- Email: [email protected]
Suppression of the in vitro immune response by isolated mouse IgG subclasses
Ma/ax/or Pergamon
Immunology, Vol. 17, pp. 1599-1602. Press Ltd. 1980. Printed in Great Britain.
PRELIMINARY SUPPRESSION
OF
THE
IN VITRO
R.W.
IMMUNE
Milne,
M.P.
COMMUNICATION RESPONSE
Chalon
and
BY
ISOLATED
J.P.
MOUSE
IgG
SUBCLASSES
Vaerman
International Institute of Cellular and Universite Catholique de Louvain, Molecular Pathology, Unit of Experimental Medicine, 75, avenue Hippocrate B-1200 Brussels, Belgium. (First received
21 April
1980; in revised form 18 August
1980)
Abstract
Mouse IgGl, IgG2a and IgG2b antibodies against sheep red blood cells are equally effective, on a weight basis, in suppressing the primary in vitro immune response of normal mouse spleen cells against sheep erythrocytes. Significant suppression was obtained with as little as 10 ng of antibody per culture.
Introduction
It has been reported that mouse anti-sheep red blood cell (SRBC) antibodies of the IgGl subclass suppressed the in vivo (Murgita and Vas, 1972) and in vitro (Gordon and Murgita, 1975) immune response to SRBC whereas those of We have previously reported that thegG2 subclass enhanced the response. the IgGl fraction from mice immunized intragastrically with SRBC appeared slightly more effective than the IgG2a or IgGPb fractions of similar antibody content in mediating suppression of a primary in vitro plaque-forming cell (PFC) response; no enhancement by IgG2a or IgG?b was observed (Chalon et al,, 1979). In the present communication we have attempted to clarify the respective roles of the three major IgG subclasses in antibody-mediated feedback by testing the ability of IgGl, IgG2a and IgG2b from serum of mice hyperimmunized with SRBC to influence an in vitro PFC response.
Materials
and
Methods
IgG subclasses. Mouse IgGl, IgG2a and IgG2b were prepared from normal and SRBC-hyperimmunized mice by elution from Sepharose:bound staphylococcal Protein A with a stepwise pH gradient (Ey et al., 1978) followed by passage on antibodycoated columns (Chalon et al., 1979). The anti-SRBC concentration was measured by radioimmunoassay (Chalon et al., 1979) and immunoglobulin concentration by radial immunodiffusion (Mancini et al., 1965; Chalon et al., 1979). IgG3 was not obtained in desirable amount and purity to be included in this study. Culture conditions. Normal mouse spleen cells were cultured in Marbrook Chambers (Mat-brook, 1967) under the conditions described previously (Chalon et al., 1979). A quantity of each isolated IgG subclass, which contained a known concentration of anti-SRBC antibody was added to test cultures. The corresponding control cultures received the same concentration of the isolated normal IgG subclass. Cultures were tested for PFC at 4 days (Cunningham and Szenberg, 1967). The cultures were done in triplicate and the PFC assay in duplicate.
R. W. MILNE et al.
1600
The numbers of PFC found in test (containing anti-SRBC IgG) or control (containing normal IgGj cultures are expressed as a percentage of the number of PFC found in cultures which did not contain mouse inmunoglobulins.
Results
The concentration of anti-SRBC antibody and the Ig content of the isolated IgG subclasses are shown in Table 1. In the case of IgGl and IgGZb the anti-SRBC antibody represents approximately 10 % of the total Ig present in the fraction, whereas, in the case of IgGZa it is much lower (2 %). For the 3 IgG subclasses SRBC antibody per culture,
tested, suppression was evident at 10 ng and virtually complete at 300 ng/culture
of anti(Fig. 1).
Fig. 1 Suppression of the primary in vitro PFC response by IgGl (IJ---@, IgG2a (H), and IgG2b (ti) anti-SRBC antibody The control cultures contained subclasses. a quantity of normal IgG of the indicated subclass equal to that of the total mouse IgG in the corresponding test cultures. The numbers of PFC in test and control cultures are expressed as percentage of the numbers of PFC in cultures devoid of mouse Ig. Each point is the mean _t SD of three experiments.
ng ANTI-SRBC
/CULTURE
The relative efficiency of the 3 IgG subclasses in mediating suppression was not significantly different. This has been confirmed in 3 separate experiments. The normal non-specific IgG did not significantly influence the anti-SRBC response at any dose.
Discussion
Previously we have reported that IgGl anti-SRBC antibodies appeared to be slightly more efficient in suppressing an in vitro anti-SRBC direct PFC response than those of the IgG2a or IgGPb subclasses (Chalon et ai., 1979). The IgG fractions had been isolated from the serum of mice immunized intragastrically with SRBC and the concentration of anti-SRBC in the fractions was very low (approximately 500 ng/ml) relative to the total IgG (0.2 to 4 mg/ml). In the present study, we have isolated the IgG subclasses from mice hyperimmunized with SRBC. Due to the higher anti-SRBC activity (Table l), the concentration of antibody could be determined more accurately.
1601
Suppression of Immune Response by Mouse IgG Subclasses Table Anti-SRBC Ig
and
sample
Ig
concentrations
from
Fraction
1 in
isolated
SRBC-immunized
mice
IgGl
mice
)
550
IgG2a IgG2b Normal
subclasses
Anti-SRBC
tug/ml
__~---
IgG
6.8
30 20
E
IgGI IgG2a
< 0.05 < 0.05
4.6 2.0
IgG2b
i
0.2
0.05
The concentrations of anti-SRBC antibody causing suppression were similar However to those previously observed (> 10 ng/culture) (Chalon et al., 1979). the 3 subclasses -appeared to suppress the in vitro PFC response similarly. Gordon and Murgita (1975) have reported that IgGl anti-SRBC antibodies suppressed an in vitro immune response to SRBC whereas the response was IgG2 was not separated into In their study, enhanced by IgG2 antibodies. and the IgG3 was not removed, in contrast with IgG2a and IgG2b fractions, The concentration of anti-SRBC in their IgG our sample (Chalon et al., 1979). fractions was estimated to be 25 % of the total IgG content, or even 50 to 83 % these class-specific However, in their in vivo study (Murgita and Vas, 1972). antibody contents were measured by a method (Nash and Heremans, 1969) devised for soluble antigens in animals having low total Ig levels (previously germWe believe that these antibody contents were largely overestimated. It free). is indeed surprizing that mice who received four injections of SRBC would have This up to 25 % or more of their total IgG with specific anti-SRBC activity. could represent a major factor involved in the discrepancies with our results. Murgita and Vas (1972) found that, in vivo, IgG2a also had rather In addition, strong suppressive (80-90 %) activity, at least at high concentrations. their in vitro enhancing effect of IgG2a was claimed to be Furthermore, insensitive to a lo-fold increase or a lOOO-fold decrease in SRBC dosage, as These data well as to be mediated by a SRBC-IgG2a antigen-antibody complex. in our opinion, contradictory. are, our IgG2a sample could be contamined only with 0.5 % of IgGl (Chalon Finally, it is therefore excluded that our inhibition by IgG2a was due et al., 1979); to contamination with IgGl. In both our previous (Chalon et al., 1979) and present studies we have observed a dose-dependent suppression by both IgG2a and IgG2b and have not seen any enhancement. two types of feed-back control mechanisms by antibody to the Recently, The first requires immunogen have been reviewed and compared (Hoffmann, 1980). high concentrations of antibody and is based on the masking of the antigenic determinants, rendering them inaccessible to the sensitive lymphocytes. The second acts at low antibody concentration and presumably interferes with This cooperation the cooperation between helper cells and precursor B cells. may be inhibited by the binding of the antigen, via the Fc portion of the antibody, to numerous non-specific FcR + cells, rendering this antigen unable to direct the proper association of B cells with helper cells (T cells and macrophages). The observed suppression by the 3 IgG subclasses could involve the second mechanism just described, although other possibilities, such as the induction of suppressor T cells, could be envisaged. It is relatively surprizing that the 3 major mouse IgG subclasses would behave identically in this respect.
Acknowledgements
Supported by grant 3.4504.70 from the Fonds de la Recherche Bruxelles. One of us, R.W.M., was recipient of an Medicale, The technical assistance of Mrs. J. Naze-De Mets is gratefully
Scientifique fellowship. acknowledged.
ICP
R. W. MILNE et al. References
Chalon, effect
M.P., Milne, of serum from
Cunningham, technique
R.W. & Vaerman, orally immunized
(1979) J.P. In vitro immunosuppressive mice. Eur, J. Immunol. 9, 747.
A-J. & Szenberg, A. (1968) Further for detecting single antibody-forming
improvements
cells.
in
the
plaque
ImnunoloqyA
14,
Ey, P.L., Prowse, S.J. & Jenkin, C.R. (1978) Isolation of pure IgGl, and IgG2b immunoglobulins from mouse serum using protein A-sepharose. Immunochemistry 15, 429. Gordon, J. & Murgita, R.A. (1975) Suppression in vitro immune response by different classes 15, 392. Hoffmann, M.K. (1980) helper cells. Immunol. Mancini, J. quantitation 2, 235.
Antibody regulates Rev. 49, 79.
Carbonara, A.O. of antigens by
Marbrook, J. (1967) Lancet ii, 1279.
Primary
the
and augmentation of antibodies.
cooperation
of
599.
IgGZa
of the primary Cell. Immunol.
B cells
with
& Heremans J.F. (1965) Immunochemical single radial immunodiffusion. Immunochemistry
immune
response
in
cultures
of
spleen
cells.
Murgita, R.A. & Vas, S.I. (1972) Specific antibody-mediated effect on the immune response : suppression and augmentation of the primary immune response in mice by different classes of antibodies. Immunology 22, 319.
J.F. (1969) A quantitative antibody-binding method for D.R. & Heremans, Nash, the determination of specific antibody within different immunoglobulin classes. Immunology 17, 685.
Immunology, Vol. 17, pp. 1599-1602. Press Ltd. 1980. Printed in Great Britain.
PRELIMINARY SUPPRESSION
OF
THE
IN VITRO
R.W.
IMMUNE
Milne,
M.P.
COMMUNICATION RESPONSE
Chalon
and
BY
ISOLATED
J.P.
MOUSE
IgG
SUBCLASSES
Vaerman
International Institute of Cellular and Universite Catholique de Louvain, Molecular Pathology, Unit of Experimental Medicine, 75, avenue Hippocrate B-1200 Brussels, Belgium. (First received
21 April
1980; in revised form 18 August
1980)
Abstract
Mouse IgGl, IgG2a and IgG2b antibodies against sheep red blood cells are equally effective, on a weight basis, in suppressing the primary in vitro immune response of normal mouse spleen cells against sheep erythrocytes. Significant suppression was obtained with as little as 10 ng of antibody per culture.
Introduction
It has been reported that mouse anti-sheep red blood cell (SRBC) antibodies of the IgGl subclass suppressed the in vivo (Murgita and Vas, 1972) and in vitro (Gordon and Murgita, 1975) immune response to SRBC whereas those of We have previously reported that thegG2 subclass enhanced the response. the IgGl fraction from mice immunized intragastrically with SRBC appeared slightly more effective than the IgG2a or IgGPb fractions of similar antibody content in mediating suppression of a primary in vitro plaque-forming cell (PFC) response; no enhancement by IgG2a or IgG?b was observed (Chalon et al,, 1979). In the present communication we have attempted to clarify the respective roles of the three major IgG subclasses in antibody-mediated feedback by testing the ability of IgGl, IgG2a and IgG2b from serum of mice hyperimmunized with SRBC to influence an in vitro PFC response.
Materials
and
Methods
IgG subclasses. Mouse IgGl, IgG2a and IgG2b were prepared from normal and SRBC-hyperimmunized mice by elution from Sepharose:bound staphylococcal Protein A with a stepwise pH gradient (Ey et al., 1978) followed by passage on antibodycoated columns (Chalon et al., 1979). The anti-SRBC concentration was measured by radioimmunoassay (Chalon et al., 1979) and immunoglobulin concentration by radial immunodiffusion (Mancini et al., 1965; Chalon et al., 1979). IgG3 was not obtained in desirable amount and purity to be included in this study. Culture conditions. Normal mouse spleen cells were cultured in Marbrook Chambers (Mat-brook, 1967) under the conditions described previously (Chalon et al., 1979). A quantity of each isolated IgG subclass, which contained a known concentration of anti-SRBC antibody was added to test cultures. The corresponding control cultures received the same concentration of the isolated normal IgG subclass. Cultures were tested for PFC at 4 days (Cunningham and Szenberg, 1967). The cultures were done in triplicate and the PFC assay in duplicate.
R. W. MILNE et al.
1600
The numbers of PFC found in test (containing anti-SRBC IgG) or control (containing normal IgGj cultures are expressed as a percentage of the number of PFC found in cultures which did not contain mouse inmunoglobulins.
Results
The concentration of anti-SRBC antibody and the Ig content of the isolated IgG subclasses are shown in Table 1. In the case of IgGl and IgGZb the anti-SRBC antibody represents approximately 10 % of the total Ig present in the fraction, whereas, in the case of IgGZa it is much lower (2 %). For the 3 IgG subclasses SRBC antibody per culture,
tested, suppression was evident at 10 ng and virtually complete at 300 ng/culture
of anti(Fig. 1).
Fig. 1 Suppression of the primary in vitro PFC response by IgGl (IJ---@, IgG2a (H), and IgG2b (ti) anti-SRBC antibody The control cultures contained subclasses. a quantity of normal IgG of the indicated subclass equal to that of the total mouse IgG in the corresponding test cultures. The numbers of PFC in test and control cultures are expressed as percentage of the numbers of PFC in cultures devoid of mouse Ig. Each point is the mean _t SD of three experiments.
ng ANTI-SRBC
/CULTURE
The relative efficiency of the 3 IgG subclasses in mediating suppression was not significantly different. This has been confirmed in 3 separate experiments. The normal non-specific IgG did not significantly influence the anti-SRBC response at any dose.
Discussion
Previously we have reported that IgGl anti-SRBC antibodies appeared to be slightly more efficient in suppressing an in vitro anti-SRBC direct PFC response than those of the IgG2a or IgGPb subclasses (Chalon et ai., 1979). The IgG fractions had been isolated from the serum of mice immunized intragastrically with SRBC and the concentration of anti-SRBC in the fractions was very low (approximately 500 ng/ml) relative to the total IgG (0.2 to 4 mg/ml). In the present study, we have isolated the IgG subclasses from mice hyperimmunized with SRBC. Due to the higher anti-SRBC activity (Table l), the concentration of antibody could be determined more accurately.
1601
Suppression of Immune Response by Mouse IgG Subclasses Table Anti-SRBC Ig
and
sample
Ig
concentrations
from
Fraction
1 in
isolated
SRBC-immunized
mice
IgGl
mice
)
550
IgG2a IgG2b Normal
subclasses
Anti-SRBC
tug/ml
__~---
IgG
6.8
30 20
E
IgGI IgG2a
< 0.05 < 0.05
4.6 2.0
IgG2b
i
0.2
0.05
The concentrations of anti-SRBC antibody causing suppression were similar However to those previously observed (> 10 ng/culture) (Chalon et al., 1979). the 3 subclasses -appeared to suppress the in vitro PFC response similarly. Gordon and Murgita (1975) have reported that IgGl anti-SRBC antibodies suppressed an in vitro immune response to SRBC whereas the response was IgG2 was not separated into In their study, enhanced by IgG2 antibodies. and the IgG3 was not removed, in contrast with IgG2a and IgG2b fractions, The concentration of anti-SRBC in their IgG our sample (Chalon et al., 1979). fractions was estimated to be 25 % of the total IgG content, or even 50 to 83 % these class-specific However, in their in vivo study (Murgita and Vas, 1972). antibody contents were measured by a method (Nash and Heremans, 1969) devised for soluble antigens in animals having low total Ig levels (previously germWe believe that these antibody contents were largely overestimated. It free). is indeed surprizing that mice who received four injections of SRBC would have This up to 25 % or more of their total IgG with specific anti-SRBC activity. could represent a major factor involved in the discrepancies with our results. Murgita and Vas (1972) found that, in vivo, IgG2a also had rather In addition, strong suppressive (80-90 %) activity, at least at high concentrations. their in vitro enhancing effect of IgG2a was claimed to be Furthermore, insensitive to a lo-fold increase or a lOOO-fold decrease in SRBC dosage, as These data well as to be mediated by a SRBC-IgG2a antigen-antibody complex. in our opinion, contradictory. are, our IgG2a sample could be contamined only with 0.5 % of IgGl (Chalon Finally, it is therefore excluded that our inhibition by IgG2a was due et al., 1979); to contamination with IgGl. In both our previous (Chalon et al., 1979) and present studies we have observed a dose-dependent suppression by both IgG2a and IgG2b and have not seen any enhancement. two types of feed-back control mechanisms by antibody to the Recently, The first requires immunogen have been reviewed and compared (Hoffmann, 1980). high concentrations of antibody and is based on the masking of the antigenic determinants, rendering them inaccessible to the sensitive lymphocytes. The second acts at low antibody concentration and presumably interferes with This cooperation the cooperation between helper cells and precursor B cells. may be inhibited by the binding of the antigen, via the Fc portion of the antibody, to numerous non-specific FcR + cells, rendering this antigen unable to direct the proper association of B cells with helper cells (T cells and macrophages). The observed suppression by the 3 IgG subclasses could involve the second mechanism just described, although other possibilities, such as the induction of suppressor T cells, could be envisaged. It is relatively surprizing that the 3 major mouse IgG subclasses would behave identically in this respect.
Acknowledgements
Supported by grant 3.4504.70 from the Fonds de la Recherche Bruxelles. One of us, R.W.M., was recipient of an Medicale, The technical assistance of Mrs. J. Naze-De Mets is gratefully
Scientifique fellowship. acknowledged.
ICP
R. W. MILNE et al. References
Chalon, effect
M.P., Milne, of serum from
Cunningham, technique
R.W. & Vaerman, orally immunized
(1979) J.P. In vitro immunosuppressive mice. Eur, J. Immunol. 9, 747.
A-J. & Szenberg, A. (1968) Further for detecting single antibody-forming
improvements
cells.
in
the
plaque
ImnunoloqyA
14,
Ey, P.L., Prowse, S.J. & Jenkin, C.R. (1978) Isolation of pure IgGl, and IgG2b immunoglobulins from mouse serum using protein A-sepharose. Immunochemistry 15, 429. Gordon, J. & Murgita, R.A. (1975) Suppression in vitro immune response by different classes 15, 392. Hoffmann, M.K. (1980) helper cells. Immunol. Mancini, J. quantitation 2, 235.
Antibody regulates Rev. 49, 79.
Carbonara, A.O. of antigens by
Marbrook, J. (1967) Lancet ii, 1279.
Primary
the
and augmentation of antibodies.
cooperation
of
599.
IgGZa
of the primary Cell. Immunol.
B cells
with
& Heremans J.F. (1965) Immunochemical single radial immunodiffusion. Immunochemistry
immune
response
in
cultures
of
spleen
cells.
Murgita, R.A. & Vas, S.I. (1972) Specific antibody-mediated effect on the immune response : suppression and augmentation of the primary immune response in mice by different classes of antibodies. Immunology 22, 319.
J.F. (1969) A quantitative antibody-binding method for D.R. & Heremans, Nash, the determination of specific antibody within different immunoglobulin classes. Immunology 17, 685.