Archives of Virology 49, 89--92 (1975) © by Springer-Verlag 1975
Heterologous Gamma Globulin as Interferon Inducer: Differences in the Properties of A99regated and Aggregate-Free Gamma Globulin Preparations Brief Report By
LUDMILLAPRIIMXGI, S. Ion:s, and A. KULBERG Institute of Epidemiolog2z, Microbiology and I-tygiene, Tallinn; N.F. Gamateya Institute of Epidemiology and Microbiology, Moscow', U.S.S.R. Accepted May 27, 1975
Summary Purified bovine and rabbit gamma globulin preparations are capable of inducing interferon in C3HA mice. Their aggregated form is a more effective inducer than native or aggregate-free form. , I t has been shown that protein antigens including g a m m a globulin preparations are capable of inducing interferon (2, 5, 11). In our previous experiments it has also been confirmed that antiviral substance appearing in the sera of mice in response to the injection of heterologous g a m m a globulin had the main properties of interferon as a) the absence of direct action on the virus, b) species-specific activity, e) resistance to low p g , d) sensitivity to trypsin etc. (i2). According to the preliminary data (KULBr:RG et al.), aggregated heterologous g a m m a globulin injected into mice is a more effective inducer of serum interferon than aggregate free preparation of the same protein. These findings appear to be related to the observation t h a t aggregated form of g a m m a globulin is highly immunogenic, whereas its aggregate free form usually induces immunological paralysis (4). Therefore it is of some interest to examine whether the aggregate free g a m m a globulin is capable of producing specific unresponsiveness to interferon induction. Purified bovine (BGG) and rabbit (RGG) gamma globulins (Serva) were used in this study as inducers of interferon. To prepare heat-aggregated gamma globulin, protein solution (10 mg/ml) in 0.15 :~ phosphate buffered saline (PBS) p g 7.6 was incubated at 63 ° C for 20 minutes in a water b a t h and then cooled immediately. Heated g a m m a globulin was fraetionated by eentrifugation on the swing-out rotor of the VAC-601 ultracentrifuge at t05,000×g for 2 hours according to ABDO~T and I~ICHTSR (1). The upper third of supernat~nt was separated and used as aggregate free (AF) fraction, The rest of the supernatant was discarded, the
90
LUI)MILLA PP~IIMXGI, S. IOKS, and A. KULBERG:
pellet was r e s u s p e n d e d in P B S (pH 8.0) a n d e m p l o y e d as a g g r e g a t e d (A) g a m m a globulin. P r o t e i n c o n c e n t r a t i o n in t h e p r e p a r a t i o n s was m e a s u r e d b y a b s o r b a n c e a t 280 nm. P r i o r to p r o t e i n d e t e r m i n a t i o n in A - f r a c t i o n it was dissolved in 0.1 N NaOH. G a m m a globulin p r e p a r a t i o n s were i n j e c t e d into m a l e C 3 t I A mice (weighing 20 g) i n t r a p e r i t o n e a l l y in t h e dose of 0.5 m g in 0.5 m l PBS. E a c h p r e p a r a t i o n was s i m u l t a n e o u s l y a d m i n i s t e r e d to a g r o u p of 4 mice. 18 hours l a t e r :mice were killed a n d sera from a n i m a l s of each group pooled. The interferon t i t r e of sera was d e t e r m i n e d w i t h e y t o p a t h i c effect r e d u c t i o n teehnique, using L cell eultures a n d 100 TCDs0 vesicular s t o m a t i t i s virus ( I n d i a n a strain). Several b o v i n e g a m m a globulin p r e p a r a t i o n s r e p e a t e d l y used in this s t u d y were f o u n d to induce i n t e r f e r o n p r o d u c t i o n in C3 H A mice, A - f r a c t i o n being a m o s t effective one (Table 1). Similar results were o b t a i n e d w i t h R G G . Table 1. Induction of Serum Interferon in Mice by Bovine Gamma Globulin Preparations BGG preparations
Interferon titre a
Native AF-fraction A-fraction
32 64 512
Interferon titre in this and following table is expressed as the reciprocal of highest dilution of the serum sample which reduced the cytopathie effect of the 100TCDs0 vesicular stomatitis virus on L cells by 50 per cent. The values are average from at least two titrations. Table 2. Titre of Interferon in Mice after Repeated Injections of Different Gamma Globulin Preparations
1st injection
2nd injection
Intervals between injeetions (days)
BGG BGG BGG BGG
Native AF-fraction AF-fraction AF-fraetion
BGG BGG BGG BGG
A-fraction AF-fraction A-fraction A-fraction
14 14 7 14
256 16 22 52
BGG BGG RGG RGG
AF-fraetion AF-fraction AF-fraction AF-fraction
RGG RGG BGG BGG
A-fraction A-fraction A-fraction A-fraction
7 14 7 14
128 256 182 182
G a m m a globulin preparations
Interferon titre
To find o u t w h e t h e r a n injection of g a m m a globulin could p r e v e n t interferon p r o d u c t i o n u p o n r e p e a t e d a d m i n i s t r a t i o n of this protein, different p r e p a r a t i o n s of t h e g a m m a globulin were i n j e c t e d into mice a t i n t e r v a l s of 7 or 14 d a y s . V~Then mice were first i n j e c t e d w i t h A F - f r a c t i o n of BGG, t h e i n j e c t i o n of A - f r a c t i o n of B G G r e s u l t e d in s u b s t a n t i a l l y lower i n t e r f e r o n p r o d u c t i o n t h a n after a single
I-Ieterologous Gamma Globulin as Interferon Inducer
91
injection of A-fraction or an injection of A-fraction followed by the administration of native BGG. On the o~her hand, in mice which had been sensitized with AFfraction of t~GG, and then challenged with A-fraction of BGG or vice versa, the interferon production was comparable to that after a single injection of A-fraction (Tables 1 and 2). Thus AF-fraetion of g a m m a globulin could produce prolonged unresponsiveness to the g a m m a globulin of the same origin. I t is known t h a t viral and non-viral interferonogens can_ induce unresponsiveness either to the same or a different interferonogen (6, 7, 8, 10, 14). I n this respect heterologous g a m m a globulin is different because a) aggregate free g a m m a globulin, being a weak inducer of interferon, can still elicit tolerance and b) the resulting tolerance is highly specific. These observations evoke some interest since it has been demonstrated that aggregate free g a m m a globulin can induce specific immunological tolerance whereas aggregated g a m m a globulin possesses strong immunogenic activity (4). Therefore it seems likely that cells which produce interferon in response to antigen (e. g. heterologous g a m m a globulin), belong to lymphoeytes preeommitted to this antigen and immunological paralysis of immurtoeomloentent cells is associated with their inability to produce interferon. Because of an ever increasing evidence of interferon participation in immune responses (3, 13), it will be important to evaluate interferon function in inducing and terminating immunological tolerance.
References I. ABDOU, N. I., RICHTER,, M. : The secondary antibody response in tissue culture. IV. Studies of the in rive and in vitro antigenicity of native aggregate-free and aggregated human gamma-globulin in rabbits. Immunology 18, 833--841 (1970). 2. BELADI, I.: Interferon induction by adenoviruses in wJtro and in vivo. Prec. Microbiol. Res. Group Hung. Acad. Sci. 4, 29--33 (1972). 3. CHESTER, T., PA~eKE~, ]~., ~{ER~GAN, T. : Suppression of mouse antibody producting spleen cells by various interferon preparations. Nature 246, 5428, 92--94 (1973). ~. I)I~ESSER, D. W., ~ITCHISOI~, I~. A. : The mechanism of immunological paralysis. Adv. Immunol. 8, 129---181 (1968). 5. FALeOFF, i%. : Some properties of virus and immunoinduced human lymphocyte interferons. J. gen. ¥irol. 16, 251--253 (1972). 6. GII~O~, D. J., SCH~IDT, J. P., PINDAI
Heterologous Gamma Globulin as Interferon Inducer: Differences in the Properties of A99regated and Aggregate-Free Gamma Globulin Preparations Brief Report By
LUDMILLAPRIIMXGI, S. Ion:s, and A. KULBERG Institute of Epidemiolog2z, Microbiology and I-tygiene, Tallinn; N.F. Gamateya Institute of Epidemiology and Microbiology, Moscow', U.S.S.R. Accepted May 27, 1975
Summary Purified bovine and rabbit gamma globulin preparations are capable of inducing interferon in C3HA mice. Their aggregated form is a more effective inducer than native or aggregate-free form. , I t has been shown that protein antigens including g a m m a globulin preparations are capable of inducing interferon (2, 5, 11). In our previous experiments it has also been confirmed that antiviral substance appearing in the sera of mice in response to the injection of heterologous g a m m a globulin had the main properties of interferon as a) the absence of direct action on the virus, b) species-specific activity, e) resistance to low p g , d) sensitivity to trypsin etc. (i2). According to the preliminary data (KULBr:RG et al.), aggregated heterologous g a m m a globulin injected into mice is a more effective inducer of serum interferon than aggregate free preparation of the same protein. These findings appear to be related to the observation t h a t aggregated form of g a m m a globulin is highly immunogenic, whereas its aggregate free form usually induces immunological paralysis (4). Therefore it is of some interest to examine whether the aggregate free g a m m a globulin is capable of producing specific unresponsiveness to interferon induction. Purified bovine (BGG) and rabbit (RGG) gamma globulins (Serva) were used in this study as inducers of interferon. To prepare heat-aggregated gamma globulin, protein solution (10 mg/ml) in 0.15 :~ phosphate buffered saline (PBS) p g 7.6 was incubated at 63 ° C for 20 minutes in a water b a t h and then cooled immediately. Heated g a m m a globulin was fraetionated by eentrifugation on the swing-out rotor of the VAC-601 ultracentrifuge at t05,000×g for 2 hours according to ABDO~T and I~ICHTSR (1). The upper third of supernat~nt was separated and used as aggregate free (AF) fraction, The rest of the supernatant was discarded, the
90
LUI)MILLA PP~IIMXGI, S. IOKS, and A. KULBERG:
pellet was r e s u s p e n d e d in P B S (pH 8.0) a n d e m p l o y e d as a g g r e g a t e d (A) g a m m a globulin. P r o t e i n c o n c e n t r a t i o n in t h e p r e p a r a t i o n s was m e a s u r e d b y a b s o r b a n c e a t 280 nm. P r i o r to p r o t e i n d e t e r m i n a t i o n in A - f r a c t i o n it was dissolved in 0.1 N NaOH. G a m m a globulin p r e p a r a t i o n s were i n j e c t e d into m a l e C 3 t I A mice (weighing 20 g) i n t r a p e r i t o n e a l l y in t h e dose of 0.5 m g in 0.5 m l PBS. E a c h p r e p a r a t i o n was s i m u l t a n e o u s l y a d m i n i s t e r e d to a g r o u p of 4 mice. 18 hours l a t e r :mice were killed a n d sera from a n i m a l s of each group pooled. The interferon t i t r e of sera was d e t e r m i n e d w i t h e y t o p a t h i c effect r e d u c t i o n teehnique, using L cell eultures a n d 100 TCDs0 vesicular s t o m a t i t i s virus ( I n d i a n a strain). Several b o v i n e g a m m a globulin p r e p a r a t i o n s r e p e a t e d l y used in this s t u d y were f o u n d to induce i n t e r f e r o n p r o d u c t i o n in C3 H A mice, A - f r a c t i o n being a m o s t effective one (Table 1). Similar results were o b t a i n e d w i t h R G G . Table 1. Induction of Serum Interferon in Mice by Bovine Gamma Globulin Preparations BGG preparations
Interferon titre a
Native AF-fraction A-fraction
32 64 512
Interferon titre in this and following table is expressed as the reciprocal of highest dilution of the serum sample which reduced the cytopathie effect of the 100TCDs0 vesicular stomatitis virus on L cells by 50 per cent. The values are average from at least two titrations. Table 2. Titre of Interferon in Mice after Repeated Injections of Different Gamma Globulin Preparations
1st injection
2nd injection
Intervals between injeetions (days)
BGG BGG BGG BGG
Native AF-fraction AF-fraction AF-fraetion
BGG BGG BGG BGG
A-fraction AF-fraction A-fraction A-fraction
14 14 7 14
256 16 22 52
BGG BGG RGG RGG
AF-fraetion AF-fraction AF-fraction AF-fraction
RGG RGG BGG BGG
A-fraction A-fraction A-fraction A-fraction
7 14 7 14
128 256 182 182
G a m m a globulin preparations
Interferon titre
To find o u t w h e t h e r a n injection of g a m m a globulin could p r e v e n t interferon p r o d u c t i o n u p o n r e p e a t e d a d m i n i s t r a t i o n of this protein, different p r e p a r a t i o n s of t h e g a m m a globulin were i n j e c t e d into mice a t i n t e r v a l s of 7 or 14 d a y s . V~Then mice were first i n j e c t e d w i t h A F - f r a c t i o n of BGG, t h e i n j e c t i o n of A - f r a c t i o n of B G G r e s u l t e d in s u b s t a n t i a l l y lower i n t e r f e r o n p r o d u c t i o n t h a n after a single
I-Ieterologous Gamma Globulin as Interferon Inducer
91
injection of A-fraction or an injection of A-fraction followed by the administration of native BGG. On the o~her hand, in mice which had been sensitized with AFfraction of t~GG, and then challenged with A-fraction of BGG or vice versa, the interferon production was comparable to that after a single injection of A-fraction (Tables 1 and 2). Thus AF-fraetion of g a m m a globulin could produce prolonged unresponsiveness to the g a m m a globulin of the same origin. I t is known t h a t viral and non-viral interferonogens can_ induce unresponsiveness either to the same or a different interferonogen (6, 7, 8, 10, 14). I n this respect heterologous g a m m a globulin is different because a) aggregate free g a m m a globulin, being a weak inducer of interferon, can still elicit tolerance and b) the resulting tolerance is highly specific. These observations evoke some interest since it has been demonstrated that aggregate free g a m m a globulin can induce specific immunological tolerance whereas aggregated g a m m a globulin possesses strong immunogenic activity (4). Therefore it seems likely that cells which produce interferon in response to antigen (e. g. heterologous g a m m a globulin), belong to lymphoeytes preeommitted to this antigen and immunological paralysis of immurtoeomloentent cells is associated with their inability to produce interferon. Because of an ever increasing evidence of interferon participation in immune responses (3, 13), it will be important to evaluate interferon function in inducing and terminating immunological tolerance.
References I. ABDOU, N. I., RICHTER,, M. : The secondary antibody response in tissue culture. IV. Studies of the in rive and in vitro antigenicity of native aggregate-free and aggregated human gamma-globulin in rabbits. Immunology 18, 833--841 (1970). 2. BELADI, I.: Interferon induction by adenoviruses in wJtro and in vivo. Prec. Microbiol. Res. Group Hung. Acad. Sci. 4, 29--33 (1972). 3. CHESTER, T., PA~eKE~, ]~., ~{ER~GAN, T. : Suppression of mouse antibody producting spleen cells by various interferon preparations. Nature 246, 5428, 92--94 (1973). ~. I)I~ESSER, D. W., ~ITCHISOI~, I~. A. : The mechanism of immunological paralysis. Adv. Immunol. 8, 129---181 (1968). 5. FALeOFF, i%. : Some properties of virus and immunoinduced human lymphocyte interferons. J. gen. ¥irol. 16, 251--253 (1972). 6. GII~O~, D. J., SCH~IDT, J. P., PINDAI
