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Evidences proving the intercalation hypothesis of the C-mediated complex release activity (CRA)
Inmunuchemisfr~, Vol. IS. pp. 33S-337 $3 Pqamon Press Ltd. 1978. Printed in Great Britain
EVIDENCES PROVING THE INTERCALATION HYPOTHESIS OF THE C-MEDIATED COMPLEX RELEASE ACTIVITY (CRA) l%‘A RAJNAVOLGYI,*
G. FiiST,t JULIA EMBER,* G. A. MEDGYESIt and J. GERGELY*
* Department of Immunology, E&v& L&and University, t National Institute of Haematology and Blood Transfusion, (Received 22 June 1977; received for publication
G&l 2131, Hungary Budapest, 1502, Hungary
I9 October 1977)
Abstract-Pre-incubation of BSA-anti-BSA precipitates with C Iq or bivalent Staphylococcus protein A inhibits the solubilization of the precipitates due to the complement dependent complex release activitv (CRA) of fresh sera. The inhibition was dose dependent. The results support the intercalation hypothesis of ‘CRA.
INTRODUCTION
Miller and Nussenzweig (1975) reported about the release of soluble antigen-antibody complexes containing C3 and C4 fragments following the treatment of immune precipitate with fresh serum. The solubilization is complement mediated (CRA, or Cmediated complex release activity) and occurs either in a less efficient way through the alternative pathway alone or through the combined activity of the classical and alternative pathways. The solubilization may be the consequence of an interaction between C3 and C3 convertase generated on the immune complexes. An explanation for the CRA is the ’ intercalation ’ hypothesis developed by Miller and Nussenzweig which postulates that the C3b binds to the immune complexes and this disrupts the lattice. The observation that monovalent binding of large protein molecules to the antibodies in a precipitate has a complex solubilizing effect (Czop etaf., 1976) favors this hypothesis. It was supposed that Clq, a large molecule with multiple binding sites for IgG must have an opposite effect, that is, it has to diminish the solubilization of the complexes. The aim of the present study was to check whether Clq and Staphylococcus protein (SPA) both interacting with the Fc part of complexed IgG inhibit the CRA of normal human serum.. MATERIALS
BSA antigen and anti-BSA
AND METHODS
antibody preparations
BSA preparates were purified by gel filtration on Sephadex G-200 column and labeled with lZ51by the chloramin T method of Bocci et a/. (1964).The specific activity of the labeled samples were 30-60 &i/mg. Anti-BSA antibodies from sera of two hyperimmunized rabbits were eluted from BSA-coated CNBr-activated Sepharose-4B columns. Antibody No. I was eluted with 0.2 MglycineHCI buffer pH 2.4, antibody No. 2 with0.35 Mglycine-HCI buffer pH 2.4 containing 0.1 M glucose and 0.5 M NaCI. The eluted fractions were immediately neutralized with 0.015 M NaOH and dialyzed against buffered saline. The
elution procedures were carried out at + 4°C. The avidity index of antibodies was determined by the Farr technique using the method of Celada et al. (1969). Antibody Nos. I and 2 have an avidity index of 0.71 and 0.9 respectively. Investigation of CRA
Immune complexes near to the equivalence zone were incubated overnight, and the precipitate was washed three times with cold buffered saline and once with PBS containing 7 x 10m4mol Ca’+ and 5 x IO-’ mol Mg*+. Finally the precipitate was resuspended by a syringe and 27-gauge needle. 40 tg immune precipitate was incubated for 30 min at room temperature with different volumes (100-400 pi) of 200 pg/ml Clq solution prepared from human serum by the method of Yonemasu and Stroud (1971). In other experiments 27 cg immune precipitate was incubated with 2-75 pg SpA kindly donated by Dr. J. SjGquist. After
incubation the amount of unabsorbed SpA was measured by a radial diffusion method using IgG containing agar gel (Medgyesi et al., submitted for publication). The preincubated immune complexes were washed twice with cold saline and their complement dependent complex release activity was investigated at 37°C using 4 ml fresh human serum and 3 ml buffered saline containing 7 x IO-” mol Ca’+ and 5 x 10V4mol Mg2+, pH 7.4. As a control the
same amount of immune complex was incubated with the same volume of heat treated (56”C, 30 min) human serum. 1 ml samples at different time intervals were put and the reaction was blocked with 2 ml ice-cold saline, containing 2% SRBC as indicator of effectivity of centrifugation. After centrifugation of the samples (2000 revlmin, 15 min) the radioactivity of the supernatant was checked. Immune precipitates formed at two-fold antibody excess and the same precipitates pre-incubated with different amounts of SpA were also compared by complement consuming capacity (Kabat & Mayer, 1961) using 27 cg precipitate samples. RESULTS
Preincubation of increasing amounts of C lq with the immune precipitates resulted in an inhibition of immune complex solubilization. Figure 1 shows the results of one typical experiment. During the initial period the complex release is slow and the presence of Clq did not influence the rate of solubilization 335
336
RVA RAJNAVGLGYI
et ~1.
Table I. The complement activation by 27 pg immune precipitate IC, formed at two-fold antibody excess. the same amount of immune precipitate + various amounts of SpA, and by SpA added alone to the serum
I
I
I
I
I
I
5
IO
2G
40
60
min
Fig. 1. The inhibition effect of Clq in the complement dependent complex release activity of immune complexes formed at the equivalence zone of antibody No. I. 40 gg washed immune precipitate was incubated with 200 (x--x);400(A---r);and800(0 0) tg Clq for 20 min at room temperature. (O--O) Indicates the per cent solubilization of immune precipitate at presence of fresh serum without preincubation with Clq. (c--.-W) Solubilization of immune complexes in the presence of heat inactivated serum.
IC, /I8
SPA P8
27 27 27 27 -
2.6 5.3 10.2 2.6 5.3 10.6
Consumed CH,,, 67 73 70 70 14 2s 37
during this lag phase (shown to be complement dependent by Takahashi et al., 1976). The steepness of the CRA curves was considerably diminished by the presence of C lq in the later phase of the process (called spontaneous release by Takahashi et al.. 1976). The quantity of solubilized immune precipitate decreased with the increasing amount of Clq. A dose dependent inhibitory effect was also observed in five different experiments when the immune precipitates were preincubated with increasing amounts of SpA. The results of one typical experiment can be seen in Fig. 2. SpA exerted a similar effect when precipitate was formed by antibody No. 1 (not shown on the figure). Since SpA binding to immune precipitates was found to interfere with complement dependent CRA, we tested the possible influence of SpA on the complement activating capacity of these immune precipitates. Doses of SpA having a significant inhibitory effect on the CRA did not influence the complement activation of immune complexes (Table 1). Cross reactivity was found between HSA and BSA (Weigle & McConahey, 1962). However, heat inactivated serum induced only a minimal immune complex solubilization effect in each experiment (Figs. 1 and 2), proving that immune complex solubilization by the human serum was not due to the effect of the cross reacting HSA present in extreme excess. DlSCUSSlON
min
Fig. 2. The inhibition effect of SpA in the complement dependent complex release activity of immune precipitate formed at two-fold antibody excess of antibody No. 2. On 27pgwashedimmuneprecipitate2.6(~ --x);5.3(~-A); and 10.2 (0.. .O) pg SpA was absorbed after an hour incubation at room temperature and washed three times with cold PBS. (O----O ) Indicates the per cent solubilization of the same immune precipitate at presence of fresh serum without preincubation with SpA. (~.-.a) Solubilization of immune complexes in the presence of heat inactivated serum.
The complement dependent complex release activity described by Miller and Nussenzweig may have a great importance influencing biological and pathological processes triggered by the immune complexes. The mechanism of the complement dependent solubilization of immune precipitates is not yet clarified. Nussenzweig and coworkers (reviewed by Takahashi et al., 1976) postulated that the solubilization may be the consequence of binding of C3b fragments to the immune complexes. Experiments using Fab fragments of different origin (Czop et al., 1976) demonstrated the solubilization of BSAanti-BSA complexes by both Fab anti-Fab and Fab anti-Fc while non-specific Fab was without effect. On the other hand (Fab’), fragments of anti-IgG antibodies made the immune precipitates more soluble. These model experiments point to the correctness of the intercalation hypothesis which
337
c-Mediated Complex Release Activity that the monovalent binding of large polypeptides to the complex can disrupt the lattice. Our present results proved that components with multiple binding sites interacting with the complexed IgG inhibit the solubilizing effect of CRA. It was supposed that Clq should function like antiimmunoglobulin (Fab’), and decrease the solubility of aggregates by further cross linking of IgG. In our experiments 200400 pg doses of Clq decreased the solubilization of BSA-anti-BSA precipitates when the Clq preincubated immune complexes were mixed with fresh serum. The amount of Clq used was perhaps not suficient to saturate the binding sites on the complexed antibody for the first complement component, i.e. the triggering of CRA by the complexes was not inhibited. SpA interacts with the Fc part of IgG, and it is at least bivalent in this reaction (SjGquist et al., 1972). The observed inhibition of complex release might be due to either the stabilization of the complex by cross linking of the Fc-regions or by influencing the complement activation by the complex. Sjwuist and Stalenheim (1969) reported that SpAIgG complexes are capable of activating complement, but inhibition of complement activation was observed when SpA was added to complexed IgG (Kronvall & Gewurz, 1970). The doses of SpA having an inhibitory effect in CRA were significantly less than those necessary for inhibiting the complement activating capacity of immune complexes. Kronvall and Gewurz (1970) found such an effect when 200 pg SpA was added to 32 pg aggregated human IgG in contrast to the 2-75 pg SpA and 27-30 gg immune precipitate in this study. postulates
Since both the C Iq and bivalent SpA inhibited the solubilization of immune precipitates and since this inhibitory effect was dose dependent we conclude that macromolecules having at least two binding sites for immunoglobulin Fc-region may compensate the disrupting effect of the C3b fragments produced during the complement activation. Cross linking of antibodies in the complex exerts a stabilizing effect, whereas monovalent binding loosen the lattice by intercalation leading to the complex solubilization phenomenon (Miller & Nussenzweig, 1975; Czop & Nussenzweig, 1976).
REFERENCES
Bocci V. (1964) Boll. Sot. ital. Biol. sper. 40, 89. Celada F., Schmidt D. & Strom R. (1969) Immunology 17, 183.
Czop J. & Nussenzweig V. (1976) J. exp. Med. 143, 615. Kabat E. A. & Meyer M. M. (1961) In Experimental Immunochemistrv (2nd edn). Springfield, Illindis.
Charles
C. Thomas,
Kronvall G. & Gewurz H. (1970) C/in. exp. Immunol. 7, 211. Miller G. W. & Nussenzweig V. (1975) Proc. natn Acad. Sci., U.S.A. 72, 418. SjGquist J. & Stalenheim G. (1969) J. Immun. 103, 467. Sjijquist J.. Meloun S. & Hjelm H. (1972) Eur. J. Biothem. 29, 572. Takahashi M., Czop J., Ferreira A. & Nussenzweig V. (1976) Transplantation Rev. 32, I2 I Weigle W. 0. & McConahey P. J. (1962) J. Immrrn. 88, 121. Yonemasu K. & Stroud R. M. (1971) J. fmmun. 106, 304.
EVIDENCES PROVING THE INTERCALATION HYPOTHESIS OF THE C-MEDIATED COMPLEX RELEASE ACTIVITY (CRA) l%‘A RAJNAVOLGYI,*
G. FiiST,t JULIA EMBER,* G. A. MEDGYESIt and J. GERGELY*
* Department of Immunology, E&v& L&and University, t National Institute of Haematology and Blood Transfusion, (Received 22 June 1977; received for publication
G&l 2131, Hungary Budapest, 1502, Hungary
I9 October 1977)
Abstract-Pre-incubation of BSA-anti-BSA precipitates with C Iq or bivalent Staphylococcus protein A inhibits the solubilization of the precipitates due to the complement dependent complex release activitv (CRA) of fresh sera. The inhibition was dose dependent. The results support the intercalation hypothesis of ‘CRA.
INTRODUCTION
Miller and Nussenzweig (1975) reported about the release of soluble antigen-antibody complexes containing C3 and C4 fragments following the treatment of immune precipitate with fresh serum. The solubilization is complement mediated (CRA, or Cmediated complex release activity) and occurs either in a less efficient way through the alternative pathway alone or through the combined activity of the classical and alternative pathways. The solubilization may be the consequence of an interaction between C3 and C3 convertase generated on the immune complexes. An explanation for the CRA is the ’ intercalation ’ hypothesis developed by Miller and Nussenzweig which postulates that the C3b binds to the immune complexes and this disrupts the lattice. The observation that monovalent binding of large protein molecules to the antibodies in a precipitate has a complex solubilizing effect (Czop etaf., 1976) favors this hypothesis. It was supposed that Clq, a large molecule with multiple binding sites for IgG must have an opposite effect, that is, it has to diminish the solubilization of the complexes. The aim of the present study was to check whether Clq and Staphylococcus protein (SPA) both interacting with the Fc part of complexed IgG inhibit the CRA of normal human serum.. MATERIALS
BSA antigen and anti-BSA
AND METHODS
antibody preparations
BSA preparates were purified by gel filtration on Sephadex G-200 column and labeled with lZ51by the chloramin T method of Bocci et a/. (1964).The specific activity of the labeled samples were 30-60 &i/mg. Anti-BSA antibodies from sera of two hyperimmunized rabbits were eluted from BSA-coated CNBr-activated Sepharose-4B columns. Antibody No. I was eluted with 0.2 MglycineHCI buffer pH 2.4, antibody No. 2 with0.35 Mglycine-HCI buffer pH 2.4 containing 0.1 M glucose and 0.5 M NaCI. The eluted fractions were immediately neutralized with 0.015 M NaOH and dialyzed against buffered saline. The
elution procedures were carried out at + 4°C. The avidity index of antibodies was determined by the Farr technique using the method of Celada et al. (1969). Antibody Nos. I and 2 have an avidity index of 0.71 and 0.9 respectively. Investigation of CRA
Immune complexes near to the equivalence zone were incubated overnight, and the precipitate was washed three times with cold buffered saline and once with PBS containing 7 x 10m4mol Ca’+ and 5 x IO-’ mol Mg*+. Finally the precipitate was resuspended by a syringe and 27-gauge needle. 40 tg immune precipitate was incubated for 30 min at room temperature with different volumes (100-400 pi) of 200 pg/ml Clq solution prepared from human serum by the method of Yonemasu and Stroud (1971). In other experiments 27 cg immune precipitate was incubated with 2-75 pg SpA kindly donated by Dr. J. SjGquist. After
incubation the amount of unabsorbed SpA was measured by a radial diffusion method using IgG containing agar gel (Medgyesi et al., submitted for publication). The preincubated immune complexes were washed twice with cold saline and their complement dependent complex release activity was investigated at 37°C using 4 ml fresh human serum and 3 ml buffered saline containing 7 x IO-” mol Ca’+ and 5 x 10V4mol Mg2+, pH 7.4. As a control the
same amount of immune complex was incubated with the same volume of heat treated (56”C, 30 min) human serum. 1 ml samples at different time intervals were put and the reaction was blocked with 2 ml ice-cold saline, containing 2% SRBC as indicator of effectivity of centrifugation. After centrifugation of the samples (2000 revlmin, 15 min) the radioactivity of the supernatant was checked. Immune precipitates formed at two-fold antibody excess and the same precipitates pre-incubated with different amounts of SpA were also compared by complement consuming capacity (Kabat & Mayer, 1961) using 27 cg precipitate samples. RESULTS
Preincubation of increasing amounts of C lq with the immune precipitates resulted in an inhibition of immune complex solubilization. Figure 1 shows the results of one typical experiment. During the initial period the complex release is slow and the presence of Clq did not influence the rate of solubilization 335
336
RVA RAJNAVGLGYI
et ~1.
Table I. The complement activation by 27 pg immune precipitate IC, formed at two-fold antibody excess. the same amount of immune precipitate + various amounts of SpA, and by SpA added alone to the serum
I
I
I
I
I
I
5
IO
2G
40
60
min
Fig. 1. The inhibition effect of Clq in the complement dependent complex release activity of immune complexes formed at the equivalence zone of antibody No. I. 40 gg washed immune precipitate was incubated with 200 (x--x);400(A---r);and800(0 0) tg Clq for 20 min at room temperature. (O--O) Indicates the per cent solubilization of immune precipitate at presence of fresh serum without preincubation with Clq. (c--.-W) Solubilization of immune complexes in the presence of heat inactivated serum.
IC, /I8
SPA P8
27 27 27 27 -
2.6 5.3 10.2 2.6 5.3 10.6
Consumed CH,,, 67 73 70 70 14 2s 37
during this lag phase (shown to be complement dependent by Takahashi et al., 1976). The steepness of the CRA curves was considerably diminished by the presence of C lq in the later phase of the process (called spontaneous release by Takahashi et al.. 1976). The quantity of solubilized immune precipitate decreased with the increasing amount of Clq. A dose dependent inhibitory effect was also observed in five different experiments when the immune precipitates were preincubated with increasing amounts of SpA. The results of one typical experiment can be seen in Fig. 2. SpA exerted a similar effect when precipitate was formed by antibody No. 1 (not shown on the figure). Since SpA binding to immune precipitates was found to interfere with complement dependent CRA, we tested the possible influence of SpA on the complement activating capacity of these immune precipitates. Doses of SpA having a significant inhibitory effect on the CRA did not influence the complement activation of immune complexes (Table 1). Cross reactivity was found between HSA and BSA (Weigle & McConahey, 1962). However, heat inactivated serum induced only a minimal immune complex solubilization effect in each experiment (Figs. 1 and 2), proving that immune complex solubilization by the human serum was not due to the effect of the cross reacting HSA present in extreme excess. DlSCUSSlON
min
Fig. 2. The inhibition effect of SpA in the complement dependent complex release activity of immune precipitate formed at two-fold antibody excess of antibody No. 2. On 27pgwashedimmuneprecipitate2.6(~ --x);5.3(~-A); and 10.2 (0.. .O) pg SpA was absorbed after an hour incubation at room temperature and washed three times with cold PBS. (O----O ) Indicates the per cent solubilization of the same immune precipitate at presence of fresh serum without preincubation with SpA. (~.-.a) Solubilization of immune complexes in the presence of heat inactivated serum.
The complement dependent complex release activity described by Miller and Nussenzweig may have a great importance influencing biological and pathological processes triggered by the immune complexes. The mechanism of the complement dependent solubilization of immune precipitates is not yet clarified. Nussenzweig and coworkers (reviewed by Takahashi et al., 1976) postulated that the solubilization may be the consequence of binding of C3b fragments to the immune complexes. Experiments using Fab fragments of different origin (Czop et al., 1976) demonstrated the solubilization of BSAanti-BSA complexes by both Fab anti-Fab and Fab anti-Fc while non-specific Fab was without effect. On the other hand (Fab’), fragments of anti-IgG antibodies made the immune precipitates more soluble. These model experiments point to the correctness of the intercalation hypothesis which
337
c-Mediated Complex Release Activity that the monovalent binding of large polypeptides to the complex can disrupt the lattice. Our present results proved that components with multiple binding sites interacting with the complexed IgG inhibit the solubilizing effect of CRA. It was supposed that Clq should function like antiimmunoglobulin (Fab’), and decrease the solubility of aggregates by further cross linking of IgG. In our experiments 200400 pg doses of Clq decreased the solubilization of BSA-anti-BSA precipitates when the Clq preincubated immune complexes were mixed with fresh serum. The amount of Clq used was perhaps not suficient to saturate the binding sites on the complexed antibody for the first complement component, i.e. the triggering of CRA by the complexes was not inhibited. SpA interacts with the Fc part of IgG, and it is at least bivalent in this reaction (SjGquist et al., 1972). The observed inhibition of complex release might be due to either the stabilization of the complex by cross linking of the Fc-regions or by influencing the complement activation by the complex. Sjwuist and Stalenheim (1969) reported that SpAIgG complexes are capable of activating complement, but inhibition of complement activation was observed when SpA was added to complexed IgG (Kronvall & Gewurz, 1970). The doses of SpA having an inhibitory effect in CRA were significantly less than those necessary for inhibiting the complement activating capacity of immune complexes. Kronvall and Gewurz (1970) found such an effect when 200 pg SpA was added to 32 pg aggregated human IgG in contrast to the 2-75 pg SpA and 27-30 gg immune precipitate in this study. postulates
Since both the C Iq and bivalent SpA inhibited the solubilization of immune precipitates and since this inhibitory effect was dose dependent we conclude that macromolecules having at least two binding sites for immunoglobulin Fc-region may compensate the disrupting effect of the C3b fragments produced during the complement activation. Cross linking of antibodies in the complex exerts a stabilizing effect, whereas monovalent binding loosen the lattice by intercalation leading to the complex solubilization phenomenon (Miller & Nussenzweig, 1975; Czop & Nussenzweig, 1976).
REFERENCES
Bocci V. (1964) Boll. Sot. ital. Biol. sper. 40, 89. Celada F., Schmidt D. & Strom R. (1969) Immunology 17, 183.
Czop J. & Nussenzweig V. (1976) J. exp. Med. 143, 615. Kabat E. A. & Meyer M. M. (1961) In Experimental Immunochemistrv (2nd edn). Springfield, Illindis.
Charles
C. Thomas,
Kronvall G. & Gewurz H. (1970) C/in. exp. Immunol. 7, 211. Miller G. W. & Nussenzweig V. (1975) Proc. natn Acad. Sci., U.S.A. 72, 418. SjGquist J. & Stalenheim G. (1969) J. Immun. 103, 467. Sjijquist J.. Meloun S. & Hjelm H. (1972) Eur. J. Biothem. 29, 572. Takahashi M., Czop J., Ferreira A. & Nussenzweig V. (1976) Transplantation Rev. 32, I2 I Weigle W. 0. & McConahey P. J. (1962) J. Immrrn. 88, 121. Yonemasu K. & Stroud R. M. (1971) J. fmmun. 106, 304.