h a t , th e o r i g i n . o f
t h e m a s t c e l l c o u l d n o t p o s s i b l y , b e t h e t h y m u s ..
t o e l i c i t a PCA r e a c t i o n ,
P r itc h a r d 's
.
.
B ecause th e
I f ..nude s k i n : was a b l e
t h e r e w o u ld b e good e v i d e n c e i n s u p p o r t o f
c o n c lu sio n . .
; ... ■
...
■.
,
;
T he f o l l o w i n g e x p e r i m e n t s w ere, d e s i g n e d t o e v a l u a t e . t h e r o l e o f
.
t h e th y m u s i n Ig E p r o d u c t i o n .
•
.
v .
.
;
•'
■.
.
' ... .
' /-V'
MATERIALS AND METHODS
.A n im a ls . . B a lb / c J m ice w ere o b t a i n e d from th e J a c k s o n L a b o r a to r y ,
B ar H a rb o r, M ain e.
C o n g e n ita lly ath y m ic
t h e i r p h e n o t y p i c a l Iy norm al l i t t e r m a t e s
(n u d e ) m ice ( n u /n u )
(3 1 ) and
(NLM; + / n u o r + / + ) w e r e
o b t a i n e d fro m t h e M o n tan a S t a t e U n i v e r s i t y C o lo n y i n w h ic h t h e m u ta n t
gene, nu,
i s b e in g p la c e d o n to a B a lb /c b ackground by b a c k e ro s s - i n t e r ­
c r o s s m a t i n g sc h e m e ( 3 2 ) .
B a lb /c J,
v a rio u s
n u d e a n d NLM m i c e ,
1 - 3 m o n t h s o f a g e , w e r e im m u n iz e d by
s c h e m e s d e s i g n e d t o i n d u c e t h e p r o d u c t i o n o f HTA.
No a t t e m p t
w as m ade t o d i s t i n g u i s h b e t w e e n t h e HTA r e s p o n s e o f m a l e s a n d f e m a l e s .
CFW f e m a l e m i c e ,
1 -3 m onths o f a g e ,
o b t a i n e d fro m C a rw o rth F a m s ,
P o r t a g e , M ic h ., w e re u se d a s serum r e c i p i e n t s
i n PCA a s s a y s .
A ll a n im a ls w e re m a in ta in e d i n a c l e a n e n v iro n m e n t and w ere g iv e n
a c i d i f i e d - c h l o r i n a t e d w a t e r a n d a u t o c l a v e d P u r i n a 5010 f e e d .
A n tig en s.
Hen e g g a l b u m i n (E A ) , 5 - t i m e s c r y s t a l l i z e d , w a s o b t a i n e d
fro m M ile s L a b o r a t o r i e s T n c . ,
s to r e d a t -20°C .
K ankakee,
F or im m u n iz a tio n s,
111.
The c r y s t a l l i z e d EA was
EA w as d isso lv e d a t th e a p p ro p ri-
a te .c o n c e n tr a tio n , in s a l i n e o r p h o sp h ate b u ffe re d s a l i n e
(P B S).
EA s o l u t i o n , w a s p r e p a r e d f o r . e a c h e x p e r i m e n t .
;
■ C ru d e A s c a r i s E x t r a c t .
.
D r. H e r b e r t J . M orse,
M i c r o b i a l Im m u n ity , N a t i o n a l I n s t i t u t e s
of. H e a lth ,
F resh
1 1 1 ,f L a b o r a to r y o f
B .e t h e s d a , M a r y l a n d ,
10
m ade a n d p r o v i d e d
th e cru d e a s c a r i s
e x t r a c t (C A E).
h o m o g en izin g s a lin e - w a s h e d A s c a r is o rg a n is m s ;
CAE w as p r e p a r e d b y
d ia ly z in g
t h e ho m o g en ate
a g a i n s t s e v e r a l c h a n g e s o f PH 8 . 2 b o r a t e b u f f e r e d s a l i n e a n d c l a r i f y i n g
d i a l y s a t e by c e n t r i f u g a t i o n
( 1 0 , 0 0 0 x G - 30 m i n u t e s ) .
in sm all a lo q u a te s ' a t -20°C . ■ For u s e ,
CAE w as s t o r e d
CAE w as th a w e d a n d d i l u t e d
to th e
d e sire d c o n c e n tra tio n in s a lin e .
D N P -A scaris.
D i n i t r o p h e n y l a t e d c r u d e A s c a r i s e x t r a c t (DNP-CAE)
w a s a l s o m ade a n d p r o v i d e d b y D r . H. M o r s e .
w ith a s c a r i s e x t r a c t in th e r a t i o
V i-a n tig e n
sity ,
P o c a te llo ,
U n iv e rsity .
o f 48 DNP/ 1 0
5
d a lto n s.
( V i ) w as p u r i f i e d b y D r . F . J a r v i s ,
Idaho,
p h o re sis p ro ced u re
am in u ro n ic a c id
T he DNP g r o u p w as c o m b in e d
(33).
by a c o n t i n u o u s - f l o w p a p e r c u r t a i n e l e c t r o ­
T h is a n tig e n ,
a p o ly m e r.o f N -a c e ty l g a la c to s -
( 3 3 ) , w as p r o v i d e d b y D r . J .
T. U lr ic h o f Idaho S ta t e
T he l y o p h i l i z e d V i w as s t o r e d a t 4 ° C .
m e n t s , V i w as d i s s o l v e d
A d ju v an ts.
Idaho S ta te U n iv er­
fo r use in e x p e r i - .
in s a lin e ..
A lum .w as, p r e p a r e d a s f o l l o w s :
ammonium s u l f a t e . ( Humco L a b o r a t o r y ,
75 g r a m s o f alu m in u m
.
T e x a r k a n a , T e x a s ) w a s a d d e d t o 180
m l o f d i s t i l l e d w a t e r a n d t h e n 75 m l o f. I N NaOH w as s l o w l y a d d e d w i t h
c o n tin u o u s m ix in g .
ste rile
T he r e s u l t i n g p r e c i p i t a t e was W ashed 8 t i m e s w i t h . •
d i s t i l l e d w a t e r a n d w as. f i n a l l y r e s u s p e n d e d i n . s t e r i l e
l e d w a t e r a t a c o n c e n t r a t i o n o f 60 m g / m l .
d is til­
T h i s a d j u v a n t w as m i x e d b y
p i p e t t i n g w ith th e d i f f e r e n t a n tig e n s u sed p r i o r to im m u n iz a tio n ^ .
.
P e rtu ssis E x tra c t.
A s a lin e e x tra c t of B o rd e te lla p e r tu s s is
(P E )
w as m ade ( 9 ) a n d p r o v i d e d by D r . J o h n M unoz, R o cky M o u n t a i n L a b o r a t o r y ,
H a m ilto n , M o n tan a.
T he l y o p h i I i z e d e x t r a c t was s t o r e d a t 4°C a n d was
d isso lv e d
f o r u s e as an a d j u v a n t .
in s a lin e
A t t i m e s i t w as m ix e d w i t h
a n t i g e n b e f o r e im m u n iz a tio n .
L ip o p o ly sacc h arid e
(L P S ) w as e x t r a c t e d
f r o m E s c h e r i c h i a c o l i 0113
b y t h e h o t p h e n o l - w a t e r m e t h o d ( 3 4 ) a n d w a s p r o v i d e d b y D r . J . A.
R u d b a c h , U n i v e r s i t y o f M o n t a n a , M i s s o u l a , M t;
LPS w as d i s s o l v e d i n PBS
a t a c o n c e n t r a t i o n o f I .m g /m l a n d s t o r e d a t -ZO0 C u n t i l d i l u t e d i n PBS
fo r use in ex p erim en ts.
C o m p l e t e F r e u n d ' s A d j u v a n t (CFA) w as p u r c h a s e d f r o m D i f c o L a b o r a ­
to rie s,
D e tro it,
M ich ig an .
A n tily m p h o c y te Serum .
R a b b i t a n t i - m o u s e l y m p h o c y t e s e r u h (RAMLS),
l o t 1 3 0 9 6 , w as p u r c h a s e d f r o m 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 ,
M a ry la n d .
O th er, l o t s w ere n o t u se d i n t h i s
N e m a to d e s.
ex p e rim en t.
.
I n some e x p e r i m e n t s d e s i g n e d t o p o t e n t i a t e HTA p r o d u c ­
t i o n , m ice w ere i n j e c t e d
a d a p te d s t r a i n
B eth esd a,-
s u b c u t a n e o u s l y . w i t h 600 Lo l a r v a e o f a m o u s e - .',
o f th e n em ato d e N i p p o s tr o n g y lu s b r a s i l i e n s i s
I r r a d i a t i o n .•
(3 5 ).
M ic e a n d r a t s w e r e g i v e n 4 0 0 - 4 5 0 r a d w h o l e - b o d y
i r r a d i a t i o n u s i n g a P i c k e r 60 C o b a l t t h e r a p y u n i t .
To. i n s u r e u n i f o r m
12
dose d u rin g i r r a d i a t i o n ,
on to p o f a r o t a t i n g
th e m ice w ere h e ld i n a p l e x i g l a s s ca g e p la c e d
tu rn ta b le .
T he d o s e r a t e was a p p r o x i m a t e l y 75
r a d / m ii i .
S p le n e c to m y .
S p l e e n s w e r e re m o v e d s u r g i c a l l y
rI
a p p r o x im a te ly s i x w eeks o f a g e .
p r o p e r d o se o f N e b u to l ( 3 6 ) .
b e f o re rem o v al.
fro m B a l b / c m ice a t
T he m i c e w e r e a n e s t h e t i z e d w i t h t h e
T he s p l e e n w as l i g a t e d a n d c a u t e r i z e d
T he p e r i t o n e u m a n d s k i n w e r e s u t u r e d s e p a r a t e l y .
C are
w as t a k e n n o t t o t e a r t h e s p l e e n a n d n o t t o l e a v e a n y s p l e e n r e m n a n t s
in th e m ice.
Thymus G r a f t s .
One m o n t h o l d n u d e m i c e w e r e a n e s t h e t i z e d w i t h
N e b u t o l (3 6 ). p r i o r . t o i m p l a n t a t i o n o f two t o f o u r th y m u s g l a n d s fro m
donors le s s
th an th r e e days o f ag e.
f a s c ia in th e a x i l l a r y
u s e d a s th y m u s d o n o r s .
I m p l a n t s w e r e m ade u n d e r t h e
r e g i o n o f t h e . n u d e . . E i t h e r B a l b / c o r NLM w e r e
O n ly t h y m u s - g r a f t e d n u d e s t h a t o u t w a r d ly
a p p e a re d h e a l t h y w ere u s e d . i n e x p e r im e n ts .
Thymus C e l l I n j e c t i o n s .
T h y m o c y te p r e p a r a t i o n s ,'w e r e m ade '.by .
s c r e e n i n g young a d u l t B a lb /c thym uses th r o u g h s t a i n l e s s
s c r e e n s i n t o PBS c o n t a i n i n g
s t e e l 8 0 -m e s h
1% f e t a l c a l f s e r u m (P B S - F C S ) .
t h y n i o c y t e s w e r e t h e n w a s h e d o n c e w i t h P B S -F C S.
The
V i a b i l i t y w as d e t e r -
■■- : g
r■
m in ed by a T ry p a n , B lu e . E x c l u s io n t e s t , , and 1 .4 x 1 0 v i a b l e c e l l s c o n - /
t a in e d i n 0 ,7 ml w ere i n j e c t e d
nude m ice.
s lo w ly .in to
'
th e t a i l y ein . o f . r e c i p i e n t
-
-'
-v ■
.
13
Im m u n izatio n P r o c e d u r e s .
To o b t a i n HTA, m i c e w e r e g i v e n v a r y i n g
d o s e s o f a n t i g e n a n d a d j u v a n t on' d a y 0 .
a l w a y s m ix e d p r i o r
to im m u n iz a tio n .
A n t i g e n a n d a d j u v a n t w e re
CFA w a s m i x e d w i t h a n t i g e n w i t h a
d o u b le s y r i n g e w h ile a l l o t h e r a d j u v a n t s u s e d w ere m ixed w i t h a
p ip e tte .
The i n j e c t i o n s w e re u s u a l l y g i v e n i n t r a p e r i t o n e a l - l y ,
alth o u g h
t h e r e w e r e some e x p e r i m e n t s i n w h i c h t h e r o u t e o f i n j e c t i o n was i n t r a ­
venous.
T he a n i m a l s w e r e b l e d o n d a y s 10 a n d 20 a f t e r i n i t i a l
im m u n iz a tio n
o f a n t i g e n a l o n e b e c a u s e I fou n d t h a t a d j u v a n t h a s a d e l e t e r i o u s e f f e c t
on a n t i b o d y p r o d u c t i o n i f g i v e n a t t h i s
b l e d on day 30,
tim e .
T he a n i m a l s w e r e t h e n
10 d a y s a f t e r t h e b o o s t e r i n j e c t i o n ;
S erum was c o l l e c ­
t e d from i n d i v i d u a l a n im a ls and s t o r e d s e p a r a t e l y a t -3 0 °C b e c a u s e I
and o t h e r s
(3 7 ) h a v e fo u n d t h a t r e a g i n i c a n t i b o d y d e n a t u r e s a t 0°C .
P a s s i v e C u tan eo u s A n a p h y la x is .
A p o o l w as m ade f r o m t h e s e r u m o f
i n d iv id u a l an im als w ith in a g iv en e x p e rim e n ta l g ro u p .
j ■
d ilu tio n s
T w o -fo ld s e r i a l
■.
o f t h e s e r u m p o o l w e r e m ade i n s a l i n e . ■ 0 . 0 5 m l o f a g i v e n
•
.
/
.
s e r u m d i l u t i o n w a s i n j e c t e d i n t r a d e r m a l l y o n t h e b a c k o f a r e c i p i e n t ..
CFW m o u s e .
T he h a i r w as c l i p p e d f r o m t h e b a c k o f r e c i p i e n t m i c e 24
h o u rs p r i o r to th e in tra d e rm a l i n j e c t i o n .
in je c te d
R e c i p i e n t m ice w ere
i n t r a v e n o u s l y w i t h 0 .2 m l o f a n t i g e n s o l u t i o n
( 1% s o l u t i o n o f
EA, 0 . 2 mg DNP-CAE o r 0 . 2 mg CAE) i n s a l i n e c o n t a i n i n g 0 . 5 % . E v a n s B l u e
dye.
T h i r t y m i n u t e s a f t e r i n t r a v e n o u s c h a l l e n g e w i t h a n t i g e n and. E v a n s
B lu e th e a n im a ls w ere k i l l e d
by c e r v i c a l , d i s l o c a t i o n a n d t h e s k i n on th e
b a c k w as i n c i s e d a n d i n v e r t e d .
A p o s i t i v e r e a c t i o n w as n o t e d b y a b l u e
c i r c u l a r a re a o f th e s k in a t th e i n j e c t i o n
t a l m easurem ents w e re r e c o r d e d .
site .
An a r e a l e s s
V e r t i c a l and h o r i z o n ­
t h a n 4mm x 4fnm w as r e c o r d e d
a s a n e g a t i v e r e a c t i o n , w h e r e a s a n a r e a g r e a t e r o r e q u a l t o 4mm x 4mm
w as r e c o r d e d a s a p o s i t i v e r e a c t i o n .
R e a c tio n s w ere g iv e n a s u b j e c t i v e
r a t i n g o f 1+ t o 4 + - d e t e r m i n e d b y t h e d e p t h o f c o l o r ,
b ein g th e d e e p e s t b lu e .
a 4+ r e a c t i o n
T he e n d p o i n t w as d e f i n e d a s t h e l a s t d i l u t i o n
g iv in g a p o s itiv e r e a c tio n .
I t sh o u ld be n o te d
assay.
If
t h a t two d i f f e r e n t HTAs a r e m e a s u r e d b y t h e PCA
t h e r e c i p i e n t a n i m a l s a r e a l l o w e d . t o w a i t tw o h o u r s b e f o r e
c h a l l e n g e w i t h a n t i g e n and E vans B lu e d y e ,
is heat sta b le ,
th e serum .
th e serum .
IgG].
h a s a s h o r t d u r a t i o n i n t h e s k i n and a lo n g d u r a t i o n i n
In c o n tr a s t,
i f a n t i g e n and Evans B lue dye a r e i n j e c t e d
h ours a f t e r sk in s e n s i t i z a t i o n ,
heat la b ile ,
t h e n IgG ^ i s d e t e c t e d .
72
t h e PCA r e a c t i o n d e t e c t s I g E , w h i c h i s
h a s a l o n g e r d u r a t i o n i n s k i n b u t a. s h o r t e r d u r a t i o n i n
RESULTS
Ig E P r o d u c t i o n i n
th e M ouse.
H eat s e n s i t i v i t y
to u s e to d i s t i n g u i s h b etw een h e a t - s t a b l e
Thus,
is
a good p r o p e r t y
IgG ^ and h e a t - l a b i l e
IgE .
i t w as d e t e r m i n e d f r o m h e a t i n g u n d i l u t e d s e r u m t h a t t h e 2 - h o u r
PCA r e a c t i o n r e m a i n e d w h i l e t h e 7 2 - h o u r PCA d i m i n i s h e d .
T h i s same
''i
r e s u l t w as f o u n d i n s e r a f r o m b o t h a n t i g e n s u s e d f o r i m m u n i z a t i o n and
from serum c o l l e c t e d
a f t e r a p r i m a r y i m m u n i z a t i o n o n d a y 10 and a
s e c o n d a r y i m m u n i z a t i o n c o l l e c t e d o n d a y 30 ( T a b l e I ) .
n o te d t h a t th e 7 2 -h o u r re s p o n s e rem ain ed i f
T hese r e s u l t s
It
s h o u ld be
t h e s e r u m w as n o t h e a t e d .
s u g g e s t t h a t th e a n tib o d y o b se rv e d a t 7 2 -h o u rs i s IgE .
B ecause o f th e s e r e s u l t s ,
I r o u t i n e l y u s e d t h e 7 2 - h o u r PCA r e s p o n s e a s a
m easure o f IgE .
P r e l i m i n a r y S t u d i e s on t h e A b i l i t y o f D i f f e r e n t A n t i g e n s to E l i c i t
a n Ig E R e s p o n s e i n M i c e .
t i o n s w ere t e s t e d
fo r th e ir a b ility
B a lb /c m ice (T a b le I I ) .
fa ile d
20.
S e v e r a l d i f f e r e n t a n t i g e n - a d j u v a n t com bina­
t o e l i c i t Ig E a n t i b o d y p r o d u c t i o n i n
M ic e im m u n iz e d w i t h 10 ^ g CAE p l u s 50 jug PE
t o p r o d u c e Ig E ( l i n e A ) ,
e v e n w hen b o o s t e d w i t h 10 Aig CAE o n d a y
N e g a t i v e r e s u l t s w e r e a l s o o b t a i n e d w i t h 125 Jig EA p l u s 50 Jug PE
( l i n e B) a n d 1 0 ' p g CAE p l u s
w ere n o t e f f e c t i v e .
I jig LPS ( l i n e C ) ; a g a i n ,
b o o ste r in je c tio n s
CAE, h o w e v e r , w a s a g o o d a n t i g e n t o e l i c i t Ig E w hen
u s e d w i t h e i t h e r CFA o r a l u m a d j u v a n t s .
100 jug CAE p l u s
20 mg a lu m
( l i n e D) g a v e 72 h r PCA r e a c t i o n s o n d a y 10 a f t e r a p r i m a r y ' i m m u n iz a ­
t i o n a n d d a y 30 a f t e r a s e c o n d a r y i m m u n i z a t i o n .
A s l i g h t l y h ig h er
TABLE I
E f f e c t s o f h e a t i n g a hom ocyt o t r o p i c a n t i b o d y p o s i t i v e serum from B a lb /c m ic e ,
m i n u t e s a t 56°C a s d e m o n s t r a t e d by p a s s i v e c u t a n e o u s a n a p h y l a x i s r e a c t i o n s
H e ated
Im m u n izatio n
Day 0
Im m u n izatio n
Day 20
10 Jjg EAa , A l b
10 Jig EA
+
sam e^
same
sam e
none
same
same
+
+
+
-
same
10 p g CAEd ,CFAe
same
same
N o. o f
Serum
in Pool
5
5
7
5
5
7
Day Serum
C o lle c te d
2 hr
256c
30
30
72 h r
72 h r
2 hr
72 h r
72 h r
negS
128
64
neg
128
20
20
20
a EA = h e n e g g a l b u m i n
c 256 = t i t e r r e c i p r o c a l o f s e r u m p o o l
dCAE = c r u d e a s c a r i s e x t r a c t
e CFA = c o m p l e t e F r e u n d s a d j u v a n t i n e q u a l v o l u m e a s a n t i g e n
f s a m e = sam e s e r u m a s a b o v e
hpCA = p a s s i v e c u t a n e o u s a n a p h y l a x i s
S e n s i t i z a t i o n PCA^1T i t e r o f
P e rio d
S erum P o o l
30
^ A l = 10 mg a lu m
S n e g = n o t d e t e c t e d a t 1 /8 d i l u t i o n
30
17
tite r
on d a y 10 w as o b t a i n e d i f
10 mg a l u m ( l i n e E ) .
o n d a y s 10 a n d 2 0 ,
t h e d o s e w as r e d u c e d t o 1 0 p g GAE p l u s
When CFA w as u s e d
( l i n e F ) , t h e m ic e p ro d u c e d IgE
and a l s o a f t e r a b o o s t e r i n j e c t i o n .
t i t e r s w e r e o b t a i n e d w i t h 10 u g DNP-GAE p l u s
V e r y good p r i m a r y
10 mg a lu m ( l i n e G ) , and
a l s o a f t e r a b o o s t e r i n j e c t i o n o f DNP-CAE.
Egg a l b u m i n w i t h a l u m a s a d j u v a n t w as b y f a r
to u s e and a l s o e l i c i t e d h i g h t i t e r s
o p t i m a l d o s e w as 10 jig EA p l u s
t h e e a s i e s t com pound
o f Ig E o n d a y s 1 0 ,
10 mg a l u m ( l i n e H ) .
20 a n d 3 0 .
The
CFA w as a l s o e f f e c ­
t i v e w i t h 125. jjg EA i n p r o d u c t i o n o f Ig E o n d a y s 20 a n d 30 a l t h o u g h t h e
tite rs
a r e m uch l o w e r t h a n i f a lu m w as u s e d a s a d j u v a n t . .
A fte r a l l
p lu s
th e s e t e s t s w ere c o m p le te d ,
i t was d e c i d e d t h a t 10 j u g . EA .
10 mg a l u m ( l i n e L ) w as t h e b e s t a n t i g e n - a d j u v a n t c o m b i n a t i o n w i t h
w h i c h t o o b s e r v e . t h e Ig E r e s p o n s e .
Thymus D e p e n d e n c y o f t h e I g E R e s p o n s e .
An a t t e m p t w as made t o
■
d e t e r m i n e w h e t h e r o r n o t Ig E e x h i b i t s . t h e u s u a l t h y m u s - d e r i v e d c e l l
( T - c e l l ) . - b u r s a - e q u i v a l e n t (B c e l l )
c o lla b o ra tio n , re q u ire d
t i o n o f o t h e r im m u n o g lo b u lin c l a s s e s .
f o r pro d u c-
.
T o . t e s t f o r s u c h ■a r e q u i r e m e n t ,
a t t e m p t s w e r e m ade t o i n d u c e Ig E p r o d u c t i o n i n t h e n u d e m o u s e ( T a b l e
III).
B ecause t h i s
an im al la c k s T c e l l s
from b i r t h , . ' i t p r o v i d e s an
e x c e l l e n t sy ste m i n w h ich to o b s e r v e .th y m u s d e p e n d e n c y .
NLM m i c e w e r e im m u n iz e d w i t h 10 jug EA p l u s
w i t h 10 jJ g .EA o n d a y 2 0 .
'
B o th n u d e .a n d .
10 m g ' a l u m , . a n d w e r e b o o s t e d
T h e NLM r e s p o n d e d o n d a y s 1 0 ,
.
20 arid .30 w i t h .
TABLE I I
E f f e c tiv e n e s s o f d i f f e r e n t a n tig e n - a d ju v a n t co m b in atio n s i n je c te d i n t r a p e r i t o n e a l l y
i n e l i c i t i n g Ig E p r o d u c t i o n i n B a L b /c m i c e ___________________________________________________
L i n e A n t i g e n - A d j u v a n t Com­
b i n a t i o n s Dav O
A
10 Mg CAEb , 50 jug PEc
B
C
D
E
F
G
H
I
Im m u n izatio n
Dav 20
10 Ug CAE
No. o f Serum
in Pool
4
125 u g EAe , 50 Ug PE
125 u g EA
10 u g CAE, I p g LPS^
10 Ug CAE
100 jug CAE, 20 mg AlS 100 Ug CAE
10 Ug CAE
10 jug CAE, 10 mg Al
10 u g CAE, CFA1
10 u g CAE
10 jug DNP-CAEJ, 1 0 mg Al. 10 u g DNP-CAE
10 Ug EA, 10 mg Al
10 Ug EA
10 u g EA
125 u g EA, CFA
5
4
4
4
4
4
4
4
a PCA = p a s s i v e c u t a n e o u s a n a p h y l a x i s
^CAE = c r u d e a s c a r i s e x t r a c t
c PE = s a l i n e e x t r a c t o f B o r d e t e I l a p e r t u s s i s
^ n e g = n o t d e t e c t e d a t 1 /2 d i l u t i o n
e EA = h e n e g g a l b u m i n
^LPS = l i p o p o l y s a c c h a r i d e
S a I = a lu m
hg = t i t e r r e c i p r o c a l o f serum p o o l
iCFA = c o m p l e t e F r e u n d s a d j u v a n t
J d n p -CAE = d i n i t r o p h e n y l a t e d c r u d e a s c a r i s e x t r a c t
72 h r PCAa T i t e r o f Serum P o o l
Day 10
Day 20
Day 30
nega
neg
neg
neg
neg
neg
neg
neg
neg
8h
neg
128
16
neg
128
64
128
32
64
32
256
64
16
128
16
neg
8
TABLE I I I
Thymus D e p e n d e n c y o f t h e Ig E R e s p o n s e
L in e
_______
A
B
C
D
E
F
G
H
I
J
K
L
M
N
0
Im m u n izatio n
Im m u n izatio n
G roup
Tim e o f
Day 10
________ Day 20_____________________ G r a f t ________
10 p g EA6 , A l c 10 p g EA
NLMd
NAe
Nug
sam e^
same
NA
Nu-TCh 2 m o n t h s
same
same
NLM
NA
10 p g EA
10 p g EA, Al
NA
Nu
sam e
same
NA
B a lb /c
10 p g EA, Al
10 j i g EA
7 m on th s
Nu-TGk
same
same
NA
B alb /c
10 p g EA
10 p g EA, Al
Nu-TC
sam e
2 m o n th s
same
Al
NLM
NA
10 j i g CAE,
10 p g CAE
NA
Nu
same
sam e
Nu-TG
same
4 -6 m on th s
same
NA
B alb /c
10 p g EA, Al
10 p g EA
Nu-TC
same
7 m o n th s
same
Nu-TG
6 -1 2 m o n th s
same
same
PCAa T i t e r o f S erum P o o l
Day 10 Day 20
Day 30
321
negJ
neg
64
neg
64
32
64
neg
128
neg
neg
16
neg
16
32
neg
neg
8
neg
32
128
128
neg
32
neg
neg
8
neg
4
32
neg
16
32
neg
32
n o t done
128
16
16
neg
2
n o t done
n o t done
n o t done
a PCA = p a s s i v e c u t a n e o u s a n a p h y l a x i s
^Nu = n u d e ,
^EA = h e n e g g a l b u m i n
^1Nu-TC = n u d e w i t h thym us c e l l s
c A l = 10 mg a lu m
^NLM = n o r m a l l i t t e r m a t e
^32 = t i t e r
to nude
e NA = n o t a p p l y
^sam e = sam e s e r u m a s a b o v e
a t h y m i c m ouse
r e c i p r o c a l o f serum p o o l
^ n e g = n o t d e t e c t e d a t 1 /2 d i l u t i o n
Ir
Nu-TG = n u d e w i t h thy m u s g r a f t
20
p ro d u c tio n o f IgE .
T h u s , NLM r e s p o n d e d a f t e r a p r i m a r y i m m u n i z a t i o n a s
s e e n o n d a y s 10 a n d .20 a n d a l s o a f t e r a s e c o n d a r y i m m u n i z a t i o n a s s e e n
o n d a y 30 ( l i n e A ) .
T he n u d e m i c e , h o w e v e r , m ade n o s u c h r e s p o n s e s
( l i n e B ).
R e p e titio n s o f t h i s ex p e rim en t y ie ld e d s i m il a r r e s u l t a
(T a b le I I I ,
'
lin e ;. D a n d F ) .
A t t e m p t s w e r e a l s o m ade t o i n d u c e Ig E p r o d u c t i o n i n n u d e m i c e w i t h
an o th er a n tig e n ,
p l u s 10 mg a l u m .
CAE ( T a b l e I I I ) .
T h e i m m u n i z a t i o n d o s e w as 10 jug CAE .
A g a i n , NLM r e s p o n d e d o n d a y s 1 0 ,
d u c t i o n o f Ig E a n t i b o d y ,
20 a n d 30 w i t h p r o ­
and a g a in , n u d e m ice f a i l e d
to resp o n d to th e
a n t i g e n even a f t e r se c o n d a ry im m u n iz a tio n .
I g E P r o d u c t i o n i n Nude M ic e R e c o n s t i t u t e d w i t h Thymus C e l l s .
B ecause n u d e m ice f a i l e d
t o p r o d u c e I g E i n r e s p o n s e t o EA a n d a lu m ,
t h e i r r e s p o n s e w as c h e c k e d f o l l o w i n g r e c o n s t i t u t i o n w i t h
thy m u s c e l l s
fro m n o rm a l B a l b / c m ic e .
i n j e c t i o n s w ere m ade,
a lu m ( T a b l e . I l l ,
t h e thy m u s c e l l
t h e m i c e w e r e im m u n iz e d w i t h 10 p g EA p l u s 10 mg ■
litte C ).
respond u n t i l a f t e r
Two m o n t h s a f t e r
1 . 4 x 10^
Thymus c e l l - i n j e c t e d n u d e m i c e d i d n o t
t h e s e c o n d a r y i m m u n i z a t i o n o f 10 jig BA.
The t i t e r
o f t h e r e s p o n s e w as n o t a s h i g h a s t h e B a l b / c c o n t r o l a l t h o u g h t h e r e was!
a resp o n se
n u d e m i c e o.
(1 6 ) i n c o n t r a s t to th e t o t a l
la c k o f a r e s p o n s e i n norm al
T h i s e x p e r i m e n t was r e p e a t e d w i t h s i m i l a r r e s u l t s , ( l i n e I ) , .
'
21
Ig E P r o d u c t i o n i n Nude M ic e R e c o n s t i t u t e d w i t h Thymus G l a n d s .
Nude m i c e w e r e a l s o i m p l a n t e d s u b c u t a n e o u s l y w i t h 2 t o 4 th y m u s g l a n d s
t o m ake th e m i m m u n o l o g i c a l l y c o m p e t e n t .
Seven m onths l a t e r ,
a n d B a l b / c c o n t r o l s w e r e im m u n iz e d w i t h 10 p g EA p l u s
0 a n d 10 jag EA a l o n e o n d a y 2 0 .
d u c t i o n o f Ig E o n day's 1 0 ,
m ice w h ich had r e c e i v e d
10 a n d 20 ( l i n e G ) .
t h e s e m ice
10 mg a lu m o n d a y
The B a l b / c m i c e r e s p o n d e d w i t h p r o ­
20 a n d 30 ( T a b l e I I I ,
lin e F ).
The n u d e
t h e th y m u s g l a n d s a l s o r e s p o n d e d w e l l o n d a y s
No t e s t w as d o n e o n d a y 3 0 .
e x p e r im e n t n u d e m ice t h a t r e c e i v e d
i m m u n i z a t i o n w i t h 10 jag EA p l u s
th y m u s g l a n d s 6 t o 12 m o n t h s b e f o r e
10 mg a lu m m ade a n I g E r e s p o n s e s i m i l a r
to th e re s p o n se o f th e B a lb /c c o n t r o l s .
o n d a y s 10 a n d 20 ( T a b l e I I I ,
In a rep eated
S i m i l a r r e s p o n s e w as o b s e r v e d
l i n e s M and 0 ) .
T h y m u s-im p !an ted nude m ice w ere a l s o
10 mg a lu m 4 t o 6 m o n t h s a f t e r g r a f t i n g .
im m u n iz e d w i t h 10 jug CAE p l u s .
In t h i s c a s e ,
t h e y made no
r e s p o n s e o n d a y s 10 a n d 20 b u t d i d m ake a s l i g h t r e s p o n s e a f t e r
sec o n d a ry im m u n iz a tio n .
(T a b le I I I ,
T he B a l b / c c o n t r o l s r e s p o n d e d o n a l l
th re e days
l i n e s J and L ) .
I g E R e s p o n s e of. Nude M ic e t o . 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 .
Nude
m ic e , a n d NLM w e r e im m u n iz e d w i t h I jag V i m i x e d w i t h 10 mg a l u m . V i is.
know n t o b e a th y m u s i n d e p e n d e n t a n t i g e n f o r t h e IgM r e s p o n s e . A t t e m p t s
w e r e m ade t o i n d u c e I g E p r o d u c t i o n i n n u d e m i c e w i t h t h i s
fo rtu n a te ly ,
V i d o e s n o t s t i m u l a t e Ig E p r o d u c t i o n i n NLM.
a n tig e n .
I t d id
Un­
22
n o t e l i c i t Ig E p r o d u c t i o n i n n u d e m i c e ( T a b l e I V ) . '
The Thymus a s t h e O r i g i n o f t h e C e l l s R e s p o n s i b l e f o r PCA R e a c t i o n s .
M o ta ( 6 ) h a s s u g g e s t e d t h a t t h e c e l l r e s p o n s i b l e f o r t h e a n a p h y l a t i c
re a c tio n in r a ts
and m ice i s
and a n t i b o d y s t i m u l a t e s
th e m ast c e l l ;
th e m ast c e l l
m e d i a t o r s o f t h e PCA r e a c t i o n ,
th e co m b in atio n o f a n tig e n
to r e le a s e
th e p h a rm a c o lo g ic a l
h i s t a m i n e and s e r o t o n i n
i f n u d e m i c e w e r e a b l e t o p r o d u c e a PCA r e a c t i o n
T herefore,
th e o r i g i n o f th e m ast
c e l l c o u l d n o t b e t h e th y m u s a s o t h e r s h a v e s u g g e s t e d
To t e s t t h i s h y p o t h e s i s ,
(6).
(28,
2 9).
n u d e m i c e a n d CFW m i c e w e r e s e n s i t i z e d
w i t h s e r u m known t o b e p o s i t i v e
f o r a PCA r e a c t i o n .
As show n i n T a b l e V,
n u d e m i c e i n a l l c a s e s g a v e a PCA r e a c t i o n a t l e a s t a s g o o d a s t h e CFW
c o n tro ls
f o r b o t h 72 h r a n d 2 h r PCA r e a c t i o n s .
E f f e c t s o f I m m u n o s u p p r e s s i v e A g e n t s o n t h e IgE R e s p o n s e i n M i c e .
Tada d e m o n s tra te d t h a t r a t s
show ed a n e n h a n c e d p r o d u c t i o n o f IgE when
t r e a t e d w i t h 400 r a d X - i r r a d i a t i o n
(1 8 ),
s p l e n e c t o m y ( 1 6 ) , . some immuno­
s u p p re s s iv e drugs
(1 9 ) and a n t i t h y m o c y t e t r e a t m e n t . ( 2 0 ) .
m ade t o show t h i s
sam e e n h a n c e m e n t e f f e c t i n m i c e .
A tte m p ts w ere
B a l b / c m ice w ere
i n j e c t e d w i t h 0 . 3 m l ALS 4 d a y s a f t e r i m m u n i z a t i o n , w i t h 10 jig EA a n d 10
mg a l u m .
( T a b le V I,
A d ecreased
t i t e r was o b s e r v e d o n d a y s 1 0 , .2 0 ,
30 a n d 40
l i n e s A a n d B) i n a n i m a l s t h a t w e r e t r e a t e d . w i t h ALS.
S i m i l a r r e s u l t s w e r e o b t a i n e d when t h i s w as r e p e a t e d c h a n g i n g o n l y t h e
t i m e o f a d m i n i s t r a t i o n o f ALS.
When ALS w as a d m i n i s t e r e d a t t h e t i m e , o f
TABLE IV
Ig E r e s p o n s e o f m i c e t o a th y m u s i n d e p e n d e n t a n t i g e n ,
Vi
Im m u n izatio n
Day 10
G roup
PCAa T i t e r o f Serum P o o l
Day 10
Day 20
I Aig V i , A l b
NLMc
n e g a tiv e ^
n e g a tiv e
sam e a s a b o v e 6
Nu^
n e g a tiv e
n e g a tiv e
a PCA = p a s s i v e c u t a n e o u s a n a p h y l a x i s
^ A l = 10 mg a lu m
c NLM = n o r m a l l i t t e r m a t e o f n u d e
^ n e g a t i v e = n o t d e t e c t e d a t a 1 /2 d i l u t i o n
e sam e a s a b o v e = d e n o t e s t h e same s e r u m a s a b o v e
% u = c o n g e n i t a l l y a t h y m i c n u d e m ouse
TABLE V
P a s s i v e c u t a n e o u s a n a p h y l a x i s r e s p o n s e o f n u d e 3 an d CFW m i c e t o hom o cy t o t r o p i c
a n t i b o d y p o s i t i v e s e r u m _____________________________________________________________________
R e c i p i e n t Mouse
N o. o f Serum
in Pool
N ude*
CFW*
N ude*
CFW*
5
5
5
5
N ude**
CFW**
P erio d o f
S e n sitiz a tio n
72 h o u r s
72 h o u r s
NDb
ND
16
16
64c
64
64
32
4
72 h o u r s
64
64
4
72 h o u r s
64
64
2 h ours
2 h ours
anude = c o n g e n i t a l l y a th y m ic nude m ice
^ND = n o t d o n e
c 64 = t i t e r
PCAd T i t e r o f Serum P o o l
Day 10
Day 3 0 e
r e c i p r o c a l o f serum p o o l
^PCA = p a s s i v e c u t a n e o u s a n a p h y l a x i s
= d e n o t e s s e r u m p o o l #1
* * = d e n o t e s s e r u m p o o l #2
TABLE V I
Thymus r e g u l a t i o n o f t h e I g E r e s p o n s e ______________________________________________ _
L in e
B
Im m u n izatio n
Day 10____________
10 Aig EAb j A l c
sam e 6
C
D
E
F
G
H
I
J
same
same
10 Alg CAEJ , Al
same
10 AJg CAE, Al
same
10 >ig BA, Al
same
A
I m m u n i z a t i o n G roup
N o. Serum PCAa T i t e r o f Serum P o o l
Day 20____________________ i n P o o l _____ Day 10
20_____ 3 0 * 40
7
64d
B alb /c
10 Aig EA
128
128
32
ALSf
same
7
4
16
32 32
7
4 0 0 Rg
negh
same
32
16
4
same
7
64
64
32
8
sK
NLMk
10 Aig CAE
4
128
32
16
ALS
4
neg
4
same
neg
CAE
NLM
5
2
n
e
g
neg
10 Aig
ALS
5
8
sam e
4
2
NLM
4
64
10 Aig EA
8
32
ALS
4
sam e
neg
neg
4
a PCA = p a s s i v e c u t a n e o u s a n a p h y l a x i s
^EA = h e n e g g a l b u m i n
c A l = 10 mg a lu m
^64 = t i t e r r e c i p r o c a l o f serum p o o l
e sam e = sam e s e r u m a s a b o v e
^ALS = r a b b i t a n t i - m o u s e l y m p h o c y t e s e r u m
8 4 0 0 R = 400 r a d X - i r r a d i a t i o n
^ n e g = n o t d e t e c t e d a t 1 /2 d i l u t i o n
i-Spx = s p l e n e c t o m y
JCAE = c r u d e a s c a r i s e x t r a c t
^cNLM = n o r m a l l i t t e r m a t e t o n u d e
* = d e n o t e s serum a f t e r se c o n d im m u n iz a tio n
26
o f i m m u n i z a t i o n w i t h 10 jig EA a n d 10 mg a l u m ,
t h e Ig E r e s p o n s e was
a b o l i s h e d o n d a y s 10 a n d 20 a n d b a r e l y d e t e c t a b l e on d a y 30 ( l i n e s
J).
I and
When ALS w as a d m i n i s t e r e d a t t h e sam e t i m e a s i m m u n i z a t i o n w i t h 10
jug CAE p l u s
10 mg a l u m ,
e x p e r im e n t ( T a b le V I,
a s l i g h t l y h i g h e r t i t e r w as o b s e r v e d f o r o n e
l i n e s G a n d H) w h e r e a s a much l o w e r t i t e r was
o b s e rv e d i n a n o th e r e x p e rim e n t (T a b le V I,
irra d ia tio n
resp o n se.
( l i n e C) a n d s p l e n e c t o m y
From t h e s e d a t a ,
l i n e E and F ) „
400 r a d X-
( l i n e D) a l s o d e c r e a s e d
t h e IgE
i t can b e s e e n t h a t im m u n o su p p ressiv e
a g e n t s d e p r e s s t h e Ig E r e s p o n s e i n s t e a d o f e n h a n c i n g i t .
E f f e c t of I n f e c tio n w ith N ip p o stro n g lu s b r a s i l i e n s i s
R esponse.
p o te n tia te d
O th er i n v e s t i g a t o r s
(2 6 ,
27) h a v e d e m o n s tra te d i n r a t s a
Ig E r e s p o n s e t o s e v e r a l d i f f e r e n t a n t i g e n s , f o l l o w i n g
nem ato d e i n f e c t i o n .
sponse in m ice.
An a t t e m p t w as m ade t o f i n d t h i s p o t e n t i a t e d r e ­
M ic e w e r e i n o c u l a t e d
s u b c u t a n e o u s l y . w i t h 600 l a r v a e o f
th e p a r a s i t e N ip p o stro n g y lu s b r a s i l i e n s i s
i m m u n i z a t i o n o f 10 jug EA p l u s 10 mg a l u m .
la rv a e serv ed as c o n t r o l s .
10,
20,
o n t h e IgE
te n days a f t e r i n i t i a l
B a l b / c m ice w i t h o u t i n j e c t e d
S e r a w e r e t e s t e d b y t h e PCA m e t h o d on d a y s
30 a n d 40 ( T a b l e V I I ) ,
T he m i c e r e c e i v e d a. s e c o n d a r y i m m u n i - ;
■ z a t i b h o n d a y 20 f o l l o w i n g c o l l e c t i o n o f s e r u m . . M ic e t h a t r e c e i v e d , t h e '
600 l a r v a e show ed a s l i g h t l y h i g h e r t i t e r o h d a y . 30 ( 2 5 6 v s 128.).
B e c a u s e t h e r e w as o n l y o n e d i l u t i o n
th e c o n tr o ls ,
se p a ra tin g
t h e . worm i n f e c t e d f r o m
th e s e d a t a are. n o t v e r y m e a n in g f u l.
tio n can be seen th ro u g h o u t t h i s
ex p e rim en t.
It
One d i l u t i o n v a r i a ­
s h o u l d b e n o t e d , how-
;. ,
v
.
TABLE V I I
E f f e c t s o n Ig E p r o d u c t i o n o f i n f e c t i o n w i t h N i p p o s t r o n g y l u s b r a s i l i e n s i s t e n d a y s
a f t e r i n i t i a l i m m u n i z a t i o n w i t h 10 /ig e g g a l b u m i n a n d 10 mg a lu m _____________________
G roup
N o. o f S erum i n P o o l
7 2 - h r PCAa T i t e r o f Serum P o o l
Day 10 Day 20 Day 30*
*Day 40
B alb /c
7
64b
B a l b / c w i t h worm
in fe c tio n 0
7
64
128
128
64
64
256
32
a PCA = p a s s i v e c u t a n e o u s a n a p h y l a x i s
^64 = t i t e r
r e c i p t o c a l o f serum p o o l
c worm i n f e c t i o n = 600 l a r v a e o f t h e worm N i p p o s t r o n g y l u s b r a s i l i e n s i s w e r e
i n j e c t e d su b cu tan e o u s
* = m i c e r e c e i v e d a s e c o n d i m m u n i z a t i o n on d a y 20 o f 10 /Ug e g g a l b u m i n
28
ev er,
m ice.
t h a t t h e . PCA r e a c t i o n s w e r e o f a d e e p e r b l u e i n t h e worm, i n f e c t e d
-
-i
DISCUSSION
B e c a u s e t h e m o u s e p r o d u c e s two d i f f e r e n t h o m o c y t o t r o p i c a n t i ­
b o d ies,
i t w as n e c e s s a r y t o m ake c e r t a i n
w as w o r k i n g w as Ig E a n d n o t IgG i .
se n sitiz e d
IgG ^ i s p r e s e n t i n t h e s k i n o f
a n i m a l s two h o u r s a f t e r s e n s i t i z a t i o n a n d i s h e a t r e s i s t a n t ■
f o r 30 m i n u t e s a t 5 6 ° C .
Ig E , on t h e o t h e r h a n d ,
f o r lo n g e r p e r io d s o f tim e ,
heat la b ile
t h a t th e a n t i b o d y w ith w hich I
i s p r e s e n t in- t h e s k i n
b e y o n d .72 h o u r s a f t e r s e n s i t i z a t i o n ,
a t 56°C f o r 30 m i n u t e s .
and i s
To m ake s u r e t h e IgG ^ was n o t
p e rsistin g
in
t h e s k i n f o r 72 h o u r s a n d t h u s g i v i n g f a l s e , p o s i t i v e
re a c tio n s,
t h e u n d i l u t e d s e r u m w as h e a t e d a t 56°C f o r 30 m i n u t e s ,
and
r e c i p i e n t a n i m a l s w e r e c h a l l e n g e d w i t h a n t i g e n a n d E v a n s B l u e d y e 72
h ours a f t e r s e n s i t i z a t i o n .
w hich i n d i c a t e d
d e te c te d
if
No r e a c t i o n w as o b s e r v e d a t t h i s
t h a t a t 72 h o u r s o n l y Ig E w as p r e s e n t .
t h e s e r u m w as n o t h e a t e d .
tim e ,
Ig E was o n l y
From t h e s e o b s e r v a t i o n s ,
I knew
t h e a n t i b o d y b e i n g o b s e r v e d was I g E .
• S e v e r a l d i f f e r e n t a n t i g e n s w ere u s e d t o e l i c i t an IgE r e s p o n s e .
A l t h o u g h o t h e r s h a v e b e e n a b l e t o o b t a i n a r e s p o n s e u s i n g PE a s -an
a d ju v a n t (2 5 ,
8,. 9 ) ,
I w as n o t . . O f t e n t i m e s w i t h , t h i s a d j u v a n t ,
was b l u i n g o f t h e e n t i r e
IgG ^ r e s p o n s e .
10 mg a l u m .
th e re
s k i n , w h i c h p r o b a b l y w as a h i n d i c a t i o n , o f a n
The b e s t i m m u n i z i n g a g e n t , t r i e d w as .10 tig. EA m ix e d w i t h
CAE w as h o t a r e l i a b l e a n t i g e n .
e l i c i t a good r e s p o n s e ,
S o m e tim e s CAE w o u ld
b u t a t o t h e r t i m e s i t w o u ld n o t .
DNP-CAE w a s a n
e x c e l l e n t im m u n o g e n . . I t w as a l s o m i x e d w i t h , a lu m p r i o r . ; t o i m m u n i z a t i o n .
30
O th er i n v e s t i g a t o r s have im p lie d
d e p e n d e n t (1 7 ,
tio n
2 1 ).
th y m u s d e p e n d e n t .
t h a t n e o n a t a l th y m e c to m y r e s u l t s
n o t c o m p le te ly e lim in a te
T -c e lls
Tada e t a l
(2 1 ) fou n d
i n a d e c r e a s e d Ig E r e s p o n s e .
I t may
t h e Ig E r e s p o n s e b e c a u s e w i t h n e o n a t a l th y m e c ­
a p o s s i b i l i t y o f l e a v i n g some th y m u s c e l l s
e i t h e r due to f a i l u r e
thym us
The d a t a I h a v e c o l l e c t e d p r o v i d e s g o o d v e r i f i c a ­
t h a t t h e Ig E r e s p o n s e i s
tomy t h e r e i s
t h a t t h e IgE r e s p o n s e i s
in th e an im a l,
t o re m o v e a l l o f t h e g l a n d o r t o s e e d i n g o f
to o th e r o rg a n s b e f o r e b i r t h .
For th ese re a so n s,
th e nude
m o u s e o f f e r s a n i d e a l s y s t e m i n w h i c h t o o b s e r v e th y m u s d e p e n d e n c y
b e c a u s e t h i s a n i m a l l a c k s th y m u s c e l l s
w i t h 10 jug EA a n d 10 mg a lu m f a i l e d
from b i r t h .
N ude m i c e im m u n iz e d
to p ro d u c e any d e t e c t a b l e IgE ,
w h e r e a s , NLM p r o d u c e d Ig E i n h i g h q u a n t i t i e s .
Nude m i c e a l s o
fa ile d
to
p r o d u c e a n y I g E w hen im m u n iz e d w i t h 10 jug CAE m ix e d w i t h 10 mg a l u m .
T he f a i l u r e o f t h e n u d e m o u s e t o p r o d u c e Ig E p r o v i d e s a g o o d i n d i c a t i o n
o f t h e th y m u s d e p e n d e n c y o f t h e Ig E r e s p o n s e .
t h e n u d e m o u s e d o e s n o t p r o d u c e Ig E i s
To s a y t h a t t h e r e a s o n
t h a t i t h a s . n o thym us,
it
is
n e c e s s a r y t o show t h a t l a c k o f a th y m u s i s
th e o n ly re a s o n th e nude
m ouse f a i l e d
th is,
to p ro d u ce IgE .
To e s t a b l i s h
th y m u s g l a n d s w e r e
i m p l a n t e d i n n u d e m i c e b e f o r e t h e y w e r e im m u n iz e d w i t h 10 ju g EA a n d 10
mg a l u m .
If
B a lb /c m ouse,
th e n u d es w ere f i r s t
i m p l a n t e d w i t h a th y m u s f r o m a NLM o r .
t h e y w e r e a b l e t o p r o d u c e I g E ; w i t h o u t t h e th y m u s g r a f t s
fro m n o rm a l a n i m a l s ,
th e y , c o u l d n o t p r o d u c e a n y I g E .
in je c tio n s a lso re s to re d
t h e r e s p o n s e t o some n u d e s .
Thymus c e l l ,
P erh ap s i f
th e
31
m i c e w e r e t o h a v e m o re t i m e b e t w e e n th y m u s c e l l i n j e c t i o n a n d i m m u n iz a ­
t i o n , . t h e y w o u ld a l l h a v e r e s p o n d e d w i t h p r o d u c t i o n o f I g E .
d a ta ,
o n e may c o n c l u d e t h a t t h e Ig E r e s p o n s e i s
From t h e s e
th y m u s d e p e n d e n t .
I a t t e m p t e d t o i n d u c e Ig E p r o d u c t i o n i n n u d e m i c e . w i t h V i ,
a n t i g e n known t o b e th y m u s i n d e p e n d e n t f o r t h e IgM r e s p o n s e .
an
N e i t h e r . ■'■
n u d e m i c e n o r t h e i r NLM r e s p o n d e d w i t h p r o d u c t i o n o f I g E t o t h i s
a n tig e n .
I t ca n be c o n c lu d e d t h a t Vi does n o t in d u c e d e t e c t a b l e l e v e l s
o f IgE i n n u d e o r n o rm a l m ic e .
A lth o u g h n u d e m ice w ere u n a b le to p ro d u c e Ig E ,
th e y a ls o
la c k c e l l s
M o ta ( 6 ) h a s c a l l e d
c a p ab le of e l i c i t i n g
th ese c e l l s
c o m b i n e w i t h Ig E a n t i b o d y a n d ,
re le a se
th e p o s s i b i l i t y
th a t
a PCA r e a c t i o n w as c h e c k e d .
"m ast c e l l s . "
T h ey a r e
th e c e l l s w h ich
in th e p re s e n c e o f th e s p e c if ic
a n tig e n ,.
t h e p h a r m a c o l o g i c a l m e d i a t o r s o f t h e PCA r e a c t i o n , w h i c h i s
p rim a rily
s e r o t o n i n i n th e m ouse.
s e n s i t i z e d w ith serum c o l l e c t e d
PCA r e a c t i o n .
B o t h n u d e m i c e a n d CFW c o n t r o l s w e r e
f r o m a n i m a l s known t o b e p o s i t i v e f o r a..
B e c a u s e t h e n u d e m i c e r e a c t e d a s w e l l a s t h e CFW c o n t r o l s j
i t w as c o n c l u d e d t h a t t h e y do h a v e t h e c e l l s n e c e s s a r y f o r PCA r e a c t ­
i o n s . , I n a s m u c h a s n u d e m i c e l a c k a; f u n c t i o n a l th y m u s , , i t was, a l s o c o n - .
elu d ed
t h a t t h e o r i g i n , o f s u c h a c e l l . , i s : n o t . t h e . .thymus, a n d i t
th y m u s -d e p e n d e n t.c e ll ty p e .
• ..
.
is not a .
•.
S e v e r a l i n v e s t i g a t o r s h a v e show n t h a t a g e n t s , w h i c h a r e im m unosup­
p r e s s i v e ' . f o r . o t h e r t y p e s o f a n t i b o d y h a v e a n e n h a n c i n g e f f e c t o n Ig E
'
:
32
p ro d u c tio n .
19,
20).
ALS.
Tada and c o - w o rk e r s fo u n d t h i s
18,
( 1 1 ) show ed s i m i l a r r e s u l t s when m i c e
w e r e t r e a t e d w i t h 450 r a d X - i r r a d i a t i o n .
w e r e g i v e n ALS o r ''X-i r r a d i a t e d .
p ro d u c tio n o f IgE .
I n my e x p e r i m e n t , B a l b / c m i c e
N e i t h e r o f t h e s e show ed a n i n c r e a s e i n
S p l e n e c t o m y o f B a l b / c m i c e a l s o sh o w ed no e n h a n c i n g
In m ost c a s e s ,
Ig E p r o d u c t i o n .
stra te
(1 6 ,
W h i t e a n d Holm ( 2 5 ) f o u n d e n h a n c e m e n t o f Ig E i n r a t s g i v e n
K in d a n d M a c e d o - S o b r i n h o
e ffe c t.
to be t r u e in r a t s
im m u n o su p p ressiv e a g e n ts c a u s e d a d e c r e a s e i n
T he i m m u n o s u p p r e s s i v e a g e n t s I u s e d d i d n o t demon­
t h a t t h e th y m u s h a s a n e g a t i v e r e g u l a t o r y e f f e c t o n IgE p r o ­
d u c tio n .
It
B a lb /c m ice,
sh o u ld be n o te d
and a n o t h e r s t r a i n
B a lb /c m ice w ere s e l e c t e d
resp o n ses;
t h a t th e s e e x p e rim e n ts w ere done w ith
o f m i c e may show d i f f e r e n t r e s u l t s .
f o r t h e s e s t u d i e s b e c a u s e t h e y make good Ig E
p e r h a p s t h e y m ake g o o d r e s p o n s e s b e c a u s e t h e y l a c k s u p p r e s s
sor T -c e lls .
If
th is
is
th e c a se ,
o n e w o u l d n o t e x p e c t im m u n o s u p p r e s ­
s i v e t r e a t m e n t s t o e n h a n c e Ig E p r o d u c t i o n i n t h i s
stra in .
S uch
t r e a t m e n t m i g h t , h o w e v e r, e n h a n c e Ig E p r o d u c t i o n i n s t r a i n s w h ich ,
n o r m a l l y m ake p o o r Ig E r e s p o n s e s .
A p o t e n t i a t e d TgE r e s p o n s e h a s b e e n o b s e r v e d i n r a t s
n em ato d e i n f e c t i o n
(2 6 ,
2 7 ).
I n my s t u d y ,
fo llo w in g
B a lb /c m ice w ere i n o c u l a t e d
s u b c u t a n e o u s l y w i t h 600 l a r v a e o f t h e n e m a t o d e N i p p o s t r o n g y l u s
b ra silie n sis
t e n d a y s a f t e r i m m u n i z a t i o n w i t h 10 jig EA a n d 10 mg a l u m .
No m e a n i n g f u l p o t e n t i a t e d r e s p o n s e w as o b s e r v e d i n t h e m i c e t h a t
re ceiv ed
t h e worm i n f e c t i o n .
The PCA r e a c t i o n s ,
h o w e v e r , w e r e a much
33
d eep er b lu e th an in
th e c o n tr o l.a n im a ls .
T he t i t e r s
i n f e c t e d and th e c o n t r o l s w ere n o t m e a n i n g f u l.
th is
su b je c t is needed.
.
b e t w e e n t h e worm
M ore e x p e r i m e n t a t i o n o n
SUMMARY
, T h e r e g u l a t o r y e f f e c t s o f t h e th y m u s g l a n d on t h e p r o d u c t i o n o f
re a g in ic
i m m u n o g l o b u l i n E ( I g E ) were, s t u d i e d u s i n g c o n g e n i t a l l y
ath y m ic (n u d e) m ice,
m ice and B a l b / c m ic e .
p h e n o ty p ic a lIy norm al l i t t e r m a t e s
A n tig en s p e c i f i c
by t h e p a s s i v e c u t a n e o u s a n a p h y l a x i s
(NLM) o f n u d e
Ig E w as q u a n t i t a t e d
(PCA) a s s a y .
i n CFW m i c e
Alum , c o m p l e t e
F re u n d s a d j u v a n t and s a l i n e e x t r a c t o f B o r d e t e l l a p e r t u s s i s w ere u s e d
as a d ju v a n ts.
Nude m i c e f a i l e d
to p ro d u ce s p e c if ic
Ig E f o l l o w i n g b o t h
p r im a r y and s e c o n d a r y im m u n iz a tio n w i t h h en egg a lb u m in
asc a ris
groups.
e x t r a c t and c r u d e a s c a r i s
In c o n tr a s t,
(E A ) , c r u d e
e x t r a c t co u p led to d in itr o p h e n y l
NLM a n d B a l b / c m i c e made s t r o n g I g E r e s p o n s e s .
Nude m i c e i m p l a n t e d w i t h t h y m u s - g l a n d s f r o m n e o n a t a l B a l b / c o r NLM
d o n o r s m ade Ig E r e s p o n s e s e q u a l t o t h e r e s p o n s e s o f NLM m i c e .
d a ta in d ic a te
c e lls
th a t th ere is
These
an a b s o l u t e r e q u ir e m e n t f o r th y m u s -d e riv e d
i n th e f o r m a tio n by m ice o f s p e c i f i c I g E . ■
Nude m i c e w e r e p a s s i v e l y s e n s i t i z e d
p o sitiv e
i n t r a d e r m a l l y w i t h an IgE -
■
s e r u m ; w hen c h a l l e n g e d i n t r a v e n o u s l y w i t h s p e c i f i c a n t i g e n ,
t h e s e n u d e m i c e d e m o n s t r a t e d ' s t r o n g PCA r e a c t i o n s .
th e re fo re ,
It
is c lea r,
t h a t a l t h o u g h n u d e m i c e c a n n o t m ake s p e c i f i c Ig E ,. t h e y do,
h a v e t h e m e c h a n i s m n e c e s s a r y t o e l i c i t PCA r e a c t i o n s ' ;
A t t e m p t s w e r e m ade t o show a n e g a t i v e r e g u l a t o r y e f f e c t o f . t h e th y m u s o n I g E p r o d u c t i o n b y u s i n g s p l e n e c t o m i z e d ,
a n t i l y m p h o c y t e s e r u m - t r e a t e d mice-.-
'60
•
C o-ir r a d ia te d , o r
No e n h a n c e m e n t o f Ig E p r o d u c t i o n
•
35
w as o b s e r v e d i n t h e s e e x p e r i m e n t s .
o th e rs u sin g r a t s ,
I n c o n t r a s t t o r e s u l t s o b t a i n e d by-
i n f e c t i o n o f im m u n iz e d m i c e w i t h t h e n e m a t o d e
N ip p o stro n g y lu s b r a s i l i e n s i s
r,’
d id n o t p o t e n t i a t e
sp e c ific
.
Ig E p r o d u c t i o n .
BIBLIOGRAPHY
1.
I s h i z a k a , K „, a n d T . I s h i z a k a .
1970.
T he s i g n i f i c a n c e o f immuno­
g l o b u l i n E i n r e a g i n i c h y p e r s e n s i t i v i t y . . Ann. A l l e r g y 28: 1 8 9 - 2 0 2 .
2.
S t a n w o r t h , D. R .
1971.
Im m u n o g lo b u lin E ( r e a g i n )
N a tu re .
1971. 2 3 3 :3 1 0 -3 1 6 .
3.
M o ta , I . , a n d A. P e r i n i .
1970.
S u s c e p t i b l e hom ocyt o t r o p i c a n t i ­
body in .th e g u in e a p i g .
L ife S c i.
9 :9 2 3 -9 3 0 .
4.
M o ta ,
5.
M o ta , I . , D. Wong a n d E . H. S a d u n .
1 9 6 8 . M ouse H o m o c y t o t f o p i c
an tib o d ie s.
I.
S p e c i f i c d i f f e r e n t i a t i o n b e tw e e n m ouse 7 s^ and
re a g in -lik e an tib o d ie s.
L ife Sci P t. 2.
7 :1 2 8 9 -1 2 9 3 .
6.
M o ta, I .
1964.
The m ech an ism o f a n a p h y l a x i s .
I.
P r o d u c t i o n and
b i o l o g i c a l p r o p e r t i e s o f 'm a s t c e l l s e n s i t i z i n g 1 a n tib o d y .
Im m u n o lo g y 7 : 6 8 1 - 6 9 9 .
7.
R e v e l t e l l a , R . , and Z . O v a r y .
1969.
R eag in ic a n tib o d y in d i f f e r e n t
m ouse s t r a i n s .
Im m u n o lo g y 1 7 : 4 5 - 5 4 .
8.
C l a u s e n , C. R . , J . Munoz a n d R 1 K. B e rg m a n .
1970.
A re a g in ic
o f a n tib o d y s t i m u l a t e d by e x t r a c t s o f B o r d e t e l l a p e r t u s s i s in
in b re d s t r a i n s o f m ice.
J.
Im m u n o l. . 1 0 4 : 3 1 2 - 3 1 9 .
9.
C l a u s e n , C. R . , J . Munoz a n d R. K. B e rg m a n .
1969.
R e a g in ic -ty p e
a n tib o d y i n m ice s t i m u l a t e d by e x t r a c t s o f B b r d e t e l l a p e r t u s s i s .
J.
Im m u n o l.
1 0 3 :7 6 8 -7 7 7 .
10.
I.
1961.
M ast c e l l
ly tic
a n tib o d y .
N a tu re .
and a l l e r g y .
1 9 2 :4 8 0 8 -4 8 1 4 .
ty p e
B a r t h o l o m a e u s , W. N . , a n d D. K e a s t .
1972.
R e a g in ic a n tib o d y to
tum our and a l l o a n t i g e n s i n m i c e .
N a tu r e -N e W B i o .
123 9 :2 0 6 -2 0 7 .
11.
K i n d , L . S . ,.and B.. M a c e d o - S o b r i n h o .
1973. R e a g in ic . a n tib o d y form a1
t i o r i i n m i c e i n j e c t e d w i t h r a b b i t a n t i - m o u s e , t h y m o c y t e s e r u m ,. I n t .
. A r c h . . A l l e r g y A ppI .
Im m u n o l.
4 5 :7 8 0 -7 8 8 .
12.
S t a n w o r t h , D. R . 1 9 7 3 . . I m m e d i a t e h y p e r s e n s i t i v i t y , ' t h e m o l e c u l a r .
b a s i s o f th e a l l e r g i c re s p o n s e , p . 170. N o rth -H o lla n d P u b lis h in g
Com pany, A m s te rd a m a n d L o n d o n .
13.
P e r i n i , A . , . a n d I . M ota. ,1 9 7 3 .
T h e p r o d u c t i o n o f I g E a n d IgG
a n t i b o d i e s . i n ' g u i n e a - p i g s im m u n iz e d w i t h . a n t i g e n , a n d b a c t e r i a l
iip o p o ly sa c c h a rid e s.
Im m u n o lo g y
2 5 :2 9 7 -3 0 5 .
37
14.
K a t z , D. H . , T . Hamzoka a n d B. B e n a c e r r a f .
1973.
In d u c tio n o f
im m u n o lo g ic a l t o l e r a n c e i n bone m a rr o w -d e r iv e d ly p m h o c y te s o f th e
IgE a n t i b o d y c l a s s .
P r o c . N a t. A cad. S c i .
7 0 :2 7 7 6 -2 7 8 0 .
15.
S a u e r b r o n n M a i a , L . C,., N. M. V as and, E . M. V a s .
1974. • E f f e c ts o f
s o l u b l e a n t i g e n on IgE r e s p o n s e s i n t h e m ouse.
In t.
A rch.
A lle r g y . , 4 6 :3 3 9 -3 4 4 .
16.
O kum ura, K . , a n d T . T a d a .
1971.
R e g u l a t i o n o f hom ocyt o t r o p i c a n t i ­
body fo rm a tio n in th e r a t .
III.
E f f e c t o f th y m e c to m y and
sp le n e c to m y .
J . I m m u n o l.
1 0 6 :1 0 1 9 -1 0 2 5 .
17.
M ic h a e l, J . G . , and I . L. B e r n s t e i n .
r e a g i n i c a n tib o d y fo rm a tio n in m ice.
18.
T a d a , T . , M. T a n i g u c h i a n d K. O k u m u ra .
1971.
cy to t r e p i c a n tib o d y fo rm a tio n in th e r a t . I I .
X -i r r a d i a t i o n .
J . Im m u n o l.
1 0 6 :1 0 1 2 -1 0 1 8 .
19.
T a n i g u c h i , ' M ., . and T. T a d a .
1971.
R e g u la tio n o f h o m o cy to tro p ic
a n tib o d y fo rm a tio n in th e r a t .
I V . ■ E f f e c t o f v a r i o u s .im munosup­
p re s siv e d ru g s.
J . Im m u n o l.
1 0 7 :5 7 9 -5 8 5 .
1973.
Thymus d e p e n d e n c e o f
J . Im m u n o l. ' H I : 1 6 0 0 - 1 6 0 1 .
R e g u l a t i o n o f.h o m o ­
E ffect of
2 0 . . Okumura,. K . , a n d T . . T a d a .
1974.
E f f e c t o f a n t i th y m o c y te serum on
r e a g i n i c a n tib o d y fo rm a tio n in th e r a t .
Im m u n o lo g y . 2 6 : 2 5 7 - 2 6 8 .
21.
T a d a , - T . , a n d K. O k u m u ra .
1971.
R e g u la tio n , o f hom ocyt o t r o p i c
a n tib o d y fo rm a tio n in th e r a t .
I.
F e e d - b a c k r e g u l a t i o n by
p a s s iv e ly a d m in iste re d a n tib o d y . .J .
Im m u n o l.
106:1 0 0 2 -1 0 1 1 .
22.
O kum ura, K . , a n d T . T a d a .
1971.
R e g u l a t i o n of, h o m o c y t o t r o p i c
a n tib o d y fo rm a tio n in th e r a t .
V I.
In h ib ito ry e ffe c t of
th y m o cy tes on th e h o m o c y to tro p ic a n tib o d y r e s p o n s e . . J .
Im m u n o l.
107:1682-1689.
23.
24.
T a d a , T .^ a n d K. O kum ura .
1 9 7 1 .. R e g u l a t i o n o f h o m o c y to tr o p ic
a n tib o d y .fo rm a tio n in th e r a t .
V.
C e ll c o o p e ra tio n in ' th e . a n t i ­
hap te n h o m o c y to tro p ic a n tib o d y r e s p o n s e .
J . Im m u n o l,
1 0 7 :1 1 3 7 -.
. 1145.
• .
'V"
O kum ura, K,., a n d . T . T a d a .
1974.
R e g u la tio n o f h o m o cy to tro p ic
a n tib o d y fo rm a tio n in th e r a t .
I X 0 F u r t h e r . c h a r a c t e r i z a t i o n o f..;,
t h e a n t i g e n - s p e c i f i c i n h i b i t o r y T c e l l - f a c t o r .- i n . h a p t e n - s p e c i f i c ’
h o m o cy to tro p ic a n tib o d y resp o n se^
J . . Im m u n o l... 1 1 2 : 7 8 3 - 7 9 1 »
38
25.
W h i t e , G. J . , a n d M. S . Holm .
1973.
In d u c tio n o f re a g in
s y n t h e s i s i n t h e r a t w i t h a s s o c i a t e d and d i s s o c i a t e d h em o cy an in :
E f f e c t o f a n tily m p h o c y te serum .
J . Im m u n o l.
1 1 0 :3 2 7 -3 3 4 .
26.
B l o c h , K. J . , J . L . Ohman, J . W a l t i n a n d , R. W. C y g a n .
1973.
P o te n tia te d re a g in resp o n se:
i n i t i a t i o n w ith m in u te doses o f
a n t i g e n a n d a lu m f o l l o w e d b y i n f e c t i o n w i t h N i p p o s t r o n g y l u s
b ra silie n sis. J.
Im m u n o l.
1 1 0 :1 9 7 -2 0 4 .
27.
J a r r e t t , E . E . , D. H e n d e r s o n , P . R i l e y a n d R. G. W h i t e .
1972.
T he e f f e c t o f v a r i o u s a d j u v a n t r e g i m e n s a n d o f n e m a t o d e i n f e c t i o n
on th e r e a g i n i c a n tib o d y r e s p o n s e to egg a lb u m in i n t h e r a t .
In t.
A rch.
A lle rg y .
4 2 :7 7 5 -7 8 1 .
28.
C sa b a , G ., L . S u r j a n , J . F i s c h e r , J . K is s and I . T o r o .
19 6 9 ;
On t h e m e c h a n i s m o f m a s t c e l l f o r m a t i o n .
A c ta b i o l . A cad. S c i .
hung.
2 0 :5 7 -6 3 .
29.
G i n s b e r g , H.
1963.
The i n v i t r o d i f f e r e n t i a t i o n and c u l t u r e o f
n o r m a l m a s t c e l l s fro m m ouse th y m u s.
A nn. N 0 Y . A c a d 0 S c i .
1 0 3 :2 0 -2 5 .
30.
V i k l i c k y , V . , P . Sim a a n d H. P r i t c h a r d .
1973.
On t h e o r i g i n o f
m ast c e l l s in a d u lt l i f e .
F o lia . B io l. P rague.
1 9 :2 4 7 -2 5 1 .
31.
R ygaard, J .
m u tan t nude.
32.
B i I l i n g h a m , R. E . , a n d W. K. S i l v e r s .
tra n sp la n ta tio n .
P re n tic e -H a ll, In c .,
33.
T a n d y , M ., R. J . T r a p a n i , M. E . W e b s t e r a n d F . G. J a r v i s .
1963.
I m m u n o l o g ic a l , p r o p e r t i e s o f V i a n t i g e n i s o l a t e d , b y c h e m i c a l
f r a c t i o n a t i o n and by e l e c t r o p h o r e s i s .
T e x . R e p . B i o l . Med.
2 1 :2 1 4 -2 2 9 .
34.
W e stp h a I , 0 . , 0 . L u d e r i t z and F . B i s t e r .
1952,
e x t r a c t i o n von b a c t e r i e n m it P h en o l/W ass e r .
Z.
7 :1 4 8 -1 5 5 .
35.
W e s t p h a l , 0 . , a n d A. C 0 T o d d .
b r a s i l i e n s i s to th e m ouse.
J.
36.
P i l g r i m , I . , a n d K. DeOme.
1955.
In tra p e rito n e a l p e n to b a rb ito l
a n e s th e s ia in m ice.
E x p . Med.
S u rg .
1 3 :4 0 1 -4 0 3 .
1973.
Thymus a n d s e l f :
I m m u n o b i o lo g y o f t h e m ouse
F . A. D. L . , C o p e n h a g e n , D e n m a rk .
1971.
The im m u n o b io lo g y o f
E n g le w o o d C l i f f s , N. J .
U ber dee
n atu rfo rsc h
1966.
A d a p ta tio n o f N ip p o stro n g y Iu s
P a ra site .
5 2 :2 3 3 -2 3 6 .
1
1T378
Sch59
cop. 2
DATE
S c h n e l l e r , R h e t Lucy
R e g u la to ry e f f e c t s o f
t h e t h y m u s o n t h e Ig E
re s p o n s e o f m ice.
IS SU ED TO
I
ScJiS
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