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Induction of interferon by Streptococcus pyogenes extracellular products
hnmunoh)g.v Letters. 5 (1982) 323-326
Elsevier BiomedicalPress
INDUCTION
OF INTERFERON
BY STREPTOCOCCUS
EXTRACELLULAR
PYOGENES
PRODUCTS
J.-M. CAVAILLON***, Y. RIVIERE**, J. SVAB**, L. M O N T A G N I E R * * and J. E. ALOUF* * Uniti, des Antigi,nes Bactkriens (ERA CNRS no. 794). and ** Unitb aeOncoh)gie Virale (ER CNRS no. 147). lnstitut Pasteur. 28 rue du Dr. Roux. 75015 Paris. France
(Received 2 August 1982) (Modified version receivedand accepted 29 September 1982)
1. Summary l,ymphocyte-activating streptococcal exoproteins, which were previously characterized, have been tested for their capacity to induce interferon in vitro. Two out of the 3 different streptococcal fractions, studied on mice splenocytes, were shown to elicit the production of a significant amount of interferon. A large proportion of the interferon detected in the supernatants from mice activated spleen cells was acidlabile interferon. The highest level of interferon titer was obtained with the streptococcal fraction identified as the erythrogenic toxin.
2. Introduction A wide variety of immunity mediators (lymphokines, monokines . . . . ) are detected in vitro, in the supernatants of cells stimulated by mitogens. One of them, interferon, referred to as "immune interferon" may be induced by different activators such as lectins (concanavalin A, phytohaemagglutinin), antibodics (OKT 3 monoclonal antibody), allogenic cells or bacteria. In fact, among the wide variety of bacterial species, the capacity to induce interferon in vitro, has only been reported for a few defined macromolecules, such as lipopolysaccharides from E w h e r i K(v words: interferon mitogcns streptococcalexoproteins
*** Present address to which correspondenceshould be sent: Unit~: d'lmmuno-Allergie, lnstitut Pasteur, Paris. 0000 0000.82.0000 0000:S0.00 © ElsevierBiomedical Press
chia coli [ 1,2] or S a l m o n e l l a enteritidis [3], purified
protein derivative from M . v c o b a c t e r i u m [4], staphylococcal protein A [5] and staphylococcal enterotoxin A [6,7]. We have previously reported [8,9] the purification and some immunological properties of 3 different extracellular products, called 3,, x and e, purified from group A S t r e p t o c o c c u s p r o g e n e s culture supernatant fluids. To different extents, the 3 fractions were mitogenic for mice spleen cells and polyclonal activators for the antibody secretion. The most potent mitogen was the x-fraction (pl = 4.8) identified as the erythrogenic toxin and the most potent polyclonal activator was the ",/-fraction (pl = 4.2). The 3'fraction and the e-fraction (pl = 10.3) possessed adjuvant properties whereas the x-fraction suppressed an in vitro antibody immune response. The different immunological mediators induced by these different streptococcal fractions are currently being investigated. Here we report their capacity to induce in vitro synthesis of interferon.
3. Materials and methods 3. I. S t r e p t o c o c c a l . f r a c t i o n s
The purification of the extracellular streptococcal fractions has been previously described [8]. The last step of purification was a preparative isoelectrofocusing leading to the separation of 5 different fractions. Only the mitogenic fractions have been studied, i.e. 3' (pl =4.2), r (pl =4.8), and ~ (pl = 10.3). Con A (Miles) was used as a positive control. 323
3.2. Mice spleen cells culture CBA mice (8 i I weeks old) purchased from Iffa Credo (France) were used. Spleen cell suspensions were prepared in 199 medium (Inst. Pasteur). Cells were cultured in microtiter flat-bottomed plates (Nunclon delta, 96 wells). Each well contained 5 × 105 splenocytes in RPMi-1640 medium (M. A. Bioproducts, U.S.A.) supplemented with streptomycin (100 #g,'ml), penicillin (100 I.U.;ml) and 2% decomplemented fetal calf serum (BioM6rieux). Cultures were incubated at 37 ° C in an atmosphere of 5% C O r 9 5 % air (Heraeus incubator); 24 96 b later, the culture supernatants were collected, centrifuged ( 15 rain, 3000 rpm) and kept at - 70 ° C.
As shown in Fig. 1, the streptococcal fractions dtlfered in their capacity to induce interferon in mouse spleen cells. The highest level of interferon production was obtained with the K-fraction, whereas the efraction induced a very low amount of interferon; the y-fraction had an intermediate potentiality. Fluctuations of interferon production were quite negligible within the period tested (i.e. 24 96 h). A high proportion of the induced interferon seemed to be of ",/-type, since it lost its activity after a pH 2 treatment (Table I). This interferon was species specific since it had no activity on human M RC5 cells. The capacity of the supernatants to induce two interferon-dependent enzyme activities, i.e. 2 5A syn-
3.3. Interferon titration Interferon activity in the supernatants of the cell cultures was measured in a microassay using mouse 1,929 cells and vesicular stomatitis virus, as previously described [10]. Acid treatment (24 h, 4 ° C) of cell culture supernatants was done by addition of HCI (I N) to lower the pH to 2. The samples were then neutralized with NaOH (I N) before titration for interferon activity. One international unit (NIH, National Institutes of Health, U.S.A) of mouse interferon was equivalent to 30 effective laboratory units [10].
=; T
.
z
J
g ,.=,
_J
4. Results
0
Before performing interferon titration in the supernatants of activated cells, we established that no direct anti-viral activity was due to the streptococcal extracellular fractions themselves.
HOUR
S
Fig. I. Kinetics of interferon induced by 10 ,ag of the three different streptococcal fractions (A, K; m. y: o, ~).
Table 1 Titers of interferon induced by the 3 streptococcalfractions and Con A. before and after acidic treatment Activators
none
D o s e s ( p.g)a
3'
K
e
I
10
100
I
l0
100
Con A 1
10
100
I
initial
'<1
150
200
400
150
3200
1200
<1
12.5
6.25
1600
after pH 2 treatment
<1
0
9
19
12
50
400
<1
I
1.2
200
Interferon titers~ ~g/5 X 105CBA mice spleen cells/0.2 ml. b NIH units/ml; interferon detected in 96 h-culture supernatants. 324
•C •
.
.
"..
•
i:.:
•
.
:
1:::23 1 : 2 3
:
• . .:
¸
] .::i
1:23
°
::a
.:
Fig. 2. The protein kinase activity was characterized by the phosphorylation of a 67,000 d polypcptide in the presence of [~2P]A'I"P, and autoradiography as previously described [10]. An autoradiograph of a stained gel is shown. Mouse 1.929 cells were incubated with culture medium (C. control), reference mouse interferon (or ~ /3) (M.IFN), or interferon from the supernatants of mouse spleen cells cultures stimulated with the r or ",/fractions; doses were I, 10, and I(10 IFN units• ml reftered as I, 2 and 3, respectively.
thetase and protein kinase, was studied in vitro as previously described [ 10]. The level of the protein kinase activity was determined on mouse L929 cells treated with the supernatants of mouse spleen cells stimulated by the x and 333streptococcal extracellular fractions (Fig. 2). The enhancement of the enzyme activity further confirmed the interferon activity. Similar results were obtained with 2-5A synthetase activity.
5. Discussion The stimulation of lymphocytes is a complex phenomenon in which the participation of other cells and mediators are often involved. Interleukins I and 2 play a major role in the cell interactions, but other lymphokines, such as interferon may participate to modulate the cell activation [11,12]. Furthermore, the production of interferon induced by lymphocytes activators might be particularly relevant when considering the products of pathogenic bacteria as the
source of induction. Thus, to further investigate the different immunological properties of 3 different streptococcal extracellular products active on lymphocytcs, we studied their capacity to induce interferon. The x- and 3"-fractions induced significant amounts of interferon, whereas the ~-fraction was a very weak inducer. As the y- and ~-fractions possessed similar mitogenic properties on mice spleen cells [8], there is no direct relationship between the mitogenicity of the streptococcal fractions and their capacity to induce interferon. On the other hand, as already suggested for the staphylococcal enterotoxin A which is an inhibitor of the antibody immune response [13,14], one may evoke a possible relationship between the high level of interferon induced by the x-fraction and the capacity of this fraction to suppress an in vitro antibody immune response [9], since an inhibitory property on antibody secretion has been reported for interferon [ 15,16]. Among all the intact bacteria or bacterial products which induce in vivo or in vitro interferon [17] very little was known about the capacity of Streptococcus p.l'ogenes itself or its extracellular products to elicit the production of interferon; few positive tests have been reported in human with streptolysin 0 [18] and Saito et al. [19] recently reported the capacity of a preparation of Streptococcus pyogenes to induce mainly ",/-type interferon. The data reported here clearly establish the capacity of two different streptococcal exoproteins to elicit the secretion of mainly immune interferon by mouse spleen cells.
Acknowledgements This investigation was supported by the lnstitut National de la Sant~ et de |a Recherche M6dicale (INSERM, Contrat de Recherche libre no. 801001). We thank Doctor A. Hovanessian for helpful discussion.
References [1] Matisovfi, E., Bfitorov/t, E., hackovi~:. V. and Boreck~', 1,. (1970) Acta Virol. 14. 1 7. [2] Maehara, N. and Ho, M. (1977) Infect. lmmun. 15, 78 83. [3] De Clercq. E. and Merigan, T. C. (1969),I. Immunol. 103. 899 905.
325
[4] Epstein, E. B.. Cline, M. J. and Merigan, T. C. (1971) Cell. Immunol. 2, 602 613. [5] Ratliff, T. 1,., McCool. R. I:.. and Catalona. W. J. (1981) Cell. lmmunol. 57. I 12. [6] l,angford. M. P., Georgiades, J. A., Stanton, G. J.. l)ianzani, F. and Johnson, H. M. (1979) Infect. lmmun. 26, 36--41. [7] Archer, D. 1,.. Smith, B. G., Ulrich, J. T. and .lohnson, H. M. (1979) Cell. Immunol. 48. 420 426. [8] Cavaillon, J.-M. and Alouf, J. E. (1982) Immunol. Lett. 5, 317 322. [9] Cavaillon, J.-M.. Leclerc, ('. and Alouf, J. E. (1982) submitted. [10] Hovanessian. A. G. and Riviere. Y. (1980) Ann. Virol. (Inst. Pasteur) 131E, 501 516. [11] Blomgren. H. and Einhorn, S. (1981) Int. Arch. Allergy Appl. Immunol. 66. 173 178.
326
[12] Yamamoto. J. K.. Farrar. W. I,. and Johnson, H. M. (1982) (?ell. Immnnol. 66. 333 341. 113] Smith, B.G. and Johnson, H. M.(1975) J. lmmunol. 115, 575 578. 114] Johnson, H. M.. Stanton, G. J. and Baron, S. (1977) Proc. Soc. Exp. Biol. Med. 154, 138 141. [ 15] Gisler, R. H., Lindhal, A. and Gresser, I. (1974) J. Immunol. 113, 438 444. [16] Johnson. H. M.. Smith, B. G. and Baron, S. (1975)J. lmmunol. 114, 403 409. [17] Stewart, W. I'. (1980) in: The Interferon System, SpringerVerlag, Wien New York, pp. 27 57. [18] Friedman. R. M. and Cooper, It. I.. (1967) I'roc. Soc. Exp. Biol. Med. 125. 901 905. [19[ Saito, M., Ebina. ]'.. Koi. M., Yamaguchi, T., Kaw.ade, Y. and Ishida, N. (1982) (.'ell. Immunol. 68. 187 192.
Elsevier BiomedicalPress
INDUCTION
OF INTERFERON
BY STREPTOCOCCUS
EXTRACELLULAR
PYOGENES
PRODUCTS
J.-M. CAVAILLON***, Y. RIVIERE**, J. SVAB**, L. M O N T A G N I E R * * and J. E. ALOUF* * Uniti, des Antigi,nes Bactkriens (ERA CNRS no. 794). and ** Unitb aeOncoh)gie Virale (ER CNRS no. 147). lnstitut Pasteur. 28 rue du Dr. Roux. 75015 Paris. France
(Received 2 August 1982) (Modified version receivedand accepted 29 September 1982)
1. Summary l,ymphocyte-activating streptococcal exoproteins, which were previously characterized, have been tested for their capacity to induce interferon in vitro. Two out of the 3 different streptococcal fractions, studied on mice splenocytes, were shown to elicit the production of a significant amount of interferon. A large proportion of the interferon detected in the supernatants from mice activated spleen cells was acidlabile interferon. The highest level of interferon titer was obtained with the streptococcal fraction identified as the erythrogenic toxin.
2. Introduction A wide variety of immunity mediators (lymphokines, monokines . . . . ) are detected in vitro, in the supernatants of cells stimulated by mitogens. One of them, interferon, referred to as "immune interferon" may be induced by different activators such as lectins (concanavalin A, phytohaemagglutinin), antibodics (OKT 3 monoclonal antibody), allogenic cells or bacteria. In fact, among the wide variety of bacterial species, the capacity to induce interferon in vitro, has only been reported for a few defined macromolecules, such as lipopolysaccharides from E w h e r i K(v words: interferon mitogcns streptococcalexoproteins
*** Present address to which correspondenceshould be sent: Unit~: d'lmmuno-Allergie, lnstitut Pasteur, Paris. 0000 0000.82.0000 0000:S0.00 © ElsevierBiomedical Press
chia coli [ 1,2] or S a l m o n e l l a enteritidis [3], purified
protein derivative from M . v c o b a c t e r i u m [4], staphylococcal protein A [5] and staphylococcal enterotoxin A [6,7]. We have previously reported [8,9] the purification and some immunological properties of 3 different extracellular products, called 3,, x and e, purified from group A S t r e p t o c o c c u s p r o g e n e s culture supernatant fluids. To different extents, the 3 fractions were mitogenic for mice spleen cells and polyclonal activators for the antibody secretion. The most potent mitogen was the x-fraction (pl = 4.8) identified as the erythrogenic toxin and the most potent polyclonal activator was the ",/-fraction (pl = 4.2). The 3'fraction and the e-fraction (pl = 10.3) possessed adjuvant properties whereas the x-fraction suppressed an in vitro antibody immune response. The different immunological mediators induced by these different streptococcal fractions are currently being investigated. Here we report their capacity to induce in vitro synthesis of interferon.
3. Materials and methods 3. I. S t r e p t o c o c c a l . f r a c t i o n s
The purification of the extracellular streptococcal fractions has been previously described [8]. The last step of purification was a preparative isoelectrofocusing leading to the separation of 5 different fractions. Only the mitogenic fractions have been studied, i.e. 3' (pl =4.2), r (pl =4.8), and ~ (pl = 10.3). Con A (Miles) was used as a positive control. 323
3.2. Mice spleen cells culture CBA mice (8 i I weeks old) purchased from Iffa Credo (France) were used. Spleen cell suspensions were prepared in 199 medium (Inst. Pasteur). Cells were cultured in microtiter flat-bottomed plates (Nunclon delta, 96 wells). Each well contained 5 × 105 splenocytes in RPMi-1640 medium (M. A. Bioproducts, U.S.A.) supplemented with streptomycin (100 #g,'ml), penicillin (100 I.U.;ml) and 2% decomplemented fetal calf serum (BioM6rieux). Cultures were incubated at 37 ° C in an atmosphere of 5% C O r 9 5 % air (Heraeus incubator); 24 96 b later, the culture supernatants were collected, centrifuged ( 15 rain, 3000 rpm) and kept at - 70 ° C.
As shown in Fig. 1, the streptococcal fractions dtlfered in their capacity to induce interferon in mouse spleen cells. The highest level of interferon production was obtained with the K-fraction, whereas the efraction induced a very low amount of interferon; the y-fraction had an intermediate potentiality. Fluctuations of interferon production were quite negligible within the period tested (i.e. 24 96 h). A high proportion of the induced interferon seemed to be of ",/-type, since it lost its activity after a pH 2 treatment (Table I). This interferon was species specific since it had no activity on human M RC5 cells. The capacity of the supernatants to induce two interferon-dependent enzyme activities, i.e. 2 5A syn-
3.3. Interferon titration Interferon activity in the supernatants of the cell cultures was measured in a microassay using mouse 1,929 cells and vesicular stomatitis virus, as previously described [10]. Acid treatment (24 h, 4 ° C) of cell culture supernatants was done by addition of HCI (I N) to lower the pH to 2. The samples were then neutralized with NaOH (I N) before titration for interferon activity. One international unit (NIH, National Institutes of Health, U.S.A) of mouse interferon was equivalent to 30 effective laboratory units [10].
=; T
.
z
J
g ,.=,
_J
4. Results
0
Before performing interferon titration in the supernatants of activated cells, we established that no direct anti-viral activity was due to the streptococcal extracellular fractions themselves.
HOUR
S
Fig. I. Kinetics of interferon induced by 10 ,ag of the three different streptococcal fractions (A, K; m. y: o, ~).
Table 1 Titers of interferon induced by the 3 streptococcalfractions and Con A. before and after acidic treatment Activators
none
D o s e s ( p.g)a
3'
K
e
I
10
100
I
l0
100
Con A 1
10
100
I
initial
'<1
150
200
400
150
3200
1200
<1
12.5
6.25
1600
after pH 2 treatment
<1
0
9
19
12
50
400
<1
I
1.2
200
Interferon titers~ ~g/5 X 105CBA mice spleen cells/0.2 ml. b NIH units/ml; interferon detected in 96 h-culture supernatants. 324
•C •
.
.
"..
•
i:.:
•
.
:
1:::23 1 : 2 3
:
• . .:
¸
] .::i
1:23
°
::a
.:
Fig. 2. The protein kinase activity was characterized by the phosphorylation of a 67,000 d polypcptide in the presence of [~2P]A'I"P, and autoradiography as previously described [10]. An autoradiograph of a stained gel is shown. Mouse 1.929 cells were incubated with culture medium (C. control), reference mouse interferon (or ~ /3) (M.IFN), or interferon from the supernatants of mouse spleen cells cultures stimulated with the r or ",/fractions; doses were I, 10, and I(10 IFN units• ml reftered as I, 2 and 3, respectively.
thetase and protein kinase, was studied in vitro as previously described [ 10]. The level of the protein kinase activity was determined on mouse L929 cells treated with the supernatants of mouse spleen cells stimulated by the x and 333streptococcal extracellular fractions (Fig. 2). The enhancement of the enzyme activity further confirmed the interferon activity. Similar results were obtained with 2-5A synthetase activity.
5. Discussion The stimulation of lymphocytes is a complex phenomenon in which the participation of other cells and mediators are often involved. Interleukins I and 2 play a major role in the cell interactions, but other lymphokines, such as interferon may participate to modulate the cell activation [11,12]. Furthermore, the production of interferon induced by lymphocytes activators might be particularly relevant when considering the products of pathogenic bacteria as the
source of induction. Thus, to further investigate the different immunological properties of 3 different streptococcal extracellular products active on lymphocytcs, we studied their capacity to induce interferon. The x- and 3"-fractions induced significant amounts of interferon, whereas the ~-fraction was a very weak inducer. As the y- and ~-fractions possessed similar mitogenic properties on mice spleen cells [8], there is no direct relationship between the mitogenicity of the streptococcal fractions and their capacity to induce interferon. On the other hand, as already suggested for the staphylococcal enterotoxin A which is an inhibitor of the antibody immune response [13,14], one may evoke a possible relationship between the high level of interferon induced by the x-fraction and the capacity of this fraction to suppress an in vitro antibody immune response [9], since an inhibitory property on antibody secretion has been reported for interferon [ 15,16]. Among all the intact bacteria or bacterial products which induce in vivo or in vitro interferon [17] very little was known about the capacity of Streptococcus p.l'ogenes itself or its extracellular products to elicit the production of interferon; few positive tests have been reported in human with streptolysin 0 [18] and Saito et al. [19] recently reported the capacity of a preparation of Streptococcus pyogenes to induce mainly ",/-type interferon. The data reported here clearly establish the capacity of two different streptococcal exoproteins to elicit the secretion of mainly immune interferon by mouse spleen cells.
Acknowledgements This investigation was supported by the lnstitut National de la Sant~ et de |a Recherche M6dicale (INSERM, Contrat de Recherche libre no. 801001). We thank Doctor A. Hovanessian for helpful discussion.
References [1] Matisovfi, E., Bfitorov/t, E., hackovi~:. V. and Boreck~', 1,. (1970) Acta Virol. 14. 1 7. [2] Maehara, N. and Ho, M. (1977) Infect. lmmun. 15, 78 83. [3] De Clercq. E. and Merigan, T. C. (1969),I. Immunol. 103. 899 905.
325
[4] Epstein, E. B.. Cline, M. J. and Merigan, T. C. (1971) Cell. Immunol. 2, 602 613. [5] Ratliff, T. 1,., McCool. R. I:.. and Catalona. W. J. (1981) Cell. lmmunol. 57. I 12. [6] l,angford. M. P., Georgiades, J. A., Stanton, G. J.. l)ianzani, F. and Johnson, H. M. (1979) Infect. lmmun. 26, 36--41. [7] Archer, D. 1,.. Smith, B. G., Ulrich, J. T. and .lohnson, H. M. (1979) Cell. Immunol. 48. 420 426. [8] Cavaillon, J.-M. and Alouf, J. E. (1982) Immunol. Lett. 5, 317 322. [9] Cavaillon, J.-M.. Leclerc, ('. and Alouf, J. E. (1982) submitted. [10] Hovanessian. A. G. and Riviere. Y. (1980) Ann. Virol. (Inst. Pasteur) 131E, 501 516. [11] Blomgren. H. and Einhorn, S. (1981) Int. Arch. Allergy Appl. Immunol. 66. 173 178.
326
[12] Yamamoto. J. K.. Farrar. W. I,. and Johnson, H. M. (1982) (?ell. Immnnol. 66. 333 341. 113] Smith, B.G. and Johnson, H. M.(1975) J. lmmunol. 115, 575 578. 114] Johnson, H. M.. Stanton, G. J. and Baron, S. (1977) Proc. Soc. Exp. Biol. Med. 154, 138 141. [ 15] Gisler, R. H., Lindhal, A. and Gresser, I. (1974) J. Immunol. 113, 438 444. [16] Johnson. H. M.. Smith, B. G. and Baron, S. (1975)J. lmmunol. 114, 403 409. [17] Stewart, W. I'. (1980) in: The Interferon System, SpringerVerlag, Wien New York, pp. 27 57. [18] Friedman. R. M. and Cooper, It. I.. (1967) I'roc. Soc. Exp. Biol. Med. 125. 901 905. [19[ Saito, M., Ebina. ]'.. Koi. M., Yamaguchi, T., Kaw.ade, Y. and Ishida, N. (1982) (.'ell. Immunol. 68. 187 192.