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Analysis and modulation of the immune response of mice to acetylcholine receptor by anti-idiotypes
Immunology Letters, 9 (1985) 331 336
Elsevier lmlet 582
ANALYSIS
AND MODULATION OF THE IMMUNE RESPONSE OF MICE TO ACETYLCHOLINE RECEPTOR BY ANTI-IDIOTYPES Miry C. SOUROUJON, Dora BARCHAN and Sara FUCHS Department of Chemical Immunology, The Weizmann Institute of Science. Rehovot 76100, Israel
(Received 27 December1984) (Accepted 30 December1984)
I. Summary
2. Introduction
Anti-idiotypes were raised in mice against three well-characterized anti-acetylcholine receptor (AChR) monoclonal antibodies (mcAbs), as well as against polyclonal mouse anti-AChR antibodies. In binding experiments, the anti-idiotypic antibodies inhibited the binding of AChR only to the immunizing idiotype. However, a less restricted specificity was found in in vivo experiments. Mice producing anti-idiotypes were challenged with AChR and the idiotypic composition of their anti-AChR response was analysed using specific rabbit anti-idiotypic antibodies. It was found that preimmunization with a certain idiotype leads to the preferential suppression of this particular idiotype in the polyclonal response to AChR. However, preimmunization with either polyclonal or monoclonal anti-AChR antibodies resuited in a reduction of the overall anti-Torpedo AChR and anti-muscle AChR titers. This reduction was greater than would be expected from the representation of each of the respective idiotypes in the polyclonal anti-AChR serum, and may imply that in addition to the immunizing idiotype other antiAChR idiotypes are also suppressed. Our results suggest that anti-idiotypes may have a potential for the modulation of the autoimmune response directed against AChR in myasthenia.
Acetylcholine receptor (AChR) is the main autoantigen in the autoimmune disease myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG) [1]. The ideal therapy in MG would be to eliminate or to turn off the specific immune response towards AChR. One of the possible therapeutic approaches is based on the production of specific anti-idiotypic antibodies. Suppression of idiotype expression by the administration of anti-idiotypes has been demonstrated in some systems [2-4]. Long-term suppression of the total antibody production was reported in only a few cases [4]. The use of passively transferred anti-idiotypes as regulators of autoimmune diseases has been reported previously: exogenous anti-idiotypic antibodies were shown to suppress autoimmune tubulointerstitial nephritis [5] and spontaneous autoimmune thyroiditis [6]. On the other hand, administration of rabbit antiserum against a dominant idiotype of anti-DNA antibodies failed to suppress anti-DNA antibodies in lupus prone mice [7]. Recently, Hahn and Ebling [8] have succeeded to delay the onset of nephritis by intraperitoneal administration of a syngeneic antiDNA monoclonal antibody (mcAb) into (NZBX NZW)F~ mice, probably by anti-idiotype formation. We have previously shown that anti-idiotypes raised against polyclonal anti-AChR antibodies isolated from a rabbit with EAMG were successful in preventing the initial development of EAMG and
Key words: acetylcholinereceptor monoclonalantibodies regulation - anti-idiotypes
0165 2478 / 85 / $ 3.30 © 1985 ElsevierScience Publishers B.V.(BiomedicalDivision)
331
possibly in modifying existing EAMG [9]. On the other hand, Lennon and Lambert have found that anti-idiotypes prepared against monoclonal antiAChR antibodies were unsuccessful in preventing the development of EAMG in rats [10]. It is possible that in cases in which anti-idiotypes did not exert the desired suppressive effect, the idiotypes selected for the elicitation of anti-idiotypes were not of the adequate specificity. Indeed, one of the problems of the anti-idiotype approach is posed by the heterogeneity of anti-AChR antibodies. Therefore, to inhibit the autoimmune response, one would need to induce anti-idiotypes with specificity for many or at least the most pathologically important anti-AChR idiotypes. In this study we demonstrate that the humoral anti-AChR response in mice can be suppressed to various degrees by prior administration of either polyclonal or monoclonal anti-AChR antibodies, probably via the formation of antiidiotypic antibodies. It appears that "vaccination" with either of three mcAbs studied, suppressed not only the idiotype borne by the immunizing mcAb but also affected the total anti-AChR response.
and mcAb 5.34) were elicited in rabbits as described elsewhere (M. C. Souroujon, D. Barchan and S. Fuchs, submitted for publ.). Anti-idiotypic antibodies were elicited in A K R / J female mice, aged 6 wk by injection in the footpads with 25 50 #g of the different idiotypes in complete Freund's adjuvant (CFA) (1 : 1). Each group consisting of 10 mice received either the immunoglobulin fraction precipitated from the ascitic fluids of (BALB/BL)Fj containing mcAbs 5.5, 5.14 or 5.34, or pooled affinity purified C57BL/6 anti-AChR antibodies. A control group was injected with pooled normal mouse immunoglobulins (NMIgs). Booster injections of the idiotypes in CFA were given intradermally at 10-day intervals. Four immunizations were required before a significant amount of anti-idiotypic antibodies could be monitored. Pooled anti-idiotypic antibodies prepared in rabbits and mice against the monoclonal and polyclonal anti-AChR antibodies were exhaustively adsorbed on a Sepharose-NMIg column prepared from (BALB/BL)F 1 and C57BL/6 mice, respectively. The latter step was done in order to remove anti-mouse activity from the rabbit anti-idiotypes and anti-allotypic activity from the mouse anti-idiotypes.
3. Materials and Methods
3.1. A ChR preparations Torpedo AChR was purified as described elsewhere [11], and iodinated by the chloramine-T method [12]. Mouse muscle AChR was extracted in 1% Triton X-100 from denervated hind legs of C57BL/6 mice, essentially as described previously [13]. Denervation of the mice was performed by bilateral removal of a 3 mm segment from the sciatic nerve, 14 days prior to the extraction of the muscle receptor. For RIA with Triton extracted muscle AChR, the extract was preincubated with an excess of JzsI-et-bungarotoxin (t~-Bgt), labelled by the lactoperoxidase method [14]. The muscle AChR complexed with 125I-a-Bgt served as antigen in the radioimmunoassay as described previously [13]. 3.2. Preparation of anti-idiotypic antibodies The preparation and characterization of the antiAChR mcAbs used in this study were described elsewhere [13, 15]. Anti-idiotypes against monoclonal anti-AChR antibodies (mcAb 5.5; mcAb 5.14 332
3.3. Radioimmunoassay For radioimmunoassay 25 #1 of 125I-AChR or 100 #1 of 1251-c~-Bgtcomplexed with muscle AChR, diluted in 0.01 M Tris-HC1 buffer, pH 7.4, containing 0.1% Triton X-100 and 1 rag/ml bovine serum albumin, were incubated for 30 min at 37°C with 25 #1 of the anti-AChR mcAb diluted in PBS, containing 5% normal mouse serum. Goat anti-mouse immunoglobulin (100 #1) was added and incubation was allowed for 30 min at 37 °C and then overnight at 4°C. The precipitates formed were centrifuged, washed twice and precipitated radioactivity was counted in a 7-counter. For inhibition of the radioimmunoassay, the anti-AChR antibodies (in a dilution that bound 15-25% of the 125I-AChR) were incubated for 1 h with varying concentrations of the different rabbit or mouse anti-idiotypes (in a volume of 25 #1 containing I0% normal rabbit or mouse serum) before the addition of J25I-AChR.
4. Results
4.1. Specificity of anti-idiotypic antibodies produced in mice The immunoglobulin fraction of three anti-AChR mcAbs (5.5, 5.14 and 5.34) as well as of affinity-purified pooled C57BL/6 anti-AChR antibodies were injected into A K R / J mice to produce anti-idiotypes. The properties of the anti-AChR mcAbs were described elsewhere [13, 15]. All three mcAbs which represent distinct antigenic specificities were shown to be directed to the c~-subunit of the receptor, and to cross-react with skeletal muscle AChR. Their effect on muscle AChR function and metabolism was previously studied [13]. Mice immunized with the anti-AChR antibodies developed anti-idiotypes following 4 injections of idiotypes as was measured by their ability to specifically inhibit the binding of AChR to the respective idiotypes. Anti-idiotypes produced against purified polyclonal C57BL/6 anti-AChR antibodies, inhibited AChR binding to the preparation of polyclonal anti-AChR antibodies used for immunization by 16% (data not shown); these anti-idiotypes did not inhibit the binding of a non-related antigen to its specific antibodies prepared in C57BL/6 mice. As can be seen in Fig. i, each anti-idiotypic serum, produced against the mcAbs inhibited the binding of AChR to the immunizing idiotype as was previously found for rabbit anti-idiotypes (M. C. Souroujon, D. Barchan and S. Fuchs, submitted for publ.). This
_
E
g ~o 3o ~-" 20
~ ~o 10-3
10-2
10-1 10-3 10-2 10-1 10-3 10-2 mouse anti-idiotypes (dtlution)
10-1
Fig. 1. Inhibition of 1251-AChR binding to monoclonal antiAChR antibodies by mouse anti-idiotypes. Inhibition by: (A)anti-5.5; (B) anti-5.14; (C) anti-5.34. Antibody inhibited: mcAb 5.5 (e---e); mcAb 5.14 ( A - - ~ ) ; mcAb 5.34 (O O). Anti-idiotypic sera were drawn from individual mice 12 wk following first immunization with the respective idiotype. The amount of mcAb used was that which binds 20% of the 1251-AChR added. Vertical bars represent SE.
specificity was also confirmed by the binding in solid phase radioimmunoassay of the anti-idiotypes to the (Fab')2 fragment of the immunizing idiotype (data not shown). 4.2. Regulation of the anti-A ChR response by
preimmunization with idiotypes We have examined the idiotypic expression as well as the total anti-AChR response in mice following preimmunization with different idiotypes. AKR/J mice were injected each with one of the various anti-AChR antibodies and for control with pooled NMIgs. Twelve weeks following the first immunization with the idiotype these mice were challenged with 10 #g of Torpedo AChR in CFA. At the time of challenge with Torpedo AChR all mice had comparable anti-idiotypic titers (see Fig. 1), and showed no detectable anti-AChR activity. The mice were bled individually 32 days after the challenge. It was of interest to test whether preimmunization of mice with a certain idiotype leads to a preferential suppression of this particular idiotype. In control mice preimmunized with NM|gs and then challenged with AChR, the three idiotypes tested (5.5, 5.14 and 5.34) were expressed to the extent of 12, 32 and 14% of the total anti-AChR response, respectively (Table !). The expression of the idiotypes was deduced from the inhibition of AChR binding to the mouse anti-AChR sera by rabbit anti-idiotypes specific for the idiotypes borne by mcAbs 5.5, 5.14 and 5.34 (M. C. Souroujon, D. Barchan and S. Fuchs, submitted for publ.). As seen in Table 1, the 5.14 idiotype is expressed to a significantly higher extent (32%) than that of mcAb 5.5 and 5.34 (12 and 14%, respectively). Preimmunization with a certain idiotype led to a suppression of the immunizing idiotype as compared with the expression of that particular idiotype in control mice (Table 1). Also, in some cases there was some effect on the expression of additional idiotypes; thus, preimmunization with mcAb 5.14 and 5.34 mutually suppressed the idiotypes of each other to a certain extent. The idiotype borne by mcAb 5.14 was partially suppressed by preimmunization with all three idiotypes tested. Mice preimmunized with mcAb 5.5 had elevated levels of the 5.34 idiotype. A possible explanation for this latter result 333
Table 1 Effect of preimmunization of mice with anti-AChR mcAbs on idiotypic expression a Preimmunization with NMIg 5.5 5.14 5.34
ldiotype expression (%)b 5.5 idiotype
5.14 idiotype
5.34 idiotype
12.0+_0.8 8.1 ±0.4 d 13.4& 0.2 14.3 ± 0.3
32.4± 1.5 23.4_+2.1 d 9.3±0.4 c 17. l ± 0.9 d
14.0±2.0 27. I ±2.1 c 8.5± 1.9d 1.4 ± 0.9 c
For this experiment, rabbit anti-idiotypes elicited against mcAbs 5.5, 5.14 and 5.34 (M. C. Souroujon, D. Barchan and S. Fuchs, submitted for publ.) were used as inhibitors of AChR binding to mouse anti-AChR sera of the different groups. b The numbers represent the percentage of inhibition of A C h R binding to mouse anti-AChR antibodies by a high excess of each of the respective rabbit antiidiotypes. % Inhibition was calculated as follows: a
(I
cpm in the presence of anti-idiotypes cpm in the presence of normal rabbit serum
)× 100
The results are the mean value of ten mice± SD. c Significantly different from the control group - P<0.001. d Significantly different from the control group P<0.05.
m a y b e t h a t t h e s u p p r e s s i o n o f s o m e i d i o t y p e s is
AChR
compensated
m u s c l e . T h e m e a n t i t e r s a r e g i v e n in T a b l e 2. A s c a n
b y t h e e l e v a t i o n o f o t h e r i d i o t y p e s in
the polyclonal response to AChR. distinct idiotypes on the overall humoral
with
anti-AChR
response to
of mice with polyclonal
a n t i b o d i e s r e s u l t e d in e s s e n t i a l l y a n a b o l -
ishment (98% reduction) of the anti-AChR
For that, the individual sera of mice chal-
lenged with AChR
and from denervated mouse
be seen, preimmunization
We then tested the effect of preimmunization AChR.
from Torpedo
as compared
were scored for titer towards
to mice preimmunized
t e r e s t i n g l y , p r e t r e a t m e n t w i t h e i t h e r m c A b s 5.5, 5.14
Table 2 A n t i - A C h R titer in mice preimmunized with idiotypes Idiotype injected
normal mouse lg mouse anti-AChR antibodies mcAb 5.5 Ig mcAb5.14 Ig mcAb 5.34 Ig
Anti-Torpedo A C h R
Anti-muscle A C h R
Antibody titer× 10 7a
Antibody titer× 10 9
100.0+ 18.0 1.85:0.5 b 30.0+ 4.8 c 40.0+ 6.1 c 4.6± 0.6 b
Suppression of titer (%)
Suppression of titer (%)
0
8.65:0.4
0
98 70 60 95
0.2+0.1 b 2.3+0.2 c 2.0+0.4 c 0.5±0.2 b
99 74 77 94
a Titer is given in moles A C h R precipitated by l l serum. The results are the mean values of l0 mice± SD. Statistical analysis of the difference in antibody activity between control and idiotype injected groups was performed using Student's t-test. b Significantly lower than the control group P<0.001. c Significantly lower than the control group - P<0.05. 334
response
with NMIgs. In-
or 5.34 resulted in a higher reduction of the antiAChR titer than would be expected from the representation of each of the respective idiotypes in mouse anti-AChR sera (Table 1, control group). Thus, a decrease of 95, 70 and 60% in the anti-AChR titer was obtained following immunization with mcAb 5.34, 5.5 and 5.14, respectively (see Table 2).
5. Discussion
In this study we have prepared and characterized anti-idiotypic antibodies towards anti-AChR monoclonal and polyclonal antibodies in allogeneic mice. We have analyzed the representation of different idiotypes in the anti-AChR response of mice and tested the feasibility of anti-idiotypes for regulating the humoral response to AChR. Rabbit anti-idiotypic antibodies against anti-AChR mcAbs have been recently prepared in our laboratory (M. C. Souroujon, D. Barchan and S. Fuchs, submitted for publ.). We have used these heterologous anti-idiotypic antibodies as specific reagents to analyze the expression of the respective idiotypes in the immune response of mice to AChR. It was shown that in the sera of mice preimmunized with a certain idiotype and challenged later with AChR, this particular idiotype is significantly suppressed, although other idiotypes seem to be affected to some extent as well. Interestingly, vaccination with different idiotypes resulted in a marked decrease of the total antiAChR titer. Preimmunization with polyclonal mouse anti-AChR antibodies suppressed most of the anti-AChR humoral response. Moreover, the suppression in the anti-AChR titer following vaccination with mcAbs 5.34, 5.14 and 5.5 was much greater than that expected from the representation of their idiotypes in mouse anti-AChR sera. It seems that a narrower idiotypic specificity is observed in binding and inhibition experiments performed in vitro, than in the in vivo regulation of the anti-AChR response. For example, the idiotype of mcAb 5.34 is expressed in about 14% of the anti-AChR antibodies (Table I), but leads to a 95% decrease in the total anti-AChR response (Table 2). It is of course important to find out whether "vaccination" with idiotypes can protect mice from autoimmune myasthenia, induced by active immuniza-
tion with AChR. Since clinical signs of EAMG are rather inconsistent in mice, we measured the anti"self,' AChR titer as a parameter of an autoimmune response. It was found that anti-"self,' AChR was also decreased and that there was a good correlation between the reduction of anti-Torpedo and anti"self' AChR titers. It appears that not all idiotypes have a similar regulating capacity, and that there is no correlation between the representation of a certain idiotype and its suppressive effect. For instance, preimmunization with mcAb 5.34 was more effective in reducing the anti-AChR titer than was preimmunization with mcAb 5.14, although mcAb 5.14 is more widely represented in the polyclonal anti-AChR serum. In this line, Lennon and Lambert [10] have reported that preimmunization of rats with a common antiAChR idiotype, did not affect the overall titer towards AChR. Two possible mechanisms for the suppressive effect by anti-idiotypes could be a clonal deletion of B cells and T cell regulation. We have demonstrated here that preimmunization with idiotypes affects not only the expression of the immunizing idiotype but suppresses the overall anti-AChR response. We therefore favor the explanation of the regulation via T cells; as T cell receptor idiotypes are known to be less heterogeneous than B cell receptor idiotypes [2, 3, 16 19]. The manipulation by anti-idiotypes on the level of T cells could thus affect the overall response to the antigen and not only the expression of the specific idiotype. Simple neutralization of idiotype by anti-idiotypic antibody is probably inadequate to explain the mechanism of suppression. It appears that the small amount of anti-idiotype present in the sera of the mice is greatly exceeded by the amount of idiotype produced by the immunized control animals. Our results provide evidence that the humoral response to AChR can be manipulated by the antiidiotypic approach, in spite of the complexity and heterogeneity of this immune response. Moreover, the use of the appropriate anti-idiotypes may have a potential in the regulation of the autoimmune response directed against the receptor in myasthenia.
335
Acknowledgements T h i s r e s e a r c h was s u p p o r t e d b y the M u s c u l a r D y s t r o p h y A s s o c i a t i o n o f A m e r i c a , the U n i t e d S t a t e s - I s r a e l B i n a t i o n a l Science F o u n d a t i o n ( B S F ) a n d t h e Los A n g e l e s C h a p t e r o f M y a s t h e n i a G r a v i s Foundation.
References [1] Fuchs, S. (1980) Trends Biochem. Sci. 5, 259. [2] Nissonof, A., Ju, S. T. and Owen, F. L. (1977) lmmunol. Rev. 34, 89. [3] Eichmann, K. (1978) Adv. Immunol. 26, •95. [4] Cosenza, H., Julius, M. H. and Augustin, A. A. (1977) lmmunol. Rev. 150, 67. [5] Brown, C. A., Corey, K. and Colvin, R. B, (1979) J. lmmunol. 123, 2102. [6] Zanetti, M. and Bigazzi, P. E. (1981) Eur. J. lmmunol. I l, 187. [7] Teitelbaum, D., Rauch, J., Stollar, B. D. and Schwartz, R. S. (1984) J. Immunol. 132, 1282.
336
[8] Hahn, B. H: and Ebting, F. M. (1983) J. Clin. Invest. 71, 1728. [9] Fuchs, S,, Bartfeld, D., Mochly-Rosen, D., Souroujon, M. and Feingold, C. (1981) Ann. N.Y. Acad. Sci. 377, I10. [10] Lennon~ V, A. and Lambert, E. H. (1981) Ann. N.Y. Acad. Sci. 377, 77. [11] Aharonov, A., Tarrab-Hazdai, R., Silman, 1. and Fuchs, S. (1977) Immunochemistry 14, 129. [12] Hunter, W. M. (1978) in: Handbook of Experimental Immunology (D. M. Weir, Ed.) p. 14.1, Blackwelk Oxford. [13] Souroujon, M. C., Mochly-Rosen, D., Gordon, A. S. and Fuchs, S. (1983) Muscle and Nerve 6, 303. [14] Marchalonis, J. J. (1969) Biochem. J. 113,299. [15] Mochly-Rosen, D. and Fuchs, S. (1981) Biochemistry 20,
5920. [16] Binz, H. and Wigzell, H, (1977) Cont. Top. lmmunobiol. 7, 113. [17] Krawinkel, U., Cramer, M., Melchers, 1., lmanishi-Kari, T. and Rajewsky, K. J. (1978) J. Exp. Med. 147, 1341. [18] Mozes, E. and Haimovich, J. (1979) Nature (London) 276, 56. [19] Sablitzky, F. and Rajewsky, K. (1984) EMBO 3.3, 3005.
Elsevier lmlet 582
ANALYSIS
AND MODULATION OF THE IMMUNE RESPONSE OF MICE TO ACETYLCHOLINE RECEPTOR BY ANTI-IDIOTYPES Miry C. SOUROUJON, Dora BARCHAN and Sara FUCHS Department of Chemical Immunology, The Weizmann Institute of Science. Rehovot 76100, Israel
(Received 27 December1984) (Accepted 30 December1984)
I. Summary
2. Introduction
Anti-idiotypes were raised in mice against three well-characterized anti-acetylcholine receptor (AChR) monoclonal antibodies (mcAbs), as well as against polyclonal mouse anti-AChR antibodies. In binding experiments, the anti-idiotypic antibodies inhibited the binding of AChR only to the immunizing idiotype. However, a less restricted specificity was found in in vivo experiments. Mice producing anti-idiotypes were challenged with AChR and the idiotypic composition of their anti-AChR response was analysed using specific rabbit anti-idiotypic antibodies. It was found that preimmunization with a certain idiotype leads to the preferential suppression of this particular idiotype in the polyclonal response to AChR. However, preimmunization with either polyclonal or monoclonal anti-AChR antibodies resuited in a reduction of the overall anti-Torpedo AChR and anti-muscle AChR titers. This reduction was greater than would be expected from the representation of each of the respective idiotypes in the polyclonal anti-AChR serum, and may imply that in addition to the immunizing idiotype other antiAChR idiotypes are also suppressed. Our results suggest that anti-idiotypes may have a potential for the modulation of the autoimmune response directed against AChR in myasthenia.
Acetylcholine receptor (AChR) is the main autoantigen in the autoimmune disease myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG) [1]. The ideal therapy in MG would be to eliminate or to turn off the specific immune response towards AChR. One of the possible therapeutic approaches is based on the production of specific anti-idiotypic antibodies. Suppression of idiotype expression by the administration of anti-idiotypes has been demonstrated in some systems [2-4]. Long-term suppression of the total antibody production was reported in only a few cases [4]. The use of passively transferred anti-idiotypes as regulators of autoimmune diseases has been reported previously: exogenous anti-idiotypic antibodies were shown to suppress autoimmune tubulointerstitial nephritis [5] and spontaneous autoimmune thyroiditis [6]. On the other hand, administration of rabbit antiserum against a dominant idiotype of anti-DNA antibodies failed to suppress anti-DNA antibodies in lupus prone mice [7]. Recently, Hahn and Ebling [8] have succeeded to delay the onset of nephritis by intraperitoneal administration of a syngeneic antiDNA monoclonal antibody (mcAb) into (NZBX NZW)F~ mice, probably by anti-idiotype formation. We have previously shown that anti-idiotypes raised against polyclonal anti-AChR antibodies isolated from a rabbit with EAMG were successful in preventing the initial development of EAMG and
Key words: acetylcholinereceptor monoclonalantibodies regulation - anti-idiotypes
0165 2478 / 85 / $ 3.30 © 1985 ElsevierScience Publishers B.V.(BiomedicalDivision)
331
possibly in modifying existing EAMG [9]. On the other hand, Lennon and Lambert have found that anti-idiotypes prepared against monoclonal antiAChR antibodies were unsuccessful in preventing the development of EAMG in rats [10]. It is possible that in cases in which anti-idiotypes did not exert the desired suppressive effect, the idiotypes selected for the elicitation of anti-idiotypes were not of the adequate specificity. Indeed, one of the problems of the anti-idiotype approach is posed by the heterogeneity of anti-AChR antibodies. Therefore, to inhibit the autoimmune response, one would need to induce anti-idiotypes with specificity for many or at least the most pathologically important anti-AChR idiotypes. In this study we demonstrate that the humoral anti-AChR response in mice can be suppressed to various degrees by prior administration of either polyclonal or monoclonal anti-AChR antibodies, probably via the formation of antiidiotypic antibodies. It appears that "vaccination" with either of three mcAbs studied, suppressed not only the idiotype borne by the immunizing mcAb but also affected the total anti-AChR response.
and mcAb 5.34) were elicited in rabbits as described elsewhere (M. C. Souroujon, D. Barchan and S. Fuchs, submitted for publ.). Anti-idiotypic antibodies were elicited in A K R / J female mice, aged 6 wk by injection in the footpads with 25 50 #g of the different idiotypes in complete Freund's adjuvant (CFA) (1 : 1). Each group consisting of 10 mice received either the immunoglobulin fraction precipitated from the ascitic fluids of (BALB/BL)Fj containing mcAbs 5.5, 5.14 or 5.34, or pooled affinity purified C57BL/6 anti-AChR antibodies. A control group was injected with pooled normal mouse immunoglobulins (NMIgs). Booster injections of the idiotypes in CFA were given intradermally at 10-day intervals. Four immunizations were required before a significant amount of anti-idiotypic antibodies could be monitored. Pooled anti-idiotypic antibodies prepared in rabbits and mice against the monoclonal and polyclonal anti-AChR antibodies were exhaustively adsorbed on a Sepharose-NMIg column prepared from (BALB/BL)F 1 and C57BL/6 mice, respectively. The latter step was done in order to remove anti-mouse activity from the rabbit anti-idiotypes and anti-allotypic activity from the mouse anti-idiotypes.
3. Materials and Methods
3.1. A ChR preparations Torpedo AChR was purified as described elsewhere [11], and iodinated by the chloramine-T method [12]. Mouse muscle AChR was extracted in 1% Triton X-100 from denervated hind legs of C57BL/6 mice, essentially as described previously [13]. Denervation of the mice was performed by bilateral removal of a 3 mm segment from the sciatic nerve, 14 days prior to the extraction of the muscle receptor. For RIA with Triton extracted muscle AChR, the extract was preincubated with an excess of JzsI-et-bungarotoxin (t~-Bgt), labelled by the lactoperoxidase method [14]. The muscle AChR complexed with 125I-a-Bgt served as antigen in the radioimmunoassay as described previously [13]. 3.2. Preparation of anti-idiotypic antibodies The preparation and characterization of the antiAChR mcAbs used in this study were described elsewhere [13, 15]. Anti-idiotypes against monoclonal anti-AChR antibodies (mcAb 5.5; mcAb 5.14 332
3.3. Radioimmunoassay For radioimmunoassay 25 #1 of 125I-AChR or 100 #1 of 1251-c~-Bgtcomplexed with muscle AChR, diluted in 0.01 M Tris-HC1 buffer, pH 7.4, containing 0.1% Triton X-100 and 1 rag/ml bovine serum albumin, were incubated for 30 min at 37°C with 25 #1 of the anti-AChR mcAb diluted in PBS, containing 5% normal mouse serum. Goat anti-mouse immunoglobulin (100 #1) was added and incubation was allowed for 30 min at 37 °C and then overnight at 4°C. The precipitates formed were centrifuged, washed twice and precipitated radioactivity was counted in a 7-counter. For inhibition of the radioimmunoassay, the anti-AChR antibodies (in a dilution that bound 15-25% of the 125I-AChR) were incubated for 1 h with varying concentrations of the different rabbit or mouse anti-idiotypes (in a volume of 25 #1 containing I0% normal rabbit or mouse serum) before the addition of J25I-AChR.
4. Results
4.1. Specificity of anti-idiotypic antibodies produced in mice The immunoglobulin fraction of three anti-AChR mcAbs (5.5, 5.14 and 5.34) as well as of affinity-purified pooled C57BL/6 anti-AChR antibodies were injected into A K R / J mice to produce anti-idiotypes. The properties of the anti-AChR mcAbs were described elsewhere [13, 15]. All three mcAbs which represent distinct antigenic specificities were shown to be directed to the c~-subunit of the receptor, and to cross-react with skeletal muscle AChR. Their effect on muscle AChR function and metabolism was previously studied [13]. Mice immunized with the anti-AChR antibodies developed anti-idiotypes following 4 injections of idiotypes as was measured by their ability to specifically inhibit the binding of AChR to the respective idiotypes. Anti-idiotypes produced against purified polyclonal C57BL/6 anti-AChR antibodies, inhibited AChR binding to the preparation of polyclonal anti-AChR antibodies used for immunization by 16% (data not shown); these anti-idiotypes did not inhibit the binding of a non-related antigen to its specific antibodies prepared in C57BL/6 mice. As can be seen in Fig. i, each anti-idiotypic serum, produced against the mcAbs inhibited the binding of AChR to the immunizing idiotype as was previously found for rabbit anti-idiotypes (M. C. Souroujon, D. Barchan and S. Fuchs, submitted for publ.). This
_
E
g ~o 3o ~-" 20
~ ~o 10-3
10-2
10-1 10-3 10-2 10-1 10-3 10-2 mouse anti-idiotypes (dtlution)
10-1
Fig. 1. Inhibition of 1251-AChR binding to monoclonal antiAChR antibodies by mouse anti-idiotypes. Inhibition by: (A)anti-5.5; (B) anti-5.14; (C) anti-5.34. Antibody inhibited: mcAb 5.5 (e---e); mcAb 5.14 ( A - - ~ ) ; mcAb 5.34 (O O). Anti-idiotypic sera were drawn from individual mice 12 wk following first immunization with the respective idiotype. The amount of mcAb used was that which binds 20% of the 1251-AChR added. Vertical bars represent SE.
specificity was also confirmed by the binding in solid phase radioimmunoassay of the anti-idiotypes to the (Fab')2 fragment of the immunizing idiotype (data not shown). 4.2. Regulation of the anti-A ChR response by
preimmunization with idiotypes We have examined the idiotypic expression as well as the total anti-AChR response in mice following preimmunization with different idiotypes. AKR/J mice were injected each with one of the various anti-AChR antibodies and for control with pooled NMIgs. Twelve weeks following the first immunization with the idiotype these mice were challenged with 10 #g of Torpedo AChR in CFA. At the time of challenge with Torpedo AChR all mice had comparable anti-idiotypic titers (see Fig. 1), and showed no detectable anti-AChR activity. The mice were bled individually 32 days after the challenge. It was of interest to test whether preimmunization of mice with a certain idiotype leads to a preferential suppression of this particular idiotype. In control mice preimmunized with NM|gs and then challenged with AChR, the three idiotypes tested (5.5, 5.14 and 5.34) were expressed to the extent of 12, 32 and 14% of the total anti-AChR response, respectively (Table !). The expression of the idiotypes was deduced from the inhibition of AChR binding to the mouse anti-AChR sera by rabbit anti-idiotypes specific for the idiotypes borne by mcAbs 5.5, 5.14 and 5.34 (M. C. Souroujon, D. Barchan and S. Fuchs, submitted for publ.). As seen in Table 1, the 5.14 idiotype is expressed to a significantly higher extent (32%) than that of mcAb 5.5 and 5.34 (12 and 14%, respectively). Preimmunization with a certain idiotype led to a suppression of the immunizing idiotype as compared with the expression of that particular idiotype in control mice (Table 1). Also, in some cases there was some effect on the expression of additional idiotypes; thus, preimmunization with mcAb 5.14 and 5.34 mutually suppressed the idiotypes of each other to a certain extent. The idiotype borne by mcAb 5.14 was partially suppressed by preimmunization with all three idiotypes tested. Mice preimmunized with mcAb 5.5 had elevated levels of the 5.34 idiotype. A possible explanation for this latter result 333
Table 1 Effect of preimmunization of mice with anti-AChR mcAbs on idiotypic expression a Preimmunization with NMIg 5.5 5.14 5.34
ldiotype expression (%)b 5.5 idiotype
5.14 idiotype
5.34 idiotype
12.0+_0.8 8.1 ±0.4 d 13.4& 0.2 14.3 ± 0.3
32.4± 1.5 23.4_+2.1 d 9.3±0.4 c 17. l ± 0.9 d
14.0±2.0 27. I ±2.1 c 8.5± 1.9d 1.4 ± 0.9 c
For this experiment, rabbit anti-idiotypes elicited against mcAbs 5.5, 5.14 and 5.34 (M. C. Souroujon, D. Barchan and S. Fuchs, submitted for publ.) were used as inhibitors of AChR binding to mouse anti-AChR sera of the different groups. b The numbers represent the percentage of inhibition of A C h R binding to mouse anti-AChR antibodies by a high excess of each of the respective rabbit antiidiotypes. % Inhibition was calculated as follows: a
(I
cpm in the presence of anti-idiotypes cpm in the presence of normal rabbit serum
)× 100
The results are the mean value of ten mice± SD. c Significantly different from the control group - P<0.001. d Significantly different from the control group P<0.05.
m a y b e t h a t t h e s u p p r e s s i o n o f s o m e i d i o t y p e s is
AChR
compensated
m u s c l e . T h e m e a n t i t e r s a r e g i v e n in T a b l e 2. A s c a n
b y t h e e l e v a t i o n o f o t h e r i d i o t y p e s in
the polyclonal response to AChR. distinct idiotypes on the overall humoral
with
anti-AChR
response to
of mice with polyclonal
a n t i b o d i e s r e s u l t e d in e s s e n t i a l l y a n a b o l -
ishment (98% reduction) of the anti-AChR
For that, the individual sera of mice chal-
lenged with AChR
and from denervated mouse
be seen, preimmunization
We then tested the effect of preimmunization AChR.
from Torpedo
as compared
were scored for titer towards
to mice preimmunized
t e r e s t i n g l y , p r e t r e a t m e n t w i t h e i t h e r m c A b s 5.5, 5.14
Table 2 A n t i - A C h R titer in mice preimmunized with idiotypes Idiotype injected
normal mouse lg mouse anti-AChR antibodies mcAb 5.5 Ig mcAb5.14 Ig mcAb 5.34 Ig
Anti-Torpedo A C h R
Anti-muscle A C h R
Antibody titer× 10 7a
Antibody titer× 10 9
100.0+ 18.0 1.85:0.5 b 30.0+ 4.8 c 40.0+ 6.1 c 4.6± 0.6 b
Suppression of titer (%)
Suppression of titer (%)
0
8.65:0.4
0
98 70 60 95
0.2+0.1 b 2.3+0.2 c 2.0+0.4 c 0.5±0.2 b
99 74 77 94
a Titer is given in moles A C h R precipitated by l l serum. The results are the mean values of l0 mice± SD. Statistical analysis of the difference in antibody activity between control and idiotype injected groups was performed using Student's t-test. b Significantly lower than the control group P<0.001. c Significantly lower than the control group - P<0.05. 334
response
with NMIgs. In-
or 5.34 resulted in a higher reduction of the antiAChR titer than would be expected from the representation of each of the respective idiotypes in mouse anti-AChR sera (Table 1, control group). Thus, a decrease of 95, 70 and 60% in the anti-AChR titer was obtained following immunization with mcAb 5.34, 5.5 and 5.14, respectively (see Table 2).
5. Discussion
In this study we have prepared and characterized anti-idiotypic antibodies towards anti-AChR monoclonal and polyclonal antibodies in allogeneic mice. We have analyzed the representation of different idiotypes in the anti-AChR response of mice and tested the feasibility of anti-idiotypes for regulating the humoral response to AChR. Rabbit anti-idiotypic antibodies against anti-AChR mcAbs have been recently prepared in our laboratory (M. C. Souroujon, D. Barchan and S. Fuchs, submitted for publ.). We have used these heterologous anti-idiotypic antibodies as specific reagents to analyze the expression of the respective idiotypes in the immune response of mice to AChR. It was shown that in the sera of mice preimmunized with a certain idiotype and challenged later with AChR, this particular idiotype is significantly suppressed, although other idiotypes seem to be affected to some extent as well. Interestingly, vaccination with different idiotypes resulted in a marked decrease of the total antiAChR titer. Preimmunization with polyclonal mouse anti-AChR antibodies suppressed most of the anti-AChR humoral response. Moreover, the suppression in the anti-AChR titer following vaccination with mcAbs 5.34, 5.14 and 5.5 was much greater than that expected from the representation of their idiotypes in mouse anti-AChR sera. It seems that a narrower idiotypic specificity is observed in binding and inhibition experiments performed in vitro, than in the in vivo regulation of the anti-AChR response. For example, the idiotype of mcAb 5.34 is expressed in about 14% of the anti-AChR antibodies (Table I), but leads to a 95% decrease in the total anti-AChR response (Table 2). It is of course important to find out whether "vaccination" with idiotypes can protect mice from autoimmune myasthenia, induced by active immuniza-
tion with AChR. Since clinical signs of EAMG are rather inconsistent in mice, we measured the anti"self,' AChR titer as a parameter of an autoimmune response. It was found that anti-"self,' AChR was also decreased and that there was a good correlation between the reduction of anti-Torpedo and anti"self' AChR titers. It appears that not all idiotypes have a similar regulating capacity, and that there is no correlation between the representation of a certain idiotype and its suppressive effect. For instance, preimmunization with mcAb 5.34 was more effective in reducing the anti-AChR titer than was preimmunization with mcAb 5.14, although mcAb 5.14 is more widely represented in the polyclonal anti-AChR serum. In this line, Lennon and Lambert [10] have reported that preimmunization of rats with a common antiAChR idiotype, did not affect the overall titer towards AChR. Two possible mechanisms for the suppressive effect by anti-idiotypes could be a clonal deletion of B cells and T cell regulation. We have demonstrated here that preimmunization with idiotypes affects not only the expression of the immunizing idiotype but suppresses the overall anti-AChR response. We therefore favor the explanation of the regulation via T cells; as T cell receptor idiotypes are known to be less heterogeneous than B cell receptor idiotypes [2, 3, 16 19]. The manipulation by anti-idiotypes on the level of T cells could thus affect the overall response to the antigen and not only the expression of the specific idiotype. Simple neutralization of idiotype by anti-idiotypic antibody is probably inadequate to explain the mechanism of suppression. It appears that the small amount of anti-idiotype present in the sera of the mice is greatly exceeded by the amount of idiotype produced by the immunized control animals. Our results provide evidence that the humoral response to AChR can be manipulated by the antiidiotypic approach, in spite of the complexity and heterogeneity of this immune response. Moreover, the use of the appropriate anti-idiotypes may have a potential in the regulation of the autoimmune response directed against the receptor in myasthenia.
335
Acknowledgements T h i s r e s e a r c h was s u p p o r t e d b y the M u s c u l a r D y s t r o p h y A s s o c i a t i o n o f A m e r i c a , the U n i t e d S t a t e s - I s r a e l B i n a t i o n a l Science F o u n d a t i o n ( B S F ) a n d t h e Los A n g e l e s C h a p t e r o f M y a s t h e n i a G r a v i s Foundation.
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