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Primary and secondary IgG are equally efficient immunosuppressors in relation to antigen binding capacity
Immunology Letters, 17 (1988) 189-194
Elsevier IML 01008
Primary and secondary IgG are equally efficient immunosuppressors in relation to antigen binding capacity Birgitta H e y m a n I a n d Lars P i l s t r 6 m 2 IDepartment of Immunology, BMC, and 2Department of Zoophysiology, Uppsala University, Uppsala, Sweden
(Received 12 October 1987; accepted 28 October 1987)
1. Summary
2. Introduction
Secondary, hyperimmune IgG antibodies can suppress the humoral immune response against the relevant antigen. Whether IgG antibodies derived from a primary antigen response also have this capacity is not clear, although the role of primary IgG is of great interest in a physiological situation. In this study we compared the in vivo immunosuppressive potential of primary and secondary IgG anti-SRBC (sheep erythrocytes) on the primary anti-SRBC PFC response in CBA/Ca mice. Both primary and secondary IgG antibodies are potent immunosuppressors causing more than 99% specific suppression. Preparations of primary and secondary IgG antibodies which, measured by an ELISA method, were shown to bind to SRBC to the same extent, also had very similar immunosuppressive potency. This emphasizes the strong correlation between the antigen binding and the immunosuppressive capacities of IgG antibodies.
IgG antibodies can act as efficient specific inhibitors of the antibody response [1, 2]. Most investigators o f this phenomenon have utilized IgG antibodies derived from hyperimmune sera. With the advent of the monoclonal antibody technique it became evident that the ability o f individual monoclonal IgG antibodies to suppress the immune response varied considerably [2, 3]. Some antibodies were totally nonsuppressive regardless of concentration whereas others suppressed to the same extent as polyclonal IgG. An explanation was offered in two independent studies where the ability o f individual monoclonal antibodies to bind to the antigen was shown to correlate with their immunosuppressive capacity [2, 3]. This correlation was independent of subclass o f the IgG antibodies. In a physiological situation the effect of primary IgG is of major interest but studies of the immunosuppressive role of primary IgG are scarce and show conflicting results. IgG1 antibodies derived from a primary anti-SRBC response have been reported to be inactive as immunosuppressors, whereas secondary IgG1 anti-SRBC antibodies efficiently suppressed the anti-SRBC response in mice [4]. Walker and Siskind [5], on the other hand, showed that both primary and secondary IgG antiDNP antibodies from rabbit were able to suppress the antibody response to DNP-BGG, but the high affinity, secondary antibodies were more efficient than the low affinity, primary antibodies. Whether primary SRBC-specific IgG is unable to act as an immunosuppressor due to qualitative differences from secondary IgG or whether there is
Key words: IgG; Immunosuppression;ELISA; Antigen binding Abbreviations: BGG, bovine gammaglobulin; BSS, Hank's
balanced salt solution; DNP, dinitrophenyl; ELISA, enzyme linked immunosorbentassay; HRBC, horse erythrocytes;NP, nitrophenyl; PBS, phosphate buffered saline; PFC, plaque forming cells; SRBC, sheep erythrocytes. Correspondence to: Birgitta Heyman, Dept. of Immunology,
BMC, Box 582, S-751 23 Uppsala, Sweden.
0165-2478 / 88 / $ 3.50 © 1988 ElsevierSciencePublishers B.V.(BiomedicalDivision)
189
merely a quantitative difference between them is the subject of the present investigation. It is demonstrated that primary IgG anti-SRBC is able to suppress more than 99% of a primary anti-SRBC response. When primary and secondary IgG antibodies were compared at the same level of binding to SRBC they were equally potent immunosuppressors, thus suggesting a quantitative rather than qualitative difference.
3. Materials and Methods
3.1. Mice Male CBA/Ca mice, 10-16 weeks old, from Anticimex, Stockholm, were used. 3.2. Antigens SRBC and HRBC were purchased from the National Veterinary Institute, Hhtunaholm, Sweden, as blood in sterile Alsever's solution and stored at 4 °C. Before use the erythrocytes were washed three times with PBS or BSS.
3.5. Plaque assay A modified version of Jerne's hemolytic plaque assay [8] was used: 25/~l 10% erythrocyte suspension, 100/zl appropriately diluted spleen cell suspension and 25 #1 guinea pig complement, diluted 1:5, was added to 300/~l 0.5% agarose solution (Agarose A 37, Kebo Laboratories, Stockholm, Sweden) at 45 °C and plated on 76 × 25 mm glass slides. All dilutions were made in BSS. The plates were incubated for 3 h at 37 °C and plaques were counted 'blindly'. The results are expressed as logl0 PFC per spleen and as the geometrical mean of each group. 3.6. ELISA Binding of the antibodies was assayed in an ELISA [9]. Briefly, erythrocytes were fixed to ELISA plates by glutaraldehyde. Antibodies, Protein A alkaline phosphatase and substrate were added consecutively with intermediate washings. The plates were read in a Titertec Multiscan microtiter plate reader (Flow Laboratories). The results are expressed as absorbance at 405 nm per 25 ~I antibody preparation.
3.3. Antibodies Primary IgG antibodies were derived from CBA/Ca mice immunised i.p. with 0.1 ml of 10% SRBC suspension in PBS. Seven days later the mice were bled. Secondary IgG antibodies were produced by immunising CBA/Ca mice i.p. three times at 14 day intervals with 0.1 ml 10070SRBC suspension in PBS. The mice were bled 10 days after the last injection. The anti-SRBC antisera were purified by affinity chromatography on Protein A-Sepharose by the method of Ey et al. which allows separation of all murine IgG subclasses [6]. Part of the eluted fraction containing primary IgG antibodies was further purified by passage over a Con-A-Sepharose column to deplete the sera of contaminating IgMantibodies [7]. The antibodies were dialysed against PBS.
4.1. Primary IgG is an efficient immunosuppressor The ability of primary IgG anti-SRBC to suppress the immune response in mice immunized with antibody followed by SRBC was investigated. Primary IgG eluted from a Protein A - Sepharose column as well as primary IgG further purified by passage over a Con A-Sepharose column (thus depleting the preparation of contaminating IgM antibodies [7]) were able to suppress the anti-SRBC response more than 99% (Table 1). There wa~no enhancing capacity of contaminating IgM anti-SRBC [1] in the IgG preparations purified only by Protein A - Sepharose chromatography. This antibody fraction was used throughout the rest of the experiments.
3.4. Immunisations Groups of at least four mice were given 0.1 ml of the antibody preparations in PBS in their tail veins 1 h prior to the intravenous injection of erythrocytes in 0.1 ml PBS. Control mice received only erythrocytes. The mice were killed five days later and their spleens tested in plaque assays.
4.2. Comparison of primary and secondary lgG: antigen binding and immunosuppression Next we compared the immunosuppressive potency of primary IgG to that of secondary IgG, which is a well known inhibitor of the antibody response. The protein concentrations of the primary and secondary IgG preparations, both purified by Pro-
190
4. Results
Table 1 Suppression of anti-SRBC response by primary anti-SRBC. Antibody (#g/mouse)
PFC anti-SRBCa
07o of Control b
IgG (820)d (82) IgG (820)e (82) Control
2.00+0.35 (100) 0.6 1.70+0.0 (50) 0.3 1.92 + 0.45 (84) 0.5 2.19+0.36 (156) 1.0 4.19+0.45 (15511) 100.0
1.2
IgG-
/x 1.0
P vs Cc 0.001 0.001 0.001 0.001
o
0.8
~
0.6
E
~
0.4
e~
Mice were immunized with primary IgG and 106 SRBC. The PFC response against SRBC was measured five days later. a Log10 PFC anti-SRBC/spleen +_ S.D. Figures within brackets = geometrical mean values of groups of five mice. b Percent of the control group (immunized with antigen only). c p value versus control group. d This IgG preparation was eluted from a Protein A-Sepharose column and reconstituted to the same volume as the initial serum. e Same as d but also passed over a Con A - Sepharose column to remove IgM.
0.2 [
0.01
0.1
1
......
10
l
100
IgG concentration (pg/ml) Fig. 1. Binding of primary (circles)and secondary (triangles)IgG anti-SRBC to SRBC in ELISA.
100000
tein A chromatography, were d e t e r m i n e d by measuring the a b s o r b a n c e at 280 nm. A s s u m i n g that an abs o r b a n c e o f 1.5 corresponds to a n IgG c o n c e n t r a t i o n o f 1 m g / m l the two p r e p a r a t i o n s c o n t a i n e d 1.8 mg (primary) a n d 8.2 mg (secondary) I g G / m l . Since the m e t h o d used for p u r i f i c a t i o n does n o t allow separation o f anti-SRBC-specific from non-specific IgG it is n o t possible to make a m o l a r c o m p a r i s o n o f the antigen-specific IgG c o n t e n t o f the p r i m a r y a n d secondary a n t i b o d y preparations. I n the following they are c o m p a r e d o n the basis o f total IgG content, i.e., protein c o n c e n t r a t i o n . Assayed in ELISA, secondary I g G b i n d s more efficiently to SRBC t h a n does p r i m a r y IgG. The p r i m a r y I g G a n t i b o d i e s reached h a l f their m a x i m u m b i n d i n g at 3 . 5 / z g / m l (A405 = 0.25) whereas the corr e s p o n d i n g value for s e c o n d a r y IgG is 0.5 # g / m l (A405 = 0.52) (Fig. 1). The capacity o f the two fractions to suppress a n in vivo a n t i - S R B C response was compared. The results o f two experiments are s u m m a r i z e d in Fig. 2 where the i m m u n e response ( P F C / s p l e e n ) is plotted against the a m o u n t o f p r i m a r y a n d s e c o n d a r y IgG injected into mice. A l t h o u g h b o t h p r i m a r y a n d s e c o n d a r y IgG a n t i b o d i e s are suppressive, secondary I g G is more efficient calculated per mg protein injected. Two/zg o f the s e c o n d a r y IgG p r e p a r a t i o n
1oooo t~ i1 t~
"6 ~.
1000
100
i
0.1
i
.......
I
.....
1.0 Dose
,,,i
........
10 of IgG
/ mouse
i
100 (pg)
Fig. 2. PFC responsein CBA/Ca mice injectedi.v.with syngeneic polyclonal IgG-anfibodies derived from primary (circles) or hyperimmune (triangles) anti-SRBC antisera and followed by an i.v. immunization of 4x 106 (Exp. 1, open symbols) or 2.3 x 106 (Exp. 2, filled symbols) SRBC. The direct anti-SRBC PFC response was assayed 5 days later and compared to that of the control groups (squares) immunized with antigen only. The results are expressed as number of PFC per spleen and each dot represents the geometrical mean of a group of five animals. The bars denote S.D. The PFC is plotted as a function of the amount of IgG injected into each mouse. The line denoting 99°70suppression is calculated from a mean of the control groups in the two experiments. 191
could suppress approx. 99% of control PFC, while a dose of approx. 80 #g of primary IgG was necessary for the same suppressive acitivity. Thus, the dose required for equal suppressive effect is 40-fold higher for primary than for secondary IgG. This could be compared to the 4 - 5 fold higher total IgG content of the primary IgG preparations (8.2 versus 1.8 mg/ml in the secondary antibody preparation). However, regardless of the nature of the injected antibodies, there was an apparent relation between the binding to the antigen in ELISA and suppression of the antibody response (Fig.. 3). It is apparent that the more efficiently the antibodies bind to SRBC in the ELISA, the more potent they are as immunosup-
100000
pressors and vice versa. In Exp. 1 (open symbols) one primary and one secondary IgG preparation had exactly the same binding value (absorbance = 0.72) and also showed very similar immunosuppressive capacities (99.3°70 and 99.5070, respectively). In Exp. 2 (filled symbols) three pairs of primary and secondary IgG were matched for equal binding to SRBC. The pair having the highest binding capacity (absorbance approx. 0.53) suppressed the anti-SRBC response to a very similar degree: 90070 (primary) and 86°70 (secondary IgG). The pair with intermediate binding (absorbance approx. 0.36) suppressed 6307o and 77070, respectively. The weakest binders (absorbance 0.18) did not show any significant suppression. Throughout the experiments the antigenspecificity of the immunosuppression was confirmed by immunising with and assaying the response against HRBC, which does not crossreact with SRBC at the antibody level [10]. No significant suppression of the anti-HRBC response could be found in any of the experiments (data not shown).
r"
4~ ~(37)
m ¢X
5. Discussion
10000
(5)
o LL n
0
z
~X~
(14)
,
\x 10)
1000
(°-2)I
100 I
0
I
I
I
I
I
I
0.2 0.4 0.6 0.8 1.0 1.2 Abs0rbance a t 405nm
Fig. 3. PFC response in CBA/Ca mice to SRBC (ordinate) when pretreated with syngeneic polyclonal anti-SRBC IgG antibodies derived from a primary (circles) or hyperimmune (triangles) antiserum as a function of their binding to~the antigen in ELISA (abscissa). Experimental details are the same as in Fig. 2. The lines indicate the regression from Exp. 1 (open symbols) and Exp. 2 (filled symbols). The dotted lines indicate the regression line when the results of the two experiments are pooled. The figure at each dot represents the percentage in comparison to the corresponding control group (squares). Groups with no suppression are not included in the calculations.
192
The present study is an attempt to elucidate the role of primary IgG in feedback immunosuppression. This is of great interest in a physiological situation since primary IgG is produced when an antigen for the first time induces an immune response in an animal. The results show that primary IgG antiSRBC antibodies can be extremely efficient immunosuppressors causing more than 99070 suppression of a primary anti-SRBC response (Table 1). When the suppressive ability of primary IgG was compared to that of secondary IgG anti-SRBC, there was a striking similarity in their inhibitory effect when compared at the same binding levels to the antigen (Fig. 3). Based on the total IgG content, however, secondary IgG is a much more potent suppressor than primary IgG (Fig. 2). It is likely that the primary IgG anti-SRBC preparation contains proportionally more nonspecific IgG, which accounts for at least part of this difference. However, the suppressive effect based on protein amounts differs 40 times while the protein concentration differs only 4 - 5 times (as mentioned in Section 4) indicating that the secondary IgG is more efficient
also on a molar basis. This is most likely due to an increase in avidity in the secondary IgG, which is indicated by the difference in binding to the antigen in ELISA (Fig. 1). We believe that the failure to demonstrate any immunosuppressive effect by primary IgG in earlier studies [4] is explained by the use of too-low antibody concentrations rather than by an inherent inability of primary IgG to induce immunosuppression. Apart from establishing a role for primary IgG also in suppression of the response to a particulate antigen, the present findings support earlier data [2, 3] on the importance of strong antigen-antibody binding for the initiation of efficient immunosuppression. It also points to the usefulness of ELISA in predicting the immunosuppressive capacity of IgG by measuring levels of antibody binding to the antigen.
Acknowledgements The excellent technical assistance by Ms Imma Brogren and the critical review of the manuscript by Professor Jan Andersson are gratefully ac-
knowledged. This work was supported by the Swedish Medical Research Council, grant No. B86-16X-O7492-O1A to B.H. and No. B88-16X-06016-08 to Jan Andersson as well as grant No. 85-4141 from the Swedish Board for Technical Development to Jan Andersson.
References [1] Henry, C. and Jerne, N. (1968) J. Exp. Med. 128, 133-152. [2] Heyman, B. and Wigzell, H. (1984) J. Immunol. 132, 1136-1143. [3] Bruggemann, M. and Rajewsky, K. (1982) Cell. Immunol. 71, 365-373. [4] Couthinho, A. and Forni, L. (1981) Ann. Immunol. (Inst. Pasteur) 132C, 131-144. [5] Walker, J. G. and Siskind, G. W. (1968) Immunology 14, 21-28. [6] Ey, P. L., Prowse, S. J. and Jenkin. C. R. (1978) Immunochemistry 15,429-436, [7] Weinstein, Y., Givol, D. and Strausbauch, P. H. (1972) J. Immunol. 109, 1402-1409. [8] Jerne, N. K. and Nordin, A. A. (1963) Science (Wash., D. C.) 140, 405. [9] Heyman, B., Holmquist, G., Borwell, P. and Heyman, U. (1984) J. Immunol. Methods 68, 193-204. [10] Heyman, B., Andrighetto, G. and Wigzell, H. (1982) J. Exp. Med. 155, 994-1009.
193
Elsevier IML 01008
Primary and secondary IgG are equally efficient immunosuppressors in relation to antigen binding capacity Birgitta H e y m a n I a n d Lars P i l s t r 6 m 2 IDepartment of Immunology, BMC, and 2Department of Zoophysiology, Uppsala University, Uppsala, Sweden
(Received 12 October 1987; accepted 28 October 1987)
1. Summary
2. Introduction
Secondary, hyperimmune IgG antibodies can suppress the humoral immune response against the relevant antigen. Whether IgG antibodies derived from a primary antigen response also have this capacity is not clear, although the role of primary IgG is of great interest in a physiological situation. In this study we compared the in vivo immunosuppressive potential of primary and secondary IgG anti-SRBC (sheep erythrocytes) on the primary anti-SRBC PFC response in CBA/Ca mice. Both primary and secondary IgG antibodies are potent immunosuppressors causing more than 99% specific suppression. Preparations of primary and secondary IgG antibodies which, measured by an ELISA method, were shown to bind to SRBC to the same extent, also had very similar immunosuppressive potency. This emphasizes the strong correlation between the antigen binding and the immunosuppressive capacities of IgG antibodies.
IgG antibodies can act as efficient specific inhibitors of the antibody response [1, 2]. Most investigators o f this phenomenon have utilized IgG antibodies derived from hyperimmune sera. With the advent of the monoclonal antibody technique it became evident that the ability o f individual monoclonal IgG antibodies to suppress the immune response varied considerably [2, 3]. Some antibodies were totally nonsuppressive regardless of concentration whereas others suppressed to the same extent as polyclonal IgG. An explanation was offered in two independent studies where the ability o f individual monoclonal antibodies to bind to the antigen was shown to correlate with their immunosuppressive capacity [2, 3]. This correlation was independent of subclass o f the IgG antibodies. In a physiological situation the effect of primary IgG is of major interest but studies of the immunosuppressive role of primary IgG are scarce and show conflicting results. IgG1 antibodies derived from a primary anti-SRBC response have been reported to be inactive as immunosuppressors, whereas secondary IgG1 anti-SRBC antibodies efficiently suppressed the anti-SRBC response in mice [4]. Walker and Siskind [5], on the other hand, showed that both primary and secondary IgG antiDNP antibodies from rabbit were able to suppress the antibody response to DNP-BGG, but the high affinity, secondary antibodies were more efficient than the low affinity, primary antibodies. Whether primary SRBC-specific IgG is unable to act as an immunosuppressor due to qualitative differences from secondary IgG or whether there is
Key words: IgG; Immunosuppression;ELISA; Antigen binding Abbreviations: BGG, bovine gammaglobulin; BSS, Hank's
balanced salt solution; DNP, dinitrophenyl; ELISA, enzyme linked immunosorbentassay; HRBC, horse erythrocytes;NP, nitrophenyl; PBS, phosphate buffered saline; PFC, plaque forming cells; SRBC, sheep erythrocytes. Correspondence to: Birgitta Heyman, Dept. of Immunology,
BMC, Box 582, S-751 23 Uppsala, Sweden.
0165-2478 / 88 / $ 3.50 © 1988 ElsevierSciencePublishers B.V.(BiomedicalDivision)
189
merely a quantitative difference between them is the subject of the present investigation. It is demonstrated that primary IgG anti-SRBC is able to suppress more than 99% of a primary anti-SRBC response. When primary and secondary IgG antibodies were compared at the same level of binding to SRBC they were equally potent immunosuppressors, thus suggesting a quantitative rather than qualitative difference.
3. Materials and Methods
3.1. Mice Male CBA/Ca mice, 10-16 weeks old, from Anticimex, Stockholm, were used. 3.2. Antigens SRBC and HRBC were purchased from the National Veterinary Institute, Hhtunaholm, Sweden, as blood in sterile Alsever's solution and stored at 4 °C. Before use the erythrocytes were washed three times with PBS or BSS.
3.5. Plaque assay A modified version of Jerne's hemolytic plaque assay [8] was used: 25/~l 10% erythrocyte suspension, 100/zl appropriately diluted spleen cell suspension and 25 #1 guinea pig complement, diluted 1:5, was added to 300/~l 0.5% agarose solution (Agarose A 37, Kebo Laboratories, Stockholm, Sweden) at 45 °C and plated on 76 × 25 mm glass slides. All dilutions were made in BSS. The plates were incubated for 3 h at 37 °C and plaques were counted 'blindly'. The results are expressed as logl0 PFC per spleen and as the geometrical mean of each group. 3.6. ELISA Binding of the antibodies was assayed in an ELISA [9]. Briefly, erythrocytes were fixed to ELISA plates by glutaraldehyde. Antibodies, Protein A alkaline phosphatase and substrate were added consecutively with intermediate washings. The plates were read in a Titertec Multiscan microtiter plate reader (Flow Laboratories). The results are expressed as absorbance at 405 nm per 25 ~I antibody preparation.
3.3. Antibodies Primary IgG antibodies were derived from CBA/Ca mice immunised i.p. with 0.1 ml of 10% SRBC suspension in PBS. Seven days later the mice were bled. Secondary IgG antibodies were produced by immunising CBA/Ca mice i.p. three times at 14 day intervals with 0.1 ml 10070SRBC suspension in PBS. The mice were bled 10 days after the last injection. The anti-SRBC antisera were purified by affinity chromatography on Protein A-Sepharose by the method of Ey et al. which allows separation of all murine IgG subclasses [6]. Part of the eluted fraction containing primary IgG antibodies was further purified by passage over a Con-A-Sepharose column to deplete the sera of contaminating IgMantibodies [7]. The antibodies were dialysed against PBS.
4.1. Primary IgG is an efficient immunosuppressor The ability of primary IgG anti-SRBC to suppress the immune response in mice immunized with antibody followed by SRBC was investigated. Primary IgG eluted from a Protein A - Sepharose column as well as primary IgG further purified by passage over a Con A-Sepharose column (thus depleting the preparation of contaminating IgM antibodies [7]) were able to suppress the anti-SRBC response more than 99% (Table 1). There wa~no enhancing capacity of contaminating IgM anti-SRBC [1] in the IgG preparations purified only by Protein A - Sepharose chromatography. This antibody fraction was used throughout the rest of the experiments.
3.4. Immunisations Groups of at least four mice were given 0.1 ml of the antibody preparations in PBS in their tail veins 1 h prior to the intravenous injection of erythrocytes in 0.1 ml PBS. Control mice received only erythrocytes. The mice were killed five days later and their spleens tested in plaque assays.
4.2. Comparison of primary and secondary lgG: antigen binding and immunosuppression Next we compared the immunosuppressive potency of primary IgG to that of secondary IgG, which is a well known inhibitor of the antibody response. The protein concentrations of the primary and secondary IgG preparations, both purified by Pro-
190
4. Results
Table 1 Suppression of anti-SRBC response by primary anti-SRBC. Antibody (#g/mouse)
PFC anti-SRBCa
07o of Control b
IgG (820)d (82) IgG (820)e (82) Control
2.00+0.35 (100) 0.6 1.70+0.0 (50) 0.3 1.92 + 0.45 (84) 0.5 2.19+0.36 (156) 1.0 4.19+0.45 (15511) 100.0
1.2
IgG-
/x 1.0
P vs Cc 0.001 0.001 0.001 0.001
o
0.8
~
0.6
E
~
0.4
e~
Mice were immunized with primary IgG and 106 SRBC. The PFC response against SRBC was measured five days later. a Log10 PFC anti-SRBC/spleen +_ S.D. Figures within brackets = geometrical mean values of groups of five mice. b Percent of the control group (immunized with antigen only). c p value versus control group. d This IgG preparation was eluted from a Protein A-Sepharose column and reconstituted to the same volume as the initial serum. e Same as d but also passed over a Con A - Sepharose column to remove IgM.
0.2 [
0.01
0.1
1
......
10
l
100
IgG concentration (pg/ml) Fig. 1. Binding of primary (circles)and secondary (triangles)IgG anti-SRBC to SRBC in ELISA.
100000
tein A chromatography, were d e t e r m i n e d by measuring the a b s o r b a n c e at 280 nm. A s s u m i n g that an abs o r b a n c e o f 1.5 corresponds to a n IgG c o n c e n t r a t i o n o f 1 m g / m l the two p r e p a r a t i o n s c o n t a i n e d 1.8 mg (primary) a n d 8.2 mg (secondary) I g G / m l . Since the m e t h o d used for p u r i f i c a t i o n does n o t allow separation o f anti-SRBC-specific from non-specific IgG it is n o t possible to make a m o l a r c o m p a r i s o n o f the antigen-specific IgG c o n t e n t o f the p r i m a r y a n d secondary a n t i b o d y preparations. I n the following they are c o m p a r e d o n the basis o f total IgG content, i.e., protein c o n c e n t r a t i o n . Assayed in ELISA, secondary I g G b i n d s more efficiently to SRBC t h a n does p r i m a r y IgG. The p r i m a r y I g G a n t i b o d i e s reached h a l f their m a x i m u m b i n d i n g at 3 . 5 / z g / m l (A405 = 0.25) whereas the corr e s p o n d i n g value for s e c o n d a r y IgG is 0.5 # g / m l (A405 = 0.52) (Fig. 1). The capacity o f the two fractions to suppress a n in vivo a n t i - S R B C response was compared. The results o f two experiments are s u m m a r i z e d in Fig. 2 where the i m m u n e response ( P F C / s p l e e n ) is plotted against the a m o u n t o f p r i m a r y a n d s e c o n d a r y IgG injected into mice. A l t h o u g h b o t h p r i m a r y a n d s e c o n d a r y IgG a n t i b o d i e s are suppressive, secondary I g G is more efficient calculated per mg protein injected. Two/zg o f the s e c o n d a r y IgG p r e p a r a t i o n
1oooo t~ i1 t~
"6 ~.
1000
100
i
0.1
i
.......
I
.....
1.0 Dose
,,,i
........
10 of IgG
/ mouse
i
100 (pg)
Fig. 2. PFC responsein CBA/Ca mice injectedi.v.with syngeneic polyclonal IgG-anfibodies derived from primary (circles) or hyperimmune (triangles) anti-SRBC antisera and followed by an i.v. immunization of 4x 106 (Exp. 1, open symbols) or 2.3 x 106 (Exp. 2, filled symbols) SRBC. The direct anti-SRBC PFC response was assayed 5 days later and compared to that of the control groups (squares) immunized with antigen only. The results are expressed as number of PFC per spleen and each dot represents the geometrical mean of a group of five animals. The bars denote S.D. The PFC is plotted as a function of the amount of IgG injected into each mouse. The line denoting 99°70suppression is calculated from a mean of the control groups in the two experiments. 191
could suppress approx. 99% of control PFC, while a dose of approx. 80 #g of primary IgG was necessary for the same suppressive acitivity. Thus, the dose required for equal suppressive effect is 40-fold higher for primary than for secondary IgG. This could be compared to the 4 - 5 fold higher total IgG content of the primary IgG preparations (8.2 versus 1.8 mg/ml in the secondary antibody preparation). However, regardless of the nature of the injected antibodies, there was an apparent relation between the binding to the antigen in ELISA and suppression of the antibody response (Fig.. 3). It is apparent that the more efficiently the antibodies bind to SRBC in the ELISA, the more potent they are as immunosup-
100000
pressors and vice versa. In Exp. 1 (open symbols) one primary and one secondary IgG preparation had exactly the same binding value (absorbance = 0.72) and also showed very similar immunosuppressive capacities (99.3°70 and 99.5070, respectively). In Exp. 2 (filled symbols) three pairs of primary and secondary IgG were matched for equal binding to SRBC. The pair having the highest binding capacity (absorbance approx. 0.53) suppressed the anti-SRBC response to a very similar degree: 90070 (primary) and 86°70 (secondary IgG). The pair with intermediate binding (absorbance approx. 0.36) suppressed 6307o and 77070, respectively. The weakest binders (absorbance 0.18) did not show any significant suppression. Throughout the experiments the antigenspecificity of the immunosuppression was confirmed by immunising with and assaying the response against HRBC, which does not crossreact with SRBC at the antibody level [10]. No significant suppression of the anti-HRBC response could be found in any of the experiments (data not shown).
r"
4~ ~(37)
m ¢X
5. Discussion
10000
(5)
o LL n
0
z
~X~
(14)
,
\x 10)
1000
(°-2)I
100 I
0
I
I
I
I
I
I
0.2 0.4 0.6 0.8 1.0 1.2 Abs0rbance a t 405nm
Fig. 3. PFC response in CBA/Ca mice to SRBC (ordinate) when pretreated with syngeneic polyclonal anti-SRBC IgG antibodies derived from a primary (circles) or hyperimmune (triangles) antiserum as a function of their binding to~the antigen in ELISA (abscissa). Experimental details are the same as in Fig. 2. The lines indicate the regression from Exp. 1 (open symbols) and Exp. 2 (filled symbols). The dotted lines indicate the regression line when the results of the two experiments are pooled. The figure at each dot represents the percentage in comparison to the corresponding control group (squares). Groups with no suppression are not included in the calculations.
192
The present study is an attempt to elucidate the role of primary IgG in feedback immunosuppression. This is of great interest in a physiological situation since primary IgG is produced when an antigen for the first time induces an immune response in an animal. The results show that primary IgG antiSRBC antibodies can be extremely efficient immunosuppressors causing more than 99070 suppression of a primary anti-SRBC response (Table 1). When the suppressive ability of primary IgG was compared to that of secondary IgG anti-SRBC, there was a striking similarity in their inhibitory effect when compared at the same binding levels to the antigen (Fig. 3). Based on the total IgG content, however, secondary IgG is a much more potent suppressor than primary IgG (Fig. 2). It is likely that the primary IgG anti-SRBC preparation contains proportionally more nonspecific IgG, which accounts for at least part of this difference. However, the suppressive effect based on protein amounts differs 40 times while the protein concentration differs only 4 - 5 times (as mentioned in Section 4) indicating that the secondary IgG is more efficient
also on a molar basis. This is most likely due to an increase in avidity in the secondary IgG, which is indicated by the difference in binding to the antigen in ELISA (Fig. 1). We believe that the failure to demonstrate any immunosuppressive effect by primary IgG in earlier studies [4] is explained by the use of too-low antibody concentrations rather than by an inherent inability of primary IgG to induce immunosuppression. Apart from establishing a role for primary IgG also in suppression of the response to a particulate antigen, the present findings support earlier data [2, 3] on the importance of strong antigen-antibody binding for the initiation of efficient immunosuppression. It also points to the usefulness of ELISA in predicting the immunosuppressive capacity of IgG by measuring levels of antibody binding to the antigen.
Acknowledgements The excellent technical assistance by Ms Imma Brogren and the critical review of the manuscript by Professor Jan Andersson are gratefully ac-
knowledged. This work was supported by the Swedish Medical Research Council, grant No. B86-16X-O7492-O1A to B.H. and No. B88-16X-06016-08 to Jan Andersson as well as grant No. 85-4141 from the Swedish Board for Technical Development to Jan Andersson.
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