Regulation of IgE antibody responses in mice
by Judith Estelle Klein Manning
A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF
PHILOSOPHY In Microbiology
Montana State University
© Copyright by Judith Estelle Klein Manning (1977)
Abstract:
The validity of the heterologous (rat) passive cutaneous anaphylaxis (PCA) test to assess mouse IgE
responses was confirmed by comparing the 72 hr homologous PCA test with the 24 hr heterologous
PCA test, and by the diminution in titer when undiluted serum was heated at 56°C for 90 min. Earlier
work, showing that dilution of the serum prior to heating abrogated the effect of heating on the skin
sensitizing activity of IgE, was confirmed. Treatment with an anti-helminthic agent improved the
homologous, 72 hr PCA reactions in certain mice probably due to a decline In endogenous anti-worm
IgE levels as a result of the elimination of pinworms.- Neonatally initiated anti-μ treatment suppressed
subsequent IgE responses to Infection with N. brasiliensis showing that IgE forming cells are derived
from the same precursors I cell(s) as are IgM, IgG and IgA forming cells.
The induction and regulation of IgE responses in Balb/c mice were studied, using the 24 hr
heterologous PCA test. Immunization with one intraperitonea 1 (IP) injection of either 1 ù, 10 μg, 100
μg or 1000 μg doses of ovalbumin (OVA), each with 1 mg alum, Induced high PCA and passive
hemagglutination (PHA) antibody titers that persisted for at least 56 days. At 100 μg and 1000 μg
doses, a lag in the peak IgE response was seen. When B_. pertussis vaccine was used instead of alum, a
PHA antibody was Induced but IgE antibody could not be detected. Immunization by one IP injection
of DNPn c-OVA + I mg alum Induced a transient anti-DNP IgE response anda persistent anti-OVA IgE
response. In contrast, Immunization with DNPgif-OVA induced a persistent anti-DNP IgE response but
no ant I-OVA IgE could be detected. Immunization with multiple, IP injections of normal rabbit serum
and OVA, without adjuvant, Induced an IgE antibody response.
When mice, primed with one IP Injection of 10μg OVA + I mg alum, were given 3, intravenous
injections of 100 μg OVA on days 3, 5, and 7 their PCA antibody titers and the number of IgE forming
cells In the spleen declined to undetectable levels by day 16, whereas their PHA antibody titers did not
change. Secondary IgE responses were also suppressed by this treatment. The route of administration
of this treatment was shown to be an important factor In producing this effect. This suppression could
be transferred to normal animals by spleen cells from suppressed animals, suggesting that this
treatment Induced suppressor cells. REGULATION OF IgE ANTIBODY RESPONSES IN MICE
by
JUDITH ESTELLE KLEIN MANNING
A th e s is sub m itted In p a r t i a l f u l f i l l m e n t
o f th e requirem ents f o r th e degree
of
DOCTOR OF PHILOSOPHY
In
M ic ro b io lo g y
Approved:
ys
a
C h a irp e rs o n , G raduate Committee
7 ft
Head^ M ajor Department
G raduate Dean
MONTANA STATE UNIVERSITY
Bozeman, Montana
May, 1977
m
ACKNOWLEDGMENTS
The m ajor p o rtio n o f th e re se a rch f o r t h is th e s is was done o f f
campus.
T h is was a unique and e d if y in g e x p e rie n c e and I am g r a te ­
f u l t o a l l those whose c o o p e ra tio n made i t p o s s ib le .
I
w ish t o extend a s p e c ia l thanks t o D r. Norman D. Reed f o r
co n se n tin g t o be my m ajor p ro fe s s o r under th ese c o n d itio n s and f o r
h is e x te n s iv e support In th e supply o f rese a rch m a t e r ia ls .
I am
g r a t e f u l , a ls o , f o r th e many e x tr a th in g s th a t he d id so th a t th is
arrangem ent could be s u c c e s s fu l.
I w is h , a ls o , t o acknowledge th e
e x c e lle n t p ro fe s s io n a l example s e t by him .
He is a s c h o la r and a
good s c i e n t i s t .
I wish t o thank D r. David T . Berman, o f th e Departm ent o f V e t­
e r in a r y S c ie n c e , U n iv e r s ity o f W is co n sin , Madison f o r p ro v id in g
la b o r a to ry space and equipment th a t a llo w e d me to pursue my th e s is
re s e a rc h and f o r h is c o o p e ra tio n , f l e x i b i l i t y and h e lp fu l d is c u s ­
sio n s d u rin g th e tim e I was employed by him .
I w ish t o th an k my husband. D r. Dean D. Manning, f o r a ls o p ro ­
v id in g
la b o ra to ry space and equipment d u rin g th e f i n a l stages o f my
s tu d y , f o r p ro v id in g th e a n tis e ru m f o r our c o lla b o r a t iv e study and
f o r h is
lo y a l support and encouragement.
T h is re se a rch was supported by U .S . P u b lic H e a lth S e rv ic e
g ra n ts Al
10384, Al
12854 and CA 17531.
Iv
TABLE OF CONTENTS
Page
V I T A . . . ; ..............................................................................................................
Ii
ACKNOWLEDGMENTS....... ....................................................................................................M i
TABLE OF CONTENTS.....................................................................................
LIST OF TABLES..................................
iv
v ll
ABSTRACT.................................................................................................
Ix
INTRODUCTION...................................................................................................................
S tatem en t o f T h e s is ........................................................................................
I
9
MATERIALS AND METHODS...........................................................................
10
A n im a ls ........................................................................................................
10
A n tig e n s .....................................................................................
A d ju v a n ts ..............................................................................................................
10
II
12
Im m u n izatio n s ..................................................................
Serum c o l l e c t i o n ................................................................
12
A s s ay s .....................................................................................
13
A n tI-M tr e a tm e n t......................................................................
15
Worm I n f e c t i o n ......................................................................................
I&
RESULTS.........................................................................
In d u c tio n o f h cm ocytotropic a n tib o d ie s in m i c e . . . . . .............
17
The h e te ro lo g o u s ( r a t ) PCA assay f o r mouse Ig E ....... .................
17
E f f e c t o f an a n t I -h e lm in th ag en t on th e homologous PCA
r e a c tio n in ICR m ic e ........................................................................
18
V
Page
Suppression o f 8gE a n tib o d y fo rm a tio n by tre a tm e n t w ith
a n t i -n a n tis e ru m ........................................................................................ 20
E f f e c t o f m u lt ip le in je c tio n s o f MRS on subsequent IgE
a n tib o d y fo rm a tio n t o im m unization w ith ovalbum in and
alu m ....................................
24
K in e tic s o f th e PCA and PHA responses o f mice immunized
w ith 10 Hg OVA + I mg alum ................................................................. 25
Dose response stud I e s . . . . . ...................................................................... 28
IgE responses t o DNP-conjugates o f o v a lb u m in ........................... 29
Immune response t o v a rio u s doses o f ovalbum in using B.
p e rtu s s is as a d ju v a n t...........................................................................
35
PCA a n tib o d y responses t o o th e r a n tig e n s using alum
as a d ju v a n t ..................................................................................................... 35
In d u c tio n o f IgE responses by m u lt ip le in je c tio n s o f
a n tig e n w ith o u t a d ju v a n t................................
35
C o n firm a tio n o f p re v io u s f in d in g s ...........................
40
M aintenance o f d e s e n s it iz a t io n ...........................................................
44
E f f e c t o f d e s e n s itiz a t io n on IgE a n tib o d y -s e c re tin g
c e l I s ..................................
44
E f f e c t o f g iv in g m u lt ip le , h ig h doses o f a n tig e n w it h ­
o u t alum by th e IP r o u te ..................................................
45
E f f e c t o f g iv in g an o p tim a lly immunogenic dose o f a n t i ­
gen as a d e s e n s itiz a t io n tr e a tm e n t......................................
46
A comparison o f m u lt ip le and s in g le tre a tm e n ts f o r
d e s e n s i t i z a t io n . ...........................................................................................46
E f f e c t o f g iv in g o th e r p ro te in s o r h a p te n -p ro te in con­
ju g a te s In tr a v e n o u s ly ............. .................................
53
v$
Page
E f f e c t o f d e s e n s itiz a t io n tre a tm e n t on secondary
IgE responses............ ........................... ................................................. . .
53.
S up p ressive e f f e c t o f sp leen c e l l s from s e n s it iz e d d e s e n s itiz e d a n im a ls ...........................................................................
56
DISCUSSION.........................
61
SUMMARY.......................
74
LITERATURE CITED................................................................................
76
LIST OF TABLES
T a b le
Page
I
A comparison between th e homologous and h e te r o lo ­
gous p a s s iv e cutaneous a n a p h y la x is (PCA) te s ts
f o r mouse IgE a n tib o d y a g a in s t ovalbum in................................... 21
11
E f f e c t o f h e a t on p a s s iv e cutaneous a n a p h y la x is
(PCA) t i t e r s o f mouse a n tis e ru m ....................... ................................22
III
IV
V
Vl
Vl I
E f f e c t o f an a n ti-h e lm in t h a g e n t on th e homolo­
gous p a s s iv e cutaneous a n a p h y la x is (PCA)r e a c t io n ..................... 23
IgE a n t I -worm responses in m ice t r e a te d from b i r t h
w ith a n t i a n t i s e r u m ............................... ............................ . . . . . . 2 6
E f f e c t o f n e o n a ta lI y I n i t i a t e d normal r a b b it serum
(NRS) tre a tm e n t on a n t I -o v a lbumin responses in
m ic e ...........................
27
Long term m aintenance o f immune responses t o alum
adsorbed ovalbum in In B a lb /c m ic e .....................................
30
IgE responses t o v a rio u s doses o f ovalbum in adsorbed
t o alum ....................
.31
V lll
P as s iv e h e m a g g lu tin a tio n a n tib o d y responses t o
v a rio u s doses o f ovalbum in adsorbed to alum .............. . . . . . . . 3 2
IX
IgE a n t I -h a p te n and a n t i - c a r r i e r responses o f
B a lb /c m ice immunized w ith l i g h t l y d I n l t r o p h e n y la te d ovalbum in (DNPQ g -O V A )............... ; ........................ . . . 3 6
X
Xl
IgE a n t i h a p te n -c a r r ie r c o n ju g a te , a n ti-h a p te n and
a n t i - c a r r i e r responses o f B a lb /c mice Immunized
w ith h e a v ily d i n itro p h e n y Ia te d ovalbumin
(DNP24 - O V A ) . . . .......................................
A n tI -o v a lbtmiln responses In mice immunized w ith
v a rio u s doses o f ovalbum in and B o r t e t e lla
p e r t u s s i s . ................................................
..3 7
38
vlH
XH
Immune responses o f m ice immunized by neonatal Iy
i n i t i a t e d , m u lt ip le , in je c tio n s o f normal
r a b b it serum (MRS) w ith o u t a d ju v a n t ............................... ............. 4 1
X lll
IgE responses o f m ice immunized by a d u lt i n i t i a t e d
m u lt ip le in je c tio n s o f normal r a b b it serum (NRS)
w ith o u t a d ju v a n t ........................................................................................42
XIV
E f f e c t o f in tra v e n o u s , m u lt ip le in je c tio n s o f
s o lu b le a n tig e n on th e a n t I -o v a I bumin responses
o f m ice s e n s itiz e d p re v io u s ly w ith an I „pe in ­
j e c t i o n o f a n tig e n + alum .............................. .................... ............. .. .4 7
XV
E f f e c t o f in tra v e n o u s , m u lt ip le in je c tio n s o f
s o lu b le ovalbum in on IgE r e s p o n s e s . . . . ......................... ............. 48
XVI
H eterologous a d o p tiv e cutaneous a n ap h y la x is
re a c tio n s in s e n s itiz e d and sensi t I z e d -d e sens i 11 zed m ic e ......................................... ; ................................................49
X V II
E ffe c t o f m u lt ip le , in tr a p e r ito n e a I in je c tio n s
o f s o lu b le a n tig e n on a n t I -o v a I bumin responses
a t day 16 ..........; ............. .................................................................... .. .50
X V III
E ffe c t o f g iv in g m u lt ip le in tra ve n o u s in je c tio n ,
o f low doses o f s o lu b le a n tig e n on d e s e n s it iz a t io n ............ 51
XIX
A comparison o f th e e f f e c t o f m u lt ip le in je c tio n s
and a s in g le in je c t io n o f ovalbum in on desens I t I z a t i o n . . . . ................................................ .................... ........................... .52
XX
E f f e c t o f a d m in is te rin g o th e r p ro te in s o r h a p te n p r o te in c o n ju g ates in tra v e n o u s ly on th e a n t i ­
ovalbum in PCA responses o f m ice p re v io u s ly
s e n s itiz e d w ith a I urn-adsorbed o v a lb u m in ............................. . . . 5 4
XXI
X X II
Secondary responses o f m ice d e s e n s itiz e d w ith
m u l t i p l e , h ig h doses o f ovalbum in given
in tra v e n o u s ly .............. .....................................................................5 7
S uppressive e f f e c t s o f s p lee n c e l l s from desen­
s i t i z e d anim als on IgE a n t I -o v a I bumin responses
when tr a n s fe r r e d t o norma I , syngeneic m i c e . . . . . . ................ 60
ABSTRACT
The v a l i d i t y o f th e h e te ro lo g o u s ( r a t ) p a ss iv e cutaneous ana­
p h y la x is (PCA) t e s t t o assess mouse IgE responses was confirm ed by
comparing th e 72 hr homologous PCA t e s t w ith th e 24 h r h eterolo gous
PCA t e s t , and by th e d im in u tio n In t i t e r when u n d ilu te d serum was
heated a t 56 eC fo r. 90 m in.
E a r l i e r w o rk, showing t h a t d i l u t i o n o f
th e serum p r i o r to h e a tin g a b ro g ated th e e f f e c t o f h e a tin g on th e
s k in s e n s it iz in g a c t i v i t y o f Ig E , was c o n firm e d . Treatm en t w ith
an a n t i- h e lm in t h ic a g en t improved th e homologous, 72 h r PCA re a c ­
tio n s in c e r t a in m ice p ro b ab ly due t o a d e c lin e In endogenous an­
ti-w o rm IgE le v e ls as a r e s u lt o f th e e lim in a tio n o f pinworms,N e o n a ta lly i n i t i a t e d a n t I
tre a tm e n t suppressed subsequent
IgE responses t o In fe c t io n w ith N. bras 11 lens is showing th a t IgE
fo rm in g c e l l s a r e d e riv e d from th e same p recu rso rs I c e l l ( s ) as a re
IgM, IgG and IgA fo rm in g c e l l s .
The In d u c tio n and r e g u la tio n o f IgE responses in B a lb /c mice
w ere s tu d ie d , using th e 24 h r h e te ro lo g o u s PCA t e s t .
Im m unization
w ith one in tr a p e r ito n e a I ( IP ) in je c t io n o f e it h e r I fig , IO fig,
100 fig o r 1000 fig doses o f ovalbum in (OVA), each w ith I mg alum .
Induced high PCA and p a s s iv e h e m a g g lu tin a tio n (PHA) a n tib o d y t i ­
t e r s th a t p e rs is te d f o r a t le a s t $6 d ays. A t 100 fig and 1000 fig
doses, a lag in th e peak IgE response was seen. When Bi, p e rtu s s is
v a c c in e was used In s te a d o f alum , a PHA a n tib o d y was Induced but
IgE a n tib o d y could not be d e te c te d .
Im m unization by one IP in je c ­
t io n o f DNPn c -OVA + I mg alum Induced a t r a n s ie n t a n t i -DNP IgE
response a n d a p e r s is te n t anti-O V A IgE response.
In c o n tr a s t ,
Im m unization w ith DNPg^-OVA induced a pars I s t e n t . a n t i -DNP IgE r e ­
sponse but no a n t I -OVA IgE could be d e te c te d .
Im m unization w ith
m u l t i p l e , IP in je c tio n s o f normal r a b b it serum and OVA, w ith o u t- ,
a d ju v a n t. Induced an IgE a n tib o d y response.
When m ice , prim ed w ith one IP In je c t io n o f 10 fig OVA + I mg
alu m , were g iv e n 3 , in tra ve n o u s In je c tio n s o f 100 fig OVA on days
3 , 5 , and 7 t h e i r PCA a n tib o d y t i t e r s and th e number o f IgE form ing
c e l l s in th e sp leen d e c lin e d to u n d e te c ta b le le v e ls by day 16 ,
whereas t h e i r PHA a n tib o d y t i t e r s d id n o t change.
Secondary IgE
responses w ere a ls o suppressed by t h i s tre a tm e n t. The ro u te o f
a d m in is tr a tio n o f t h i s tre a tm e n t was shown t o be an im p o rtan t f a c ­
t o r in producing t h is e f f e c t . T h is suppression could be tr a n s fe rr e d
t o normal a n im als by s p le e n c e l l s from suppressed a n im a ls , suggest­
ing th a t t h is tre a tm e n t induced suppressor c e l l s .
INTRODUCTION
In humans, th e ’ 's k in s e n s it iz in g f a c t o r " p re se n t in th e serum
o f hay fe v e r p a tie n ts has been c l e a r l y
a d is tin c t
i d e n t i f i e d as b elongin g to
Immunoglobulin c la s s , d esig n a te d as IgE f o r
its a b ilit y
t o bind p u r i f i e d ragweed a n tig e n (a n tig e n E) in ra d io im m u n o e le c trc p h o re s is ( I ) .
O ther names f o r t h is a n tib o d y in c lu d e :
re a g in , homo-
c y t o tr o p ic a n tib o d y , and P-K a n tib o d y .
IgE d i f f e r s
from o th e r known Immunoglobulins in physico ch em ical,
a n tig e n ic and b io lo g ic a l p r o p e r tie s .
I t c o n ta in s
12% c a rb o h y d ra te ,
has a s e d im e n ta tio n c o e f f i c i e n t o f 8 . OS and a m o le c u la r w eight o f
a p p ro x im a te ly 1 9 0 ,0 0 0 .
It
Is p re s e n t In serum a t much lower concen­
t r a t io n s than o th e r im m unoglobulins, w ith an average c o n c e n tra tio n
o f 0 .3 n g /m l.
E le v a te d serum le v e ls a re p re se n t in in d iv id u a ls who
a r e a to p ic or in fe c te d w ith worms.
E p s ilo n heavy ch ain s have unique
a n tig e n ic d e te rm in a n ts and do not possess any o f th e m ajor a n tig e n ic
d e te rm in a n ts p re s e n t in th e o th e r fo u r known Immunoglobulin classes
(I).
The most d i s t i n c t i v e p ro p e rty o f IgE is i t s a b i l i t y
to bind to
mast c e l l s f o r up t o s e v e ra l weeks a t l e a s t , w ith th e re le a s e o f
h is ta m in e and o th e r p h a rm a c o lo g ic a lly a c t iv e substances from these
c e l l s upon in t e r a c t io n o f th e c e l l bound IgE w ith a n tig e n ( 1 , 2 ) .
T h is p ro p e rty is th e b a s is f o r most o f th e assays f o r t h i s a n tib o d y
in anim als and* u n t i l r e c e n t ly ,
In humans.
The e f f e c t s o f h is ta m in e
2
and o th e r pharm acological Iy a c t iv e substances a re th e cause o f th e
symptoms exp e rien ce d by a l l e r g i c
in d iv id u a ls .
T h is c e l l b in d in g
a c t i v i t y can be d e stro yed by h e a tin g a t 56°C f o r 2 -4 hours ( I ,
The dog is th e o n ly o th e r sp ecies
2 ).
in which spontaneous a l l e r ­
g ic symptoms s im ila r to those found in hay fe v e r p a tie n ts have been
o b served.
d e te c te d
A s k in s e n s it iz in g f a c t o r s im ila r to human IgE has been
in serum from th e s e anim als ( 3 ) .
However, heat l a b i l e ,
h o m o cytotropic a n tib o d ie s which rem ain bound to s k in mast c e l l s f o r
long p e rio d s o f tim e ( i . e . ,
72 hours) can be d e l ib e r a t e ly induced
in many sp ec ie s and a re con sid ered th e anim al c o u n te rp a rt to human
IgE ( I ,
2, 4 ).
These a n tib o d ie s have been d e te c te d in many o th e r
mammals in c lu d in g monkeys, dogs, m ice , r a t s , r a b b it s , c a t t l e , sheep
p ig s and guinea pigs ( 4 ) , and in a m a rs u p ia l, th e quokka ( 5 ) .
The
p hysicochem ical p r o p e rtie s o f th e s e hom ocytotropic a n tib o d ie s a re
s im ila r to human IgE ( I ,
2 , 4 ) and in some cases, c r o s s -r e a c t im­
m u nological Iy w ith anti-hum an IgE ( 6 , 7 ) .
.
The d e lib e r a t e in d u c tio n o f IgE (c o n tra s te d w ith th e " a to p ic "
c o n d itio n ) re q u ire s a s u it a b le com bination o f A n tig e n , dose o f an­
t ig e n , a d ju v a n t,
im m unization schedule and species ( 4 ) .
It
Is gen­
e r a l l y agreed th a t f o r most a n tig e n s some a d ju v a n t is re q u ire d f o r
IgE in d u c tio n .
A d ju van ts commonly used t o induce IgE responses a re
aluminum h y d ro xid e g el
(alum ) and v a c c in es o r s o lu b le e x tr a c ts o f
B o r d e te ila p e rtu s s is
(4 ).
and concanavaI i n A (con A)
B a c te r ia l
I Spopolysaceharides (LPS)
( 8 ) have a ls o been used, as w e ll as
F re u n d 9S com plete a d ju v a n t, b u t th e l a t t e r
a d ju v a n t f o r
IgE (4 )„
ju v a n t -s p e c ie s
(4 )
is not co n sid e re d a good .
E xperim en tal o b s e rv a tio n s on th e v a rio u s a d ­
in te r a c tio n s suggest t h a t th e mechanisms r e g u la tin g
IgE p ro d u c tio n may d i f f e r both f o r sp ec ie s and f o r a d ju v a n t ( 4 ) .
One a p p a re n t e x c e p tio n t o t h i s requirem ent f o r a d ju v a n t is th e
in d u c tio n o f a n t i- h e lm in t h ic
Ig E .
Some in v e s tig a to r s b e lie v e th a t
l i v e worm in fe c t io n may p ro v id e a d ju v a n t a c tio n because worm ex­
t r a c t s , by th e m s elv es , cannot induce th e IgE response ( 4 ) .
Another
p o s s ib le e x c e p tio n is th e In d u c tio n o f IgE a n tib o d ie s t o h e te r o lo ­
gous serum by im m unization w ith one in tr a p e r ito n e a I ( I P )
in je c t io n
o f h e te ro lo g o u s serum in th e form o f a n ti-ly m p h o c y te serum ( 9 ) .
E xp erim en tal e vid en ce has f i r m l y e s ta b lis h e d th a t c o lla b o r a tio n
between bone-marrow d e riv e d (B) and th ym us-derived (T )
lymphocytes
is re q u ire d f o r th e in d u c tio n o f c e r t a in a n tib o d y responses ( 10) .
The im portance o f a t h i r d c e l l ty p e , th e macrophage, has a ls o been
reco g n ize d ( 1 1 ) .
S e v e ra l lin e s o f e vid en ce dem onstrate th a t s im ila r
c e l l u l a r events a r e a ls o necessary f o r
IgE p ro d u c tio n as o u tlin e d
below .
I)
Using h a p te n -c a r r ie r system s,
i t has been shown th a t T c e l l s a re
prim ed w ith c a r r i e r and then ' in t e r a c t w ith B c e l l s t o form h a p te n s p e c if ic
IgE in both a d o p tiv e t r a n s f e r experim ents (1 2 -1 5 ) and hi
4
v i t r o experim ents ( 16) .
2)
.
Rats thymectomlzed w it h in 24 hours o f b i r t h cannot produce IgE
a n tib o d ie s
3)
.
(1 7 ).
L e th a l Iy ir r a d ia t e d r a ts r e c o n s titu te d w ith thymus and bone mar­
row c e l l s produce a low le v e l o f IgE whereas 'those r e c e iv in g bone
marrow a lo n e do not ( 1 8 ) .
4)
C o n g e n ita l Iy athym ic (n u /n u ) m ice cannot produce d e te c ta b le le v ­
e ls o f IgE t o ovalbum in unless r e c o n s titu te d w ith thymocytes (1 9 ) or
thymus glands ( 20 ) .
Some in v e s tig a to r s have suggested a heterogenous p o p u la tio n o f
h e lp e r T c e l l s ; one s p e c if ic f o r
IgE and one s p e c if ic f o r
O th ers suggest one p o p u la tio n o f h e lp e r I
IgG (1 6 ) „
c e l l s f o r both Immunoglobu^
I i n c la s s e s w ith a d i f f e r e n t i a l s u s c e p t i b i li t y to T c e l l
re g u la to ry
in flu e n c e s o f th e re s p e c tiv e c la s s e s o f a n tib o d y fo rm in g B c e lls
(2 1 ).
More r e c e n tly a c r i t i c a l
d u c tio n o f h e lp e r I
c e lls fo r
r o l e f o r adherent c e l l s
in th e In ­
IgE p ro d u c tio n has been shown ( 2 2 ) .
Two d i f f e r e n t p a tte rn s o f IgE a n tib o d y p ro d u c tio n have been
re c o g n iz e d .
One is t r a n s ie n t , n o n -b o o stab le and u s u a lly th e r e s u lt
o f Im m unization w ith a high dose o f a n tig e n .
shown In s e v e ra l sp ecies ( 4 ) .
T h is p a tte r n has been
The o th e r is s u s ta in e d f o r r e l a t i v e l y
long p e rio d s o f tim e and b o o s ta b le .
T h is p a tte r n
is c h a r a c t e r is t ic
o f IgE p ro d u c tio n to p o lle n a n tig e n s in humans w ith hay fe v e r ( 2 3 ) ,
.
5
t o h e lm in th a n tig e n s as a r e s u lt o f p a r a s ite in fe c t io n
( 2 4 ) , and in
m ice immunized w ith a low dose o f a n tig e n adsorbed t o alum ( 25 ) .
I n t e r e s t in th e management o f c l i n i c a l a ll e r g y has prompted
s tu d ie s o f r e g u la to ry mechanisms In IgE a n tib o d y fo rm a tio n .
d iv id u a ls w ith hay f e v e r ,
tite r s
In in ­
i t has been shown th a t serum IgE a n tib o d y
p e r s is t and a re enhanced a f t e r c o n ta c t w ith a n tig e n durin g
hay fe v e r season ( 2 3 ) .
H y p o s e n s itiz a tio n tre a tm e n t o f a ll e r g y con­
s is t s o f m u lt ip le in je c tio n s o f m inute doses o f a lle r g e n over long
p e rio d s o f tim e .
As a r e s u lt o f t h is tre a tm e n t IgG a n tib o d y le v e ls
a r e in creased and secondary IgE t i t e r s a r e e v e n tu a lly suppressed.
I t was thought th a t t h is tre a tm e n t Induced a b lo c k in g a n tib o d y , p re ­
sumably IgG , th a t competed w ith
IgE f o r b in d in g o f a lle r g e n .
How­
e v e r , a n a ly s is o f t i t e r s o f b lo c k in g a n tib o d y in c e r t a in tr e a te d pa­
t i e n t s f a i l e d to support t h is h yp o th esis ( 2 3 ) .
T h is evidence cou­
p le d w ith th e f a c t t h a t IgG Is not p re s e n t in v e ry g r e a t q u a n titie s
in r e s p ir a to r y s e c r e tio n s , th e s i t e where th e y would be most e f f e c ­
tiv e
in competing f o r a n tig e n , suggested th a t an In c re a s e in b lo c k ­
ing a n tib o d y may not be re s p o n s ib le f o r th e e f f e c t o f h y p p s e n s itiz a ;
t io n on c l i n i c a l symptoms.
An a l t e r n a t i v e h ypothesis was suggested,
nam ely, th a t a change in th e memory c e l l p o p u la tio n , p a r t i c u l a r l y
In T c e l l s , o ccurred w ith th e subsequent suppression o f th e secon­
dary IgE response ( 2 3 ) .
6
The tr a n s ie n t p a tte r n o f IgE a n tib o d y fo rm a tio n In c e r t a in
lab o ­
r a to r y a n im als p ro v id ed one means o f s tu d y in g th e suppression o f
t h i s c la s s o f a n tib o d y .
Using th e r a t as an e x p e rim e n ta l anim al and
a unique im m unization schedule to induce IgE , th e te rm in a tin g event
in th e tr a n s ie n t p a tte r n o f response was shown to in v o lv e r e g u la tio n
a t th e T c e l l
le v e l.
In t h is m odel,
IgE responses to a d in itro p h e n y l
c o n ju g a te o f A s c a ris suum e x t r a c t (DNP-Asc) were induced by a high
dose o f DNP-Asc ( I mg)
in je c te d w ith a B. p e r tu s s is v a c c in e v ia the
fo o tp a d s , on day 0 , fo llo w e d by an in tra m u s c u la r In je c t io n o f 0 .5 mg
o f DNP-Asc on day 5 ( 2 6 ) .
c lin e d by day 2 1 .
by T c e l l s
IgE t i t e r s
peaked by day 14 and then de­
The lin e s o f evidence f o r th e r e g u la tio n o f IgE
in t h is system In c lu d e enhancement and m aintenance o f
high IgE le v e ls beyond day 21 by a n t i thymocyte serum (ATS) (2 7 ) ;
a d u lt thymectomy ( 17)> splenectom y ( 17) and x - i r r a d i a t i o n
a d d it io n ,
( 28 ) .
In
i t was shown th a t th e IgE responses prolonged by these
means could be suppressed by tr a n s f e r o f sp len o c y te s o r thymocytes
from anim als hyper immunized w ith c a r r i e r a n tig e n ( 2 9 ) .
O ther c la s se s
o f a n tib o d y w ere not s im i l a r ly a f f e c t e d by these tre a tm e n ts .
Suppression o f IgE p ro d u c tio n by p a ss iv e a d m in is tr a tio n o f an­
tig e n s p e c if ic
IgG a n tib o d y has a ls o been dem onstrated In th e r a t .
( 26 ) and in th e r a b b it ( 30 ) .
It
is
in te r e s tin g to n o te th a t In th e
r a b b i t , p a s s iv e a d m in is tr a tio n o f a n t ig e n - s p e c if ic
IgM a n tib o d y e n -
7
haneed IgE fo rm a tio n ( 3 1 ) .
A somewhat d i f f e r e n t e x p e rim e n ta l model f o r IgE responses e x is ts
in th e mouse.
When mice a re immunized w ith a low dose o f a n tig e n in
co m b in ation w ith alum th e y g iv e a high and p e r s is te n t IgE response
(2 5 ).
tio n
T h is model most c lo s e ly resembles th e p a tte r n o f IgE produc­
in hay fe v e r p a t ie n t s .
S e v e ra l examples o f r e g u la tio n o f IgE
a n tib o d y fo rm a tio n In t h is model have been dem onstrated.
The a b i l i t y o f mice to form IgE a n tib o d y , as w e ll as IgG a n t i ­
body, to low doses o f a n tig e n ( 0.1 jxg -l jug) in alum appears t o be
under g e n e tic c o n t r o l, dependent on H-2 ty p e (3 2 ) and comparable to
th e ty p e o f Immune response r e g u la tio n observed w ith th e s y n th e tic ,
branched p o ly p e p tid e s
(TG )-AL and (HG)-AL ( 3 3 ) .
S t r a in d iffe r e n c e s
t o h ig h e r doses o f a n tig e n (100 *ig) were not as a p p are n t ( 3 2 ) ,
When
h a p te n -p ro te in c o n ju g ates were used, t h i s s t r a in d if f e r e n c e was shown
t o be r e la t e d t o c a r r i e r p r o t e in , sug g estin g th a t th e g e n e tic c o n tro l
o ccu rred a t th e T c e l l
le v e l
(3 2 ).
A nother ty p e o f g e n e tic c o n tr o l,
u n re la te d to H-2 ty p e and s p e c if ic f o r
3 4 ).
IgE has been observed (3 2 ,
Thus, th e SJL s t r a in o f mouse produces high t i t e r s
o f IgG an­
tib o d y a g a in s t many d i f f e r e n t a n tig e n s bu t low le v e ls o f Ig E , even
w ith h ig h doses o f a n tig e n .
The IgE t i t e r s o f these m ice can be en­
hanced by using Con A as a d ju v a n t In s te a d o f alum ( 3 5 ) .
ly ,
A d d itio n a l­
I t has been shown t h a t th e low re a g in p ro d u c tio n is due t o th e
8
presence o f n o n -s p e c if ic suppressor I
A n o th er means o f r e g u la tin g
Is by amount o f a n tig e n .
c e l is ( 36 ) .
IgE a n tib o d y in th e mouse system
Whereas low doses o f alum -adsorbed a n t i ­
gen induce a p e r s is te n t response, high doses o f alum -adsorbed an­
tig e n
induce a tr a n s ie n t response ( 2 5 ) .
A nother ty p e o f r e g u la tio n
has been observed in m ice immunized w ith a low dose o f alum -adsorbed
a n tig e n .
Maia e t a l ,
(3 7 ) re p o rte d th e s e le c t iv e d e p ressio n o f IgE
a n tib o d y fo rm a tio n t o ovalbum in In D B A /IJ m ice,
i f , a f t e r being im­
munized w ith an o p tim a l dose o f ovalbum in ( 0.1 # jg - 1 .0 Mg) and alum,
( I R ) , m ice were g iv e n In tra v e n o u s ( I V )
in je c tio n s o f 100 MQ o f s o lu ­
b le ovalbum in on days 3 » 5 , 7 > t h e i r r e a g in lc response was near n o r­
mal on days 8 , 9 ,
10 but had d e c lin e d by day 16.
c o n tin u e d t o make r e a g in lc a n tib o d y .
C o n tr o l. anim als
The IgG response, as measured,
by th e 2 hour homologous PCA t e s t , was n o t a f f e c t e d .
T h is p ro to c o l
is s im ila r to h y p o s e n s itiz a tio n th e ra p y In hay fe v e r p a t ie n t s .
Tada
(4 ) has suggested th a t t h i s phenomenon may re p re s e n t an example o f
to le r a n c e In IgE fo rm in g B c e l l s .
Bach and B ra s h le r ( 3 8 ) , re p o rtin g
a s im ila r phenomenon produced by m u lt ip le IV in je c tio n s o f a c e ty la te d o v a la lb u m ln , s p e c u la te th a t t h e i r system may re p re s e n t th e f o r ­
m ation o f suppressor T c e l l s .
More r e c e n tly i t has been dem onstrated
th a t t h i s ty p e o f suppression can be tr a n s fe r r e d t o normal anim als
v ia s p le n ic T lymphocytes ( 3 9 ) .
In a d d it io n ,
i t was subsequently
shown t h a t th e s e suppressor c e l l s could be g enerated j_n v i t r o only
9
i f a d h ere n t c e lt s were absen t from th e c u ltu r e ( 22 ) .
STATEMENT OF THESIS
The purpose o f t h is study was t o examine th e r e g u la tio n o f IgE
a n tib o d y p ro d u c tio n in m ice and to r e l a t e t h is to c e l l u l a r events^
The study was focused p r im a r ily on th e B a lb /c s t r a in
in o rd e r to
a v o id th e c o m p lic a tio n s a r is in g from s t r a in d iffe r e n c e s
in re g u la ­
t i o n o f IgE s y n th e s is .
The e x p e rim e n ta l approach c o n s is te d o f :
1)
An exa m in atio n o f th e r e la t io n s h ip o f Ig E -p ro d u c in g c e l l s
t o o th e r c la s s e s , o f a n tib o d y -p ro d u c in g c e l l s by te s t in g th e e f f e c t
o f n e o n a tal Iy
2)
in itia te d ant I
on subsequent IgE a n tib o d y responses
A comparison o f th e v a rio u s ways in which IgE responses
can be induced and r e g u la te d .
3)
An a tte m p t t o e lu c id a te th e mechanism(s) re s p o n s ib le fo r
th e s e le c t iv e suppression o f IgE responses by th re e in traven o u s In ­
je c t io n s o f high doses o f s o lu b le a n tig e n .
MATERIALS AND METHODS
A nim als
B a lb /c mice were ra is e d
in our own la b o ra to ry f a c i l i t i e s .
C3H/HeJ m ice were o b ta in e d from Jackson L a b o ra to rie s , Bar H arb o r,
M a in e.
ICR mice were o b ta in e d from th e Research A nim als Resources
C e n te r, U n iv e r s ity o f W is co n sin , M adison.
CF^ mice w ere purchased
from Carw orth Farms, P o rta g e , M ic h ig a n .
O utbred a lb in o r a ts were purchased from ARS Sprague-Dawley or
H o ltzm an, C o ., M adison, W isco n sin .
A n tig e n s
The fo llo w in g a n tig e n s were o b ta in e d from Calbiochem (San
D ie g o , C a l i f o r n i a ) :
Ovalbum in,
(OVA) 5X c r y s ta l 11z e d ,
and th e fo llo w in g d in ltr o p h e n y la te d (DNP-) p ro te in s :
l o t #387039
DNP-o v a I bumin
(DNP24 -O V A ), DNP-bovihe serum album in (DNPgg-BSA), DNP-Keyhole lim ­
p e t hemocyanin (DNPgyg-KLH).
S u b s c rip ts r e f e r t o th e averag e num­
b er o f groups per m o lecu le o f p r o t e in .
In th e case o f KLH, t h is
was based on a m o le c u la r w e ig h t o f 2 x IO ^.
O ther a n tig e n s and t h e i r commercial sources a r e :
normal ra b ­
b i t serum (NRS), C olorado Serum C o .; o v in e a lb u m in , Sigma; ovine
gamma g lo b u lin , P en tex .
Ragweed e x t r a c t was a g i f t from D r. E. Rau, E ndocrine Labora­
t o r i e s , M adison, W is co n sin .
N tp p o stro n q ylu s b ra s ? Iie n s t s a n tig e n was prepared a cc o rd in g to
th e method o f O g ilv ie ( 4 0 ) .
L i g h t ly d i n Jtro p h en yla te d ovalbum in (DNPooJ-OVA) was prepared
by th e method o f E isen (4 1 ) as m o d ifie d by Is h lz a k a and Okudalra
(4 2 ).
B r i e f l y , 50 mg each o f d I n itro b e n z e n e s u lfo n ic a c id , 2X r e -
c r y s t a l l i z e d , and o f sodium c a rb o n a te were d is s o lv e d in 6 ml d is ­
tille d
w a te r .
To t h i s ,
100 mg o f OVA were added and th e m ix tu re
s t i r r e d f o r 3 - 1 / 2 h r a t room te m p e ra tu re .
The r e s u lt in g c o n ju g ate
was d ia ly s e d a g a in s t s a l i n e , a t 4 ° C f o r s e v e ra l days (changed a t
le a s t 5 X ).
The number o f DNP groups per m olecule o f OVA was d e te r ­
mined s p e c tro p h o to m e trSeal Iy (4 3 ) by m easuring absorbance a t 360 nm
f o r DNP and 280 nm f o r OVA.
The p r o te in c o n c e n tra tio n was e stim a te d
by lo w erin g th e observed OD28 Q re a d in g by 38 . 5% to c o r r e c t f o r th e
a b s o rp tio n a t 280 nm by DNP ( 4 3 ) .
A d ju v an ts
P re p a ra tio n o f A l (0H)^ gel
(A lu m ):
F if t e e n g o f alum (p u r­
chased from a lo c a l d ru g s to re ) was d is s o lv e d In 180 ml o f d i s t i l l e d
w a te r .
To t h i s s o lu t io n , 75 ml o f
continuous m ix in g .
d i s t i l l e d w a te r.
IN NaOH was added dropw ise w ith
The r e s u lt in g p r e c i p i t a t e was washed 6 - 8X w ith
Dry w eig h t d e te rm in a tio n s were done on th e f i n a l
s o lu t io n .
B o r d e te iIa p e rtu s s is v a c c in e was purchased from El I L i l l y ,
12
C o .,
In d ia n a p o l I s ,
In d ia n a (c o n tro l #8AL31A).
Im m unizations
The s ta n d ard im m unization used t o induce a good IgE response
was 10 ng OVA mixed w ith
I mg alum g iv e n IR on day 0 .
■ ■
The OVA. was
f r e s h ly prepared each tim e , by d is s o lv in g k mg OVA In TOO ml s a lin e .
An equal volume o f t h is s o lu tio n was mixed w ith an equal volume o f
alum (4 m g /m l), and 0 .5 ml o f t h is m ix tu re was in je c t e d .
For those
exp erim ents in which a d i f f e r e n t a n tig e n was used o r th e amount o f
a n tig e n v a r ie d , a s im i l a r p ro to c o l was fo llo w e d w ith th e a p p ro p ria te ,
a d ju s tm e n ts .
in some e x p e rim e n ts , anim als re c e iv e d ,
in a d d itio n t o th e s ta n -
!;
dard Im m u n izatio n , th re e IV In je c tio n s o f a n tig e n w ith o u t a d ju v a n t
on days 3 » 5 , 7 .
In th e s e e x p e rim e n ts , th e term s e n s itiz e d
is used
f o r th ose an im a ls r e c e iv in g th e s tan d ard im m unization; th e term sen­
s i t i z e d -d e s e n s itiz e d
is used f o r th o se r e c e iv in g th e a d d itio n a l
tr e a tm e n t.
O th er im m unization p ro to c o ls a r e d e sc rib e d in th e t e x t .
Serum co l le c t io n
A t th e a p p ro p ria te tim es a f t e r
le c te d from th e t a l l
Im m unization, blood was c o l­
in to p l a s t i c m ic ro c e n trifu g e tubes to which I
drop o f s a lin e had been added.
The c lo t t e d blood was rimmed and
'
l e f t f o r s e v e ra l hours o r o v e rn ig h t a t 4 ” C.
The serum was c o l l e c t -
; I
:|
]
13
ed a f t e r c e n t r if u g a t io n and s to re d In 1 /4 or 1/2 dram v ia l s a t
-20°C o r -7 0 °C .
Serum s to re d In t h is manner d is p la y e d e q u iv a le n t
PCA t i t e r s 9 months a f t e r c o l le c t i o n .
Assays
IgE responses were assayed by th e p a ss iv e cutaneous a n a p h y la x is
(PCA) t e s t perfo rm ed , f o r most e x p e rim e n ts ,
in r a ts
(4 4 , 4 5 ) .
For
each serum, o r serum p o o l, a 0 .1 0 ml a liq u o t o f each o f a s e rie s o f
tw o fo ld d i lu t io n s was In je c te d
backs o f t e s t r a t s .
t i n e l y made.
in tra d e rm a l Iy (ID.)
in to th e shaved
A p p ro xim ately, 40 in je c tio n s per r a t were rou­
The r a ts were c h a lle n g e d 24 hr l a t e r by Intravenous
in je c t io n o f 5 mg a n tig e n d is s o lv e d in 0 .5 ml 1% E van's b lu e .
When
a ss a y in g f o r PCA responses a g a in s t MRS, th e c h a lle n g e a n tig e n con­
s is te d o f 2 ml o f NRS in which 5 mg E van's b lu e dye was d is s o lv e d .
S i m i l a r l y , f o r PQA responses a g a in s t N lp p o s tro n q y lu s a n tig e n s , 5 mg
o f E van's b lu e dye was d is s o lv e d in 1-2 ml o f . a s a lin e s o lu tio n con­
t a in in g 2 ,0 0 0 worm e q u iv a le n ts .
T h i r t y m inutes a f t e r
IV c h a lle n g e ,
th e r a ts were s a c r if ic e d and th e re a c tio n s read on th e underside o f
th e s k in .
A p o s it iv e re a c tio n was In d ic a te d by a d is c r e t e c i r c l e
o f b lu e .
For both th e ID and IV In je c t io n s , th e r a ts were sedated by an
in tra m u s c u la r in je c t io n o f 0 .0 4 - 0 .0 6 ml I n n o v a r-v e t ( P i t man-Moore
C o .).
14
When th e PCA t e s t was perform ed In m ice, 0 .0 5 ml o f a serum
d i l u t i o n was in je c t e d ,
were c h a lle n g e d
ID ,
in to th e shaved backs o f m ice .
The mice
IV , 72 h r l a t e r , w ith 0 .2 ml 1% E van's b lu e dye,
s a lin e , c o n ta in in g 1-2 mg o f a n tig e n .
In
Only one ID in je c t io n per
mouse was made.
IgE a n tib o d y -p ro d u c in g c e l l s were measured by th e h eterolo gous
a d o p tiv e cutaneous a n a p h y la x is (HACA) t e s t ( 4 6 ) .
T h is procedure is
s im i l a r to th e PCA assay except th a t 0 .1 ml a liq u o t s o f th e appro­
p r i a t e c e l l suspensions were In je c te d
in to th e shaved backs o f r a t s .
A n tib o d ie s o th e r than those o f th e IgE c la s s were assayed by
p a ss iv e h e m a g g lu tin a tio n (PHA) using e ry th ro c y te s coated w ith OVA.
Two d i f f e r e n t methods f o r c o a tin g e ry th ro c y te s were used.
In one
method ( 4 7 ) , OVA was coupled t o guinea p ig e ry th ro c y te s w ith c a rb o d iim id e .
To 3 ml o f PBS in which 30 mg o f OVA was d is s o lv e d was
added 0 .0 5 ml o f washed packed guinea p ig e r y th r o c y te s .
To th is
was added 50 mg I - e t h y l -3 ( 3 -d im e th y la m i n o p ro p y l)-C a r b o d iimide hy­
d r o c h lo rid e (Sigma) d is s o lv e d
in 0 .5 ml PBS.
T h is m ix tu re was l e f t
a t room te m p e ra tu re f o r I hr w ith o c ca s io n al m ix in g .
The c e lls
w ere then washed 3X and resuspended in m o d ifie d A I s e v ie r 's s o lu tio n
(4 8 ) t o a p p ro x im a te ly 0.5% .
In th e o th e r method ( 4 9 ) , OVA was coupled t o sheep e r y th r o ­
c y te s w ith chromium c h lo r id e (C r C lg ).
(1 0 mg/ml
S tock s o lu tio n s o f CrCIg
in d i s t i l l e d w a te r) and OVA ( I mg/ml In s a lin e ) were p r e -
15
pa re d „
For th e c o u p lin g p ro c ed u re , th e s to c k CrCl^ s o lu tio n was
d ilu t e d 1 /2 0 in s a lin e .
To 0 .2 ml o f C rC lg s o lu tio n was added 0 .2
ml o f OVA s o lu tio n and 0 .2 ml washed packed sheep e r y th r o c y te s .
T h is m ix tu re was l e f t a t room te m p e ra tu re f o r 5 m in u te s , and then
th e c e l l s were washed 4X in s a l i n e .
c e l l s were d ilu t e d
A f t e r th e la s t wash th e packed
1 /6 0 in m o d ifie d A l s e v i e r gS s o lu tio n and s to re d .
For use in assays a f u r t h e r
1/10 d i l u t i o n o f th e c e l l s was made
( f i n a l d i l u t i o n = 1/ 600 ) .
PHA t i t e r s were determ ined by m i c r o t it e r using p l a s t i c V-bottom
m i c r o t it e r p la t e s , 25 n I d i l u t i n g
loops and 25 p i p ip e t t e droppers.
M o d ifie d A I s e v i e r gS s o lu tio n was used as a d i l u e n t .
A ll
t i t e r s a re
expressed as th e r e c ip r o c a l o f th e la s t d i l u t i o n t© g iv e a p o s itiv e
r e s u lt.
A n t l- p tre a tm e n t
The a n t i -p a n tis e ru m was p repared by D r. Dean D. Manning, U n i­
v e r s i t y o f W is co n sin , M adison, as d e s c rib e d p re v io u s ly ( 5 0 ) .
in 14 hr o f b i r t h , each o f th r e e l i t t e r s was d iv id e d
groups.
One group In each l i t t e r was in je c te d
W ith ­
In to th re e
IP w ith 0 .0 5 ml o f
a n t i - p serum, th e o th e r two r e c e iv in g an id e n tic a l
in je c t io n o f NRS
o r p h o s p h a te -b u ffe re d s a lin e (PB S); f u r t h e r in je c tio n s o f 0 .0 5 ,
0 . 07 , 0 . 0 7 , 0 . 0 8 , and 0 .0 8 ml were made on days 2 , 4 , 6 , 8 , and 10 ,
r e s p e c t iv e ly .
T h e r e a ft e r a l l m ice re c e iv e d a 0 .1 0 ml in je c t io n o f
16
PBS o r d ilu t e d NRS o r a n t I
serum (b o th d ilu t e d
1 :2 .5
in PBS)
e v e ry Monday, Wednesday, and F rid a y u n t i l te rm in a tio n o f th e e x p e ri
m ent,
Worm in fe c t io n
A t a p p ro x im a te ly 40 days o f age a l l mice re c e iv e d a subcutane­
ous in je c t io n o f 300 t h ir d - s t a g e ( in f e c t io u s )
adapted s t r a in o f N. bras 11 lens is ( S I ) ,
la rv a e o f a mouse-
2 wk l a t e r a l l mice were
r e in f e c te d w ith 300 such la rv a e and 13 days t h e r e a f t e r were exsan­
g u in a te d r e t r o - o r b i t a l I y .
RESULTS
In d u c tio n o f hoanocytotropic a n tib o d ie s
P rev io u s s tu d ie s
in th is
in mice
la b o ra to ry have e s ta b lis h e d th a t 10
Hg OVA '+ 10 mg alum g iv en IP to B a lb /c mice is v e ry e f f e c t i v e fo r
in d u c tio n o f 72 hr PCA a n tib o d y using th e homologous PCA assay ( 2 0 ) .
T h is was confirm ed and i t was shown th a t I mg o f alum was j u s t as
e f f e c t i v e as 10 mg.
10 days a f t e r
in a d d it io n , C3H/HeJ mice gave a PCA t i t e r , a t
im m unization, th a t was s im ila r to B a lb /c m ice.
The h e te ro lo g o u s ( r a t )
PCA assay f o r mouse IqE
A lthoug h IgE is con sid ered a hom ocytotropic a n tib o d y ,
shown th a t human IgE could bind to monkey mast c e l l s
l a r l y , v a rio u s
(5 2 ).
i t was
S im i­
lin e s o f evidence have shown th a t mouse Ig E , but not
lgG||, w i l l bind t o r a t mast c e l l s
(4 4 , 4 $ , 5 3 , 5 4 ) .
ing is s e le c t iv e f o r mouse Ig E , when r a t s ,
S in ce th is bind
in s tea d o f m ice , a re used
as th e r e c ip ie n ts f o r th e p a ss iv e t r a n s f e r o f mouse a n tis e ru m in
th e PCA assay a la t e n t p e rio d o f 2 -7 2 hours can be employed ( 4 5 ) .
A comparison between th e homologous and h etero lo g o u s ( r a t ) PCA
assays is shown in T a b le I .
S im ila r PCA t i t e r s were observed w ith
th e two a s s a y s , a lth o u g h th e t i t e r
d ilu tio n
In th e homologous assay is one
lo w er.
To f u r t h e r t e s t th e a b i l i t y
mouse Ig E ,
o f th e r a t PCA assay to, measure
I examined th e heat s e n s i t i v i t y o f th e PCA a n tib o d y .
It
had been re p o rte d p re v io u s ly ( 55 ) th a t d i l u t i n g serum p r io r to heat
18
tre a tm e n t a t 56°C f o r 30 m inutes would p ro te c t th e IgE s k in s e n s i­
t i z i n g a c t i v i t y from h e at d e s tr u c tio n .
S ince these w orkers were
using th e homologous RCA assay I re te s te d t h is o b s e rv a tio n using
th e h e te ro lo g o u s
( r a t ) PCA assay w ith a longer h e a tin g tim e .
The h e at tre a tm e n t was c a r r ie d out as fo llo w s :
quots were removed from a serum pool from 10 m ice.
Two 0 .1 ml a l i ­
One a liq u o t was
p la ce d in a 10 x 75 mm t e s t tu b e , corked t i g h t l y and th e tube was
th en p laced in a 56°C w a te r bath f o r 90 m in u te s.
was d ilu t e d
1:8 in PBS and d iv id e d
The o th e r a liq u o t
In to 2 p o rtio n s .
One o f these
was heated as above w h ile th e o th e r was placed in th e c o ld .
A fte r
th e in c u b a tio n p e rio d , f u r t h e r d ilu t io n s o f th e samples were made
and te s te d f o r PCA a c t i v i t y
II.
in r a t s .
The r e s u lts a re shown in T a b le
The PCA t i t e r was d im in is h e d from 256 to < 4 f o r pool
I and from
2048 to 4 f o r pool 2 by h e a tin g serum b e fo re d i l u t i o n w h ile serum
d ilu t e d p r i o r to h e a tin g showed o n ly a s li g h t re d u c tio n in t i t e r .
These r e s u lt s c o n firm t h a t th e a n tib o d y measured by th e r a t PCA a s ­
say is h e a t l a b i l e and th a t d i l u t i o n o f th e a n tib o d y p r io r t o heat
tre a tm e n t p ro te c ts
i t from d e s tr u c tio n .
E f f e c t o f an a n ti-h e lm in t h agent on th e homologous PCA re a c tio n in
I CR mice
A comparison between v a rio u s outb red mice as r e c ip ie n ts fo r th e
homologous PCA t e s t produced an in t e r e s t in g fin d in g .
I t was noted
19
th a t ICR m ice o b ta in e d from th e Research Anim als Resources C e n te r,
U n iv e r s ity o f W is co n sin , M adison, c o n s is te n tly gave v e ry poor or
n e g a tiv e PCA re a c tio n s w ith a n tis e ru m th a t produced s tro n g re a c ­
tio n s
in CF| mice o b ta in e d c o m m e rc ia lly .
J a rre tt e t a l .
re p o rte d th a t N ip p o s tro n q y iu s bras 11 le n s is
in fe c t io n
(5 6 ) have
in r a ts had an
In h ib it o r y e f f e c t on subsequent homologous PCA ass a ys .
I hypothe­
s iz e d th a t a s im ila r e f f e c t due to p I nworm in fe c t io n m ight be r e ­
s p o n s ib le f o r th e e f f e c t on PCA re a c tio n s seen in ICR m ice.
A p r e lim in a r y t e s t o f t h is h yp o th esis was made in c o lla b o r a ­
t io n w ith D r. Ralph Anslow , U n iv e r s ity o f W is co n sin .
m ice w ere found t o have a high pinworm burden,
S ince th e I CR
i t was decided to ad
m in is t e r an a n ti-h e lm in t h agent t o th e s e mice and t e s t th e e f f e c t
o f such tre a tm e n t on t h e i r r e a c t i v i t y
in th e PCA a ss a y.
A group o f r e t i r e d fe m a le b re ed e r mice was s e p a ra te d from th e
main colony and d iv id e d
Dodge L a b o r a to r ie s ,
In to 2 groups.
I n c . , F o r t Dodge,
A s o lu tio n o f D yrex R (F o rt
Iowa) a t a c o n c e n tra tio n o f
2 .5 gm per l i t e r o f w a te r was a d m in is te re d as th e s o le source o f
d rin k in g w a te r f o r 14 days t o one group w h ile th e o th e r group was
g iv e n p la in w a te r.
A p p ro x im a te ly two weeks a f t e r th e tre a tm e n t
p e rio d s e v e ra l m ice from each group were s a c r if ic e d and t h e i r
t e s tin e s examined f o r p inworms.
in ­
The tr e a te d group was a p p a re n tly
f r e e o f worms, whereas th e u n tre a te d group had worms.
Two months
20
la te r ,
th e m ice w ere used as r e c ip ie n t s
The r e s u lt s
fe r e n c e
f o r th e hom ologous PCA t e s t .
shown in T a b le I I in d ic a t e t h a t th e r e
is a d e f i n i t e d i f ­
in th e q u a l i t y and i n t e n s i t y o f th e PCA le s io n between th e
tw o g ro u p s .
The PCA le s io n s o f th e t r e a t e d g ro u p w ere la r g e r , d a r ­
k e r b lu e and more d is c r e t e th a n th o s e o f th e u n tre a te d g ro u p .
th e
When
in t e s t in e s o f th e s e m ice w ere e xam ine d , p inworms c o u ld n o t be
d e te c te d
in . e ith e r g ro u p .
t y c o u ld n o t be r e la t e d
A lth o u g h
in t h i s c a s e , th e PCA r e a c t i v i ­
t o pinw orm b urde n a t th e tim e o f th e a ssay
t h e r e was a s tr o n g c o r r e l a t i o n betw een a n t i- h e lm in t h tre a tm e n t and
PCA r e a c t io n s .
th e
T h is s t r o n g ly s u g g e s ts t h a t th e p o o r r e a c t i v i t y o f
ICR m ice was due t o a worm I n f e c t io n .
S u p p re s s io n o f IqE a n tib o d y fo r m a tio n by tre a tm e n t w it h a n t i - u
a n tis e ru m
I t was o f in t e r e s t to know w hether th e ontog enic development
o f IgE form ing c e l l s
is r e la te d to IgM b e a rin g p re c u rs o ra l c e l l s ,
as a r e th e c e l l s form ing o th e r c la s s e s o f im m unoglobulins.
way o f s tu d y in g th is
r e la t io n s h ip
l i n fo rm a tio n by n e o n atal Iy
is by suppression o f immunoglobu­
i n i t i a t e d a n t i - # tre a tm e n t.
ment has been shown to suppress th e fo rm a tio n o f IgM,
and IgA in m ice ( 5 7 ) .
One
Such t r e a t ­
Ig G ^
IgGg,
K ishim o to and Is h iz a k a (5 8 ) showed th a t
a n t i - # a n tib o d ie s cannot suppress secondary IgE responses o f r a b b it
c e l l s j_n v i t r o .
T h e r e fo r e ,
i t was decided to t e s t th e e f f e c t o f jm
21
TABLE I
A comparison between th e homologous and h eterolo gous pa ss iv e
cutaneous a n a p h y la x is
(PCA) te s ts f o r mouse IgE
a n tib o d y a g a in s t ovalbumin
Mouse a n t is e r a 3
______ PCA t i t e r s
Mouse
Rat
I
128
256
Pool 2
128
256
Pool
aT i t e r s were done on a serum pool from 3 -5 m ice.
Mice were immu­
n iz e d w ith 10 jug ovalbum in + I mg alum IP on day 0 and b le d 10 days
la te r .
22
TABLE I I
E f f e c t o f h e a t3 on p a s s iv e c u ta n e o u s a n a p h y la x is
t it e r s
Serum poo1c
from 10 mice
(PCA)*3
o f mouse a n tis e ru m
1 :8 d i l u t i o n
unheated
U n d ilu te d s e rum heated
1 :8 d il u t i o n
heated
I
256
<4
128
Pool 2
2048
4
1024
Pool
a 56°C f o r 90 m in u te s.
kpCA t e s t perform ed in r a t s .
cM ice were immunized w ith 10 fig OVA + I mg alum ,
IP .
23
TABLE I I I
E f f e c t o f an a n t i- h e lm in t h a g e n t on th e hom ologous
p a s s iv e c u ta n e o u s a n a p h y la x is
(PCA) r e a c tio n
PCA a c t i v i t y 9
In t e n s it y
C o n tro l^
2+
+
T re ated ^
D iam eter
15 x 15
unmeasurable
D e s c rip tio n
d is c r e te
b a r e ly v i s i b l e
1+
12 x 10
+
10 x 10
d i f f u s e , s p lo tc h y
+
12 x 15
d i f f u s e , S plotchy
2+
12 x 15
d if f u s e
4+
20 x 15
d is c r e te
3+
20 x 25
d is c r e te
3+
20 x 20
d is c r e te
3+
20 x 25
d is c r e te
d is c r e te
aA n tiseru m used f o r p a ss iv e t r a n s f e r was a pool o f serum ob tain ed
from mice immunized w ith 10 jug ovalbum in + I mg alum .
^ T re a te d anim als were g iv en Dyrex R in d rin k in g w a te r f o r 14 days.
C o n tro l group was l e f t u n tre a te d . A p p ro x im ately 2 months l a t e r th e
m ice were te s te d f o r a b i l i t y to show PCA r e a c tio n s .
24
v iv o , a n t I -/it tre a tm e n t on th e a b i l i t y o f mice to g iv e a subsequent
IgE a n tib o d y response to 11. b ra s i I ie n s is
a tte m p ts to u t i l i z e
in fe c t io n
(5 9 ).
E a rly
th e ovalbum in-alum model were f r u s t r a t e d by th e
fre q u e n t suppression o f a n t i - o v a I bumin responses by m u lt ip le
tio n s o f NRS (see b e lo w ).
Dwyer e t a l .
in je c ­
W h ile th e s e s tu d ie s were in p ro g re ss ,
(6 0 ) re p o rte d th a t they f a i l e d
to suppress IgE r e ­
sponses to im m unization w ith ovalbum in and alum by neonatal Iy i n i ­
t i a t e d a n t i -M tre a tm e n t.
The r e s u lts o f th is study a r e shown in T a b le IV .
th a t a n im als r e c e iv in g a n t I-M tre a tm e n t f a i l e d
It
is c le a r
to produce d e te c t­
a b le le v e ls o f PCA a n tib o d y t o worms, whereas those re c e iv in g e it h e r
PBS o r NRS made high le v e ls o f s p e c if ic PCA a n tib o d y .
m ice were indeed a c t iv e l y
in fe c te d w ith worms was determ ined by
worm egg counts in th e fe c es
s ev e re p a n s p e c ific humoral
T h at a l l
( 51 ) .
In a d d it io n , th e achievem ent o f
immunosuppression as a r e s u lt o f a n t i -/Lt
tre a tm e n t was confirm ed by assay o f serum immunoglobulin le v e ls o f
IgM,
IgG1,
IgG2 , and IgA ( 5 9 ) .
E f f e c t o f m u lt ip le
m a tio n t o
in je c t io n s o f NRS on su bse q ue n t IqE a n tib o d y f o r ­
im m u n iz a tio n w it h o v a lb u m in and alum .
D u rin g th e s tu d ie s o f th e e f f e c t o f a n t i -/Lt tre a tm e n t on IgE
a n tib o d y fo r m a tio n th e p r e lim in a r y o b s e r v a tio n was made, t h a t
c o n t r o l a n im a ls r e c e iv in g NRS tr e a tm e n t t h e
in
IgE re s p o n s e , when t e s t -
25
ed 10 days a f t e r one IP in je c t io n o f OVA and alum , was suppressed.
T h is phenomenon was in v e s tig a te d f u r t h e r .
fe re n t l i t t e r s
in T a b le V .
g iven m u lt ip le
The r e s u lts from 2 d i f ­
in je c tio n s o f NRS from b i r t h a re shown
In a l l anim als re c e iv in g PBS tre a tm e n t, th e PCA and IHA
responses were h ig h , whereas in th e NRS tr e a te d anim als most o f th e
responses f o r both PCA and PHA a n tib o d y were e it h e r u n d e te c ta b le or
s ig n if ic a n tly
low ered.
However, th e s u p p res s ive e f f e c t was not always p re s e n t to th e
same d e g ree .
One anim al
in th e NRS tr e a te d group showed a PCA t i ­
t e r o f 128 and PHA t i t e r o f 64 which can probably be considered
normal and one anim al showed a PCA t i t e r o f 64 and PHA t i t e r o f 16
which is o n ly s l i g h t l y
lower than th a t o b ta in e d n o rm a lly but which
p ro b a b ly re p re s e n ts a s ig n if ic a n t su p p res s io n .
The o th e r responses
in t h is group c l e a r l y a re suppressed.
Thus,
i t was shown th a t NRS tre a tm e n t d id have a su ppressive
e f f e c t on th e IgE a n tib o d y response, bu t t h is e f f e c t was v a r ia b le
in i t s e xp re s sio n and was not s e le c t iv e f o r IgE; suppression o f PHA
responses p a r a lle le d th a t o f IgE .
S im ila r r e s u lts have been ob­
served by a n o th e r in v e s tig a to r (G . M. Lang, personal com m unication).
K in e tic s o f th e PCA and PHA responses o f mice immunized w ith 10 jug
OVA + I mg alum
Vaz e t a l .
(2 5 ) showed th a t one IP in je c t io n o f a low dose o f
26
TABLE IV
IgE a n ti-w o rm responses in mice tr e a te d
from b i r t h w ith a n t i -ju a n tise ru m
A n ti-w o rm PCA t i t e r s 3
I
PBS t r e a t e d *3
NRS tr e a t e d *1
A n t i- u tr e a te d
512
128
<4
512
128
<4
32
128
0
256
256
0
256
512
0
256
32
0
512
0
256
a PCA = p a s s iv e cutaneous a n a p h y la x is assay perform ed in r a t s .
^M ice re c e iv e d e it h e r PBS, NRS or a n t i- M , IP , acc o rd in g to th e f o l ­
low ing schedule:
Days 0 , 2 -0 ,0 5 m l; days 4 , 6 - 0 ,0 7 m l; days 8 , 100 .0 8 m l; t h e r e a f t e r , every Monday, Wednesday and F r id a y -0 .1 0 m l.
A t a p p ro x im a te ly 40 days a l l mice re c e iv e d 300 t h ir d - s t a g e ( i n f e c ­
t io u s ) la rv a e o f N. b r a s i I i e n s l s . 2 wk l a t e r a l l mice were r e in ­
fe c te d w ith th e same dose o f la rv a e and b le d 13 days l a t e r .
27
TABLE V
E ffe c t o f n e o n a ta lIy i n i t i a t e d
normal r a b b it serum (NRS)
tre a tm e n t on a n t i - o v a I bumin responses in mice
Treatm ent®
PBS
NRS
PCA^
PHA^
512
512
512
256
512
128
256
512
256
512
<4
2
<4
2
<4
4
16
2
64
16
128
64
a M ice re c e iv e d e it h e r NRS o r PBS, IP , acco rd in g to th e fo llo w in g
s ch e d u le :
Days 0 , 2 -0 .0 5 m l; day 5 - 0 .0 7 m l; day 8 - 0 .0 8 or 0 .1 0
m l; days 13, 18, 2 3 , 2 8 , 3 5 , 3 7 , 4 2 - 0 .1 0 m l. Day 0 = day o f b i r t h .
^Mice were p re b le d on day 3 6 ; immunized on day 37 w ith 10 jug OVA +
10 mg alum , IP ; b le d on day 4 7 . R e s u lts re p re s e n t in d iv id u a l serum
t i t e r s , o f 2 d i f f e r e n t l i t t e r s , on day 4 7 .
28
a n tig e n w ith alum was a r e l i a b l e method o f inducing high and p e r­
s is t e n t IgE t i t e r s
munized w ith
V I,
in m ice.
T h is was confirm ed f o r B a lb /c mice im­
10 jug OVA + I mg alum .
From th e r e s u lts shown in T a b le
i t can be seen th a t PCA a n tib o d y is p re se n t a t peak le v e ls a t
day 10 and is m a in ta in e d a t th a t le v e l f o r a t le a s t 5 months.
PHA a n tib o d y
A
is a ls o induced and m a in ta in e d .
Dose response s tu d ie s
Vaz e t a I .
m ice ,
(2 5 ) have a ls o re p o rte d t h a t ,
in c e r t a in s tr a in s o f .
im m unization w ith a 100 jug dose o f OVA + 10-20 mg o f alum ex­
e r te d a su p p res s ive e f f e c t on IgE responses.
In c o n tra s t to the
high and p e r s is te n t IgE response d is p la y e d by mice immunized w ith
low doses ( 0 .1 - 1 jug) o f alum -adsorbed OVA, mice immunized w ith a
hig h dose (100 jug) o f alum -adsorbed OVA showed a somewhat lower ho­
mologous PCA t i t e r
th a t d e c lin e d , to v e ry low or u n d e te c ta b le le v ­
e l s , by day 21 .
S in ce th e s e in v e s tig a to r s d id not use B a lb /c m ice in t h e i r
s tu d y ,
I te s te d th e e f f e c t o f one in je c t io n o f d i f f e r e n t doses of
OVA on th e a n tib o d y responses o f t h is s t r a in to see i f a s im ila r
ty p e o f r e g u la tio n could be dem onstrated.
In these s tu d ie s , the
amount o f alum was kep t c o n s ta n t ( I mg) and th e PCA t i t e r s were as­
sayed by th e h e te ro lo g o u s ( r a t ) PCA t e s t .
The r e s u lt s o f th e PCA and PHA t i t e r s
a re shown in T a b les V ll
29
and V I I I ,
21,
r e s p e c tiv e ly .
No evidence o f re g u la tio n o f Ig E , a t day
is seen a t high doses o f a n tig e n .
1000 4 g, a l l
a s li g h t
Over th e dosage range o f I -
groups show a high t i t e r a t days 21 and 2 8 .
There is
lag in th e peak IgE response, w ith h ig h e r doses o f a n tig e n ,
but t h is e f f e c t
is gone by day 2 1 .
A s im ila r lag has been observed
w ith high doses o f c e r t a in o th e r a n tig e n s and may re p re s e n t a t r a n ­
s ie n t n e u t r a liz a t io n o f IgE a n tib o d y by excess a n tig e n .
A lag in
peak PHA responses was not seen.
A second experim ent ( r e s u lt s not shown) confirm ed th e p e r s is ­
te n c e o f th e IgE response, even a t high doses o f a n tig e n ; a t day 5&
a f t e r mice were immunized w ith
PCA t i t e r s
10 or 1000 #xg OVA + I mg alum , th e
o b ta in e d were s i m i l a r , a t both dosage le v e ls , and to the
r e s p e c tiv e responses on day 14.
day 21 th e re was v e ry l i t t l e
It
is
in te r e s tin g t o note th a t by
d iffe r e n c e between th e t i t e r s
o b ta in e d ,
in both th e PCA and PHA a s s a y s , when 10-1000 ng o f a n tig e n was used
fo r
im m unization.
IgE responses to DNP-conjuqates o f ovalbum in
S tu d ie s
in th e r a t , on IgE a n tib o d y responses to d i n i tro p h e n y -
Ia te d A s c a ris e x tr a c ts
(DNP-Asc) showed th a t th e le v e l o f anti-D N P
IgE was in v e rs e ly r e la te d to th e number o f DNP groups lin k e d to th e
c a r r ie r
(6 l ) .
Thus,
im m unization w ith 4 x IO""** moles DNP/mg c a r ­
r i e r produced an ave ra g e an ti-D N P PCA t i t e r o f 13, whereas I x IO"**
30
TABLE Vl
Long term m aintenance o f immune responses to alum adsorbed
ovalbumin®
in B a ib /c mice
Type of
a n tib o d y
Day 10
PCAb
256°
512
512
128
PHAb
128
512
1024
1024
Day 21
Day 56
Day 150
aM ice were immunized w ith one IP in je c t io n o f 10 ng ovalbum in +
I mg alum .
bPCA = p a s s iv e cutaneous a n a p h y la x is .
PHA = p a s s iv e h e m a g g lu tin a tio n .
c Each r e s u lt was o b ta in e d from a serum pool from 5 m ice.
TABLE V l l
IgE responses to v a rio u s doses o f ovalbum in adsorbed to alum
Dose o f
ovalbum in 8
Day 10
Day 14
Day 21
Day 28
256 b
256
128
256
10 ng
512
512
512
1024
100 Hg
8
64
512
1024
1000 Hg
<8
256
512
1024
I ug
a M ice were g iven one IP in je c t io n o f 0 .2 5 ml c o n ta in in g th e in d ic a te d amount o f
ovalbum in in s a lin e mixed w ith an equal volume o f a s o lu tio n c o n ta in in g I mg alum.
^Each r e s u lt was o b ta in e d from a serum pool from 4 m ice.
TABLE V l l l
P as s iv e h e m a g g lu tin a tio n a n tib o d y responses t o V ario u s
doses o f ovalbum in adsorbed to alum
Dose o f
ovalbumin®
I 49
Day 10
64b
Day 14
Day 21
Day 28
256
512
256
10 fig
512
1024
1024
1024
100 49
512
512
1024
2048
I 000 4 g
256
512
512
2048
a M ice were g iv e n one IP in je c t io n o f 0 .2 5 ml c o n ta in in g th e in d ic a te d amount o f
ovalbum in in s a lin e mixed w ith an equal volume o f a s o lu tio n c o n ta in in g I mg o f alum.
^Each r e s u lt was o b ta in e d from a serum pool from 4 m ice.
33
moles DNP/mg c a r r i e r gave a t i t e r o f 103.
The a n t I - c a r r i e r r e ­
sponses fo llo w e d th e same p a tte r n .
C e r ta in s tu d ie s on a n ti-h a p te n and a n t i - c a r r i e r
IgE responses
in th e mouse have re v e a le d a dichotomy in these responses.
Sehon (6 2 ) re p o rte d t h a t when mice w ere immunized w ith
Lee and
I uq o f
h e a v ily d in itr o p h e iiy la te d OVA (DNPgg-OVA) w ith alum th e PCA a n t i ­
body response produced could be e l i c i t e d
in th e PCA r e c ip ie n t by
c h a lle n g e w ith DNPgg-OVA but not w ith OVA.
Is h iz a k a and O kudaira (4 2 ) showed a dosage e f f e c t ,
th e c a r r i e r , on a n ti-h a p te n
r e la te d to
IgE responses using a l i g h t l y d i n l t r o -
p h e n y la te d c o n ju g a te o f OVA (DNPg ^-OVA) f o r im m unization.
In
DBA/gJ, m ice which produce high and p e r s is te n t IgE responses to m i­
n u te doses ( 0 . 0 5 - 0 . 2 jxg) o f OVA, th e y showed th a t im m unization w ith
low doses o f DNPg ^-OVA produced high and p e r s is te n t a n t i -DNP r e ­
sponses, whereas h ig h e r doses (10 uq) produced much low er t i t e r s ,
which were t r a n s ie n t .
A nti-O VA PCA responses d id not p a r a l l e l th e a n tl-D N P responses;
a n t I -OVA a n tib o d y fo rm a tio n was p e r s is te n t alth o u g h th e le v e l o f
a n tib o d y was lower a t h ig h e r doses ( 4 2 ) .
c a r r i e r , a t a l l doses, r e s u lte d
to
In a d d it io n , p rim in g w ith
in a secondary a n t i - c a r r i e r response
im m unization w ith DNPg^-OVA whereas th e an tl-D N P response was
enhanced d r a c c e le ra te d when anim als were primed w ith a low dose o f
34
c a r r i e r but suppressed when th e y were primed w ith a high dose ( 4 2 ) .
The a n ti-h a p te n and a n t i - c a r r i e r
to
IgE responses o f B a ib /c mice
im m unization w ith e it h e r DNPq ^-O V A o r DNP2Zfl-OVA w ere compared
using th e h e te ro lo g o u s ( r a t ) PCA a s s a y .
When mice were immunized
w ith DNPq ij-OVA and I mg alum (T a b le IX) t h e i r a n ti-D N P responses
were tr a n s ie n t and in v e rs e ly r e la te d t o dose.
The a n t I -OVA r e ­
sponses were high and p e r s is t e n t , s im ila r to th a t o b ta in e d w ith un­
c o n ju g ated OVA, except t h a t I *zg DNPq ^-OVA produced a 4 - f o l d h ig h ­
e r t i t e r than 100 p g .
When mice were immunized w ith DNPg^-OVA and I mg alum (T a b le X)
th e o b s e rv a tio n o f Lee and Sehon (6 2 ) was confirm ed and extended.
A nim als immunized w ith
1-100 #g o f a n tig e n produced both a n t i- D N P ^ -
OVA and a n ti-D N P responses th a t were high and p e r s is t e n t .
However,
a n im a ls Immunized w ith 1000 pg o f a n tig e n show no d e te c ta b le a n t i ­
body.
Anti-O VA a n tib o d y could not be d e te c te d in any o f th e ex­
p e rim e n ta l groups.
When a 1 /8 d i l u t i o n o f an a n t I -OVA serum th a t had d is p la y e d a
t i t e r o f 256 upon c h a lle n g e w ith ovalbum in, was p a s s iv e ly tr a n s ­
fe r r e d t o a r a t , th e subsequent c h a lle n g e w ith 5 mg DNP2Zt-OVA was
a b le t o e l i c i t a PCA response f o r th a t a n tis e ru m .
th a t a t
T h is
le a s t some o f th e B c e l l a n tig e n ic d e te rm in a n ts
were p re s e n t on th e DNP2Zii-OVA m o le c u le .
in d ic a te s
f o r OVA
35
Immune response to v a rio u s doses o f ovalbum in using f3. p e rtu s s is
as a d ju v a n t
B a lb /c mice were g iv e n one IP In je c t io n o f 0 .2 5 ml o f a s o lu ­
t io n c o n ta in in g
10,
100, o r 1000 tig OVA + 0 .2 5 ml o f B. p e rtu s s is
v a c c in e e q u iv a le n t to 10*0 organism s.
T a b le X I .
The r e s u lts a re shown in
M ice so immunized produced a PHA a n tib o d y , but a p p a re n t­
ly no PCA a n tib o d y .
PCA a n tib o d y responses to o th e r a n tig e n s using alum as a d ju v a n t
O th er a lu m -p r e c ip ita te d substances which were a b le to Induce
PCA a n tib o d ie s , a t days 14 and 2 8 , by im m unization w ith one IP in ­
j e c t i o n w ere:
I)
ragweed a n tig e n a t doses o f I 4 9 ,
10 /tig, and 100
p g , and 2) NRS and normal sheep serum when 0 .2 5 ml o f e it h e r u n d i­
lu te d o r d ilu t e d
1:5 in s a lin e was used.
When u n d ilu te d serum was
used, a la g In th e peak response was seen s im ila r to th a t observed
w ith h ig h doses o f OVA.
Im m unization w ith 10 jizg, 100 jug, or 1000 fig
doses o f o v in e album in o r OGG p r e c ip it a t e d w ith alum d id not p ro ­
duce d e te c ta b le le v e ls o f PCA a n tib o d y .
In d u c tio n o f IqE responses by m u lt ip le In je c tio n s o f a n tig e n w ith ­
o u t a d ju v a n t
It
is g e n e r a lly accepted th a t In o rd e r t o Induce IgE responses
in la b o ra to ry a n im als an " a d ju v a n t" is re q u ire d .
th is
is th e in d u c tio n o f s p e c if ic
An e x c e p tio n to
IgE a n tib o d y as a r e s u l t . o f worm
TABLE IX
IgE a n ti-h a p te n and a n t i - c a r r i e r responses o f B a lb /c mice immunized w ith
l i g h t l y d in itr o p h e n y la te d ovalbum in (DNPq ^-OVA)
PCA t i t e r s *1
A n ti -DNP
Dose o f
a n t i qena
LA
U
N
I Aig
Day 14
A n ti--ovalbum in
Day 36
Day 14
<8
2048
512
Day 36
10 /tig
64
<8
512
256
100 Aig
<8
<8
64
128
aMice were given one IP in je c tio n of 0.25 ml of the indicated dose of DNPq r=0VA in
s a lin e mixed w ith an equal volume of a solution containing I mg alum.
kpCA (p a s s iv e cutaneous a n a p h y la x is ) te s ts were perform ed in r a ts which were c h a l­
lenged IV w ith 5 mg o f e it h e r DNP-BSA o r ovalbum in.
c Each r e s u lt was o b ta in e d from a serum pool from 3 m ice.
TABLE X
IgE a n ti h ap te n -c a rrier conjugate, an ti-h ap ten and a n t i- c a r r ie r responses of Balb/c
mice immunized w ith h e a v ily di n i trophenylated ovalbumin (DNPg^-OVA)
PCA t I t e r s b
A n ti DNPgjt-OVA
Dose o f
a n tig e n 3
A n ti -DNP
Day 14
Day 56
128°
128
256
10 Mg
256
128
100 jug
256
1000 wg
<2
I M9
Day 14
Day 56
A n ti ovalbum in
Day I 4
Day 56
256
<8
<8
256
256
<8
<8
256
>8
128
<8
<8
<4
<4
<4
<8
<8
aH ic e were g iv e n one IR in je c t io n o f 0 .2 5 ml o f th e in d ic a te d dose o f DNPq 5 -OVA in
s a lin e mixed w ith an equal volume o f a s o lu tio n c o n ta in in g I mg alum .
bpCA (p a s s iv e cutaneous a n a p h y la x is ) t e s ts w ere perform ed in r a ts which were c h a l­
lenged IV w ith 5 mg o f e i t h e r DNPgjt -OVA, DNP-BSA o r ovalbum in.
c Each r e s u lt was o b ta in e d from a serum pool from 4 m ice.
TABLE Xl
A n ti-o v a lb u m in responses in m ice immunized w ith v a rio u s
doses o f ovalbum in arid B o rd e te i Ia p e rtu s s is
Day 10
Dose o f
ovalbum in 3
PCA
PHA
CTl
a
O
<4b
100 Hg
1000 fig
Day 21
Day 14
PCA
PHA
PCA
16
<4
NDc
<4
<4
32
<4
ND
<4
128
<4
ND
Day 28
PCA
PHA
16
<4
ND
<4
16
<4
ND
<4
128
<4
ND
PHA
aM ice were g iv e n one IP I n je c t io n o f 0 .2 5 ml ovalbum in in s a lin e mixed w ith an equal
volume EL p e r tu s s is v a c c in e e q u iv a le n t to a p p ro x im a te ly 10 ' " organism s.
^ R e s u lts were o b ta in e d from a serum pool from 3 m ice.
cNot done.
39
in f e c t io n ( 4 0 ) .
One r e s u l t o f l i v e worm in fe c t io n may be th e r e ­
peated re le a s e o f low doses o f worm a n tig e n s d u rin g th e v a rio u s
stag es o f th e in f e c t io n .
to p ic p e o p le .
S im ila r a n t Ig e n lz a tio n may occur in a -
I th e r e fo r e c o n sid ered th e p o s s i b i l i t y th a t re p e a t­
ed Im m unization w ith a n tig e n a lo n e m ight overcome th e requirem ent
f o r a d ju v a n t
in th e in d u c tio n o f IgE responses.
Two d i f f e r e n t
im m unization p ro to c o ls were used.
The f i r s t was
s ta r te d on th e day o f b i r t h and g iv en a p p ro x im a te ly e ve ry 2-3 days
as o u tlin e d
in T a b le X I I .
From th e r e s u l t s ,
it
is a p p aren t th a t a
h e a t l a b i l e , PCA a n tib o d y response could be e l i c i t e d by neonatal Iy
in it i a t e d , m u ltip le ,
IP ,
in je c tio n s o f NRS.
o f p r e c i p i t a t i o n a n tib o d y were a ls o form ed.
ished th e PCA t i t e r ,
R e la t iv e ly high le v e ls
Heat tre a tm e n t d im in ­
but not th e p r e c i p i t i n t i t e r .
However, th e re
seemed to be a p o rtio n o f th e PCA a n tib o d y th a t is r e s is t a n t to
h e a tin g .
A s im ila r response was o b ta in e d when mice re c e iv e d neonatal Iy
i n i t i a t e d m u lt ip le
in je c tio n s o f low doses o f ovalbum in (0 .0 2 -1 0
Mg) a t a p p ro x im a te ly w eekly I n t e r v a l s .
A pool o f serum from 7 mice
gave an a n t i - o v a I bumin t i t e r o f 64 a t day 4 8 , 7 days a f t e r th e la s t
i n je c t io n .
In th e second p ro to c o l th e im m unizations were i n i t i a t e d
a d u lt a n im a ls .
in
A t o t a l o f s ix In je c tio n s o f 0 .2 5 ml was a d m in is ­
40
IP a t v a rio u s
in te r v a ls .
The mice were assayed a t day 18 , 4
days a f t e r th e la s t
in j e c t i o n .
The r e s u lt s , shown in T a b le X l l l
te re d
were s im ila r t o those o b ta in e d w ith n e o n atal Iy i n i t i a t e d
t io n s .
in je c ­
A pool o f serum from group I was te s te d f o r h e at s e n s i t i v i ­
t y ; th e u n tre a te d pool gave a t i t e r o f 128 , whereas th e heated pool
gave a t i t e r o f < 16 ,
a d d it io n a l
I t was o r i g i n a l l y thought th a t by g iv in g an
Im m unization o f OVA + alu m , t o group I I ,
t o s tim u la te
a n ti-o v a lb u m in responses, th e a n t I -NRS response m ight have been po­
te n tia te d .
However, w it h in th e tim e course o f t h is experim ent th is
d id not occur and in f a c t , th e re appears to be a s l i g h t suppression.
T hese r e s u lt s c l e a r l y
in d ic a t e t h a t ,
in m ic e , m u l t i p l e
in je c ­
t io n s o f a n t ig e n , w it h o u t an a d ju v a n t, can e l i c i t PCA resp on se s t h a t
m ost p r o b a b ly a r e o f th e IgE c la s s .
The fo llo w in g experim ents w ere designed t o exam ine,
d e ta il,
th e phenomenon re p o rte d by Maia e t a l . (3 7 )
in more
in which mul­
t i p l e high doses o f a n tig e n , w ith o u t alu m , given IV , re s u lte d
in
th e s e le c t iv e suppression o f IgE responses.
C o n fir m a tio n o f p re v io u s f in d in g s
B a lb /c mice w ere s e n s itiz e d on day 0 w ith one IP in je c t io n o f
10 ug OVA + I mg alum fo llo w e d by a d e s e n s itiz in g tre a tm e n t, on
days 3» 5 , 7 which c o n s is te d o f th e in tra ve n o u s In je c t io n o f e it h e r
s a lin e
(c o n tro l a n im a ls ),
100 fig OVA w ith o u t alum or 1000 pg OVA
41
TABLE Xl I
Immune responses o f mice immunized by n e o n a tal Iy
I n i t i a t e d , m u lt ip le ,
In je c tio n s o f normal
r a b b it serum (MRS) w ith o u t adjuvant®
A n tI-N R S re sp on se s
PCAb
U n tre a te d
H ea te d c
Unheated
H eatedc
32
32
ND
I
64
2
128
i
Mouse
no.
P r e c ip itin
64
3
64
16
64
64
4
64
16
32
32
5
32
4
64
64
6
16
8
64
64
CL
8
aM ice re c e iv e d NRS, IP , acc o rd in g t o th e fo llo w in g sch ed u le: Days
0 , 2 - 0 .0 5 m l; day 5 - 0 .0 7 m l; day 8 - 0 .0 8 or 0 .1 0 m l; days 13 , 18,
2 3 , 2 8 , 3 5 , 3 7 , 4 2 - 0 .1 0 ml (day 0 => day o r b i r t h ) .
They were bled
on day 3 6 .
kpCA = p a s s iv e cutaneous a n a p h y la x is perform ed in r a t s .
cU n d ilu te d serum samples were heated a t 56°C f o r 90 m in u te s.
dNot done.
42
TABLE X l l l
IgE responses o f mice immunized by a d u lt I n i t i a t e d m u ltip le
in je c tio n s o f normal r a b b it serum (NRS) w ith o u t a d ju v a n t
AntI-WRS PCA responses 3
Group I *5
128
128
16
64
Group I l k* c
32
8
32
64
a PCA = p a s s iv e cutaneous a n a p h y la x is perform ed in r a t s .
a r e from sera c o lle c te d on day 18 .
R esu lts
^ H ic e in both groups re c e iv e d 0 .2 5 ml NRS. IP , on days 0 , 2 , 4 , 7 ,
11 and 14 (day 0 =» day o f f i r s t I n j e c t i o n ) .
cMice in group 11 re c e iv e d ,
OVA + I mg alum on day 9 .
in a d d it io n , one IP In je c t io n o f 10 pg
43
w ith o u t alum each day.
These 2 dosages were te s te d because I was
using 10 -1 OOX g r e a te r s e n s it iz in g dose f o r B a lb /c m ice than had
been used in th e o r ig in a l s tu d y .
As can be seen from th e re s u lts
In T a b le X IV , tre a tm e n t w ith both 100 ng and 1000 ng OVA re s u lte d
in th e suppression o f th e PCA response by day 2 1 , w h ile th e group
r e c e iv in g s a lin e had high t i t e r s .
W ith th e 100 ng dose, th e IgE r e ­
sponse is a t near normal le v e ls on day 10 , whereas w ith th e 1000 jug
dose th e response is much lo w e r, p ro b a b ly in d ic a tin g th a t th e sup­
p re s s io n was expressed a l i t t l e
sooner w ith th e h ig h e r dose.
The
PHA response d id not appear t o be s i m i l a r l y a ffe c te d by th e t r e a t ­
m ent.
Subsequent experim ents w ith a s im ila r design w ere c a r r ie d o u t.
100-200 ng OVA f o r th e d e s e n s itiz in g tre a tm e n t was r o u t in e ly used.
From th e r e s u lts o f th e s e experim ents i t can be concluded th a t
d e s e n s it iz a t io n
is a r e p e a ta b le , v a l i d e xp e rim e n ta l f in d i n g .
hundred o r 200 /ug Is e q u a lly e f f e c t i v e
One
in inducing d e s e n s itiz a t io n .
By making comparisons between d i f f e r e n t e xp e rim e n ts ,
i t appeared
t h a t te r m in a tio n o f th e IgE response occurred by day 16 and r e ­
mained a t
le a s t through day 2 4 .
A ls o , th e e xp ressio n o f desensI -
tiz a t .io n appeared to be p ro g re s s iv e .
A t day 9 th e PCA t i t e r s were
n o rm a l, a t day 10 some m ice began t o show a lower PCA t i t e r and by
day 11 most anim als showed s ig n if ic a n t supp ressio n .
M aintenance o f d e s e n s itiz a t io n
When serum from th e anim als discussed above was te s te d a t 77
days a f t e r s e n s i t i z a t i o n , th e PCA responses o f th e groups re c e iv in g
e ith e r
100 fig o r 1000 jzg OVA were s t i l l <8 w h ile th e c o n tro l group
was s t i l l
making PCA a n tib o d y .
was long l i v e d .
T h is suggested th a t th e phenomenon
In a n o th e r e x p e rim e n t,
sponses were te s te d ,
in which In d iv id u a l r e ­
i t was seen t h a t th e d e s e n s itiz e d s t a t e was
n o t u n ifo rm ly m a in ta in e d in a n im als t r e a te d w ith 100 ixg OVA.
c a l r e s u lt s a re shown In T a b le XV.
m a ls , which re c e iv e d m u lt ip le ,
T y p i­
Bt can be seen t h a t in some a n i ­
IV in je c tio n s o f 100 fig OVA, PCA
a n tib o d y was d e te c ta b le on day 52 even though t h e i r response had
p r e v io u s ly dropped to u n d e te c ta b le le v e ls by day 16.
T h is has been
observed in o th e r e x p e rim e n ts .
E f f e c t o f desens I t I z a t ion on IqE a n tib o d y -s e c re tin g c e l l s
In o rd e r to d e te rm in e w hether th e suppression o f IgE a n tib o d y
responses induced by tre a tm e n t w ith m u lt ip le high doses o f a n tig e n
was due t o suppression o f a n tib o d y s e c re tio n a n d /o r fo rm a tio n or to
n e u t r a li z a t i o n o f a n tib o d y by excess a n tig e n , th e WCA t e s t (34)
was em ployed.
For t h i s t e s t , an im a ls were s a c r if ic e d a t v a rio u s
tim es a f t e r s e n s it iz a t io n and s in g le c e l l suspensions o f t h e i r
s p le e n s , thymuses and bone marrows p repared in PBS.
The c e l l p re ­
p a ra tio n s were washed 2X w ith PBS and resuspended t o an a p p ro p ria te
45
-
c o n c e n tra tio n .
made.
•
; ■
.
.
V ia b le c e l l counts by Trypan b lu e e x c lu s io n were
T w o -fo ld d i lu t io n s o f t h i s p re p a ra tio n were made and 0 .1 ml
a liq u o t s were in je c te d
in to th e s k in o f r a ts which were c h alle n g ed
24 h r l a t e r w ith E van's b lu e dye and a n tig e n .
The r e s u lts using c e l l s from s e n s itiz e d anim als a r e shown In
T a b le X V I.
IgE s e c re tin g c e l l s w ere p re se n t in th e spleens and
bone marrows, bu t not th e thymuses o f s e n s itiz e d m ice a t days 17
and 3 2 .
T h is c onfirm ed p re v io u s work ( 3 4 ) .
The r e s u l t s , using
c e l l s from d e s e n s itiz e d a n im a ls , a re shown in T a b le X V i.
i t can
be seen th a t a t th e c e l l c o n c e n tra tio n s , te s te d , no IgE s e c re tin g
c e l l s were d e te c te d in e it h e r s p le e n o r bone marrow.
C o n sid erin g
t h a t th e number o f c e l l s which gave a n e g a tiv e r e a c tio n was 100f o l d g r e a te r f o r sp leen c e l l s and 5-10 f o ld g r e a te r f o r bone marrow
c e ils
than th e le a s t number o f c e l l s , from th e s e n s itiz e d group,
t h a t gave a p o s it iv e r e a c tio n , th e s e r e s u lts s tro n g ly suggested
t h a t th e suppression o f IgE re s p o n s e s ,d u e t o in tra ve n o u s in je c ­
t i o n o f a n tig e n , was th e r e s u lt o f a la c k o f IgE a n t I b o d y -s e c re ting
c e l l s and not to n e u t r a liz a t io n o f a n tib o d y by excess a n tig e n .
■E f f e c t o f g iv in g m u l t i p l e , high doses o f a n tig e n ,w ith o u t alum by
th e IP ro u te
From th e r e s u lts shown In T a b le X V I I ,
I t can be seen th a t when
3 In je c tio n s o f high doses o f a n tig e n a re g iv e n IP in s te a d o f IV ,
46
th e IgE a n tib o d y responses a re o n ly s l i g h t l y d im in is h e d when com­
pared w ith th e responses o f a n im als re c e iv in g th e h ig h .d o ses IV .
T h is
is t r u e when e it h e r
je c tio n s .
These r e s u lts
100 /ug o r 1000 /ug was used f o r th e IP in ­
in d ic a te th a t th e ro u te o f a d m in is tr a tio n
o f th e d e s e n s itiz in g tre a tm e n t was v e ry Im p o rta n t.
Thus, th e e x ­
t e n t o f Suppression by IP in je c t io n was not n e a rly as g r e a t as th a t
Induced by a low dose o f a n tig e n g iv en in tra v e n o u s ly (s e e b e lo w ).
E f f e c t o f g iv in g an o p tim a lly immunogenic dose o f a n tig e n as a desens I t i z a t ion tre a tm e n t
T a b le X V M I shows th e responses o f anim als g iv en 10 <Ltg OVA IV ,
compared t o 100 pg OVA, IV .
I t can be seen th a t 10 ng was a b le to
induce a s ig n if ic a n t suppression o f PCA t i t e r s .
T h is supports th e
h y p o th e s is t h a t an Im p o rta n t re q u ire m e n t f o r d e s e n s itiz a t io n
is th e
ro u te o f in j e c t i o n .
A comparison o f m u lt ip le and s in g le tre a tm e n ts f o r desens 51 5za.t I on
T a b le XIX shows th e e f f e c t o f g iv in g one IV in je c t io n o f 300
Ug o f OVA on e it h e r day 3 , day 5 , o r day 7 .
one s in g le
in je c t io n
I t can be seen th a t
induced a s ig n if ic a n t suppression in PCA t i ­
te r s b u t not as g re a t as th a t o b ta in e d w ith m u lt ip le in je c t io n s .
T h e re d i d n 't seem to be much d if f e r e n c e among s in g le in je c tio n s
g iv e n on d i f f e r e n t days.
47
TABLE XIV
E ffe c t o f in tra v e n o u s , m u lt ip le
in je c tio n s o f s o lu b le a n tig e n on
th e a n t I -ova I bumin responses o f mice s e n s itiz e d p re v io u s ly
w ith an l . p .
in je c t io n o f a n tig e n + alum
Day 10
T reatm en t
Day 0 - Days 3 , 5 , 7
PCA
Sens 111z e d -s a 11nea
512 d
Sens 11 1zed-desens 11 1zed^
100 fig
Sens 111ze d -d e s e n s i 1 1zedc
1000 uq
Day 21
PHA
PCA
PHA
64
256
512
128
512
<4
256
32
512
<4
512
a M ice were s e n s itiz e d on Day 0 by an in tr a p e r ito n e a I in je c t io n o f
0 .2 5 ml ovalbum in in s a lin e mixed w ith an equal volume o f a s o lu ­
t i o n c o n ta in in g I mg alum . On days 3 , 5 , 7 they were given an in ­
traven ous in je c t io n o f s a lin e .
^M ice were tr e a te d as above except th e y were given 100 /ng s o lu b le
ovalbum in in s te a d o f s a lin e on days 3 , 5 , 7 .
cM lce were tr e a te d as above, except th e y were g iv en 1000 ug s o lu ­
b le ovalbum in on days 3 , 5 , 7 .
^ R e s u lts were o b ta in e d from a serum pool from 3 m ice.
48
TABLE XV
E f f e c t o f In tra v e n o u s , m u lt ip le
in je c tio n s o f s o lu b le
ovalbum in on IgE responses
T reatm en t
S e n s itiz e d 3
Day 10
Day 16
Dav 52
256
1024
1024
1024
64
1024
256
512
512
512
1024
256
512
256
512
512
256
256
S e n s itiz e d d e s e n s itiz e d 6
128
128
64
128
256
64
128
32
32
16
8
512
512
512
128
128
256
256
128
512
256
256
256
<8
<8
<8
<8
<8
<8
<8
<8
<8
<8
<8
64
64
32
32
32
32
16
<8
<8
<8
<8
aM ice were s e n s itiz e d on day 0 by an l . p . in je c t io n o f 0 .2 5 ml
ovalbum in In s a lin e mixed w ith an equal volume o f a s o lu tio n con­
t a in in g I mg alum .
ljM lce were tr e a te d as above, except th e y were g iv en th re e l . v .
je c t io n s o f 100 Mg egg album in w ith o u t alum on days 3 , 5» 7 .
In ­
49
TABLE XVI
H eterologous a d o p tiv e cutaneous a n a p h y la x is re a c tio n s
in s e n s itiz e d and s e n s ltlz e d -d e s e n s I t l z e d mice
No. o f c e l l s re q u ire d f o r a p o s it iv e re a c tio n
Day 17
Day 32
S e n s itiz e d 3
Spleen
7 .5
X
105
4 .6 x IO5
Bone marrow
6 .0
X
IO6
2 .5 x IO6
>5
X
IO7
>3 x IO7
Spleen
> 5 .2
X
IO7
> 6 .2 x IO7
Bone marrow
> 3 .2
X
IO7
> 3 .5 x 10?
Thymus
> 4 .5
X
IO7
>2 x IO7
Thymus
Sens i 11 zed-desens 111 zed*1
a M ice were immunized w ith one IP In je c t io n o f 10 fig OVA + I mg alum
on day 0 .
^Mlce were immunized w ith one IP in je c t io n o f 10 ^g OVA + I mg alum
on day 0 and su bsequently g iv en th r e e 100 fig doses o f OVA w ith o u t
alum on days 3 , 5 , 7 .
50
TABLE XVM
E ffe c t o f m u lt ip le ,
ln t r a p e r lt o n e a l
in je c t io n s o f s o lu b le
a n tig e n on a n t i - o v a lb u m in re sp on se s a t day 16
T reatment®
PCA
PHA
S e n s it iz a t io n
+ s a lin e
256
1024
128
2048
256
2048
<4
2048
<4
1024
4
4096
128
1024
64
4096
64
4096
256
4096
64
4096
32
2048
S e n s it iz a t io n
+ 100 ng IV
S e n s it iz a t io n +
100 ng IP
S e n s it iz a t io n +
1000 ng IP
a On day 0 , a l l mice w ere g iv en one IP in je c t io n o f 10 jug OVA + I mg
alu m . On days 3 , 5 , 7 th e y were t r e a te d as In d ic a te d .
i,
51
TABLE XVI I I
E f f e c t o f g iv in g m u lt ip le
in tra ve n o u s
in je c t io n o f low doses
o f s o lu b le a n tig e n on d e s e n s itiz a t io n
A nt?-ova I bumIn responses^
Treatm ent®
S e n s it iz a t io n
+ 100 ug IV
S e n s it iz a t io n
+ 10 fig IV
PHAd
Exp. I
512
256
256
ND
ND
ND
Exp. 2
256
128
256
1024
2048
2048
CX
a
S e n s it iz a t io n
+ s a lin e
PCAc
<4
<4
<4
ND
ND
ND
Exp. 2
<4
<4
<4
2048
1024
4og6
Exp. I
64
32
16
ND
ND
ND
Exp. 2
8
16
32
4096
4096
4096
aA l l m ice were s e n s itiz e d on Day 0 w ith one IP in je c t io n o f 10 ixg
OVA + I mg alum . On days 3* 5 , 7 th e y were given in traven o u s in ­
je c t io n s o f e it h e r s a l i n e , 100 /zg OVA o r 10 /ug OVA.
^ R e s u lts a r e from In d iv id u a l mice b le d on day 16 (E xp.
(E xp . 2 ) .
c PCA = p a s s iv e cutaneous a n a p h y la x is .
clPHA = p a s s iv e h e m a g g lu tin a tio n .
I)
o r 19
52
TABLE XIX
A comparison o f th e e f f e c t o f m u lt ip le In je c tio n s and a s in g le
i n je c t io n o f ovalbum in on d e s e n s itiz a t io n
T reatm ent3
D av(s) o f tre a tm e n t
PCA responses
on day I 6
S a lin e
3, 5, 7
512,
128, 256
100 jug OVA
3, 5, 7
< 4 , < 4 , <4
300 jug OVA
3
6 4 , 16, 16
300 jug OVA
5
3 2 , 6 4 , ND
300 ug OVA
7
128, 3 2 , 8
aA l l tre a tm e n ts were g iv e n IV .
On day 0 , a l l mice were g iv en one
IR In je c t io n o f 10 jug OVA * I mg alu m ,
.
53
E f f e c t o f g SvSng o th e r p ro te in s o r h a p te n -p ro te in co n ju g ates
in -
t ravenous Iy
In o rd e r to d e te rm in e w hether th e suppression observed in sen­
s i t i z e d mice th a t were g iv en th r e e subsequent IV in je c tio n s o f 100
Ug OVA m ight p o s s ib ly be due t o a n o n -s p e c ific e f f e c t , perhaps
s im ila r t o t h a t observed w ith m u lt ip le
substances were in je c te d
in je c tio n s o f MRS, o th e r
IV In a s im i l a r p r o to c o l.
The r e s u lt s ,
shown in T a b le XX, showed th a t n e ith e r 100 ng sheep album in nor
100 fig DNP-KLH had any s u p p res s ive e f f e c t on th e PCA responses of
m ice p re v io u s ly s e n s itiz e d w ith 10 jug OVA + I mg alum .
were e q u iv o c a l when 100 jug DNPg^-OVA was g iv e n .
The r e s u lts
One mouse out of
th r e e showed a s ig n if ic a n t suppression o f PCA a c t i v i t y , whereas th e
mean PCA v a lu e f o r th e o th e r two was o n ly one tube low er than th a t
f o r th e a n im als g iv e n no tre a tm e n t.
However, even DNPg^-OVA was
not a b le t o suppress th e PCA responses t o th e e x te n t t h a t 100 jug
OVA c o u ld .
E f f e c t o f desens i t I z a t ion tre a tm e n t on secondary IgE responses
The e f f e c t o f d e s e n s itiz a t io n on a subsequent IgE secondary
response was te s te d by g iv in g a second IP In je c t io n o f 10 jug OVA +
I mg alum t o s e n s itiz e d and s e n s itiz e d -d e s e n s itiz e d m ice .
The sec­
ond Im m unization was a d m in is te re d a t two d i f f e r e n t In t e r v a ls a f t e r
th e p rim a ry I n j e c t i o n .
The r e s u lts a re shown In T a b le X X I.
When a
TABLE XX
E f f e c t o f a d m in is te rin g o th e r p ro te in s o r h a p te n -p ro te in conjugates
in tra v e n o u s ly on th e a n t i - o v a lbumln PCA responses o f mice
p re v io u s ly s e n s itiz e d w ith alum -adsorbed ovalbum in
T re atm en t3
on days 3 , 5 , 7
PCA responses*’
on day 16-21
Experim ent I
256
S a lin e
<8
100 ng OVA
256
100 ng sheep album in
Experim ent 2
S a lin e
100 Hg OVA
100 Hg DNP2lt-OVA
100 Hg DNP-KLH
128, 5 1 2 , 256
< 4 , < 4 , <4
128,
128, 32
5 1 2 , 5 1 2 , 512
3On day 0 , a l l m ice re c e iv e d 10 Hg OVA + I mg alu m , IP . On days 3 ,
5 , 7 th e y re c e iv e d , in tra v e n o u s ly , th e In d ic a te d substances.
^ R e s u lts f o r e xperim ent I a r e from a serum pool from 3 m ice c o l le c t
ed on day 16. R e s u lts f o r exp erim en t 2 a re from in d iv id u a l serum
samples c o lle c te d on day 2 1 .
secondary im m unization was g iv en to s e n s itiz e d a n im als on day 29
(Group I )
th e re was an 8 - f o l d
in c re a s e in RCA t i t e r a t f i r s t , which
i
decreases one d i l u t i o n
l a t e r on.
m in is te re d on day 50 (Group I I I )
te r.
When th e second c h a lle n g e was ad­
th e re was a 4 - f o l d
In c re a s e in t i ­
I do not c o n s id e r th a t th e s e in creases a re d i f f e r e n t from each
o th e r , however, th e y do re p re s e n t s ig n if ic a n t in c re a se s when com­
pared t o th e group th a t d id not r e c e iv e a second im m unisation (Group
II).
On th e o th e r hand, th e p a tte r n o f secondary responses was
somewhat d i f f e r e n t in sens 111ze d -d e s e n s i t Ized mic e .
In Group IV ,
w hich re c e iv e d a second im m unization on day 2 9 , th e PCA Increased
by o n ly one d i l u t i o n , whereas in Group V I , which re c e iv e d a second
im m unization on day 5 0 , th e PCA t i t e r
th e t i t e r was s t i l l
s ig n if ic a n tly
sponse in th e s e n s itiz e d group.
increased 3 2 - f d l d .
However,
lower than th e re s p e c tiv e r e ­
In Group V , which d id not re c e iv e
any secondary im m u n izatio n , th e PCA t i t e r
Increased s l i g h t l y be­
tween days 29 and 5 0 .
Thus,
i t appears t h a t .
In d e s e n s itiz e d a n im a ls , th e e x te n t o f
in c re a s e in secondary PCA t i t e r s depends on when th e second immuni­
z a tio n was g iv en in r e la t i o n t o th e I n i t i a l s e n s it iz a t io n and desens itlz a tlo n
tre a tm e n t.
The secondary response was h ig h e r , when th e
t im e .i n t e r v a l was In creased between d e s e n s itiz a t io n tre a tm e n t and
secondary c h a lle n g e .
T h is was, a p p a r e n tly , not tr u e f o r anim als
56
which had been s e n s itiz e d o n ly .
The PHA re sp o n se s d id n o t seem t o be s i m i l a r l y
( d a ta n o t sh o w n ).
A l l g ro u p s r e c e iv in g a s e c o n d a ry c h a lle n g e
showed a s i g n i f i c a n t
c re a s e was s i m i l a r
in flu e n c e d
in c re a s e in t i t e r and th e e x te n t o f t h a t in ­
in a l l c a s e s .
S u p p re ss ive e f f e c t o f sp leen c e l l s from s e n s itiz e d -d e s e n s itiz e d
an im als
Experim ents were undertaken t o see i f th e t r a n s f e r o f spleen
c e l l s from s e n s itiz e d -d e s e n s it iz e d a n im als to normal anim als would
suppress th e subsequent IgE response o f th e r e c i p i e n t .
Two s ep a r­
a t e experim ents were s e t up.
In e xperim ent I , 2 groups o f donors
were s e n s itiz e d on day 0 w ith
10 ^g OVA + I mg alum .
7 one group re c e iv e d an in traven o u s
On days 3» 5 ,
In je c t io n o f 100 %g OVA a lo n e .
A t day 18, th e a n im als were b le d and t h e i r sera assayed f o r PCA
a c tiv ity .
A t day 19, 4 anim als from th e s e n s itlz e d -d e s e n s itIzed
group which had u n d e te c ta b le le v e ls o f PCA a n tib o d y (< 4 ) and 4 a n i ­
mals from th e s e n s itiz e d group (PCA > 64) were s a c r if ic e d and s in g le
cell
suspensions o f t h e i r spleens w ere prepared in PBS c o n ta in in g
1% f e t a l c a l f serum.
A f t e r I wash In t h is medium, th e c e l l s from
each group were pooled and a v ia b le count was made.
2 .5 x 10® c e l l s
in je c te d
A p p ro x im ately
in a volume o f 0 . 3 5 - 0 .5 0 m l, from each group, were
IV in to normal r e c ip ie n ts which were immunized th e f o llo w -
57
TABLE XXI
Secondary responses o f mice d e s e n s itiz e d w ith m u lt ip le ,
high doses o f ovalbum in g iv e n In tra v e n o u s ly
Group
I
T reatm en t
Day o f second
im m unization^
S e n s it iz a t io n 8
29
PCA a n t i - o v a I bumin responses
Day 29
Day 36
Day 50
Day 57
256d
2048
1024
1024
11
SI
NONE
256
512
512
512
111
Il
50
512
512
512
2048
29
64
128
128
128
NONE
<4
8
16
16
50
<4
16
16
512
IV
S e n s it1 z a tio n -b
desens i t I z a t ton
"
V
Vl
Il
aA l l m ice re c e iv e d 10 Hg OVA *
I mg alu m .
bA I l m ice re c e iv e d 10 jitg OVA + I mg alum ,
OVA, w ith o u t alum , IV,, on days 3 , 5 , 7 .
IP , on day 0 .
IP , on day 0 and 100 Jjtg
c H lc e re c e iv e d e it h e r s a lin e or 100 txg OVA + I mg alu m , IP , on th e
day in d ic a te d .
R e s u lts a r e from a serum pool from 3 m ice.
58
Ing day w ith
10 ng OVA + I mg alum .
The a n t I -OVA IgE a n tib o d y r e ­
sponses o f th e r e c ip ie n ts a r e shown In T a b le X X II.
I t can be seen
t h a t th e PCA responses o f a n im als r e c e iv in g d e s e n s itiz e d c e l l s a re
s ig n if ic a n tly
c e lls .
lower than those o f a n im als r e c e iv in g s e n s itiz e d
When th e means o f th e Iogg v a lu e s f o r th ese two groups
w ere a n a ly ze d by S tu d e n t's t t e s t , th e d iffe r e n c e s between th e two
groups w ere s t a t i s t i c a l l y s ig n if ic a n t
(p < 0 . 05 ) .
In th e second e x p e rim e n t, 3 groups o f donors were used.
groups w ere primed on day 0 w ith
5 , 7 one group re c e iv e d
10 pg OVA + I mg alu m .
On days 3 ,
IV in je c tio n s o f 100 ^g OVA and a n o th e r
group re c e iv e d IV In je c tio n s o f 1000 iug OVA.
c e iv e d no tre a tm e n t.
A ll
The t h i r d group r e ­
A t day 19, th e spleens and thymuses o f a l l 3
groups were prepared in a manner s im i l a r t o th a t d e s c rib e d above.
F o r each group o f donors, a s p leen c e l l suspension c o n ta in in g 2 .5
x 10® o r 2 .5 x 10? c e l l s
in 0 .5 ml was prepared and in je c t e d ,
In to normal r e c ip ie n t s .
A d d i t i o n a l l y , a n o th e r group o f r e c ip ie n ts
IV ,
O
re c e iv e d an IV i n je c t io n o f 2 x 10° thymocytes from th e same group
o f donors.
The a n t I -OVA PCA responses o f r e c ip ie n ts re c e iv in g
s p lee n c e l l s a r e shown in T a b le X X iI
(E xperim ent 2 ) .
The PCA r e ­
sponses o f a n im als re c e iv in g 2 .5 x 10® sp leen c e l l s from anim als
t r e a te d w ith
1000 ug OVA were 8 - f o l d
low er than those o f anim als
r e c e iv in g th e same number o f s e n s itiz e d c e l l s .
When 1 0 -fo ld less
59
c e l l s from th e same donors were t r a n s f e r r e d , th e PCA responses were
4 -fo ld
low er than those o f s e n s itiz e d a n im a ls , however th e re were
o n ly 2 a n im als l e f t
in t h is e x p e rim e n ta l group.
O
The PCA responses o f th e 2 m ice re c e iv in g 2 .5 x 10
from donors d e s e n s itiz e d w ith
sp leen c e l l s
100 jug doses o f OVA w ere s im ila r to
th o se o f e xp e rim en t I and s i g n i f i c a n t l y
m ice r e c e iv in g s e n s itiz e d c e l l s .
lower than th e responses o f
When 1 0 -fo ld les s c e l l s from t h is
group w ere t r a n s f e r r e d , th e s u p p res s ive e f f e c t was not seen.
o
When 2 x 10
thymocytes from each o f th e th re e groups were
tr a n s fe r r e d th e PCA responses o f serum pools from 3 r e c ip ie n ts fo r
each gro u p , were id e n t ic a l
(PCA t i t e r ° 256 in a l l 3 c a s e s ).
These r e s u lts showed th a t th e suppression o f IgE by 3 ,
IV in ­
je c t io n s o f a n tig e n was t r a n s f e r r a b le to normal m ice by spleen
c e l l s , but not by thym ocytes.
-
:
60
.
■
TABLE XXI I
S u p p ressive e f f e c t s o f sp leen c e l l s from d e s e n s itiz e d anim als
on IgE a n t i - o v a lbumin responses when tr a n s fe r r e d
t o norm al, syngeneic mice
T re atm en t o f donors
o f s p lee n c e l Is a
No. c e l l s
t r a n s fe r r e d
PCA responses o f r e c ip ie n ts ^
'_______ Day 14-15__________
S e n s itiz e d
2 .5 x 10®
256, 512, 256
Sensi t iz e d -d e s e n s it iz e d -100 *tg
2 .5 x 10®
Experim ent I
32,
128 , 64
Experim ent 2
S e n s itiz e d
S e n s itiz e d -d e s e n s it iz e d -100 ug
S e n s it iz e d -d e s e n s it iz e d -
1000 ng
2 .5 x 10®
5 1 2 , 5 1 2 , 512
2 .5 x IO 7
5 1 2 , 5 1 2 , 512
2 .5 x 10®
128, 6 4 , ND
2 .5 x IO 7
2 56 , 2 5 6 , 512
2 .5 x 10®
6 4 , 6 4 , 64
2 .5 x IO 7
128,
128,
128
aA l I m ice w ere g iv en 10 tig OVA + I mg alu m , IP , on day O0 In th e
s e n s itiz e d -d e s e n s it iz e d groups, m ice w ere g iv en 3 , IV in je c tio n s
o f th e in d ic a te d doses o f OVA on days 3 , 5 , 7 .
®0ne day a f t e r c e l l t r a n s f e r , a l l
+ I mg alu m , IP .
r e c ip ie n ts re c e iv e d 10 ng OVA
DISCUSSION
The s t u d ie s on th e h e te ro lo g o u s
( r a t ) PCA t e s t c o n firm e d p re ­
v io u s f in d i n g s and h e lp e d e s t a b lis h th e v a l i d i t y o f t h i s a ssay f o r
mouse Ig E e
In a d d i t i o n , th e
I n h i b i t o r y e f f e c t o f d i l u t i o n on th e
h e a t s e n s i t i v i t y o f th e m ast c e l l b in d in g a b i l i t y
was c o n firm e d and e x te n d e d .
o f PCA a n tib o d y
In th e s tu d y by C la u se n e t a l .
th e PCA a n tib o d y re s p o n s e was Ind u ced by im m u n iz a tio n w it h
(5 5 )
125
ug
OVA p lu s a s a lin e e x t r a c t o f B. p e r t u s s is and a ssa ye d by th e hom olo­
gous PCAe
My s t u d ie s e x te n d e d t h i s o b s e r v a tio n t o PCA a n tib o d y i n ­
duced by im m u n iz a tio n w it h a lu m -a d s o rb e d a n tig e n and a ssa ye d by th e
h e te ro lo g o u s PCA a s s a y .
a 30 m in h e a tin g t im e .
A ls o , s in c e C la u sen e t a l .
(5 5 ) em ployed
I t m ig h t be a rg u e d t h a t by d i l u t i n g
o u t th e
a n t ib o d y , a lo n g e r h e a tin g tim e w o u ld be r e q u ir e d t o a c h ie v e th e
same e f f e c t .
My s t u d ie s showed t h a t even when th e h e a tin g tim e
was e x te n d e d t o 90 m in th e e f f e c t was s t i l l
se e n .
A re c e n t paper (6 3 ) has re p o rte d th a t th e re a r e 2 a n tib o d ie s
in mouse a n tis e ru m , a c t iv e in th e h etero lo g o u s ( r a t ) PCA assay when
a 2 h r l a t e n t p e rio d was used, one h e a t -s ta b le and one h e a t - l a b i l e .
(The h e a t -s ta b le a n tib o d y d id not re a c t w ith e it h e r a n ti-Ig G ^ or a n t i
Ig G g .)
T h is c o n c lu s io n was based on I ) a la c k o f c o r r e la t io n o f 48
h r h o m ocytotropic a n tib o d y a c t i v i t y w ith 2 h r h e te r o c y to tr o p ic ( r a t )
a c t i v i t y , 2 ) th e presence o f a h e at r e s is t a n t a n tib o d y t h a t is more
a c t iv e In th e h e te ro lo g o u s assay and 3 ) th e presence o f h e te r e c y to -
62
t r o p ic
( r a t ) a n tib o d ie s
a n tib o d y a c t iv e
in mouse a n tis e ru m known not t o c o n ta in PCA
in th e 48 hr homologous a ss a y.
I t should be noted
th a t in th e s e s tu d ie s , th e h e at tre a tm e n t was a p p lie d t o a 1:5 d i l u ­
t i o n o f serum which could account f o r th e presence o f th e 1'h e a t-r e ­
s is t a n t " a n tib o d y in some o f th e a n tis e r a te s te d .
The d iffe r e n c e s
between th e 48 h r h cm ocytotropic and 2 h r h e te r o c y to tr o p ic a n tib o d y
w ere n o t more than 2 -4 f o l d .
r e s is t a n t PCA a n tib o d y
The a p p are n t g r e a te r amount o f heat
in th e 2 h r h etero lo g o u s assay when compared
w ith th e 4 8 hr homologous assay can be accounted f o r by th e d i f f e r ­
ence in tim e .
I t has been shown t h a t a lth o u g h th e h a l f - l i f e o f IgE
a n tib o d y in th e s k in was a p p ro x im a te ly 7 -1 4 days ( I ,
6 4 ) , th e g r e a t­
e s t r a t e o f decrease occurred w ith th e f i r s t 3 - 4 days ( I ) .
The p re ­
sence o f a PCA a n tib o d y In serum from SJL m ice, th a t was a c t iv e in
.
th e 2 h r h e te ro lo g o u s bu t not th e 4 8 h r homologous t e s t and which
appears t o be t o t a l l y h e a t r e s i s t a n t ,
is n o te w o rth y .
However, th ese
in v e s tig a to r s f a i l e d t o compare 48 h r homologous assay r e s u lts w ith
4 8 hr h e te ro lo g o u s assay r e s u l t s .
l a t e n t p e rio d was employed.
In a l l o f my s tu d ie s , a 24 hr
I b e lie v e th a t o th e r s tu d ie s (4 4 , 4 5 ) ,
as w e ll as my own, have e s ta b lis h e d th e v a l i d i t y o f th e h e te r o lo ­
gous ( r a t ) PCA assay t o measure mouse IgE a n tib o d y .
The a m e lio r a tio n o f th e PCA le s io n caused by IgE a n tib o d y ,
in
ICR m ice , due t o tre a tm e n t w ith an a n t i- h e lm in t h ic a g e n t has Im p o r-
63
tanfc c o n s id e ra tio n s f o r th e use o f th e PCA assay.
It
is v e ry l i k e ­
ly th a t th e poor PCA r e a c t i v i t y observed b e fo re tre a tm e n t was due
t o a c o m p e titiv e
i n h ib it io n o r b lo c k in g o f th e b in d in g o f th e pas­
s iv e l y tr a n s fe r r e d
worm I n f e c t io n .
IgE by endogenous IgE produced as a r e s u lt o f
Is h iz a k a e t a l .
(6 5 ) have shown, v e ry c o n v in c in g ly ,
t h a t when p e r ito n e a l mast c e l l s were incubated w ith
'^ ! - la b e le d
r a t E myeloma p r o t e in , th e number o f IgE m olecules combined w ith
mast c e l l s
Is o la te d from N. b ra s H I e n s l s
In fe c te d r a t was about 10%
o f t h a t bound to mast c e l l s from normal r a t s .
The d e te c tio n o f p Inworms Sn th e e xp e rim e n ta l a n im als b e fo re
tre a tm e n t suggests s tro n g ly th a t t h i s p a r a s ite was re s p o n s ib le fo r
th e e f f e c t seen.
A lthoug h i t has not been re p o rte d ,
i t seems re a ­
so n ab le t o assume t h a t , based on o th e r p a r a s ite systems ( 66 ) ,
fo rm a tio n occurs as a r e s u lt o f p Inworm In f e c t io n .
It
IgE
Is f e l t th a t
th e o b s e rv a tio n s re p o rte d h e re may re p re s e n t an extrem e case f o r
th e fo llo w in g reasons:
I)
th e anim al c olony from which th e e x p e r i­
m ental a n im als were o b ta in e d was n e ith e r m onitored nor tr e a te d f o r
p inworms; 2 ) because r e t i r e d fe m a le breed mice were used, th e worm
burden and hence, t h e i r
IgE le v e ls may have been e x c e p tio n a lly high
due to th e In h ib it o r y e f f e c t s o f pregnancy and l a c t a t io n on worm e x ­
p u ls io n ( 67 ) .
The o b s e rv a tio n th a t measurements o f PCA a c t i v i t y may be i n -
64
f Iuenced by p inworm in fe c t io n
is e x tre m e ly Im p o rtan t c o n s id e rin g
th e u b iq u ity o f pinworm In fe c tio n s
In anim al c o lo n ie s .
However,
some commercial anim al s u p p lie rs do m o n ito r t h e i r c o lo n ie s f o r
p I nworms and a p p ly a p p r o p r ia te means t o c o n tro l th e in fe c t io n s .
The suppression o f IgE a n tib o d y fo rm a tio n by n e o n a ta lIy
in i­
t i a t e d tre a tm e n t w ith a n t i - u a n tis e ru m p ro v id es c o n v in c in g evidence
th a t
Ig E -fo rm in g c e l l s ,
l i k e those producing IgM,
Ig A , a r is e from IgM b e a rin g p re c u rs o r® I c e l l s .
c o u rs e , t h a t th e a n t I
Ig G j,
It
IgGg and
Is p o s s ib le , o f
ser um used in th e s e s tu d ie s c o n ta in ed un­
d e te c te d tra c e s o f a n t i - £ a c t i v i t y which m ight be re s p o n s ib le f o r
th e observed s u p p res s io n .
T h a t s it u a t io n seems u n l i k e l y , however,
in v ie w o f th e degree o f p u r it y o f IgM used to induce fo rm a tio n o f
th e a n tis e ru m and th e subsequent e x te n s iv e a b s o rp tio n o f t h i s a n t i ­
serum w ith o th e r im m unoglobulins.
e f f e c t is
I t seems more l i k e l y th a t th e
in f a c t due t o a n t i a n t i b o d i e s .
p re s s io n o f IgE re sp o n s iv en e s s,
lik e
I t may be th a t sup­
IgA s u p p res s io n , re q u ire s a
r e l a t i v e l y high le v e l o f s u p p re s s iv e tre a tm e n t.
T h is could account
f o r th e la c k o f IgE suppression observed by Dwyer e t a l . ( 6 0 ) .
The supp ressio n o f anti-O V A responses by n e o n a ta lIy
NRS tre a tm e n t proved t o be v a r ia b le
known what in flu e n c e s t h i s .
It
in itia te d
in I t s exp re s sio n and i t
is not
Is c le a r however, t h a t th e e f f e c t
Is not r e s t r i c t e d t o th e PCA response b u t extends t o th e PHA response
65
in a p a r a l l e l m anner.
g e n ic c o m p e titio n
th e c e i l s
T h is e f f e c t may be due t o some ty p e o f a n t i ­
(6 8 ) o r t o a d i r e c t e f f e c t , p erha p s c y t o t o x ic , on
r e q u ir e d f o r th e g e n e r a tio n o f an immune re s p o n s e .
t h e r s tu d y
F u r­
is needed. -
The r e s u lts o f th e s tu d ie s on im m unization o f B a lb /c mice w ith
one IP in je c t io n o f low doses ( I and 10 jug) o f OVA and I mg alum
c o n firm ed th a t t h is procedure was a h ig h ly r e l i a b l e means o f induc­
ing h ig h and p e r s is te n t le v e ls o f IgE a n tib o d y .
The r e s u lt s , when
h ig h e r doses o f OVA (100 and 1000 ug) w ith I mg alum were used,
showed th a t t h is procedure a ls o Induced a high and p e r s is te n t IgE
response.
These r e s u lts d i f f e r e d from th a t o f Vaz e t a t .
( 2 5 ) , who
showed t h a t h ig h doses o f OVA Induced a low and tr a n s ie n t IgE r e ­
sponse.
The d iffe r e n c e s observed, may be due to d iffe r e n c e s
s t r a in s o f mouse used a n d /o r t o d iffe r e n c e s
used.
Vaz e t a l .
In th e
in th e amount o f alum
(2 5 ) used SW- 5 5 , SWR/J and C57BL/6J mice and 10-
20 mg alum w ith h ig h e r doses o f OVA whereas I used B a lb /c mice and
I mg alum .
From in fo rm a tio n in th e Handbook o f E xperim en tal
munology ( 69 )
i t appears th a t a p p ro x im a te ly 50 49 o f p r o te in
maximum amount th a t can be p r e c ip it a t e d by I mg alum .
when I
Immunized w ith
im­
is th e
T h e re fo re ,
100 o r 1000 yg o f OVA + I mg alu m , th e a n i­
mals were p ro b a b ly re c e iv in g a p p ro x im a te ly 50 49 o f alum p r e c ip i­
ta te d a n tig e n + 50 o r 950 pg o f n o n -p re c Ip it a t e d a n tig e n .
66
Presumably th e n , th e alum p r e c ip it a t e d a n tig e n remained as a
depot w it h in th e a n im a l, whereas th e r e s t o f the a n tig e n , th e nonp r e c ip it a t e d p o r t io n , a c te d as any o th e r s o lu b le a n tig e n and be­
came a v a i l a b l e to th e c i r c u l a t i o n , c r e a tin g a high c ir c u l a t i n g con­
c e n t r a t io n o f a n tig e n , which was then m e ta b o lize d and e x c re te d nor­
m a lly .
T h e r e fo r e , th e a c tu a l dose o f a n tig e n th a t p e rs is te d w ith in
th e depot was p ro b a b ly about 50 u g f no m a tte r how much more was
g iv e n i n i t i a l l y w ith
I mg alum .
T h is may not have been enough an­
tig e n to a c h ie v e th e ty p e o f high dose re g u la tio n observed by Vaz
et a l.
(2 5 ).
The d iffe r e n c e s observed between IgE a n t I -DNP responses p ro ­
duced in response t o e it h e r l i g h t l y d in ltro p h e n y la te d OVA o r h e a v i­
ly d in itr o p h e n y la te d OVA a re in t e r e s t in g .
Immunogenic doses o f
DNP0 tj-OVA induced a tr a n s ie n t IgE a n t i -DNP response and th e t i t e r s
were in v e rs e ly r e la te d t o dose w h ile immunogenic doses o f DNPg^-OVA
induced a high and p e r s is te n t response.
T h is d if f e r e n c e between th e tr a n s ie n t and p e r s is te n t n a tu re o f
th e responses is p ro b a b ly b e st e x p la in e d on th e b a s is o f s tu d ie s by
K laus and M it c h e ll
( 69 )
In which th e y showed a d i s t i n c t d iffe r e n c e
In th e m etabolism o f DNPg-BSA and DNPgQ-BSA.
DNPgQ-BSA p e r s is t e d ,.
In v iv o , f o r long p e rio d s o f tim e , was re ta in e d m a in ly in th e spleen
and l i v e r and was e f f i c i e n t l y ta k en up by macrophages, in v i t r o .
67
b u t degraded v e ry sllowHy.
ONPg-BSAs on th e o th e r hand was e x c re te d
r a p id ly , was more b ro a d ly d is t r ib u t e d
in v a rio u s organs and was not
ta k e n up as e f f i c i e n t l y by macrophages as DNPg0-BSA.
Thus, i t
is
th o u g ht th a t perhaps th e IgE responses produced as a r e s u lt o f im­
m u n iz a tio n w ith DNPq
not r e ta in e d
jj-OVA
a re tr a n s ie n t because t h is a n tig e n is
In v iv o f o r v e ry long and th e r e fo r e is not a v a ila b le
t o p ro v id e a c o n s ta n t s tim u lu s f o r
IgE B c e l l s .
An a d d itio n a l e x p la n a tio n , th e fo rm a tio n o f suppressor T c e l l s ,
may be c o n s id e re d .
Recent work by Is h iz a k a and Adachi
(2 2 ) showed
t h a t th e Jji v i t r o c u lt u r e o f s p le n ic lym phocytes, w ith a n tig e n ,
th e absence o f macrophages, re s u lte d
in
In th e fo rm a tio n o f T sup­
p re s s o r c e l l s f o r IgE and IgG a n ti-h a p te n responses whereas c u l­
t u r in g on a n tig e n b e a rin g macrophages re s u lte d
T h e lp e r c e l l s .
in th e fo rm a tio n o f
Thus, suppressor T c e l l s may be-form ed as a r e s u lt
o f im m unization w ith DNP0 ^g-OVA b u t not w ith DNPg^-OVA because o f
th e d iffe r e n c e s
In th e e f f e c t on macrophages between th e two con­
ju g a te s .
One o th e r f a c t o r th a t m ight be in flu e n c in g th e responses to th e
DNP-c o n ju g a te s may be r e la t e d t o th e d iffe r e n c e s
ses o f a n tib o d y induced by th ese a n tig e n s .
in th e o th e r c la s ­
Klaus and Cross (7 0 )
showed th a t DNPg-BSA e l i c i t e d m a in ly IgG a n tib o d y and immunological
memory w h ile DNPg0 -BSA Induced a p rim ary IgM response, l i t t l e
IgG
68
a n tib o d y and poor memory.
As mentioned above, a n t ig e n - s p e c if ic
enhanced IgE fo rm a tio n in th e r a b b it (3 1 ) and a n t ig e n - s p e c if ic
suppressed IgE fo rm a tio n in th e r a t ( 2 6 ) .
c a r e fu l
IgM
IgG
However, one must be
in a p p ly in g o b s e rv a tio n s on IgE fo rm a tio n in one species to
a n o th e r ( 4 ) .
A ls o th e r e g u la to ry e f f e c t o f p a s s iv e ly a d m in is te re d
IgG a n tib o d y on IgE fo rm a tio n in m ice has been shown t o be d i f f e r ­
e n t from th a t observed in th e r a t ( 7 1 ) .
f a c to r s
I t may be t h a t a l l o f these
in flu e n c e th e IgE responses t o th e d i f f e r e n t DNP-OVA c o n ju ­
g a te s ; however f u r t h e r work Is r e q u ire d .
The dichotom y In a n ti-h a p te n and a n t i - c a r r i e r
observed by o th e rs
(4 2 , 58) was c o n firm e d .
IgE responses
Mice Immunized w ith
DNP0 i 5 -OVA produced high and p e r s is te n t a n t I -OVA IgE responses.
It
may appear th a t t h is response c o n tr a d ic ts th e e x p la n a tio n advanced
above f o r th e tr a n s ie n t n a tu re o f th e a n t I -DNP response because i t
im p lie s th a t a n tig e n
macrophages.
is re ta in e d j_n v iv o and p ro b ab ly processed by
However,
I t seems p ro b a b le th a t a t such a low d e n s ity
o f hapten c o n ju g a tio n , th e re a re many OVA m olecules which do not
c o n ta in any DNP groups and hence would be processed as " n a tiv e " alum
p r e c ip it a t e d OVA and produce a t y p ic a l
o f DNP24~0VA t o induce an anti-O V A
IgE response.
The f a i l u r e
IgE response can b e s t be ex­
p la in e d by a masking o f o r a c o n fo rm a tio n a l change in th e n a tiv e
OVA d e te rm in a n ts .
69
The la c k o f a d e te c ta b le PCA a n tib o d y response in m ice I immu­
n iz e d w ith OVA and B_0 p e rtu s s is v a c c in e was s u r p r is in g because o f
p re v io u s work In which t h is a d ju v a n t fa v o re d th e p ro d u c tio n o f IgE
(4 ).
However, 1$, p e rtu s s is vac c in es a r e a p p a re n tly u n r e lia b le fo r
t h i s purpose and o n ly c e r t a in p re p a ra tio n s a re a b le t o induce IgE
fo rm a tio n t o s p e c if ic a n tig e n s
(4 ).
The in d u c tio n o f IgE responses w ith o u t a d ju v a n t, by m u lt ip le
in je c tio n s o f a n tig e n has not been re p o rte d p re v io u s ly f o r la b o ra ­
to r y a n im a ls .
T h is
Is a s ig n if ic a n t o b s e rv a tio n because i t may
re p re s e n t a model f o r
IgE a n tib o d y in d u c tio n th a t Is more a k in to
th e means by which a l l e r g i c
s in g le
th is
in d iv id u a ls become s e n s itiz e d than th e
in je c t io n o f a low dose o f alum -adsorbed a n tig e n .
regimen Induced high IgE responses.
c ip itin
Although
I t a ls o Induced high p re ­
responses, when MRS was used, which is not observed in th e
a to p ic c o n d itio n ,
I b e lie v e th a t I f much lower doses o f a n tig e n
a r e used then th e p a tte r n o f a n tib o d y responses w ith re g ard to im­
m unoglobulin c la s s would be a lt e r e d such th a t th e p r e c i p i t i n r e ­
sponse would be c o n s id e ra b ly d im in ish ed and th e IgE response en­
hanced.
I s p e c u la te t h a t ,
In o rd e r t o induce IgE a n tib o d y fo rm a tio n ,
re p ea te d s tim u la tio n o f th e c e l l s
T o r B o r b o th )
is r e q u ire d .
In v o lv e d in IgE fo rm a tio n ( e it h e r
T h is Is so th a t a c e r t a in minimum
70
number o f IgE producing c e l l s a r e formed In o rd e r t o a t t a i n high
enough serum le v e ls o f IgE a n tib o d y th a t can be d e te c te d by the
PCA a s s a y .
T h is could be a ch ie v ed e it h e r by g iv in g alum -adsorbed
a n tig e n o r m u lt ip le
In je c tio n s o f a n tig e n s .
Macrophage p ro cessin g has been shown to be im p o rtan t In the
fo rm a tio n o f h e lp e r T c e l l s f o r
IgE responses ( 2 2 ) .
A lthough th e
mechanism by which alum -adsorbed a n tig e n s tim u la te s
IgE fo rm a tio n
is not known, Tada (4 ) s p e c u la te s th a t sm all doses o f alum -adsorbed
a n tig e n a r e p h ag o cytized by macrophages and re le a s e d
ly
In an extrem e­
Immunogenic form to s tim u la te c e r t a in p o p u la tio n s o f T o r B
c e lls .
Presum ably, alum -adsorbed a n tig e n s a re taken up v e ry e f ­
f i c i e n t l y by macrophages.
It
Is not known to what e x te n t s o lu b le
a n tig e n s , such as those used in m u lt ip le in je c t io n s ,
in comparison
t o alum -adsorbed a n tig e n , a re processed by macrophages.
I t may be
th a t by g iv in g re p ea te d In je c tio n s o f s o lu b le a n tig e n th e r e q u ir e ­
ment f o r macrophage Involvem ent is overcome o r a l t e r n a t i v e l y , th a t
macrophage pro cessin g o f s o lu b le a n tig e n is s tim u la te d by t h is r e g i ­
men.
From th e s tu d ie s on th e e f f e c t o f m u lt ip le , h ig h doses o f an­
t ig e n , g iv e n IV on days 3 , 5 , and 7 a f t e r p r io r im m unization w ith
10 /Ltg OVA + I mg alu m ,
r e s u lt s
IP I t can be concluded th a t t h i s tre a tm e n t
in th e s e le c t iv e suppression o f IgE responses t o OVA and
71
t h a t t h is e f f e c t can be tr a n s fe r r e d t o normal anim als by s p le n ic
c e lls .
W h ile th e s e s tu d ie s were in p ro g re s s , o th e r w orkers re p o rte d
t h a t a d m in is tr a tio n o f 3 ,
100 jug, IV In je c tio n s o f urea d e n atu rated
OVA (UD-QVA) g iv e n on days 3» 5 , and 7 a f t e r
im m unization w ith a
low dose o f a lu m -p r e c ip ita te d OVA, suppressed anti-O V A
IgE and IgG
responses and r e s u lte d in th e fo rm a tio n o f suppressor T c e l l s
(3 9 ).
T h e ir system was unique in th a t UD-OVA c o n ta in ed o n ly th e T - c e ll
s tim u la tin g d e te rm in a n ts o f n a tiv e OVA; th e n a tiv e OVA B- c e l l stim u
la t i n g d e te rm in a n ts w ere lo s t by th e urea tre a tm e n t ( 7 3 ) .
In l i g h t o f th e s e s tu d ie s .
3,
I t seems re as o n a b le t o assume th a t
100 #zg, IV in je c tio n s o f n a tiv e OVA a ls o induces th e fo rm a tio n
o f suppressor T c e l l s , however th e evidence f o r t h is
Is s t i l l
in ­
d i r e c t , supported m a in ly by th e f a c t t h a t suppression can be tr a n s ­
f e r r e d by s p le n ic c e l l s .
One main d if f e r e n c e between th e UD-OVA and th e n a tiv e OVA used
in my s tu d ie s
OVA.
is th e presence o f B c e l l d e te rm in a n ts on th e n a tiv e
For t h is reaso n , o th e r a l t e r n a t i v e hypotheses t o e x p la in th e
desens 111z a t i on observed in my system m ight be c o n s id e re d .
One such h y p o th e s is Is th a t th e m u lt ip le , high dose in je c tio n s
o f s o lu b le a n tig e n Induce a h ig h a f f i n i t y
IgG a n tib o d y which then
e x e r ts a s u p p re s s iv e e f f e c t on th e IgE response.
I t has been r e ­
72
p o rte d th a t p a s s iv e ly a d m in is te re d , a n t ig e n - s p e c if ic
IgG a n tib o d y
from hyperimmune a n im als w i l l e x e r t a s u p p ressive e f f e c t on IgE
responses t o th e same a n tig e n (7 1 )«
The e f f e c t was t r a n s ie n t how­
e v e r , and o n ly la s te d u n t i l th e tr a n s fe r r e d a n tib o d y had been c a t a b p llz e d .
A ls o , th e suppression was most e f f e c t i v e when th e pas­
s iv e a n tib o d y was tr a n s fe r r e d w ith in 2k hr o f a c t iv e
im m unization.
The p a s s iv e ly a d m in is te re d a n tib o d y could not te rm in a te an e x is tin g
IgE response o r suppress a secondary IgE response.
These o b s e rv a tio n s a r e in c o n s is te n t w ith th e events seen when
m ice a r e d e s e n s itiz e d w ith m u lt ip le In je c tio n s o f n a tiv e OVA, be­
cause both a p r e - e x is t in g and th e secondary IgE response were shown
t o be suppressed by th e tre a tm e n t.
lenge o f a n tig e n
A ls o , when a secondary c h a l­
Is g iv e n t o s e n s itiz e d a n im a ls , high le v e ls o f IgE
a n tib o d y a r e m a in ta in e d in th e presence o f e x tre m e ly h ig h le v e ls o f
PHA a n tib o d y .
The fo re g o in g argues a g a in s t th e h y p o th e s is th a t th e
observed suppression o f IgE responses by a d m in is tr a tio n o f m u ltip le
IV In je c tio n s o f a n tig e n is due t o an IgG a n tib o d y m ediated mecha­
nism .
A nother h yp o th e s is th a t m ight be considered Is th e s e le c t iv e
in d u c tio n o f IgE B c e l l to le r a n c e .
However, In th e case o f p ro te in
a n tig e n s , th e in d u c tio n o f to le ra n c e in p r e s e n s itiz e d B c e l l s has
proved t o be d i f f i c u l t t o accom plish ( 7 * 0 .
Some re c e n t s tu d ie s
73
have shown th a t to le ra n c e In IgE B c e l l s could be a c h ie v e d , even
a f t e r p r i o r s e n s i t i z a t i o n , by DNP coupled to non-immunogenic c a r ­
r ie r s
( 6 2 , 7 5 ) , and th a t th e dose o f to le ro g e n re q u ire d f o r IgE
to le r a n c e in d u c tio n was le s s than t h a t re q u ire d f o r
in d u c tio n ( 7 5 ) .
It
IgG to le ra n c e
is not known w hether t h is dosage e f f e c t a p p lie s
to th e in d u c tio n o f IgE B c e l l to le r a n c e to p r o te in a n tig e n s ,
. A lth o u g h th e in d u c tio n o f B c e l l to le ra n c e cannot be ru le d
o u t, th e f a c t th a t th e suppression o f Ig E , which Is
m u lt ip le ,
induced by
IV in je c tio n s o f a n tig e n can be tr a n s fe r r e d t o o th e r a n i ­
mals by s p le n ic c e l l s makes t h is mechanism as th e o n ly e x p la n a tio n
o f d e s e n s itiz a t io n h ig h ly u n lik e ly and suggests v e ry s tr o n g ly th e
presence o f a suppressor c e l l .
SUM MARY
The r e s u lt s o f t h i s th e s is can be summarized as fo llo w s :
1)
The suppression o f IgE a n tib o d y responses by neonatal Iy
i n i t i a t e d a n t i - # tre a tm e n t showed th a t Ig E -fo rm in g c e l l s a r e de­
r iv e d from th e same p re c u rs o rs ! c e l ! as a r e th e c e l l s form ing
o th e r c la s s e s o f Im m unoglobulins.
2)
V a rio u s ways o f inducing and r e g u la tin g
B a lb /c m ice were compared.
IgE responses in
Im m unization w ith alum -adsorbed OVA
o ver a w ide dosage range Induced a h ig h and p e r s is te n t IgE response.
F a i lu r e t o show a tr a n s ie n t IgE response t o high doses o f a n tig e n
may be r e la t e d t o th e I n a b i l i t y o f I mg alum to c o m p le te ly re a c t
w ith an excess o f p r o t e in .
Im m unization w ith alum -adsorbed DMP-
o v a Ibumln c o n ju g ates produced 2 d i f f e r e n t p a tte rn s o f IgE responses
r e la t e d t o th e hapten d e n s ity .
T h is was thought t o be r e la te d to
th e way In which th e s e c o n ju g ates may be m e ta b o liz e d .
Im m unization
w ith one in je c t io n o f OVA w ith B. p e rtu s s is f a i l e d t o induce IgE
resp o nses.
A new means o f Inducing IgE responses by m u lt ip le IP
in je c tio n s o f a n tig e n was shown.
A ls o , tre a tm e n t w ith n e o n a ta lIy
I n i t i a t e d m u lt ip le in je c tio n s o f MRS suppressed subsequent immune
responses t o Im m unization w ith alum -adsorbed OVA.
3)
The s e le c t iv e suppression o f IgE responses by 3 ,
IV in je c ­
tio n s o f high doses o f s o lu b le a n tig e n g iv en on days 3 , 5 , and 7
a f t e r p r i o r s e n s it iz a t io n w ith 10 fig OVA + I mg alum was shown to
75
be due, a t
th e s p le e n .
le a s t In p a r t to th e g e n e ra tio n o f suppressor c e l l s
In
Both a n tib o d y t i t e r s and th e number o f IgE producing
c e l l s were reduced to u n d e te c ta b le le v e ls by t h is tre a tm e n t.
T h is
phenomenon was a n tig e n s p e c if ic and v e ry dependent on th e ro u te o f
a d m in is tr a tio n .
Whereas 100 (ig o f s o lu b le a n tig e n was v e ry sup­
p re s s iv e when g iv e n IV , th e same o r 1 0 -fo ld h ig h e r doses were o n ly
s l i g h t l y s u p p re s s iv e when g iv en IP ,
g e n ic dose, 10 jug, g iv en IV ,
However, an o p tim a lly
Immuno­
Induced s ig n if ic a n t s u p p res s io n .
ondary responses were a ls o a f f e c t e d by t h is tre a tm e n t.
Sec­
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(Klein) 19^0R e g u l a t i o n o f IgE
anti
b o d y responses in mice.
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