IMMUNOLOGICAL COMMUNICATIONS, 5 ( 5 1 , 455-468 ( 1 9 7 6 )
INHIBITION OF B LYMPHOCYTE ACTIVATION BY INTERACTION WITH Fc RECEPTORS
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J.L. Ryan and P.A. Henkart Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 Abstract Much early work indicated that specific antibody can play an inhibitory role in the immune response. This inhibitory activity was found to be dependent on an intact Fc portion o f the antibody used, both in vivo and in_ vitro. _ Thus a role for lymphocyte Fc receptors in regulation of the immune response was suggested. However, soluble antigen-antibody complexes, which bind to Fc receptors, do not appear to inhibit B cell activation. Recent experiments have demonstrated that antigen-antibody complexes immobilized on a surface strikingly inhibit LPS induced B cell mitogenesis and polyclonal antibody synthesis. Mechanisms for Fc receptor-mediated inhibition of B cell activation have been considered, and a model proposed to explain many of these findings as well as allow for antigen specific inhibition. I. A.
Introduction
Purpose and Scope Surface receptors on lymphocytes have proven vital in defining
lymphocyte subpopulations with different functional roles in the immune response. The precise physiological role of these surface receptors has provided the central problem in much recent research, but has been difficult to establish.
For example, it appears evident that the B cell surface
immunoglobulin binds antigen, but the direct consequence of this binding is unclear and a considerable controversy exists on the question of whether such binding itself results in a signal being transmitted to the cell interior (1).
In this review we will concentrate on the function of
B lymphocyte Fc receptors; specifically we will consider the evidence that
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RYAN AND HENKART
t h e s e r e c e p t o r s p l a y an i n h i b i t o r y r o l e i n r e g u l a t i n g B c e l l a c t i v a t i o n . Most of t h e experiments c i t e d have been done u s i n g murine s p l e e n c e l l systems. B.
The Fc Receptor Fc r e c e p t o r s a r e d e f i n e d by t h e i r a b i l i t y t o bind t h e Fc p o r t i o n
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of IgG immunoglobulin which has been complexed w i t h a n t i g e n , o r has been aggregated by h e a t o r o t h e r means ( 2 , 3 ) .
S i n g l e IgG molecules are bound
t o t h e lymphocyte Fc r e c e p t o r w i t h a markedly lower a f f i n i t y ( 2 ) .
Fc
r e c e p t o r s a r e d e t e c t e d c h i e f l y by monitoring t h e b i n d i n g of antigen-antibody complexes by autoradiography o r immunofluorescence ( 4 , 5 ) , by b i n d i n g o f f l u o r e s c e n t l a b e l l e d h e a t aggregated IgG ( 3 ) , o r by forming r o s e t t e s w i t h antibody coated e r y t h r o c y t e s (EA r o s e t t e s ) (6).
These r e c e p t o r s do n o t
bind t o IgG F ( a b ' ) 2 fragments which l a c k t h e Fc p o r t i o n of t h e a n t i b o d y molecule, nor t o i n t a c t a n t i b o d i e s of o t h e r c l a s s e s (2).
They have been
found on B lymphocytes i n t h e mouse ( 2 , 7 ) , human ( 3 ) , and o t h e r s p e c i e s and have r e c e n t l y been d e s c r i b e d on normal T lymphocytes i n t h e mouse (8).
Fc
r e c e p t o r s have a l s o been found on many o t h e r t y p e s of c e l l s i n c l u d i n g macrophages.
I t seems q u i t e p o s s i b l e a t t h i s p o i n t t h a t t h e r e may be d i f -
f e r e n t t y p e s of Fc r e c e p t o r s on d i f f e r e n t c e l l s .
A r e c e n t review of lympho-
c y t e Fc r e c e p t o r s i s recommended f o r a d e t a i l e d d i s c u s s i o n ( 9 ) .
11. Antibody Mediated Suppression of t h e Immune Response I n a v a r i e t y of o l d e r experiments i t w a s c o n v i n c i n g l y shown t h a t p a s s i v e l y administered antibody could i n an animal given a n t i g e n .
s p e c i f i c a l l y s u p p r e s s a n antibody r e s p o n s e
The e a r l y work i n t h i s a r e a h a s been reviewed
I,
by Uhr and Moller (10).
These experiments have given r i s e t o t h e s u g g e s t i o n
t h a t one e s s e n t i a l a s p e c t of t h e i n v i v o r e g u l a t i o n of a n t i b o d y production
is a n e g a t i v e feedback system i n which c i r c u l a t i n g a n t i b o d y molecules s p e c i f i c a l l y i n h i b i t t h e d i f f e r e n t i a t i o n of new a n t i b o d y s e c r e t i n g c e l l s .
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B LYMPHOCYTE ACTIVATION
More r e c e n t l y , t h e s t u d i e s o f S i n c l a i r a n d h i s c o l l e a g u e s h a v e b e e n o f c r i t i c a l importance i n t h i s a r e a b e c a u s e t h e y have e s t a b l i s h e d t h e r e q u i r e ment f o r a n i n t a c t Fc p o r t i o n o f t h e a n t i b o d y m o l e c u l e t o s u p p r e s s immune r e s p o n s e s b o t h i n v i v o and i n v i t r o ( 1 1 , 1 2 , 1 3 ) .
F o r e x a m p l e , s i x week o l d
mice were immunized w i t h s h e e p e r y t h r o c y t e s a n d v a r y i n g d o s e s of F ( a b ' )
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o r IgC, anti--SRRC a n t i b o d i e s .
2
When t h e s p l e e n s were a s s a y e d f o r anti-SRBC
p l a q u e s 5 d a y s l a t e r , i t w a s f o u n d t h a t t h e LgC, a n t i b o d y w a s much m o r e e f f e c t i v e i n d e c r e a s i n g t h e r e s p o n s e t h a n t h e F ( a b ' ) 2 a n t i b o d y (12). was a l s o f o u n d t h a t o n l y IgC and n o t F ( a b ' ) r e s p o n s e which was a l r e a d y underway.
2
It
fragments could i n h i b i t a
Thus t h e y s u g g e s t e d t h a t t h e i n h i b i -
t o r y a c t i v i t y t h a t was s e e n i n t h e a n i m a l t r e a t e d w i t h a n t i g e n p l u s F ( a b ' ) 2 a n t i b o d y was l i k e l y d u e t o i n c r e a s e d a n t i g e n d e s t r u c t i o n a n d t h a t t h e i n t a c t IgC was a c t i n g by i n a c t i v a t i n g t h e a n t i b o d y - f o r m i n g p o p u l a t i o n by i n t e r a c t i o n w i t h a n t i g e n - a n t i b o d y
c e l l precursor
complexes.
To e x p l a i n t h e s e d a t a , S i n c l a i r a n d Chan p r o p o s e d a t r i p a r t i t e i n a c t i v a t i o n model f e a t u r i n g a n i n t e r a c t i o n b e t w e e n a n t i g e n , a n t i b o d y , a n d a n t i g e n - s e n s i t i v e c e l l i n w h i c h a n e g a t i v e s i g n a l i s d e l i v e r e d v i a t h e Fc p o r t i o n of t h e a n t i b o d y molecule ( 1 2 ) . S t u d i e s showing a n t i g e n - s p e c i f i c
s u p p r e s s i o n of i n v i t r o immune
r e s p o n s e s h a v e a l s o b e e n r e p o r t e d where t h e s u p p r e s s i o n was d e p e n d e n t o n a n i n t a c t Fc f r a g m e n t i n t h e a n t i b o d y m o l e c u l e (13,14,15,16).
'These
studies i n d i c a t e t h a t t h e spleen c e l l s being cultured contain a l l t h e e l e m e n t s n e c e s s a r y t o a c h i e v e t h e s u p p r e s s i o n a n d e l i m i n a t e many mechanisms p o s s i b l e i n t h e i n v i v o e x p e r i m e n t s s u c h as a l t e r a t i o n s i n r e n a l t u b u l a r function (17).
K a p p l e r e t a l . (16) h a v e d e m o n s t r a t e d t h a t t h e r e i s a n
a n t i g e n s p e c i f i c , d e t e r m i n a n t n o n s p e c i f i c s u p p r e s s i o n o f t h e immune r e s p o n s e o f mouse s p l e e n c e l l s t o h e t e r o l o g o u s e r y t h r o c y t e s which w a s n o t m e d i a t e d by F ( a b ' )
2
antibodies.
They s u g g e s t e d t h a t Fc r e c e p t o r s may b e
RYAN AND HENKART
458
f u n c t i o n i n g t o bind antigen-antibody complexes r e n d e r i n g t h e a n t i g e n inaccessible t o B c e l l precursors.
111.
I n h i b i t i o n of B Lymphocyte A c t i v a t i o n by Antibody
B lymphocyte Fc r e c e p t o r s seem an obvious c a n d i d a t e f o r mediating t h e
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s u p p r e s s i v e e f f e c t s d i s c u s s e d above.
One major conceptual d i f f i c u l t y i s
t h a t t h e s e r e c e p t o r s recognize t h e Fc p o r t i o n of complexed antibody molec u l e s r e g a r d l e s s of t h e i r a n t i g e n b i n d i n g s p e c i f i c i t y w h i l e t h e s u p p r e s s i v e effects are antigen specific. below.
T h i s problem w i l l be more f u l l y d i s c u s s e d
Two o t h e r l i n e s of evidence have l e d t o s p e c u l a t i o n r e g a r d i n g t h e
r o l e of Fc r e c e p t o r s i n r e g u l a t i o n of t h e immune response.
F i r s t , was t h e
demonstration t h a t t h e r e i s a c l o s e r e l a t i o n s h i p on t h e lymphocyte c e l l s u r f a c e between t h e Fc r e c e p t o r and Ia a n t i g e n s ( 1 8 ) . coded f o r by genes which e i t h e r a r e t h e same a s
OK
Since I a a n t i g e n s a r e
c l o s e l y l i n k e d t o immune
response genes, i t seemed p o s s i b l e t h a t t h e Fc r e c e p t o r may p l a y a r o l e i n r e g u l a t i n g t h e immune response (19).
Secondly, it a p p e a r s t h a t a n a s s o c i -
a t i o n between Fc r e c e p t o r s and membrane immunoglobulin can be induced by binding of l i g a n d s t o t h e l a t t e r molecules (20.21).
Although many q u e s t i o n s
remain r e g a r d i n g t h e s t r u c t u r e o f Fc r e c e p t o r s and t h e i r s u r f a c e r e l a t i o n s h i p s w i t h o t h e r c e l l markers, t h i s evidence a l s o s u g g e s t s t h a t t h e Fc r e c e p t o r s may p l a y an important r o l e in t h e immune response.
A.
Study of B Lymphocyte A c t i v a t i o n
I n o r d e r t o e s t a b l i s h t h a t t h e B lymphocyte Fc r e c e p t o r is c a p a b l e of i n h i b i t i n g B c e l l a c t i v a t i o n i t is u s e f u l t o study systems i n which t h e l a t t e r p r o c e s s i s being measured and i n which t h e e f f e c t of o t h e r c e l l t y p e s can be minimized.
This is important because of t h e presence of Fc
r e c e p t o r s on macrophages and some T c e l l s .
B c e l l s can be a c t i v a t e d i n a
Polyclonal f a s h i o n by a g r e a t many compounds i n c l u d i n g b a c t e r i a l l i p o p o l y s a c c h a r i d e , p u r i f i e d p r o t e i n d e r i v a t i v e of M. t u b e r c u l o s i s . and o t h e r poly-
459
B LYMPHOCYTE ACTIVATION
anions (22).
Such polyclonal activation stimulates B cells of all antigenic
specificities to undergo an increase in DNA synthesis, divide, and differentiate into antibody-forming cells. DNA synthesis (mitogenesis) is conveniently measured by incorporation of radiolabelled DNA precursors while differentiation is usually measured using the plaque assay developed by Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/01/15 For personal use only.
Jerne and Nordin ( 2 3 ) .
The polyclonal activation of B cells by compounds
such as LPS appears to mimic the natural activation of specific B cell clones by antigen and has been studied as a convenient model for the natural event. The advantage of using these systems is that it is possible to focus on B cells in isolation from other cells which are not required for the response being studied. One disadvantage is that antigen specificity is lost.
B.
Lack of Activation of B Cells by Complexes There was some early success in activating human lymphocytes using
soluble and particulate antigen-antibody complexes ( 2 4 ) .
Attempts to extend
11
this work, however, have met with little success. Moller and Coutinho (25) investigated the ability of both the Fc receptor and the C'3 receptor to act as activating mechanisms f o r mouse splenic B cells. They found that heterologous erythrocytes coated with 75 rabbit antibody did not activate mouse spleen cells for polyclonal antibody activity.
Other experiments showed
that soluble complexes were also unable to induce any B cell activation under a variety of conditions. These results have been corraborated in other laboratories. Ramasamy ( 2 6 ) used soluble immune complexes of ferritin-anti-ferritin in an attempt to activate nude spleen cell cultures. No increase in DNA synthesis was observed. Ryan et al. (27.28) attempted to stimulate DNA synthesis in human lymphocyte cultures using immobilized antigen-antibody complexes without success and further showed that neither soluble complexes nor immobilized complexes stimulated DNA synthesis in ,I
mouse spleen cell cultures. Thus with the exception of Moiler's early
460
RYAN AND HENKART
results ( 2 4 ) . all attempts to activate B cells via interaction with Fc receptors have been unsuccessful. C.
Inhibition of B Cell Activation by Complexes Given the failure of attempts to stimulate B cells by binding of
ligand to the Fc receptor, the question of whether such binding can prevent 9,
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activation of the cell by other means has been asked.
Moller and Coutinho
(25) found that EA rosetted lymphocytes would not suppress the polyclonal
antibody synthesis induced by LPS.
Furthermore, they demonstrated that
soluble antigen-antibody complexes over a broad range o f concentrations exerted no inhibitory effect on the polyclonal antibody response of nude More recently, Ramasamy (26) investigated the ability
spleen cells to LPS.
of soluble antigen-antibody complexes and aggregated immunoglobulin to inhibit LPS induced mitogenesis in nude spleen cell culture.
Under no con-
ditions did the binding o f complexed immunoglobulin interfere with LPS induced B cell activation. In contrast to these reports the results o f Ryan et al. ( 2 8 , 2 9 ) have shown that under the appropriate conditions antigen-antibody complexes can be strongly suppressive to B lymphocyte activation.
They confirmed the
11
earlier results of Moller and Coutinho (25) showing that soluble complexes did not lead to an inhibition of B cell activation, but further showed that if cells were cultured on a surface coated with immobilized antigen-antibody complexes ( 3 0 ) , B cell activation as measured by both mitogenesis and production of antibody forming cells was markedly inhibited.
The observed
inhibition was attributed to the Fc receptor since similar immobilized complexes prepared from IgA antibody or F(ab')2
antibody were inactive in
suppressing either function. The inhibitory effect of immobilized complexes was observed for all B cell activators tested including LPS, pneumococcal polysaccharide SIII, poly T:C, PPD, and 8 BrcGMP.
The ability
of complexes to inhibit LPS induced DNA synthesis was later confirmed by
461
B LYMPHOCYTE ACTIVATION Stout and Herzenberg (31).
These investigators used large molecular weight
romplexcs to mediate the inhibition. D.
Coordinated Interactions Between s I g and the Fc Receptor The search for a physiological role for surface immunoglobulin has
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it has been found that, with certain notable exreptions ( 3 2 , 3 3 ) , antiimmunoglobulin antibodies that combine with lymphocyte surface immunoglobulin do not lead to activation of B cells ( 2 6 , 3 4 ) .
Attempts to block B cell
activation have been successful, but at present remain controversial. Andersson et al. ( 3 4 ) reported that B cell activation induced by LPS, PPD, and FCS and measured by plaque-forming cell production was inhibited by anti-immunoglobulin
(K,
A, or u)or its F(ab')*
fragments. The same sera,
however, failed to block LPS induced mitogenesis.
Conversely, Schrader
( 3 5 ) , found that anti-immunoglobulin sera (polyvalent anti-IgG) did indeed
suppress LPS induced mitogenesis. Very recently, Sidman ( 3 6 ) has confirmed and extended the findings of Schrader (35).
He found that anti-immunoglob-
ulin inhibited LPS mitogensis, but that F(ab')* mediate this inhibition. the first ( 3 4 ) .
fragments were unable to
These two reports ( 3 5 , 3 6 ) are in conflict with
The latter data suggests that a coordinated binding
between surface immunoglobulin and Fc receptors or aggregation of Fc receptors induced by the binding of antibody to surface immunoglobulin may be required to inhibit B cell activation.
E. Role of the T Cell Fc Receptor Since T-B collaboration is a critical event for most antigen-induced B cell activation ( 3 7 ) , and Fc receptors have been described on a subpopulation of T c e l l s
(a),
a possible role for T cell Fc receptors regulating B
cell activation must be considered. It has been proposed that antigenantibody complexes may block Fc receptor positive T cells and prevent their interaction with other cellular species ( 3 1 ) .
This inference is based on
462
RYAN AND HENKART
the ability of complexes to partially inhibit the Concanavalin A response of spleen cells (28,31).
This inhibition of the Con A response was found to be
dependent on an intact Fc fragment of the antibody used to create the complexes ( 2 8 ) .
Further evidence for the role of T cell Fc receptors in
regulating B cell activation has been provided by Fridman and his associates They have found that alloantigen activated T cells release a
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(38,39).
factor which combines with the Fc fragment of antigen complexed IgG.
It
has been suggested that this factor (immunoglobulin-binding factor, IBF) may be identical to the T cell Fc receptor. -in vitro
IBF has been shown to inhibit
19s antibody formation to both T-dependent and T-independent anti-
gens ( 4 0 ) .
IBF has further been shown to be capable of suppressing 7s anti-
body response and of being neutralized by aggregated mouse IgG (39).
It is
not clear at this time whether IBF reacts directly with B cells. Thus there is increasing evidence that T cell Fc receptors may play a role in inhibiting B cell activation. IV. Mechanism of Inhibition of B Cell Activation by the Fc Receptor Many questions remain unanswered regarding the molecular events involved in the inhibition of B cell activation by the Fc receptor. It is perhaps too much to expect to understand how B cell activation is inhibited while the most basic aspects of the molecular mechanism of the activation itself are still highly controversial. Nevertheless, a number of questions must be considered regarding the relevant experiments in this area. Pirst, why is effective inhibition observed only with immobilized or very large molecular weight complexes but not with soluble complexes? While there is precedent for B cells responding to immobilized, but not soluble stimuli ( 4 1 , 4 2 ) , the mechanism remains unclear.
Furthermore it is most relevant to
question whether the observed nonspecific inhibition of B cell activation
B LYMPHOCYTE ACTIVATION
463
by Fc receptors can indeed explain the antibody mediated suppression of the immune response which is antigen specific. We have proposed (28) that both of these problems could be explained by a variant of the tripartite model proposed by Chan and Sinclair (12).
The model assumes that a B lymphocyte
may become blocked from being activated by a coordinated binding of the
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antigen to the sIg receptor and an 1gG antibody from the medium bound to this antigen by another determinant. The Fc portion of this complexed antibody would then bind to the lymphocyte surface by interaction with Fc receptors to complete the inactivating signal.
This is diagrammed below
(Fig. 1). Several aspects of the model deserve comment. 1) Antigen specificity is provided for by sIg which may operate not only as a passive focussing device, but as a necessary part of the inhibitory event. 2) The crosslinking of membrane receptors which occurs may be a crucial aspect of the inhibition and may be mimicked by the immobilized complexes which were observed to inhibit effectively.
3 ) The recent experimental data of Sidman
( 3 6 ) would certainly support this model.
Regardless of the correctness of this model there are a number of different levels at which inhibition of B lymphocyte activation via Fc receptors may take place.
1) Blockage of the surface binding event(s) which trigger the cell to become activated. Ryan et al. (28), have argued from indirect evidence that this is not the case. They were unable to demonstrate blocking of Fc receptors by any of a series of B cell activators as measured by competition for binding with that aggregated immunoglobulin.
In the more general case
of antigen and T cell stimulation of the B cell, this mechanism remains a possibility. 2) The activation could be blocked by an uncoupling of the linkage between surface receptor binding and cytoplasmic events. One possibility
RYAN AND HENKART
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464
Figure 1: Model for B cell inactivation by the Fc receptor. Antigen is bound to the B cell surface by sIg. An IgG antibody from the circulation binds to the same antigen molecule, and the Fc portion of this antibody binds to the Fc receptor.
The cell
could be inactivated by a "one signal model" involving the Fc receptor alone,
OK
by a "two signal model'' which requires
signals from both sIg and the Fc receptor.
along these lines would be a "freezing" of surface membrane mobility as envisioned by Edelman ( 4 3 ) .
However, no experimental evidence yet Supports
this idea. A related mechanism would involve immobilizing B cell surface components.
The concept of a redistribution of surface components being
critical for B cell activation has been fully discussed ( 4 4 , 4 5 ) .
Melchers
and Andersson ( 4 6 ) have presented evidence that stimulation of B cells by
LPS results in the aggregation of surface IgM. This aggregation rapidly and lasts for more than 10 hours.
OCCUKS
They suggest that this redistri-
bution of sIg is a critical event for activation. While the role, if any,
465
B LYMPHOCYTE ACTIVATION
of Fc r e c e p t o r s i n augmenting o r i n h i b i t i n g s u r f a c e r e d i s t r i b u t i o n i s u n c l e a r , t h e p o s s i b i l i t y e x i s t s t h a t t h e i n h i b i t o r y e v e n t i n d u c e d by i m m o b i l i z e d o r l a r g e m o l e c u l a r w e i g h t complexes may b e r e l a t e d t o p r e v e n t i o n o f t h e movement of s u r f a c e m o l e c u l e s s u c h t h a t a t r i g g e r i n g e v e n t c a n n o t take place.
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3)
The i n h i b i t i o n i s a r e s u l t o f a c e n t r a l i n h i b i t o r y s i g n a l g e n e r a t e d
by b i n d i n g t o t h e Fc r e c e p t o r under a p p r o p r i a t e c o n d i t i o n s ( p e r h a p s b i n d i n g a l s o w i t h sIg).
The n a t u r e of s u c h c e n t r a l ( c y t o p l a s m i c ) s i g n a l s r e m a i n s
unknown, b u t one a t t r a c t i v e s u g g e s t i o n i s t h e p r o p o s a l t h a t c e l l d i v i s i o n is c o n t r o l l e d by t h e i n t r a c e l l u l a r r a t i o of cGMp t o CAMP ( 4 7 ) .
In support
o f t h i s i d e a , Watson ( 4 9 ) found t h a t t h e i n t r a c e l l u l a r l e v e l o f cGMP i n c r e a s e s r a p i d l y a f t e r t h e a d d i t i o n o f LPS t o c u l t u r e s p e a k i n g a t a b o u t 15 minutes.
D u r i n g t h i s t i m e t h e CAMP l e v e l s remained r e l a t i v e l y c o n s t a n t .
T h i s i n c r e a s e d r a t i o of cGMP t o CAMP h a s been p r o p o s e d t o b e t h e s i g n a l f o r B c e l l activation.
The f i n d i n g t h a t cGMP d e r i v a t i v e s a r e m i t o g e n i c f o r mouse
B lymphocytes when added e x o g e n o u s l y w h i l e CAMP d e r i v a t i v e s are i n h i b i t o r y
( 4 9 ) is i n accord with t h i s concept. antigen-antibody
S i n c e i t was found t h a t i m m o b i l i z e d
complexes a l s o i n h i b i t t h e m i t o g e n i c e f f e c t of 8 BrcGMP
( 2 9 ) , i t may b e a r g u e d t h a t t h e s e i n h i b i t o r y e f f e c t s a r e o c c u r r i n g v i a a central inhibitory signal.
Recent r e s u l t s i n o u r l a b o r a t o r y (50) i n d i c a t e
t h a t t h e i n t e r a c t i o n of mouse s p l e e n c e l l s w i t h i m m o b i l i z e d complexes r e s u l t s i n a p p r o x i m a t e l y a 50% i n c r e a s e i n t h e i n t r a c e l l u l a r l e v e l of CAMP w i t h i n 30 m i n u t e s .
This may r e p r e s e n t a c e n t r a l " o f f " s i g n a l d e l i v e r e d t o
t h e c e l l v i a Fc r e c e p t o r s . S t u d i e s on t h e f u n c t i o n a l r o l e of t h e lymphocyte Fc r e c e p t o r i n t h e immune r e s p o n s e a r e s t i l l i n t h e i r i n f a n c y .
The d a t a r e v i e w e d h e r e l e n d
p r o m i s e t h a t f u r t h e r i n v e s t i g a t i o n w i l l b e r e w a r d i n g a n d c o n t i n u e t o prov i d e some of t h e most e x c i t i n g problems i n c e l l u l a r immunology.
RYAN AND HENKART
466 References II
1.
Coutinho, A. and M o l l e r , G . , Scand. J . Immunol., 3:133, 1974.
2.
Basten, A.,
S p r e n t , J . and M i l l e r , J.F.A.P.,
N a t u r e , New B i o l . , 235:178,
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/01/15 For personal use only.
1972. 3.
D i c k l e r , H.B.
4.
Basten, A . , M i l l e r , J.F.A.P.,
and Kunkel, H . G . ,
J. Exp. Med., 136:191, 1972. S p r e n t , J . and Pye, J . , J . Exp. Med.,
135:610, 1972. 5.
Henkart. P.A.
A r b e i t , R.D.,
and D i c k l e r , H.B.,
i n I n V i t r o Methods i n
C e l l Mediated Immunity, e d i t e d by B. Bloom and J. David, v o l . 11, i n press. 6.
Yoshida, T.O.
7.
P a r a s k e v a s , F., Lee, S.T., Orr, K.B.
and A n d e r s o n , B . ,
Scand. J . Immunol.,
1:401, 1972.
and I s r a e l s , L.C., J. Immunol.,
108:1319, 1972. 8.
S t o u t , R.P. and Herzenberg, L.A.,
9.
Dickler, H.B.,
Adv. Immunol.,
J . Exp. Med., 142:611. 1975.
i n p r e s s , 1976.
11
10.
Uhr, J . W .
11.
S i n c l a i r , N.R.,
12.
Chan, P.L.
and S i n c l a i r , N.R.,
St.C.,
Immunol.,
21:967, 1971.
13.
Lees, R.K.
and S i n c l a i r , N.R.,
St.C.,
Immunol.,
24:735,
14.
Wason, W.M.
and F i t c h , F.W.,
15.
Abrahams, S . , P h i l l i p s . R.A.
and M o l l e r , G . , St.C.,
Adv. Immunol., 8:81, 1968.
J. Exp. Med., 129:1183, 1969.
J. Immunol., 110:1427, and Miller, R . G . ,
1973.
1973.
J . Exp. Med., 137:870,
1973. 16.
Kappler. J.W.. Van d e r Haven, A . , Dharmarajan, V. and Hoffman, M . , J. Immunol.,
111:1228, 1973.
17.
S p i e g e l b e r g , H. and Weigle, W.O.,
18.
D i c k l e r , H.B.
19.
Sachs, D.H.
and Sachs, D.H., and D i c k l e r , H . B . ,
J . Exp. Med.,
123:999, 1966.
J. Exp. Med., 140:779, 1974. T r a n s p l a n t . Rev., 23:159,
1975.
B LYMPHOCYTE ACTIVATION
467
20. Forni, L. and Pernis, B., in Membrane Receptor o f Lymphocytes, edited by M. Seligmann, J . L . Preud'homme and F.M. Kourilsky, p. 193, North Holland, Amsterdam, 1975. 21.
Unanue, E.R. and Abbas, A.K., in Membrane Receptor o f Lymphocytes, edited by M. Seligmann, J.L. Preud'homme and F.M. Kourilsky, p. 281,
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/01/15 For personal use only.
North Holland, Amsterdam, 1975. 11
22.
Coutinho, A. and Moller, G., Nature, New Biol., 245:12, 1973.
23.
Jerne, N.K. and Nordin, A.A., Science, 140:405, 1963.
24. Moller, G.,
Clin. Exp. Immunol., 4:65, 1969.
I*
25.
Moller, G. and Coutinho, A., J . Exp. Med., 141:647, 1975.
26. Ramasamy, R., Immunol., 30:559, 1976. 21.
Ryan, J.L. and Henkart, P.A., unpublished results.
28.
Ryan, J.L., Arbeit, R.D., Dickler, H.B. and Henkart, P.A., J . Exp. Med., 142:814, 1975.
29.
Ryan, J . L . and Henkart, P.A., submitted for publication.
30. Henkart, P.A. and Alexander, E., manuscript in preparation. 31. Stout, R.D. and Herzenberg, L.A., J. Exp. Med., 142:1041, 1975. 32. Sell, S . , Rowe, D . S . and Gell, P.G.H., J . Exp. Med. 122:823, 1965. 33. Weber, W.T., Transplantation, 24:113, 1975. 34. Andersson, J . , Bullock, W.W. and Melchers, F., Eur. J . Immunol., 4:715, 1974. 35.
Schrader, J.W., J . Immunol., 115:323, 1975.
36.
Sidman, C.L., Fed. Proc. Abst., 35:820, 1976.
37. Miller. J.F.A.P., Mitchell, G.F., Davies, A.J.S., Claman, H.N., Chaperon, E.A. and Taylor, R.B., Transplant. Rev., 1:1, 1969. 38. Fridman, W. and Goldstein, P., Cell. Immunol., 11:442, 1974. 39.
Gisler, R.H. and Fridman, W., Cell. Immunol., 23:99, 1976.
40. Gisler, R.H. and Fridrnan, W., J . Exp. Med., 142:507, 1975.
468
RYAN AND HENKART
41.
Greaves, M.F. and Bauminger, S.,
42.
Feldmann, M., Greaves, M., Parker, P.C. and Rittenberg, M.B., Eur. J.
Nature, New Biol., 2 3 5 : 6 7 , 1 9 7 2 .
Immunol., 4 : 5 9 1 , 1 9 7 4 . 43.
Wang, J . L . ,
McClaine, D.A. and Edelman, G.M., Proc. Nat. Acad. Science,
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/01/15 For personal use only.
72:1917, 1975. 44.
Greaves, M. and Janossy, G . , Transplant. Rev., 1 1 : 8 7 , 1 9 7 2 .
45.
Unanue, E. and Karnovsky, M., Transplant. Rev., 1 4 : 1 8 4 , 1 9 7 3 .
46.
Melchers, F. and Andersson, J., Transplant. Rev., 1 4 : 7 6 , 1 9 7 4 .
47.
Goldberg, N.D., Haddox, M.K., Nicol, S . E . , Glass, D.B., Sanford, C.H., Kuehl, F.A. and Estensen, R., Adv. Cyclic. Nucl. Res., 5 : 3 0 7 , 1 9 7 5 .
48,
Watson, J., J . Exp. Med., 1 4 1 : 9 7 , 1 9 7 5 .
49.
Weinstein, Y., Segal, S. and Melmon, K.L., J . Immunol., 115:112, 1 9 7 5 .
50.
Ryan, J . L . and Henkart, P.A., unpublished results.
INHIBITION OF B LYMPHOCYTE ACTIVATION BY INTERACTION WITH Fc RECEPTORS
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/01/15 For personal use only.
J.L. Ryan and P.A. Henkart Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 Abstract Much early work indicated that specific antibody can play an inhibitory role in the immune response. This inhibitory activity was found to be dependent on an intact Fc portion o f the antibody used, both in vivo and in_ vitro. _ Thus a role for lymphocyte Fc receptors in regulation of the immune response was suggested. However, soluble antigen-antibody complexes, which bind to Fc receptors, do not appear to inhibit B cell activation. Recent experiments have demonstrated that antigen-antibody complexes immobilized on a surface strikingly inhibit LPS induced B cell mitogenesis and polyclonal antibody synthesis. Mechanisms for Fc receptor-mediated inhibition of B cell activation have been considered, and a model proposed to explain many of these findings as well as allow for antigen specific inhibition. I. A.
Introduction
Purpose and Scope Surface receptors on lymphocytes have proven vital in defining
lymphocyte subpopulations with different functional roles in the immune response. The precise physiological role of these surface receptors has provided the central problem in much recent research, but has been difficult to establish.
For example, it appears evident that the B cell surface
immunoglobulin binds antigen, but the direct consequence of this binding is unclear and a considerable controversy exists on the question of whether such binding itself results in a signal being transmitted to the cell interior (1).
In this review we will concentrate on the function of
B lymphocyte Fc receptors; specifically we will consider the evidence that
455 Copyright 0 1976 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electron~cor mechanical, including photocopying. microfilming, and recording, or by any information storageand retrievalsystem. w~thoutpermission in writing from the publisher.
456
RYAN AND HENKART
t h e s e r e c e p t o r s p l a y an i n h i b i t o r y r o l e i n r e g u l a t i n g B c e l l a c t i v a t i o n . Most of t h e experiments c i t e d have been done u s i n g murine s p l e e n c e l l systems. B.
The Fc Receptor Fc r e c e p t o r s a r e d e f i n e d by t h e i r a b i l i t y t o bind t h e Fc p o r t i o n
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of IgG immunoglobulin which has been complexed w i t h a n t i g e n , o r has been aggregated by h e a t o r o t h e r means ( 2 , 3 ) .
S i n g l e IgG molecules are bound
t o t h e lymphocyte Fc r e c e p t o r w i t h a markedly lower a f f i n i t y ( 2 ) .
Fc
r e c e p t o r s a r e d e t e c t e d c h i e f l y by monitoring t h e b i n d i n g of antigen-antibody complexes by autoradiography o r immunofluorescence ( 4 , 5 ) , by b i n d i n g o f f l u o r e s c e n t l a b e l l e d h e a t aggregated IgG ( 3 ) , o r by forming r o s e t t e s w i t h antibody coated e r y t h r o c y t e s (EA r o s e t t e s ) (6).
These r e c e p t o r s do n o t
bind t o IgG F ( a b ' ) 2 fragments which l a c k t h e Fc p o r t i o n of t h e a n t i b o d y molecule, nor t o i n t a c t a n t i b o d i e s of o t h e r c l a s s e s (2).
They have been
found on B lymphocytes i n t h e mouse ( 2 , 7 ) , human ( 3 ) , and o t h e r s p e c i e s and have r e c e n t l y been d e s c r i b e d on normal T lymphocytes i n t h e mouse (8).
Fc
r e c e p t o r s have a l s o been found on many o t h e r t y p e s of c e l l s i n c l u d i n g macrophages.
I t seems q u i t e p o s s i b l e a t t h i s p o i n t t h a t t h e r e may be d i f -
f e r e n t t y p e s of Fc r e c e p t o r s on d i f f e r e n t c e l l s .
A r e c e n t review of lympho-
c y t e Fc r e c e p t o r s i s recommended f o r a d e t a i l e d d i s c u s s i o n ( 9 ) .
11. Antibody Mediated Suppression of t h e Immune Response I n a v a r i e t y of o l d e r experiments i t w a s c o n v i n c i n g l y shown t h a t p a s s i v e l y administered antibody could i n an animal given a n t i g e n .
s p e c i f i c a l l y s u p p r e s s a n antibody r e s p o n s e
The e a r l y work i n t h i s a r e a h a s been reviewed
I,
by Uhr and Moller (10).
These experiments have given r i s e t o t h e s u g g e s t i o n
t h a t one e s s e n t i a l a s p e c t of t h e i n v i v o r e g u l a t i o n of a n t i b o d y production
is a n e g a t i v e feedback system i n which c i r c u l a t i n g a n t i b o d y molecules s p e c i f i c a l l y i n h i b i t t h e d i f f e r e n t i a t i o n of new a n t i b o d y s e c r e t i n g c e l l s .
451
B LYMPHOCYTE ACTIVATION
More r e c e n t l y , t h e s t u d i e s o f S i n c l a i r a n d h i s c o l l e a g u e s h a v e b e e n o f c r i t i c a l importance i n t h i s a r e a b e c a u s e t h e y have e s t a b l i s h e d t h e r e q u i r e ment f o r a n i n t a c t Fc p o r t i o n o f t h e a n t i b o d y m o l e c u l e t o s u p p r e s s immune r e s p o n s e s b o t h i n v i v o and i n v i t r o ( 1 1 , 1 2 , 1 3 ) .
F o r e x a m p l e , s i x week o l d
mice were immunized w i t h s h e e p e r y t h r o c y t e s a n d v a r y i n g d o s e s of F ( a b ' )
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o r IgC, anti--SRRC a n t i b o d i e s .
2
When t h e s p l e e n s were a s s a y e d f o r anti-SRBC
p l a q u e s 5 d a y s l a t e r , i t w a s f o u n d t h a t t h e LgC, a n t i b o d y w a s much m o r e e f f e c t i v e i n d e c r e a s i n g t h e r e s p o n s e t h a n t h e F ( a b ' ) 2 a n t i b o d y (12). was a l s o f o u n d t h a t o n l y IgC and n o t F ( a b ' ) r e s p o n s e which was a l r e a d y underway.
2
It
fragments could i n h i b i t a
Thus t h e y s u g g e s t e d t h a t t h e i n h i b i -
t o r y a c t i v i t y t h a t was s e e n i n t h e a n i m a l t r e a t e d w i t h a n t i g e n p l u s F ( a b ' ) 2 a n t i b o d y was l i k e l y d u e t o i n c r e a s e d a n t i g e n d e s t r u c t i o n a n d t h a t t h e i n t a c t IgC was a c t i n g by i n a c t i v a t i n g t h e a n t i b o d y - f o r m i n g p o p u l a t i o n by i n t e r a c t i o n w i t h a n t i g e n - a n t i b o d y
c e l l precursor
complexes.
To e x p l a i n t h e s e d a t a , S i n c l a i r a n d Chan p r o p o s e d a t r i p a r t i t e i n a c t i v a t i o n model f e a t u r i n g a n i n t e r a c t i o n b e t w e e n a n t i g e n , a n t i b o d y , a n d a n t i g e n - s e n s i t i v e c e l l i n w h i c h a n e g a t i v e s i g n a l i s d e l i v e r e d v i a t h e Fc p o r t i o n of t h e a n t i b o d y molecule ( 1 2 ) . S t u d i e s showing a n t i g e n - s p e c i f i c
s u p p r e s s i o n of i n v i t r o immune
r e s p o n s e s h a v e a l s o b e e n r e p o r t e d where t h e s u p p r e s s i o n was d e p e n d e n t o n a n i n t a c t Fc f r a g m e n t i n t h e a n t i b o d y m o l e c u l e (13,14,15,16).
'These
studies i n d i c a t e t h a t t h e spleen c e l l s being cultured contain a l l t h e e l e m e n t s n e c e s s a r y t o a c h i e v e t h e s u p p r e s s i o n a n d e l i m i n a t e many mechanisms p o s s i b l e i n t h e i n v i v o e x p e r i m e n t s s u c h as a l t e r a t i o n s i n r e n a l t u b u l a r function (17).
K a p p l e r e t a l . (16) h a v e d e m o n s t r a t e d t h a t t h e r e i s a n
a n t i g e n s p e c i f i c , d e t e r m i n a n t n o n s p e c i f i c s u p p r e s s i o n o f t h e immune r e s p o n s e o f mouse s p l e e n c e l l s t o h e t e r o l o g o u s e r y t h r o c y t e s which w a s n o t m e d i a t e d by F ( a b ' )
2
antibodies.
They s u g g e s t e d t h a t Fc r e c e p t o r s may b e
RYAN AND HENKART
458
f u n c t i o n i n g t o bind antigen-antibody complexes r e n d e r i n g t h e a n t i g e n inaccessible t o B c e l l precursors.
111.
I n h i b i t i o n of B Lymphocyte A c t i v a t i o n by Antibody
B lymphocyte Fc r e c e p t o r s seem an obvious c a n d i d a t e f o r mediating t h e
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s u p p r e s s i v e e f f e c t s d i s c u s s e d above.
One major conceptual d i f f i c u l t y i s
t h a t t h e s e r e c e p t o r s recognize t h e Fc p o r t i o n of complexed antibody molec u l e s r e g a r d l e s s of t h e i r a n t i g e n b i n d i n g s p e c i f i c i t y w h i l e t h e s u p p r e s s i v e effects are antigen specific. below.
T h i s problem w i l l be more f u l l y d i s c u s s e d
Two o t h e r l i n e s of evidence have l e d t o s p e c u l a t i o n r e g a r d i n g t h e
r o l e of Fc r e c e p t o r s i n r e g u l a t i o n of t h e immune response.
F i r s t , was t h e
demonstration t h a t t h e r e i s a c l o s e r e l a t i o n s h i p on t h e lymphocyte c e l l s u r f a c e between t h e Fc r e c e p t o r and Ia a n t i g e n s ( 1 8 ) . coded f o r by genes which e i t h e r a r e t h e same a s
OK
Since I a a n t i g e n s a r e
c l o s e l y l i n k e d t o immune
response genes, i t seemed p o s s i b l e t h a t t h e Fc r e c e p t o r may p l a y a r o l e i n r e g u l a t i n g t h e immune response (19).
Secondly, it a p p e a r s t h a t a n a s s o c i -
a t i o n between Fc r e c e p t o r s and membrane immunoglobulin can be induced by binding of l i g a n d s t o t h e l a t t e r molecules (20.21).
Although many q u e s t i o n s
remain r e g a r d i n g t h e s t r u c t u r e o f Fc r e c e p t o r s and t h e i r s u r f a c e r e l a t i o n s h i p s w i t h o t h e r c e l l markers, t h i s evidence a l s o s u g g e s t s t h a t t h e Fc r e c e p t o r s may p l a y an important r o l e in t h e immune response.
A.
Study of B Lymphocyte A c t i v a t i o n
I n o r d e r t o e s t a b l i s h t h a t t h e B lymphocyte Fc r e c e p t o r is c a p a b l e of i n h i b i t i n g B c e l l a c t i v a t i o n i t is u s e f u l t o study systems i n which t h e l a t t e r p r o c e s s i s being measured and i n which t h e e f f e c t of o t h e r c e l l t y p e s can be minimized.
This is important because of t h e presence of Fc
r e c e p t o r s on macrophages and some T c e l l s .
B c e l l s can be a c t i v a t e d i n a
Polyclonal f a s h i o n by a g r e a t many compounds i n c l u d i n g b a c t e r i a l l i p o p o l y s a c c h a r i d e , p u r i f i e d p r o t e i n d e r i v a t i v e of M. t u b e r c u l o s i s . and o t h e r poly-
459
B LYMPHOCYTE ACTIVATION
anions (22).
Such polyclonal activation stimulates B cells of all antigenic
specificities to undergo an increase in DNA synthesis, divide, and differentiate into antibody-forming cells. DNA synthesis (mitogenesis) is conveniently measured by incorporation of radiolabelled DNA precursors while differentiation is usually measured using the plaque assay developed by Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/01/15 For personal use only.
Jerne and Nordin ( 2 3 ) .
The polyclonal activation of B cells by compounds
such as LPS appears to mimic the natural activation of specific B cell clones by antigen and has been studied as a convenient model for the natural event. The advantage of using these systems is that it is possible to focus on B cells in isolation from other cells which are not required for the response being studied. One disadvantage is that antigen specificity is lost.
B.
Lack of Activation of B Cells by Complexes There was some early success in activating human lymphocytes using
soluble and particulate antigen-antibody complexes ( 2 4 ) .
Attempts to extend
11
this work, however, have met with little success. Moller and Coutinho (25) investigated the ability of both the Fc receptor and the C'3 receptor to act as activating mechanisms f o r mouse splenic B cells. They found that heterologous erythrocytes coated with 75 rabbit antibody did not activate mouse spleen cells for polyclonal antibody activity.
Other experiments showed
that soluble complexes were also unable to induce any B cell activation under a variety of conditions. These results have been corraborated in other laboratories. Ramasamy ( 2 6 ) used soluble immune complexes of ferritin-anti-ferritin in an attempt to activate nude spleen cell cultures. No increase in DNA synthesis was observed. Ryan et al. (27.28) attempted to stimulate DNA synthesis in human lymphocyte cultures using immobilized antigen-antibody complexes without success and further showed that neither soluble complexes nor immobilized complexes stimulated DNA synthesis in ,I
mouse spleen cell cultures. Thus with the exception of Moiler's early
460
RYAN AND HENKART
results ( 2 4 ) . all attempts to activate B cells via interaction with Fc receptors have been unsuccessful. C.
Inhibition of B Cell Activation by Complexes Given the failure of attempts to stimulate B cells by binding of
ligand to the Fc receptor, the question of whether such binding can prevent 9,
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activation of the cell by other means has been asked.
Moller and Coutinho
(25) found that EA rosetted lymphocytes would not suppress the polyclonal
antibody synthesis induced by LPS.
Furthermore, they demonstrated that
soluble antigen-antibody complexes over a broad range o f concentrations exerted no inhibitory effect on the polyclonal antibody response of nude More recently, Ramasamy (26) investigated the ability
spleen cells to LPS.
of soluble antigen-antibody complexes and aggregated immunoglobulin to inhibit LPS induced mitogenesis in nude spleen cell culture.
Under no con-
ditions did the binding o f complexed immunoglobulin interfere with LPS induced B cell activation. In contrast to these reports the results o f Ryan et al. ( 2 8 , 2 9 ) have shown that under the appropriate conditions antigen-antibody complexes can be strongly suppressive to B lymphocyte activation.
They confirmed the
11
earlier results of Moller and Coutinho (25) showing that soluble complexes did not lead to an inhibition of B cell activation, but further showed that if cells were cultured on a surface coated with immobilized antigen-antibody complexes ( 3 0 ) , B cell activation as measured by both mitogenesis and production of antibody forming cells was markedly inhibited.
The observed
inhibition was attributed to the Fc receptor since similar immobilized complexes prepared from IgA antibody or F(ab')2
antibody were inactive in
suppressing either function. The inhibitory effect of immobilized complexes was observed for all B cell activators tested including LPS, pneumococcal polysaccharide SIII, poly T:C, PPD, and 8 BrcGMP.
The ability
of complexes to inhibit LPS induced DNA synthesis was later confirmed by
461
B LYMPHOCYTE ACTIVATION Stout and Herzenberg (31).
These investigators used large molecular weight
romplexcs to mediate the inhibition. D.
Coordinated Interactions Between s I g and the Fc Receptor The search for a physiological role for surface immunoglobulin has
a more detailed history than that for the Fc receptor. In a parallel fashion Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/01/15 For personal use only.
it has been found that, with certain notable exreptions ( 3 2 , 3 3 ) , antiimmunoglobulin antibodies that combine with lymphocyte surface immunoglobulin do not lead to activation of B cells ( 2 6 , 3 4 ) .
Attempts to block B cell
activation have been successful, but at present remain controversial. Andersson et al. ( 3 4 ) reported that B cell activation induced by LPS, PPD, and FCS and measured by plaque-forming cell production was inhibited by anti-immunoglobulin
(K,
A, or u)or its F(ab')*
fragments. The same sera,
however, failed to block LPS induced mitogenesis.
Conversely, Schrader
( 3 5 ) , found that anti-immunoglobulin sera (polyvalent anti-IgG) did indeed
suppress LPS induced mitogenesis. Very recently, Sidman ( 3 6 ) has confirmed and extended the findings of Schrader (35).
He found that anti-immunoglob-
ulin inhibited LPS mitogensis, but that F(ab')* mediate this inhibition. the first ( 3 4 ) .
fragments were unable to
These two reports ( 3 5 , 3 6 ) are in conflict with
The latter data suggests that a coordinated binding
between surface immunoglobulin and Fc receptors or aggregation of Fc receptors induced by the binding of antibody to surface immunoglobulin may be required to inhibit B cell activation.
E. Role of the T Cell Fc Receptor Since T-B collaboration is a critical event for most antigen-induced B cell activation ( 3 7 ) , and Fc receptors have been described on a subpopulation of T c e l l s
(a),
a possible role for T cell Fc receptors regulating B
cell activation must be considered. It has been proposed that antigenantibody complexes may block Fc receptor positive T cells and prevent their interaction with other cellular species ( 3 1 ) .
This inference is based on
462
RYAN AND HENKART
the ability of complexes to partially inhibit the Concanavalin A response of spleen cells (28,31).
This inhibition of the Con A response was found to be
dependent on an intact Fc fragment of the antibody used to create the complexes ( 2 8 ) .
Further evidence for the role of T cell Fc receptors in
regulating B cell activation has been provided by Fridman and his associates They have found that alloantigen activated T cells release a
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(38,39).
factor which combines with the Fc fragment of antigen complexed IgG.
It
has been suggested that this factor (immunoglobulin-binding factor, IBF) may be identical to the T cell Fc receptor. -in vitro
IBF has been shown to inhibit
19s antibody formation to both T-dependent and T-independent anti-
gens ( 4 0 ) .
IBF has further been shown to be capable of suppressing 7s anti-
body response and of being neutralized by aggregated mouse IgG (39).
It is
not clear at this time whether IBF reacts directly with B cells. Thus there is increasing evidence that T cell Fc receptors may play a role in inhibiting B cell activation. IV. Mechanism of Inhibition of B Cell Activation by the Fc Receptor Many questions remain unanswered regarding the molecular events involved in the inhibition of B cell activation by the Fc receptor. It is perhaps too much to expect to understand how B cell activation is inhibited while the most basic aspects of the molecular mechanism of the activation itself are still highly controversial. Nevertheless, a number of questions must be considered regarding the relevant experiments in this area. Pirst, why is effective inhibition observed only with immobilized or very large molecular weight complexes but not with soluble complexes? While there is precedent for B cells responding to immobilized, but not soluble stimuli ( 4 1 , 4 2 ) , the mechanism remains unclear.
Furthermore it is most relevant to
question whether the observed nonspecific inhibition of B cell activation
B LYMPHOCYTE ACTIVATION
463
by Fc receptors can indeed explain the antibody mediated suppression of the immune response which is antigen specific. We have proposed (28) that both of these problems could be explained by a variant of the tripartite model proposed by Chan and Sinclair (12).
The model assumes that a B lymphocyte
may become blocked from being activated by a coordinated binding of the
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antigen to the sIg receptor and an 1gG antibody from the medium bound to this antigen by another determinant. The Fc portion of this complexed antibody would then bind to the lymphocyte surface by interaction with Fc receptors to complete the inactivating signal.
This is diagrammed below
(Fig. 1). Several aspects of the model deserve comment. 1) Antigen specificity is provided for by sIg which may operate not only as a passive focussing device, but as a necessary part of the inhibitory event. 2) The crosslinking of membrane receptors which occurs may be a crucial aspect of the inhibition and may be mimicked by the immobilized complexes which were observed to inhibit effectively.
3 ) The recent experimental data of Sidman
( 3 6 ) would certainly support this model.
Regardless of the correctness of this model there are a number of different levels at which inhibition of B lymphocyte activation via Fc receptors may take place.
1) Blockage of the surface binding event(s) which trigger the cell to become activated. Ryan et al. (28), have argued from indirect evidence that this is not the case. They were unable to demonstrate blocking of Fc receptors by any of a series of B cell activators as measured by competition for binding with that aggregated immunoglobulin.
In the more general case
of antigen and T cell stimulation of the B cell, this mechanism remains a possibility. 2) The activation could be blocked by an uncoupling of the linkage between surface receptor binding and cytoplasmic events. One possibility
RYAN AND HENKART
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464
Figure 1: Model for B cell inactivation by the Fc receptor. Antigen is bound to the B cell surface by sIg. An IgG antibody from the circulation binds to the same antigen molecule, and the Fc portion of this antibody binds to the Fc receptor.
The cell
could be inactivated by a "one signal model" involving the Fc receptor alone,
OK
by a "two signal model'' which requires
signals from both sIg and the Fc receptor.
along these lines would be a "freezing" of surface membrane mobility as envisioned by Edelman ( 4 3 ) .
However, no experimental evidence yet Supports
this idea. A related mechanism would involve immobilizing B cell surface components.
The concept of a redistribution of surface components being
critical for B cell activation has been fully discussed ( 4 4 , 4 5 ) .
Melchers
and Andersson ( 4 6 ) have presented evidence that stimulation of B cells by
LPS results in the aggregation of surface IgM. This aggregation rapidly and lasts for more than 10 hours.
OCCUKS
They suggest that this redistri-
bution of sIg is a critical event for activation. While the role, if any,
465
B LYMPHOCYTE ACTIVATION
of Fc r e c e p t o r s i n augmenting o r i n h i b i t i n g s u r f a c e r e d i s t r i b u t i o n i s u n c l e a r , t h e p o s s i b i l i t y e x i s t s t h a t t h e i n h i b i t o r y e v e n t i n d u c e d by i m m o b i l i z e d o r l a r g e m o l e c u l a r w e i g h t complexes may b e r e l a t e d t o p r e v e n t i o n o f t h e movement of s u r f a c e m o l e c u l e s s u c h t h a t a t r i g g e r i n g e v e n t c a n n o t take place.
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3)
The i n h i b i t i o n i s a r e s u l t o f a c e n t r a l i n h i b i t o r y s i g n a l g e n e r a t e d
by b i n d i n g t o t h e Fc r e c e p t o r under a p p r o p r i a t e c o n d i t i o n s ( p e r h a p s b i n d i n g a l s o w i t h sIg).
The n a t u r e of s u c h c e n t r a l ( c y t o p l a s m i c ) s i g n a l s r e m a i n s
unknown, b u t one a t t r a c t i v e s u g g e s t i o n i s t h e p r o p o s a l t h a t c e l l d i v i s i o n is c o n t r o l l e d by t h e i n t r a c e l l u l a r r a t i o of cGMp t o CAMP ( 4 7 ) .
In support
o f t h i s i d e a , Watson ( 4 9 ) found t h a t t h e i n t r a c e l l u l a r l e v e l o f cGMP i n c r e a s e s r a p i d l y a f t e r t h e a d d i t i o n o f LPS t o c u l t u r e s p e a k i n g a t a b o u t 15 minutes.
D u r i n g t h i s t i m e t h e CAMP l e v e l s remained r e l a t i v e l y c o n s t a n t .
T h i s i n c r e a s e d r a t i o of cGMP t o CAMP h a s been p r o p o s e d t o b e t h e s i g n a l f o r B c e l l activation.
The f i n d i n g t h a t cGMP d e r i v a t i v e s a r e m i t o g e n i c f o r mouse
B lymphocytes when added e x o g e n o u s l y w h i l e CAMP d e r i v a t i v e s are i n h i b i t o r y
( 4 9 ) is i n accord with t h i s concept. antigen-antibody
S i n c e i t was found t h a t i m m o b i l i z e d
complexes a l s o i n h i b i t t h e m i t o g e n i c e f f e c t of 8 BrcGMP
( 2 9 ) , i t may b e a r g u e d t h a t t h e s e i n h i b i t o r y e f f e c t s a r e o c c u r r i n g v i a a central inhibitory signal.
Recent r e s u l t s i n o u r l a b o r a t o r y (50) i n d i c a t e
t h a t t h e i n t e r a c t i o n of mouse s p l e e n c e l l s w i t h i m m o b i l i z e d complexes r e s u l t s i n a p p r o x i m a t e l y a 50% i n c r e a s e i n t h e i n t r a c e l l u l a r l e v e l of CAMP w i t h i n 30 m i n u t e s .
This may r e p r e s e n t a c e n t r a l " o f f " s i g n a l d e l i v e r e d t o
t h e c e l l v i a Fc r e c e p t o r s . S t u d i e s on t h e f u n c t i o n a l r o l e of t h e lymphocyte Fc r e c e p t o r i n t h e immune r e s p o n s e a r e s t i l l i n t h e i r i n f a n c y .
The d a t a r e v i e w e d h e r e l e n d
p r o m i s e t h a t f u r t h e r i n v e s t i g a t i o n w i l l b e r e w a r d i n g a n d c o n t i n u e t o prov i d e some of t h e most e x c i t i n g problems i n c e l l u l a r immunology.
RYAN AND HENKART
466 References II
1.
Coutinho, A. and M o l l e r , G . , Scand. J . Immunol., 3:133, 1974.
2.
Basten, A.,
S p r e n t , J . and M i l l e r , J.F.A.P.,
N a t u r e , New B i o l . , 235:178,
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/01/15 For personal use only.
1972. 3.
D i c k l e r , H.B.
4.
Basten, A . , M i l l e r , J.F.A.P.,
and Kunkel, H . G . ,
J. Exp. Med., 136:191, 1972. S p r e n t , J . and Pye, J . , J . Exp. Med.,
135:610, 1972. 5.
Henkart. P.A.
A r b e i t , R.D.,
and D i c k l e r , H.B.,
i n I n V i t r o Methods i n
C e l l Mediated Immunity, e d i t e d by B. Bloom and J. David, v o l . 11, i n press. 6.
Yoshida, T.O.
7.
P a r a s k e v a s , F., Lee, S.T., Orr, K.B.
and A n d e r s o n , B . ,
Scand. J . Immunol.,
1:401, 1972.
and I s r a e l s , L.C., J. Immunol.,
108:1319, 1972. 8.
S t o u t , R.P. and Herzenberg, L.A.,
9.
Dickler, H.B.,
Adv. Immunol.,
J . Exp. Med., 142:611. 1975.
i n p r e s s , 1976.
11
10.
Uhr, J . W .
11.
S i n c l a i r , N.R.,
12.
Chan, P.L.
and S i n c l a i r , N.R.,
St.C.,
Immunol.,
21:967, 1971.
13.
Lees, R.K.
and S i n c l a i r , N.R.,
St.C.,
Immunol.,
24:735,
14.
Wason, W.M.
and F i t c h , F.W.,
15.
Abrahams, S . , P h i l l i p s . R.A.
and M o l l e r , G . , St.C.,
Adv. Immunol., 8:81, 1968.
J. Exp. Med., 129:1183, 1969.
J. Immunol., 110:1427, and Miller, R . G . ,
1973.
1973.
J . Exp. Med., 137:870,
1973. 16.
Kappler. J.W.. Van d e r Haven, A . , Dharmarajan, V. and Hoffman, M . , J. Immunol.,
111:1228, 1973.
17.
S p i e g e l b e r g , H. and Weigle, W.O.,
18.
D i c k l e r , H.B.
19.
Sachs, D.H.
and Sachs, D.H., and D i c k l e r , H . B . ,
J . Exp. Med.,
123:999, 1966.
J. Exp. Med., 140:779, 1974. T r a n s p l a n t . Rev., 23:159,
1975.
B LYMPHOCYTE ACTIVATION
467
20. Forni, L. and Pernis, B., in Membrane Receptor o f Lymphocytes, edited by M. Seligmann, J . L . Preud'homme and F.M. Kourilsky, p. 193, North Holland, Amsterdam, 1975. 21.
Unanue, E.R. and Abbas, A.K., in Membrane Receptor o f Lymphocytes, edited by M. Seligmann, J.L. Preud'homme and F.M. Kourilsky, p. 281,
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/01/15 For personal use only.
North Holland, Amsterdam, 1975. 11
22.
Coutinho, A. and Moller, G., Nature, New Biol., 245:12, 1973.
23.
Jerne, N.K. and Nordin, A.A., Science, 140:405, 1963.
24. Moller, G.,
Clin. Exp. Immunol., 4:65, 1969.
I*
25.
Moller, G. and Coutinho, A., J . Exp. Med., 141:647, 1975.
26. Ramasamy, R., Immunol., 30:559, 1976. 21.
Ryan, J.L. and Henkart, P.A., unpublished results.
28.
Ryan, J.L., Arbeit, R.D., Dickler, H.B. and Henkart, P.A., J . Exp. Med., 142:814, 1975.
29.
Ryan, J . L . and Henkart, P.A., submitted for publication.
30. Henkart, P.A. and Alexander, E., manuscript in preparation. 31. Stout, R.D. and Herzenberg, L.A., J. Exp. Med., 142:1041, 1975. 32. Sell, S . , Rowe, D . S . and Gell, P.G.H., J . Exp. Med. 122:823, 1965. 33. Weber, W.T., Transplantation, 24:113, 1975. 34. Andersson, J . , Bullock, W.W. and Melchers, F., Eur. J . Immunol., 4:715, 1974. 35.
Schrader, J.W., J . Immunol., 115:323, 1975.
36.
Sidman, C.L., Fed. Proc. Abst., 35:820, 1976.
37. Miller. J.F.A.P., Mitchell, G.F., Davies, A.J.S., Claman, H.N., Chaperon, E.A. and Taylor, R.B., Transplant. Rev., 1:1, 1969. 38. Fridman, W. and Goldstein, P., Cell. Immunol., 11:442, 1974. 39.
Gisler, R.H. and Fridman, W., Cell. Immunol., 23:99, 1976.
40. Gisler, R.H. and Fridrnan, W., J . Exp. Med., 142:507, 1975.
468
RYAN AND HENKART
41.
Greaves, M.F. and Bauminger, S.,
42.
Feldmann, M., Greaves, M., Parker, P.C. and Rittenberg, M.B., Eur. J.
Nature, New Biol., 2 3 5 : 6 7 , 1 9 7 2 .
Immunol., 4 : 5 9 1 , 1 9 7 4 . 43.
Wang, J . L . ,
McClaine, D.A. and Edelman, G.M., Proc. Nat. Acad. Science,
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/01/15 For personal use only.
72:1917, 1975. 44.
Greaves, M. and Janossy, G . , Transplant. Rev., 1 1 : 8 7 , 1 9 7 2 .
45.
Unanue, E. and Karnovsky, M., Transplant. Rev., 1 4 : 1 8 4 , 1 9 7 3 .
46.
Melchers, F. and Andersson, J., Transplant. Rev., 1 4 : 7 6 , 1 9 7 4 .
47.
Goldberg, N.D., Haddox, M.K., Nicol, S . E . , Glass, D.B., Sanford, C.H., Kuehl, F.A. and Estensen, R., Adv. Cyclic. Nucl. Res., 5 : 3 0 7 , 1 9 7 5 .
48,
Watson, J., J . Exp. Med., 1 4 1 : 9 7 , 1 9 7 5 .
49.
Weinstein, Y., Segal, S. and Melmon, K.L., J . Immunol., 115:112, 1 9 7 5 .
50.
Ryan, J . L . and Henkart, P.A., unpublished results.
